U.S. patent application number 13/497195 was filed with the patent office on 2012-07-26 for polypeptides and uses thereof.
Invention is credited to Martina Kalle, Nils Martin Malmsten, Praveen Papareddy, Victoria Rydengard, Artur Schmidtchen, Bjorn Ulrik Walse.
Application Number | 20120189673 13/497195 |
Document ID | / |
Family ID | 41278086 |
Filed Date | 2012-07-26 |
United States Patent
Application |
20120189673 |
Kind Code |
A1 |
Kalle; Martina ; et
al. |
July 26, 2012 |
POLYPEPTIDES AND USES THEREOF
Abstract
The present invention provides polypeptides comprising or
consisting of an amino acid sequence derived from a naturally
occurring protein which modulates blood coagulation, or a fragment,
variant, fusion or derivative thereof, or a fusion of said
fragment, variant or derivative thereof, for use in the treatment
or prevention of inflammation and/or excessive coagulation of the
blood. Related aspects of the invention provide isolated
polypeptides comprising or consisting of an amino acid sequence of
SEQ ID NOs: 1 to 11, or a fragment, variant, fusion or derivative
thereof, or a fusion of said fragment, variant or derivative
thereof which exhibit an anti-inflammatory and/or anti-coagulant
activity, together with isolated nucleic acid molecules, vectors
and host cells for making the same. Additionally provided are
pharmaceutical compositions comprising a polypeptide of the
invention, as well as methods of use of the same in the treatment
and/or prevention of inflammation and/or excessive coagulation.
Inventors: |
Kalle; Martina; (Lund,
SE) ; Malmsten; Nils Martin; (Taby, SE) ;
Papareddy; Praveen; (Lund, SE) ; Rydengard;
Victoria; (Vellinge, SE) ; Schmidtchen; Artur;
(Lund, SE) ; Walse; Bjorn Ulrik; (Lund,
SE) |
Family ID: |
41278086 |
Appl. No.: |
13/497195 |
Filed: |
September 22, 2010 |
PCT Filed: |
September 22, 2010 |
PCT NO: |
PCT/GB2010/001781 |
371 Date: |
March 20, 2012 |
Current U.S.
Class: |
424/400 ;
514/1.4; 514/1.5; 514/1.7; 514/14.5; 514/2.3; 530/324 |
Current CPC
Class: |
A61P 29/00 20180101;
A61P 11/08 20180101; A61P 37/06 20180101; A61P 37/08 20180101; A61P
33/00 20180101; A61P 17/00 20180101; A61K 38/57 20130101; A61P
17/02 20180101; A61P 1/18 20180101; A61P 31/04 20180101; A61P 31/12
20180101; A61P 11/06 20180101; A61K 31/55 20130101; A61K 38/00
20130101; A61P 9/00 20180101; A61P 1/04 20180101; A61P 5/00
20180101; A61P 17/04 20180101; A61P 31/10 20180101; A61P 19/02
20180101; A61P 39/02 20180101; A61P 1/02 20180101; A61P 11/00
20180101; A61P 13/02 20180101; A61P 31/00 20180101; C07K 14/8121
20130101; A61K 45/06 20130101; A61P 27/02 20180101; A61K 31/573
20130101; A61P 11/02 20180101; A61P 43/00 20180101; A61P 1/16
20180101; A61P 7/02 20180101; A61P 1/00 20180101; A61K 31/573
20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/400 ;
514/14.5; 514/1.4; 514/1.5; 514/2.3; 514/1.7; 530/324 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61P 7/02 20060101 A61P007/02; A61P 29/00 20060101
A61P029/00; C07K 14/81 20060101 C07K014/81; A61P 11/00 20060101
A61P011/00; A61P 31/00 20060101 A61P031/00; A61P 11/06 20060101
A61P011/06; A61K 38/57 20060101 A61K038/57; A61P 5/00 20060101
A61P005/00 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 22, 2009 |
GB |
0916578.8 |
Claims
1. A method for treating or preventing inflammation and/or
excessive coagulation of the blood in a patient, the method
comprising administering to the patient a therapeutically effective
amount of a polypeptide comprising an amino acid sequence derived
from a tissue factor pathway inhibitor (TFPI), or a fragment,
variant, fusion or derivative thereof, or a fusion of said
fragment, variant or derivative thereof, wherein the tissue factor
pathway inhibitor is selected from the group consisting of TFPI-1
and TFPI-2, and wherein the fragment, variant, fusion or derivative
exhibits an anti-inflammatory and/or anti-coagulant activity.
2-14. (canceled)
15. The method according to claim 1, wherein the tissue factor
pathway inhibitor is TFPI-1.
16. The method according to claim 15, wherein the TFPI-1 is Swiss
Port Accession No. P10646.
17. The method according to claim 1 wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO:4: TABLE-US-00010
"GGL27": [SEQ ID NO: 4] GGLIKTKRKRKKQRVKIAYEEIFVKNM
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID
NO:4.
18. The method according to claim 17, wherein the polypeptide
consists of the amino acid sequence of SEQ ID NO:4.
19. The method according to claim 1, wherein the tissue factor
pathway inhibitor is TFPI-2.
20. The method according to claim 19 wherein the TFPI-2 is Swiss
Port Accession No. P48307.
21. The method according to claim 19, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO:8: TABLE-US-00011
"EDC34": [SEQ ID NO: 8] EDCKRACAKALKKKKKMPKLRFASRIRKIRKKQF
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID
NO:8.
22. The method according to claim 21, wherein the polypeptide
consists of the amino acid sequence of SEQ ID NO:8.
23-48. (canceled)
49. The method according to claim 1, wherein said inflammation
and/or or excessive coagulation of the blood in a patient is
associated with a disease, condition or indication selected from
the following: i) Acute systemic inflammatory disease, with or
without an infective component, such as systemic inflammatory
response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic
shock. Other generalized or localized invasive infective and
inflammatory disease, including erysipelas, meningitis, arthritis,
toxic shock syndrome, diverticulitis, appendicitis, pancreatitis,
cholecystitis, colitis, cellulitis, burn wound infections,
pneumonia, urinary tract infections, postoperative infections, and
peritonitis; ii) Chronic inflammatory and or infective diseases,
including cystic fibrosis, COPD and other pulmonary diseases,
gastrointestinal disease including chronic skin and stomach
ulcerations, other epithelial inflammatory and or infective disease
such as atopic dermatitis, oral ulcerations (aphtous ulcers),
genital ulcerations and inflammatory changes, parodontitis, eye
inflammations including conjunctivitis and keratitis, external
otitis, mediaotitis, genitourinary inflammations; iii)
Postoperative inflammation. Inflammatory and coagulative disorders
including thrombosis, DIC, postoperative coagulation disorders, and
coagulative disorders related to contact with foreign material,
including extracorporeal circulation, and use of biomaterials.
Furthermore, vasculitis related inflammatory disease, as well as
allergy, including allergic rhinitis and asthma; iv) Excessive
contact activation and/or coagulation in relation to, but not
limited to, stroke; or v) Excessive inflammation in combination
with antimicrobial treatment.
50-55. (canceled)
56. An isolated polypeptide comprising an amino acid sequence
derived from a tissue factor pathway inhibitor (TFPI), or a
fragment, variant, fusion or derivative thereof, or a fusion of
said fragment, variant or derivative thereof, which polypeptide
exhibits an anti-inflammatory and/or anti-coagulant activity,
wherein the tissue factor pathway inhibitor is selected from the
group consisting of TFPI-1 and TFPI 2, with the proviso that the
polypeptide is not a naturally occurring protein.
57-70. (canceled)
71. The polypeptide according to claim 56, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO:4: TABLE-US-00012
"GGL27": [SEQ ID NO: 4] GGLIKTKRKRKKQRVKIAYEEIFVKNM
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID
NO:4.
72. The polypeptide according to claim 71, wherein the polypeptide
consists of the amino acid sequence of SEQ ID NO:4.
73-74. (canceled)
75. The polypeptide according to claim 56, wherein the polypeptide
comprises the amino acid sequence of SEQ ID NO:8: TABLE-US-00013
"EDC34": [SEQ ID NO: 8] EDCKRACAKALKKKKKMPKLRFASRIRKIRKKQF
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID
NO:8.
76. The polypeptide according to claim 75, wherein the polypeptide
consists of the amino acid sequence of SEQ ID NO:8.
77-102. (canceled)
103. A pharmaceutical composition comprising a polypeptide
according to claim 56 together with a pharmaceutically acceptable
excipient, diluent, carrier, buffer or adjuvant.
104. The pharmaceutical composition according to claim 103, wherein
said composition is suitable for administration via a route
selected from the group consisting of topical, ocular, nasal,
pulmonar, buccal, parenteral (intravenous, subcutaneous,
intrathecal and intramuscular), oral, vaginal and rectal.
105. The pharmaceutical composition according to claim 103, wherein
said composition is suitable for administration via an implant.
106. The pharmaceutical composition according to claim 103, wherein
the pharmaceutical composition is associated with a device or
material to be used in medicine.
107. (canceled)
108. The pharmaceutical composition according to claim 103, wherein
the pharmaceutical composition is coated, painted, sprayed or
otherwise applied to a suture, prosthesis, implant, wound dressing,
catheter, lens, skin graft, skin substitute, fibrin glue or
bandage.
109-137. (canceled)
Description
FIELD OF THE INVENTION
[0001] The present invention relates to novel polypeptides derived
from naturally occurring proteins which modulates blood coagulation
and their use in the treatment and prevention of inflammation
and/or excessive coagulation. In particular, the invention provides
polypeptides comprising or consisting of an amino acid sequence of
SEQ ID NOs: 1 to 11, or a fragment, variant, fusion or derivative
thereof, or a fusion of said fragment, variant or derivative
thereof, for use in medicine, e.g. the treatment or prevention of
inflammation and/or excessive coagulation.
INTRODUCTION
[0002] The innate immune system, largely based on antimicrobial
peptides, provides a first line of defense against invading
microbes (Lehrer, R. I. and T. Ganz, Curr Opin Hematol, 2002. 9(1):
p. 18-22; Harder, J., R. Glaser, and J. M. Schroder, J Endotoxin
Res, 2007. 13(6): p. 317-38; Zasloff, M., Nature, 2002. 415(6870):
p. 389-95; Tossi, A., L. Sandri, and A. Giangaspero, Biopolymers,
2000. 55(1): p. 4-30; Yount, N. Y., et al., Biopolymers, 2006).
During recent years it has become increasingly evident that many
cationic and amphipathic antimicrobial peptides, such as defensins
and cathelicidins, are multifunctional, also mediating
immunomodulatory roles and angiogenesis (Zanetti, M., J Leukoc
Biol, 2004. 75(1): p. 39-48; Elsbach, P., J Clin Invest, 2003.
111(11): p. 1643-5; Ganz, T., Defensins: antimicrobial peptides of
innate immunity. Nat Rev Immunol, 2003. 3(9): p. 710-20), thus
motivating the recent and broader definition host defense peptides
(HDP) for these members of the innate immune system. The family of
HDPs has recently been shown to encompass various bioactive
peptides with antimicrobial activities, including proinflammatory
and chemotactic chemokines (Cole, A. M., et al., J Immunol, 2001.
167(2): p. 623-7), neuropeptides (Brogden, K. A., Nat Rev
Microbiol, 2005. 3(3): p. 238-50), peptide hormones (Kowalska, K.,
D. B. Carr, and A. W. Lipkowski, Life Sci, 2002. 71(7): p. 747-50;
Mor, A., M. Amiche, and P. Nicolas, Biochemistry, 1994. 33(21): p.
6642-50), growth factors (Malmsten, M., et al., Growth Factors,
2007. 25(1): p. 60-70), the anaphylatoxin peptide C3a (Nordahl, E.
A., et al., Natl Acad Sci USA, 2004. 101(48): p. 16879-84;
Pasupuleti, M., et al., J Biol Chem, 2007. 282(4): p. 2520-8), and
kininogen-derived peptides (Frick, I. M., et al., Embo J, 2006.
25(23): p. 5569-78; Nordahl, E. A., et al., Domain 5 of high
molecular weight kininogen is antibacterial. J Biol Chem, 2005.
280(41): p. 34832-9; Rydengard, V., E. Andersson Nordahl, and A.
Schmidtchen, Zinc potentiates the antibacterial effects of
histidine-rich peptides against Enterococcus faecalis. Febs J,
2006. 273(11): p. 2399-406).
[0003] The coagulation cascade also represents a fundamental system
activated in response to injury and infection (Davie, E. W. and J.
D. Kulman, Semin Thromb Hemost, 2006. 32 Suppl 1: p. 3-15; Bode,
W., Semin Thromb Hemost, 2006. 32 Suppl 1: p. 16-31). Through a
series of cascade-like proteinase activation steps, thrombin is
formed, leading to fibrinogen degradation and clot formation. The
coagulation cascade is controlled by various regulatory proteins,
such as the serine proteinase inhibitors (or "serpins") heparin
cofactor II (HCII), antithrombin III (ATIII) and protein C
inhibitor, as well as by tissue factor proteinase inhibitor (TFPI).
Furthermore, histidine-rich glycoprotein may modulate coagulation
by interacting with fibrinogen as well as plasminogen.
[0004] The present invention seeks to provide new polypeptide
agents, derived from molecules involved in hemostasis, for use in
medicine, for example in the treatment or prevention of
inflammation and/or excessive coagulation.
SUMMARY OF THE INVENTION
[0005] A first aspect of the invention provides a polypeptide
comprising or consisting of an amino acid sequence derived from a
naturally occurring protein which modulates blood coagulation
(other than heparin cofactor II or thrombin), or a fragment,
variant, fusion or derivative thereof, or a fusion of said
fragment, variant or derivative thereof, for use in the treatment
or prevention of inflammation and/or excessive coagulation, wherein
the fragment, variant, fusion or derivative exhibits an
anti-inflammatory and/or anti-coagulant activity.
[0006] The invention derives from the unexpected discovery by the
inventors that naturally occurring proteins which modulate blood
coagulation comprise "cryptic peptides" within their C-terminal,
and/or other internal regions, which exhibit anti-inflammatory
activity. It is believed that such peptides may be `released` by
cleavage of the parent peptidase holoprotein in response to
wounding and other physiological challenges. Thus, the polypeptides
of the invention constitute a novel and previously undisclosed
class of HDPs, which have therapeutic potential against disorders
and conditions associated with inflammation.
[0007] By "naturally occurring protein which modulates blood
coagulation" we include all naturally occurring proteins which
modulates blood coagulation which modulate, either positively or
negatively, the blood coagulation process. Such modulatory activity
may be determined by methods well known in the art, for example
using the activated partial thromboplastin time (aPTT) test,
prothrombin time (PT) test or the thrombin clotting time (TCT)
test. Furthermore, specific measurements of prekallikrein
activation or the activity of Factor X and other coagulation
factors may be performed. It will be appreciated by persons skilled
in the art that the naturally occurring protein may modulate blood
coagulation directly or indirectly.
[0008] Advantageously, the naturally occurring protein which
modulates blood coagulation is a human protein.
[0009] By an amino acid sequence "derived from" a naturally
occurring protein which modulates blood coagulation, we mean that
the amino acid sequence is found within the amino acid sequence of
the naturally occurring protein. For example, in one embodiment the
amino acid sequence may be from the C-terminal region of a
naturally occurring protein which modulates blood coagulation. By
"C-terminal region" we mean that the one hundred amino acids
adjacent the C-terminus of the naturally occurring protein. In
another embodiment, the amino acid sequence may be from an internal
region of up to 100 amino acids of a naturally occurring protein
which modulates blood coagulation.
[0010] By "anti-inflammatory activity" we mean an ability to reduce
or prevent one or more biological processes associated with
inflammatory events. Such anti-inflammatory activity of
polypeptides may be determined using methods well known in the art,
for example by measuring LPS-induced release of pro-inflammatory
cytokines from macrophages (e.g. TNF.alpha., IL-6, IF-.gamma.), or
neutrophils (see Examples below). Other relevant assays comprise
effects of lipoteichoic acid, zymosan, DNA, RNA, flagellin or
peptidoglycan in the above systems as well as determination of
regulation at the transcriptional level (e.g. Gene-array, qPCR
etc). Furthermore, dendritic cell activation or activation of
thrombocytes may also be used as a measure of anti-inflammatory
activity.
[0011] By "anti-coagulant activity" we mean an ability to increase
the prothrombin time (PT), the thrombin clotting time (TCT) and/or
the activated partial thromboplastin time (aPTT). Alternatively,
peripheral blood mononuclear cells (PBMNC)s can be stimulated by E.
coli LPS with or without the peptide and tissue factor and clot
formation followed after addition of human plasma, or clotting
times for whole blood can be measured.
[0012] It will be appreciated by persons skilled in the art that
the invention encompasses polypeptides comprising or consisting of
an amino acid sequence derived from a naturally occurring protein
which modulates blood coagulation, as well as fragments, variants,
fusions and derivatives of such amino acid sequence which retain an
anti-inflammatory activity. Preferably, however, the polypeptide is
not a naturally occurring protein, e.g. a holoprotein (although it
will, of course, be appreciated that the polypeptide may constitute
an incomplete portion or fragment of a naturally occurring
protein).
[0013] In one embodiment of the polypeptides of the invention, the
polypeptide comprises a heparin-binding domain. By "heparin-binding
domain" we mean an amino acid sequence within the polypeptide which
is capable of binding heparin under physiological conditions. Such
sequences often comprise XBBXB and XBBBXXB (where B=basic residue
and X=hydropathic or uncharged residue), or clusters of basic amino
acids (XBX, XBBX, XBBBX). Spacing of such clusters with non-basic
residues (BXB, BXXB) may also occur. Additionally, a distance of
approximately 20 .ANG. between basic amino acids constitutes a
prerequisite for heparin-binding.
[0014] It will be appreciated by persons skilled in the art that
the naturally occurring protein which modulates blood coagulation
may be from a human or non-human source. For example, the naturally
occurring protein which modulates blood coagulation may be derived
(directly or indirectly) from a non-human mammal, such as an ape
(e.g. chimpanzee, bonobo, gorilla, gibbon and orangutan), monkey
(e.g. macaque, baboon and colobus), rodent (e.g. mouse, rat) or
ungulates (e.g. pig, horse and cow).
[0015] In preferred embodiments of the polypeptides of the
invention, the naturally occurring protein which modulates blood
coagulation is a human protein.
[0016] Thus, the naturally occurring protein which modulates blood
coagulation may be selected from the group consisting of serine
proteinase inhibitors (serpins), tissue factor pathway inhibitors
(such as TFPI-1 and TFPI-2) and histidine-rich glycoprotein
(HRG).
[0017] In one embodiment, the naturally occurring protein which
modulates blood coagulation is a serpin.
[0018] For example, the serpin may be anti-thrombin III (ATIII;
e.g. see Swiss Port Accession No. P01008).
[0019] Thus, the polypeptide may comprise or consist of the amino
acid sequence of SEQ ID NO:1 or 2:
TABLE-US-00001 "FFF21": [SEQ ID NO: 1] FFAKLNCRLYRKANKSSKLV
"AKL22": [SEQ ID NO: 2] AKLNCRLYRKANKSSKLVSANR
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory activity and/or anti-coagulant of SEQ ID NO:1 or
2.
[0020] In an alternative embodiment, the serpin may be protein C
inhibitor (PCI; e.g. see Swiss Port Accession No. P05154).
[0021] For example, the polypeptide may comprise or consist of the
amino acid sequence of SEQ ID NO:3:
TABLE-US-00002 "SEK20": [SEQ ID NO: 3] SEKTLRKWLKMFKKRQLELY
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID
NO:3.
[0022] In a further embodiment, the naturally occurring protein
which modulates blood coagulation is tissue factor pathway
inhibitor-1 (TFPI-1; e.g. see Swiss Port Accession No. P10646).
[0023] For example, the polypeptide may comprise or consist of the
amino acid sequence of any one of SEQ ID NOS:4 to 6:
TABLE-US-00003 "GGL27": [SEQ ID NO: 4] GGLIKTKRKRKKQRVKIAYEEIFVKNM
"LIK17": [SEQ ID NO: 5] LIKTKRKRKKQRVKIAY "TKR22": [SEQ ID NO: 6]
TKRKRKKQRVKIAYEEIFVKNM
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of any one of SEQ
ID NOS:4 to 6.
[0024] In a still further alternative embodiment, the naturally
occurring protein which modulates blood coagulation is tissue
factor pathway inhibitor 2 (TFPI-2; e.g. see Swiss Port Accession
No. P48307).
[0025] For example, the polypeptide may comprise or consist of the
amino acid sequence of SEQ ID NO:7 or 8:
TABLE-US-00004 "ALK25": [SEQ ID NO: 7] ALKKKKKMPKLRFASRIRKIRKKQF
"EDC34": [SEQ ID NO: 8] EDCKRACAKALKKKKKMPKLRFASRIRKIRKKQF
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID NO:7 or
8.
[0026] In a still further alternative embodiment, the naturally
occurring protein which modulates blood coagulation is
histidine-rich glycoprotein (HRG; e.g. see Swiss Port Accession No.
P04196).
[0027] For example, the polypeptide may comprise or consist of the
amino acid sequence of SEQ ID NO:9:
TABLE-US-00005 [SEQ ID NO: 9]
(X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5).sub.n,
wherein [0028] X.sub.1 and X.sub.4 independently represent G, P, L,
I, F, T, V, Y or W; [0029] X.sub.2, X.sub.3 and X.sub.5
independently represent H, R or K; and [0030] `n` is an integer
from 2 to 6 or a fragment, variant, fusion or derivative thereof,
or a fusion of said fragment, variant or derivative thereof, which
retains an anti-inflammatory and/or anti-coagulant activity of SEQ
ID NO:9.
[0031] Thus, the polypeptide may comprise or consist of the amino
acid sequence of SEQ ID NO:10:
TABLE-US-00006 [SEQ ID NO: 10] (GHHPH).sub.n,
where `n` is an integer from 2 to 6 or a fragment, variant, fusion
or derivative thereof, or a fusion of said fragment, variant or
derivative thereof, which retains an anti-inflammatory and/or
anti-coagulant activity of SEQ ID NO:10.
[0032] For example, the polypeptide may comprise or consist of the
amino acid sequence of SEQ ID NO:11:
TABLE-US-00007 "GHH25": [SEQ ID NO: 11]
GHHPHGHHPHGHHPHGHHPHGHHPH
or a fragment, variant, fusion or derivative thereof, or a fusion
of said fragment, variant or derivative thereof, which retains an
anti-inflammatory and/or anti-coagulant activity of SEQ ID
NO:11.
[0033] It will be appreciated by persons skilled in the art that
the term `amino acid`, as used herein, includes the standard twenty
genetically-encoded amino acids and their corresponding
stereoisomers in the `D` form (as compared to the natural `L`
form), omega-amino acids other naturally-occurring amino acids,
unconventional amino acids (e.g., .alpha.,.alpha.-disubstituted
amino acids, N-alkyl amino acids, etc.) and chemically derivatised
amino acids (see below).
[0034] When an amino acid is being specifically enumerated, such as
`alanine` or `Ala` or `A`, the term refers to both L-alanine and
D-alanine unless explicitly stated otherwise.
[0035] Other unconventional amino acids may also be suitable
components for polypeptides of the present invention, as long as
the desired functional property is retained by the polypeptide. For
the peptides shown, each encoded amino acid residue, where
appropriate, is represented by a single letter designation,
corresponding to the trivial name of the conventional amino
acid.
[0036] In one embodiment, the polypeptides of the invention
comprise or consist of L-amino acids.
[0037] Where the polypeptide comprises an amino acid sequence
according to a reference sequence (for example, SEQ ID NOs: 1 to
11), it may comprise additional amino acids at its N- and/or
C-terminus beyond those of the reference sequence, for example, the
polypeptide may comprise additional amino acids at its N-terminus.
Likewise, where the polypeptide comprises a fragment, variant or
derivative of an amino acid sequence according to a reference
sequence, it may comprise additional amino acids at its N- and/or
C-terminus.
[0038] In a further embodiment the polypeptide comprises or
consists of a fragment of the amino acid sequence according to a
reference sequence (for example, SEQ ID NOs: 1 to 11). Thus, the
polypeptide may comprise or consist of at least 5 contiguous amino
acid of the reference sequence, for example at least 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 contiguous amino
acid of SEQ ID NOS: 1 to 11.
[0039] In one embodiment the polypeptide fragment commences at an
amino acid residue selected from amino acid residues 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 of SEQ ID NO:1.
Alternatively/additionally, the polypeptide fragment may terminate
at an amino acid residue selected from amino acid residues 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 and of SEQ ID
NO:1.
[0040] It will be appreciated by persons skilled in the art that
the polypeptide of the invention may comprise or consist of a
variant of the amino acid sequence according to a reference
sequence (for example, SEQ ID NO: 1), or fragment of said variant.
Such a variant may be non-naturally occurring.
[0041] By `variants` of the polypeptide we include insertions,
deletions and substitutions, either conservative or
non-conservative. For example, conservative substitution refers to
the substitution of an amino acid within the same general class
(e.g. an acidic amino acid, a basic amino acid, a non-polar amino
acid, a polar amino acid or an aromatic amino acid) by another
amino acid within the same class. Thus, the meaning of a
conservative amino acid substitution and non-conservative amino
acid substitution is well known in the art. In particular we
include variants of the polypeptide which exhibit an
anti-inflammatory activity.
[0042] In a further embodiment the variant has an amino acid
sequence which has at least 50% identity with the amino acid
sequence according to a reference sequence (for example, SEQ ID NO:
1) or a fragment thereof, for example at least 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or at least 99%
identity.
[0043] The percent sequence identity between two polypeptides may
be determined using suitable computer programs, for example the GAP
program of the University of Wisconsin Genetic Computing Group and
it will be appreciated that percent identity is calculated in
relation to polypeptides whose sequences have been aligned
optimally.
[0044] The alignment may alternatively be carried out using the
Clustal W program (as described in Thompson et at, 1994, Nuc. Acid
Res. 22:4673-4680, which is incorporated herein by reference).
[0045] The parameters used may be as follows: [0046] Fast pairwise
alignment parameters: K-tuple(word) size; 1, window size; 5, gap
penalty; 3, number of top diagonals; 5. Scoring method: x percent.
[0047] Multiple alignment parameters: gap open penalty; 10, gap
extension penalty; 0.05. [0048] Scoring matrix: BLOSUM.
[0049] Alternatively, the BESTFIT program may be used to determine
local sequence alignments.
[0050] In one embodiment, amino acids from the above reference
sequences may be mutated in order to reduce proteolytic degradation
of the polypeptide, for example by I, F to W modifications (see
Stromstedt et al, Antimicrobial Agents Chemother 2009, 53,
593).
[0051] In a further embodiment, the polypeptide comprises or
consists of an amino acid sequence from a serpin other than ATIII
which corresponds to the sequence of SEQ ID NO: 1 ("FFF21") of
ATIII. Examples of such sequences are shown in Table 1 below.
TABLE-US-00008 TABLE 1 Selected regions of human serpin family
sequence alignment. The highlighted region of SEQ ID NO: 1
("FFF21") of anti-thrombin III (P01008) corresponds to amino acids
153-173 sp|P01008|ANT3_HUMAN
-----KTSDQIHFFFAKLNCRLYR-KANKSSKLVSANRLFGDKSLTFNETYQDISELVYG [SEQ
ID NO: 12] P01011|AACT_HUMAN
-----TSEAEIHQSFQHLLRTL-N-QSSDELQLSMGNAMFVKEQLSLLDRFTEDAKRLYG [SEQ
ID NO: 13] Q86WD7|SPA9_HUMAN
-----TPESAIHQGFQHLVHSL-T-VPSKDLTLKMGSALFVKKELQLQANFLGNVKRLYE [SEQ
ID NO: 14] tr|Q2T9J2|Q2T9J2_HUMAN
-----TPESAIHQGFQHLVHSL-T-VPSKDLTLKMGSALFVKKELQLQANFLGNVKRLYE [SEQ
ID NO: 15] Q9UIV8|SPB13_HUMAN
EKEVIENTEAVHQQFQKFLTEI-S-KLTNDYELNITNRLFGEKTYLFLQKYLDYVEKYYH [SEQ
ID NO: 16] sp|O75635|SPB7_HUMAN
NSS--NSQSGLQSQLKRVFSDI-N-ASHKDYDLSIVNGLFAEKVYGFHKDYIECAEKLYD [SEQ
ID NO: 17] sp|O75830|SPI2_HUMAN
------SAGEEFFVLKSFFSAI-S-EKKQEFTFNLANALYLQEGFTVKEQYLHGNKEFFQ [SEQ
ID NO: 18] tr|Q8TCE1|Q8TCE1_HUMAN
-----KTSDQIHFFFAKLNCRLYR-KANKSSKLVSANRLFGDKSLTF------------- [SEQ
ID NO: 19] unk|VIRT7736|Blast_submission
-----KTSDQIHFFFAKLNCRLYR-KANKSSKLVSANRLFGDKSLTFNETYQDISELVYG [SEQ
ID NO: 20] sp|P01009|A1AT_HUMAN
-----IPEAQIHEGFQELLRTL-N-QPDSQLQLTTGNGLFLSEGLKLVDKFLEDVKKLYH [SEQ
ID NO: 21] sp|P05120|PAI2_HUMAN
ILQA-QAADKIHSSFRSLSSAI-N-ASTGNYLLESVNKLFGEKSASFREEYIRLCQKYYS [SEQ
ID NO: 22] sp|P05121|PAI1_HUMAN
-------DKGMAPALRHLYKEL-M-GPWNKDEISTTDAIFVQRDLKLVQGFMPHFFRLFR [SEQ
ID NO: 23] sp|P05154|IPSP_HUMAN
-----SSEKELHRGFQQLLQEL-N-QPRDGFQLSLGNALFTDLVVDLQDTFVSAMKTLYL [SEQ
ID NO: 24] sp|P05155|IC1_HUMAN
--------TCVHQALKGFTT----------KGVTSVSQIFHSPDLAIRDTFVNASRTLYS [SEQ
ID NO: 25] sp|P05543|THBG_HUMAN
-----TPMVEIQHGFQHLICSL-N-FPKKELELQIGNALFIGKHLKPLAKFLNDVKTLYE [SEQ
ID NO: 26] sp|P61640|THBG_PANTR
-----TPMVEIQHGFQHLICSL-N-FPKKELELQIGNALFIGKHLKPLAKFLNDVKTLYE [SEQ
ID NO: 27] tr|Q8IVC0|Q8IVC0_HUMAN
-----YEITTIHNLFRKLTHRL-F-RRNFGYTLRSVNDLYIQKQFPILLDFKTKVREYYF [SEQ
ID NO: 28] sp|P07093|GDN_HUMAN
---------GVGKILKKINKAI-V-SKKNKDIVTVANAVFVKNASEIEVPFVTRNKDVFQ [SEQ
ID NO: 29] sp|P08697|A2AP_HUMAN
----------SGPCLPHLLSRL-C-QDLGPGAFRLAARMYLQKGFPIKEDFLEQSEQLFG [SEQ
ID NO: 30] tr|Q8N5U7|Q8N5U7_HUMAN
----------SGPCLPHLLSRL-C-QDLGPGAFRLAARMYLQKGFPIKEDFLEQSEQLFG [SEQ
ID NO: 31] sp|P20848|A1ATR_HUMAN
-----TPEAKIHECFQQVLQAL-S-RPDTRLQLTTGSSLFVNKSMKLVDTFLEDTKKLYH [SEQ
ID NO: 32] sp|P29508|SPB3_HUMAN
TYHV-DRSGNVEHQFQKLLTEF-N-KSTDAYELKIANKLFGEKTYLFLQEYLDAIKKFYQ [SEQ
ID NO: 33] sp|P48594|SPB4_HUMAN
TYHV-DRSGNVHHQFQKLLTEF-N-KSTDAYELKIANKLFGEKTYQFLQEYLDAIKKFYQ [SEQ
ID NO: 34] tr|Q5K634|Q5K634_HUMAN
TYHV-DRSGNVHHQFQKLLTEF-N-KSTDAYELKIANKLFGEKTYQFLQEYLDAIKKFYQ [SEQ
ID NO: 35] tr|Q5K684|Q5K684_HUMAN
TYHV-DRSGNVHHQFQKLLTEF-N-KSTDAYELKIANKLFGEKTYLFLQEYLDAIKKFYQ [SEQ
ID NO: 36] tr|Q9BYF7|Q9BYF7_HUMAN
TYHV-DRSGNVHHQFQKLLTEF-N-KSTDAYELKIANKLFGEKTYQFLQEYLDAIKKFYQ [SEQ
ID NO: 37] sp|P29622|KAIN_HUMAN
-----LSESDVHRGFQHLLHTL-N-LPGHGLETRVGSALFLSHNLKFLAKFLNDTMAVYE [SEQ
ID NO: 38] sp|P30740|ILEU_HUMAN
---------EVHSRFQSLNADI-N-KRGASYILKLANRLYGEKTYNFLPEFLVSTQKTYG [SEQ
ID NO: 39] sp|P36952|SPB5_HUMAN
--------KDIPFGFQTVTSDV-N-KLSSFYSLKLIKRLYVDKSLNLSTEFISSTKRPYA [SEQ
ID NO: 40] sp|P48595|SPB10_HUMAN
EFNL-SNSEEIHSDFQTLISEI-L-KPNDDYLLKTANAIYGEKTYAFHNKYLEDMKTYFG [SEQ
ID NO: 41] sp|P50453|SPB9_HUMAN
-------EEDIHRAFQSLLTEV-N-KAGTQYLLRTANRLFGEKTCQFLSTFKESCLQFYH [SEQ
ID NO: 42] tr|Q5TD03|Q5TD03_HUMAN
-------EEDIHRAFQSLLTEV-N-KAGTQYLLRTANRLFGEKTCQFLSTFKESCLQFYH [SEQ
ID NO: 43] sp|P50454|SERPH_HUMAN
-------DEEVHAGLGELLRSL-SNSTARNVTWKLGSRLYGPSSVSFADDFVRSSKQHYN [SEQ
ID NO: 44] sp|Q6UXR4|SPA13_HUMAN
------PEEEIQEGFWDLLIRL-R-GQGPRLLLTMDQRRFSGLGARAN------------ [SEQ
ID NO: 45] sp|Q86U17|SPA11_HUMAN
------PEADIHQGFRSLLHTL-A-LPSPKLELKVGNSLFLDKRLKPRQHYLDSIKELYG [SEQ
ID NO: 46] sp|Q8IW75|SPA12_HUMAN
--------KDLHEGFHYIIHEL-T-QKTQDLKLSIGNTLFIDQRLQPQRKFLEDAKNFYS [SEQ
ID NO: 47] sp|Q96P15|SPB11_HUMAN
SPKC-SQAGRIHSEFGVEFSQI-N-QPDSNCTLSIANRLYGTKTMAFHQQYLSCSEKWYQ [SEQ
ID NO: 48] sp|Q96P63|SPB12_HUMAN
GSLN-NESGLVSCYFGQLLSKL-D-RIKTDYTLSIANRLYGEQEFPICQEYLDGVIQFYH [SEQ
ID NO: 49] sp|Q99574|NEUS_HUMAN
------EE--FSFLKEFSN-MV-T-AKESQYVMKIANSLFVQNGFHVNEEFLQMMKKYFN [SEQ
ID NO: 50] sp|Q9UK55|ZPI_HUMAN
------KPGLLPSLFKGLR-ET-L-SRNLELGLTQGSFAFIHKDFDVKETFFNLSKRYFD [SEQ
ID NO: 51] tr|A5Z2A5|A5Z2A5_HUMAN
------KPGLLPSLFKGLR-ET-L-SRNLELGLTQGSFAFIHKDFDVKETFFNLSKRYFD [SEQ
ID NO: 52] sp|P35237|SPB6_HUMAN
-----GGGGDIHQGFQSLLTEV-N-KTGTQYLLRVANRLFGEKSCDFLSSFRDSCQKFYQ [SEQ
ID NO: 53] sp|P50452|SPB8_HUMAN
------KDGDIHRGFQSLLSEV-N-RTGTQYLLRTANRLFGEKTCDFLPDFKEYCQKFYQ [SEQ
ID NO: 54] tr|Q8N178|Q8N178_HUMAN
------KDGDIHRGFQSLLSEV-N-RTGTQYLLRTANRLFGEKTCDFLPDFKEYCQKFYQ [SEQ
ID NO: 55]
[0052] In an alternative embodiment, the polypeptide comprises or
consists of an amino acid sequence from a serpin other than PCI
which corresponds to the sequence of SEQ ID NO: 3 ("SEK20") of PCI.
Examples of such sequences are shown in Table 2 below.
TABLE-US-00009 TABLE 2 Selected regions of human serpin family
sequence alignment. The highlighted region of SEQ ID NO: 3
("SEK20") of anti-thrombin III (P015154) corresponds to amino acids
283-302 sp|P05154|IPSP_HUMAN
VPYQGNATALFI-LPSE-----GKMQQVENGLSEKTLRKWLK---MFKK----RQL-ELY [SEQ
ID NO: 56] P01011|AACT_HUMAN
LKYTGNASALFIL-PDQ-----DKMEEVEAMLLPETLKRWRD---SLEF----REIGELY [SEQ
ID NO: 57] Q66WD7|SPA9_HUMAN
MDYKGDAVAFFVL-PSK-----GKMRQLEQALSARTLRKWSH---SLQK----RWI-EVF [SEQ
ID NO: 58] tr|Q2T9J2|Q2T9J2_HUMAN
MDYKGDAVAFFVL-PSK-----GKMRQLEQALSARTLRKWSH---SLQK----RWI-EVF [SEQ
ID NO: 59] Q9UIV8|SPB13_HUMAN
IPYKNNDLSMFVLLPNDI----DGLEKIIDKISPEKLVEWTSP-GHMEE----RKV-NLH [SEQ
ID NO: 60] sp|O75635|SPB7_HUMAN
LRYNGG-INMYVLLPE------NDLSEIENKLTFQNLMEWTNP-RRMTS----KYV-EVF [SEQ
ID NO: 61] sp|O75830|SPI2_HUMAN
LSYKGDEFSLIIILPAEG----MDIEEVEKLITAQQILKWLS---EMQE----EEV-EIS [SEQ
ID NO: 62] sp|P01008|ANT3_HUMAN
LPFKGDDITMVLILPKPE----KSLAKVEKELTPEVLQEWLD---ELEE----MML-VVH [SEQ
ID NO: 63] unk|VIRT7736|Blast_submission
LPFKGDDITMVLILPKPE----KSLAKVEKELTPEVLQEWLD---ELEE----MML-VVH [SEQ
ID NO: 64] sp|P01009|A1AT_HUMAN
MKYLGNATAIF-FLPDE-----GKLQHLENELTHDIITKFLE---NEDR----RSA-SLH [SEQ
ID NO: 65] sp|P05120|PAI2_HUMAN
LPYAGD-VSMFLLLPDEIADVSTGLELLESEITYDKLNKWTSK-DKMAE----DEV-EVY [SEQ
ID NO: 66] sp|P05121|PAI1_HUMAN
LPYHGDTLSMFIAAPYEKE---VPLSALTNILSAQLISHWKG---NMTR----LPR-LLV [SEQ
ID NO: 67] sp|P05155|IC1_HUMAN
LQLSHN-LSLVILVPQNLK---HRLEDMEQALSPSVFKAIMEKLEMSKF----QPT-LLT [SEQ
ID NO: 68] sp|P05543|THBG_HUMAN
MDYSKNALALFVL-PKE-----GQMESVEAAMSSKTLKKWNR---LLQK----GWV-DLF [SEQ
ID NO: 69] sp|P61640|THBG_PANTR
MDYSKNALALFVL-PKE-----GQMESVEAAMSSKTLKKWNR---LLQK----GWV-DLF [SEQ
ID NO: 70] tr|Q8IVC0|Q8IVC0_HUMAN
LEYVGG-ISMLIVVPHKM----SGMKTLEAQLTPGVVERWQK---SMTN----RTR-EVL [SEQ
ID NO: 71] sp|P07093|GDN_HUMAN
LPYHGESISMLIALPTESS---TPLSAIIPHISTKTIDSWMS---IMVP----KRV-QVI [SEQ
ID NO: 72] sp|P08697|A2AP_HUMAN
FPFKNNMS-FVVLVPTHFE---WNVSQVLANLSWDTLHP-----PLVWE----RPT-KVR [SEQ
ID NO: 73] tr|Q8N5U7|Q8N5U7_HUMAN
FPFKNNMS-FVVLVPTHFE---WNVSQVLANLSWDTLHP-----PLVWE----RPT-KVR [SEQ
ID NO: 74] sp|P20848|A1ATR_HUMAN
QHYVGNATAFFIL-PDP-----KKMWQLEEKLTYSHLENIQR---AFDI----RSI-NLH [SEQ
ID NO: 75] sp|P29508|SPB3_HUMAN
IPYKGKDLSMIVLLPNEI----DGLQKLEEKLTAEKLMEWTSL-QNMRE----TRV-DLH [SEQ
ID NO: 76] sp|P48594|SPB4_HUMAN
IPYKGKDLSMIVLLPNEI----DGLQKLEEKLTAEKLMEWTSL-QNMRE----TCV-DLH [SEQ
ID NO: 77] tr|Q5K634|Q5K634_HUMAN
IPYKGKDLSMIVLLPNEI----DGLQKLEEKLTAEKLMEWTSL-QNMRE----TRV-DLH [SEQ
ID NO: 78] tr|Q5K684|Q5K684_HUMAN
IPYKGKDLSMIVLLPNEI----DGLQKLEEKLTAEKLMEWTSL-QNMRE----TCV-DLH [SEQ
ID NO: 79] tr|Q9BYF7|Q9BYF7_HUMAN
IPYKGKDLSMIVLLPNEI----DGLQKLEEKLTAEKLMEWTSL-QNMRE----TCV-DLH [SEQ
ID NO: 80] sp|P29622|KAIN_HUMAN
MDYKGDATVFFI-LPNQ-----GKMREIEEVLTPEMLMRWNN---LLRKRNEYKKL-ELH [SEQ
ID NO: 81] sp|P30740|ILEU_HUMAN
LPYQGEELSMVILLPDDIEDESTGLKKIEEQLTLEKLHEWTKP-ENLDF----IEV-NVS [SEQ
ID NO: 82] sp|P36952|SPH5_HUMAN
LPFQNKHLSMFILLPKDVEDESTGLEKIEKQLNSESLSQWTNP-STMAN----AKV-KLS [SEQ
ID NO: 83] sp|P48595|SPB10_HUMAN
LYYKSRDLSLLILLPE----DINGLEQLEKAITYEKLNEWTSA-DMMEL----YEV-QLH [SEQ
ID NO: 84] sp|P50453|SPB9_HUMAN
LPYARKELSLLVLLPDD----GVELSTVEKSLTFEKLTAWTKP-DCMKS----TEV-EVL [SEQ
ID NO: 85] tr|Q5TD03|Q5TD03_HUMAN
LPYARKELSLLVLLPDD----GVELSTVEKSLTFEKLTAWTKP-DCMKS----TEV-EVL [SEQ
ID NO: 86] sp|P50454|SERPH_HUMAN
MPLAHKLSSLIILMPHH----VEPLERLEKLLTKEQLKIWMG---KMQK----KAV-AIS [SEQ
ID NO: 87] sp|Q6UXR4|SPA13_HUMAN
MDHAGNTTTFFI-FPNR-----GKMRHLEDALLPETLIKWDS---LLRT----REL-DFH [SEQ
ID NO: 88] sp|Q86U17|SPA11_HUMAN
IEYRGNALALLV-LPDP-----GKMKQVEAALQPQTLRKWGQ---LLLP----SLL-DLH [SEQ
ID NO: 89] sp|Q8IW75|SPA12 _HUMAN
IPYQKN-ITAIFILPDE-----GKLKHLEKGLQVDTFSRWKT---LLSR----RVV-DVS [SEQ
ID NO: 90] sp|Q96P15|SPB11_HUMAN
LPYVNNKLSMIILLPV----GIANLKQIEKQLNSGTFHEWTSS-SNMME----REV-EVH [SEQ
ID NO: 91] sp|Q96P63|SPB12_HUMAN
MRYTKGKLSMFVLLPSHSKDNLKGLEELERKITYEKMVAWSSS-ENMSE----ESV-VLS [SEQ
ID NO: 92] tr|Q3SYB5|Q3SYB5_HUMAN
MRYTKGKLSMFVLLPSHSKDNLKGLEELERKITYEKMVAWSSS-ENMSE----ESV-VLS [SEQ
ID NO: 93] sp|Q99574|NEUS_HUMAN
IPYEGDEISMML-VLSRQ---EVPLATLEPLVKAQLVEEWAN---SVKK----QKV-EVY [SEQ
ID NO: 94] sp|Q9UK55|ZPI_HUMAN
LPYQGNATMLVV-LMEKM----GDHLALEDYLTTDLVETWLR---NMKT----RNM-EVF [SEQ
ID NO: 95] tr|A5Z2A5|A5Z2A5_HUMAN
LPYQGNATMLVV-LMEKM----GDHLALEDYLTTDLVETWLR---NMKT----RNM-EVF [SEQ
ID NO: 96] sp|P35237|SPB6_HUMAN
LPYVGKELNMIIMLPDE----TTDLRTVEKELTYEKFVEWTRL-DMMDE----EEV-EVS [SEQ
ID NO: 97] tr|Q8IXH2|Q8IXH2_HUMAN
LPYVGKELNMIIMLPDE----TTDLRTVEKELTYEKFVEWTRL-DMMDE----EEV-EVS [SEQ
ID NO: 98] sp|P50452|SPB8_HUMAN
LPYVEEELSMVILLPDD----NTDLAVVEKALTYEKFKAWTNS-EKLTK----SKV-QVF [SEQ
ID NO: 99] tr|Q8N178|Q8N178_HUMAN
LPYVEEELSMVILLPDD----NTDLAVKE------------------------------- [SEQ
ID NO: 100]
[0053] By a sequence which "corresponds" to SEQ ID NO: 1 or 3, in
relation to Tables 1 and 2 above, we include that the polypeptide
corresponds to the equivalent amino acid sequence within a
different human serpin, i.e. which polypeptide exhibits the maximum
sequence identity with SEQ ID NO: 1 or 3 (for example, as measured
by a GAP or BLAST sequence comparison). Typically, the
corresponding polypeptide will be the same length as the reference
sequence (i.e. SEQ ID NO: 1 or 3).
[0054] Variants may be made using the methods of protein
engineering and site-directed mutagenesis well known in the art
using the recombinant polynucleotides (see example, see Molecular
Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell,
2000, Cold Spring Harbor Laboratory Press, which is incorporated
herein by reference).
[0055] In one embodiment, the polypeptide comprises or consists of
an amino acid which is a species homologue of any one of the above
amino acid sequences (e.g. SEQ ID NOS: 1 to 11). By "species
homologue" we include that the polypeptide corresponds to the same
amino acid sequence within an equivalent protein from a non-human
species, i.e. which polypeptide exhibits the maximum sequence
identity with of any one of SEQ ID NOS: 1 to 11 (for example, as
measured by a GAP or BLAST sequence comparison). Typically, the
species homologue polypeptide will be the same length as the human
reference sequence (i.e. SEQ ID NOS: 1 to 11).
[0056] In a still further embodiment, the polypeptide comprises or
consists of a fusion protein.
[0057] By `fusion` of a polypeptide we include an amino acid
sequence corresponding to a reference sequence (for example, SEQ ID
NO: 1, or a fragment or variant thereof) fused to any other
polypeptide. For example, the said polypeptide may be fused to a
polypeptide such as glutathione-S-transferase (GST) or protein A in
order to facilitate purification of said polypeptide. Examples of
such fusions are well known to those skilled in the art. Similarly,
the said polypeptide may be fused to an oligo-histidine tag such as
His6 or to an epitope recognised by an antibody such as the
well-known Myc tag epitope. In addition, fusions comprising a
hydrophobic oligopeptide end-tag may be used. Fusions to any
variant or derivative of said polypeptide are also included in the
scope of the invention. It will be appreciated that fusions (or
variants or derivatives thereof) which retain desirable properties,
such as an anti-inflammatory activity, are preferred.
[0058] The fusion may comprise a further portion which confers a
desirable feature on the said polypeptide of the invention; for
example, the portion may be useful in detecting or isolating the
polypeptide, or promoting cellular uptake of the polypeptide. The
portion may be, for example, a biotin moiety, a streptavidin
moiety, a radioactive moiety, a fluorescent moiety, for example a
small fluorophore or a green fluorescent protein (GFP) fluorophore,
as well known to those skilled in the art. The moiety may be an
immunogenic tag, for example a Myc tag, as known to those skilled
in the art or may be a lipophilic molecule or polypeptide domain
that is capable of promoting cellular uptake of the polypeptide, as
known to those skilled in the art.
[0059] It will be appreciated by persons skilled in the art that
the polypeptide of the invention may comprise one or more amino
acids that are modified or derivatised, for example by PEGylation,
amidation, esterification, acylation, acetylation and/or
alkylation.
[0060] As appreciated in the art, pegylated proteins may exhibit a
decreased renal clearance and proteolysis, reduced toxicity,
reduced immunogenicity and an increased solubility [21, 22].
Pegylation has been employed for several protein-based drugs
including the first pegylated molecules asparaginase and adenosine
deaminase [22, 23].
[0061] In order to obtain a successfully pegylated protein, with a
maximally increased half-life and retained biological activity,
several parameters that may affect the outcome are of importance
and should be taken into consideration. The PEG molecules may
differ, and PEG variants that have been used for pegylation of
proteins include PEG and monomethoxy-PEG. In addition, they can be
either linear or branched [24]. The size of the PEG molecules used
may vary and PEG moieties ranging in size between 1 and 40 kDa have
been linked to proteins [24-27]. In addition, the number of PEG
moieties attached to the protein may vary, and examples of between
one and six PEG units being attached to proteins have been reported
[24, 26]. Furthermore, the presence or absence of a linker between
PEG as well as various reactive groups for conjugation have been
utilised. Thus, PEG may be linked to N-terminal amino groups, or to
amino acid residues with reactive amino or hydroxyl groups (Lys, H
is, Ser, Thr and Tyr) directly or by using .gamma.-amino butyric
acid as a linker. In addition, PEG may be coupled to carboxyl (Asp,
Glu, C-terminal) or sulfhydryl (Cys) groups. Finally, Gln residues
may be specifically pegylated using the enzyme transglutaminase and
alkylamine derivatives of PEG has been described [25].
[0062] It has been shown that increasing the extent of pegylation
results in an increased in vivo half-life. However, it will be
appreciated by persons skilled in the art that the pegylation
process will need to be optimised for a particular protein on an
individual basis.
[0063] PEG may be coupled at naturally occurring disulphide bonds
as described in WO 2005/007197. Disulfide bonds can be stabilised
through the addition of a chemical bridge which does not compromise
the tertiary structure of the protein. This allows the conjugating
thiol selectivity of the two sulphurs comprising a disulfide bond
to be utilised to create a bridge for the site-specific attachment
of PEG. Thereby, the need to engineer residues into a peptide for
attachment of to target molecules is circumvented.
[0064] A variety of alternative block copolymers may also be
covalently conjugated as described in WO 2003/059973. Therapeutic
polymeric conjugates can exhibit improved thermal properties,
crystallisation, adhesion, swelling, coating, pH dependent
conformation and biodistribution. Furthermore, they can achieve
prolonged circulation, release of the bioactive in the proteolytic
and acidic environment of the secondary lysosome after cellular
uptake of the conjugate by pinocytosis and more favourable
physicochemical properties due to the characteristics of large
molecules (e.g. increased drug solubility in biological fluids).
Block copolymers, comprising hydrophilic and hydrophobic blocks,
form polymeric micelles in solution. Upon micelle disassociation,
the individual block copolymer molecules are safely excreted.
[0065] Chemical derivatives of one or more amino acids may also be
achieved by reaction with a functional side group. Such derivatised
molecules include, for example, those molecules in which free amino
groups have been derivatised to form amine hydrochlorides,
p-toluene sulphonyl groups, carboxybenzoxy groups,
1-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatised to form salts, methyl and
ethyl esters or other types of esters and hydrazides. Free hydroxyl
groups may be derivatised to form O-acyl or O-alkyl derivatives.
Also included as chemical derivatives are those peptides which
contain naturally occurring amino acid derivatives of the twenty
standard amino acids. For example: 4-hydroxyproline may be
substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine and ornithine for lysine.
Derivatives also include peptides containing one or more additions
or deletions as long as the requisite activity is maintained. Other
included modifications are amidation, amino terminal acylation
(e.g. acetylation or thioglycolic acid amidation), terminal
carboxylamidation (e.g. with ammonia or methylamine), and the like
terminal modifications.
[0066] It will be further appreciated by persons skilled in the art
that peptidomimetic compounds may also be useful. Thus, by
`polypeptide` we include peptidomimetic compounds which have an
anti-inflammatory activity. The term `peptidomimetic` refers to a
compound that mimics the conformation and desirable features of a
particular peptide as a therapeutic agent.
[0067] For example, the polypeptides of the invention include not
only molecules in which amino acid residues are joined by peptide
(--CO--NH--) linkages but also molecules in which the peptide bond
is reversed. Such retro-inverso peptidomimetics may be made using
methods known in the art, for example such as those described in
Meziere et al. (1997) J. Immunol. 159, 3230-3237, which is
incorporated herein by reference. This approach involves making
pseudopeptides containing changes involving the backbone, and not
the orientation of side chains. Retro-inverse peptides, which
contain NH--CO bonds instead of CO--NH peptide bonds, are much more
resistant to proteolysis. Alternatively, the polypeptide of the
invention may be a peptidomimetic compound wherein one or more of
the amino acid residues are linked by .alpha.-y(CH.sub.2NH)-- bond
in place of the conventional amide linkage.
[0068] In a further alternative, the peptide bond may be dispensed
with altogether provided that an appropriate linker moiety which
retains the spacing between the carbon atoms of the amino acid
residues is used; it may be advantageous for the linker moiety to
have substantially the same charge distribution and substantially
the same planarity as a peptide bond.
[0069] It will be appreciated that the polypeptide may conveniently
be blocked at its N- or C-terminal region so as to help reduce
susceptibility to exoproteolytic digestion.
[0070] A variety of uncoded or modified amino acids such as D-amino
acids and N-methyl amino acids have also been used to modify
mammalian peptides. In addition, a presumed bioactive conformation
may be stabilised by a covalent modification, such as cyclisation
or by incorporation of lactam or other types of bridges, for
example see Veber et al., 1978, Proc. Natl. Acad. Sci. USA 75:2636
and Thursell et al., 1983, Biochem. Biophys. Res. Comm. 111:166,
which are incorporated herein by reference.
[0071] A common theme among many of the synthetic strategies has
been the introduction of some cyclic moiety into a peptide-based
framework. The cyclic moiety restricts the conformational space of
the peptide structure and this frequently results in an increased
specificity of the peptide for a particular biological receptor. An
added advantage of this strategy is that the introduction of a
cyclic moiety into a peptide may also result in the peptide having
a diminished sensitivity to cellular peptidases.
[0072] Thus, exemplary polypeptides of the invention comprise
terminal cysteine amino acids. Such a polypeptide may exist in a
heterodetic cyclic form by disulphide bond formation of the
mercaptide groups in the terminal cysteine amino acids or in a
homodetic form by amide peptide bond formation between the terminal
amino acids. As indicated above, cyclising small peptides through
disulphide or amide bonds between the N- and C-terminal region
cysteines may circumvent problems of specificity and half-life
sometime observed with linear peptides, by decreasing proteolysis
and also increasing the rigidity of the structure, which may yield
higher specificity compounds. Polypeptides cyclised by disulphide
bonds have free amino and carboxy-termini which still may be
susceptible to proteolytic degradation, while peptides cyclised by
formation of an amide bond between the N-terminal amine and
C-terminal carboxyl and hence no longer contain free amino or
carboxy termini. Thus, the peptides of the present invention can be
linked either by a C--N linkage or a disulphide linkage.
[0073] The present invention is not limited in any way by the
method of cyclisation of peptides, but encompasses peptides whose
cyclic structure may be achieved by any suitable method of
synthesis. Thus, heterodetic linkages may include, but are not
limited to formation via disulphide, alkylene or sulphide bridges.
Methods of synthesis of cyclic homodetic peptides and cyclic
heterodetic peptides, including disulphide, sulphide and alkylene
bridges, are disclosed in U.S. Pat. No. 5,643,872, which is
incorporated herein by reference. Other examples of cyclisation
methods includes cyclization through click chemistry, epoxides,
aldehyde-amine reactions, as well as and the methods disclosed in
U.S. Pat. No. 6,008,058, which is incorporated herein by
reference.
[0074] A further approach to the synthesis of cyclic stabilised
peptidomimetic compounds is ring-closing metathesis (RCM). This
method involves steps of synthesising a peptide precursor and
contacting it with an RCM catalyst to yield a conformationally
restricted peptide. Suitable peptide precursors may contain two or
more unsaturated C--C bonds. The method may be carried out using
solid-phase-peptide-synthesis techniques. In this embodiment, the
precursor, which is anchored to a solid support, is contacted with
a RCM catalyst and the product is then cleaved from the solid
support to yield a conformationally restricted peptide.
[0075] Another approach, disclosed by D. H. Rich in Protease
Inhibitors, Barrett and Selveson, eds., Elsevier (1986), which is
incorporated herein by reference, has been to design peptide mimics
through the application of the transition state analogue concept in
enzyme inhibitor design. For example, it is known that the
secondary alcohol of staline mimics the tetrahedral transition
state of the scissile amide bond of the pepsin substrate.
[0076] In summary, terminal modifications are useful, as is well
known, to reduce susceptibility by proteinase digestion and
therefore to prolong the half-life of the peptides in solutions,
particularly in biological fluids where proteases may be present.
Polypeptide cyclisation is also a useful modification because of
the stable structures formed by cyclisation and in view of the
biological activities observed for cyclic peptides.
[0077] Thus, in one embodiment the polypeptide of the first aspect
of the invention is linear. However, in an alternative embodiment,
the polypeptide is cyclic.
[0078] It will be appreciated by persons skilled in the art that
the polypeptides of the invention may be of various lengths.
Typically, however, the polypeptide is between 10 and 200 amino
acids in length, for example between 10 and 150, 15 and 100, 15 and
50, 15 and 30, 17 and 30, or 17 and 28 amino acids in length. For
example, the polypeptide may be at least 17 amino acids in
length.
[0079] As stated at the outset, anti-inflammatory activity is a
feature common to the polypeptides of the invention. In one
embodiment, the polypeptides are capable of inhibiting the release
of one or more pro-inflammatory cytokines from human
monocyte-derived macrophages, such as monocyte-derived macrophages,
including macrophage inhibitory factor, TNF-alpha, HMGB1, C5a,
IL-17, IL-8, MCP-1, IFN-gamma, 11-6, IL-1b, IL-12. Antiinflammatory
IL-10 may be unaffected or transiently increased.
[0080] Other markers may also be affected: These include tissue
factor on monocytes and endothelial cells, procalcitonin, CRP,
reactive oxygen species, bradykinin, nitric oxide, PGE1, platelet
activating factor, arachidonic acid metabolites, MAP kinase
activation.
[0081] In particular, the polypeptide may exhibit anti-inflammatory
activity in one or more of the following models: [0082] (i) in
vitro macrophage models using LPS, LTA, zymosan, flagellin, dust
mites, viral or bacterial DNA or RNA, or peptidoglycan as
stimulants; [0083] (ii) in vivo mouse models of endotoxin shock;
[0084] (iii) in vivo infection models, either in combination with
antimicrobial therapy, or given alone.
[0085] In a further embodiment of the invention, the polypeptide
exhibits anticoagulant activity.
[0086] By "anti-coagulant activity" we mean an ability to reduce or
prevent coagulation (i.e. the clotting of blood) or an associated
signal or effect. Such activity may be determined by methods well
known in the art, for example using the activated partial
thromboplastin time (aPTT) test, prothrombin time (PT) test or the
thrombin clotting time (TCT) test. Furthermore, specific
measurements of prekallikrein activation or the activity of Factor
X and other coagulation factors may be performed. It will be
appreciated by skilled persons that the polypeptide may inhibit the
extrinsic coagulation pathway and/or the intrinsic coagulation
pathway. However, in a preferred embodiment, the polypeptide
inhibits (at least in part) the intrinsic coagulation pathway.
[0087] In a still further embodiment of the invention, the
polypeptide exhibits Toll-like receptor (TLR) blocking activity.
Such receptor blocking activity can be measured using methods well
known in the art, for example by analysis of suitable down-stream
effectors, such as iNOS, nuclear factor kappa B and cytokines.
[0088] By virtue of possessing an anti-inflammatory activity, the
polypeptides of the first aspect of the invention are intended for
use in the treatment or prevention of inflammation.
[0089] By "treatment or prevention of inflammation" we mean that
the polypeptide of the invention is capable of preventing or
inhibiting (at least in part) one or more symptom, signal or effect
constituting or associated with inflammation.
[0090] It will be appreciated by persons skilled in the art that
inhibition of inflammation may be in whole or in part. In a
preferred embodiment, the polypeptide is capable of inhibiting one
or more markers of inflammation by 20% or more compared to cells or
individuals which have not been exposed to the polypeptide, for
example by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.
[0091] Advantageously, the polypeptides of the invention are
capable of treating or preventing inflammation selectively.
[0092] By `selectively` we mean that the polypeptide inhibits or
prevents inflammation to a greater extent than it modulates other
biological functions. Preferably, the polypeptide or fragment,
variant, fusion or derivative thereof inhibits or prevents
inflammation only.
[0093] However, in a further embodiment, the polypeptide also (or
alternatively) inhibits or prevents coagulation of the blood. As
above, it will be appreciated by persons skilled in the art that
inhibition of coagulation may be in whole or in part. In a
preferred embodiment, the polypeptide is capable of inhibiting one
or more measures and.or markers of coagulation by 20% or more
compared to cells or individuals which have not been exposed to the
polypeptide, for example by at least 30%, 40%, 50%, 60%, 70%, 80%,
90% or more.
[0094] In one embodiment, the polypeptides are for use in the
treatment or prevention of inflammation associated with (i.e.
caused by or merely co-presenting with) an infection.
[0095] In preferred but non-limiting embodiments of the invention,
the polypeptides are for use in the treatment or prevention of a
disease, condition or indication selected from the following:
[0096] i) Acute systemic inflammatory disease, with or without an
infective component, such as systemic inflammatory response
syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic shock.
Other generalized or localized invasive infective and inflammatory
disease, including erysipelas, meningitis, arthritis, toxic shock
syndrome, diverticulitis, appendicitis, pancreatitis,
cholecystitis, colitis, cellulitis, burn wound infections,
pneumonia, urinary tract infections, postoperative infections, and
peritonitis. [0097] ii) Chronic inflammatory and or infective
diseases, including cystic fibrosis, COPD and other pulmonary
diseases, gastrointestinal disease including chronic skin and
stomach ulcerations, other epithelial inflammatory and or infective
disease such as atopic dermatitis, oral ulcerations (aphtous
ulcers), genital ulcerations and inflammatory changes,
parodontitis, eye inflammations including conjunctivitis and
keratitis, external otitis, mediaotitis, genitourinary
inflammations. [0098] iii) Postoperative inflammation. Inflammatory
and coagulative disorders including thrombosis, DIC, postoperative
coagulation disorders, and coagulative disorders related to contact
with foreign material, including extracorporeal circulation, and
use of biomaterials. Furthermore, vasculitis related inflammatory
disease, as well as allergy, including allergic rhinitis and
asthma. [0099] iv) Excessive contact activation and/or coagulation
in relation to, but not limited to, stroke. [0100] v) Excessive
inflammation in combination with antimicrobial treatment. The
antimicrobial agents used may be administred by various routes;
intravenous (iv), intraarterial, intravitreal, subcutaneous (sc),
intramuscular (im), intraperitoneal (ip), intravesical,
intratechal, epidural, enteral (including oral, rectal, gastric,
and other enteral routes), or topically, (including dermal, nasal
application, application in the eye or ear, eg by drops, and
pulmonary inhalation). Examples of agents are penicillins,
cephalosporins, carbacephems, cephamycins, carbapenems,
monobactams, aminoglycosides, glycopeptides, quinolones,
tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents
include iodine, silver, copper, clorhexidine, polyhexanide and
other biguanides, chitosan, acetic acid, and hydrogen peroxide.
[0101] For example, the polypeptides may be for use in the
treatment or prevention of an acute inflammation, sepsis, acute
respiratory distress syndrome (ARDS), chronic obstructive pulmonary
disease (COPD), cystic fibrosis, wounds, asthma, allergic and other
types of rhinitis, cutaneous and systemic vasculitis, thrombosis
and/or disseminated intravascular coagulation (DIC).
[0102] In one embodiment, the polypeptide exhibits both
anti-inflammatory and anti-coagulant activity and may be used in
the concomitant treatment or prevention of inflammation and
coagulation. Such polypeptides may be particularly suited to the
treatment and prevention of conditions where the combined
inhibition of both inflammatory and coagulant processes is
desirable, such as sepsis, chronic obstructive pulmonary disorder
(COPD), thrombosis, DIC and acute respiratory distress syndrome
(ARDS). Furthermore, other diseases associated with excessive
inflammation and coagulation changes may benefit from treatment by
the polypeptides, such as cystic fibrosis, asthma, allergic and
other types of rhinitis, cutaneous and systemic vasculitis.
[0103] In a further embodiment, the polypeptides of the invention
are for use in combination with one or more additional therapeutic
agent. For example, the polypeptides of the invention may be
administered in combination with antibiotic agents,
anti-inflammatory agents, immunosuppressive agents and/or
antiseptic agents, as well as vasoactive agents and/or
receptor-blockers or receptor agonists. The antimicrobial agents
used may be applied iv, sc, im, intratechal, per os, or topically.
Examples of agents are penicillins, cephalosporins, carbacephems,
cephamycins, carbapenems, monobactams, aminoglycosides,
glycopeptides, quinolones, tetracyclines, macrolides, and
fluoroquinolones. Antiseptic agents include iodine, silver, copper,
clorhexidine, polyhexanide and other biguanides, chitosan, acetic
acid, and hydrogen peroxide. For example, the peptides of the
invention may serve as adjuvants to antiseptic treatments, for
example silver/PHMB treatment of wounds to quench LPS effects.
[0104] Thus, the peptides of the invention may serve as adjuvants
(for blocking inflammation) to complement antibiotic, antiseptic
and/or antifungal treatments of internal and external infections
(such as erysipelas, lung infections including fungal infections,
sepsis, COPD, wounds, and other epithelial infections). Likewise,
the peptides of the invention may serve as adjuvants to antiseptic
treatments, for example silver/PHMB treatment of wounds to quench
LPS effects.
[0105] In one embodiment, the polypeptides of the invention are for
use in combination with a steroid, for example a glucocorticoid
(such as dexamethasone).
[0106] A second, related aspect of the invention provides an
isolated polypeptide comprising or consisting of an amino acid
sequence derived from a naturally occurring protein which modulates
blood coagulation, or a fragment, variant, fusion or derivative
thereof, or a fusion of said fragment, variant or derivative
thereof, which polypeptide exhibits an anti-inflammatory and/or
anti-coagulant activity, wherein the naturally occurring protein
which modulates blood coagulation is selected from the group
consisting of serpins (other than heparin cofactor II),
histidine-rich glycoprotein (HRG) and tissue factor pathway
inhibitors (such as TFPI-1 and TFPI-2), with the proviso that the
polypeptide is not a naturally occurring protein (e.g.
holoprotein).
[0107] By "naturally occurring protein" in this context we mean
that the polypeptide is synthesized de novo. However, fragments of
such naturally occurring holoproteins generated in vivo are not
excluded.
[0108] It will be appreciated by persons skilled in the art that
terms such as fragment, variant, fusion or derivative should be
construed as discussed above in relation to the first aspect of the
invention.
[0109] In one embodiment, the polypeptide comprises or consists of
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 1 to 11, or a fragment, variant, fusion or derivative of said
sequence, or a fusion of said fragment, variant or derivative
thereof. For example, the polypeptide may comprise or consist of an
amino acid sequence selected from the group consisting of SEQ ID
NOS: 1 to 11.
[0110] It will be appreciated by persons skilled in the art that
the optional features discussed above in relation to the
polypeptides of the first aspect of the invention are also of
relevance to the related polypeptides of the second aspect of the
invention.
[0111] For example, in one preferred embodiment the polypeptide is
capable of inhibiting the release of one or more pro-inflammatory
cytokines from human monocyte-derived macrophages (such as IL-6,
IFN-gamma, TNF-alpha, IL-12, IL-1 and/or IL-18).
[0112] In another preferred embodiment, the polypeptide exhibits
anticoagulant activity.
[0113] The present invention also includes pharmaceutically
acceptable acid or base addition salts of the above described
polypeptides. The acids which are used to prepare the
pharmaceutically acceptable acid addition salts of the
aforementioned base compounds useful in this invention are those
which form non-toxic acid addition salts, i.e. salts containing
pharmacologically acceptable anions, such as the hydrochloride,
hydrobromide, hydroiodide, nitrate, sulphate, bisulphate,
phosphate, acid phosphate, acetate, lactate, citrate, acid citrate,
tartrate, bitartrate, succinate, maleate, fumarate, gluconate,
saccharate, benzoate, methanesulphonate, ethanesulphonate,
benzenesulphonate, p-toluenesulphonate and pamoate [i.e.
1,1'-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among
others.
[0114] Pharmaceutically acceptable base addition salts may also be
used to produce pharmaceutically acceptable salt forms of the
polypeptides. The chemical bases that may be used as reagents to
prepare pharmaceutically acceptable base salts of the present
compounds that are acidic in nature are those that form non-toxic
base salts with such compounds. Such non-toxic base salts include,
but are not limited to those derived from such pharmacologically
acceptable cations such as alkali metal cations (e.g. potassium and
sodium) and alkaline earth metal cations (e.g. calcium and
magnesium), ammonium or water-soluble amine addition salts such as
N-methylglucamine-(meglumine), and the lower alkanolammonium and
other base salts of pharmaceutically acceptable organic amines,
among others.
[0115] It will be appreciated that the polypeptides of the
invention may be lyophilised for storage and reconstituted in a
suitable carrier prior to use, e.g. through freeze drying, spray
drying, spray cooling, or through use of particle formation
(precipitation) from supercritical carbon dioxide. Any suitable
lyophilisation method (e.g. freeze-drying, spray drying, cake
drying) and/or reconstitution techniques can be employed. It will
be appreciated by those skilled in the art that lyophilisation and
reconstitution can lead to varying degrees of activity loss and
that use levels may have to be adjusted upward to compensate.
Preferably, the lyophilised (freeze dried) polypeptide loses no
more than about 1% of its activity (prior to lyophilisation) when
rehydrated, or no more than about 5%, 10%, 20%, 25%, 30%, 35%, 40%,
45%, or no more than about 50% of its activity (prior to
lyophilisation) when rehydrated.
[0116] Methods for the production of polypeptides of the invention
are well known in the art.
[0117] Conveniently, the polypeptide is or comprises a recombinant
polypeptide. Suitable methods for the production of such
recombinant polypeptides are well known in the art, such as
expression in prokaryotic or eukaryotic hosts cells (for example,
see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory
Manual, Third Edition, Cold Spring Harbor, N.Y., the relevant
disclosures in which document are hereby incorporated by
reference).
[0118] Polypeptides of the invention can also be produced using a
commercially available in vitro translation system, such as rabbit
reticulocyte lysate or wheatgerm lysate (available from Promega).
Preferably, the translation system is rabbit reticulocyte lysate.
Conveniently, the translation system may be coupled to a
transcription system, such as the TNT transcription-translation
system (Promega). This system has the advantage of producing
suitable mRNA transcript from an encoding DNA polynucleotide in the
same reaction as the translation.
[0119] It will be appreciated by persons skilled in the art that
polypeptides of the invention may alternatively be synthesised
artificially, for example using well known liquid-phase or solid
phase synthesis techniques (such as t-Boc or Fmoc solid-phase
peptide synthesis).
[0120] Thus, included within the scope of the present invention are
the following: [0121] (a) a third aspect of the invention provides
an isolated nucleic acid molecule which encodes a polypeptide
according to the second aspect of the invention; [0122] (b) a
fourth aspect of the invention provides a vector (such as an
expression vector) comprising a nucleic acid molecule according to
the third aspect of the invention; [0123] (c) a fifth aspect of the
invention provides a host cell comprising a nucleic acid molecule
according to the third aspect of the invention or a vector
according to the fourth aspect of the invention; and [0124] (d) a
sixth aspect of the invention provides a method of making a
polypeptide according to the second aspect of the invention
comprising culturing a population of host cells according to the
fifth aspect of the invention under conditions in which said
polypeptide is expressed, and isolating the polypeptide
therefrom.
[0125] A seventh aspect of the invention provides a pharmaceutical
composition comprising a polypeptide according to the first aspect
of the invention together with a pharmaceutically acceptable
excipient, diluent or carrier.
[0126] As used herein, `pharmaceutical composition` means a
therapeutically effective formulation for use in the treatment or
prevention of disorders and conditions associated with
inflammation.
[0127] As used herein, `pharmaceutical composition` means a
therapeutically effective formulation for use in the treatment or
prevention of disorders and conditions associated with
inflammation.
[0128] Additional compounds may also be included in the
pharmaceutical compositions, such as other peptides, low molecular
weight immunomodulating agents, receptor agonists and antagonists,
and antimicrobial agents. Other examples include chelating agents
such as EDTA, citrate, EGTA or glutathione.
[0129] The pharmaceutical compositions may be prepared in a manner
known in the art that is sufficiently storage stable and suitable
for administration to humans and animals.
[0130] The pharmaceutical compositions may be lyophilised, e.g.
through freeze drying, spray drying, spray cooling, or through use
of particle formation from supercritical particle formation.
[0131] By "pharmaceutically acceptable" we mean a non-toxic
material that does not decrease the effectiveness of the biological
activity of the active ingredients, i.e. the anti-inflammatory
polypeptide(s). Such pharmaceutically acceptable buffers, carriers
or excipients are well-known in the art (see Remington's
Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack
Publishing Company (1990) and handbook of Pharmaceutical
Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press
(2000).
[0132] The term "buffer" is intended to mean an aqueous solution
containing an acid-base mixture with the purpose of stabilising pH.
Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS,
Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate,
glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO,
BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine,
HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO,
TAPS, TABS, TAPSO and TES.
[0133] The term "diluent" is intended to mean an aqueous or
non-aqueous solution with the purpose of diluting the peptide in
the pharmaceutical preparation. The diluent may be one or more of
saline, water, polyethylene glycol, propylene glycol, ethanol or
oils (such as safflower oil, corn oil, peanut oil, cottonseed oil
or sesame oil).
[0134] The term "adjuvant" is intended to mean any compound added
to the formulation to increase the biological effect of the
peptide. The adjuvant may be one or more of colloidal silver or
gold, or of zinc, copper or silver salts with different anions, for
example, but not limited to fluoride, chloride, bromide, iodide,
tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate,
glycolate, citrate, borate, tartrate, and acetates of different
acyl composition. The adjuvant may also be cationic polymers such
as cationic cellulose ethers, cationic cellulose esters,
deacetylated hyaluronic acid, chitosan, cationic dendrimers,
cationic synthetic polymers such as poly(vinyl imidazole), and
cationic polypeptides such as polyhistidine, polylysine,
polyarginine, and peptides containing these amino acids.
[0135] The excipient may be one or more of carbohydrates, polymers,
lipids and minerals. Examples of carbohydrates include lactose,
sucrose, mannitol, and cyclodextrines, which are added to the
composition, e.g., for facilitating lyophilisation. Examples of
polymers are starch, cellulose ethers, cellulose,
carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl
cellulose, ethylhydroxyethyl cellulose, ethyl cellulose, methyl
cellulose, propyl cellulose, alginates, carageenans, hyaluronic
acid and derivatives thereof, polyacrylic acid, polysulphonate,
polyethylenglycol/polyethylene oxide,
polyethyleneoxide/polypropylene oxide copolymers,
polyvinylalcohol/polyvinylacetate of different degree of
hydrolysis, poly(lactic acid), poly(glycholic acid) or copolymers
thereof with various composition, and polyvinylpyrrolidone, all of
different molecular weight, which are added to the composition,
e.g. for viscosity control, for achieving bioadhesion, or for
protecting the active ingredient (applies to A-C as well) from
chemical and proteolytic degradation. Examples of lipids are fatty
acids, phospholipids, mono-, di-, and triglycerides, ceramides,
sphingolipids and glycolipids, all of different acyl chain length
and saturation, egg lecithin, soy lecithin, hydrogenated egg and
soy lecithin, which are added to the composition for reasons
similar to those for polymers. Examples of minerals are talc,
magnesium oxide, zinc oxide and titanium oxide, which are added to
the composition to obtain benefits such as reduction of liquid
accumulation or advantageous pigment properties.
[0136] The pharmaceutical composition may also contain one or more
mono- or di-sacharides such as xylitol, sorbitol, mannitol,
lactitiol, isomalt, maltitol or xylosides, and/or
monoacylglycerols, such as monolaurin. The characteristics of the
carrier are dependent on the route of administration. One route of
administration is topical administration. For example, for topical
administrations, a preferred carrier is an emulsified cream
comprising the active peptide, but other common carriers such as
certain petrolatum/mineral-based and vegetable-based ointments can
be used, as well as polymer gels, liquid crystalline phases and
microemulsions.
[0137] It will be appreciated that the pharmaceutical compositions
may comprise one or more polypeptides of the invention, for example
one, two, three or four different peptides. By using a combination
of different peptides the anti-inflammatory effect may be
increased.
[0138] As discussed above, the polypeptide may be provided as a
salt, for example an acid adduct with inorganic acids, such as
hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid,
phosphoric acid, perchloric acid, thiocyanic acid, boric acid etc.
or with organic acid such as formic acid, acetic acid, haloacetic
acid, propionic acid, glycolic acid, citric acid, tartaric acid,
succinic acid, gluconic acid, lactic acid, malonic acid, fumaric
acid, anthranilic acid, benzoic acid, cinnamic acid,
p-toluenesulfonic acid, naphthalenesulfonic acid, sulfanilic acid
etc. Inorganic salts such as monovalent sodium, potassium or
divalent zinc, magnesium, copper calcium, all with a corresponding
anion, may be added to improve the biological activity of the
antimicrobial composition.
[0139] The pharmaceutical compositions of the invention may also be
in the form of a liposome, in which the polypeptide is combined, in
addition to other pharmaceutically acceptable carriers, with
amphipathic agents such as lipids, which exist in aggregated forms
as micelles, insoluble monolayers and liquid crystals. Suitable
lipids for liposomal formulation include, without limitation,
monoglycerides, diglycerides, sulfatides, lysolecithin,
phospholipids, saponin, bile acids, and the like. Suitable lipids
also include the lipids above modified by poly(ethylene glycol) in
the polar headgroup for prolonging bloodstream circulation time.
Preparation of such liposomal formulations is can be found in for
example U.S. Pat. No. 4,235,871.
[0140] The pharmaceutical compositions of the invention may also be
in the form of biodegradable microspheres. Aliphatic polyesters,
such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA),
copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and
polyanhydrides have been widely used as biodegradable polymers in
the production of microshperes. Preparations of such microspheres
can be found in U.S. Pat. No. 5,851,451 and in EP 213 303.
[0141] The pharmaceutical compositions of the invention may also be
formulated with micellar systems formed by surfactants and block
copolymers, preferably those containing poly(ethylene oxide)
moieties for prolonging bloodstream circulation time.
[0142] The pharmaceutical compositions of the invention may also be
in the form of polymer gels, where polymers such as starch,
cellulose ethers, cellulose, carboxymethylcellulose,
hydroxypropylmethyl cellulose, hydroxyethyl cellulose,
ethylhydroxyethyl cellulose, ethyl cellulose, methyl cellulose,
propyl cellulose, alginates, chitosan, carageenans, hyaluronic acid
and derivatives thereof, polyacrylic acid, polyvinyl imidazole,
polysulphonate, polyethylenglycol/polyethylene oxide,
polyethylene-oxide/polypropylene oxide copolymers,
polyvinylalcohol/polyvinylacetate of different degree of
hydrolysis, and polyvinylpyrrolidone are used for thickening of the
solution containing the peptide. The polymers may also comprise
gelatin or collagen.
[0143] Alternatively, the polypeptides of the invention may be
dissolved in saline, water, polyethylene glycol, propylene glycol,
ethanol or oils (such as safflower oil, corn oil, peanut oil,
cottonseed oil or sesame oil), tragacanth gum, and/or various
buffers.
[0144] The pharmaceutical composition may also include ions and a
defined pH for potentiation of action of anti-inflammatory
polypeptides.
[0145] The compositions of the invention may be subjected to
conventional pharmaceutical operations such as sterilisation and/or
may contain conventional adjuvants such as preservatives,
stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.,
e.g., as disclosed elsewhere herein.
[0146] It will be appreciated by persons skilled in the art that
the pharmaceutical compositions of the invention may be
administered locally or systemically. Routes of administration
include topical, ocular, nasal, pulmonary, buccal, parenteral
(intravenous, subcutaneous, and intramuscular), oral, vaginal and
rectal. Also administration from implants is possible. Suitable
preparation forms are, for example granules, powders, tablets,
coated tablets, (micro) capsules, suppositories, syrups, emulsions,
microemulsions, defined as optically isotropic thermodynamically
stable systems consisting of water, oil and surfactant, liquid
crystalline phases, defined as systems characterised by long-range
order but short-range disorder (examples include lamellar,
hexagonal and cubic phases, either water- or oil continuous), or
their dispersed counterparts, gels, ointments, dispersions,
suspensions, creams, aerosols, droplets or injectable solution in
ampoule form and also preparations with protracted release of
active compounds, in whose preparation excipients, diluents,
adjuvants or carriers are customarily used as described above. The
pharmaceutical composition may also be provided in bandages,
plasters or in sutures or the like.
[0147] In preferred embodiments, the pharmaceutical composition is
suitable for parenteral administration or topical
administration.
[0148] In alternative preferred embodiments, the pharmaceutical
composition is suitable for pulmonary administration or nasal
administration.
[0149] For example, the pharmaceutical compositions of the
invention can be administered intranasally or by inhalation and are
conveniently delivered in the form of a dry powder inhaler or an
aerosol spray presentation from a pressurised container, pump,
spray or nebuliser with the use of a suitable propellant, e.g.
dichlorodifluoromethane, trichlorofluoro-methane,
dichlorotetrafluoro-ethane, a hydrofluoroalkane such as
1,1,1,2-tetrafluoroethane (HFA 134A3 or
1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or
other suitable gas. In the case of a pressurised aerosol, the
dosage unit may be determined by providing a valve to deliver a
metered amount. The pressurised container, pump, spray or nebuliser
may contain a solution or suspension of the active compound, e.g.
using a mixture of ethanol and the propellant as the solvent, which
may additionally contain a lubricant, e.g. sorbitan trioleate.
Capsules and cartridges (made, for example, from gelatin) for use
in an inhaler or insufflator may be formulated to contain a powder
mix of a polypeptide of the invention and a suitable powder base
such as lactose or starch.
[0150] Aerosol or dry powder formulations are preferably arranged
so that each metered dose or `puff` contains at least 0.1 mg of a
polypeptide of the invention for delivery to the patient. It will
be appreciated that the overall daily dose with an aerosol will
vary from patient to patient, and may be administered in a single
dose or, more usually, in divided doses throughout the day.
[0151] The pharmaceutical compositions will be administered to a
patient in a pharmaceutically effective dose. By "pharmaceutically
effective dose" is meant a dose that is sufficient to produce the
desired effects in relation to the condition for which it is
administered. The exact dose is dependent on the, activity of the
compound, manner of administration, nature and severity of the
disorder, age and body weight of the patient different doses may be
needed. The administration of the dose can be carried out both by
single administration in the form of an individual dose unit or
else several smaller dose units and also by multiple
administrations of subdivided doses at specific intervals.
[0152] The pharmaceutical compositions of the invention may be
administered alone or in combination with other therapeutic agents,
such as additional antibiotic, anti-inflammatory,
immunosuppressive, vasoactive and/or antiseptic agents (such as
anti-bacterial agents, anti-fungicides, anti-viral agents, and
anti-parasitic agents).
[0153] Examples of suitable antibiotic agents include penicillins,
cephalosporins, carbacephems, cephamycins, carbapenems,
monobactams, aminoglycosides, glycopeptides, quinolones,
tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents
include iodine, silver, copper, clorhexidine, polyhexanide and
other biguanides, chitosan, acetic acid, and hydrogen peroxide.
Likewise, the pharmaceutical compositions may also contain
additional anti-inflammatory drugs, such as steroids and
macrolactam derivatives.
[0154] In one embodiment, the pharmaceutical compositions of the
invention are administered in combination with a steroid, for
example a glucocorticoid (such as dexamethasone).
[0155] It will be appreciated by persons skilled in the art that
the additional therapeutic agents may be incorporated as part of
the same pharmaceutical composition or may be administered
separately.
[0156] In one embodiment of the seventh aspect of the invention,
the pharmaceutical composition is associated with` a device or
material to be used in medicine (either externally or internally).
By `associated with` we include a device or material which is
coated, impregnated, covalently bound to or otherwise admixed with
a pharmaceutical composition of the invention (or polypeptide
thereof).
[0157] For example, the composition may be coated to a surface of a
device that comes into contact with the human body or component
thereof (e.g. blood), such as a device used in by-pass surgery,
extracorporeal circulation, wound care and/or dialysis. Thus, the
composition may be coated, painted, sprayed or otherwise applied to
or admixed with a suture, prosthesis, implant, wound dressing,
catheter, lens, skin graft, skin substitute, fibrin glue or
bandage, etc. In so doing, the composition may impart improved
anti-inflammatory and/or anti-coagulant properties to the device or
material.
[0158] Preferably, the device or material is coated with the
pharmaceutical composition of the invention (or the polypeptide
component thereof). By `coated` we mean that the pharmaceutical
composition is applied to the surface of the device or material.
Thus, the device or material may be painted or sprayed with a
solution comprising a pharmaceutical composition of the invention
(or polypeptide thereof). Alternatively, the device or material may
be dipped in a reservoir of a solution comprising a polypeptide of
the invention.
[0159] Advantageously, the device or material is impregnated with a
pharmaceutical composition of the invention (or polypeptide
thereof). By `impregnated` we mean that the pharmaceutical
composition is incorporated or otherwise mixed with the device or
material such that it is distributed throughout.
[0160] For example, the device or material may be incubated
overnight at 4.degree. C. in a solution comprising a polypeptide of
the invention. Alternatively, a pharmaceutical composition of the
invention (or polypeptide thereof) may be immobilised on the device
or material surface by evaporation or by incubation at room
temperature.
[0161] In an alternative embodiment, a polypeptide of the invention
is covalently linked to the device or material, e.g. at the
external surface of the device or material. Thus, a covalent bond
is formed between an appropriate functional group on the
polypeptide and a functional group on the device or material. For
example, methods for covalent bonding of polypeptides to polymer
supports include covalent linking via a diazonium intermediate, by
formation of peptide links, by alkylation of phenolic, amine and
sulphydryl groups on the binding protein, by using a poly
functional intermediate e.g. glutardialdehyde, and other
miscellaneous methods e.g. using silylated glass or quartz where
the reaction of trialkoxysilanes permits derivatisation of the
glass surface with many different functional groups. For details,
see Enzyme immobilisation by Griffin, M., Hammonds, E. J. and
Leach, C. K. (1993) In Technological Applications of Biocatalysts
(BIOTOL SERIES), pp. 75-118, Butterworth-Heinemann. See also the
review article entitled `Biomaterials in Tissue Engineering` by
Hubbell, J. A. (1995) Science 13:565-576.
[0162] In a preferred embodiment, the device or material comprise
or consists of a polymer. The polymer may be selected from the
group consisting of polyesters (e.g. polylactic acid, polyglycolic
acid or poly lactic acid-glycolic acid copolymers of various
composition), polyorthoesters, polyacetals, polyureas,
polycarbonates, polyurethanes, polyamides) and polysaccharide
materials (e.g. cross-linked alginates, hyaluronic acid,
carageenans, gelatines, starch, cellulose derivatives).
[0163] Alternatively, or in addition, the device or material may
comprise or consists of metals (e.g. titanium, stainless steel,
gold, titanium), metal oxides (silicon oxide, titanium oxide)
and/or ceramics (apatite, hydroxyapatite).
[0164] Such materials may be in the form of macroscopic
solids/monoliths, as chemically or physicochemically cross-linked
gels, as porous materials, or as particles.
[0165] Thus, the present invention additionally provides devices
and materials to be used in medicine, to which have been applied a
polypeptide of the invention or pharmaceutical composition
comprising the same.
[0166] Such devices and materials may be made using methods well
known in the art.
[0167] An eighth aspect of the invention provides a polypeptide
according to the second aspect of the invention or a pharmaceutical
composition according to the seventh aspect of the invention for
use in medicine.
[0168] In preferred embodiments, the polypeptide according to the
second aspect of the invention or the pharmaceutical composition
according to the seventh aspect of the invention are for use:
[0169] (a) the treatment and/prevention of acute and/or chronic
inflammation; [0170] (b) the treatment and/prevention of microbial
infection (e.g. bacterial infection); [0171] (c) the modulation of
blood coagulation; and/or [0172] (d) the treatment of wounds.
[0173] For example, the polypeptide according to the second aspect
of the invention or the pharmaceutical composition according to the
seventh aspect of the invention may be for use in the treatment
and/prevention of a disease, condition or indication selected from
the following: [0174] i) Acute systemic inflammatory disease, with
or without an infective component, such as systemic inflammatory
response syndrome (SIRS), ARDS, sepsis, severe sepsis, and septic
shock. Other generalized or localized invasive infective and
inflammatory disease, including erysipelas, meningitis, arthritis,
toxic shock syndrome, diverticulitis, appendicitis, pancreatitis,
cholecystitis, colitis, cellulitis, burn wound infections,
pneumonia, urinary tract infections, postoperative infections, and
peritonitis. [0175] ii) Chronic inflammatory and or infective
diseases, including cystic fibrosis, COPD and other pulmonary
diseases, gastrointestinal disease including chronic skin and
stomach ulcerations, other epithelial inflammatory and or infective
disease such as atopic dermatitis, oral ulcerations (aphtous
ulcers), genital ulcerations and inflammatory changes,
parodontitis, eye inflammations including conjunctivitis and
keratitis, external otitis, mediaotitis, genitourinary
inflammations. [0176] iii) Postoperative inflammation. Inflammatory
and coagulative disorders including thrombosis, DIC, postoperative
coagulation disorders, and coagulative disorders related to contact
with foreign material, including extracorporeal circulation, and
use of biomaterials. Furthermore, vasculitis related inflammatory
disease, as well as allergy, including allergic rhinitis and
asthma. [0177] iv) Excessive contact activation and/or coagulation
in relation to, but not limited to, stroke. [0178] v) Excessive
inflammation in combination with antimicrobial treatment.
[0179] The antimicrobial agents used may be administred by various
routes; intravenous (iv), intraarterial, intravitreal, subcutaneous
(sc), intramuscular (im), intraperitoneal (ip), intravesical,
intratechal, epidural, enteral (including oral, rectal, gastric,
and other enteral routes), or topically, (including dermal, nasal
application, application in the eye or ear, eg by drops, and
pulmonary inhalation). Examples of agents are penicillins,
cephalosporins, carbacephems, cephamycins, carbapenems,
monobactams, aminoglycosides, glycopeptides, quinolones,
tetracyclines, macrolides, and fluoroquinolones. Antiseptic agents
include iodine, silver, copper, clorhexidine, polyhexanide and
other biguanides, chitosan, acetic acid, and hydrogen peroxide.
[0180] Thus, the polypeptides may be for use in the treatment or
prevention of an acute inflammation, sepsis, acute respiratory
distress syndrome (ARDS), chronic obstructive pulmonary disease
(COPD), cystic fibrosis, wounds, asthma, allergic and other types
of rhinitis, cutaneous and systemic vasculitis, thrombosis and/or
disseminated intravascular coagulation (DIC).
[0181] A related ninth aspect of the invention provides the use of
a polypeptide according to the second aspect of the invention or a
pharmaceutical composition according to the seventh aspect of the
invention in the preparation of a medicament for the treatment or
prevention of inflammation and/or excessive coagulation (as
described above).
[0182] A tenth aspect of the invention provides a method for
treating or preventing inflammation and/or coagulation in a
patient, the method comprising administering to the patient a
therapeutically-effective amount of a polypeptide according to the
second aspect of the invention or a pharmaceutical composition
according to the seventh aspect of the invention (as described
above). In preferred but non-limiting embodiments, the method is
for the treatment or prevention of an acute inflammation, sepsis,
acute respiratory distress syndrome (ARDS), chronic obstructive
pulmonary disease (COPD), cystic fibrosis, asthma, allergic and
other types of rhinitis, cutaneous and systemic vasculitis,
thrombosis and/or disseminated intravascular coagulation (DIC).
[0183] Persons skilled in the art will further appreciate that the
uses and methods of the present invention have utility in both the
medical and veterinary fields. Thus, the polypeptide medicaments
may be used in the treatment of both human and non-human animals
(such as horses, dogs and cats). Advantageously, however, the
patient is human.
[0184] Preferred aspects of the invention are described in the
following non-limiting examples, with reference to the following
figures:
[0185] FIG. 1: NO-blocking effects of C-terminal peptides of
TFPI.
[0186] RAW 264.7 macrophages were stimulated with 10 ng/ml E. coli
LPS, and 10 .mu.M of the peptides GGL27, LIK17 and TKR22 were
added. NO was measured using Griess reagent.
[0187] FIG. 2: NO-blocking effects of peptides of heparin cofactor
II.
[0188] RAW macrophages were stimulated with 10 ng/ml E. coli LPS,
and the peptides KYE28, NLF20, and KYE21, were added at the
indicated doses. LL-37 is presented as positive control. NO was
measured using Griess reagent.
[0189] FIG. 3: Anti-inflammatory effects of peptides of heparin
cofactor II.
[0190] KYE28, KYE21 and NLF20 blocks NO production of RAW264.7
macrophages stimulated with various microbial products. Cells were
subjected to the indicated concentrations of E. coli LPS,
lipoteichoic acid (LTA) and peptidoglycan (PGN) from S. aureus as
well as zymosan A from Saccharosmyces cerevisiae. NO production
with or without 10 .mu.M GKY25 was determined by using the Griess
reagent.
[0191] FIG. 4: HRG is LPS-binding.
[0192] 2 and 5 .mu.g HRG was applied onto a nitrocellulose
membrane, followed by incubation with iodinated LPS in 10 mM Tris,
pH 7.4 or 10 mM MES, pH 5.5, with or without 0.15M NaCl.
Radioactivity was visualized using a phosphorimager system.
[0193] FIG. 5: HRG increases LPS mediated NO release from murine
macrophages.
[0194] RAW macrophages were incubated with 100 ng/ml LPS with or
without LL-37 and HRG (2 and 10 .mu.M) for 24 hours. The
supernatant was aspirated and NO was measured using Griess method.
HRG significantly increased LPS mediated NO release in a dose
dependent way (n=6, p=0.001).
[0195] FIG. 6: Antibodies against TLR4 block NO release.
[0196] RAW macrophages were incubated with 100 ng/ml LPS with or
without HRG (20M) and polyclonal anti mouse TRL4 (5 .mu.g/ml) for
24 hours. Supernatant was aspirated and NO was measured using
Griess method. Anti mouse TLR4 were able to significantly block LPS
and HRG mediated NO release. (n=6, p=0.001).
[0197] FIG. 7: Increased survival in LPS induced sepsis in
Hrg.sup.-/- mice.
[0198] Increased survival of Hrg-/- mice after LPS challenge.
Wildtype (dashed line) and Hrg.sup.-/- (solid line) mice were
injected i.p. with E. coli LPS and survival of the animals was
followed for seven days. Mice lacking HRG (n=8) showed a
significantly increased survival compared with wildtype animals
(n=9, p=0.002).
[0199] FIG. 8: Visual observation scores 18 hours after LPS
challenge.
[0200] Status of wt and Hrg.sup.-/- mice was visual observed
(ruffled fur, hunched and pre-mortal) 18 hours post LPS challenge.
Wildtype mice were visually significantly sicker Status of wt and
Hrg.sup.-/- mice was visual observed (ruffled fur [black], hunched
[light grey] and pre-mortal [dark grey]) 18 hours post LPS
challenge. Wildtype mice were visually significantly sicker
compared with Hrg.sup.-/- mice, p=0.001 n=10.
[0201] FIG. 9: Vascular leakage is decreased in lungs of
Hrg.sup.-/- mice after LPS challenge compared to wildtype
animals.
[0202] Lungs of wt and Hrg.sup.-/- mice, were analyzed by scanning
electron microscopy after LPS injection i.p. A) wildtype B) Hrg-/-
C) wt, 24 hours post-treatment of LPS D) Hrg.sup.-/-, 24 hours
post-treatment of LPS. Scale bar represents 50 .mu.m. A
representative lung section is shown.
[0203] FIG. 10: GHH25 inhibits HRG and LPS induced responses in
murine macrophages.
[0204] RAW macrophages were incubated with 100 ng/ml LPS with or
without HRG (2 .mu.M) and GHH25 (10 and 100 .mu.M) for 24 hours.
The supernatant was aspirated and NO was measured using Griess
method. GHH25 significantly decreased LPS and HRG mediated NO
release in a dose dependent way. (n=6, p=0.001).
[0205] FIG. 11: GHH25 inhibits HRG and LPS induced responses in
human macrophages.
[0206] Human macrophages were incubated with 10 ng/ml LPS with or
without HRG (2 .mu.M) and GHH25 (10 and 100 .mu.M) for 24 hours.
Supernatant was aspirated and TNF-.alpha. was measured.
[0207] FIG. 12: Improved health status and increased survival in
LPS induced sepsis after treatment with GHH25.
[0208] Increased survival of wildtype mice after treatment with
GHH25. Wildtype animals were injected i.p. with E. coli LPS, and 1
mg GHH25 (dashed line) or buffer only (solid line) was injected 30
minutes after. Survival of the animals was followed for seven days.
Mice treated with GHH25 (n=13) showed a significantly increased
survival when compared with untreated animals (n=12, p=0.03).
[0209] FIG. 13: Visual observation scores 18 hours after LPS
challenge.
[0210] Status of wt and Hrg.sup.-/- mice was visual observed
(ruffled fur, hunched and) 18 hours post LPS challenge. Wildtype
mice were visually significantly sicker Status of wt and
Hrg.sup.-/- mice was visual observed (ruffled fur [black], hunched
[light grey] and pre-mortal [dark grey]) 18 hours post LPS
challenge. Wildtype mice were visually significantly more sick
compared with Hrg.sup.-/- mice, p=0.004 n=12.
[0211] FIG. 14: C-terminal peptides of TFPI block coagulation.
(A)
[0212] The C-terminal peptides of TFPI; GGL27, LIK17, as well as
TKR22 impair the intrinsic pathway of coagulation in normal human
plasma. This was determined by measuring the activated partial
thromboplastin time (aPTT). GGL27 and LIK17 also affected
prothrombin time (PT) monitoring the extrinsic pathway of
coagulation. The thrombin clotting time (TCT), measuring thrombin
induced fibrin network formation, were not significantly affected
by the peptides (B) GGL-27 impairs coagulation in a dose dependent
manner monitored by measuring the aPTT, PT and TCT in normal human
plasma.
[0213] FIG. 15: Peptides of heparin-cofactor II block
coagulation.
[0214] KYE28 and NLF20 impair the intrinsic pathway of coagulation
in normal human plasma determined by measuring the activated
partial thromboplastin time (aPTT). KYE21 shows only minor blocking
of the aPTT. Other parts of the coagulation system, as judged by
the prothrombin time (PT) monitoring the extrinsic pathway of
coagulation, and the thrombin clotting time (TCT), measuring
thrombin induced fibrin network formation, were not significantly
affected.
[0215] FIG. 16: Cartoon illustrating the structure of TFPI.
[0216] Cleavage points by enzymes are indicated.
[0217] FIG. 17: Antimicrobial activities of TFPI-derived
peptides.
[0218] Antimicrobial activity of selected peptides (at 100 .mu.M in
RDA) against the indicated microbes. For determination of
antimicrobial activities, E. coli ATCC 25922, S. aureus ATCC 29213
isolates (4.times.10.sup.6 cfu) or C. parapsilosis ATCC 90018
(1.times.10.sup.5 cfu) was inoculated in 0.1% TSB agarose gel. Each
4 mm-diameter well was loaded with 6 .mu.l of peptide. The zones of
clearance correspond to the inhibitory effect of each peptide after
incubation at 37.degree. C. for 18-24 h (mean values are presented,
n=3).
[0219] FIG. 18: Antibacterial effects of TFPI-derived peptides.
[0220] Effects of TFPI-derived peptides and LL-37 against E. coli,
P. aeruginosa, and S. aureus in viable count assays.
2.times.10.sup.8 cfu/ml of bacteria were incubated in 50 .mu.l with
peptides at the indicated concentrations in 10 mM Tris, pH 7.4
buffer (Tris), or in 0.15 m NaCl, 10 mM Tris, pH 7.4 containing
normal or heat-inactivated 20% human plasma (n=3, SD is
indicated).
[0221] FIG. 19: Kinetic analysis.
[0222] The time-dependence of bacterial killing by TFPI-derived
peptides (at 6 [ left panel] and 30 .mu.M [right panel]) in 0.15 m
NaCl, 10 mM Tris, pH 7.4 containing 20% plasma was analyzed by
viable count assays using E. coli.
[0223] FIG. 20: Effects on bacterial membranes.
[0224] (A) Permeabilizing effects of peptides on P. aeruginosa and
E. coli. (A) Bacteria were incubated with the indicated peptides
and permeabilization was assessed using the impermeant probe FITC.
(B) Electron microscopy analysis. P. aeruginosa and S. aureus
bacteria was incubated for 2 h at 37.degree. C. with 30 .mu.M of
GKY25 and LL-37 and analysed with electron microscopy. Scale bar
represents 1 .mu.m. Control; Buffer control.
[0225] FIG. 21: Structure of TFPI peptide LIK17.
[0226] Helical content of the TFPI-derived C-terminal peptide in
presence of negatively charged liposomes (DOPE/DOPG). LIK17
structure was largely unaffected by the addition of liposomes.
[0227] FIG. 22: CD spectra of LIK17 in Tris-buffer and in presence
of LPS. For control, CD spectra for buffer and LPS alone are also
presented.
[0228] FIG. 23: Effects of the indicated peptides on liposome
leakage.
[0229] The membrane permeabilizing effect was recorded by measuring
fluorescence release of carboxyfluorescein from DOPE/DOPG
(negatively charged) liposomes. The experiments were performed in
10 mM Tris-buffer, pH 7.4. Values represents mean of triplicate
samples.
[0230] FIG. 24: Activities on eukaryotic cells
[0231] Hemolytic effects of the indicated peptides. The cells were
incubated with different concentrations of the peptides, 2% Triton
X-100 (Sigma-Aldrich) served as positive control. The absorbance of
hemoglobin release was measured at .lamda. 540 nm and is expressed
as % of Triton X-100 induced hemolysis (note the scale of the
y-axis). Effects of LL-37 are shown for comparison.
[0232] FIG. 25: HaCaT keratinocytes were subjected to the indicated
TFPI-peptides as well as LL-37. Cell permeabilizing effects were
measured by the LDH based TOX-7 kit. LDH release from the cells was
monitored at .lamda. 490 nm and was plotted as % of total LDH
release.
[0233] FIG. 26: The MIT-assay was used to measure viability of
HaCaT keratinocytes in the presence of the indicated peptides (at
60 .mu.M). In the assay, MTT is modified into a dye, blue formazan,
by enzymes associated with metabolic activity. The absorbance of
the dye was measured at .lamda. 550 nm.
[0234] FIG. 27: Antimicrobial activities of heparin cofactor
II-derived peptides.
[0235] Antimicrobial activity of selected peptides (at 100 .mu.M)
in RDA against the indicated microbes. For determination of
antimicrobial activities, E. coli ATCC 25922, S. aureus ATCC 29213
isolates (4.times.10.sup.6 cfu) or C. parapsilosis ATCC 90018
(1.times.10.sup.5 cfu) was inoculated in 0.1% TSB agarose gel. Each
4 mm-diameter well was loaded with 6 .mu.l of peptide. The zones of
clearance correspond to the inhibitory effect of each peptide after
incubation at 37.degree. C. for 18-24 h (mean values are presented,
n=3).
[0236] FIG. 28: (upper) Antibacterial effects of heparin cofactor
II-derived peptides and LL-37 against E. coli in viable count
assays. 2.times.10.sup.6 cfu/ml of bacteria were incubated in 50
.mu.l with peptides at the indicated concentrations in 10 mM Tris,
pH 7.4 buffer (Tris), or in 0.15 m NaCl, 10 mM Tris, pH 7.4
containing 20% human plasma (n=3, SD is indicated). (lower) The
time-dependence of bacterial killing by heparin cofactor II-derived
peptides (at 6 and 30 .mu.M) in 0.15 m NaCl, 10 mM Tris, pH 7.4
containing 20% plasma was analyzed by viable count assays using E.
coli. LL-37 (30 .mu.M) was used for comparison.
[0237] FIG. 29: Effects on bacterial membranes.
[0238] (A) Permeabilizing effects of peptides on P. aeruginosa and
E. coli. (A) Bacteria were incubated with the indicated peptides
and permeabilization was assessed using the impermeant probe FITC.
(B) Electron microscopy analysis. P. aeruginosa and S. aureus
bacteria was incubated for 2 h at 37.degree. C. with 30 .mu.M of
NLF20 and LL-37 and analysed with electron microscopy. Scale bar
represents 1 .mu.m. Control; Buffer control.
[0239] FIG. 30: Structure and effects on liposomes.
[0240] (A) Helical content of the heparin cofactor II-derived
C-terminal peptides in presence of negatively charged liposomes
(DOPE/DOPG). (B) Effects of NLF20 on liposome leakage. The membrane
permeabilizing effect was recorded by measuring fluorescence
release of carboxyfluorescein from DOPE/DOPG (negatively charged)
liposomes. (C) CD spectra of NLF20 in Tris-buffer and in presence
of LPS. For control, CD spectra for buffer and LPS alone are also
presented. The experiments were performed in 10 mM Tris-buffer, pH
7.4. Values represents mean of triplicate samples
[0241] FIG. 31: Effects of NLF20 in an animal model of P.
aeruginosa sepsis.
[0242] The thrombin HCII peptide NLF20 significantly increases
survival. Mice were i.p. injected with P. aeruginosa bacteria,
followed by subcutaneous injection of NLF20 or buffer only, after 1
h and then with intervals of 24 h for the three following days.
Treatment with the peptide significantly increased survival.
[0243] FIG. 32: Activities on eukaryotic cells
[0244] (A) Hemolytic effects of the indicated peptides. The cells
were incubated with different concentrations of the peptides, 2%
Triton X-100 (Sigma-Aldrich) served as positive control. The
absorbance of hemoglobin release was measured at .lamda. 540 nm and
is expressed as % of Triton X-100 induced hemolysis (note the scale
of the y-axis). Effects of LL-37 are shown for comparison. (B)
HaCaT keratinocytes were subjected to the indicated HCII-peptides
as well as LL-37. Cell permeabilizing effects were measured by the
LDH based TOX-7 kit. LDH release from the cells was monitored at
.lamda. 490 nm and was plotted as % of total LDH release. (C) The
MTT-assay was used to measure viability of HaCaT keratinocytes in
the presence of the indicated peptides (at 60 .mu.M). In the assay,
MTT is modified into a dye, blue formazan, by enzymes associated
with metabolic activity. The absorbance of the dye was measured at
.lamda. 550 nm. Right panel shows results in presence of 20% plasma
(hemolysis) or 20% serum (LDH and MTT).
[0245] FIG. 33: Antithrombin III-derived peptide FFF21 blocks
coagulation.
[0246] FFF21 impairs the intrinsic pathway of coagulation in normal
human plasma determined by measuring the activated partial
thromboplastin time (aPTT). Other parts of the coagulation system,
as judged by the prothrombin time (PT) monitoring the extrinsic
pathway of coagulation, and the thrombin clotting time (TCT),
measuring thrombin induced fibrin network formation, were not
significantly affected.
[0247] FIG. 34. Antimicrobial activities of TFPI-derived
peptides
[0248] (A) Cartoon illustrating the structure of TFPI. Enzymatic
cleavage sites are indicated. (B) Antimicrobial activity (using RDA
of selected peptides against the indicated microbes. For
determination of antimicrobial activities, E. coli ATCC 25922, P.
aeruginosa ATCC 27853, S. aureus ATCC 29213 or B. subtilis ATCC
6633 isolates (4.times.10.sup.6 cfu) or C. albicans ATCC 90028 and
C. parapsilosis ATCC 90018 (1.times.10.sup.5 cfu) were inoculated
in 0.1% TSB agarose gel. Each 4 mm-diameter well was loaded with 6
.mu.l of peptide (at 100 .mu.M). The zones of clearance correspond
to the inhibitory effect of each peptide after incubation at
37.degree. C. for 18-24 h (mean values are presented, n=3). (C)
Antibacterial effects of the TFPI-derived peptides and LL-37
against E. coli ATCC 25922 in viable count assays. 2.times.10.sup.6
cfu/ml of bacteria were incubated in 50 .mu.l with peptides at the
indicated concentrations in 10 mM Tris, pH 7.4 buffer, and the cfu
were determined.
[0249] FIG. 35. Effects on bacterial membranes
[0250] (A) Binding of TFPI to bacterial surfaces. The indicated
bacteria (1-2.times.10.sup.9 cfu/ml) were incubated with 3 .mu.M of
TAMRA labeled GGL27 peptide in 500 .mu.l human plasma and samples
were analyzed by FACS. (B) Permeabilizing effects of peptides on E.
coli. Bacteria were incubated with the indicated peptides and
permeabilization was assessed using the impermeant probe FITC. (C)
Electron microscopy analysis. P. aeruginosa bacteria were incubated
for 2 h at 37.degree. C. with 30 .mu.M of LIK17 and LL-37 and
visualized by negative staining. Scale bar represents 1 .mu.m.
Control; Buffer control.
[0251] FIG. 36. Structure and effects on liposomes
[0252] (A) Effects of the indicated peptides on liposome leakage.
The membrane permeabilizing effect was recorded by measuring
fluorescence release of carboxyfluorescein from DOPE/DOPG
(negatively charged) liposomes. The experiments were performed in
10 mM Tris-buffer. Values represents mean of triplicate samples.
(B) Kinetics of CF release from liposomes. 1 .mu.M of peptides was
used. (C) Helical content of the TFPI-derived C-terminal LIK16 and
GGL27 peptides in presence of negatively charged liposomes
(DOPE/DOPG). LIK17 and GGL27 structure was largely unaffected by
the addition of liposomes. (D) CD spectra of LIK17 and GGL27 in
Tris-buffer and in presence of LPS. For control, CD spectra for
buffer and LPS alone are also presented.
[0253] FIG. 37. Activities on eukaryotic cells
[0254] (A) Hemolytic effects of the indicated peptides. The cells
were incubated with different concentrations of the peptides, 2%
Triton X-100 (Sigma-Aldrich) served as positive control. The
absorbance of hemoglobin release was measured at .lamda. 540 nm and
is expressed as % of Triton X-100 induced hemolysis (note the scale
of the y-axis). Effects of LL-37 are shown for comparison. (B)
Upper panel: HaCaT keratinocytes were subjected to the indicated
TFPI-peptides as well as LL-37. Cell permeabilizing effects were
measured by the LDH based TOX-7 kit. LDH release from the cells was
monitored at .lamda. 490 nm and was plotted as % of total LDH
release. Lower panel: The MU-assay was used to measure viability of
HaCaT keratinocytes in the presence of the indicated peptides (at
60 .mu.M). In the assay, MU is modified into a dye, blue formazan,
by enzymes associated with metabolic activity. The absorbance of
the dye was measured at .lamda. 550 nm.
[0255] FIG. 38. Activities of C-terminal TFPI-derived peptides
[0256] (A) Antibacterial effects of the TFPI-derived peptides and
LL-37 against E. coli or P. aeruginosa in viable count assays.
2.times.10.sup.6 cfu/ml of bacteria were incubated in 50 with
peptides at the indicated concentrations in 10 mM Tris, 0.15 M
NaCl, pH 7.4 (buffer), or in 0.15 m NaCl, 10 mM Tris, pH 7.4
containing 20% human citrate plasma (CP), or in the same buffer but
with heat-inactivated human citrate plasma (HCP) (n=3, SD is
indicated). (B) The indicated bacterial isolates were subjected to
GGL27 and LIK17 at 3 .mu.M in buffer, native plasma (CP), or
heat-inactivated plasma (HCP) for 2 h and the number of cfu was
determined.
[0257] FIG. 39. The C-terminal TFPI peptide GGL27 enhances C1q, C3a
and MAC binding to E. coli
[0258] (A) E. coli ATCC 25922 and P. aeruginosa 15159 bacteria were
washed, resuspended, and incubated with citrate plasma either alone
or supplemented with GGL27 (at 3 .mu.M) for 30 min or 1 h at
37.degree. C. The bacterial cells were collected, washed with PBS,
and bound proteins and corresponding supernatants were subjected to
Tris-Tricine SDS-PAGE under reducing conditions, followed by
immunoblotting with antibodies recognizing C1q or C5b-9. CP;
citrate plasma, S; supernatant or unbound bacterial, P; pellet or
material bound to bacterial cells. (B) As in (A), but antibodies
against the C3a were used (25). (C) (left panel) Comparison of the
mean proportion of bacteria positive for C1q/C3a binding in citrate
plasma (control; black columns) and in plasma supplemented with
GGL27 (gray columns). (right panel) Comparative degree of C1q and
C3a binding to E. coli and P. aeruginosa strains, expressed as
means of the fluorescence index (Fl; proportion of bacteria
positive for C1q/C3a multiplied by the mean intensity of C1q/C3a
binding). (*; p<0.05; **; p<0.01, ***; p<0.001, t-test).
(D) Examples of flow cytometry histograms of C1q/C3a binding to E.
coli and P. aeruginosa in citrate plasma and in plasma supplemented
with GGL27.
[0259] FIG. 40. Identification of TFPI in human skin and
wounds.
[0260] (A) Immunohistochemical identification of TFPI in normal
skin, acute wound (AW) and in chronic venous leg ulcer tissue
(chronic wounds; CW). Skin biopsies were taken from normal skin
(normal skin; n=3), acute wounds (AW-1 and AW-2; 5 and 8 days after
wounding, respectively) and from the wound edges of patients with
chronic venous ulcers (CW; n=3). Representative sections are shown.
In normal skin TFPI is detected mainly in the basal layers of
epidermis. TFPI in AW and CW are ubiquitously found in all
epidermal layers. Scale bar is 100 .mu.m. (B) TFPI derived peptides
are found in human wounds. Visualization of binding of C-terminal
TFPI peptides to bacteria found in fibrin slough from a P.
aeruginosa infected chronic wound surface. The peptides bind to
fibrin (A), bacteria (B, C), and bacteria inside a macrophage (D).
In (E) and (F), TFPI and C3a peptides were visualized by immunogold
using gold-labeled antibodies of different sizes, specific for C3a
(10 nm) and C-termini of TFPI (20 nm), respectively. Evaluation of
50 bacterial profiles showed that .about.70% of TFPI-molecules were
associated with C3a. See inset for exemplification.
[0261] FIG. 41. In vitro and in vivo effects of GGL27
[0262] (A) 2.times.10.sup.6 cfu/ml of E. coli were incubated in 50
.mu.l with GGL27 peptide at the indicated concentrations in 10 mM
Tris, pH 7.4 buffer (Buffer), or in 0.15 m NaCl, 10 mM Tris, pH 7.4
containing 20% human citrate plasma (CP), or in the same buffer but
with mice citrate plasma (Balb/c or C57B/6) (n=3, SD is indicated).
(B) The GGL27 peptide prolongs survival in E. coli and P.
aeruginosa infected mice. Mice were injected i.p. with E. coli or
P. aeruginosa bacteria. In the E. coli infection model GGL27 (200
.mu.g) or buffer alone was injected i.p. after 30 min. (n=10 in
each group, P<0.001, Kaplan-Meier Survival Analysis Log-Rank
test). In the P. aeruginosa infection model GGL27 (500 .mu.g) or
buffer alone was injected s.c. after 1 h (n=8 in each group,
P<0.001, Kaplan-Meier Survival Analysis Log-Rank test). (C)
GGL27 inhibits NO production. RAW264.7 mouse macrophages were
stimulated with LPS from E. coli (left panel) or P. aeruginosa
(right panel) in presence of GGL27 or the two control peptides
GGL27(S) and DSE25 at the indicated concentrations. The difference
between GGL27 and the control peptides is statistically significant
(P<0.01). (D) GGL27 significantly increases survival in
LPS-induced shock. Mice were injected with E. coli LPS followed by
intraperitoneal administration of GGL27 (500 .mu.g). Buffer and the
peptides GGL27(S) and DSE25 (500 .mu.g) served as controls.
Survival was followed for 7 days. (GGL27; n=16, buffer; n=15,
GGL27(S); n=8, DSE25; n=8. The difference between GGL27 and buffer,
or the control peptides is significant, P<0.01, Kaplan-Meier
Survival Analysis Log-Rank test). * indicates that the lines for
GGL27(S), DSE25, and buffer are overlaid. (E) GGL27 attenuates
proinflammatory cytokines. In a separate experiment, mice were
sacrificed 20 h after i.p. injection of LPS followed by treatment
as above with GGL27 (500 .mu.g), buffer or the control peptides
GGL27(S) or DSE25, and the indicated cytokines were analyzed in
blood (Control; n=8; GGL27, n=12; GGL27(S), n=7; DSE25; n=8). In
all cases, the difference between GGL27-treated animals and buffer
was significant. P values for the respective cytokines are IL-6;
0.0023, TNF-.alpha.; 0.0018, IFN-.gamma.; 0.0002, MCP-1; 0.0002,
IL-10; 0.0023. There was a significant difference between controls
and GGL27(S) with respect to IFN-.gamma.. (F) Lungs were analyzed
20 h after LPS injection i.p. followed by treatment with GGL27 (500
.mu.g) or buffer. Histochemical analysis shows marked attenuation
of inflammatory changes in GGL27-treated lungs (a representative
lung section is shown).
[0263] FIG. 42. Evolution of TFPI
[0264] The phylogenetic tree and sequence similarities show that
TFPI in Homo sapiens, Pongo abelii and Sus scrofa are closely
related.
[0265] FIG. 43
[0266] The peptide GGL27, and the control peptides GGL27(S), having
the central K/R residues replaced by S, and the peptide DSE25, from
the N-terminus of TFPI were analyzed for antimicrobial activities
against the indicated bacteria and fungi. The inhibitory zones of
the peptides are indicated. For determination of antimicrobial
activities, bacteria (4.times.10.sup.6 cfu) or fungi
(1.times.10.sup.5 cfu) were inoculated in 0.1% TSB agarose gel.
Each 4 mm-diameter well was loaded with 6 .mu.l of peptide (at 100
.mu.M). The zones of clearance correspond to the inhibitory effect
of each peptide after incubation at 37.degree. C. for 18-24 h (mean
values are presented, n=3). *indicates zero values.
[0267] FIG. 44
[0268] Bacteria were incubated with GGL27 and permeabilization was
assessed using the impermeant probe FITC.
[0269] FIG. 45
[0270] The time-dependence of bacterial killing by the indicated
TFPI-derived peptides (at 30 .mu.M) in 0.15 M NaCl, 10 mM Tris, pH
7.4 containing 20% plasma was analyzed by viable count assays using
E. coli and P. aeruginosa.
[0271] FIG. 46. HRG is LPS-binding
[0272] 2 and 5 .mu.g HRG was applied onto a nitrocellulose
membrane, followed by incubation with iodinated LPS in 10 mM Tris,
pH 7.4 or 10 mM MES, pH 5.5, with or without 0.15M NaCl.
Radioactivity was visualized using a phosphorimager system.
[0273] FIG. 47. HRG increases LPS mediated NO release from murine
macrophages.
[0274] RAW macrophages were incubated with 100 ng/ml LPS with or
without LL-37 and HRG (2 and 10 .mu.M) for 24 hours. The
supernatant was aspirated and NO was measured using Griess method.
HRG significantly increased LPS mediated NO release in a dose
dependent way (n=6, p=0.001).
[0275] FIG. 48. Antibodies against TLR4 block NO release
[0276] RAW macrophages were incubated with 100 ng/ml LPS with or
without HRG (2 .mu.M) and polyclonal anti mouse TRL4 (5 .mu.g/ml)
for 24 hours. Supernatant was aspirated and NO was measured using
Griess method. Anti mouse TLR4 were able to significantly block LPS
and HRG mediated NO release. (n=6, p=0.001).
[0277] FIG. 49. Increased survival in LPS induced sepsis in
Hrg.sup.-/- mice
[0278] Increased survival of Hrg-/- mice after LPS challenge.
Wildtype (dashed line) and Hrg.sup.-1 (solid line) mice were
injected i.p. with E. coli LPS and survival of the animals was
followed for seven days. Mice lacking HRG (n=8) showed a
significantly increased survival compared with wildtype animals
(n=9, p=0.002).
[0279] FIG. 50. Visual observation scores 18 hours after LPS
challenge
[0280] Status of wt and Hrg.sup.-/- mice was visual observed
(ruffled fur, hunched and) 18 hours post LPS challenge. Wildtype
mice were visually significantly sicker Status of wt and
Hrg.sup.-/- mice was visual observed (ruffled fur [black], hunched
[light grey] and pre-mortal [dark grey]) 18 hours post LPS
challenge. Wildtype mice were visually significantly sicker
compared with Hrg.sup.-/- mice, p=0.001 n=10.
[0281] FIG. 51. Vascular leakage is decreased in lungs of
Hrg.sup.-/- mice after LPS challenge compared to wildtype
animals
[0282] Lungs of wt and Hrg.sup.-/- mice, were analyzed by scanning
electron microscopy after LPS injection i.p. A) wildtype B) Hrg-/-
C) wt, 24 hours post-treatment of LPS D) Hrg.sup.-/-, 24 hours
post-treatment of LPS. Scale bar represents 50 .mu.m. A
representative lung section is shown.
[0283] FIG. 52. GHH25 inhibits HRG and LPS induced responses in
murine macrophages
[0284] RAW macrophages were incubated with 100 ng/ml LPS with or
without HRG (2 .mu.M) and GHH25 (10 and 100 .mu.M) for 24 hours.
The supernatant was aspirated and NO was measured using Griess
method. GHH25 significantly decreased LPS and HRG mediated NO
release in a dose dependent way. (n=6, p=0.001).
[0285] FIG. 53. GHH25 inhibits HRG and LPS induced responses in
human macrophages
[0286] Human macrophages were incubated with 10 ng/ml LPS with or
without HRG (2 .mu.M) and GHH25 (10 and 100 .mu.M) for 24 hours.
Supernatant was aspirated and TNF-.alpha. was measured.
[0287] FIG. 54. Improved health status and increased survival in
LPS induced sepsis after treatment with GHH25
[0288] Increased survival of wildtype mice after treatment with
GHH25. Wildtype animals were injected i.p. with E. coli LPS, and 1
mg GHH25 (dashed line) or buffer only (solid line) was injected 30
minutes after. Survival of the animals was followed for seven days.
Mice treated with GHH25 (n=13) showed a significantly increased
survival when compared with untreated animals (n=12, p=0.03).
[0289] FIG. 55. Visual observation scores 18 hours after LPS
challenge
[0290] Status of wt and Hrg.sup.-/- mice was visual observed
(ruffled fur, hunched and) 18 hours post LPS challenge. Wildtype
mice were visually significantly sicker Status of wt and
Hrg.sup.-/- mice was visual observed (ruffled fur [black], hunched
[light grey] and pre-mortal [dark grey]) 18 hours post LPS
challenge. Wildtype mice were visually significantly more sick
compared with Hrg.sup.-/- mice, p=0.004 n=12.
[0291] FIG. 56. Antithrombin III-derived peptide FFF21 blocks
coagulation
[0292] FFF21 impairs the intrinsic pathway of coagulation in normal
human plasma determined by measuring the activated partial
thromboplastin time (aPTT). Other parts of the coagulation system,
as judged by the prothrombin time (PT) monitoring the extrinsic
pathway of coagulation, and the thrombin clotting time (TCT),
measuring thrombin induced fibrin network formation, were not
significantly affected.
EXAMPLES
Example A
Introduction
Heparin Cofactor II
[0293] Serpins are a group of proteins with similar structures that
were first identified as a set of proteins able to inhibit
proteases. The acronym serpin was originally coined because many
serpins inhibit chymotrypsin-like serine proteases (serine protease
inhibitors). The first members of the serpin superfamily to be
extensively studied were the human plasma proteins antithrombin and
antitrypsin, which play key roles in controlling blood coagulation
and inflammation, respectively.
[0294] Structural studies on serpins have revealed that inhibitory
members of the family undergo an unusual conformational change,
termed the Stressed to Relaxed (S to R) transition. This
conformational mobility of serpins provides a key advantage over
static lock-and-key protease inhibitors. In particular, the
function of inhibitory serpins can be readily controlled by
specific cofactors like heparin. The archetypal example of this
situation is antithrombin, which circulates in plasma in a
relatively inactive state. Upon binding a high-affinity heparin
pentasaccharide sequence within long-chain heparin, antithrombin
undergoes a conformational change, exposing key residues important
for the mechanism. The heparin pentasaccharide-bound form of
antithrombin is, thus, a more effective inhibitor of thrombin and
factor Xa. Furthermore, both of these coagulation proteases contain
binding sites (called exosites) for heparin. Heparin, therefore,
also acts as a template for binding of both protease and serpin,
further dramatically accelerating the interaction between the two
parties. After the initial interaction, the final serpin complex is
formed and the heparin moiety is released.
[0295] Peptides corresponding to the heparin binding sites in these
proteins possess antibacterial, anti-inflammatory and
anti-coagulation properties.
Tissue Factor Pathway Inhibitor (TFPI)
[0296] Tissue factor pathway inhibitor (or TFPI) is a Kunitz-type
proteinase inhibitor which reversibly inhibits the tissue
factor-factor VII (TF-VII) complex in a factor X (FX) dependent
manner, leading to inhibition of both FX and FIX activation. TFPI
consists of a highly negatively-charged amino-terminus, three
tandemly-linked Kunitz-type domains, and a highly
positively-charged carboxy-terminus. In plasma, TFPI exists in both
full-length and variably C-terminal truncated forms [28]. The first
and second Kunitz domains are involved in binding and inhibition of
the TF-VII complex and factor Xa, respectively [29]. The third
Kunitz domain may via its cationic residues, including amino acid
sequences at the C-terminal end, interact with heparin [30]. This
C-terminal region has also been implicated in interaction with
plasma lipoproteins, thrombospondin-1, clearance receptors ([31]),
lipopolysaccharide [32] and may inhibit cell growth [33] as well as
blood coagulation [34], [35]. Since various C-terminally truncated
forms exist in vivo, a potential role of proteolysis of the
C-terminus has been implicated, and data indicate that TFPI can be
cleaved by various proteinases such as thrombin [36], plasmin [37],
and matrix metalloptoteinase-8 [38], releasing C-terminal
fragments. Upregulators of TFPI expression include endotoxin, IL-1,
TNF-.alpha., platelet-derived growth factor, heparin, and basic
fibroblast growth factor, all physiological mechanisms involved in
infection, inflammation, and growth [31].
[0297] The above reported multifunctionality of TFPI, and presence
of an exposed cationic and heparin-binding C-terminus made us raise
the question whether the C-terminal region of TFPI could exert a
direct antimicrobial activity. We here show that C-terminal TFPI
peptides may indeed directly kill both Gram-negative and
Gram-positive bacteria and fungi. Furthermore, evidence is
presented that the peptides may, also exert anticoagulant and
antiinflammatory effects.
Histidine-Rich Glycoprotein and GHH25
[0298] The antimicrobial plasma protein Histidine-rich glycoprotein
(HRG) protects the host against systemic microbial infections in
vivo. Here, we show that HRG is a pattern recognition molecule, by
binding LPS and increasing LPS mediated TLR-4 response in the host
cells.
[0299] LPS interacts with CD14, a receptor on macrophages,
monocytes and neutrophils, the interaction is increased by plasma
protein LBP (LPS-binding protein). The LPS/CD14 complex leads to
activation of toll-like receptor (TLR) 4 which in turn activates
the MyD88-dependent or -independent pathways [39]. The LPS-response
can also be LBP-independent [40]. There is no difference in
TNF-.alpha. release in vivo between wildtype and LBP-deficient mice
after LPS injection, suggesting that a protein with LBP-like
capabilities may be responsible [41]. Activation of TLR4 then in
turn leads to an increase of phagocytic activity of mononuclear
phagocytes, a cascade of released cytokines and nitric oxide (NO),
which initiate and support the inflammatory response. NO is
produced by a group of enzymes called nitric oxide synthases [42]
and involved in many biological processes such as protecting the
host against microbes, parasitic worms and tumours and regulate
blood pressure, high levels of NO can instead be cytotoxic and
destroy endogenous tissue.
[0300] The LPS-induced response is essential for the host defense,
but an overwhelming response can instead lead to sepsis. Cytokines
and NO are identified to be major contributors to the development
of septic shock with symptoms as fever, coagulant activity, septic
shock, multiple organ failure and in worst cases death of the host
[43]. This is supported by the fact that inducible NOS (iNOS)
mutant mice where resistant to LPS-induced mortality [44]. The
susceptibility of different animal species to the toxicity of LPS
is highly variable, for example humans are very sensitive [45, 46]
comparing to mice that are fairly resistant [47]. A common
misunderstanding is that sepsis is controlled by only
pro-inflammatory mediators. The early stages of sepsis is dominated
by pro-inflammatory mediators such as TNF-.alpha., NO, bradykinin,
thrombin and histamine, whereas anti-inflammatory mediators, such
as IL-6, activated protein C, antithrombin and granulocyte
colony-stimulating factor, are most widely occurring during the
later stages [48].
[0301] The treatment of sepsis today includes administration of
intravenous fluids, broad-spectrum antibiotics, protective lung
ventilation, glucocorticoids, insulin therapy and recombinant human
activator protein C (APC). Many attempts have been done to
neutralize LPS or inhibit LPS-mediated activation of host immune
cells, such as blocking cytokine activity with antibodies, blocking
NO production [49] and by using antimicrobial peptides as
neutralizers of LPS [50].
[0302] The aim of this study was to investigate the effects of an
abundant plasma protein, HRG, on LPS-mediated responses. HRG is a
67 kDa plasma protein synthesized in the liver and was first
described in 1972 [51, 52]. HRG can also be released upon thrombin
activation from .alpha.-granules of thrombocytes, and is able to
cover the surface of a fibrin clot. Together with fetuins and
kininogens, HRG belongs to the cystatin superfamily. The most
distinguishing characteristic of the protein is the histidine-rich
domain of the protein contains a conserved GHHPH repeat [53]. HRG
has been shown to interact in vitro with a diverse group of
ligands, like heparin, plasminogen, fibrinogen, thrombospondin,
heme, IgG, FcgR and C1q. In vivo-studies demonstrates that HRG is
an anticoagulant and antifibrinolytic modifier [54], has an
inhibitory effects on tumour vascularization [55] and plays an
important role in the innate immunity as a antimicrobial protein
[56].
[0303] Herein, we demonstrate that HRG is a potent pro-inflammatory
protein, by enhancing LPS induced NO and cytokines in vitro. In
vivo experiment showed that Hrg-/- mice were markedly resistant
against LPS induced sepsis. Our data clearly demonstrate that HRG
contributes to LPS toxicity in experimental endotoxemia. However,
the data also demonstrate an antiinflammatory effect of the
peptides fragments of HRG, such as "GHH25".
Materials and Methods
Histidine-Rich Glycoprotein.
[0304] Synthetic peptide GHH25 (GHHPHGHHPHGHHPHGHHPHGHHPH [SEQ ID
NO:11]) was from Biopeptide (San Diego, Calif., USA). Polyclonal
rabbit anti mouse TRL4 was purchased from GeneTex (Irvine, Calif.,
USA). The cytometric bead array, mouse inflammation kit was from BD
Biosciences (Stockholm, Sweden). Escherichia coli LPS (0111:B4) was
purchased from Sigma (St Lois, Mo.).
[0305] Purification of Human HRG. Serum HRG was purified as
described before [57]. Briefly, human serum were gently shaken at
4.degree. C. overnight with nickel-nitrilotriacetic acid (Ni-NTA)
agarose, washed with phosphate-buffered saline (PBS, pH 7.4).
Elution was first performed with PBS containing 80 mM imidazole to
elute unspecifically bound proteins, and then with PBS containing
500 mM imidazole to elute purified HRG. The protein was dialyzed,
freeze-dried and the concentration was determined using the
Bradford method [58].
[0306] Radioiodination of heparin and LPS. The radioiodination of
heparin (from porcine intestinal mucosa, Sigma-Aldrich) was
performed as described previously [59]. The iodination of LPS was
performed as described by Ulevitch [60]. 1 mg Escherichia coli
0111:B4 LPS was incubated in 50 mM p-OH benzimidate in borate
buffer, pH 8, over night at 4.degree. C., and then dialyzed against
PBS, pH 7.4. LPS was then radiolabelled with .sup.125I using the
chloramine T method, and unlabelled .sup.125I was then removed by
dialysis.
[0307] Heparin and LPS-binding assay. 1, 2 and 5 .mu.g of the
synthetic peptides in 100 .mu.l PBS pH 7.4 were applied onto
nitrocellulose membranes (Hybond-C, Amersham Biosciences) using a
slot-blot apparatus. Membranes were blocked for 1 h at room
temperature with 2% bovine serum albumin in PBS pH 7.4 and then
incubated with radiolabelled LPS (.about.40 .mu.gmL.sup.-1,
0.13.times.10.sup.6 cpm.mu.g.sup.-1) or radiolabelled heparin
(.about.10 .mu.gmL.sup.-1, 0.4.times.10.sup.6 cpm.mu.g.sup.-1) for
1 h at room temperature in PBS, pH 7.4. Unlabeled heparin (6 mg/ml)
was added for competition of binding. The membranes were washed 3
times in PBS, pH 7.4. A Bas 2000 radio-imaging system (Fuji Film,
Tokyo, Japan) was used to visualize radioactivity.
[0308] Cell culture. Murine macrophage cell line, RAW 264.7 (kindly
provided by Dr. H Bjorkbacka) were grown in Dulbeccos Modified
Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum
(FCS). All experiments were performed under serum free
conditions.
[0309] Nitric oxide induction in RAW macrophages. Confluent cells
were harvested and transferred to 96-wells plate
(3.5.times.10.sup.5/well). After adhesion cells were washed with
phenol red-free DMEM (Gibco). E. Coli LPS (100 ng/ml), LL-37 (2 or
10 .mu.M) or HRG (2 or 10 .mu.M) was preincubated at 37.degree. C.
for 30 minutes and then transferred to the cells. For inhibition of
NO induction, 5 .quadrature.g/ml anti mouse TLR4 antibody, 10 or
100 .quadrature.M GHH25 or 100 .quadrature.g/ml heparin were used.
The cells were stimulated for 24 hours and nitric oxide was
determined using the Griess chemical method [61].
[0310] TNF-.alpha. release from human macrophages. Human
monocyte-derived macrophages (hMDMs) were obtained from peripheral
blood mononuclear cells (PBMCs) obtained from the blood of healthy
donors using a Lymphoprep (Axis-Shield PoC AS) density gradient.
PBMCs were seeded at concentrations of 3.times.10.sup.6 cells/well
into 24-well plates and cultured in RPMI1640 medium supplemented
with 10% heat-inactivated autologous human plasma, 2 mM
L-glutamine, and 50 .mu.l/ml Antibiotic-Antimycotic (Gibco) in a
humidified atmosphere of 5% CO.sub.2. After 24 h, non-adherent
cells were removed and adherent monocytes were differentiated to
macrophages for 10 days, with fresh medium changes every second
day. The cells were stimulated for 24 hours with 10 ng/ml of LPS
with or without HRG (2 .mu.M) and GHH25 (100 .mu.M) under
serum-free conditions. After stimulation the supernatant was
aspirated and TNF-.alpha. was measured using the TNF-.alpha. human
ELISA kit (Invitrogen).
[0311] Animal experiments. The original knockout mice
129/B6-HRG.sup.tm1wja1 were crossed with C57BL/6 mice (Taconic) for
14 generations to obtain uniform genetic background. These
HRG-deficient mouse strain was called B6-HRG.sup.tm1wja1 following
ILAR (Institute of Laboratory Animal Resources) rules. Wildtype
C57BL/6 control mice and C57BL/6 Hrg-/- mice (8-12 weeks, 27+/-4 g)
were bred in the animal facility at Lund University. C57BL/6
Hrg.sup.-/-, lacks the translation start point of exon 1 of the Hrg
gene [54]. Animals were housed under standard conditions of light
and temperature and had free access to standard laboratory chow and
water. In order to induce sepsis, 18 .quadrature.g/g Escherichia
coli 0111:B4 LPS were injected intraperitoneally into C57BL/6 or
C57BL/6 Hrg-/- mice, divided into weight and sex matched groups.
Survival and status was followed during seven days.
[0312] For treatment with GHH25 peptide, 1 mg of the peptide
(diluted in 10 mM Tris, pH 7.4) or buffer only was injected
intraperitoneal 30 minutes after LPS-challenge and survival and
status was then followed.
TFPI and Heparin Cofactor II
[0313] Peptides. The TFPI and HCII-derived peptides were
synthesized by Biopeptide Co., San Diego, USA, with the exception
of LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES [SEQ ID NO: 101]),
which was obtained from Innovagen AB, Lund, Sweden. The purity
(>95%) of these peptides was confirmed by mass spectral analysis
(MALDI-ToF Voyager).
[0314] Microorganisms. Bacterial isolates Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus
ATCC 29213, Bacillus subtilis ATCC 6633, Candida albicans ATCC
90028 and Candida parapsilosis ATCC 90018 and were obtained from
the Department of Bacteriology, Lund University Hospital.
[0315] Viable count analysis. E. coli ATCC 25922 bacteria were
grown to mid-logarithmic phase in Todd-Hewitt (TH) medium. Bacteria
were washed and diluted in 10 mM Tris, pH 7.4 containing 5 mM
glucose. E. coli ATCC 25922 (50 .mu.l; 2.times.10.sup.6 cfu/ml)
were incubated, at 37.degree. C. for 2 h with peptides at the
indicated concentrations. Other experiments with the TFPI-peptides
and LL-37 were performed in 10 mM Tris, pH 7.4, containing also
0.15 M NaCl, with normal or heat inactivated 20% citrate-plasma
(PP). Serial dilutions of the incubation mixture were plated on TH
agar, followed by incubation at 37.degree. C. overnight and cfu
determination.
[0316] Radial diffusion assay. Essentially as described earlier
[62, 63], bacteria were grown to mid-logarithmic phase in 10 ml of
full-strength (3% w/v) trypticase soy broth (TSB)
(Becton-Dickinson). The microorganisms were then washed once with
10 mM Tris, pH 7.4. Subsequently, 4.times.10.sup.6 cfu were added
to 15 ml of the underlay agarose gel, consisting of 0.03% (w/v)
TSB, 1% (w/v) low electroendosmosis type (EEO) agarose
(Sigma-Aldrich) and 0.02% (v/v) Tween 20 (Sigma-Aldrich). The
underlay was poured into a O 144 mm petri dish. After agarose
solidification, 4 mm-diameter wells were punched and 6 .mu.l
peptide solution of required concentration added to each well.
Plates were incubated at 37.degree. C. for 3 h to allow peptide
diffusion. The underlay gel was then covered with 15 ml of molten
overlay (6% TSB and 1% Low-EEO agarose in distilled H.sub.2O).
Antimicrobial activity of a peptide was visualized as a zone of
clearing around each well after 18-24 h of incubation at 37.degree.
C.
[0317] Fluorescence microscopy. The impermeant probe FITC
(Sigma-Aldrich, St. Louis, USA) was used for monitoring of
bacterial membrane permeabilization. S. aureus ATCC 29213 bacteria
were grown to mid-logarithmic phase in TSB medium. Bacteria were
washed and resuspended in buffer (10 mM Tris, pH 7.4, 0.15M NaCl, 5
mM glucose) to yield a suspension of 1.times.10.sup.7 CFU/ml. 100
.mu.l of the bacterial suspension was incubated with 30 .mu.M of
the respective peptides at 30.degree. C. for 30 min. Microorganisms
were then immobilized on poly (L-lysine)-coated glass slides by
incubation for 45 min at 30.degree. C., followed by addition onto
the slides of 200 .mu.l of FITC (6 .mu.g/ml) in buffer and a final
incubation for 30 min at 30.degree. C. The slides were washed and
bacteria fixed by incubation, first on ice for 15 min, then in room
temperature for 45 min in 4% paraformaldehyde. The glass slides
were subsequently mounted on slides using Prolong Gold antifade
reagent mounting medium (Invitrogen, Eugene, USA). Bacteria were
visualized using a Nikon Eclipse TE300 (Nikon, Melville, USA)
inverted fluorescence microscope equipped with a Hamamatsu C4742-95
cooled CCD camera (Hamamatsu, Bridgewater, USA) and a Plan
Apochromat .times.100 objective (Olympus, Orangeburg, USA).
Differential interference contrast (Nomarski) imaging was used for
visualization of the microbes themselves.
[0318] Hemolysis assay. EDTA-blood was centrifuged at 800 g for 10
min, whereafter plasma and buffy coat were removed. The
erythrocytes were washed three times and resuspended in PBS, pH 7.4
to get a 5% suspension. The cells were then incubated with
end-over-end rotation for 60 min at 37.degree. C. in the presence
of peptides (60 .mu.M). 2% Triton X-100 (Sigma-Aldrich) served as
positive control. The samples were then centrifuged at 800 g for 10
min and the supernatant was transferred to a 96 well microtiter
plate. The absorbance of hemoglobin release was measured at .lamda.
540 nm and is in the plot expressed as % of TritonX-100 induced
hemolysis.
[0319] Lactate dehydrogenase (LDH) assay. HaCaT keratinocytes were
grown to confluency in 96 well plates (3000 cells/well) in
serum-free keratinocyte medium (SFM) supplemented with bovine
pituitary extract and recombinant EGF (BPE-rEGF) (Invitrogen,
Eugene, USA). The medium was then removed, and 100 .mu.l of the
peptides investigated (at 60 .mu.M, diluted in SFM/BPE-rEGF or in
keratinocyte-SFM supplemented with 20% human serum) were added. The
LDH-based TOX-7 kit (Sigma-Aldrich, St. Louis, USA) was used for
quantification of LDH release from the cells. Results represent
mean values from triplicate measurements, and are given as
fractional LDH release compared to the positive control consisting
of 1% Triton X-100 (yielding 100% LDH release).
[0320] Slot-blot assay. LPS binding ability of the peptides were
examined by slot-blot assay. Peptides (2 and 5 .mu.g) were bound to
nitrocellulose membrane (Hybond-C, GE Healthcare BioSciences, UK),
pre-soaked in PBS, by vacuum. Membranes were then blocked by 2 wt %
BSA in PBS, pH 7.4, for 1 h at RT and subsequently incubated with
.sup.125I-labelled LPS (40 .mu.g/mL; 0.13.times.10.sup.6 cpm/.mu.g)
or .sup.125I-labelled heparin (Sigma) for 1 h at RT in 10 mM Tris,
pH 7.4, 0.15 M NaCl, or 10 mM MES, pH 5.5, 0.15 M NaCl. After LPS
binding, membranes were washed 3 times, 10 min each time in the
above buffers and visualized for radioactivity on Bas 2000
radioimaging system (Fuji, Japan).
[0321] Liposome preparation and leakage assay. The liposomes
investigated were either zwitterionic (DOPC/cholesterol 60/40
mol/mol or DOPC without cholesterol) or anionic (DOPE/DOPG 75/25
mol/mol). DOPG (1,2-Dioleoyl-sn-Glycero-3-Phosphoglycerol,
monosodium salt), DOPC (1,2-dioleoyl-sn-Glycero-3-phosphocholine),
and DOPE (1,2-dioleoyl-sn-Glycero-3-phosphoetanolamine) were all
from Avanti Polar Lipids (Alabaster, USA) and of >99% purity,
while cholesterol (>99% purity), was from Sigma-Aldrich (St.
Louis, USA). Due to the long, symmetric and unsaturated acyl chains
of these phospholipids, several methodological advantages are
reached. In particular, membrane cohesion is good, which
facilitates very stable, unilamellar, and largely defect-free
liposomes (observed from cryo-TEM) and well defined supported lipid
bilayers (observed by ellipsometry and AFM), allowing detailed
values on leakage and adsorption density to be obtained. The lipid
mixtures were dissolved in chloroform, after which solvent was
removed by evaporation under vacuum overnight. Subsequently, 10 mM
Tris buffer, pH 7.4, was added together with 0.1 M
carboxyfluorescein (CF) (Sigma, St. Louis, USA). After hydration,
the lipid mixture was subjected to eight freeze-thaw cycles
consisting of freezing in liquid nitrogen and heating to 60.degree.
C. Unilamellar liposomes of about O140 nm were generated by
multiple extrusions through polycarbonate filters (pore size 100
nm) mounted in a LipoFast miniextruder (Avestin, Ottawa, Canada) at
22.degree. C. Untrapped CF was removed by two subsequent gel
filtrations (Sephadex G-50, GE Healthcare, Uppsala, Sweden) at
22.degree. C., with Tris buffer as eluent. CF release from the
liposomes was determined by monitoring the emitted fluorescence at
520 nm from a liposome dispersion (10 mM lipid in 10 mM Tris, pH
7.4). An absolute leakage scale was obtained by disrupting the
liposomes at the end of each experiment through addition of 0.8 mM
Triton X-100 (Sigma-Aldrich, St. Louis, USA). A SPEX-fluorolog 1650
0.22-m double spectrometer (SPEX Industries, Edison, USA) was used
for the liposome leakage assay in Tris buffer in the absence and
presence of liposomes under conditions described above.
Measurements were performed in triplicate at 37.degree. C.
[0322] CD-spectroscopy. The CD spectra of the peptides in solution
were measured on a Jasco J-810 Spectropolarimeter (Jasco, U.K.).
The measurements were performed at 37.degree. C. in a 10 mm quartz
cuvet under stirring and the peptide concentration was 10 .mu.M.
The effect on peptide secondary structure of liposomes at a lipid
concentration of 100 .mu.M was monitored in the range 200-250 nm.
The only peptide conformations observed under the conditions
investigated were .alpha.-helix and random coil. The fraction of
the peptide in .alpha.-helical conformation, X.sub..alpha., was
calculated from
X.sub..alpha.=(A-A.sub.c)/(A.sub..alpha.-A.sub.c)
where A is the recorded CD signal at 225 nm, and A.sub..quadrature.
and A.sub.c are the CD signal at 225 nm for a reference peptide in
100% .alpha.-helix and 100% random coil conformation, respectively.
100% .alpha.-helix and 100% random coil references were obtained
from 0.133 mM (monomer concentration) poly-L-lysine in 0.1 M NaOH
and 0.1 M HCl, respectively [64, 65]. For determination of effects
of lipopolysaccharide on peptide structure, the peptide secondary
structure was monitored at a peptide concentration of 10 .mu.M,
both in Tris buffer and in the presence of E. coli
lipopolysaccharide (0.02 wt %) (Escherichia coli 0111:B4, highly
purified, less than 1% protein/RNA, Sigma, UK). To account for
instrumental differences between measurements the background value
(detected at 250 nm, where no peptide signal is present) was
subtracted. Signals from the bulk solution were also corrected
for.
[0323] Effects of various microbial products on macrophages in
vitro and anti-inflammatory effects by various peptides of HCII and
TFPI. 3.5.times.10.sup.5 cells were seeded in 96-well tissue
culture plates (Nunc, 167008) in phenol red-free DMEM (Gibco)
supplemented with 10% FBS and antibiotics. Following 6 hours of
incubation to permit adherence, cells were stimulated with 10 ng/mL
E. coli (0111:B4) LPS (Sigma), lipoteichoic acid, peptioglycan, or
zymosan, with and without peptides at the indicated doses (se
figure legends and figures). The levels of NO in culture
supernatants were determined after 24 hours from stimulation using
the Griess reaction [66]. Briefly, nitrite, a stable product of NO
degradation, was measured by mixing 50 .mu.l of culture
supernatants with the same volume of Griess reagent (Sigma, G4410)
and reading absorbance at 550 nm after 15 min. Phenol-red free DMEM
with FBS and antibiotics were used as a blank. A standard curve was
prepared using 0-80 .mu.M sodium nitrite solutions in ddH20.
[0324] Clotting assays. All clotting times were measured using an
Amelung coagulometer. Activated partial thromboplastin time (aPTT)
was measured by incubating the peptides diluted in sterile water at
the indicated concentrations, with 100 .mu.L citrated human plasma
for 1 minute followed by the addition of 100 .mu.L aPTT reagent
(aPTT Automate, Diagnostica Stago) for 60 seconds at 37.degree. C.
Clotting was initiated by the addition of 100 .mu.L of a 25-mM
CaCl.sub.2 solution. In the prothombrin time assay (PT), clotting
was initiated by the addition of 100 .mu.L Thrombomax with calcium
(PT reagent; Sigma-Aldrich). For measuring the thrombin clotting
time (TCT), clotting was initiated by the addition of 100 .mu.L
Accuclot thrombin time reagent (TCT reagent; Sigma-Aldrich).
Results and Conclusions
[0325] In summary, peptides reported above corresponding cryptic
heparin binding sites in serpins such as HCII and ATIII, as well as
other proteins including HRG and TFPI possess antibacterial,
anti-inflammatory and anti-coagulative properties (FIG. 1-18,
29-33). Of particular importance was the finding that the HCII
peptides blocked TLR-mediated LPS responses as well as the
intrinsic pathway of coagulation. Furthermore, the TFPI peptides
showed unique and previously undisclosed inhibitory activities on
both the intrinsic, as well as extrinsic pathways of coagulation.
The results thus illustrate that the peptides not only attenuate
bacterial infection and the related inflammatory response involving
interference with macrophage activation, but importantly also
interfere with coagulation, and therefore, show significant
therapeutic potential for sepsis, COPD and other multifactorial
diseases involving pathogenetic steps including inflammation and
coagulation.
REFERENCES
[0326] 1. Lehrer, R. I. and T. Ganz, Cathelicidins: a family of
endogenous antimicrobial peptides. Curr Opin Hematol, 2002. 9(1):
p. 18-22. [0327] 2. Harder, J., R. Glaser, and J. M. Schroder,
Review: Human antimicrobial proteins effectors of innate immunity.
J Endotoxin Res, 2007. 13(6): p. 317-38. [0328] 3. Zasloff, M.,
Antimicrobial peptides of multicellular organisms. Nature, 2002.
415(6870): p. 389-95. [0329] 4. Tossi, A., L. Sandri, and A.
Giangaspero, Amphipathic, alpha-helical antimicrobial peptides.
Biopolymers, 2000. 55(1): p. 4-30. [0330] 5. Yount, N. Y., et al.,
Advances in antimicrobial peptide immunobiology. Biopolymers, 2006.
[0331] 6. Zanetti, M., Cathelicidins, multifunctional peptides of
the innate immunity. J Leukoc Biol, 2004. 75(1): p. 39-48. [0332]
7. Elsbach, P., What is the real role of antimicrobial polypeptides
that can mediate several other inflammatory responses? J Clin
Invest, 2003. 111(11): p. 1643-5. [0333] 8. Ganz, T., Defensins:
antimicrobial peptides of innate immunity. Nat Rev Immunol, 2003.
3(9): p. 710-20. [0334] 9. Cole, A. M., et al., Cutting edge:
IFN-inducible ELR-CXC chemokines display defensin-like
antimicrobial activity. J Immunol, 2001. 167(2): p. 623-7. [0335]
10. Brogden, K. A., Antimicrobial peptides: pore formers or
metabolic inhibitors in bacteria? Nat Rev Microbiol, 2005. 3(3): p.
238-50. [0336] 11. Kowalska, K., D. B. Carr, and A. W. Lipkowski,
Direct antimicrobial properties of substance P. Life Sci, 2002.
71(7): p. 747-50. [0337] 12. Mor, A., M. Amiche, and P. Nicolas,
Structure, synthesis, and activity of dermaseptin b, a novel
vertebrate defensive peptide from frog skin: relationship with
adenoregulin. Biochemistry, 1994. 33(21): p. 6642-50. [0338] 13.
Malmsten, M., et al., Antimicrobial peptides derived from growth
factors. Growth Factors, 2007. 25(1): p. 60-70. [0339] 14. Nordahl,
E. A., et al., Activation of the complement system generates
antibacterial peptides. Proc Natl Acad Sci USA, 2004. 101(48): p.
16879-84. [0340] 15. Pasupuleti, M., et al., Preservation of
antimicrobial properties of complement peptide C3a, from
invertebrates to humans. J Biol Chem, 2007. 282(4): p. 2520-8.
[0341] 16. Frick, I. M., et al., The contact system--a novel branch
of innate immunity generating antibacterial peptides. Embo J, 2006.
25(23): p. 5569-78. [0342] 17. Nordahl, E. A., et al., Domain 5 of
high molecular weight kininogen is antibacterial. J Biol Chem,
2005. 280(41): p. 34832-9. [0343] 18. Rydengard, V., E. Andersson
Nordahl, and A. Schmidtchen, Zinc potentiates the antibacterial
effects of histidine-rich peptides against Enterococcus faecalis.
Febs J, 2006. 273(11): p. 2399-406. [0344] 19. Davie, E. W. and J.
D. Kulman, An overview of the structure and function of thrombin.
Semin Thromb Hemost, 2006. 32 Suppl 1: p. 3-15. [0345] 20. Bode,
W., The structure of thrombin: a janus-headed proteinase. Semin
Thromb Hemost, 2006. 32 Suppl 1: p. 16-31. [0346] 21. Chapman, A.
P., PEGylated antibodies and antibody fragments for improved
therapy: a review. Adv Drug Deliv Rev, 2002. 54(4): p. 531-45.
[0347] 22. Veronese, F. M. and J. M. Harris, Introduction and
overview of peptide and protein pegylation. Adv Drug Deliv Rev,
2002. 54(4): p. 453-6. [0348] 23. Veronese, F. M. and G. Pasut,
PEGylation, successful approach to drug delivery. Drug Discov
Today, 2005. 10(21): p. 1451-8. [0349] 24. Wang, Y. S., et al.,
Structural and biological characterization of pegylated recombinant
interferon alpha-2b and its therapeutic implications. Adv Drug
Deliv Rev, 2002. 54(4): p. 547-70. [0350] 25. Sato, H., Enzymatic
procedure for site-specific pegylation of proteins. Adv Drug Deliv
Rev, 2002. 54(4): p. 487-504. [0351] 26. Bowen, S., et al.,
Relationship between molecular mass and duration of activity of
polyethylene glycol conjugated granulocyte colony-stimulating
factor mutein. Exp Hematol, 1999. 27(3): p. 425-32. [0352] 27.
Chapman, A. P., et al., Therapeutic antibody fragments with
prolonged in vivo half-lives. Nat Biotechnol, 1999. 17(8): p.
780-3. [0353] 28. Lwaleed, B. A. and P. S. Bass, Tissue factor
pathway inhibitor: structure, biology and involvement in disease. J
Pathol, 2006. 208(3): p. 327-39. [0354] 29. Girard, T. J., et al.,
Functional significance of the Kunitz-type inhibitory domains of
lipoprotein-associated coagulation inhibitor. Nature, 1989.
338(6215): p. 518-20. [0355] 30. Mine, S., et al., Structural
mechanism for heparin-binding of the third Kunitz domain of human
tissue factor pathway inhibitor. Biochemistry, 2002. 41(1): p.
78-85. [0356] 31. Crawley, J. T. and D. A. Lane, The haemostatic
role of tissue factor pathway inhibitor. Arterioscler Thromb Vasc
Biol, 2008. 28(2): p. 233-42. [0357] 32. Park, C. T., A. A.
Creasey, and S. D. Wright, Tissue factor pathway inhibitor blocks
cellular effects of endotoxin by binding to endotoxin and
interfering with transfer to CD14. Blood, 1997. 89(12): p. 4268-74.
[0358] 33. Hembrough, T. A., et al., Identification and
characterization of a very low density lipoprotein receptor-binding
peptide from tissue factor pathway inhibitor that has antitumor and
antiangiogenic activity. Blood, 2004. 103(9): p. 3374-80. [0359]
34. Wesselschmidt, R., et al., Tissue factor pathway inhibitor: the
carboxy-terminus is required for optimal inhibition of factor Xa.
Blood, 1992. 79(8): p. 2004-10. [0360] 35. Ettelaie, C., et al.,
The role of the C-terminal domain in the inhibitory functions of
tissue factor pathway inhibitor. FEBS Lett, 1999. 463(3): p. 341-4.
[0361] 36. Ohkura, N., et al., A novel degradation pathway of
tissue factor pathway inhibitor: incorporation into fibrin clot and
degradation by thrombin. Blood, 1997. 90(5): p. 1883-92. [0362] 37.
Li, A. and T. C. Wun, Proteolysis of tissue factor pathway
inhibitor (TFPI) by plasmin: effect on TFPI activity. Thromb
Haemost, 1998. 80(3): p. 423-7. [0363] 38. Cunningham, A. C., et
al., Structural and functional characterization of tissue factor
pathway inhibitor following degradation by matrix
metalloproteinase-8. Biochem J, 2002. 367(Pt 2): p. 451-8. [0364]
39. Lu, Y. C., W. C. Yeh, and P. S. Ohashi, LPS/TLR4 signal
transduction pathway. Cytokine, 2008. 42(2): p. 145-51. [0365] 40.
Nakatomi, K., et al., Neutrophils responded to immobilized
lipopolysaccharide in the absence of lipopolysaccharide-binding
protein. J Leukoc Biol, 1998. 64(2): p. 177-84. [0366] 41. Wurfel,
M. M., et al., Targeted deletion of the lipopolysaccharide
(LPS)-binding protein gene leads to profound suppression of LPS
responses ex vivo, whereas in vivo responses remain intact. J Exp
Med, 1997. 186(12): p. 2051-6. [0367] 42. Kichler, A., et al.,
Histidine-rich amphipathic peptide antibiotics promote efficient
delivery of DNA into mammalian cells. Proc Natl Acad Sci USA, 2003.
100(4): p. 1564-8. [0368] 43. Karima, R., et al., The molecular
pathogenesis of endotoxic shock and organ failure. Mol Med Today,
1999. 5(3): p. 123-32. [0369] 44. Wei, X. Q., et al., Altered
immune responses in mice lacking inducible nitric oxide synthase.
Nature, 1995. 375(6530): p. 408-11. [0370] 45. Michie, H. R., et
al., Detection of circulating tumor necrosis factor after endotoxin
administration. N Engl J Med, 1988. 318(23): p. 1481-6. [0371] 46.
Sauter, C. and C. Wolfensberger, Interferon in human serum after
injection of endotoxin. Lancet, 1980. 2(8199): p. 852-3. [0372] 47.
Dinges, M. M. and P. M. Schlievert, Comparative analysis of
lipopolysaccharide-induced tumor necrosis factor alpha activity in
serum and lethality in mice and rabbits pretreated with the
staphylococcal superantigen toxic shock syndrome toxin 1. Infect
Immun, 2001. 69(11): p. 7169-72. [0373] 48. Opal, S. M., The host
response to endotoxin, antilipopolysaccharide strategies, and the
management of severe sepsis. Int J Med Microbiol, 2007. 297(5): p.
365-77. [0374] 49. Schwartz, D. and R. C. Blantz, Nitric oxide,
sepsis, and the kidney. Semin Nephrol, 1999. 19(3): p. 272-6.
[0375] 50. Kirikae, T., et al., Protective effects of a human
18-kilodalton cationic antimicrobial protein (CAP18)-derived
peptide against murine endotoxemia. Infect Immun, 1998. 66(5): p.
1861-8. [0376] 51. Haupt, H. and N. Heimburger, [Human serum
proteins with high affinity for carboxymethylcellulose. I.
Isolation of lysozyme, C1q and 2 hitherto unknown-globulins]. Hoppe
Seylers Z Physiol Chem, 1972. 353(7): p. 1125-32. [0377] 52.
Heimburger, N., et al., [Human serum proteins with high affinity to
carboxymethylcellulose. II. Physico-chemical and immunological
characterization of a histidine-rich 3,8S-2-glycoportein
(CM-protein I)]. Hoppe Seylers Z Physiol Chem, 1972. 353(7): p.
1133-40. [0378] 53. Jones, A. L., M. D. Hulett, and C. R. Parish,
Histidine-rich glycoprotein: A novel adaptor protein in plasma that
modulates the immune, vascular and coagulation systems. Immunol
Cell Biol, 2005. 83(2): p. 106-18. [0379] 54. Tsuchida-Straeten,
N., et al., Enhanced blood coagulation and fibrinolysis in mice
lacking histidine-rich glycoprotein (HRG). J Thromb Haemost, 2005.
3(5): p. 865-72. [0380] 55. Olsson, A. K., et al., A fragment of
histidine-rich glycoprotein is a potent inhibitor of tumor
vascularization. Cancer Res, 2004. 64(2): p. 599-605.
[0381] 56. Rydeng{dot over (a)}rd, V., et al., Histidine-rich
glycoprotein protects from systemic Candida infection. PLoS Pathog,
2008. 4(8): p. e1000116. [0382] 57. Rydeng{dot over (a)}rd, V., et
al., Histidine-rich glycoprotein exerts antibacterial activity.
Febs J, 2007. 274(2): p. 377-89. [0383] 58. Bradford, M. M., A
rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem, 1976. 72: p. 248-54. [0384] 59. Cheng, F.,
et al., A new method for sequence analysis of glycosaminoglycans
from heavily substituted proteoglycans reveals non-random
positioning of 4-and 6-O-sulphated N-acetylgalactosamine in
aggrecan-derived chondroitin sulphate. Glycobiology, 1992. 2(6): p.
553-61. [0385] 60. Ulevitch, R. J., The preparation and
characterization of a radioiodinated bacterial lipopolysaccharide.
Immunochemistry, 1978. 15(3): p. 157-64. [0386] 61. Park, E., et
al., Taurine chloramine inhibits the synthesis of nitric oxide and
the release of tumor necrosis factor in activated RAW 264.7 cells.
J Leukoc Biol, 1993. 54(2): p. 119-24. [0387] 62. Andersson, E., et
al., Antimicrobial activities of heparin-binding peptides. Eur
Biochem, 2004. 271(6): p. 1219-26. [0388] 63. Lehrer, R. I., et
al., Ultrasensitive assays for endogenous antimicrobial
polypeptides. J Immunol Methods, 1991. 137(2): p. 167-73. [0389]
64. Greenfield, N. and G. D. Fasman, Computed circular dichroism
spectra for the evaluation of protein conformation. Biochemistry,
1969. 8(10): p. 4108-16. [0390] 65. Sjogren, H. and S. Ulvenlund,
Comparison of the helix-coil transition of a titrating polypeptide
in aqueous solutions and at the air-water interface. Biophys Chem,
2005. 116(1): p. 11-21. [0391] 66. Pollock, J. S., et al.,
Purification and characterization of particulate
endothelium-derived relaxing factor synthase from cultured and
native bovine aortic endothelial cells. Proc Natl Acad Sci USA,
1991. 88(23): p. 10480-4.
Example B
C-Terminal Peptides of Tissue-Factor Pathway Inhibitor are Novel
Host Defense Molecules
Abstract
[0392] Tissue factor pathway inhibitor (TFPI) inhibits tissue
factor-induced coagulation, but may, via its C-terminus, also
modulate cell surface-, heparin-, and lipopolysaccharide
interactions as well as participate in growth inhibition. Here we
show that C-terminal TFPI peptide sequences are antimicrobial
against the Gram-negative bacteria Escherichia coli and Pseudomonas
aeruginosa, Gram-positive Bacillus subtilis and Staphylococcus
aureus, as well as the fungi Candida albicans and Candida
parapsilosis. Fluorescence studies of peptide-treated bacteria,
paired with analysis of peptide effects on liposomes, showed that
the peptides exerted membrane-breaking effects similar to those
seen for the "classical" human antimicrobial peptide LL-37. The
killing of E. coli, but not P. aeruginosa, by the C-terminal
peptide GGLIKTKRKRKKQRVKIAYEEIFVKNM [SEQ ID NO: 4] (GGL27), was
enhanced in human plasma and largely abolished in heat-inactivated
plasma, a phenomenon linked to generation of antimicrobial C3a and
activation of the classical pathway of complement activation.
Furthermore, GGL27 displayed anti-endotoxic effects in vitro and in
vivo in a mouse model of LPS-shock. Importantly, TFPI was found to
be expressed in the basal layers of normal epidermis, and was
markedly up-regulated in acute skin wounds as well as wound edges
of chronic leg ulcers. Furthermore, C-terminal fragments of TFPI
were associated with bacteria present in human chronic leg ulcers.
These findings suggest a new role for TFPI in cutaneous defense
against infections.
Introduction
[0393] In order to control our microbial flora, humans and
virtually all life forms are armored with rapidly acting host
defense systems based on various antimicrobial peptides (1-3). The
majority of these peptides is characterized by an amphipathic
structure, and comprise linear peptides, many of which adopt an
.alpha.-helical and amphipathic conformation upon bacterial
binding, peptides forming cysteine-linked antiparallel
.beta.-sheets, as well as cysteine-constrained loop structures. In
addition, antimicrobial peptides may be found among peptides not
displaying such ordered structures as long as these are
characterized by an over-representation of certain amino acids (1,
4-6). Although interactions with bacterial membranes are
fundamental for antimicrobial peptide function, the exact modes of
action are complex, and can be divided into membrane disruptive and
non-membrane disruptive (1, 3, 7, 8). During recent years it has
also become increasingly evident that many cationic and amphipathic
antimicrobial peptides, such as defensins and cathelicidins, are
multifunctional, also mediating immunomodulatory roles and
angiogenesis (9-11), thus motivating the recent and broader
definition host defense peptides for these members of the innate
immune system.
[0394] Tissue factor pathway inhibitor (or TFPI) is a Kunitz-type
proteinase inhibitor which reversibly inhibits the tissue
factor-factor VII (TF-VII) complex in a factor X (FX)-dependent
manner, leading to inhibition of both FX and FIX activation. TFPI
consists of a highly negatively-charged N-terminus, three
tandemly-linked Kunitz-type domains, and a highly
positively-charged C-terminus. In plasma, TFPI exists in both
full-length and various C-terminal truncated forms (12). The first
and second Kunitz domains are involved in binding and inhibition of
the TF-VII complex and factor Xa, respectively (13). The third
Kunitz domain, in turn, may interact with heparin via its cationic
C-terminal end (14). This C-terminal region has also been
implicated in interactions with plasma lipoproteins,
thrombospondin-1, clearance receptors (15), lipopolysaccharide
(16), and may also inhibit cell growth (17) and blood coagulation
(18, 19). Since various C-terminally truncated forms exist in vivo,
a potential role of proteolysis of the C-terminus has been
implicated, and data indicate that TFPI can be cleaved by various
proteinases such as thrombin (20), plasmin (21), and matrix
metalloptoteinase-8 (22), thereby releasing C-terminal fragments.
Upregulators of TFPI expression include endotoxin, IL-1,
TNF-.alpha., platelet-derived growth factor, heparin, and basic
fibroblast growth factor, all molecules involved in infection,
inflammation, and growth (15).
[0395] The above reported multifunctionality of TFPI, and presence
of an exposed cationic and heparin-binding C-terminus made us raise
the question whether the C-terminal region of TFPI could exert
antimicrobial activity. We here show that C-terminal TFPI peptides,
found to be expressed in skin wounds, and detected in fibrin of
chronic leg ulcers, may indeed function as host defense peptides.
Furthermore, killing of the Gram-negative E. coli, but not P.
aeruginosa, was markedly boosted by peptide-mediated complement
activation, including formation of the membrane attack complex
(MAC) and antimicrobial C3a.
Materials & Methods
[0396] Peptides--The TFPI-derived peptides (see FIG. 1) and the
control peptides GGL27(S) (GGLISTSSSSSSQRVKIAYEEIFVKNM [SEQ ID NO:
102]) and DSE25 (DSEEDEEHTIITDTELPPLKLMHSF [SEQ ID NO: 103]) were
synthesized by Biopeptide Co., San Diego, USA, while LL-37
(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES [SEQ ID NO: 101]), was
obtained from Innovagen AB, Lund, Sweden. The purity (>95%) of
these peptides was confirmed by mass spectral analysis (MALDI-ToF
Voyager).
[0397] Microorganisms--Bacterial isolates Escherichia coil ATCC
25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus
ATCC 29213, Bacillus subtilis ATCC 6633, Candida albicans ATCC
90028 and Candida parapsilosis ATCC 90018 were obtained from the
Department of Bacteriology, Lund University Hospital. Other
clinical isolates of E. coli and P. aeruginosa were from patients
with skin infections.
[0398] Biological materials--Fibrin slough was collected from
chronic venous leg ulcers (chronic wound slough/surface) with a
sterile spatula and immediately fixed for electron microscopy.
Tissue sections from 3 patients with chronic venous ulcers
(duration >6 months) were analysed. 4 mm biopsies were taken
from the edge of the wound and a control area on the thigh. For
acute wounds, a 4 mm biopsy was taken from the thigh of one
individual, and subsequent biopsies from the wound edges were taken
at day 5 and 8. Biopsies from normal skin (n=3, thigh) were also
taken for analysis. The research project was approved by the Ethics
committee, Lund University Hospital. Written consent was obtained
from the patients.
[0399] Radial diffusion assay--Essentially as described earlier
(23, 24), bacteria were grown to mid-logarithmic phase in 10 ml of
full-strength (3% w/v) trypticase soy broth (TSB)
(Becton-Dickinson). The microorganisms were then washed once with
10 mM Tris, pH 7.4. Subsequently, 4.times.10.sup.6 cfu were added
to 15 ml of the underlay agarose gel, consisting of 0.03% (w/v)
TSB, 1% (w/v) low electroendosmosis type (EEO) agarose
(Sigma-Aldrich) and 0.02% (v/v) Tween 20 (Sigma-Aldrich). The
underlay was poured into a O 144 mm petri dish. After agarose
solidification, 4 mm-diameter wells were punched and 6 .mu.l
peptide solution of required concentration added to each well.
Plates were incubated at 37.degree. C. for 3 h to allow peptide
diffusion. The underlay gel was then covered with 15 ml of molten
overlay (6% TSB and 1% Low-EEO agarose in distilled H.sub.2O).
Antimicrobial activity of a peptide was visualized as a clearance
zone around each well after 18-24 h of incubation at 37.degree.
C.
[0400] Viable count analysis--E. coli strains were grown to
mid-logarithmic phase in Todd-Hewitt (TH). P. aeruginosa strains
were grown in TH overnight. Bacteria were washed and diluted in 10
mM Tris, pH 7.4 containing 5 mM glucose. 2.times.10.sup.6 cfu/ml
bacteria were incubated in 50 .mu.l, at 37.degree. C. for 2 h with
the C-terminal TFPI-derived peptides GGL27, TKR22, or LIK17 at the
indicated concentrations. Additional experiments were performed in
10 mM Tris, pH 7.4, containing 0.15 M NaCl, either alone or with
20% normal or heat inactivated citrate-plasma. Serial dilutions of
the incubation mixture were plated on TH agar, followed by
incubation at 37.degree. C. overnight and cfu determination.
[0401] Flow cytometry analysis--Fifty .mu.l of bacteria
(1-2.times.10.sup.6 cfu) were incubated with 450 .mu.l of human
plasma either alone or supplemented with GGL27 (at 3 .mu.M).
Samples were incubated for 30 min or 1 h at 37.degree. C., divided
into two equal parts, centrifuged, washed with PBS, and resuspended
in 100 .mu.l PBS with rabbit polyclonal antibodies against either
LGE27 (25), a C-terminal epitope of human C3a, or rabbit polyclonal
antibodies against C1q (both at 1:100). The mixtures were
subsequently incubated for 1 h at room temperature. Bacteria were
pelleted and washed twice with PBS, incubated in 100 .mu.l PBS with
goat anti rabbit IgG FITC-labeled antibodies (1:500, Sigma) for 30
min at room temperature and washed twice with PBS. In another
experiment bacteria were incubated for 1 h in citrate plasma
together with 3 .mu.M TAMRA-labeled GGL27 peptide, then pelleted
and washed twice with PBS. Flow cytometry analysis
(Becton-Dickinson, Franklin Lakes, N.J.) was performed using a
FACS-Calibur flow cytometry system equipped with a 15 mW argon
laser turned a 488 nm. The bacterial population was selected by
gating with appropriate settings of forward scatter (FSC) and
sideward scatter (SSC).
[0402] Fluorescence microscopy--Fluorescein isothiocyanate (FITC;
Sigma-Aldrich, St. Louis, USA) was used for monitoring of bacterial
membrane permeabilization. E. coli ATCC 25922 bacteria were grown
to mid-logarithmic phase in TSB medium. Bacteria were washed and
resuspended in buffer (10 mM Tris, pH 7.4, 0.15M NaCl, 5 mM
glucose) to yield a suspension of 1.times.10.sup.7 cfu/ml. 100
.mu.l of the bacterial suspension was incubated with 30 .mu.M of
the respective peptides at 30.degree. C. for 30 min. Microorganisms
were then immobilized on poly (L-lysine)-coated glass slides by
incubation for 45 min at 30.degree. C., followed by addition onto
the slides of 200 .mu.l of FITC (6 .mu.g/ml) in buffer and a final
incubation for 30 min at 30.degree. C. The slides were washed and
bacteria fixed by incubation, first on ice for 15 min, then in room
temperature for 45 min in 4% paraformaldehyde. The glass slides
were subsequently mounted on slides using Prolong Gold antifade
reagent mounting medium (Invitrogen, Eugene, USA). Bacteria were
visualized using a Nikon Eclipse TE300 (Nikon, Melville, USA)
inverted fluorescence microscope equipped with a Hamamatsu C4742-95
cooled CCD camera (Hamamatsu, Bridgewater, USA) and a Plan
Apochromat .times.100 objective (Olympus, Orangeburg, USA).
Differential interference contrast (Nomarski) imaging was used for
visualization of the microbes themselves.
[0403] Histochemistry--For immunostaining, wound biopsies were
fixed in 10% formalin, rehydrated and embedded in paraffin.
Sections of 5 .mu.m thickness were placed on poly-lysine coated
glass slides, deparaffinized in xylene and rehydrated in graded
alcohols. The slides were then treated with Dako antigen retrieval
solution (Dako) for 40 min at 97.degree. C., and incubated for 24 h
at room temperature in a 1:50 dilution of polyclonal antibodies
against TFPI (Sigma-Aldrich, in TBS with 1% BSA, 5% goat serum,
0.05% Tween 20). After three 20 min washes in TBS with 0.05% Tween
20, the sections were incubated with alkaline phosphatase
conjugated secondary goat anti-rabbit IgG (Dako) diluted 1:1000 in
the same buffer as the primary antibody and incubated for another
24 hours, followed by three 20 min washes. Sections were developed
with Vulcan Fast Red chromogen (Biocare Medical, Concord, Calif.)
and the slides were counterstained with Harris Hematoxylin (EM
Science, Gibbstown, N.J.). For histological evaluation of lungs
derived from the in vivo LPS-models in mice, tissues were embedded
as above, sectioned and stained with hematoxylin and eosin by
routine procedures (Histocenter, Gothenburg, Sweden).
[0404] Electron Microscopy--For transmission electron microscopy
and visualization of peptide effects on bacteria, P. aeruginosa
ATCC 27853 (1-2.times.10.sup.6 cfu/sample) was incubated for 2 h at
37.degree. C. with the peptide GGL27 at 30 .mu.M. LL-37 (30 .mu.M)
was included as a control. P. aeruginosa sample suspensions were
adsorbed onto carbon-coated copper grids for 2 min, washed briefly
on two drops of water, and negatively stained on two drops of 0.75%
uranyl formate. The grids were rendered hydrophilic by glow
discharge at low pressure in air. In-vivo experiment; Fibrin slough
from patients with chronic venous ulcers (CWS) was fixed (1.5% PFA,
0.5% GA in 0.1 M phosphate buffer, pH 7.4) for 1 h at room
temperature, followed by washing with 0.1 M phosphate buffer, pH
7.4. The fixed and washed samples were subsequently dehydrated in
ethanol and further processed for Lowicryl embedding (26). Sections
were cut with a LKB ultratome and mounted on gold grids. For
immunostaining, the grids were floated on top of drops of immune
reagents displayed on a sheet of parafilm. Free aldehyde groups
were blocked with 50 mM glycine, and the grids were then incubated
with 5% (vol/vol) goat serum in incubation buffer (0.2% BSA-c in
PBS, pH 7.6) for 15 minutes. This blocking procedure was followed
by overnight incubation at 4.degree. C. with C-terminal TFPI goat
polyclonal antibodies (1 .mu.g/ml) (Abcam, UK) alone, or in
combination with rabbit polyclonal antibodies against the
C-terminal part of C3a (LGE27 antibodies) (1 .mu.g/ml) (Innovagen
AB) (25). Controls without primary antibodies were included. The
grids were washed in a large volume (200 ml) of incubation buffer,
floating on drops containing the gold conjugate reagents. For
detection of TFPI-peptides, 1 .mu.g/ml EM rabbit anti-goat IgG 10
nm Au (BBI) in incubation buffer was added and incubation performed
for 2 h at 4.degree. C. For simultaneous detection of TFPI and C3a,
1 .mu.g/ml EM rabbit anti-goat IgG 20 nm Au (BBI) and 1 .mu.g/ml EM
goat anti-rabbit IgG 10 nm Au (BBI) were used. After further washes
by an excess volume of incubation buffer, the sections were
postfixed in 2% glutaraldehyde. Finally, sections were washed with
distilled water and poststained with 2% uranyl acetate and lead
citrate. All samples were examined with a Jeol JEM 1230 electron
microscope operated at 80 kV accelerating voltage. Images were
recorded with a Gatan Multiscan 791 charge-coupled device
camera.
[0405] SDS-PAGE and immunoblotting--Human citrate plasma (450
.mu.l) was incubated with 50 .mu.l bacteria (1-2.times.10.sup.9
cfu) either alone or supplemented with the peptide GGL27 at 3
.mu.M. The mixture was incubated for 30 min or 1 h at 37.degree.
C., centrifuged, and supernatants and bacteria collected. The
bacterial pellet was washed with PBS and bound proteins eluted with
0.1 M glycine-HCl, pH 2.0. The pH of the eluted material was raised
to 7.5 with 1 M Tris. Eluted proteins were precipitated by addition
of 1 volume of trichloroacetic acid (TCA) to 4 volumes of sample,
followed by incubation for 30 min on ice and centrifugation at 15
000 g (4.degree. C. for 20 min). Precipitated material and
supernatants were dissolved in SDS sample buffer and analyzed under
reducing conditions by SDS-PAGE on 16.5% Tris-tricine gels (Clear
PAGE.TM., C.B.S Scientific). Proteins and peptides were transferred
to nitrocellulose membranes (Hybond-C). Membranes were blocked by
3% (w/v) skimmed milk, washed, and incubated for 1 h with rabbit
polyclonal antibodies against the C-terminal part of C3a (LGE27
antibodies) (1:1000), rabbit polyclonal antibodies to C1q
(1:1000)(Dako), goat polyclonal antibodies recognizing the
C-terminal TFPI sequence VKIAYEEIFVKNM [SEQ ID NO: 104] (Abeam), or
rabbit polyclonal antibodies to C5b-9 (1:1000) (Abcam, England).
The membranes were washed three times for 10 min, and incubated (1
h) with HRP-conjugated secondary antibodies (1:2000) (Dako), and
washed (3.times.10 min). Proteins were visualized by an enhanced
chemiluminescent substrate (LumiGLO.RTM.) developing system
(Upstate cell signaling solutions).
[0406] LPS effects on macrophages in vitro--3.5.times.10.sup.8
cells were seeded in 96-well tissue culture plates (Nunc, 167008)
in phenol red-free DMEM (Gibco) supplemented with 10% FBS
containing 1% Anti-Anti (Invitrogen). Following 20 hours of
incubation to permit adherence, cells were washed and stimulated
with 10 ng/ml E. coli (0111:B4) or P. aeruginosa LPS (Sigma), with
and without the peptides GGL27, GGL27(S), and DSE25 of various
doses. The levels of NO in culture supernatants were determined
after 24 hours from stimulation using the Griess reaction (27).
Briefly, nitrite, a stable product of NO degradation, was measured
by mixing 50 .mu.l of culture supernatants with the same volume of
Griess reagent (Sigma, G4410) and reading absorbance at 550 nm
after 15 min. Phenol-red free DMEM with FBS and antibiotics were
used as a blank. A standard curve was prepared using 0-80 .mu.M
sodium nitrite solutions in ddH20.
[0407] Animal infection model--Animals were housed under standard
conditions of light and temperature and had free access to standard
laboratory chow and water. P. aeruginosa 15159 bacteria were grown
to logarithmic phase (OD.sub.620.about.0.5), harvested, washed in
PBS, diluted in the same buffer to 2.times.10.sup.8 cfu/ml, and
kept on ice until injection. Hundred microliter of the bacterial
suspension was injected intraperitoneally (i.p.) into female Balb/c
mice. Sixty minutes after the bacterial injection, 0.5 mg GGL27 or
buffer alone was injected subcutaneously (s.c.) into the mice. In a
corresponding E. coli infection model, bacteria were grown to early
logarithmic phase (OD.sub.620.about.0.4), harvested, washed in PBS,
diluted in the same buffer to 1.times.10.sup.8 cfu/ml, and kept on
ice until injection. Hundred microliter of the bacterial suspension
was injected i.p. into female Balb/c mice. Thirty minutes after the
bacterial injection, 0.2 mg of GGL27 peptide or buffer alone was
injected i.p. Data from two independent experiments were
pooled.
[0408] LPS model in vivo--Male C57BL/6 mice (8-10 weeks, 22+/-5 g),
were injected intraperitoneally with 18 mg E. coli 0111:B4 LPS
(Sigma) per kg of body weight. Thirty minutes after LPS injection,
0.5 mg GGL27, GGL27(S), DSE25 or buffer alone was injected
intraperitoneally into the mice. Survival and status was followed
during seven days. For blood collection and histochemistry, mice
were sacrificed 20 h after LPS challenge, and lungs were removed
and fixed. These experiments were approved by the Laboratory Animal
Ethics Committee of Malmo/Lund.
[0409] Cytokine assay--The cytokines IL-6, IL-10, MCP-1,
INF-.gamma., and TNF-.alpha. were measured in plasma from mice
subjected to LPS (with or without peptide treatment) using the
Cytometric bead array; mouse inflammation kit (Becton Dickinson AB)
according to the manufacturer's instructions.
[0410] Hemolysis assay--EDTA-blood was centrifuged at 800 g for 10
min, whereafter plasma and buffy coat were removed. The
erythrocytes were washed three times and resuspended in PBS, pH 7.4
to get a 5% suspension. The cells were then incubated with
end-over-end rotation for 60 min at 37.degree. C. in the presence
of peptides (60 .mu.M). 2% Triton X-100 (Sigma-Aldrich) served as
positive control. The samples were then centrifuged at 800 g for 10
min and the supernatant was transferred to a 96 well microtiter
plate. The absorbance of hemoglobin release was measured at 540 nm
and expressed as % of Triton X-100 induced hemolysis.
[0411] Lactate dehydrogenase (LDH) assay--HaCaT keratinocytes were
grown to confluency in 96 well plates (3000 cells/well) in
serum-free keratinocyte medium (SFM) supplemented with bovine
pituitary extract and recombinant EGF (BPE-rEGF) (Invitrogen,
Eugene, USA). The medium was then removed, and 100 .mu.l of the
peptides investigated (at 60 .mu.M, diluted in SFM/BPE-rEGF or in
keratinocyte-SFM supplemented with 20% human serum) were added. The
LDH-based TOX-7 kit (Sigma-Aldrich, St. Louis, USA) was used for
quantification of LDH release from the cells. Results represent
mean values from triplicate measurements, and are given as
fractional LDH release compared to the positive control consisting
of 1% Triton X-100 (yielding 100% LDH release).
[0412] Liposome preparation and leakage assay--The liposomes
investigated were anionic (DOPE/DOPG 75125 mol/mol). DOPG
(1,2-Dioleoyl-sn-Glycero-3-Phosphoglycerol, monosodium salt) and
DOPE (1,2-dioleoyl-sn-Glycero-3-phoshoetanolamine) were both from
Avanti Polar Lipids (Alabaster, USA) and of >99% purity. Due to
the long, symmetric and unsaturated acyl chains of these
phospholipids, several methodological advantages are reached. In
particular, membrane cohesion is good, which facilitates very
stable, unilamellar, and largely defect-free liposomes (observed
from cryo-TEM), allowing detailed studies on liposome leakage. The
lipid mixtures were dissolved in chloroform, after which solvent
was removed by evaporation under vacuum overnight. Subsequently, 10
mM Tris buffer, pH 7.4, was added together with 0.1 M
carboxyfluorescein (CF) (Sigma, St. Louis, USA). After hydration,
the lipid mixture was subjected to eight freeze-thaw cycles
consisting of freezing in liquid nitrogen and heating to 60.degree.
C. Unilamellar liposomes of about 0140 nm were generated by
multiple extrusions through polycarbonate filters (pore size 100
nm) mounted in a LipoFast miniextruder (Avestin, Ottawa, Canada) at
22.degree. C. Untrapped CF was removed by two subsequent gel
filtrations (Sephadex G-50, GE Healthcare, Uppsala, Sweden) at
22.degree. C., with Tris buffer as eluent. CF release from the
liposomes was determined by monitoring the emitted fluorescence at
520 nm from a liposome dispersion (10 .quadrature.M lipid in 10 mM
Tris, pH 7.4). An absolute leakage scale was obtained by disrupting
the liposomes at the end of each experiment through addition of 0.8
mM Triton X-100 (Sigma-Aldrich, St. Louis, USA). A SPEX-fluorolog
1650 0.22-m double spectrometer (SPEX Industries, Edison, USA) was
used for the liposome leakage assay. Measurements were performed in
triplicate at 37.degree. C.
[0413] CD-spectroscopy--CD spectra of the peptides were measured on
a Jasco J-810 Spectropolarimeter (Jasco, U.K.). The measurements
were performed at 37.degree. C. in a 10 mm quartz cuvet under
stirring and the peptide concentration was 10 .mu.M. The effect on
peptide secondary structure of liposomes at a lipid concentration
of 100 .mu.M was monitored in the range 200-250 nm. The fraction of
the peptide in .alpha.-helical conformation, X.sub..alpha., was
calculated from
X.sub..alpha.=(A-A.sub.c)/(A.sub..alpha.-A.sub.c)
where A is the recorded CD signal at 225 nm, and A.sub..quadrature.
and A.sub.c are the CD signal at 225 nm for a reference peptide in
100% .alpha.-helix and 100% random coil conformation, respectively.
100% .alpha.-helix and 100% random coil references were obtained
from 0.133 mM (monomer concentration) poly-L-lysine in 0.1 M NaOH
and 0.1 M HCl, respectively (28, 29). For determination of effects
of lipopolysaccharide on peptide structure, the peptide secondary
structure was monitored at a peptide concentration of 10 .mu.M,
both in Tris buffer and in the presence of E. coli
lipopolysaccharide (0.02 wt %) (Escherichia coli 0111:B4, highly
purified, less than 1% protein/RNA, Sigma, UK). To account for
instrumental differences between measurements the background value
(detected at 250 nm, where no peptide signal is present) was
subtracted. Signals from the bulk solution were also corrected
for.
[0414] Phylogenetic analyses of TFPI--The TFPI amino acid sequence
was retrieved from the NCBI site. Each sequence was analyzed with
Psi-Blast (NCBI) to find the ortholog and paralog sequences.
Sequences that showed structural homology >70% were selected.
These sequences were aligned using ClustalW using Blosum 69 protein
weight matrix settings. Internal adjustments were made taking the
structural alignment into account utilizing the ClustalW interface.
The level of consistency of each position within the alignment was
estimated by using the alignment-evaluating software Tcoffee.
[0415] Statistical analysis--Bar diagrams (RDA, VCA) are presented
as mean and standard deviation, from at least three independent
experiments.
Results
[0416] To elucidate whether C-terminal peptides of TFPI possess
antimicrobial activity, we investigated the effects of defined
regions of TFPI previously reported to be generated by proteolytic
action (plasmin and thrombin), as well as the peptide
LIKTKRKRKKQRVKIAY [SEQ ID NO: 5] (LIK17) comprising the C-terminal
heparin-binding epitope of TFPI (see FIG. 34A for sequences). The
results showed that the peptides were indeed antimicrobial in
radial diffusion assays (RDA) against Gram-negative Escherichia
coli and Pseudomonas aeruginosa, Gram-positive Bacillus subtilis
and Staphylococcus aureus, as well as the fungi Candida albicans
and Candida parapsilosis (FIG. 34B). It is of note that the
peptides displayed activities comparable to that of the "classical"
AMP human cathelicidin LL-37 (FIG. 34B). In contrast, the control
peptides GGL27(S), having the central K/R residues replaced by S,
and the peptide DSE25, from the N-terminus of TFPI (see
Experimental Procedures for sequences), yielded no antimicrobial
effects against the above microbes (FIG. 43). The antibacterial
results above were further substantiated by matrix-free viable
count assays. The results from these dose-response experiments
utilizing E. coli confirmed that particularly GGL27 and LIK17
displayed significant antibacterial activity (FIG. 34C).
[0417] FACS analyses showed that plasmin-generated GGL27 avidly
bound to E. coli and P. aeruginosa in human plasma (FIG. 35A).
Studies employing the impermeant probe FITC showed that LIK17 (FIG.
35B) and GGL27 (FIG. 44) permeabilized bacterial membranes of E.
coli similarly to those seen after treatment with LL-37 (30, 31)
(FIG. 35B). Electron microscopy utilizing P. aeruginosa
demonstrated extensive membrane damage, with cell envelopes of P.
aeruginosa devoid of their cytoplasmic contents, and intracellular
material found extracellularly (FIG. 35C). Again, similar findings
were obtained with LL-37. These data suggest that the TFPI-derived
peptides act on bacterial membranes, however they do not
demonstrate the exact mechanistic events following peptide addition
to bacteria, as secondary metabolic effects on bacteria also may
trigger bacterial death and membrane destabilization. Therefore, a
liposome model was employed to study membrane permeabilization of
LIK17 and GGL27. The peptides caused CF release (FIG. 36A), thus
indicating a direct effect on lipid membranes. Kinetic analysis
showed that .about.80% of the maximal release occurred within 5-10
minutes, comparable to results obtained with LL-37 (FIG. 36B). It
is noteworthy that LIK17 and GGL27 displayed no significant
conformational changes associated with binding to liposomes (FIG.
36C) and only relatively minor ones together with E. coli LPS (FIG.
36D), the latter originating from peptide and/or LPS, contrasting
to LL-37, which showed a significant increase in helicity on
liposome binding. AMPs that kill bacteria may also exhibit
hemolytic and membrane permeabilizing activities against eukaryotic
cells. The results showed, however, that there was no hemolytic
activity of the TFPI-derived peptides at doses of 3-60 .mu.M (FIG.
37A). This contrasted to LL-37, which readily permeabilized
erythrocytes at doses >6 .mu.M. Likewise, the TFPI-derived
peptides did not permeabilize HaCaT cells, nor did they display any
significant toxicity at the concentrations studied (FIG. 37B).
[0418] Since the activity of many antimicrobial peptides is
abrogated by the presence of physiological salt as well as presence
of "biomatrices" such as plasma or serum (32, 33), we also
investigated the influence of salt and human plasma on peptide
activity. As demonstrated in FIG. 38A, the bactericidal activity of
all three TFPI-derived peptides (GGL27, TKR22, and LIK17),
particularly against E. coli, was not only retained in presence of
human plasma, but significantly enhanced. It should here be noted
that killing of E. coli by C-terminal TFPI peptides was recently
shown to be mediated via the classical pathway and linked to
formation of the membrane attack complex (MAC) (34). Consequently,
heat-inactivation of plasma abolished the potentiation, findings
compatible with previous results showing that the presence of an
intact complement system promotes the killing of this microbe.
Additionally, it should be noted that the TFPI-derived peptides
also mediated killing in heat-inactivated plasma, although at
higher concentrations (FIG. 38A), compatible with a direct
peptide-mediated antimicrobial effect, as demonstrated in FIGS. 35
and 36. Interestingly, in contrast to the findings with E. coli,
killing of P. aeruginosa was not enhanced in human plasma when
compared with physiological buffer. Furthermore, the potentiating
effect of native plasma, when compared with heat-inactivated
plasma, was less significant (notably for GGL27 and LIK17). The
above findings were generalized using a panel of E. coli and P.
aeruginosa isolates (FIG. 38B). In addition, kinetic studies
demonstrated that the bacterial killing by the peptides occurred
within 5-20 min indicating a fast direct action compatible with
many antimicrobial peptides (FIG. 45).
[0419] Western blot experiments (FIGS. 39A and 8) and FACS analyses
(FIGS. 39C and D), showed that GGL27 enhanced binding of C1q to E.
coli, resulting in increased formation of MAC (FIGS. 39A, C and D).
It also induced a significant generation of C3a (FIGS. 39B, C and
D), an anaphylatoxin previously shown to exert antimicrobial
effects under physiological conditions against various bacteria,
mediated by bacterial binding and membrane lysis (25). Taken
together, these observations provide a novel mechanism for
bacterial killing, based on initial bacterial binding of GGL27
(FIG. 35A), followed by boosting of formation and bacterial binding
of antimicrobial C3a (FIGS. 39B,C, and D), which therefore should
further add to the total antimicrobial effect induced by GGL27
(FIG. 38B). In contrast to these results, no significant C1q/MAC
alterations were induced after subjecting P. aeruginosa to GGL27
(FIGS. 39A, C and D). Considering C3a in relation to P. aeruginosa,
the western blots of bacteria-bound peptides identified minor C3a
bands, as well as peptides of higher molecular weight, containing
the C-terminal epitope of C3a. FACS analysis showed increased
generation and binding of these C3a-containing fragments to the
bacterial surface (FIGS. 39 C and D).
[0420] TFPI is mostly considered a plasma protein, but is also
expressed by endothelial cells, monocytes and macrophages (12).
Immunohistochemistry analyses showed that TFPI was expressed in
normal human skin, particularly in the basal epidermal layers (FIG.
40A). Furthermore, the molecule showed a ubiquitous expression at
the wound edges of both acute wounds and chronic leg ulcers.
Chronic leg ulcers are characterized by an excessive chronic
inflammatory state, high levels of proteinases (such as plasmin)
and frequent bacterial colonization with P. aeruginosa (35). Hence,
we analyzed fibrin slough from such an ulcer, infected with P.
aeruginosa. As demonstrated by electron microscopy and
immunogold-labeled antibodies against the C-terminal part of TFPI,
these peptide epitopes were found in association with bacterial
membranes, as well as fibrin fibers. Importantly, using double
staining, C3a was found to be particularly associated with these
TFPI-peptides (FIG. 40B). Hence, these in vivo data are compatible
with previous results (FIG. 39) on microbial binding of GGL27, and
its relation to enhanced generation of antimicrobial C3a in plasma
in vitro.
[0421] It is of note that the human TFPI peptides did not enhance
bacterial killing in mouse plasma (FIG. 41A). Nevertheless, a
prolongation of survival in the mouse infection models was observed
(FIG. 41B). In order to delineate a possible mechanism underlying
this observation, and considering the previously reported
LPS-binding property of TFPI (16), we investigated whether GGL27
could exert anti-endotoxin effects in vitro and in vivo. The
results showed indeed that the peptide inhibited LPS-mediated
NO-release from mouse-derived macrophages (RAW 264.7 cells) (FIG.
41C), and also significantly increased survival in a mouse model of
LPS shock (FIG. 41D). Analyses of cytokines 20 hours after LPS
injection, showed significant reductions of proinflammatory IL-6,
IFN-.gamma., TNF-.alpha., and MCP-1, whereas anti-inflammatory
IL-10 was increased (FIG. 41E). Furthermore, a marked reduction of
inflammation and vascular leakage in the lungs of the GGL27-treated
animals was observed (FIG. 41F). In contrast, the two control
peptides DSE25 from the N-terminal region of TFPI, and the peptide
GGL27(S), having the "core" K/R residues substituted with S, did
neither block NO release in vitro, nor significantly alter overall
cytokine release, as well as survival in vivo (FIG. 41C, D, E).
These data demonstrate a specific anti-endotoxic role of GGL27,
compatible with the observed improvement in the E. coli and P.
aeruginosa mouse infection models. Furthermore, the abrogated
anti-endotoxic activity of GGL27(S) also highlights the importance
of the central cationic K/R residues in this molecule. Taken
together, the results are indicative of differential and
host-related effects of GGL27; a host-independent anti-endotoxic
effect, and a host-dependent and complement-based antimicrobial
function, likely a logical consequence of different structural
prerequisites for LPS- and complement-interactions. In line with
this reasoning, FIG. 42 illustrates the evolution of this region of
TFPI and highlights the conservation of the cationic "core", but
also significant sequence changes between mice and men in this
C-terminal region of TFPI.
Discussion
[0422] The key findings in this study are the identification of a
dual antimicrobial activity of C-terminal TFPI peptides, based on
direct and complement-mediated bactericidal effects, the
demonstration of an anti-endotoxic effect, combined with the
identification of TFPI in skin, its upregulation during wounding,
and presence in wounds.
[0423] The presently disclosed direct antibacterial action of
C-terminal peptides of TFPI is in line with observations indicating
that heparin-binding proteins, such as complement C3 (25),
kininogen (36, 37), heparin-binding protein (38), heparin-binding
epidermal growth factor and other growth factors (39),
heparin/heparan sulfate interacting protein (40),
.beta.2-glycoprotein (41), histidine-rich glycoprotein (42), and
human thrombin (43) may, either as holoproteins or after
fragmentation, exert antimicrobial activities in vitro, and in
several cases, also in vivo (36, 37, 42). In addition,
peptide-mediated C3a generation, along with enhanced MAC formation,
represents a mechanism by which a host defense peptide may
selectively enhance microbial killing by generation of additional
complement-derived AMPs. Recent evidence showing a significant
cross-talk between the coagulation and complement systems (44)
further adds biological relevance to the facilitated generation of
C3a by the TFPI-peptides.
[0424] Considering the C-terminal region of TFPI, peptides derived
from this region resemble other linear peptides of low helical
content. For example, antimicrobial peptides derived from growth
factors also display a low helical content in presence of
membranes, reflecting their low content of features typical of
"classical" helical peptides, such as regularly interspersed
hydrophobic residues (39). Furthermore, studies on a
kininogen-derived antimicrobial peptide, HKH20
(HKHGHGHGKHKNKGKKNGKH [SEQ ID NO: 105]) (45) showed that the HKH20
peptide displays predominantly random coil conformation in buffer
and at lipid bilayers, the interactions dominated by electrostatics
(45). It is noteworthy that like the TFPI-derived peptides, both
HKH20 (36) and GKR22 (GKRKKKGKGLGKKRDPCLRKYK [SEQ ID NO: 106])
(39), a peptide derived from heparin-binding growth factor, retain
their antibacterial activity at physiological conditions. In
relation to the above, it is interesting that recent studies
indicate that certain low amphipathic peptides exhibit activity
mainly by strong electrostatic interactions, e.g., leading to
negative curvature strains, to membrane thinning, or to the
formation of lipid domains, sometimes resulting in bacterial
membrane fluidity changes affecting biological function (46).
[0425] As previously mentioned, many antimicrobial peptides,
including LL-37 (31), C3a (25), thrombin- and kininogen-derived
peptides (36, 37, 43) are released during proteolysis. Considering
TFPI, enzymes such as plasmin and thrombin release distinct
C-terminal fragments of relevance for physiological events, such as
wounding. Therefore, TFPI-derived peptides generated in fibrin
clots by thrombin (20), may contribute to antimicrobial activity
during wound healing. Furthermore, subsequent proteolysis by
plasmin (generating GGL27) (21) may further add to the spectrum of
host defense peptides released. As mentioned above, it is of note
that TFPI, in addition to its synthesis in microvascular
endothelial cells and subsequent occurrence in the endothelium,
plasma, and platelets, is also produced by the liver, and found in
monocytes and macrophages (12). Production of TFPI has also been
demonstrated in capillaries, megakaryocytes, several cell lines, as
well as neoplastic cells. This evidence combined with a marked
upregulation of TFPI during wounding of human skin, as well as its
occurrence in fibrin and on bacteria, suggests that high local
levels of endogenous TFPI peptides may occur in vivo. The finding
that the peptides were particularly active in human plasma against
E. coli, and to a lesser extent against P. aeruginosa, is
particularly relevant and interesting, in the light of the
ubiquitous occurrence of P. aeruginosa in chronic leg ulcers,
contrasting to the very sparse E. coli colonization in these wounds
(35, 47). Furthermore, based on the demonstration that bacterial
omptins (such as OmpT from E. coli) may release GGL27 fragments
from TFPI, it was recently proposed that TFPI has evolved
sensitivity to omptin-mediated proteolytic inactivation to
potentiate procoagulant responses to E. coli infection in certain
conditions (48). Our data further substantiate this hypothesis by
adding another host-response mechanism to E. coli infection;
OmpT-mediated generation of the host defense peptide GGL27.
[0426] Simultaneous control of inflammation and coagulation plays a
key role in maintaining homeostasis, and it is notable that trials
utilizing recombinant TFPI indicate that the protein protects from
E. coli induced severe sepsis (49), and furthermore, TFPI is also
under evaluation in a phase III clinical trial involving patients
with severe community acquired pneumonia. In this context, it is
interesting to note that the here observed anti-endotoxic effect of
GGL27 in vitro and in vivo may explain, at least partly, the
previously observed protective effects of TFPI during E. coli
sepsis (49). The current findings showing direct and indirect, MAC
and C3a-mediated, antimicrobial effects of the C-terminal epitope
of TFPI, GGL27, may have implications for future attempts in
designing and developing peptide-based therapeutics combating
severe infection. Finally, the finding that the TFPI-peptide did
not significantly enhance bacterial killing in mouse plasma ex vivo
as well as increase total survival during bacterial sepsis
(although a prolonged survival was observed), illustrates that
mouse models might be disadvantageous when it comes to assessing
certain aspects of the physiological roles of human C-terminal TFPI
peptides, such as those involving complement-mediated bacterial
clearance. Interestingly, whereas the TFPI-.alpha. form (having the
C-terminal cationic sequence) is the predominant form in humans,
mice appear to largely produce TFPI-.beta. (a form lacking the
C-terminal sequence) (50), an observation compatible with the above
mentioned evolution of unique C-terminal and OmpT-releasable
peptides in humans.
REFERENCES
[0427] 1. Yount, N. Y., Bayer, A. S., Xiong, Y. Q., and Yeaman, M.
R. (2006) Biopolymers 84, 435-458 [0428] 2. Zelezetsky, I., and
Tossi, A. (2006) Biochim Biophys Acta 1758, 1436-1449 [0429] 3.
Tossi, A., and Sandri, L. (2002) Curr Pharm Des 8, 743-761 [0430]
4. Powers, J. P., and Hancock, R. E. (2003) Peptides 24, 1681-1691
[0431] 5. Bulet, P., Stocklin, R., and Menin, L. (2004) Immunol Rev
198, 169-184 [0432] 6. Durr, U. H., Sudheendra, U. S., and
Ramamoorthy, A. (2006) Biochim Biophys Acta 1758, 1408-1425 [0433]
7. Brogden, K. A. (2005) Nat Rev Microbiol 3, 238-250 [0434] 8.
Lohner, K., and Blondelle, S. E. (2005) Comb Chem High Throughput
Screen 8, 241-256 [0435] 9. Zanetti, M. (2004) J Leukoc Biol 75,
39-48 [0436] 10. Elsbach, P. (2003) J Clin Invest 111, 1643-1645
[0437] 11. Ganz, T. (2003) Nat Rev Immunol 3, 710-720 [0438] 12.
Lwaleed, B. A., and Bass, P. S. (2006) J Pathol 208, 327-339 [0439]
13. Girard, T. J., Warren, L. A., Novotny, W. F., Likert, K. M.,
Brown, S. G., Miletich, J. P., and Broze, G. J., Jr. (1989) Nature
338, 518-520 [0440] 14. Mine, S., Yamazaki, T., Miyata, T., Hara,
S., and Kato, H. (2002) Biochemistry 41, 78-85 [0441] 15. Crawley,
J. T., and Lane, D. A. (2008) Arterioscler Thromb Vasc Biol 28,
233-242 [0442] 16. Park, C. T., Creasey, A. A., and Wright, S. D.
(1997) Blood 89, 4268-4274 [0443] 17. Hembrough, T. A., Ruiz, J.
F., Swerdlow, B. M., Swartz, G. M., Hammers, H. J., Zhang, L.,
Plum, S. M., Williams, M. S., Strickland, D. K., and Pribluda, V.
S. (2004) Blood 103, 3374-3380 [0444] 18. Wesselschmidt, R.,
Likert, K., Girard, T., Wun, T. C., and Broze, G. J., Jr. (1992)
Blood 79, 2004-2010 [0445] 19. Ettelaie, C., Adam, J. M., James, N.
J., Oke, A. O., Harrison, J. A., Bunce, T. D., and Bruckdorfer, K.
R. (1999) FEBS Lett 463, 341-344 [0446] 20. Ohkura, N., Enjyoji,
K., Kamikubo, Y., and Kato, H. (1997) Blood 90, 1883-1892 [0447]
21. Li, A., and Wun, T. C. (1998) Thromb Haemost 80, 423-427 [0448]
22. Cunningham, A. C., Hasty, K. A., Enghild, J. J., and Mast, A.
E. (2002) Biochem J 367, 451-458 [0449] 23. Andersson, E.,
Rydengard, V., Sonesson, A., Morgelin, M., Bjorck, L., and
Schmidtchen, A. (2004) Eur J Biochem 271, 1219-1226 [0450] 24.
Lehrer, R. I., Rosenman, M., Harwig, S. S., Jackson, R., and
Eisenhauer, P. (1991) J Immunol Methods 137, 167-173 [0451] 25.
Nordahl, E. A., Rydengard, V., Nyberg, P., Nitsche, D. P.,
Morgelin, M., Malmsten, M., Bjorck, L., and Schmidtchen, A. (2004)
Proc Natl Acad Sci USA 101, 16879-16884 [0452] 26. Carlemalm, E.,
Villiger, W., Hobot, J. A., Acetarin, J. D., and Kellenberger, E.
(1985) J Microsc 140, 55-63 [0453] 27. Pollock, J. S., Forstermann,
U., Mitchell, J. A., Warner, T. D., Schmidt, H. H., Nakane, M., and
Murad, F. (1991) Proc Natl Acad Sci USA 88, 10480-10484 [0454] 28.
Greenfield, N., and Fasman, G. D. (1969) Biochemistry 8, 4108-4116
[0455] 29. Sjogren, H., and Ulvenlund, S. (2005) Biophys Chem 116,
11-21 [0456] 30. Tossi, A., Sandri, L., and Giangaspero, A. (2000)
Biopolymers 55, 4-30 [0457] 31. Zasloff, M. (2002) Nature 415,
389-395. [0458] 32. Ganz, T. (2001) Semin Respir Infect 16, 4-10.
[0459] 33. Wang, Y., Agerberth, B., Lothgren, A., Almstedt, A., and
Johansson, J. (1998) Biol Chem 273, 33115-33118. [0460] 34. Schirm,
S., Liu, X., Jennings, L. L., Jedrzejewski, P., Dai, Y., and Hardy,
S. (2009) J Infect Dis 199, 1807-1815 [0461] 35. Lundqvist, K.,
Herwald, H., Sonesson, A., and Schmidtchen, A. (2004) Thromb
Haemost 92, 281-287 [0462] 36. Nordahl, E. A., Rydengard, V.,
Morgelin, M., and Schmidtchen, A. (2005) J Biol Chem 280,
34832-34839 [0463] 37. Frick, I. M., Akesson, P., Herwald, H.,
Morgelin, M., Malmsten, M., Nagler, D. K., and Bjorck, L. (2006)
Embo J 25, 5569-5578 [0464] 38. Pereira, H. A. (1995) J Leukoc Biol
57, 805-812 [0465] 39. Malmsten, M., Davoudi, M., Walse, B.,
Rydengard, V., Pasupuleti, M., Morgelin, M., and Schmidtchen, A.
(2007) Growth Factors 25, 60-70 [0466] 40. Meyer-Hoffert, U.,
Hornef, M., Henriques-Normark, B., Normark, S., Andersson, M., and
Putsep, K. (2008) FASEB J 22, 2427-2434 [0467] 41. Nilsson, M.,
Wasylik, S., Morgelin, M., Olin, A. I., Meijers, J. C., Derksen, R.
H., de Groot, P. G., and Herwald, H. (2008) Mol Microbiol 67,
482-492 [0468] 42. Rydengard, V., Shannon, O., Lundqvist, K.,
Kacprzyk, L., Chalupka, A., Olsson, A. K., Morgelin, M.,
Jahnen-Dechent, W., Malmsten, M., and Schmidtchen, A. (2008) PLoS
Pathog 4, e1000116 [0469] 43. Papareddy, P., Rydengard, V.,
Pasupuleti, M., Walse, B., Morgelin, M., Chalupka, A., Malmsten,
M., and Schmidtchen, A. PLoS Pathog 6, e1000857 [0470] 44. Amara,
U., Rittirsch, D., Flierl, M., Bruckner, U., Klos, A., Gebhard, F.,
Lambris, J. D., and Huber-Lang, M. (2008) Adv Exp Med Biol 632,
71-79 [0471] 45. Ringstad, L., Andersson Nordahl, E., Schmidtchen,
A., and Malmsten, M. (2007) Biophys J 92, 87-98 [0472] 46.
Yamamoto, N., and Tamura, A. (2010) Peptides 31, 794-805 [0473] 47.
Schmidtchen, A., Wolff, H., and Hansson, C. (2001) Acta Derm
Venereol 81, 406-409. [0474] 48. Yun, T. H., Cott, J. E., Tapping,
R. I., Slauch, J. M., and Morrissey, J. H. (2009) Blood 113,
1139-1148 [0475] 49. Creasey, A. A., Chang, A. C., Feigen, L., Wun,
T. C., Taylor, F. B., Jr., and Hinshaw, L. B. (1993) J Clin Invest
91, 2850-2860 [0476] 50. Maroney, S. A., Ellery, P. E., and Mast,
A. E. Thromb Res 125 Suppl 1, S52
Example C
Anticoagulative and Anti-Inflammatory Effects of Exemplary Peptides
Derived from HRG and ATIII
Histidine-Rich Glycoprotein (Hrg)
Introduction
Histidine-Rich Glycoprotein and GHH25
[0477] The antimicrobial plasma protein Histidine-rich glycoprotein
(HRG) protects the host against systemic microbial infections in
vivo. Here, we show that HRG is a pattern recognition molecule, by
binding LPS and increasing LPS mediated TLR-4 response in the host
cells.
[0478] LPS interacts with CD14, a receptor on macrophages,
monocytes and neutrophils, the interaction is increased by plasma
protein LBP (LPS-binding protein). The LPS/CD14 complex leads to
activation of toll-like receptor (TLR) 4 which in turn activates
the MyD88-dependent or -independent pathways [1]. The LPS-response
can also be LBP-independent [2]. There is no difference in
TNF-.alpha. release in vivo between wildtype and LBP-deficient mice
after LPS injection, suggesting that a protein with LBP-like
capabilities may be responsible [3]. Activation of TLR4 then in
turn leads to an increase of phagocytic activity of mononuclear
phagocytes, a cascade of released cytokines and nitric oxide (NO),
which initiate and support the inflammatory response. NO is
produced by a group of enzymes called nitric oxide synthases [4]
and involved in many biological processes such as protecting the
host against microbes, parasitic worms and tumours and regulate
blood pressure, high levels of NO can instead be cytotoxic and
destroy endogenous tissue.
[0479] The LPS-induced response is essential for the host defence,
but an overwhelming response can instead lead to sepsis. Cytokines
and NO are identified to be major contributors to the development
of septic shock with symptoms as fever, coagulant activity, septic
shock, multiple organ failure and in worst cases death of the host
[5]. This is supported by the fact that inducibleNOS (iNOS) mutant
mice where resistant to LPS-induced mortality [6]. The
susceptibility of different animal species to the toxicity of LPS
is highly variable, for example humans are very sensitive [7,8]
comparing to mice that are fairly resistant [9]. A common
misunderstanding is that sepsis is controlled by only
pro-inflammatory mediators. The early stages of sepsis is dominated
by pro-inflammatory mediators such as TNF-a, NO, bradykinin,
thrombin and histamine, whereas anti-inflammatory mediators, such
as IL-6, activated protein C, antithrombin and granulocyte
colony-stimulating factor, are most widely occurring during the
later stages [10].
[0480] The treatment of sepsis today includes administration of
intravenous fluids, broad-spectrum antibiotics, protective lung
ventilation, glycocorticoids, insulin therapy and recombinant human
activator protein C (APC). Many attempts have been done to
neutralize LPS or inhibit LPS-mediated activation of host immune
cells, such as blocking cytokine activity with antibodies, blocking
NO production [11] and by using antimicrobial peptides as
neutralizers of LPS [12].
[0481] The aim of this study was to investigate the effects of an
abundant plasma protein, HRG, on LPS-mediated responses. HRG is a
67 kDa plasma protein synthesized in the liver and was first
described in 1972 [13, 14]. HRG can also be released upon thrombin
activation from .alpha.-granules of thrombocytes, and is able to
cover the surface of a fibrin clot. Together with fetuins and
kininogens, HRG belongs to the cystatin superfamily. The most
distinguishing characteristic of the protein is the histidine-rich
domain of the protein contains a conserved GHHPH repeat [15]. HRG
has been shown to interact in vitro with a diverse group of
ligands, like heparin, plasminogen, fibrinogen, thrombospondin,
heme, IgG, FcgR and C1q. In vivo-studies demonstrates that HRG is
an anticoagulant and antifibrinolytic modifier [16], has an
inhibitory effects on tumour vascularization [17] and plays an
important role in the innate immunity as a antimicrobial protein
[18].
[0482] Herein, we demonstrate that HRG is a potent pro-inflammatory
protein, by enhancing LPS induced NO and cytokines in vitro. In
vivo experiment showed that Hrg-/- mice were markedly resistant
against LPS induced sepsis. Our data clearly demonstrate that HRG
contributes to LPS toxicity in experimental endotoxemia. The data
also demonstrate an antiinflammatory effect of the peptide
GHH25.
Materials & Methods
[0483] Synthetic peptide GHH25 (GHHPHGHHPHGHHPHGHHPHGHHPH [SEQ ID
NO: 11]) was from Biopeptide (San Diego, Calif., USA). Polyclonal
rabbit anti mouse TRL4 was purchased from GeneTex (Irvine, Calif.,
USA). The cytometric bead array, mouse inflammation kit was from BD
Biosciences (Stockholm, Sweden). Escherichia coli LPS (0111:B4) was
purchased from Sigma (St Lois, Mo.).
[0484] Purification of Human HRG. Serum HRG was purified as
described before [19]. Briefly, human serum were gently shaken at
4.degree. C. overnight with nickel-nitrilotriacetic acid (Ni-NTA)
agarose, washed with phosphate-buffered saline (PBS, pH 7.4).
Elution was first performed with PBS containing 80 mM imidazole to
elute unspecifically bound proteins, and then with PBS containing
500 mM imidazole to elute purified HRG. The protein was dialyzed,
freeze-dried and the concentration was determined using the
Bradford method [20].
[0485] Radioiodination of heparin and LPS. The radioiodination of
heparin (from porcine intestinal mucosa, Sigma-Aldrich) was
performed as described previously [21]. The iodination of LPS was
performed as described by Ulevitch [22]. 1 mg Escherichia coli
0111:B4 LPS was incubated in 50 mM p-OH benzimidate in borate
buffer, pH 8, over night at 4.degree. C., and then dialyzed against
PBS, pH 7.4. LPS was then radiolabelled with .sup.125I using the
chloramine T method, and unlabelled .sup.125I was then removed by
dialysis.
[0486] Heparin and LPS-binding assay. 1, 2 and 5 .mu.g of the
synthetic peptides in 100 .mu.l PBS pH 7.4 were applied onto
nitrocellulose membranes (Hybond-C, Amersham Biosciences) using a
slot-blot apparatus. Membranes were blocked for 1 h at room
temperature with 2% bovine serum albumin in PBS pH 7.4 and then
incubated with radiolabelled LPS (.sup..about.40 .mu.gmL.sup.-1,
0.13.times.10.sup.6 cpm.mu.g.sup.-1) or radiolabelled heparin
(.sup..about.10 .mu.gmL.sup.-1, 0.4.times.10.sup.6 cpm.mu.g.sup.-1)
for 1 h at room temperature in PBS, pH 7.4. Unlabeled heparin (6
mg/ml) was added for competition of binding. The membranes were
washed 3 times in PBS, pH 7.4. A Bas 2000 radio-imaging system
(Fuji Film, Tokyo, Japan) was used to visualize radioactivity.
[0487] Cellculture. Murine macrophage cell line, RAW 264.7 (kindly
provided by Dr. H Bjorkbacka) were grown in Dulbeccos Modified
Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal calf serum
(FCS). All experiments were performed under serum free
conditions.
[0488] Nitric oxide induction in RAW macrophages. Confluent cells
were harvested and transferred to 96-wells plate
(3.5.times.10.sup.6/well). After adhesion cells were washed with
phenol red-free DMEM (Gibco). E. Coli LPS (100 ng/ml), LL-37 (2 or
10 .mu.M) or HRG (2 or 10 .mu.M) was preincubated at 37.degree. C.
for 30 minutes and then transferred to the cells. For inhibition of
NO induction, 5 .mu.g/ml anti mouse TLR4 antibody, 10 or 100 .mu.M
GHH25 or 100 .mu.g/ml heparin were used. The cells were stimulated
for 24 hours and nitric oxide was determined using the Griess
chemical method [23].
[0489] TNF-.alpha. release from human macrophages. Human
monocyte-derived macrophages (hMDMs) were obtained from peripheral
blood mononuclear cells (PBMCs) obtained from the blood of healthy
donors using a Lymphoprep (Axis-Shield PoC AS) density gradient.
PBMCs were seeded at concentrations of 3.times.10.sup.6 cells/well
into 24-well plates and cultured in RPMI1640 medium supplemented
with 10% heat-inactivated autologous human plasma, 2 mM
L-glutamine, and 50 .mu.l/ml Antibiotic-Antimycotic (Gibco) in a
humidified atmosphere of 5% CO.sub.2. After 24 h, non-adherent
cells were removed and adherent monocytes were differentiated to
macrophages for 10 days, with fresh medium changes every second
day. The cells were stimulated for 24 hours with 10 ng/ml of LPS
with or without HRG (2 .mu.M) and GHH25 (100 .mu.M) under
serum-free conditions. After stimulation the supernatant was
aspirated and TNF-.alpha. was measured using the TNF-.alpha. human
ELISA kit (Invitrogen).
[0490] Animal experiments. The original knockout mice
129/B6-HRG.sup.tm1wja1 were crossed with C57BL/6 mice (Taconic) for
14 generations to obtain uniform genetic background. These
HRG-deficient mouse strain was called B6-HRG.sup.tm1wja1 following
ILAR (Institute of Laboratory Animal Resources) rules. Wildtype
C57BL/6 control mice and C57BL/6 Hrg-/- mice (8-12 weeks, 27+/-4 g)
were bred in the animal facility at Lund University. C57BL/6
Hrg.sup.-/-, lacks the translation start point of exon 1 of the Hrg
gene [16]. Animals were housed under standard conditions of light
and temperature and had free access to standard laboratory chow and
water. In order to induce sepsis, 18 .quadrature.g/g Escherichia
coli 0111:B4 LPS were injected intraperitoneally into C57BL/6 or
C57BL/6 Hrg-/- mice, divided into weight and sex matched groups.
Survival and status was followed during seven days.
[0491] For treatment with GHH25 peptide, 1 mg of the peptide
(diluted in 10 mM Tris, pH 7.4) or buffer only was injected
intraperitoneal 30 minutes after LPS-challenge and survival and
status was then followed.
Antithrombin III
[0492] Serpins are a group of proteins with similar structures that
were first identified as a set of proteins able to inhibit
proteases. The acronym serpin was originally coined because many
serpins inhibit chymotrypsin-like serine proteases (serine protease
inhibitors). The first members of the serpin superfamily to be
extensively studied were the human plasma proteins antithrombin and
antitrypsin, which play key roles in controlling blood coagulation
and inflammation, respectively.
[0493] Structural studies on serpins have revealed that inhibitory
members of the family undergo an unusual conformational change,
termed the Stressed to Relaxed (S to R) transition. This
conformational mobility of serpins provides a key advantage over
static lock-and-key protease inhibitors. In particular, the
function of inhibitory serpins can be readily controlled by
specific cofactors like heparin. The archetypal example of this
situation is antithrombin, which circulates in plasma in a
relatively inactive state. Upon binding a high-affinity heparin
pentasaccharide sequence within long-chain heparin, antithrombin
undergoes a conformational change, exposing key residues important
for the mechanism. The heparin pentasaccharide-bound form of
antithrombin is, thus, a more effective inhibitor of thrombin and
factor Xa. Furthermore, both of these coagulation proteases contain
binding sites (called exosites) for heparin. Heparin, therefore,
also acts as a template for binding of both protease and serpin,
further dramatically accelerating the interaction between the two
parties. After the initial interaction, the final serpin complex is
formed and the heparin moiety is released.
Materials and Methods
[0494] (as above)
Results
[0495] The FFF21 peptide (FFFAKLNCRLYRKANKSSKLV [SEQ ID NO: 1], aa
153-173 of antithrombin III, accession number P01008) displays
antibacterial and antiinflammatory properties (see FIG. 56).
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 115 <210> SEQ ID NO 1 <211> LENGTH: 21 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
1 Phe Phe Phe Ala Lys Leu Asn Cys Arg Leu Tyr Arg Lys Ala Asn Lys 1
5 10 15 Ser Ser Lys Leu Val 20 <210> SEQ ID NO 2 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Ala Lys Leu Asn Cys Arg Leu Tyr Arg Lys Ala
Asn Lys Ser Ser Lys 1 5 10 15 Leu Val Ser Ala Asn Arg 20
<210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Ser
Glu Lys Thr Leu Arg Lys Trp Leu Lys Met Phe Lys Lys Arg Gln 1 5 10
15 Leu Glu Leu Tyr 20 <210> SEQ ID NO 4 <211> LENGTH:
27 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys
Lys Gln Arg Val Lys 1 5 10 15 Ile Ala Tyr Glu Glu Ile Phe Val Lys
Asn Met 20 25 <210> SEQ ID NO 5 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 Leu Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln
Arg Val Lys Ile Ala 1 5 10 15 Tyr <210> SEQ ID NO 6
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 6 Thr Lys Arg Lys Arg Lys Lys
Gln Arg Val Lys Ile Ala Tyr Glu Glu 1 5 10 15 Ile Phe Val Lys Asn
Met 20 <210> SEQ ID NO 7 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
7 Ala Leu Lys Lys Lys Lys Lys Met Pro Lys Leu Arg Phe Ala Ser Arg 1
5 10 15 Ile Arg Lys Ile Arg Lys Lys Gln Phe 20 25 <210> SEQ
ID NO 8 <211> LENGTH: 34 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 8 Glu Asp Cys Lys Arg
Ala Cys Ala Lys Ala Leu Lys Lys Lys Lys Lys 1 5 10 15 Met Pro Lys
Leu Arg Phe Ala Ser Arg Ile Arg Lys Ile Arg Lys Lys 20 25 30 Gln
Phe <210> SEQ ID NO 9 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile,
Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (8)..(8)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(9)..(9) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile,
Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(14)..(14) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu,
Ile, Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (15)..(15) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(11)...(15) <223> OTHER INFORMATION: All amino acids are
either present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (16)..(16) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (17)..(17) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (18)..(18) <223> OTHER INFORMATION: Xaa
is His, Arg or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (20)..(20) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (16)...(20) <223> OTHER INFORMATION:
All amino acids are either present or absent <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (23)..(23)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu,
Ile, Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (25)..(25) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)...(25) <223> OTHER INFORMATION: All amino acids are
either present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (28)..(28) <223> OTHER INFORMATION: Xaa
is His, Arg or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (29)..(29) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (30)..(30) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (26)...(30) <223> OTHER INFORMATION:
All amino acids are either present or absent <400> SEQUENCE:
9 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 <210> SEQ ID NO 10 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(11)...(15) <223> OTHER INFORMATION: All amino acids are
either present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (16)...(20) <223> OTHER
INFORMATION: All amino acids are either present or absent
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)...(25) <223> OTHER INFORMATION: All amino
acids are either present or absent <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (26)...(30)
<223> OTHER INFORMATION: All amino acids are either present
or absent <400> SEQUENCE: 10 Gly His His Pro His Gly His His
Pro His Gly His His Pro His Gly 1 5 10 15 His His Pro His Gly His
His Pro His Gly His His Pro His 20 25 30 <210> SEQ ID NO 11
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 11 Gly His His Pro His Gly His
His Pro His Gly His His Pro His Gly 1 5 10 15 His His Pro His Gly
His His Pro His 20 25 <210> SEQ ID NO 12 <211> LENGTH:
54 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Lys Thr Ser Asp Gln Ile His Phe Phe Phe
Ala Lys Leu Asn Cys Arg 1 5 10 15 Leu Tyr Arg Lys Ala Asn Lys Ser
Ser Lys Leu Val Ser Ala Asn Arg 20 25 30 Leu Phe Gly Asp Lys Ser
Leu Thr Phe Asn Glu Thr Tyr Gln Asp Ile 35 40 45 Ser Glu Leu Val
Tyr Gly 50 <210> SEQ ID NO 13 <211> LENGTH: 53
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13 Thr Ser Glu Ala Glu Ile His Gln Ser Phe
Gln His Leu Leu Arg Thr 1 5 10 15 Leu Asn Gln Ser Ser Asp Glu Leu
Gln Leu Ser Met Gly Asn Ala Met 20 25 30 Phe Val Lys Glu Gln Leu
Ser Leu Leu Asp Arg Phe Thr Glu Asp Ala 35 40 45 Lys Arg Leu Tyr
Gly 50 <210> SEQ ID NO 14 <211> LENGTH: 53 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
14 Thr Pro Glu Ser Ala Ile His Gln Gly Phe Gln His Leu Val His Ser
1 5 10 15 Leu Thr Val Pro Ser Lys Asp Leu Thr Leu Lys Met Gly Ser
Ala Leu 20 25 30 Phe Val Lys Lys Glu Leu Gln Leu Gln Ala Asn Phe
Leu Gly Asn Val 35 40 45 Lys Arg Leu Tyr Glu 50 <210> SEQ ID
NO 15 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 15 Thr Pro Glu Ser Ala
Ile His Gln Gly Phe Gln His Leu Val His Ser 1 5 10 15 Leu Thr Val
Pro Ser Lys Asp Leu Thr Leu Lys Met Gly Ser Ala Leu 20 25 30 Phe
Val Lys Lys Glu Leu Gln Leu Gln Ala Asn Phe Leu Gly Asn Val 35 40
45 Lys Arg Leu Tyr Glu 50 <210> SEQ ID NO 16 <211>
LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 16 Glu Lys Glu Val Ile Glu Asn Thr Glu Ala
Val His Gln Gln Phe Gln 1 5 10 15 Lys Phe Leu Thr Glu Ile Ser Lys
Leu Thr Asn Asp Tyr Glu Leu Asn 20 25 30 Ile Thr Asn Arg Leu Phe
Gly Glu Lys Thr Tyr Leu Phe Leu Gln Lys 35 40 45 Tyr Leu Asp Tyr
Val Glu Lys Tyr Tyr His 50 55 <210> SEQ ID NO 17 <211>
LENGTH: 56 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 17 Asn Ser Ser Asn Ser Gln Ser Gly Leu Gln
Ser Gln Leu Lys Arg Val 1 5 10 15 Phe Ser Asp Ile Asn Ala Ser His
Lys Asp Tyr Asp Leu Ser Ile Val 20 25 30 Asn Gly Leu Phe Ala Glu
Lys Val Tyr Gly Phe His Lys Asp Tyr Ile 35 40 45 Glu Cys Ala Glu
Lys Leu Tyr Asp 50 55 <210> SEQ ID NO 18 <211> LENGTH:
52 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 18 Ser Ala Gly Glu Glu Phe Phe Val Leu Lys
Ser Phe Phe Ser Ala Ile 1 5 10 15 Ser Glu Lys Lys Gln Glu Phe Thr
Phe Asn Leu Ala Asn Ala Leu Tyr 20 25 30 Leu Gln Glu Gly Phe Thr
Val Lys Glu Gln Tyr Leu His Gly Asn Lys 35 40 45 Glu Phe Phe Gln 50
<210> SEQ ID NO 19 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 Lys
Thr Ser Asp Gln Ile His Phe Phe Phe Ala Lys Leu Asn Cys Arg 1 5 10
15 Leu Tyr Arg Lys Ala Asn Lys Ser Ser Lys Leu Val Ser Ala Asn Arg
20 25 30 Leu Phe Gly Asp Lys Ser Leu Thr Phe 35 40 <210> SEQ
ID NO 20 <211> LENGTH: 54 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 20 Lys Thr Ser Asp Gln
Ile His Phe Phe Phe Ala Lys Leu Asn Cys Arg 1 5 10 15 Leu Tyr Arg
Lys Ala Asn Lys Ser Ser Lys Leu Val Ser Ala Asn Arg 20 25 30 Leu
Phe Gly Asp Lys Ser Leu Thr Phe Asn Glu Thr Tyr Gln Asp Ile 35 40
45 Ser Glu Leu Val Tyr Gly 50 <210> SEQ ID NO 21 <211>
LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 21 Ile Pro Glu Ala Gln Ile His Glu Gly Phe
Gln Glu Leu Leu Arg Thr 1 5 10 15 Leu Asn Gln Pro Asp Ser Gln Leu
Gln Leu Thr Thr Gly Asn Gly Leu 20 25 30 Phe Leu Ser Glu Gly Leu
Lys Leu Val Asp Lys Phe Leu Glu Asp Val 35 40 45 Lys Lys Leu Tyr
His 50 <210> SEQ ID NO 22 <211> LENGTH: 57 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
22 Ile Leu Gln Ala Gln Ala Ala Asp Lys Ile His Ser Ser Phe Arg Ser
1 5 10 15 Leu Ser Ser Ala Ile Asn Ala Ser Thr Gly Asn Tyr Leu Leu
Glu Ser 20 25 30 Val Asn Lys Leu Phe Gly Glu Lys Ser Ala Ser Phe
Arg Glu Glu Tyr 35 40 45 Ile Arg Leu Cys Gln Lys Tyr Tyr Ser 50 55
<210> SEQ ID NO 23 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 Asp
Lys Gly Met Ala Pro Ala Leu Arg His Leu Tyr Lys Glu Leu Met 1 5 10
15 Gly Pro Trp Asn Lys Asp Glu Ile Ser Thr Thr Asp Ala Ile Phe Val
20 25 30 Gln Arg Asp Leu Lys Leu Val Gln Gly Phe Met Pro His Phe
Phe Arg 35 40 45 Leu Phe Arg 50 <210> SEQ ID NO 24
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 24 Ser Ser Glu Lys Glu Leu His
Arg Gly Phe Gln Gln Leu Leu Gln Glu 1 5 10 15 Leu Asn Gln Pro Arg
Asp Gly Phe Gln Leu Ser Leu Gly Asn Ala Leu 20 25 30 Phe Thr Asp
Leu Val Val Asp Leu Gln Asp Thr Phe Val Ser Ala Met 35 40 45 Lys
Thr Leu Tyr Leu 50 <210> SEQ ID NO 25 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 25 Thr Cys Val His Gln Ala Leu Lys Gly Phe
Thr Thr Lys Gly Val Thr 1 5 10 15 Ser Val Ser Gln Ile Phe His Ser
Pro Asp Leu Ala Ile Arg Asp Thr 20 25 30 Phe Val Asn Ala Ser Arg
Thr Leu Tyr Ser 35 40 <210> SEQ ID NO 26 <211> LENGTH:
53 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 26 Thr Pro Met Val Glu Ile Gln His Gly Phe
Gln His Leu Ile Cys Ser 1 5 10 15 Leu Asn Phe Pro Lys Lys Glu Leu
Glu Leu Gln Ile Gly Asn Ala Leu 20 25 30 Phe Ile Gly Lys His Leu
Lys Pro Leu Ala Lys Phe Leu Asn Asp Val 35 40 45 Lys Thr Leu Tyr
Glu 50 <210> SEQ ID NO 27 <211> LENGTH: 53 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
27 Thr Pro Met Val Glu Ile Gln His Gly Phe Gln His Leu Ile Cys Ser
1 5 10 15 Leu Asn Phe Pro Lys Lys Glu Leu Glu Leu Gln Ile Gly Asn
Ala Leu 20 25 30 Phe Ile Gly Lys His Leu Lys Pro Leu Ala Lys Phe
Leu Asn Asp Val 35 40 45 Lys Thr Leu Tyr Glu 50 <210> SEQ ID
NO 28 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 28 Tyr Glu Ile Thr Thr
Ile His Asn Leu Phe Arg Lys Leu Thr His Arg 1 5 10 15 Leu Phe Arg
Arg Asn Phe Gly Tyr Thr Leu Arg Ser Val Asn Asp Leu 20 25 30 Tyr
Ile Gln Lys Gln Phe Pro Ile Leu Leu Asp Phe Lys Thr Lys Val 35 40
45 Arg Glu Tyr Tyr Phe 50 <210> SEQ ID NO 29 <211>
LENGTH: 49 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 29 Gly Val Gly Lys Ile Leu Lys Lys Ile Asn
Lys Ala Ile Val Ser Lys 1 5 10 15 Lys Asn Lys Asp Ile Val Thr Val
Ala Asn Ala Val Phe Val Lys Asn 20 25 30 Ala Ser Glu Ile Glu Val
Pro Phe Val Thr Arg Asn Lys Asp Val Phe 35 40 45 Gln <210>
SEQ ID NO 30 <211> LENGTH: 48 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 30 Ser Gly
Pro Cys Leu Pro His Leu Leu Ser Arg Leu Cys Gln Asp Leu 1 5 10 15
Gly Pro Gly Ala Phe Arg Leu Ala Ala Arg Met Tyr Leu Gln Lys Gly 20
25 30 Phe Pro Ile Lys Glu Asp Phe Leu Glu Gln Ser Glu Gln Leu Phe
Gly 35 40 45 <210> SEQ ID NO 31 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 31 Ser Gly Pro Cys Leu Pro His Leu Leu Ser
Arg Leu Cys Gln Asp Leu 1 5 10 15 Gly Pro Gly Ala Phe Arg Leu Ala
Ala Arg Met Tyr Leu Gln Lys Gly 20 25 30 Phe Pro Ile Lys Glu Asp
Phe Leu Glu Gln Ser Glu Gln Leu Phe Gly 35 40 45 <210> SEQ ID
NO 32 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 32 Thr Pro Glu Ala Lys
Ile His Glu Cys Phe Gln Gln Val Leu Gln Ala 1 5 10 15 Leu Ser Arg
Pro Asp Thr Arg Leu Gln Leu Thr Thr Gly Ser Ser Leu 20 25 30 Phe
Val Asn Lys Ser Met Lys Leu Val Asp Thr Phe Leu Glu Asp Thr 35 40
45 Lys Lys Leu Tyr His 50 <210> SEQ ID NO 33 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 33 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Leu Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 34 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 34 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Gln Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 35 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 35 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Gln Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 36 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 36 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Leu Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 37 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 37 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Gln Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 38 <211>
LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 38 Leu Ser Glu Ser Asp Val His Arg Gly Phe
Gln His Leu Leu His Thr 1 5 10 15 Leu Asn Leu Pro Gly His Gly Leu
Glu Thr Arg Val Gly Ser Ala Leu 20 25 30 Phe Leu Ser His Asn Leu
Lys Phe Leu Ala Lys Phe Leu Asn Asp Thr 35 40 45 Met Ala Val Tyr
Glu 50 <210> SEQ ID NO 39 <211> LENGTH: 49 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
39 Glu Val His Ser Arg Phe Gln Ser Leu Asn Ala Asp Ile Asn Lys Arg
1 5 10 15 Gly Ala Ser Tyr Ile Leu Lys Leu Ala Asn Arg Leu Tyr Gly
Glu Lys 20 25 30 Thr Tyr Asn Phe Leu Pro Glu Phe Leu Val Ser Thr
Gln Lys Thr Tyr 35 40 45 Gly <210> SEQ ID NO 40 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 40 Lys Asp Ile Pro Phe Gly Phe Gln Thr Val
Thr Ser Asp Val Asn Lys 1 5 10 15 Leu Ser Ser Phe Tyr Ser Leu Lys
Leu Ile Lys Arg Leu Tyr Val Asp 20 25 30 Lys Ser Leu Asn Leu Ser
Thr Glu Phe Ile Ser Ser Thr Lys Arg Pro 35 40 45 Tyr Ala 50
<210> SEQ ID NO 41 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 41 Glu
Phe Asn Leu Ser Asn Ser Glu Glu Ile His Ser Asp Phe Gln Thr 1 5 10
15 Leu Ile Ser Glu Ile Leu Lys Pro Asn Asp Asp Tyr Leu Leu Lys Thr
20 25 30 Ala Asn Ala Ile Tyr Gly Glu Lys Thr Tyr Ala Phe His Asn
Lys Tyr 35 40 45 Leu Glu Asp Met Lys Thr Tyr Phe Gly 50 55
<210> SEQ ID NO 42 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42 Glu
Glu Asp Ile His Arg Ala Phe Gln Ser Leu Leu Thr Glu Val Asn 1 5 10
15 Lys Ala Gly Thr Gln Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe Gly
20 25 30 Glu Lys Thr Cys Gln Phe Leu Ser Thr Phe Lys Glu Ser Cys
Leu Gln 35 40 45 Phe Tyr His 50 <210> SEQ ID NO 43
<211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 43 Glu Glu Asp Ile His Arg Ala
Phe Gln Ser Leu Leu Thr Glu Val Asn 1 5 10 15 Lys Ala Gly Thr Gln
Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe Gly 20 25 30 Glu Lys Thr
Cys Gln Phe Leu Ser Thr Phe Lys Glu Ser Cys Leu Gln 35 40 45 Phe
Tyr His 50 <210> SEQ ID NO 44 <211> LENGTH: 52
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 44 Asp Glu Glu Val His Ala Gly Leu Gly Glu
Leu Leu Arg Ser Leu Ser 1 5 10 15 Asn Ser Thr Ala Arg Asn Val Thr
Trp Lys Leu Gly Ser Arg Leu Tyr 20 25 30 Gly Pro Ser Ser Val Ser
Phe Ala Asp Asp Phe Val Arg Ser Ser Lys 35 40 45 Gln His Tyr Asn 50
<210> SEQ ID NO 45 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45 Pro
Glu Glu Glu Ile Gln Glu Gly Phe Trp Asp Leu Leu Ile Arg Leu 1 5 10
15 Arg Gly Gln Gly Pro Arg Leu Leu Leu Thr Met Asp Gln Arg Arg Phe
20 25 30 Ser Gly Leu Gly Ala Arg Ala Asn 35 40 <210> SEQ ID
NO 46 <211> LENGTH: 52 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 46 Pro Glu Ala Asp Ile
His Gln Gly Phe Arg Ser Leu Leu His Thr Leu 1 5 10 15 Ala Leu Pro
Ser Pro Lys Leu Glu Leu Lys Val Gly Asn Ser Leu Phe 20 25 30 Leu
Asp Lys Arg Leu Lys Pro Arg Gln His Tyr Leu Asp Ser Ile Lys 35 40
45 Glu Leu Tyr Gly 50 <210> SEQ ID NO 47 <211> LENGTH:
50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 47 Lys Asp Leu His Glu Gly Phe His Tyr Ile
Ile His Glu Leu Thr Gln 1 5 10 15 Lys Thr Gln Asp Leu Lys Leu Ser
Ile Gly Asn Thr Leu Phe Ile Asp 20 25 30 Gln Arg Leu Gln Pro Gln
Arg Lys Phe Leu Glu Asp Ala Lys Asn Phe 35 40 45 Tyr Ser 50
<210> SEQ ID NO 48 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 48 Ser
Pro Lys Cys Ser Gln Ala Gly Arg Ile His Ser Glu Phe Gly Val 1 5 10
15 Glu Phe Ser Gln Ile Asn Gln Pro Asp Ser Asn Cys Thr Leu Ser Ile
20 25 30 Ala Asn Arg Leu Tyr Gly Thr Lys Thr Met Ala Phe His Gln
Gln Tyr 35 40 45 Leu Ser Cys Ser Glu Lys Trp Tyr Gln 50 55
<210> SEQ ID NO 49 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 49 Gly
Ser Leu Asn Asn Glu Ser Gly Leu Val Ser Cys Tyr Phe Gly Gln 1 5 10
15 Leu Leu Ser Lys Leu Asp Arg Ile Lys Thr Asp Tyr Thr Leu Ser Ile
20 25 30 Ala Asn Arg Leu Tyr Gly Glu Gln Glu Phe Pro Ile Cys Gln
Glu Tyr 35 40 45 Leu Asp Gly Val Ile Gln Phe Tyr His 50 55
<210> SEQ ID NO 50 <211> LENGTH: 49 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 50 Glu
Glu Phe Ser Phe Leu Lys Glu Phe Ser Asn Met Val Thr Ala Lys 1 5 10
15 Glu Ser Gln Tyr Val Met Lys Ile Ala Asn Ser Leu Phe Val Gln Asn
20 25 30 Gly Phe His Val Asn Glu Glu Phe Leu Gln Met Met Lys Lys
Tyr Phe 35 40 45 Asn <210> SEQ ID NO 51 <211> LENGTH:
51 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 51 Lys Pro Gly Leu Leu Pro Ser Leu Phe Lys
Gly Leu Arg Glu Thr Leu 1 5 10 15 Ser Arg Asn Leu Glu Leu Gly Leu
Thr Gln Gly Ser Phe Ala Phe Ile 20 25 30 His Lys Asp Phe Asp Val
Lys Glu Thr Phe Phe Asn Leu Ser Lys Arg 35 40 45 Tyr Phe Asp 50
<210> SEQ ID NO 52 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 52 Lys
Pro Gly Leu Leu Pro Ser Leu Phe Lys Gly Leu Arg Glu Thr Leu 1 5 10
15 Ser Arg Asn Leu Glu Leu Gly Leu Thr Gln Gly Ser Phe Ala Phe Ile
20 25 30 His Lys Asp Phe Asp Val Lys Glu Thr Phe Phe Asn Leu Ser
Lys Arg 35 40 45 Tyr Phe Asp 50 <210> SEQ ID NO 53
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 53 Gly Gly Gly Gly Asp Ile His
Gln Gly Phe Gln Ser Leu Leu Thr Glu 1 5 10 15 Val Asn Lys Thr Gly
Thr Gln Tyr Leu Leu Arg Val Ala Asn Arg Leu 20 25 30 Phe Gly Glu
Lys Ser Cys Asp Phe Leu Ser Ser Phe Arg Asp Ser Cys 35 40 45 Gln
Lys Phe Tyr Gln 50 <210> SEQ ID NO 54 <211> LENGTH: 52
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 54 Lys Asp Gly Asp Ile His Arg Gly Phe Gln
Ser Leu Leu Ser Glu Val 1 5 10 15 Asn Arg Thr Gly Thr Gln Tyr Leu
Leu Arg Thr Ala Asn Arg Leu Phe 20 25 30 Gly Glu Lys Thr Cys Asp
Phe Leu Pro Asp Phe Lys Glu Tyr Cys Gln 35 40 45 Lys Phe Tyr Gln 50
<210> SEQ ID NO 55 <211> LENGTH: 52 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 55 Lys
Asp Gly Asp Ile His Arg Gly Phe Gln Ser Leu Leu Ser Glu Val 1 5 10
15 Asn Arg Thr Gly Thr Gln Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe
20 25 30 Gly Glu Lys Thr Cys Asp Phe Leu Pro Asp Phe Lys Glu Tyr
Cys Gln 35 40 45 Lys Phe Tyr Gln 50 <210> SEQ ID NO 56
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 56 Val Pro Tyr Gln Gly Asn Ala
Thr Ala Leu Phe Ile Leu Pro Ser Glu 1 5 10 15 Gly Lys Met Gln Gln
Val Glu Asn Gly Leu Ser Glu Lys Thr Leu Arg 20 25 30 Lys Trp Leu
Lys Met Phe Lys Lys Arg Gln Leu Glu Leu Tyr 35 40 45 <210>
SEQ ID NO 57 <211> LENGTH: 47 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 57 Leu Lys
Tyr Thr Gly Asn Ala Ser Ala Leu Phe Ile Leu Pro Asp Gln 1 5 10 15
Asp Lys Met Glu Glu Val Glu Ala Met Leu Leu Pro Glu Thr Leu Lys 20
25 30 Arg Trp Arg Asp Ser Leu Glu Phe Arg Glu Ile Gly Glu Leu Tyr
35 40 45 <210> SEQ ID NO 58 <211> LENGTH: 46
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 58 Met Asp Tyr Lys Gly Asp Ala Val Ala Phe
Phe Val Leu Pro Ser Lys 1 5 10 15 Gly Lys Met Arg Gln Leu Glu Gln
Ala Leu Ser Ala Arg Thr Leu Arg 20 25 30 Lys Trp Ser His Ser Leu
Gln Lys Arg Trp Ile Glu Val Phe 35 40 45 <210> SEQ ID NO 59
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 59 Met Asp Tyr Lys Gly Asp Ala
Val Ala Phe Phe Val Leu Pro Ser Lys 1 5 10 15 Gly Lys Met Arg Gln
Leu Glu Gln Ala Leu Ser Ala Arg Thr Leu Arg 20 25 30 Lys Trp Ser
His Ser Leu Gln Lys Arg Trp Ile Glu Val Phe 35 40 45 <210>
SEQ ID NO 60 <211> LENGTH: 50 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 60 Ile Pro
Tyr Lys Asn Asn Asp Leu Ser Met Phe Val Leu Leu Pro Asn 1 5 10 15
Asp Ile Asp Gly Leu Glu Lys Ile Ile Asp Lys Ile Ser Pro Glu Lys 20
25 30 Leu Val Glu Trp Thr Ser Pro Gly His Met Glu Glu Arg Lys Val
Asn 35 40 45 Leu His 50 <210> SEQ ID NO 61 <211>
LENGTH: 47 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 61 Leu Arg Tyr Asn Gly Gly Ile Asn Met Tyr
Val Leu Leu Pro Glu Asn 1 5 10 15 Asp Leu Ser Glu Ile Glu Asn Lys
Leu Thr Phe Gln Asn Leu Met Glu 20 25 30 Trp Thr Asn Pro Arg Arg
Met Thr Ser Lys Tyr Val Glu Val Phe 35 40 45 <210> SEQ ID NO
62 <211> LENGTH: 48 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 62 Leu Ser Tyr Lys Gly
Asp Glu Phe Ser Leu Ile Ile Ile Leu Pro Ala 1 5 10 15 Glu Gly Met
Asp Ile Glu Glu Val Glu Lys Leu Ile Thr Ala Gln Gln 20 25 30 Ile
Leu Lys Trp Leu Ser Glu Met Gln Glu Glu Glu Val Glu Ile Ser 35 40
45 <210> SEQ ID NO 63 <211> LENGTH: 48 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
63 Leu Pro Phe Lys Gly Asp Asp Ile Thr Met Val Leu Ile Leu Pro Lys
1 5 10 15 Pro Glu Lys Ser Leu Ala Lys Val Glu Lys Glu Leu Thr Pro
Glu Val 20 25 30 Leu Gln Glu Trp Leu Asp Glu Leu Glu Glu Met Met
Leu Val Val His 35 40 45 <210> SEQ ID NO 64 <211>
LENGTH: 48 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 64 Leu Pro Phe Lys Gly Asp Asp Ile Thr Met
Val Leu Ile Leu Pro Lys 1 5 10 15 Pro Glu Lys Ser Leu Ala Lys Val
Glu Lys Glu Leu Thr Pro Glu Val 20 25 30 Leu Gln Glu Trp Leu Asp
Glu Leu Glu Glu Met Met Leu Val Val His 35 40 45 <210> SEQ ID
NO 65 <211> LENGTH: 46 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 65 Met Lys Tyr Leu Gly
Asn Ala Thr Ala Ile Phe Phe Leu Pro Asp Glu 1 5 10 15 Gly Lys Leu
Gln His Leu Glu Asn Glu Leu Thr His Asp Ile Ile Thr 20 25 30 Lys
Phe Leu Glu Asn Glu Asp Arg Arg Ser Ala Ser Leu His 35 40 45
<210> SEQ ID NO 66 <211> LENGTH: 53 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 66 Leu
Pro Tyr Ala Gly Asp Val Ser Met Phe Leu Leu Leu Pro Asp Glu 1 5 10
15 Ile Ala Asp Val Ser Thr Gly Leu Glu Leu Leu Glu Ser Glu Ile Thr
20 25 30 Tyr Asp Lys Leu Asn Lys Trp Thr Ser Lys Asp Lys Met Ala
Glu Asp 35 40 45 Glu Val Glu Val Tyr 50 <210> SEQ ID NO 67
<211> LENGTH: 49 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 67 Leu Pro Tyr His Gly Asp Thr
Leu Ser Met Phe Ile Ala Ala Pro Tyr 1 5 10 15 Glu Lys Glu Val Pro
Leu Ser Ala Leu Thr Asn Ile Leu Ser Ala Gln 20 25 30 Leu Ile Ser
His Trp Lys Gly Asn Met Thr Arg Leu Pro Arg Leu Leu 35 40 45 Val
<210> SEQ ID NO 68 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 68 Leu
Gln Leu Ser His Asn Leu Ser Leu Val Ile Leu Val Pro Gln Asn 1 5 10
15 Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser Pro Ser Val
20 25 30 Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe Gln
Pro Thr 35 40 45 Leu Leu Thr 50 <210> SEQ ID NO 69
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 69 Met Asp Tyr Ser Lys Asn Ala
Leu Ala Leu Phe Val Leu Pro Lys Glu 1 5 10 15 Gly Gln Met Glu Ser
Val Glu Ala Ala Met Ser Ser Lys Thr Leu Lys 20 25 30 Lys Trp Asn
Arg Leu Leu Gln Lys Gly Trp Val Asp Leu Phe 35 40 45 <210>
SEQ ID NO 70 <211> LENGTH: 46 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 70 Met Asp
Tyr Ser Lys Asn Ala Leu Ala Leu Phe Val Leu Pro Lys Glu 1 5 10 15
Gly Gln Met Glu Ser Val Glu Ala Ala Met Ser Ser Lys Thr Leu Lys 20
25 30 Lys Trp Asn Arg Leu Leu Gln Lys Gly Trp Val Asp Leu Phe 35 40
45 <210> SEQ ID NO 71 <211> LENGTH: 47 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
71 Leu Glu Tyr Val Gly Gly Ile Ser Met Leu Ile Val Val Pro His Lys
1 5 10 15 Met Ser Gly Met Lys Thr Leu Glu Ala Gln Leu Thr Pro Gly
Val Val 20 25 30 Glu Arg Trp Gln Lys Ser Met Thr Asn Arg Thr Arg
Glu Val Leu 35 40 45 <210> SEQ ID NO 72 <211> LENGTH:
49 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 Leu Pro Tyr His Gly Glu Ser Ile Ser Met
Leu Ile Ala Leu Pro Thr 1 5 10 15 Glu Ser Ser Thr Pro Leu Ser Ala
Ile Ile Pro His Ile Ser Thr Lys 20 25 30 Thr Ile Asp Ser Trp Met
Ser Ile Met Val Pro Lys Arg Val Gln Val 35 40 45 Ile <210>
SEQ ID NO 73 <211> LENGTH: 46 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 73 Phe Pro
Phe Lys Asn Asn Met Ser Phe Val Val Leu Val Pro Thr His 1 5 10 15
Phe Glu Trp Asn Val Ser Gln Val Leu Ala Asn Leu Ser Trp Asp Thr 20
25 30 Leu His Pro Pro Leu Val Trp Glu Arg Pro Thr Lys Val Arg 35 40
45 <210> SEQ ID NO 74 <211> LENGTH: 46 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
74 Phe Pro Phe Lys Asn Asn Met Ser Phe Val Val Leu Val Pro Thr His
1 5 10 15 Phe Glu Trp Asn Val Ser Gln Val Leu Ala Asn Leu Ser Trp
Asp Thr 20 25 30 Leu His Pro Pro Leu Val Trp Glu Arg Pro Thr Lys
Val Arg 35 40 45 <210> SEQ ID NO 75 <211> LENGTH: 46
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 75 Gln His Tyr Val Gly Asn Ala Thr Ala Phe
Phe Ile Leu Pro Asp Pro 1 5 10 15 Lys Lys Met Trp Gln Leu Glu Glu
Lys Leu Thr Tyr Ser His Leu Glu 20 25 30 Asn Ile Gln Arg Ala Phe
Asp Ile Arg Ser Ile Asn Leu His 35 40 45 <210> SEQ ID NO 76
<211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 76 Ile Pro Tyr Lys Gly Lys Asp
Leu Ser Met Ile Val Leu Leu Pro Asn 1 5 10 15 Glu Ile Asp Gly Leu
Gln Lys Leu Glu Glu Lys Leu Thr Ala Glu Lys 20 25 30 Leu Met Glu
Trp Thr Ser Leu Gln Asn Met Arg Glu Thr Arg Val Asp 35 40 45 Leu
His 50 <210> SEQ ID NO 77 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
77 Ile Pro Tyr Lys Gly Lys Asp Leu Ser Met Ile Val Leu Leu Pro Asn
1 5 10 15 Glu Ile Asp Gly Leu Gln Lys Leu Glu Glu Lys Leu Thr Ala
Glu Lys 20 25 30 Leu Met Glu Trp Thr Ser Leu Gln Asn Met Arg Glu
Thr Cys Val Asp 35 40 45 Leu His 50 <210> SEQ ID NO 78
<211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 78 Ile Pro Tyr Lys Gly Lys Asp
Leu Ser Met Ile Val Leu Leu Pro Asn 1 5 10 15 Glu Ile Asp Gly Leu
Gln Lys Leu Glu Glu Lys Leu Thr Ala Glu Lys 20 25 30 Leu Met Glu
Trp Thr Ser Leu Gln Asn Met Arg Glu Thr Arg Val Asp 35 40 45 Leu
His 50 <210> SEQ ID NO 79 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
79 Ile Pro Tyr Lys Gly Lys Asp Leu Ser Met Ile Val Leu Leu Pro Asn
1 5 10 15 Glu Ile Asp Gly Leu Gln Lys Leu Glu Glu Lys Leu Thr Ala
Glu Lys 20 25 30 Leu Met Glu Trp Thr Ser Leu Gln Asn Met Arg Glu
Thr Cys Val Asp 35 40 45 Leu His 50 <210> SEQ ID NO 80
<211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 80 Ile Pro Tyr Lys Gly Lys Asp
Leu Ser Met Ile Val Leu Leu Pro Asn 1 5 10 15 Glu Ile Asp Gly Leu
Gln Lys Leu Glu Glu Lys Leu Thr Ala Glu Lys 20 25 30 Leu Met Glu
Trp Thr Ser Leu Gln Asn Met Arg Glu Thr Cys Val Asp 35 40 45 Leu
His 50 <210> SEQ ID NO 81 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
81 Met Asp Tyr Lys Gly Asp Ala Thr Val Phe Phe Ile Leu Pro Asn Gln
1 5 10 15 Gly Lys Met Arg Glu Ile Glu Glu Val Leu Thr Pro Glu Met
Leu Met 20 25 30 Arg Trp Asn Asn Leu Leu Arg Lys Arg Asn Phe Tyr
Lys Lys Leu Glu 35 40 45 Leu His 50 <210> SEQ ID NO 82
<211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 82 Leu Pro Tyr Gln Gly Glu Glu
Leu Ser Met Val Ile Leu Leu Pro Asp 1 5 10 15 Asp Ile Glu Asp Glu
Ser Thr Gly Leu Lys Lys Ile Glu Glu Gln Leu 20 25 30 Thr Leu Glu
Lys Leu His Glu Trp Thr Lys Pro Glu Asn Leu Asp Phe 35 40 45 Ile
Glu Val Asn Val Ser 50 <210> SEQ ID NO 83 <211> LENGTH:
54 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 83 Leu Pro Phe Gln Asn Lys His Leu Ser Met
Phe Ile Leu Leu Pro Lys 1 5 10 15 Asp Val Glu Asp Glu Ser Thr Gly
Leu Glu Lys Ile Glu Lys Gln Leu 20 25 30 Asn Ser Glu Ser Leu Ser
Gln Trp Thr Asn Pro Ser Thr Met Ala Asn 35 40 45 Ala Lys Val Lys
Leu Ser 50 <210> SEQ ID NO 84 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 84 Leu Tyr Tyr Lys Ser Arg Asp Leu Ser Leu
Leu Ile Leu Leu Pro Glu 1 5 10 15 Asp Ile Asn Gly Leu Glu Gln Leu
Glu Lys Ala Ile Thr Tyr Glu Lys 20 25 30 Leu Asn Glu Trp Thr Ser
Ala Asp Met Met Glu Leu Tyr Glu Val Gln 35 40 45 Leu His 50
<210> SEQ ID NO 85 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 85 Leu
Pro Tyr Ala Arg Lys Glu Leu Ser Leu Leu Val Leu Leu Pro Asp 1 5 10
15 Asp Gly Val Glu Leu Ser Thr Val Glu Lys Ser Leu Thr Phe Glu Lys
20 25 30 Leu Thr Ala Trp Thr Lys Pro Asp Cys Met Lys Ser Thr Glu
Val Glu 35 40 45 Val Leu 50 <210> SEQ ID NO 86 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 86 Leu Pro Tyr Ala Arg Lys Glu Leu Ser Leu
Leu Val Leu Leu Pro Asp 1 5 10 15 Asp Gly Val Glu Leu Ser Thr Val
Glu Lys Ser Leu Thr Phe Glu Lys 20 25 30 Leu Thr Ala Trp Thr Lys
Pro Asp Cys Met Lys Ser Thr Glu Val Glu 35 40 45 Val Leu 50
<210> SEQ ID NO 87 <211> LENGTH: 48 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 87 Met
Pro Leu Ala His Lys Leu Ser Ser Leu Ile Ile Leu Met Pro His 1 5 10
15 His Val Glu Pro Leu Glu Arg Leu Glu Lys Leu Leu Thr Lys Glu Gln
20 25 30 Leu Lys Ile Trp Met Gly Lys Met Gln Lys Lys Ala Val Ala
Ile Ser 35 40 45 <210> SEQ ID NO 88 <211> LENGTH: 46
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 88 Met Asp His Ala Gly Asn Thr Thr Thr Phe
Phe Ile Phe Pro Asn Arg 1 5 10 15 Gly Lys Met Arg His Leu Glu Asp
Ala Leu Leu Pro Glu Thr Leu Ile 20 25 30 Lys Trp Asp Ser Leu Leu
Arg Thr Arg Glu Leu Asp Phe His 35 40 45 <210> SEQ ID NO 89
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 89 Ile Glu Tyr Arg Gly Asn Ala
Leu Ala Leu Leu Val Leu Pro Asp Pro 1 5 10 15 Gly Lys Met Lys Gln
Val Glu Ala Ala Leu Gln Pro Gln Thr Leu Arg 20 25 30 Lys Trp Gly
Gln Leu Leu Leu Pro Ser Leu Leu Asp Leu His 35 40 45 <210>
SEQ ID NO 90 <211> LENGTH: 46 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 90 Ile Pro
Tyr Gln Lys Asn Ile Thr Ala Ile Phe Ile Leu Pro Asp Glu 1 5 10 15
Gly Lys Leu Lys His Leu Glu Lys Gly Leu Gln Val Asp Thr Phe Ser 20
25 30 Arg Trp Lys Thr Leu Leu Ser Arg Arg Val Val Asp Val Ser 35 40
45 <210> SEQ ID NO 91 <211> LENGTH: 50 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
91 Leu Pro Tyr Val Asn Asn Lys Leu Ser Met Ile Ile Leu Leu Pro Val
1 5 10 15 Gly Ile Ala Asn Leu Lys Gln Ile Glu Lys Gln Leu Asn Ser
Gly Thr 20 25 30 Phe His Glu Trp Thr Ser Ser Ser Asn Met Met Glu
Arg Glu Val Glu 35 40 45 Val His 50 <210> SEQ ID NO 92
<211> LENGTH: 54 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 92 Met Arg Tyr Thr Lys Gly Lys
Leu Ser Met Phe Val Leu Leu Pro Ser 1 5 10 15 His Ser Lys Asp Asn
Leu Lys Gly Leu Glu Glu Leu Glu Arg Lys Ile 20 25 30 Thr Tyr Glu
Lys Met Val Ala Trp Ser Ser Ser Glu Asn Met Ser Glu 35 40 45 Glu
Ser Val Val Leu Ser 50 <210> SEQ ID NO 93 <211> LENGTH:
54 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 93 Met Arg Tyr Thr Lys Gly Lys Leu Ser Met
Phe Val Leu Leu Pro Ser 1 5 10 15 His Ser Lys Asp Asn Leu Lys Gly
Leu Glu Glu Leu Glu Arg Lys Ile 20 25 30 Thr Tyr Glu Lys Met Val
Ala Trp Ser Ser Ser Glu Asn Met Ser Glu 35 40 45 Glu Ser Val Val
Leu Ser 50 <210> SEQ ID NO 94 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 94 Ile Pro Tyr Glu Gly Asp Glu Ile Ser Met
Met Leu Val Leu Ser Arg 1 5 10 15 Gln Glu Val Pro Leu Ala Thr Leu
Glu Pro Leu Val Lys Ala Gln Leu 20 25 30 Val Glu Glu Trp Ala Asn
Ser Val Lys Lys Gln Lys Val Glu Val Tyr 35 40 45 <210> SEQ ID
NO 95 <211> LENGTH: 47 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 95 Leu Pro Tyr Gln Gly
Asn Ala Thr Met Leu Val Val Leu Met Glu Lys 1 5 10 15 Met Gly Asp
His Leu Ala Leu Glu Asp Tyr Leu Thr Thr Asp Leu Val 20 25 30 Glu
Thr Trp Leu Arg Asn Met Lys Thr Arg Asn Met Glu Val Phe 35 40 45
<210> SEQ ID NO 96 <211> LENGTH: 47 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 96 Leu
Pro Tyr Gln Gly Asn Ala Thr Met Leu Val Val Leu Met Glu Lys 1 5 10
15 Met Gly Asp His Leu Ala Leu Glu Asp Tyr Leu Thr Thr Asp Leu Val
20 25 30 Glu Thr Trp Leu Arg Asn Met Lys Thr Arg Asn Met Glu Val
Phe 35 40 45 <210> SEQ ID NO 97 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 97 Leu Pro Tyr Val Gly Lys Glu Leu Asn Met
Ile Ile Met Leu Pro Asp 1 5 10 15 Glu Thr Thr Asp Leu Arg Thr Val
Glu Lys Glu Leu Thr Tyr Glu Lys 20 25 30 Phe Val Glu Trp Thr Arg
Leu Asp Met Met Asp Glu Glu Glu Val Glu 35 40 45 Val Ser 50
<210> SEQ ID NO 98 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 Leu
Pro Tyr Val Gly Lys Glu Leu Asn Met Ile Ile Met Leu Pro Asp 1 5 10
15 Glu Thr Thr Asp Leu Arg Thr Val Glu Lys Glu Leu Thr Tyr Glu Lys
20 25 30 Phe Val Glu Trp Thr Arg Leu Asp Met Met Asp Glu Glu Glu
Val Glu 35 40 45 Val Ser 50 <210> SEQ ID NO 99 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 99 Leu Pro Tyr Val Glu Glu Glu Leu Ser Met
Val Ile Leu Leu Pro Asp 1 5 10 15 Asp Asn Thr Asp Leu Ala Val Val
Glu Lys Ala Leu Thr Tyr Glu Lys 20 25 30 Phe Lys Ala Trp Thr Asn
Ser Glu Lys Leu Thr Lys Ser Lys Val Gln 35 40 45 Val Phe 50
<210> SEQ ID NO 100 <211> LENGTH: 25 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 100
Leu Pro Tyr Val Glu Glu Glu Leu Ser Met Val Ile Leu Leu Pro Asp 1 5
10 15 Asp Asn Thr Asp Leu Ala Val Lys Glu 20 25 <210> SEQ ID
NO 101 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: LL-37 control peptide <400> SEQUENCE: 101
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5
10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu
Val 20 25 30 Pro Arg Thr Glu Ser 35 <210> SEQ ID NO 102
<211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: GGL27(S) control peptide <400> SEQUENCE: 102 Gly
Gly Leu Ile Ser Thr Ser Ser Ser Ser Ser Ser Gln Arg Val Lys 1 5 10
15 Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 20 25 <210>
SEQ ID NO 103 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: DSE25 control peptide <400>
SEQUENCE: 103 Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp
Thr Glu Leu 1 5 10 15 Pro Pro Leu Lys Leu Met His Ser Phe 20 25
<210> SEQ ID NO 104 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C-terminal TFPI sequence <400>
SEQUENCE: 104 Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 1
5 10 <210> SEQ ID NO 105 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: A kininogen-derived
antimicrobial peptide <400> SEQUENCE: 105 His Lys His Gly His
Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 1 5 10 15 Asn Gly Lys
His 20 <210> SEQ ID NO 106 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Heparin-binding growth
factor derived peptide <400> SEQUENCE: 106 Gly Lys Arg Lys
Lys Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro 1 5 10 15 Cys Leu
Arg Lys Tyr Lys 20 <210> SEQ ID NO 107 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 107 Gly Gly Leu Ile Lys Thr Lys Arg Lys Lys
Lys Lys Gln Pro Val Lys 1 5 10 15 Ile Thr Tyr Val Glu Thr Phe Val
Lys Lys Thr 20 25 <210> SEQ ID NO 108 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Canis lupus <400>
SEQUENCE: 108 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln
Thr Val Lys 1 5 10 15 Ile Val Tyr Glu Lys Ile Phe Val Lys Lys Leu
20 25 <210> SEQ ID NO 109 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Danio rerio <400> SEQUENCE:
109 Arg Lys Gln Ile Arg Ile Lys Thr Arg Asn Ser Asn Ile Leu Phe Arg
1 5 10 15 Ser Val <210> SEQ ID NO 110 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 110 Gly Val Val Lys Ile Gln Arg Arg Lys Ala
Pro Phe Val Lys Val Val 1 5 10 15 Tyr Glu Ser Ile Asn 20
<210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
111 Arg Ala Lys Thr Gln Arg Arg Arg Lys Ser Phe Val Lys Val Met Tyr
1 5 10 15 Glu Asn Ile His 20 <210> SEQ ID NO 112 <211>
LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 112 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg
Lys Lys Gln Arg Val Lys 1 5 10 15 Ile Ala Tyr Glu Glu Ile Phe Val
Lys Asn Met 20 25 <210> SEQ ID NO 113 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Pongo abelii
<400> SEQUENCE: 113 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg
Lys Lys Gln Arg Val Lys 1 5 10 15 Ile Ala Tyr Glu Glu Ile Phe Val
Lys Asn Met 20 25 <210> SEQ ID NO 114 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Sus scrofa <400>
SEQUENCE: 114 Asp Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln
Pro Val Lys 1 5 10 15 Ile Val Tyr Glu Glu Ile Phe Val Lys Lys Ile
20 25 <210> SEQ ID NO 115 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE:
115 Glu Gly Leu Ile Lys Thr Lys Lys Lys Lys Lys 1 5 10
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 115
<210> SEQ ID NO 1 <211> LENGTH: 21 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1 Phe
Phe Phe Ala Lys Leu Asn Cys Arg Leu Tyr Arg Lys Ala Asn Lys 1 5 10
15 Ser Ser Lys Leu Val 20 <210> SEQ ID NO 2 <211>
LENGTH: 22 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Ala Lys Leu Asn Cys Arg Leu Tyr Arg Lys Ala
Asn Lys Ser Ser Lys 1 5 10 15 Leu Val Ser Ala Asn Arg 20
<210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 3 Ser
Glu Lys Thr Leu Arg Lys Trp Leu Lys Met Phe Lys Lys Arg Gln 1 5 10
15 Leu Glu Leu Tyr 20 <210> SEQ ID NO 4 <211> LENGTH:
27 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 4 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys
Lys Gln Arg Val Lys 1 5 10 15 Ile Ala Tyr Glu Glu Ile Phe Val Lys
Asn Met 20 25 <210> SEQ ID NO 5 <211> LENGTH: 17
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 5 Leu Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln
Arg Val Lys Ile Ala 1 5 10 15 Tyr <210> SEQ ID NO 6
<211> LENGTH: 22 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 6 Thr Lys Arg Lys Arg Lys Lys
Gln Arg Val Lys Ile Ala Tyr Glu Glu 1 5 10 15 Ile Phe Val Lys Asn
Met 20 <210> SEQ ID NO 7 <211> LENGTH: 25 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
7 Ala Leu Lys Lys Lys Lys Lys Met Pro Lys Leu Arg Phe Ala Ser Arg 1
5 10 15 Ile Arg Lys Ile Arg Lys Lys Gln Phe 20 25 <210> SEQ
ID NO 8 <211> LENGTH: 34 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 8 Glu Asp Cys Lys Arg
Ala Cys Ala Lys Ala Leu Lys Lys Lys Lys Lys 1 5 10 15 Met Pro Lys
Leu Arg Phe Ala Ser Arg Ile Arg Lys Ile Arg Lys Lys 20 25 30 Gln
Phe <210> SEQ ID NO 9 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (1)..(1)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (2)..(2) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (3)..(3)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(4)..(4) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile,
Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (5)..(5) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (6)..(6)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (7)..(7) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (8)..(8)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(9)..(9) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile,
Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (10)..(10) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (11)..(11)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (12)..(12) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (13)..(13)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(14)..(14) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu,
Ile, Phe, Thr, Val, Tyr or Trp <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (15)..(15) <223>
OTHER INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(11)...(15) <223> OTHER INFORMATION: All amino acids are
either present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (16)..(16) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (17)..(17) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (18)..(18) <223> OTHER INFORMATION: Xaa
is His, Arg or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (19)..(19) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (20)..(20) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (16)...(20) <223> OTHER INFORMATION:
All amino acids are either present or absent <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr,
Val, Tyr or Trp <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (22)..(22) <223> OTHER
INFORMATION: Xaa is His, Arg or Lys <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (23)..(23)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(24)..(24) <223> OTHER INFORMATION: Xaa is Gly, Pro, Leu,
Ile, Phe, Thr, Val, Tyr or Trp <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION: (25)..(25)
<223> OTHER INFORMATION: Xaa is His, Arg or Lys <220>
FEATURE: <221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(21)...(25) <223> OTHER INFORMATION: All amino acids are
either present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (26)..(26) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (27)..(27) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (28)..(28) <223> OTHER INFORMATION: Xaa
is His, Arg or Lys <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (29)..(29) <223> OTHER
INFORMATION: Xaa is Gly, Pro, Leu, Ile, Phe, Thr, Val, Tyr or Trp
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (30)..(30) <223> OTHER INFORMATION: Xaa is His, Arg
or Lys <220> FEATURE: <221> NAME/KEY: MISC_FEATURE
<222> LOCATION: (26)...(30) <223> OTHER INFORMATION:
All amino acids are either present or absent <400> SEQUENCE:
9 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1
5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20
25 30 <210> SEQ ID NO 10 <211> LENGTH: 30 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: MISC_FEATURE <222> LOCATION:
(11)...(15) <223> OTHER INFORMATION: All amino acids are
either present or absent <220> FEATURE: <221> NAME/KEY:
MISC_FEATURE <222> LOCATION: (16)...(20) <223> OTHER
INFORMATION: All amino acids are either present or absent
<220> FEATURE: <221> NAME/KEY: MISC_FEATURE <222>
LOCATION: (21)...(25) <223> OTHER INFORMATION: All amino
acids are either present or absent <220> FEATURE: <221>
NAME/KEY: MISC_FEATURE <222> LOCATION: (26)...(30)
<223> OTHER INFORMATION: All amino acids are either present
or absent <400> SEQUENCE: 10 Gly His His Pro His Gly His His
Pro His Gly His His Pro His Gly 1 5 10 15 His His Pro His Gly His
His Pro His Gly His His Pro His 20 25 30 <210> SEQ ID NO 11
<211> LENGTH: 25 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 11 Gly His His Pro His Gly His
His Pro His Gly His His Pro His Gly 1 5 10 15 His His Pro His Gly
His His Pro His 20 25 <210> SEQ ID NO 12 <211> LENGTH:
54 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Lys Thr Ser Asp Gln Ile His Phe Phe Phe
Ala Lys Leu Asn Cys Arg 1 5 10 15 Leu Tyr Arg Lys Ala Asn Lys Ser
Ser Lys Leu Val Ser Ala Asn Arg 20 25 30 Leu Phe Gly Asp Lys Ser
Leu Thr Phe Asn Glu Thr Tyr Gln Asp Ile 35 40 45 Ser Glu Leu Val
Tyr Gly 50 <210> SEQ ID NO 13 <211> LENGTH: 53
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13 Thr Ser Glu Ala Glu Ile His Gln Ser Phe
Gln His Leu Leu Arg Thr 1 5 10 15 Leu Asn Gln Ser Ser Asp Glu Leu
Gln Leu Ser Met Gly Asn Ala Met 20 25 30 Phe Val Lys Glu Gln Leu
Ser Leu Leu Asp Arg Phe Thr Glu Asp Ala 35 40 45 Lys Arg Leu Tyr
Gly 50 <210> SEQ ID NO 14 <211> LENGTH: 53 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
14 Thr Pro Glu Ser Ala Ile His Gln Gly Phe Gln His Leu Val His Ser
1 5 10 15 Leu Thr Val Pro Ser Lys Asp Leu Thr Leu Lys Met Gly Ser
Ala Leu 20 25 30 Phe Val Lys Lys Glu Leu Gln Leu Gln Ala Asn Phe
Leu Gly Asn Val 35 40 45 Lys Arg Leu Tyr Glu 50 <210> SEQ ID
NO 15 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 15 Thr Pro Glu Ser Ala
Ile His Gln Gly Phe Gln His Leu Val His Ser 1 5 10 15 Leu Thr Val
Pro Ser Lys Asp Leu Thr Leu Lys Met Gly Ser Ala Leu 20 25 30 Phe
Val Lys Lys Glu Leu Gln Leu Gln Ala Asn Phe Leu Gly Asn Val 35 40
45 Lys Arg Leu Tyr Glu 50 <210> SEQ ID NO 16 <211>
LENGTH: 58 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 16 Glu Lys Glu Val Ile Glu Asn Thr Glu Ala
Val His Gln Gln Phe Gln 1 5 10 15 Lys Phe Leu Thr Glu Ile Ser Lys
Leu Thr Asn Asp Tyr Glu Leu Asn 20 25 30 Ile Thr Asn Arg Leu Phe
Gly Glu Lys Thr Tyr Leu Phe Leu Gln Lys 35 40 45 Tyr Leu Asp Tyr
Val Glu Lys Tyr Tyr His 50 55 <210> SEQ ID NO 17 <211>
LENGTH: 56 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 17 Asn Ser Ser Asn Ser Gln Ser Gly Leu Gln
Ser Gln Leu Lys Arg Val 1 5 10 15 Phe Ser Asp Ile Asn Ala Ser His
Lys Asp Tyr Asp Leu Ser Ile Val 20 25 30 Asn Gly Leu Phe Ala Glu
Lys Val Tyr Gly Phe His Lys Asp Tyr Ile 35 40 45 Glu Cys Ala Glu
Lys Leu Tyr Asp 50 55 <210> SEQ ID NO 18 <211> LENGTH:
52 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 18 Ser Ala Gly Glu Glu Phe Phe Val Leu Lys
Ser Phe Phe Ser Ala Ile 1 5 10 15 Ser Glu Lys Lys Gln Glu Phe Thr
Phe Asn Leu Ala Asn Ala Leu Tyr 20 25 30 Leu Gln Glu Gly Phe Thr
Val Lys Glu Gln Tyr Leu His Gly Asn Lys 35 40 45 Glu Phe Phe Gln 50
<210> SEQ ID NO 19 <211> LENGTH: 41 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 19 Lys
Thr Ser Asp Gln Ile His Phe Phe Phe Ala Lys Leu Asn Cys Arg 1 5 10
15 Leu Tyr Arg Lys Ala Asn Lys Ser Ser Lys Leu Val Ser Ala Asn Arg
20 25 30 Leu Phe Gly Asp Lys Ser Leu Thr Phe 35 40 <210> SEQ
ID NO 20 <211> LENGTH: 54
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 20 Lys Thr Ser Asp Gln Ile His Phe Phe Phe
Ala Lys Leu Asn Cys Arg 1 5 10 15 Leu Tyr Arg Lys Ala Asn Lys Ser
Ser Lys Leu Val Ser Ala Asn Arg 20 25 30 Leu Phe Gly Asp Lys Ser
Leu Thr Phe Asn Glu Thr Tyr Gln Asp Ile 35 40 45 Ser Glu Leu Val
Tyr Gly 50 <210> SEQ ID NO 21 <211> LENGTH: 53
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 21 Ile Pro Glu Ala Gln Ile His Glu Gly Phe
Gln Glu Leu Leu Arg Thr 1 5 10 15 Leu Asn Gln Pro Asp Ser Gln Leu
Gln Leu Thr Thr Gly Asn Gly Leu 20 25 30 Phe Leu Ser Glu Gly Leu
Lys Leu Val Asp Lys Phe Leu Glu Asp Val 35 40 45 Lys Lys Leu Tyr
His 50 <210> SEQ ID NO 22 <211> LENGTH: 57 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
22 Ile Leu Gln Ala Gln Ala Ala Asp Lys Ile His Ser Ser Phe Arg Ser
1 5 10 15 Leu Ser Ser Ala Ile Asn Ala Ser Thr Gly Asn Tyr Leu Leu
Glu Ser 20 25 30 Val Asn Lys Leu Phe Gly Glu Lys Ser Ala Ser Phe
Arg Glu Glu Tyr 35 40 45 Ile Arg Leu Cys Gln Lys Tyr Tyr Ser 50 55
<210> SEQ ID NO 23 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 23 Asp
Lys Gly Met Ala Pro Ala Leu Arg His Leu Tyr Lys Glu Leu Met 1 5 10
15 Gly Pro Trp Asn Lys Asp Glu Ile Ser Thr Thr Asp Ala Ile Phe Val
20 25 30 Gln Arg Asp Leu Lys Leu Val Gln Gly Phe Met Pro His Phe
Phe Arg 35 40 45 Leu Phe Arg 50 <210> SEQ ID NO 24
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 24 Ser Ser Glu Lys Glu Leu His
Arg Gly Phe Gln Gln Leu Leu Gln Glu 1 5 10 15 Leu Asn Gln Pro Arg
Asp Gly Phe Gln Leu Ser Leu Gly Asn Ala Leu 20 25 30 Phe Thr Asp
Leu Val Val Asp Leu Gln Asp Thr Phe Val Ser Ala Met 35 40 45 Lys
Thr Leu Tyr Leu 50 <210> SEQ ID NO 25 <211> LENGTH: 42
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 25 Thr Cys Val His Gln Ala Leu Lys Gly Phe
Thr Thr Lys Gly Val Thr 1 5 10 15 Ser Val Ser Gln Ile Phe His Ser
Pro Asp Leu Ala Ile Arg Asp Thr 20 25 30 Phe Val Asn Ala Ser Arg
Thr Leu Tyr Ser 35 40 <210> SEQ ID NO 26 <211> LENGTH:
53 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 26 Thr Pro Met Val Glu Ile Gln His Gly Phe
Gln His Leu Ile Cys Ser 1 5 10 15 Leu Asn Phe Pro Lys Lys Glu Leu
Glu Leu Gln Ile Gly Asn Ala Leu 20 25 30 Phe Ile Gly Lys His Leu
Lys Pro Leu Ala Lys Phe Leu Asn Asp Val 35 40 45 Lys Thr Leu Tyr
Glu 50 <210> SEQ ID NO 27 <211> LENGTH: 53 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
27 Thr Pro Met Val Glu Ile Gln His Gly Phe Gln His Leu Ile Cys Ser
1 5 10 15 Leu Asn Phe Pro Lys Lys Glu Leu Glu Leu Gln Ile Gly Asn
Ala Leu 20 25 30 Phe Ile Gly Lys His Leu Lys Pro Leu Ala Lys Phe
Leu Asn Asp Val 35 40 45 Lys Thr Leu Tyr Glu 50 <210> SEQ ID
NO 28 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 28 Tyr Glu Ile Thr Thr
Ile His Asn Leu Phe Arg Lys Leu Thr His Arg 1 5 10 15 Leu Phe Arg
Arg Asn Phe Gly Tyr Thr Leu Arg Ser Val Asn Asp Leu 20 25 30 Tyr
Ile Gln Lys Gln Phe Pro Ile Leu Leu Asp Phe Lys Thr Lys Val 35 40
45 Arg Glu Tyr Tyr Phe 50 <210> SEQ ID NO 29 <211>
LENGTH: 49 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 29 Gly Val Gly Lys Ile Leu Lys Lys Ile Asn
Lys Ala Ile Val Ser Lys 1 5 10 15 Lys Asn Lys Asp Ile Val Thr Val
Ala Asn Ala Val Phe Val Lys Asn 20 25 30 Ala Ser Glu Ile Glu Val
Pro Phe Val Thr Arg Asn Lys Asp Val Phe 35 40 45 Gln <210>
SEQ ID NO 30 <211> LENGTH: 48 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 30 Ser Gly
Pro Cys Leu Pro His Leu Leu Ser Arg Leu Cys Gln Asp Leu 1 5 10 15
Gly Pro Gly Ala Phe Arg Leu Ala Ala Arg Met Tyr Leu Gln Lys Gly 20
25 30 Phe Pro Ile Lys Glu Asp Phe Leu Glu Gln Ser Glu Gln Leu Phe
Gly 35 40 45 <210> SEQ ID NO 31 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 31 Ser Gly Pro Cys Leu Pro His Leu Leu Ser
Arg Leu Cys Gln Asp Leu 1 5 10 15 Gly Pro Gly Ala Phe Arg Leu Ala
Ala Arg Met Tyr Leu Gln Lys Gly 20 25 30 Phe Pro Ile Lys Glu Asp
Phe Leu Glu Gln Ser Glu Gln Leu Phe Gly 35 40 45 <210> SEQ ID
NO 32 <211> LENGTH: 53 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 32 Thr Pro Glu Ala Lys
Ile His Glu Cys Phe Gln Gln Val Leu Gln Ala 1 5 10 15 Leu Ser Arg
Pro Asp Thr Arg Leu Gln Leu Thr Thr Gly Ser Ser Leu 20 25 30 Phe
Val Asn Lys Ser Met Lys Leu Val Asp Thr Phe Leu Glu Asp Thr 35 40
45 Lys Lys Leu Tyr His 50 <210> SEQ ID NO 33 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens
<400> SEQUENCE: 33 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Leu Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 34 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 34 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Gln Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 35 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 35 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Gln Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 36 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 36 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Leu Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 37 <211>
LENGTH: 57 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 37 Thr Tyr His Val Asp Arg Ser Gly Asn Val
His His Gln Phe Gln Lys 1 5 10 15 Leu Leu Thr Glu Phe Asn Lys Ser
Thr Asp Ala Tyr Glu Leu Lys Ile 20 25 30 Ala Asn Lys Leu Phe Gly
Glu Lys Thr Tyr Gln Phe Leu Gln Glu Tyr 35 40 45 Leu Asp Ala Ile
Lys Lys Phe Tyr Gln 50 55 <210> SEQ ID NO 38 <211>
LENGTH: 53 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 38 Leu Ser Glu Ser Asp Val His Arg Gly Phe
Gln His Leu Leu His Thr 1 5 10 15 Leu Asn Leu Pro Gly His Gly Leu
Glu Thr Arg Val Gly Ser Ala Leu 20 25 30 Phe Leu Ser His Asn Leu
Lys Phe Leu Ala Lys Phe Leu Asn Asp Thr 35 40 45 Met Ala Val Tyr
Glu 50 <210> SEQ ID NO 39 <211> LENGTH: 49 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
39 Glu Val His Ser Arg Phe Gln Ser Leu Asn Ala Asp Ile Asn Lys Arg
1 5 10 15 Gly Ala Ser Tyr Ile Leu Lys Leu Ala Asn Arg Leu Tyr Gly
Glu Lys 20 25 30 Thr Tyr Asn Phe Leu Pro Glu Phe Leu Val Ser Thr
Gln Lys Thr Tyr 35 40 45 Gly <210> SEQ ID NO 40 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 40 Lys Asp Ile Pro Phe Gly Phe Gln Thr Val
Thr Ser Asp Val Asn Lys 1 5 10 15 Leu Ser Ser Phe Tyr Ser Leu Lys
Leu Ile Lys Arg Leu Tyr Val Asp 20 25 30 Lys Ser Leu Asn Leu Ser
Thr Glu Phe Ile Ser Ser Thr Lys Arg Pro 35 40 45 Tyr Ala 50
<210> SEQ ID NO 41 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 41 Glu
Phe Asn Leu Ser Asn Ser Glu Glu Ile His Ser Asp Phe Gln Thr 1 5 10
15 Leu Ile Ser Glu Ile Leu Lys Pro Asn Asp Asp Tyr Leu Leu Lys Thr
20 25 30 Ala Asn Ala Ile Tyr Gly Glu Lys Thr Tyr Ala Phe His Asn
Lys Tyr 35 40 45 Leu Glu Asp Met Lys Thr Tyr Phe Gly 50 55
<210> SEQ ID NO 42 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 42 Glu
Glu Asp Ile His Arg Ala Phe Gln Ser Leu Leu Thr Glu Val Asn 1 5 10
15 Lys Ala Gly Thr Gln Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe Gly
20 25 30 Glu Lys Thr Cys Gln Phe Leu Ser Thr Phe Lys Glu Ser Cys
Leu Gln 35 40 45 Phe Tyr His 50 <210> SEQ ID NO 43
<211> LENGTH: 51 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 43 Glu Glu Asp Ile His Arg Ala
Phe Gln Ser Leu Leu Thr Glu Val Asn 1 5 10 15 Lys Ala Gly Thr Gln
Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe Gly 20 25 30 Glu Lys Thr
Cys Gln Phe Leu Ser Thr Phe Lys Glu Ser Cys Leu Gln 35 40 45 Phe
Tyr His 50 <210> SEQ ID NO 44 <211> LENGTH: 52
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 44 Asp Glu Glu Val His Ala Gly Leu Gly Glu
Leu Leu Arg Ser Leu Ser 1 5 10 15 Asn Ser Thr Ala Arg Asn Val Thr
Trp Lys Leu Gly Ser Arg Leu Tyr 20 25 30 Gly Pro Ser Ser Val Ser
Phe Ala Asp Asp Phe Val Arg Ser Ser Lys 35 40 45 Gln His Tyr Asn 50
<210> SEQ ID NO 45 <211> LENGTH: 40 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 45 Pro
Glu Glu Glu Ile Gln Glu Gly Phe Trp Asp Leu Leu Ile Arg Leu 1 5 10
15 Arg Gly Gln Gly Pro Arg Leu Leu Leu Thr Met Asp Gln Arg Arg Phe
20 25 30 Ser Gly Leu Gly Ala Arg Ala Asn 35 40
<210> SEQ ID NO 46 <211> LENGTH: 52 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 46 Pro
Glu Ala Asp Ile His Gln Gly Phe Arg Ser Leu Leu His Thr Leu 1 5 10
15 Ala Leu Pro Ser Pro Lys Leu Glu Leu Lys Val Gly Asn Ser Leu Phe
20 25 30 Leu Asp Lys Arg Leu Lys Pro Arg Gln His Tyr Leu Asp Ser
Ile Lys 35 40 45 Glu Leu Tyr Gly 50 <210> SEQ ID NO 47
<211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 47 Lys Asp Leu His Glu Gly Phe
His Tyr Ile Ile His Glu Leu Thr Gln 1 5 10 15 Lys Thr Gln Asp Leu
Lys Leu Ser Ile Gly Asn Thr Leu Phe Ile Asp 20 25 30 Gln Arg Leu
Gln Pro Gln Arg Lys Phe Leu Glu Asp Ala Lys Asn Phe 35 40 45 Tyr
Ser 50 <210> SEQ ID NO 48 <211> LENGTH: 57 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
48 Ser Pro Lys Cys Ser Gln Ala Gly Arg Ile His Ser Glu Phe Gly Val
1 5 10 15 Glu Phe Ser Gln Ile Asn Gln Pro Asp Ser Asn Cys Thr Leu
Ser Ile 20 25 30 Ala Asn Arg Leu Tyr Gly Thr Lys Thr Met Ala Phe
His Gln Gln Tyr 35 40 45 Leu Ser Cys Ser Glu Lys Trp Tyr Gln 50 55
<210> SEQ ID NO 49 <211> LENGTH: 57 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 49 Gly
Ser Leu Asn Asn Glu Ser Gly Leu Val Ser Cys Tyr Phe Gly Gln 1 5 10
15 Leu Leu Ser Lys Leu Asp Arg Ile Lys Thr Asp Tyr Thr Leu Ser Ile
20 25 30 Ala Asn Arg Leu Tyr Gly Glu Gln Glu Phe Pro Ile Cys Gln
Glu Tyr 35 40 45 Leu Asp Gly Val Ile Gln Phe Tyr His 50 55
<210> SEQ ID NO 50 <211> LENGTH: 49 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 50 Glu
Glu Phe Ser Phe Leu Lys Glu Phe Ser Asn Met Val Thr Ala Lys 1 5 10
15 Glu Ser Gln Tyr Val Met Lys Ile Ala Asn Ser Leu Phe Val Gln Asn
20 25 30 Gly Phe His Val Asn Glu Glu Phe Leu Gln Met Met Lys Lys
Tyr Phe 35 40 45 Asn <210> SEQ ID NO 51 <211> LENGTH:
51 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 51 Lys Pro Gly Leu Leu Pro Ser Leu Phe Lys
Gly Leu Arg Glu Thr Leu 1 5 10 15 Ser Arg Asn Leu Glu Leu Gly Leu
Thr Gln Gly Ser Phe Ala Phe Ile 20 25 30 His Lys Asp Phe Asp Val
Lys Glu Thr Phe Phe Asn Leu Ser Lys Arg 35 40 45 Tyr Phe Asp 50
<210> SEQ ID NO 52 <211> LENGTH: 51 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 52 Lys
Pro Gly Leu Leu Pro Ser Leu Phe Lys Gly Leu Arg Glu Thr Leu 1 5 10
15 Ser Arg Asn Leu Glu Leu Gly Leu Thr Gln Gly Ser Phe Ala Phe Ile
20 25 30 His Lys Asp Phe Asp Val Lys Glu Thr Phe Phe Asn Leu Ser
Lys Arg 35 40 45 Tyr Phe Asp 50 <210> SEQ ID NO 53
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 53 Gly Gly Gly Gly Asp Ile His
Gln Gly Phe Gln Ser Leu Leu Thr Glu 1 5 10 15 Val Asn Lys Thr Gly
Thr Gln Tyr Leu Leu Arg Val Ala Asn Arg Leu 20 25 30 Phe Gly Glu
Lys Ser Cys Asp Phe Leu Ser Ser Phe Arg Asp Ser Cys 35 40 45 Gln
Lys Phe Tyr Gln 50 <210> SEQ ID NO 54 <211> LENGTH: 52
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 54 Lys Asp Gly Asp Ile His Arg Gly Phe Gln
Ser Leu Leu Ser Glu Val 1 5 10 15 Asn Arg Thr Gly Thr Gln Tyr Leu
Leu Arg Thr Ala Asn Arg Leu Phe 20 25 30 Gly Glu Lys Thr Cys Asp
Phe Leu Pro Asp Phe Lys Glu Tyr Cys Gln 35 40 45 Lys Phe Tyr Gln 50
<210> SEQ ID NO 55 <211> LENGTH: 52 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 55 Lys
Asp Gly Asp Ile His Arg Gly Phe Gln Ser Leu Leu Ser Glu Val 1 5 10
15 Asn Arg Thr Gly Thr Gln Tyr Leu Leu Arg Thr Ala Asn Arg Leu Phe
20 25 30 Gly Glu Lys Thr Cys Asp Phe Leu Pro Asp Phe Lys Glu Tyr
Cys Gln 35 40 45 Lys Phe Tyr Gln 50 <210> SEQ ID NO 56
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 56 Val Pro Tyr Gln Gly Asn Ala
Thr Ala Leu Phe Ile Leu Pro Ser Glu 1 5 10 15 Gly Lys Met Gln Gln
Val Glu Asn Gly Leu Ser Glu Lys Thr Leu Arg 20 25 30 Lys Trp Leu
Lys Met Phe Lys Lys Arg Gln Leu Glu Leu Tyr 35 40 45 <210>
SEQ ID NO 57 <211> LENGTH: 47 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 57 Leu Lys
Tyr Thr Gly Asn Ala Ser Ala Leu Phe Ile Leu Pro Asp Gln 1 5 10 15
Asp Lys Met Glu Glu Val Glu Ala Met Leu Leu Pro Glu Thr Leu Lys 20
25 30 Arg Trp Arg Asp Ser Leu Glu Phe Arg Glu Ile Gly Glu Leu Tyr
35 40 45 <210> SEQ ID NO 58 <211> LENGTH: 46
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 58 Met Asp Tyr Lys Gly Asp Ala Val Ala Phe
Phe Val Leu Pro Ser Lys 1 5 10 15 Gly Lys Met Arg Gln Leu Glu Gln
Ala Leu Ser Ala Arg Thr Leu Arg 20 25 30 Lys Trp Ser His Ser Leu
Gln Lys Arg Trp Ile Glu Val Phe 35 40 45 <210> SEQ ID NO 59
<211> LENGTH: 46
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 59 Met Asp Tyr Lys Gly Asp Ala Val Ala Phe
Phe Val Leu Pro Ser Lys 1 5 10 15 Gly Lys Met Arg Gln Leu Glu Gln
Ala Leu Ser Ala Arg Thr Leu Arg 20 25 30 Lys Trp Ser His Ser Leu
Gln Lys Arg Trp Ile Glu Val Phe 35 40 45 <210> SEQ ID NO 60
<211> LENGTH: 50 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 60 Ile Pro Tyr Lys Asn Asn Asp
Leu Ser Met Phe Val Leu Leu Pro Asn 1 5 10 15 Asp Ile Asp Gly Leu
Glu Lys Ile Ile Asp Lys Ile Ser Pro Glu Lys 20 25 30 Leu Val Glu
Trp Thr Ser Pro Gly His Met Glu Glu Arg Lys Val Asn 35 40 45 Leu
His 50 <210> SEQ ID NO 61 <211> LENGTH: 47 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
61 Leu Arg Tyr Asn Gly Gly Ile Asn Met Tyr Val Leu Leu Pro Glu Asn
1 5 10 15 Asp Leu Ser Glu Ile Glu Asn Lys Leu Thr Phe Gln Asn Leu
Met Glu 20 25 30 Trp Thr Asn Pro Arg Arg Met Thr Ser Lys Tyr Val
Glu Val Phe 35 40 45 <210> SEQ ID NO 62 <211> LENGTH:
48 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 62 Leu Ser Tyr Lys Gly Asp Glu Phe Ser Leu
Ile Ile Ile Leu Pro Ala 1 5 10 15 Glu Gly Met Asp Ile Glu Glu Val
Glu Lys Leu Ile Thr Ala Gln Gln 20 25 30 Ile Leu Lys Trp Leu Ser
Glu Met Gln Glu Glu Glu Val Glu Ile Ser 35 40 45 <210> SEQ ID
NO 63 <211> LENGTH: 48 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 63 Leu Pro Phe Lys Gly
Asp Asp Ile Thr Met Val Leu Ile Leu Pro Lys 1 5 10 15 Pro Glu Lys
Ser Leu Ala Lys Val Glu Lys Glu Leu Thr Pro Glu Val 20 25 30 Leu
Gln Glu Trp Leu Asp Glu Leu Glu Glu Met Met Leu Val Val His 35 40
45 <210> SEQ ID NO 64 <211> LENGTH: 48 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
64 Leu Pro Phe Lys Gly Asp Asp Ile Thr Met Val Leu Ile Leu Pro Lys
1 5 10 15 Pro Glu Lys Ser Leu Ala Lys Val Glu Lys Glu Leu Thr Pro
Glu Val 20 25 30 Leu Gln Glu Trp Leu Asp Glu Leu Glu Glu Met Met
Leu Val Val His 35 40 45 <210> SEQ ID NO 65 <211>
LENGTH: 46 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 65 Met Lys Tyr Leu Gly Asn Ala Thr Ala Ile
Phe Phe Leu Pro Asp Glu 1 5 10 15 Gly Lys Leu Gln His Leu Glu Asn
Glu Leu Thr His Asp Ile Ile Thr 20 25 30 Lys Phe Leu Glu Asn Glu
Asp Arg Arg Ser Ala Ser Leu His 35 40 45 <210> SEQ ID NO 66
<211> LENGTH: 53 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 66 Leu Pro Tyr Ala Gly Asp Val
Ser Met Phe Leu Leu Leu Pro Asp Glu 1 5 10 15 Ile Ala Asp Val Ser
Thr Gly Leu Glu Leu Leu Glu Ser Glu Ile Thr 20 25 30 Tyr Asp Lys
Leu Asn Lys Trp Thr Ser Lys Asp Lys Met Ala Glu Asp 35 40 45 Glu
Val Glu Val Tyr 50 <210> SEQ ID NO 67 <211> LENGTH: 49
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 67 Leu Pro Tyr His Gly Asp Thr Leu Ser Met
Phe Ile Ala Ala Pro Tyr 1 5 10 15 Glu Lys Glu Val Pro Leu Ser Ala
Leu Thr Asn Ile Leu Ser Ala Gln 20 25 30 Leu Ile Ser His Trp Lys
Gly Asn Met Thr Arg Leu Pro Arg Leu Leu 35 40 45 Val <210>
SEQ ID NO 68 <211> LENGTH: 51 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 68 Leu Gln
Leu Ser His Asn Leu Ser Leu Val Ile Leu Val Pro Gln Asn 1 5 10 15
Leu Lys His Arg Leu Glu Asp Met Glu Gln Ala Leu Ser Pro Ser Val 20
25 30 Phe Lys Ala Ile Met Glu Lys Leu Glu Met Ser Lys Phe Gln Pro
Thr 35 40 45 Leu Leu Thr 50 <210> SEQ ID NO 69 <211>
LENGTH: 46 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 69 Met Asp Tyr Ser Lys Asn Ala Leu Ala Leu
Phe Val Leu Pro Lys Glu 1 5 10 15 Gly Gln Met Glu Ser Val Glu Ala
Ala Met Ser Ser Lys Thr Leu Lys 20 25 30 Lys Trp Asn Arg Leu Leu
Gln Lys Gly Trp Val Asp Leu Phe 35 40 45 <210> SEQ ID NO 70
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 70 Met Asp Tyr Ser Lys Asn Ala
Leu Ala Leu Phe Val Leu Pro Lys Glu 1 5 10 15 Gly Gln Met Glu Ser
Val Glu Ala Ala Met Ser Ser Lys Thr Leu Lys 20 25 30 Lys Trp Asn
Arg Leu Leu Gln Lys Gly Trp Val Asp Leu Phe 35 40 45 <210>
SEQ ID NO 71 <211> LENGTH: 47 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 71 Leu Glu
Tyr Val Gly Gly Ile Ser Met Leu Ile Val Val Pro His Lys 1 5 10 15
Met Ser Gly Met Lys Thr Leu Glu Ala Gln Leu Thr Pro Gly Val Val 20
25 30 Glu Arg Trp Gln Lys Ser Met Thr Asn Arg Thr Arg Glu Val Leu
35 40 45 <210> SEQ ID NO 72 <211> LENGTH: 49
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 72 Leu Pro Tyr His Gly Glu Ser Ile Ser Met
Leu Ile Ala Leu Pro Thr 1 5 10 15 Glu Ser Ser Thr Pro Leu Ser Ala
Ile Ile Pro His Ile Ser Thr Lys 20 25 30 Thr Ile Asp Ser Trp Met
Ser Ile Met Val Pro Lys Arg Val Gln Val 35 40 45 Ile <210>
SEQ ID NO 73 <211> LENGTH: 46 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 73 Phe Pro
Phe Lys Asn Asn Met Ser Phe Val Val Leu Val Pro Thr His 1 5 10 15
Phe Glu Trp Asn Val Ser Gln Val Leu Ala Asn Leu Ser Trp Asp Thr 20
25 30 Leu His Pro Pro Leu Val Trp Glu Arg Pro Thr Lys Val Arg
35 40 45 <210> SEQ ID NO 74 <211> LENGTH: 46
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 74 Phe Pro Phe Lys Asn Asn Met Ser Phe Val
Val Leu Val Pro Thr His 1 5 10 15 Phe Glu Trp Asn Val Ser Gln Val
Leu Ala Asn Leu Ser Trp Asp Thr 20 25 30 Leu His Pro Pro Leu Val
Trp Glu Arg Pro Thr Lys Val Arg 35 40 45 <210> SEQ ID NO 75
<211> LENGTH: 46 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 75 Gln His Tyr Val Gly Asn Ala
Thr Ala Phe Phe Ile Leu Pro Asp Pro 1 5 10 15 Lys Lys Met Trp Gln
Leu Glu Glu Lys Leu Thr Tyr Ser His Leu Glu 20 25 30 Asn Ile Gln
Arg Ala Phe Asp Ile Arg Ser Ile Asn Leu His 35 40 45 <210>
SEQ ID NO 76 <211> LENGTH: 50 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 76 Ile Pro
Tyr Lys Gly Lys Asp Leu Ser Met Ile Val Leu Leu Pro Asn 1 5 10 15
Glu Ile Asp Gly Leu Gln Lys Leu Glu Glu Lys Leu Thr Ala Glu Lys 20
25 30 Leu Met Glu Trp Thr Ser Leu Gln Asn Met Arg Glu Thr Arg Val
Asp 35 40 45 Leu His 50 <210> SEQ ID NO 77 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 77 Ile Pro Tyr Lys Gly Lys Asp Leu Ser Met
Ile Val Leu Leu Pro Asn 1 5 10 15 Glu Ile Asp Gly Leu Gln Lys Leu
Glu Glu Lys Leu Thr Ala Glu Lys 20 25 30 Leu Met Glu Trp Thr Ser
Leu Gln Asn Met Arg Glu Thr Cys Val Asp 35 40 45 Leu His 50
<210> SEQ ID NO 78 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 78 Ile
Pro Tyr Lys Gly Lys Asp Leu Ser Met Ile Val Leu Leu Pro Asn 1 5 10
15 Glu Ile Asp Gly Leu Gln Lys Leu Glu Glu Lys Leu Thr Ala Glu Lys
20 25 30 Leu Met Glu Trp Thr Ser Leu Gln Asn Met Arg Glu Thr Arg
Val Asp 35 40 45 Leu His 50 <210> SEQ ID NO 79 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 79 Ile Pro Tyr Lys Gly Lys Asp Leu Ser Met
Ile Val Leu Leu Pro Asn 1 5 10 15 Glu Ile Asp Gly Leu Gln Lys Leu
Glu Glu Lys Leu Thr Ala Glu Lys 20 25 30 Leu Met Glu Trp Thr Ser
Leu Gln Asn Met Arg Glu Thr Cys Val Asp 35 40 45 Leu His 50
<210> SEQ ID NO 80 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 80 Ile
Pro Tyr Lys Gly Lys Asp Leu Ser Met Ile Val Leu Leu Pro Asn 1 5 10
15 Glu Ile Asp Gly Leu Gln Lys Leu Glu Glu Lys Leu Thr Ala Glu Lys
20 25 30 Leu Met Glu Trp Thr Ser Leu Gln Asn Met Arg Glu Thr Cys
Val Asp 35 40 45 Leu His 50 <210> SEQ ID NO 81 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 81 Met Asp Tyr Lys Gly Asp Ala Thr Val Phe
Phe Ile Leu Pro Asn Gln 1 5 10 15 Gly Lys Met Arg Glu Ile Glu Glu
Val Leu Thr Pro Glu Met Leu Met 20 25 30 Arg Trp Asn Asn Leu Leu
Arg Lys Arg Asn Phe Tyr Lys Lys Leu Glu 35 40 45 Leu His 50
<210> SEQ ID NO 82 <211> LENGTH: 54 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 82 Leu
Pro Tyr Gln Gly Glu Glu Leu Ser Met Val Ile Leu Leu Pro Asp 1 5 10
15 Asp Ile Glu Asp Glu Ser Thr Gly Leu Lys Lys Ile Glu Glu Gln Leu
20 25 30 Thr Leu Glu Lys Leu His Glu Trp Thr Lys Pro Glu Asn Leu
Asp Phe 35 40 45 Ile Glu Val Asn Val Ser 50 <210> SEQ ID NO
83 <211> LENGTH: 54 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 83 Leu Pro Phe Gln Asn
Lys His Leu Ser Met Phe Ile Leu Leu Pro Lys 1 5 10 15 Asp Val Glu
Asp Glu Ser Thr Gly Leu Glu Lys Ile Glu Lys Gln Leu 20 25 30 Asn
Ser Glu Ser Leu Ser Gln Trp Thr Asn Pro Ser Thr Met Ala Asn 35 40
45 Ala Lys Val Lys Leu Ser 50 <210> SEQ ID NO 84 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 84 Leu Tyr Tyr Lys Ser Arg Asp Leu Ser Leu
Leu Ile Leu Leu Pro Glu 1 5 10 15 Asp Ile Asn Gly Leu Glu Gln Leu
Glu Lys Ala Ile Thr Tyr Glu Lys 20 25 30 Leu Asn Glu Trp Thr Ser
Ala Asp Met Met Glu Leu Tyr Glu Val Gln 35 40 45 Leu His 50
<210> SEQ ID NO 85 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 85 Leu
Pro Tyr Ala Arg Lys Glu Leu Ser Leu Leu Val Leu Leu Pro Asp 1 5 10
15 Asp Gly Val Glu Leu Ser Thr Val Glu Lys Ser Leu Thr Phe Glu Lys
20 25 30 Leu Thr Ala Trp Thr Lys Pro Asp Cys Met Lys Ser Thr Glu
Val Glu 35 40 45 Val Leu 50 <210> SEQ ID NO 86 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 86 Leu Pro Tyr Ala Arg Lys Glu Leu Ser Leu
Leu Val Leu Leu Pro Asp 1 5 10 15 Asp Gly Val Glu Leu Ser Thr Val
Glu Lys Ser Leu Thr Phe Glu Lys 20 25 30 Leu Thr Ala Trp Thr Lys
Pro Asp Cys Met Lys Ser Thr Glu Val Glu 35 40 45
Val Leu 50 <210> SEQ ID NO 87 <211> LENGTH: 48
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 87 Met Pro Leu Ala His Lys Leu Ser Ser Leu
Ile Ile Leu Met Pro His 1 5 10 15 His Val Glu Pro Leu Glu Arg Leu
Glu Lys Leu Leu Thr Lys Glu Gln 20 25 30 Leu Lys Ile Trp Met Gly
Lys Met Gln Lys Lys Ala Val Ala Ile Ser 35 40 45 <210> SEQ ID
NO 88 <211> LENGTH: 46 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 88 Met Asp His Ala Gly
Asn Thr Thr Thr Phe Phe Ile Phe Pro Asn Arg 1 5 10 15 Gly Lys Met
Arg His Leu Glu Asp Ala Leu Leu Pro Glu Thr Leu Ile 20 25 30 Lys
Trp Asp Ser Leu Leu Arg Thr Arg Glu Leu Asp Phe His 35 40 45
<210> SEQ ID NO 89 <211> LENGTH: 46 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 89 Ile
Glu Tyr Arg Gly Asn Ala Leu Ala Leu Leu Val Leu Pro Asp Pro 1 5 10
15 Gly Lys Met Lys Gln Val Glu Ala Ala Leu Gln Pro Gln Thr Leu Arg
20 25 30 Lys Trp Gly Gln Leu Leu Leu Pro Ser Leu Leu Asp Leu His 35
40 45 <210> SEQ ID NO 90 <211> LENGTH: 46 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
90 Ile Pro Tyr Gln Lys Asn Ile Thr Ala Ile Phe Ile Leu Pro Asp Glu
1 5 10 15 Gly Lys Leu Lys His Leu Glu Lys Gly Leu Gln Val Asp Thr
Phe Ser 20 25 30 Arg Trp Lys Thr Leu Leu Ser Arg Arg Val Val Asp
Val Ser 35 40 45 <210> SEQ ID NO 91 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 91 Leu Pro Tyr Val Asn Asn Lys Leu Ser Met
Ile Ile Leu Leu Pro Val 1 5 10 15 Gly Ile Ala Asn Leu Lys Gln Ile
Glu Lys Gln Leu Asn Ser Gly Thr 20 25 30 Phe His Glu Trp Thr Ser
Ser Ser Asn Met Met Glu Arg Glu Val Glu 35 40 45 Val His 50
<210> SEQ ID NO 92 <211> LENGTH: 54 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 92 Met
Arg Tyr Thr Lys Gly Lys Leu Ser Met Phe Val Leu Leu Pro Ser 1 5 10
15 His Ser Lys Asp Asn Leu Lys Gly Leu Glu Glu Leu Glu Arg Lys Ile
20 25 30 Thr Tyr Glu Lys Met Val Ala Trp Ser Ser Ser Glu Asn Met
Ser Glu 35 40 45 Glu Ser Val Val Leu Ser 50 <210> SEQ ID NO
93 <211> LENGTH: 54 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 93 Met Arg Tyr Thr Lys
Gly Lys Leu Ser Met Phe Val Leu Leu Pro Ser 1 5 10 15 His Ser Lys
Asp Asn Leu Lys Gly Leu Glu Glu Leu Glu Arg Lys Ile 20 25 30 Thr
Tyr Glu Lys Met Val Ala Trp Ser Ser Ser Glu Asn Met Ser Glu 35 40
45 Glu Ser Val Val Leu Ser 50 <210> SEQ ID NO 94 <211>
LENGTH: 48 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 94 Ile Pro Tyr Glu Gly Asp Glu Ile Ser Met
Met Leu Val Leu Ser Arg 1 5 10 15 Gln Glu Val Pro Leu Ala Thr Leu
Glu Pro Leu Val Lys Ala Gln Leu 20 25 30 Val Glu Glu Trp Ala Asn
Ser Val Lys Lys Gln Lys Val Glu Val Tyr 35 40 45 <210> SEQ ID
NO 95 <211> LENGTH: 47 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 95 Leu Pro Tyr Gln Gly
Asn Ala Thr Met Leu Val Val Leu Met Glu Lys 1 5 10 15 Met Gly Asp
His Leu Ala Leu Glu Asp Tyr Leu Thr Thr Asp Leu Val 20 25 30 Glu
Thr Trp Leu Arg Asn Met Lys Thr Arg Asn Met Glu Val Phe 35 40 45
<210> SEQ ID NO 96 <211> LENGTH: 47 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 96 Leu
Pro Tyr Gln Gly Asn Ala Thr Met Leu Val Val Leu Met Glu Lys 1 5 10
15 Met Gly Asp His Leu Ala Leu Glu Asp Tyr Leu Thr Thr Asp Leu Val
20 25 30 Glu Thr Trp Leu Arg Asn Met Lys Thr Arg Asn Met Glu Val
Phe 35 40 45 <210> SEQ ID NO 97 <211> LENGTH: 50
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 97 Leu Pro Tyr Val Gly Lys Glu Leu Asn Met
Ile Ile Met Leu Pro Asp 1 5 10 15 Glu Thr Thr Asp Leu Arg Thr Val
Glu Lys Glu Leu Thr Tyr Glu Lys 20 25 30 Phe Val Glu Trp Thr Arg
Leu Asp Met Met Asp Glu Glu Glu Val Glu 35 40 45 Val Ser 50
<210> SEQ ID NO 98 <211> LENGTH: 50 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 98 Leu
Pro Tyr Val Gly Lys Glu Leu Asn Met Ile Ile Met Leu Pro Asp 1 5 10
15 Glu Thr Thr Asp Leu Arg Thr Val Glu Lys Glu Leu Thr Tyr Glu Lys
20 25 30 Phe Val Glu Trp Thr Arg Leu Asp Met Met Asp Glu Glu Glu
Val Glu 35 40 45 Val Ser 50 <210> SEQ ID NO 99 <211>
LENGTH: 50 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 99 Leu Pro Tyr Val Glu Glu Glu Leu Ser Met
Val Ile Leu Leu Pro Asp 1 5 10 15 Asp Asn Thr Asp Leu Ala Val Val
Glu Lys Ala Leu Thr Tyr Glu Lys 20 25 30 Phe Lys Ala Trp Thr Asn
Ser Glu Lys Leu Thr Lys Ser Lys Val Gln 35 40 45 Val Phe 50
<210> SEQ ID NO 100 <211> LENGTH: 25 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 100
Leu Pro Tyr Val Glu Glu Glu Leu Ser Met Val Ile Leu Leu Pro Asp
1 5 10 15 Asp Asn Thr Asp Leu Ala Val Lys Glu 20 25 <210> SEQ
ID NO 101 <211> LENGTH: 37 <212> TYPE: PRT <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: LL-37 control peptide <400> SEQUENCE: 101
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5
10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu
Val 20 25 30 Pro Arg Thr Glu Ser 35 <210> SEQ ID NO 102
<211> LENGTH: 27 <212> TYPE: PRT <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: GGL27(S) control peptide <400> SEQUENCE: 102 Gly
Gly Leu Ile Ser Thr Ser Ser Ser Ser Ser Ser Gln Arg Val Lys 1 5 10
15 Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 20 25 <210>
SEQ ID NO 103 <211> LENGTH: 25 <212> TYPE: PRT
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: DSE25 control peptide <400>
SEQUENCE: 103 Asp Ser Glu Glu Asp Glu Glu His Thr Ile Ile Thr Asp
Thr Glu Leu 1 5 10 15 Pro Pro Leu Lys Leu Met His Ser Phe 20 25
<210> SEQ ID NO 104 <211> LENGTH: 13 <212> TYPE:
PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: C-terminal TFPI sequence <400>
SEQUENCE: 104 Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 1
5 10 <210> SEQ ID NO 105 <211> LENGTH: 20 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: A kininogen-derived
antimicrobial peptide <400> SEQUENCE: 105 His Lys His Gly His
Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 1 5 10 15 Asn Gly Lys
His 20 <210> SEQ ID NO 106 <211> LENGTH: 22 <212>
TYPE: PRT <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Heparin-binding growth
factor derived peptide <400> SEQUENCE: 106 Gly Lys Arg Lys
Lys Lys Gly Lys Gly Leu Gly Lys Lys Arg Asp Pro 1 5 10 15 Cys Leu
Arg Lys Tyr Lys 20 <210> SEQ ID NO 107 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Oryctolagus cuniculus
<400> SEQUENCE: 107 Gly Gly Leu Ile Lys Thr Lys Arg Lys Lys
Lys Lys Gln Pro Val Lys 1 5 10 15 Ile Thr Tyr Val Glu Thr Phe Val
Lys Lys Thr 20 25 <210> SEQ ID NO 108 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Canis lupus <400>
SEQUENCE: 108 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln
Thr Val Lys 1 5 10 15 Ile Val Tyr Glu Lys Ile Phe Val Lys Lys Leu
20 25 <210> SEQ ID NO 109 <211> LENGTH: 18 <212>
TYPE: PRT <213> ORGANISM: Danio rerio <400> SEQUENCE:
109 Arg Lys Gln Ile Arg Ile Lys Thr Arg Asn Ser Asn Ile Leu Phe Arg
1 5 10 15 Ser Val <210> SEQ ID NO 110 <211> LENGTH: 21
<212> TYPE: PRT <213> ORGANISM: Mus musculus
<400> SEQUENCE: 110 Gly Val Val Lys Ile Gln Arg Arg Lys Ala
Pro Phe Val Lys Val Val 1 5 10 15 Tyr Glu Ser Ile Asn 20
<210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE:
PRT <213> ORGANISM: Rattus norvegicus <400> SEQUENCE:
111 Arg Ala Lys Thr Gln Arg Arg Arg Lys Ser Phe Val Lys Val Met Tyr
1 5 10 15 Glu Asn Ile His 20 <210> SEQ ID NO 112 <211>
LENGTH: 27 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 112 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg
Lys Lys Gln Arg Val Lys 1 5 10 15 Ile Ala Tyr Glu Glu Ile Phe Val
Lys Asn Met 20 25 <210> SEQ ID NO 113 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Pongo abelii
<400> SEQUENCE: 113 Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg
Lys Lys Gln Arg Val Lys 1 5 10 15 Ile Ala Tyr Glu Glu Ile Phe Val
Lys Asn Met 20 25 <210> SEQ ID NO 114 <211> LENGTH: 27
<212> TYPE: PRT <213> ORGANISM: Sus scrofa <400>
SEQUENCE: 114 Asp Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys Lys Gln
Pro Val Lys 1 5 10 15 Ile Val Tyr Glu Glu Ile Phe Val Lys Lys Ile
20 25 <210> SEQ ID NO 115 <211> LENGTH: 11 <212>
TYPE: PRT <213> ORGANISM: Bos taurus <400> SEQUENCE:
115 Glu Gly Leu Ile Lys Thr Lys Lys Lys Lys Lys 1 5 10
* * * * *