U.S. patent application number 16/794037 was filed with the patent office on 2020-08-13 for therapeutic trem-1 peptides.
The applicant listed for this patent is Bristol-Myers Squibb Company Universite de Lorraine. Invention is credited to Gilbert FAURE, Sebastien GIBOT, Paola PANINA, Nadia PASSINI.
Application Number | 20200254058 16/794037 |
Document ID | 20200254058 / US20200254058 |
Family ID | 1000004784519 |
Filed Date | 2020-08-13 |
Patent Application | download [pdf] |
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United States Patent
Application |
20200254058 |
Kind Code |
A1 |
FAURE; Gilbert ; et
al. |
August 13, 2020 |
THERAPEUTIC TREM-1 PEPTIDES
Abstract
A polypeptide comprising one or more sequences derived from CDR2
or CDR3 of a TREM-1 protein, characterised by the ability to treat,
ameliorate, or lessen the symptoms of conditions including sepsis,
septic shock or sepsis-like conditions and IBD.
Inventors: |
FAURE; Gilbert; (Vandoeuvre
Les Nancy, FR) ; GIBOT; Sebastien; (Vandoeuvre Les
Nancy, FR) ; PANINA; Paola; (Milan, IT) ;
PASSINI; Nadia; (Milan, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bristol-Myers Squibb Company
Universite de Lorraine |
Princeton
Nancy Cedex |
NJ |
US
FR |
|
|
Family ID: |
1000004784519 |
Appl. No.: |
16/794037 |
Filed: |
February 18, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14968479 |
Dec 14, 2015 |
10603357 |
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16794037 |
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13181323 |
Jul 12, 2011 |
9273111 |
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14968479 |
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12320707 |
Feb 2, 2009 |
8013116 |
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13181323 |
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11284086 |
Nov 22, 2005 |
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12320707 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 2319/70 20130101; C07K 2319/20 20130101; G01N 2800/065
20130101; G01N 33/5088 20130101; C07K 14/705 20130101; G01N 2800/26
20130101; A61K 47/6813 20170801; A61K 38/177 20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; G01N 33/50 20060101 G01N033/50; C07K 14/705 20060101
C07K014/705; A61K 47/68 20060101 A61K047/68 |
Foreign Application Data
Date |
Code |
Application Number |
May 19, 2005 |
JP |
2005-146848 |
Claims
1. An isolated peptide or a derivative thereof, which is capable of
acting as antagonist of the TREM-1 protein as defined by SEQ ID
NO.2, comprising SEQ ID NO. 22 or at least 3 amino acids from SEQ
ID NO. 23, wherein: (a) the polypeptide consists of: (i) a
contiguous sequence of 5 to 29 amino acids from SEQ ID NO: 2; or
(ii) a contiguous sequence of 5 to 29 amino acids from SEQ ID NO: 2
in which one amino acid is substituted conservatively with another
amino acid; or (b) the polypeptide consists of an amino acid
sequence having at least 80% sequence identity to SEQ ID NOS: 3, 4,
6 or 7; wherein the derivatives of the polypeptides of (a) or (b)
are modified by glycosylation, acetylation, pegylation,
phosphorylation, amidation, or derivatization by protecting or
blocking groups.
2. An isolated polynucleotide encoding the peptide or derivative
thereof of claim 1.
3. A vector comprising the polynucleotide of claim 2.
4. A method of treating sepsis, septic shock, or an inflammatory
disorder in a subject in need thereof comprising administering to
the subject the peptide or derivative thereof of claim 1.
5. An isolated polypeptide consisting of SEQ ID NO: 19 or SEQ ID
NO: 7.
6. An isolated polynucleotide encoding the polypeptide of claim
5.
7. A vector comprising the polynucleotide of claim 6.
8. A method of treating sepsis, septic shock, or an inflammatory
disorder in a subject in need thereof comprising administering to
the subject the polypeptide of claim 5.
9. An isolated peptide or derivative thereof, which is capable of
acting as an antagonist of the TREM-1 protein as defined by SEQ ID
NO. 1, comprising the amino acid sequence of SEQ ID NO. 20 or at
least 3 amino acids of SEQ ID NO. 21, wherein: (a) the peptide
consists of: (i) a contiguous sequence of 5 to 29 amino acids from
SEQ ID NO: 1; or (ii) a contiguous sequence of 5 to 29 amino acids
from SEQ ID NO: 1 in which one amino acid is substituted
conservatively with another amino acid; or (b) the peptide consists
of an amino acid sequence having at least 80% sequence identity to
SEQ ID NOs: 16, 17, 18 or 19; and wherein the derivatives of the
peptides of (a) or (b) are modified by glycosylation, acetylation,
pegylation, phosphorylation, amidation, or derivatization by
protecting or blocking groups.
10. An isolated polynucleotide encoding the peptide or derivative
thereof of claim 9.
11. A vector comprising the polynucleotide of claim 10.
12. A method of treating sepsis, septic shock, or an inflammatory
disorder in a subject in need thereof comprising administering to
the subject the peptide or derivative thereof of claim 9.
13. The method of claim 12, wherein the disorder comprises
inflammatory bowel disease (IBD).
14. The method of claim 12, wherein the peptide or derivative
thereof consists of: (a) a contiguous sequence of 5 to 29 amino
acids from SEQ ID NO: 1; or (b) a contiguous sequence of 5 to 29
amino acids from SEQ ID NO: 1 in which one amino acid is
substituted conservatively with another amino acid; and wherein the
derivatives of the peptides of (a) or (b) are modified by
glycosylation, acetylation, pegylation, phosphorylation, amidation,
or derivatization by protecting or blocking groups.
15. The method of claim 12, wherein the peptide or derivative
thereof consists of an amino acid sequence having at least 80%
sequence identity to SEQ ID NOs: 16, 17, 18 or 19; and wherein the
derivatives of the peptide are modified by glycosylation,
acetylation, pegylation, phosphorylation, amidation, or
derivatization by protecting or blocking groups.
16. The method of claim 15, wherein the peptide consists of an
amino acid sequence having at least 80% sequence identity to SEQ ID
NO: 19.
17. The method of claim 15, wherein the peptide consists of an
amino acid sequence having the sequence of SEQ ID NOs: 16, 17, 18
or 19, or which differs from said sequence by one or more
conservative amino acid modifications.
18. The method of claim 17, wherein the peptide consists of an
amino acid having the sequence of SEQ ID NO: 19, or which differs
from said sequence by one or more conservative modifications.
19. The method of claim 12, wherein the peptide consists of a
contiguous sequence of 5 to 29 amino acids from SEQ ID NO. 1.
20. The method of claim 12, wherein the peptide or derivative
comprises at least 3 amino acids from SEQ ID NO. 21, wherein the at
least 3 amino acids from SEQ ID NO. 21 are selected from the group
consisting of QPP, QPPK (SEQ ID NO: 24), and QPPKE (SEQ ID NO. 21).
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 14/968,479, filed Jul. 12, 2011, which is a divisional of
U.S. patent application Ser. No. 13/181,323, filed Jul. 12, 2011,
which is a continuation of U.S. patent application Ser. No.
12/320,707, filed Feb. 2, 2009, which is a continuation-in-part of
U.S. Patent Application No. 11/284,086, filed on Nov. 22, 2005,
which claims priority from United Kingdom Patent Application No.
0426146.7, filed on Nov. 29, 2004, and Japanese Patent Application
No. 2005-146848, filed on May 19, 2005. The contents of these
applications are incorporated herein by reference in their
entirety.
[0002] The content of the electronically submitted sequence listing
in ASCII text file (Name: 3338_0800007_Seqlisting_ST25; Size:
13,792 bytes; and Date of Creation: Feb. 18, 2020) is incorporated
herein by reference in its entirety.
[0003] The present invention relates to the field of immunology.
More particularly, the present invention relates to inflammation
and the use of proteins and peptides containing certain sequences
of the TREM-1 protein and their functional equivalents (referred to
herein as TREM1-peptides) in the treatment of disease, for example,
sepsis, septic shock and inflammatory bowel disease (IBD).
[0004] Sepsis constitutes a significant consumption of intensive
care resources and remains an ever-present problem in the intensive
care unit. It has been estimated that between 400 000 and 500 000
patients are so affected each year in both the USA and Europe.
Morbidity and mortality have remained high despite improvements in
both supportive and antimicrobial therapies. Mortality rates vary
from 40% for uncomplicated sepsis to 80% in those suffering from
septic shock and multi-organ dysfunction. The pathogenesis of the
conditions is now becoming better understood. Greater understanding
of the complex network of immune, inflammatory and haematological
mediators may allow the development of rational and novel
therapies.
[0005] Following an infection, innate and cognitive immune
responses develop in sequential phases that build-up in specificity
and complexity, resulting ultimately in the clearance of infectious
agents and restoration of homeostasis. The innate immune response
serves as the first line of defence and is initiated upon
activation of pattern recognition receptors, such as Toll-like
receptors (TLRs) (1, 2), by various pathogen-associated microbial
patterns (PAMPs) (3). Activation of the TLRs triggers the release
of large quantities of such cytokines as TNF-.alpha. and
IL-1.beta., which in case of such massive infections as sepsis, can
precipitate tissue injury and lethal shock (4, 5). Although
antagonists of TNF-.alpha. and IL-1.beta. appeared in this context
as possibly interesting therapeutic agents of sepsis, they have
unfortunately shown limited efficacy in clinical trials (6-8). This
could be due to the fact that these cytokines are necessary for the
clearance of infections, and that their removal would allow for
fatal bacterial growth (9-11).
[0006] Another receptor involved in, inter alia, response to
infection, triggering receptor expressed on myeloid cells-1
(TREM-1) is a member of a recently discovered family of receptors,
the TREM family, expressed on the surface of neutrophils and a
subset of monocytes. TREM receptors activate myeloid cells via
association with the adaptor molecule DAP12. Engagement of TREM-1
has been reported to trigger the synthesis of pro-inflammatory
cytokines in the presence of microbial products.
[0007] The triggering receptor expressed on myeloid cells (TREM)-1
is a recently discovered cell-surface molecule that has been
identified both on human and murine polymorphonuclear neutrophils
and mature monocytes (12). It belongs to the immunoglobulin
superfamily and activates downstream signalling pathways with the
help of an adapter protein called DAP12 (12-15). Bouchon and
co-workers have shown that the expression of TREM-1 was greatly
up-regulated on neutrophils and monocytes in the presence of such
bacteria as Pseudomonas aeruginosa or Staphylococcus aureus, both
in cell culture and in tissue samples from patients with infection
(16). In striking contrast, TREM-1 was not up-regulated in samples
from patients with non-infectious inflammatory diseases such as
psoriasis, ulcerative colitis or vasculitis caused by immune
complexes (16). Moreover, when TREM-1 is bound to its ligand, there
is a synergistic effect of LPS and an amplified synthesis of the
pro-inflammatory cytokines TNF-.alpha. and GM-CSF, together with an
inhibition of IL-10 production (17). In a murine model of
LPS-induced septic shock, blockade of TREM-1 signalling protected
the animals from death, further highlighting the crucial role of
this molecule (13, 16).
[0008] Recent studies demonstrate that TREM-1 plays a critical role
in the inflammatory response to infection (see BOUCHON et al.
(2000) J. Immunol. 164:4991-4995). Expression of TREM-1 is
increased on myeloid cells in response to both bacterial and fungal
infections in humans. Similarly, in mice the induction of shock by
lipopolysaccharide (LPS) is associated with increased expression of
TREM-1. Further, treatment of mice with a soluble TREM-1/Ig fusion
protein, as a `decoy` receptor, protects mice from death due to LPS
or E.coli.
[0009] Triggering via TREM-1 results in the production of
pro-inflammatory cytokines, chemokines and reactive oxygen species,
and leads to rapid degranulation of neutrophilic granules, and
phagocytosis. Since interfering with TREM-1 engagement leads to the
simultaneous reduction in production and secretion of a variety of
proinflammatory mediators, TREM-1 represents an attractive target
for treating chronic inflammatory disorders. Indeed, a role for
TREM-1 has been demonstrated in a variety of inflammatory
disorders, including (but not limited to) acute and chronic
inflammatory disorders, sepsis, acute endotoxemia, encephalitis,
Chronic Obstructive Pulmonary Disease (COPD), allergic inflammatory
disorders, asthma, pulmonary fibrosis, pneumonia, Community
acquired pneumonia (CAP), Ventilator associated pneumonia (VAP),
Acute respiratory infection, Acute respiratory distress syndrome
(ARDS), Infectious lung diseases, Pleural effusion, Peptic ulcer,
Helicobacter pylori infection, hepatic granulomatosis, arthritis,
rheumatoid arthritis, osteoarthritis, inflammatory osteolysis,
ulcerative colitis, psoriasis, vasculitis, autoimmune disorders,
thyroiditis, Meliodosis, (mesenteric) Ischemia reperfusion,
Filovirus infection, Infection of the urinary tract, Bacterial
meningitis, Salmonella enterica infection, Marburg and Ebola
viruses infections, and in particular Inflammatory Bowel Disease
(IBD).
[0010] Inflammatory bowel disease (IBD) covers a group of disorders
in which the intestines become inflamed (red and swollen), probably
as a result of an immune reaction of the body against its own
intestinal tissue. Two major types of IBD are described: ulcerative
colitis (UC) and Crohn disease (CD). Ulcerative colitis is limited
to the colon (large intestine). Crohn disease can involve any part
of the gastrointestinal tract from the mouth to the anus, but it
most commonly affects the small intestine and/or the colon. Both
ulcerative colitis and Crohn disease vary in the intensity and
severity during the course of the disease. When there is severe
inflammation, the disease is considered to be in an active stage,
and the person experiences a flare-up of the condition. When the
degree of inflammation is less (or absent), the person usually is
without symptoms, and the disease is considered to be in remission.
In IBD factor or factors trigger the body's immune system to
produce an inflammatory reaction in the intestinal tract that
continues without control. As a result of the inflammatory
reaction, the intestinal wall is damaged leading to bloody diarrhea
and abdominal pain.
[0011] U.S. Pat. No. 6,420,526 entitled "186 Secreted Proteins"
claims unspecified and unexemplified isolated fragments of TREM-1
containing at least 30 contiguous amino acids of human TREM-1. No
biological data relating to such fragments are provided.
[0012] As described in US2003165875A, fusion proteins between human
IgG1 constant region and the extracellular domain of mouse TREM-1
or that of human TREM-1 show an effect against endotoxemia in
mice.
[0013] The inventors have surprisingly found that certain peptides
derived from the TREM-1 protein are capable of acting as
antagonists of the TREM-1 protein and therefore have applications
in the treatment of sepsis, septic shock and inflammatory bowel
disease (IBD). The Inventors further demonstrate that the same
peptides also modulate in vivo the pro-inflammatory cascade
triggered by infection, thus inhibiting hyper-responsiveness and
death in an animal model of sepsis, and that blocking TREM-1
attenuates the symptoms of IBD in mice.
[0014] Previously, the Inventors have identified a soluble form of
TREM-1 (sTREM-1) and observed significant levels in serum samples
from septic shock patients but not controls. As also described
herein the Inventors have investigated its putative role in the
modulation of inflammation during sepsis (see Gibot et al. (2004)
Ann. Intern. Med. 141(1):9-15 and Gibot et al. (2004) N. Engl. J.
Med. 350(5):451-8).
[0015] As described herein the Inventors show that a soluble form
of TREM-1 (sTREM-1) is released in the peripheral blood during
infectious aggression in mouse. The Inventors also confirm
monocytes as a major source of sTREM, and show that synthetic
peptides mimicking a part of the extra-cellular domain of TREM-1
can modulate cytokine production by activated monocytes in
vitro.
[0016] The Inventors have observed that sTREM-1 is secreted by
monocytes activated in vitro by LPS, as well as in the serum of
animals involved in an experimental model of septic shock. Both in
vitro and in vivo, synthetic peptides mimicking a short highly
conserved domain of sTREM-1 attenuate cytokine production by human
monocytes and protect septic animals from hyper-responsiveness and
death. These peptides are efficient not only in preventing but also
in down-regulating the deleterious effects of pro-inflammatory
cytokines. These data demonstrate that in vivo modulation of TREM-1
by TREM-1 peptides is a valuable therapeutic tool for the treatment
of infection, for example sepsis or septic shock or for the
treatment of sepsis-like conditions.
[0017] Accordingly, the present invention provides methods and
compositions for the treatment of infectious disease, in
particular, sepsis and septic shock or for the treatment of
sepsis-like conditions.
[0018] Other diseases or disorders that may also be treated by the
methods and compositions of the present invention include any
inflammatory disorder (or other disorder) that is mediated by the
binding of the TREM-1 ligand to a TREM-1 receptor. Examples of
inflammatory disorders include (but are not limited to) acute and
chronic inflammatory disorders, sepsis, acute endotoxemia,
encephalitis, Chronic Obstructive Pulmonary Disease (COPD),
allergic inflammatory disorders, asthma, pulmonary fibrosis,
pneumonia, Community acquired pneumonia (CAP), Ventilator
associated pneumonia (VAP), Acute respiratory infection, Acute
respiratory distress syndrome (ARDS), Infectious lung diseases,
Pleural effusion, Peptic ulcer, Helicobacter pylori infection,
hepatic granulomatosis, arthritis, rheumatoid arthritis,
osteoarthritis, inflammatory osteolysis, ulcerative colitis,
psoriasis, vasculitis, autoimmune disorders, thyroiditis,
Meliodosis, (mesenteric) Ischemia reperfusion, Filovirus infection,
Infection of the urinary tract, Bacterial meningitis, Salmonella
enterica infection, Marburg and Ebola viruses infections, and in
particular Inflammatory Bowel Disease (IBD).
[0019] As described herein, the Inventors have determined that
several peptides of the extracellular portion of the TREM-1 protein
(see Table 1), which incorporate sequences from "CDR2" and "CDR3"
surprisingly have activity similar to previously described fusion
proteins of IgG1 constant region and the extracellular domain of
TREM-1 in models of sepsis. These peptides also have advantages
over the protein particularly in terms of cost of manufacture.
[0020] Thus, the invention provides polypeptides comprising one or
more sequences derived from CDR2 or CDR3 of a TREM-1 protein.
Preferably, said polypeptides comprise less than 30 contiguous
amino acids of said TREM-1 protein.
[0021] As shown in Table 1, examples of such peptides or
polypeptides, contain or comprise for example 15-25 amino acid
("AA") peptides from the TREM-1 protein and contain or comprise all
or part of a CDR domain (3-6 AAs) of the receptor flanked by
natural sequences from the protein that can vary in length so long
as function of the CDR-like domain is not lost. Such peptides are
derived from the TREM-1 receptor protein amino acid sequence for
example, as shown in Table 2 (human) and Table 3 (mouse).
[0022] Table 1 shows peptides derived from mouse TREM-1 "mPX" (NCBI
Reference Sequences (RefSeq) NP_067381) or human TREM-1 "hPX''"
(NCBI Reference Sequences (RefSeq) NP_061113). Underlined amino
acids span the human TREM-1 Complementarity Determining Regions
(CDR), as described by Radaev et al. 2003 Structure (Camb.) 11
(12), 1527-1535 (2003).
[0023] Table 2 shows the human TREM-1 amino acid sequence
NP_061113. Underlined amino acids span the human TREM-1
Complementarity Determining Regions (CDR) 2 (RPSKNS; [SEQ ID
NO:20]) and 3 (QPPKE [SEQ ID NO:21]), as described by Radaev et al.
2003 Structure (Camb.) 11 (12), 1527-1535 (2003).
[0024] Table 3 shows the mouse TREM-1 amino acid sequence
NP_067381. Underlined amino acids span the mouse TREM-1
Complementarity Determining Regions (CDR) 2 (RPFTRP; [SEQ ID
NO:22]) and 3 (HPPND; [SEQ ID NO:23]).
TABLE-US-00001 TABLE 1 Peptides including sequences from human and
mouse TREM-1 CDR 2 and CDR 3 hCDR 2 mP1(67-89):
LVVTQRPFTRPSEVHMGKFTLKH [SEQ ID NO: 3] hP1(67-89):
LACTERPSKNSHPVQVGRIILED [SEQ ID NO: 16] hCDR 3 mP2(114-136):
VIYHPPNDPVVLFHPVRLVVTKG [SEQ ID NO: 4] mP4(103-123):
LQVTDSGLYRCVIYHPPNDPV [SEQ ID NO: 6] mP5(103-119):
LQVTDSGLYRCVIYHPP [SEQ ID NO: 7] hP2(114-136):
VIYQPPKEPHMLFDRIRLVVTKG [SEQ ID NO: 17] hP4(103-123):
LQVEDSGLYQCVIYQPPKEPH [SEQ ID NO: 18] hP5(103-119):
LQVEDSGLYQCVIYQPP [SEQ ID NO: 19]
TABLE-US-00002 TABLE 2 Human TREM-1 amino acid sequence NP_061113 1
MRKTRLWGLL WMLFVSELRA ATKLTEEKYE LKEGQTLDVK CDYTLEKFAS SQKAWQIIRD
61 GEMPKTLACT ERPSKNSHPV QVGRIILEDY HDHGLLRVRM VNLQVEDSGL
YQCVIYQPPK 121 EPHMLFDRIR LVVTKGFSGT PGSNENSTQN VYKIPPTTTK
ALCPLYTSPR TVTQAPPKST 181 ADVSTPDSEI NLTNVTDIIR VPVFNIVILL
AGGFLSKSLV FSVLFAVTLR SFVP [SEQ ID NO: 1]
TABLE-US-00003 TABLE 3 Mouse TREM-1 amino acid sequence NP_067381 1
MRKAGLWGLL CVFFVSEVKA AIVLEEERYD LVEGQTLTVK CPFNIMKYAN SQKAWQRLPD
61 GKEPLTLVVT QRPFTRPSEV HMGKFTLKHD PSEAMLQVQM TDLQVTDSGL
YRCVIYHPPN 121 DPVVLFHPVR LVVTKGSSDV FTPVIIPITR LTERPILITT
KYSPSDTTTT RSLPKPTAVV 181 SSPGLGVTII NGTDADSVST SSVTISVICG
LLSKSLVFII LFIVTKRTFG [SEQ ID NO: 2]
[0025] Accordingly, the invention provides isolated or
recombinantly prepared polypeptides or peptides comprising or
consisting essentially of one or more sequences derived from CDR2
or CDR3 of a TREM-1 protein, or fragments, homologues, derivatives,
fusion proteins or variants of such polypeptides, as defined
herein, which are herein collectively referred to as "polypeptides
or peptides of the invention" or "TREM-1 peptides or TREM-1
polypeptides", preferably such entities comprise less than 30
contiguous amino acids of a TREM-1 protein, for example as shown in
Table 2 or Table 3. Generally where polypeptides or proteins of the
invention or fragments, homologues, derivatives, or variants
thereof are intended for use (for example treatment) in a
particular species, the sequences of CDR2 or CDR3 of a TREM-1
protein are chosen from the TREM-1 protein amino acid sequence of
that species, or if the sequence is not known, an analogous
species. For example, polypeptides or proteins of the invention for
the treatment of human disease, in particular sepsis, septic shock
or sepsis-like conditions, will comprise one or more sequences
comprising all or part of CDR2 or CDR3 from the human TREM-1
protein.
[0026] Furthermore, the invention provides isolated polypeptides or
proteins comprising an amino acid sequence that is at least about
60%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identical to the amino
acid sequence of SEQ ID NO:20, 21, 22, 23 or fragments, homologues,
derivatives, or variants thereof. The invention also provides
isolated peptides, polypeptides or proteins comprising an amino
acid sequence that comprises or consists of at least about 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, or 29 or more contiguous amino acids of a
TREM-1 protein of which 3 or more contiguous amino acids are
derived from the sequences of SEQ ID NO:20, 21, 22 or 23 (in other
words a sequence representing all, or part of CDR2 or CDR3 of a
TREM-1 protein is present in the peptide, polypeptide or protein),
or fragments, homologues, derivatives, or variants thereof. In
preferred embodiments, such peptides, polypeptides or proteins, or
fragments, homologues, derivatives or variants thereof have a
biological activity of a TREM-1 full-length protein, such as
antigenicity, immunogenicity, triggering of proinflammatory
chemokines and cytokines, mobilization of cytosolic Ca.sup.2+,
protein tyrosine-phosphorylation, mediator release, and other
activities readily assayable. Generally, such peptides,
polypeptides or proteins or fragments, homologues, derivatives or
variants thereof are capable of treating sepsis, septic shock or
sepsis-like conditions, or are active in experimental models of
sepsis, septic shock or sepsis-like conditions, for example by
acting as antagonists of the activity of the TREM-1 receptor. Such
peptides, polypeptides or proteins or fragments, homologues,
derivatives or variants thereof are characterised by the ability to
treat, ameliorate, or lessen the symptoms of sepsis, septic shock
or sepsis-like conditions.
[0027] In particular, the invention provides, a TREM-1 polypeptide
having activity against sepsis, septic shock or sepsis-like
conditions which consists of (i) a contiguous sequence of 5 to 29,
for example 15-25, amino acids corresponding to the native TREM-1
protein sequence which includes at least 3 amino acids from the
CDR2 or CDR3 sequences; or (ii) such a sequence in which one or
more (e.g. one, two or three) amino acids are substituted
conservatively with another amino acid provided, however that at
least 3 amino acids from the CDR2 or CDR3 sequences are not
substituted; or (iii) a sequence of (i) or (ii) linked at one or
both of its N and C termini to a heterologous polypeptide. For
example, in a polypeptide wherein the native TREM-1 protein
sequence is the human sequence identified as (SEQ ID NO: 1), the
CDR2 and CDR3 sequences are RPSKNS (SEQ ID NO:20) and QPPKE (SEQ ID
NO:21) respectively. In such polypeptides, the at least 3 amino
acids from the CDR2 or CDR3 sequences can be QPP, PPK, PKE, RPS,
PSK, SKN or KNS. Such polypeptides may comprise the sequence
QPPK([SEQ ID NO:24), QPPKE (SEQ ID NO:21) or RPSKNS (SEQ ID NO:20).
For example, in a polypeptide wherein the native TREM-1 protein
sequence is the mouse sequence identified as (SEQ ID NO: 2) the
CDR2 and CDR3 sequences are RPFTRP (SEQ ID NO:22) and HPPND (SEQ ID
NO:23) respectively. In such polypeptides, the at least 3 amino
acids from the CDR2 or CDR3 sequences can be HPP, PPN, PND, RPF,
PFT, FTR or TRP. Such polypeptides may comprise the sequences HPP,
HPPN (SEQ ID NO:25), HPPND (SEQ ID NO:23) or RPFTRP (SEQ ID
NO:22).
[0028] In certain embodiments, the polypeptide of the invention is
or comprises SEQ ID No. 7 which is disclosed in Gibot et al (2004)
J Exp Med 200, 1419-1426.
[0029] In certain embodiments the polypeptide of the invention
neither is nor comprises SEQ ID No. 7.
[0030] In certain embodiments the polypeptide of the invention is
or comprises a sequence selected from SEQ ID Nos. 3, 4 and 6.
[0031] In certain embodiments the polypeptide of the invention is
or comprises a sequence selected from SEQ ID Nos. 16, 17, 18 and
19.
[0032] In certain embodiments the polypeptide of the invention is
or comprises a sequence derived from CDR2.
[0033] In certain embodiments the polypeptide of the invention is
or comprises a sequence derived from CDR3.
[0034] The polypeptides or peptides of the invention are provided
for use in therapy, in particular in the treatment of sepsis,
septic shock and sepsis-like conditions, and for use in the
manufacture of a medicament for the treatment of sepsis, septic
shock and sepsis-like conditions. Further provided are compositions
and pharmaceutical compositions containing polypeptides or peptides
of the invention and methods of treatment of sepsis, septic shock
and sepsis-like conditions using polypeptides or peptides of the
invention. In addition the polypeptides or peptides of the
invention are provided for use in therapy to restore haemodynamic
parameters in sepsis, septic shock and sepsis-like conditions and
for use in the manufacture of a medicament for the treatment of
aberrant haemodynamic parameters in sepsis, septic shock and
sepsis-like conditions.
[0035] The term "triggering receptor expressed on myeloid cells" or
"TREM" refers to a group of activating receptors which are
selectively expressed on different types of myeloid cells, such as
mast cells, monocytes, macrophages, dendritic cells (DCs), and
neutrophils, and may have a predominant role in immune and
inflammatory responses. TREMs are primarily transmembrane
glycoproteins with a Ig-type fold in their extracellular domain
and, hence, belong to the Ig-SF. These receptors contain a short
intracellular domain, but lack docking motifs for signaling
mediators and require adapter proteins, such as DAP12, for cell
activation.
[0036] The term "myeloid cells" as used herein refers to a series
of bone marrow-derived cell lineages including granulocytes
(neutrophils, eosinophils, and basophils), monocytes, macrophages,
and mast cells. Furthermore, peripheral blood dendritic cells of
myeloid origin, and dendritic cells and macrophages derived in
vitro from monocytes in the presence of appropriate culture
conditions, are also included.
[0037] The term "sepsis, septic shock" or "sepsis or septic shock"
as defined herein, refers to sub-groups of systemic inflammatory
response syndrome (SIRS). The term "sepsis" is generally reserved
for SIRS when infection is suspected or proven. A pattern of
physiological variables have been shown in critically ill patients
in response to a range of insults including; trauma, burns,
pancreatitis and infection. These include inflammatory responses,
leucocytosis or severe leucopaenia, hyperthermia or hypothermia,
tachycardia and tachypnoea and have been collectively termed the
systemic inflammatory response syndrome (SIRS). This definition
emphasises the importance of the inflammatory process in these
conditions regardless of the presence of infection. Sepsis is
further stratified into severe sepsis when there is evidence of
organ hypoperfusion, made evident by signs of organ dysfunction
such as hypoxaemia, oliguria, lactic acidosis or altered cerebral
function. "Septic shock" is severe sepsis usually complicated by
hypotension, defined in humans as systolic blood pressure less than
90 mmHg despite adequate fluid resuscitation. Sepsis and SIRS may
be complicated by the failure of two or more organs, termed
multiple organ failure (MOF), due to disordered organ perfusion and
oxygenation. In addition to systemic effects of infection, a
systemic inflammatory response may occur in severe inflammatory
conditions such as pancreatitis and burns. The appearance of signs
of an inflammatory response is less well defined following
traumatic insults. In the intensive care unit, gram-negative
bacteria are implicated in 50 to 60% of sepsis cases with
gram-positive bacteria accounting for a further 35 to 40% of cases.
The remainder of cases are due to the less common causes of fungi,
viruses and protozoa.
[0038] The term "sepsis-like conditions" as used herein refers to
those states in which a patient presents with symptoms similar to
sepsis or septic shock but where an infectious agent is not the
primary or initial cause of a similar cascade of inflammatory
mediators and/or change in haemodynamic parameters as seen in cases
of sepsis, for example in patients with acute or chronic liver
failure (see Wasmuth H E, et al. J Hepatol. 2005 February; 42(2):
195-201), in cases of post-resuscitation disease after cardiac
arrest (see Adrie C et al. Curr Opin Crit Care. 2004 June;
10(3):208-12) in the treatment of sepsis-like symptoms after cancer
chemotherapy (seeTsuji E et al. Int J Cancer. 2003 Nov. 1;
107(2):303-8) in patients undergoing hyperthermic isolated limb
perfusion with recombinant TNF-alpha or similar treatments (see
Zwaveling J H et al. Crit Care Med. 1996 May; 24(5):765-70) or
sepsis-like illness in neonates (see Griffin MP et al. Pediatr Res.
2003 June; 53(6):920-6).
[0039] The term "activity against sepsis, septic shock or
sepsis-like conditions" as used herein refers to the capability of
a molecule, for example a peptide, polypeptide or engineered
antibody, to treat sepsis, septic shock or sepsis-like conditions,
or be active in experimental models of sepsis, septic shock or
sepsis-like conditions, for example by acting as an antagonist of
the activity of the TREM-1 receptor.
[0040] Typically the indication for polypeptides of the invention
is sepsis or septic-shock or Inflammatory Bowel Disease (IBD).
Other indications may include any inflammatory disorder (or other
disorder) that is mediated by the binding of the TREM-1 ligand to a
TREM-1 receptor. Examples of inflammatory disorders include (but
are not limited to) acute and chronic inflammatory disorders,
sepsis, acute endotoxemia, encephalitis, Chronic Obstructive
Pulmonary Disease (COPD), allergic inflammatory disorders, asthma,
pulmonary fibrosis, pneumonia, Community acquired pneumonia (CAP),
Ventilator associated pneumonia (VAP), Acute respiratory infection,
Acute respiratory distress syndrome (ARDS), Infectious lung
diseases, Pleural effusion, Peptic ulcer, Helicobacter pylori
infection, hepatic granulomatosis, arthritis, rheumatoid arthritis,
osteoarthritis, inflammatory osteolysis, ulcerative colitis,
psoriasis, vasculitis, autoimmune disorders, thyroiditis,
Meliodosis, (mesenteric) Ischemia reperfusion, Filovirus infection,
Infection of the urinary tract, Bacterial meningitis, Salmonella
enterica infection, Marburg and Ebola viruses infections.
[0041] The term "substantial sequence identity", when used in
connection with peptides/amino acid sequences, refers to
peptides/amino acid sequences which are substantially identical to
or similar in sequence, giving rise to a homology in conformation
and thus to similar biological activity. The term is not intended
to imply a common evolution of the sequences.
[0042] Typically, peptides/amino acid sequences having "substantial
sequence identity" are sequences that are at least 50%, more
preferably at least 80%, identical in sequence, at least over any
regions known to be involved in the desired activity. Most
preferably, no more than five residues, other than at the termini,
are different. Preferably, the divergence in sequence, at least in
the aforementioned regions, is in the form of "conservative
modifications".
[0043] To determine the percent sequence identity of two
peptides/amino acid sequences or of two nucleic acid sequences, the
sequences are aligned for optimal comparison purposes (e.g., gaps
can be introduced in one or both of a first and a second amino acid
or nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). For example,
the length of a reference sequence aligned for comparison purposes
is at least 30%, preferably at least 40%, more preferably at least
50%, even more preferably at least 60%, and even more preferably at
least 70%, 80%, or 90% of the length of the reference sequence
(e.g., when aligning a second sequence to the first amino acid
sequence which has for example 100 amino acid residues, at least
30, preferably at least 40, more preferably at least 50, even more
preferably at least 60, and even more preferably at least 70, 80 or
90 amino acid residues are aligned). The amino acid residues or
nucleotides at corresponding amino acid positions or nucleotide
positions are then compared. When a position in the first sequence
is occupied by the same amino acid residue or nucleotide as the
corresponding position in the second sequence, then the molecules
are identical at that position (as used herein amino acid or
nucleic acid "identity" is equivalent to amino acid or nucleic acid
"homology"). The percent identity between the two sequences is a
function of the number of identical positions shared by the
sequences, taking into account the number of gaps, and the length
of each gap, which need to be introduced for optimal alignment of
the two sequences. The comparison of sequences and determination of
percent identity between two sequences can be accomplished using a
mathematical algorithm. In one embodiment, the percent identity
between two amino acid sequences is determined using the Needleman
and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has
been incorporated into the GAP program in the GCG software package
(available online at the GCG website), using either a Blossom 62
matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8,
6, or 4, and a length weight of 1, 2, 3, 4, 5, or 6. In another
embodiment, the percent identity between two nucleotide sequences
is determined using the GAP program in the GCG software package
(available online at the GCG website), using a NWSgapdna.CMP matrix
and a gap weight of 40, 50, 60, 70, or 80, and a length weight of
1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity
between two amino acid or nucleotide sequences is determined using
the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989))
which has been incorporated into the ALIGN program (version 2.0),
using a PAM120 weight residue table, a gap length penalty of 12,
and a gap penalty of 4. The nucleic acid and protein sequences of
the present invention can further be used as a "query sequence" to
perform a search against public databases to identify, for example,
other family members or related sequences. Such searches can be
performed using the NBLAST and XBLAST programs (version 2.0) of
Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide
searches can be performed with the NBLAST program, score=100,
wordlength=12 to obtain nucleotide sequences homologous to NIP2b,
NIP2cL, and NIP2cS nucleic acid molecules of the invention. BLAST
protein searches can be performed with the XBLAST program,
score=50, wordlength=3 to obtain amino acid sequences homologous to
NIP2b, NIP2cL, and NIP2cS protein molecules of the invention. To
obtain gapped alignments for comparison purposes, Gapped BLAST can
be utilized as described in Altschul et al., (1997) Nucleic Acids
Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST
programs, the default parameters of the respective programs (e.g.,
XBLAST and NBLAST) can be used. These programs are publically
available on the National Center for Biotechnology Information
("NCBI") website.
[0044] The terms "protein" and "polypeptide" are used
interchangeably herein. The term "peptide" is used herein to refer
to a chain of two or more amino acids or amino acid analogues
(including non-naturally occurring amino acids), with adjacent
amino acids joined by peptide (--NHCO--) bonds. Thus, the peptides
of the invention include oligopeptides, polypeptides, proteins,
mimetopes and peptidomimetics. Methods for preparing mimetopes and
peptidomimetics are known in the art.
[0045] The terms "mimetope" and "peptidomimetic" are used
interchangeably herein. A "mimetope" of a compound X refers to a
compound in which chemical structures of X necessary for functional
activity of X have been replaced with other chemical structures
which mimic the conformation of X. Examples of peptidomimetics
include peptidic compounds in which the peptide backbone is
substituted with one or more benzodiazepine molecules (see e.g.,
James, G. L. et al. (1993) Science 260:1937-1942) and
"retro-inverso" peptides (see U.S. Pat. No. 4,522,752 to Sisto).
The terms "mimetope" and "peptidomimetic" also refer to a moiety,
other than a naturally occurring amino acid, that conformationally
and functionally serves as a substitute for a particular amino acid
in a peptide-containing compound without adversely interfering to a
significant extent with the function of the peptide. Examples of
amino acid mimetics include D-amino acids. Peptides substituted
with one or more D-amino acids may be made using well known peptide
synthesis procedures. Additional substitutions include amino acid
analogues having variant side chains with functional groups, for
example, b-cyanoalanine, canavanine, djenkolic acid, norleucine,
3-phosphoserine, homoserine, dihydroxyphenylalanine,
5-hydroxytryptophan, 1-methylhistidine, or 3-methylhistidine.
[0046] As used herein an "analogue" of a compound X refers to a
compound which retains chemical structures of X necessary for
functional activity of X, yet which also contains certain chemical
structures which differ from X. An example of an analogue of a
naturally-occurring peptide is a peptide which includes one or more
non-naturally-occurring amino acids. The term "analogue" is also
intended to include modified mimetopes and/or peptidomimetics,
modified peptides and polypeptides, and allelic variants of
peptides and polypeptides. Analogues of a peptide will therefore
produce a peptide analogue that is substantially homologous or, in
other words, has substantial sequence identity to the original
peptide. The term "amino acid" includes its art recognized meaning
and broadly encompasses compounds of formula I:
##STR00001##
[0047] Preferred amino acids include the naturally occurring amino
acids, as well as synthetic derivatives, and amino acids derived
from proteins, e.g., proteins such as casein, i.e., casamino acids,
or enzymatic or chemical digests of, e.g., yeast, an animal
product, e.g., a meat digest, or a plant product, e.g., soy
protein, cottonseed protein, or a corn steep liquor (see, e.g.,
Traders' Guide to Fermentation Media, Traders Protein, Memphis,
Tenn. (1988), Biotechnology: A Textbook of Industrial Microbiology,
Sinauer Associates, Sunderland, Mass. (1989), and Product Data
Sheet for Corn Steep Liquor, Grain Processing Corp., IO).
[0048] The term "naturally occurring amino acid" includes any of
the 20 amino acid residues which commonly comprise most
polypeptides in living systems, rarer amino acids found in fibrous
proteins (e.g., 4-hydorxyproline, 5-hydroxylysine,
--N-methyllysine, 3-methylhistidine, desmosine, isodesmosine), and
naturally occurring amino acids not found in proteins (e.g.,
-aminobutyric acid, homocysteine, homoserine, citrulline,
ornithine, canavanine, djenkolic acid, and -cyanoalanine).
[0049] The term "side chain of a naturally occurring amino acid" is
intended to include the side chain of any of the naturally
occurring amino acids, as represented by R in formula I. One
skilled in the art will understand that the structure of formula I
is intended to encompass amino acids such as proline where the side
chain is a cyclic or heterocyclic structure (e.g., in proline R
group and the amino group form a five-membered heterocyclic
ring.
[0050] The term "homologue," as used herein refers to any member of
a series of peptides or polypeptides having a common biological
activity, including antigenicity/immunogenicity and inflammation
regulatory activity, and/or structural domain and having sufficient
amino acid as defined herein. Such homologues can be from either
the same or different species of animals.
[0051] The term "variant" as used herein refers either to a
naturally occurring allelic variation of a given peptide or a
recombinantly prepared variation of a given peptide or protein in
which one or more (e.g. one, two or three) amino acid residues have
been modified by amino acid substitution, addition, or
deletion.
[0052] The term "derivative" as used herein refers to a variation
of given peptide or protein that are otherwise modified, i.e., by
covalent attachment of any type of molecule, preferably having
bioactivity, to the peptide or protein, including non-naturally
occurring amino acids.
[0053] Preferably, such homologues, variants and derivatives are
capable of treating sepsis, septic shock or sepsis-like conditions,
or are active in experimental models of sepsis, septic shock or
sepsis-like conditions, or are capable of treating IBD or other
inflammatory disorder, for example by acting as antagonists of the
activity of the TREM-1 receptor.
[0054] An "isolated" or "purified" peptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free of chemical precursors or other
chemicals when chemically synthesized.
[0055] The language "substantially free of cellular material"
includes preparations of a polypeptide/protein in which the
polypeptide/protein is separated from cellular components of the
cells from which it is isolated or recombinantly produced. Thus, a
polypeptide/protein that is substantially free of cellular material
includes preparations of the polypeptide/protein having less than
about 30%, 20%, 10%, 5%, 2.5%, or 1%, (by dry weight) of
contaminating protein. When the polypeptide/protein is
recombinantly produced, it is also preferably substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the protein preparation. When
polypeptide/protein is produced by chemical synthesis, it is
preferably substantially free of chemical precursors or other
chemicals, i.e., it is separated from chemical precursors or other
chemicals which are involved in the synthesis of the protein.
Accordingly, such preparations of the polypeptide/protein have less
than about 30%, 20%, 10%, 5% (by dry weight) of chemical precursors
or compounds other than polypeptide/protein fragment of interest.
In a preferred embodiment of the present invention,
polypeptides/proteins are isolated or purified.
[0056] In addition to the polypeptides described above,
polypeptides of the invention also encompass those polypeptides
having a common biological activity and/or structural domain and
having sufficient amino acid identity (homologues) as defined
herein. These homologues can be from either the same or different
species of animal, preferably from mammals, more preferably from
rodents, such as mouse and rat, and most preferably from human.
Preferably, they exhibit at least one structural and/or functional
feature of TREM-1, and are preferably, capable of treating sepsis,
septic shock or sepsis-like conditions, for example by acting as
antagonists of the activity of the TREM-1 receptor. Such
modifications include amino acid substitution, deletion, and/or
insertion. Amino acid modifications can be made by any method known
in the art and various methods are available to and routine for
those skilled in the art.
[0057] Additionally, in making amino acid substitutions, generally
the amino acid residue to be substituted can be a conservative
amino acid substitution (i.e. "substituted conservatively"), for
example, a polar residue is substituted with a polar residue, a
hydrophilic residue with a hydrophilic residue, hydrophobic residue
with a hydrophobic residue, a positively charged residue with a
positively charged residue, or a negatively charged residue with a
negatively charged residue. Moreover, generally, the amino acid
residue to be modified is not highly or completely conserved across
species and/or is critical to maintain the biological activities of
the peptide and/or the protein it derives from.
[0058] Peptides of the invention may be directly synthesised in any
convenient way. Generally the reactive groups present (for example
amino, thiol and/or carboxyl) will be protected during overall
synthesis. A proportion of the peptides of the invention, i. e.
those wherein the comprised amino acids are genetically coded amino
acids, will be capable of being expressed in prokaryotic and
eukaryotic hosts by expression systems well known to the man
skilled in the art. Methods for the isolation and purification of
e. g. microbially expressed peptides are also well known.
Polynucleotides which encode these peptides of the invention
constitute further aspects of the present invention. As used
herein, "polynucleotide" refers to a polymer of
deoxyribonucleotides or ribonucleotides, in the form of a separate
fragment or as a component of a larger construct, e. g. an
expression vector such as a plasmid. Polynucleotide sequences of
the invention include DNA, RNA and cDNA sequences. Due to the
degeneracy of the genetic code, of course more than one
polynucleotide is capable of encoding a particular peptide
according to the invention. When a bacterial host is chosen for
expression of a peptide, it may be necessary to take steps to
protect the host from the expressed anti-bacterial peptide. Such
techniques are known in the art and include the use of a bacterial
strain which is resistant to the particular peptide being expressed
or the expression of a fusion peptide with sections at one or both
ends which disable the antibiotic activity of the peptide according
to the invention. In the latter case, the peptide can be cleaved
after harvesting to produce the active peptide. If the peptide
incorporates a chemical modification then the activity/stability of
the expressed peptide may be low, and is only modulated by
post-synthetic chemical modification.
[0059] Furthermore, the invention also encompasses derivatives of
the polypeptides of the invention. For example, but not by way of
limitation, derivatives may include peptides or proteins that have
been modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, etc. Additionally, the derivative may contain one or
more non-classical amino acids. Those skilled in the art will be
aware of various methods for modifying peptides to increase
potency, prolong activity and/or increase half-life. In one example
(WO0210195) the modification is made via coupling through an amide
bond with at least one conformationally rigid substituent, either
at the N-terminal of the peptide, the C-terminal of the peptide, or
on a free amino or carboxyl group along the peptide chain. Other
examples of peptide modifications with similar effects are
described, for example, in WO2004029081, WO03086444, WO03049684,
WO0145746, WO0103723 and WO9101743.
[0060] The invention further provides antibodies that comprise a
peptide or polypeptide of the invention or that mimic the activity
of peptides or polypeptides of the invention. Such antibodies
include, but are not limited to: polyclonal, monoclonal,
bi-specific, multi-specific, human, humanized, chimeric antibodies,
single chain antibodies, Fab fragments, F(ab')2 fragments,
disulfide-linked Fvs, and fragments containing either a VL or VH
domain or even a complementary determining region (CDR) that
specifically binds to a polypeptide of the invention. In another
embodiment, antibodies can also be generated using various phage
display methods known in the art. Techniques to recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using
methods known in the art such as those disclosed in PCT publication
WO 92/22324; Mullinax, et al., BioTechniques, 12(6):864-869, 1992;
and Sawai, et al., 1995, AJRI 34:26-34; and Better, et al., 1988,
Science 240:10411043 (each of which is incorporated by reference in
its entirety). Examples of techniques that can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498; Huston, et al., 1991, Methods in
Enzymology 203:4688; Shu, et al., 1993, Proc. Natl. Acad. Sci. USA
90:7995-7999; and Skerra, et al., 1988, Science 240:1038-1040. For
some uses, including in vivo use of antibodies in humans and in
vitro detection assays, it may be preferable to use chimeric,
humanized, or human antibodies. A chimeric antibody is a molecule
in which different portions of the antibody are derived from
different animal species, such as antibodies having a variable
region derived from a murine monoclonal antibody and a constant
region derived from a human immunoglobulin. Methods for producing
chimeric antibodies are known in the art. See, e.g., Morrison,
1985, Science 229:1202; Oi, et al., 1986, BioTechniques 4:214;
Gillies, et al., 1989, J. Immunol. Methods 125:191-202; U.S. Pat.
Nos. 5,807,715; 4,816,567; and 4,816,397; which are incorporated
herein by reference in their entireties. Humanized antibodies are
antibody molecules from non-human species that bind the desired
antigen having one or more complementarity determining regions
(CDRs) from the non-human species and framework regions from a
human immunoglobulin molecule or in the case of the present
invention, one or more CDRs derived from a TREM-1 protein. As known
in the art, framework residues in the human framework regions can
be substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modelling of the interactions of the CDR and
framework residues to identify framework residues important for
antigen binding and sequence comparison to identify unusual
framework residues at particular positions. See, e.g., Queen, et
al., U.S. Pat. No. 5,585,089; Riechmann, et al., 1988, Nature
332:323, 1988, which are incorporated herein by reference in their
entireties. Antibodies can be humanized using a variety of
techniques known in the art including, for example, CDR-grafting
(EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539;
5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan, 1991, Molecular Immunology, 28(4/5):489-498;
Studnicka, et al., 1994, Protein Engineering, 7(6):805-814;
Roguska, et al., 1994, Proc Natl. Acad. Sci. USA 91:969-973, and
chain shuffling (U.S. Pat. No. 5,565,332), all of which are hereby
incorporated by reference in their entireties.
[0061] Completely human antibodies are particularly desirable for
therapeutic treatment of human patients. Human antibodies can be
made by a variety of methods known in the art including phage
display methods described above using antibody libraries derived
from human immunoglobulin sequences. See U.S. Pat. Nos. 4,444,887
and 4,716,111; and PCT publications WO 98/46645; WO 98/50433; WO
98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO 91/10741,
each of which is incorporated herein by reference in its entirety.
Human antibodies can also be produced using transgenic mice (see
Lonberg and Huszar (1995), Int. Rev. Immunol. 13:65-93). For a
detailed discussion of this technology for producing human
antibodies and human monoclonal antibodies and protocols for
producing such antibodies, see, e.g., PCT publications WO 98/24893;
WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598
877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;
5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and
5,939,598; which are incorporated by reference herein in their
entireties. In addition, companies such as Abgenix, Inc. (Freemont,
Calif.), Medarex (N.J.) and Genpharm (San Jose, Calif.) can be
engaged to provide human antibodies directed against a selected
antigen using technology similar to that described above.
Completely human antibodies which recognize a selected epitope can
be generated using a technique referred to as "guided selection."
In this approach a selected non-human monoclonal antibody, e.g., a
mouse antibody, is used to guide the selection of a completely
human antibody recognizing the same epitope. (Jespers et al., 1988,
Bio/technology 12:899-903). Antibodies fused or conjugated to
heterologous polypeptides may be used in in vitro immunoassays and
in purification methods (e.g., affinity chromatography) well known
in the art. See, e.g., PCT publication Number WO 93/21232; EP
439,095; Naramura, et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat.
No. 5,474,981; Gillies, et al., 1992 Proc. Natl. Acad. Sci. USA
89:1428-1432; and Fell, et al., 1991, J. Immunol. 146:2446-2452,
which are incorporated herein by reference in their entireties.
[0062] In another aspect, the present invention provides methods
for identifying a compound or ligand that binds to or modulates the
activity of a polypeptide of the invention. Such a method comprises
measuring a biological activity of the polypeptide in the presence
or absence of a test compound and identifying test compounds that
alter (increase or decrease) the biological activity of the
polypeptide.
[0063] In one embodiment, the invention provides a fusion protein
comprising a bioactive molecule and one or more domains of a
polypeptide of the invention or fragment thereof. In particular,
the present invention provides fusion proteins comprising a
bioactive molecule recombinantly fused or chemically conjugated
(including both covalent and non-covalent conjugations) to one or
more domains of a polypeptide of the invention or fragments
thereof.
[0064] The present invention further encompasses fusion proteins in
which the polypeptides of the invention or fragments thereof, are
recombinantly fused or chemically conjugated (including both
covalent and non-covalent conjugations) to heterologous
polypeptides (i.e., an unrelated polypeptide or portion thereof,
preferably at least 10, at least 20, at least 30, at least 40, at
least 50, at least 60, at least 70, at least 80, at least 90 or at
least 100 amino acids of the polypeptide) to generate fusion
proteins. The fusion does not necessarily need to be direct, but
may occur through linker sequences.
[0065] In one example, a fusion protein in which a polypeptide of
the invention or a fragment thereof can be fused to sequences
derived from various types of immunoglobulins. For example, a
polypeptide of the invention can be fused to a constant region
(e.g., hinge, CH2, and CH3 domains) of human IgG1 or IgM molecule,
(for example, as described by Hudson & Souriauso (2003) Nature
Medicine 9(1):129-134) so as to make the fused polypeptides or
fragments thereof more soluble and stable in vivo. The short
half-life of antibody fragments can also be extended by
`pegylation`, that is, a fusion to polyethylene glycol (see Leong,
S. R. et al. (2001) Cytokine 16:106-119). In one example of such
fusions, described in WO0183525, Fc domains are fused with
biologically active peptides. A pharmacologically active compound
is produced by covalently linking an Fc domain to at least one
amino acid of a selected peptide. Linkage to the vehicle increases
the half-life of the peptide, which otherwise could be quickly
degraded in vivo.
[0066] Alternatively, non-classical alternative protein scaffolds
(for example see Nygren & Skerra (2004) J Immunol Methods
290(1-2):3-28 or WO03049684) can be used to incorporate, and
replicate the properties of, the peptides of the invention, for
example by inserting peptide sequences derived from TREM-1 CDR2 or
CDR3 into a protein framework to support conformationally variable
loops having structural/functional similarities to CDR2 or CDR3 in
a fixed spatial arrangement.
[0067] Such fusion proteins or scaffold based proteins can be used
as an immunogen for the production of specific antibodies which
recognize the polypeptides of the invention or fragments thereof.
In another preferred embodiment, such fusion proteins or scaffold
based proteins can be administered to a subject so as to inhibit
interactions between a ligand and its receptors in vivo. Such
inhibition of the interaction will block or suppress certain
cellular responses involved in sepsis and septic shock.
[0068] In one aspect, the fusion protein comprises a polypeptide of
the invention which is fused to a heterologous signal sequence at
its N-terminus. Various signal sequences are commercially
available. For example, the secretory sequences of melittin and
human placental alkaline phosphatase (Stratagene; La Jolla, Calif.)
are available as eukaryotic heterologous signal sequences. As
examples of prokaryotic heterologous signal sequences, the phoA
secretory signal (Sambrook, et al., supra; and Current Protocols in
Molecular Biology, 1992, Ausubel, et al., eds., John Wiley &
Sons) and the protein A secretory signal (Pharmacia Biotech;
Piscataway, N.J.) can be listed. Another example is the gp67
secretory sequence of the baculovirus envelope protein (Current
Protocols in Molecular Biology, 1992, Ausubel, et al., eds., John
Wiley & Sons).
[0069] In another embodiment, a polypeptide of the invention can be
fused to tag sequences, e.g., a hexa-histidine peptide, such as the
tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue,
Chatsworth, Calif., 91311), among others, many of which are
commercially available. As described in Gentz, et al., 1989, Proc.
Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine
provides for convenient purification of the fusion protein. Other
examples of peptide tags are the hemagglutinin "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson, et al., 1984, Cell 37:767) and the "flag" tag
(Knappik, et al., 1994, Biotechniques 17(4):754-761). These tags
are especially useful for purification of recombinantly produced
polypeptides of the invention.
[0070] Fusion proteins can be produced by standard recombinant DNA
techniques or by protein synthetic techniques, e.g., by use of a
peptide synthesizer. For example, a nucleic acid molecule encoding
a fusion protein can be synthesized by conventional techniques
including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
reamplified to generate a chimeric gene sequence (see, e.g.,
Current Protocols in Molecular Biology, 1992, Ausubel, et al.,
eds., John Wiley & Sons). The nucleotide sequence coding for a
fusion protein can be inserted into an appropriate expression
vector, i.e., a vector which contains the necessary elements for
the transcription and translation of the inserted protein-coding
sequence. Various host-vector systems and selection systems are
known. In a specific embodiment, the expression of a fusion protein
is regulated by a constitutive promoter. In another embodiment, the
expression of a fusion protein is regulated by an inducible
promoter. In accordance with these embodiments, the promoter may be
a tissue-specific promoter. Expression vectors containing inserts
of a gene encoding a fusion protein can be identified by three
general approaches: (a) nucleic acid hybridization, (b) presence or
absence of "marker" gene functions, and (c) expression of inserted
sequences. In the first approach, the presence of a gene encoding a
fusion protein in an expression vector can be detected by nucleic
acid hybridization using probes comprising sequences that are
homologous to an inserted gene encoding the fusion protein. In the
second approach, the recombinant vector/host system can be
identified and selected based upon the presence or absence of
certain "marker" gene functions (e.g., thymidine kinase activity,
resistance to antibiotics, transformation phenotype, occlusion body
formation in baculovirus, etc.) caused by the insertion of a
nucleotide sequence encoding a fusion protein in the vector. For
example, if the nucleotide sequence encoding the fusion protein is
inserted within the marker gene sequence of the vector,
recombinants containing the gene encoding the fusion protein insert
can be identified by the absence of the marker gene function. In
the third approach, recombinant expression vectors can be
identified by assaying the gene product (i.e., fusion protein)
expressed by the recombinant. Such assays can be based, for
example, on the physical or functional properties of the fusion
protein in in vitro assay systems, e.g., binding with anti-fusion
protein antibody. For long-term, high-yield production of
recombinant proteins, stable expression is preferred. For example,
cell lines which stably express the fusion protein may be
engineered. Rather than using expression vectors which contain
viral origins of replication, host cells can be transformed with
DNA controlled by appropriate expression control elements (e.g.,
promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched medium, and then are
switched to a selective medium. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines that express the differentially expressed or pathway gene
protein. Such engineered cell lines may be particularly useful in
screening and evaluation of compounds that affect the endogenous
activity of the differentially expressed or pathway gene protein.
Once a fusion protein of the invention has been produced by
recombinant expression, it may be purified by any method known in
the art for purification of a protein, for example, by
chromatography (e.g., ion exchange, affinity, particularly by
affinity for the specific antibody, and sizing column
chromatography), centrifugation, differential solubility, or by any
other standard technique for the purification of proteins.
[0071] The present invention also provides methods for treating a
subject suffering from sepsis, septic shock or a sepsis-like
condition by administering a peptide or polypeptide of the
invention. In another embodiment, the modulator may be an antibody
which mimics the activity of a polypeptide of the invention. In
particular, the invention provides a method of treating or
ameliorating sepsis, septic shock or a sepsis-like condition in a
subject, comprising: administering a therapeutically effective
amount of a peptide or polypeptide of any one of the preceding
claims to a subject. In such methods, the peptide or polypeptide
administered can have substantial sequence identity to sequence SEQ
ID NOS: 3, 4, 6, 7, 16, 17, 18 or 19, is SEQ ID NOS: 3, 4, 6, 7,
16, 17, 18 or 19, or an active fragment, analogue or derivative of
SEQ ID NOS: 3, 4, 6, 7, 16, 17, 18 or 19 or has at least about 80%
sequence identity to SEQ ID NOS: 3, 4, 6, 7, 16, 17, 18 or 19.
[0072] In one aspect, the invention provides a method for
preventing sepsis, septic shock or sepsis-like conditions, by
administering to the subject a peptide or polypeptide of the
invention. Subjects at risk of sepsis or septic shock can be
identified by, for example, any diagnostic or prognostic assays as
known in the art (for particularly suitable methods of diagnosis,
see WO2004081233, Gibot et al. (2004) Ann Intern Med. 141 (1):9-15
and Gibot et al. (2004) N Engl J Med. 350(5):451-8. The
prophylactic agents described herein, for example, can be used to
treat a subject at risk of developing disorders such as those
previously discussed. The methods of the invention are applicable
to mammals, for example humans, non human primates, sheep, pigs,
cows, horses, goats, dogs, cats and rodents, such as mouse and rat.
Generally, the methods of the invention are to be used with human
subjects.
[0073] Furthermore, the invention provides a pharmaceutical
composition comprising a polypeptide of the present invention or an
antibody or fragments thereof that mimics a polypeptide of the
invention. The peptides, polypeptides and antibodies (also referred
to herein as "active compounds") of the invention can be
incorporated into pharmaceutical compositions suitable for
administration. Such compositions typically comprise the peptide,
protein, or antibody and a pharmaceutically acceptable carrier.
[0074] As used herein the language "pharmaceutically acceptable
diluent, carrier or excipient" is intended to include any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like,
compatible with pharmaceutical administration. The use of such
media and agents for pharmaceutically active substances is well
known in the art. Except insofar as any conventional media or agent
is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0075] The invention includes methods for preparing pharmaceutical
compositions containing a peptide or polypeptide of the invention.
Such compositions can further include additional active agents.
Thus, the invention further includes methods for preparing a
pharmaceutical composition by formulating a pharmaceutically
acceptable carrier with a peptide or polypeptide of the invention
and one or more additional active compounds.
[0076] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, transdermal (topical),
transmucosal, intraarticular, intraperitoneal, and intrapleural, as
well as oral, inhalation, and rectal administration. Solutions or
suspensions used for parenteral, intradermal, or subcutaneous
application can include the following components: a sterile diluent
such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0077] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
injectability with a syringe exists. It must be stable under the
conditions of manufacture and storage and must be preserved against
the contaminating action of microorganisms such as bacteria and
fungi. The carrier can be a solvent or dispersion medium
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyetheylene glycol, and
the like), and suitable mixtures thereof. The proper fluidity can
be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. Prevention of the
action of microorganisms can be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, ascorbic acid, thimerosal, and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0078] Sterile injectable solutions can be prepared by
incorporating the active compound (e.g., a polypeptide or antibody)
in the required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required, followed
by filtered sterilization. Generally, dispersions are prepared by
incorporating the active compound into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above. In the case of sterile
powders for the preparation of sterile injectable solutions, the
preferred methods of preparation are vacuum drying and
freeze-drying which yields a powder of the active ingredient plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0079] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient, such as starch or lactose; a disintegrating agent, such
as alginic acid, Primogel, or corn starch; a lubricant, such as
magnesium stearate or Sterotes; a glidant, such as colloidal
silicon dioxide; a sweetening agent, such as sucrose or saccharin;
or a flavoring agent, such as peppermint, methyl salicylate, or
orange flavoring.
[0080] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from a pressurized
container or dispenser which contains a suitable propellant, e.g.,
a gas such as carbon dioxide, or a nebulizer.
[0081] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art. The compounds can also be prepared in
the form of suppositories (e.g., with conventional suppository
bases such as cocoa butter and other glycerides) or retention
enemas for rectal delivery.
[0082] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0083] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0084] As defined herein, a therapeutically effective amount of
protein or polypeptide (i.e., an effective dosage) ranges from
about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25
mg/kg body weight, more preferably about 0.1 to 20 mg/kg body
weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg,
3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight.
[0085] For antibodies, the preferred dosage is 0.1 mg/kg to 100
mg/kg of body weight (generally 10 mg/kg to 20 mg/kg). If the
antibody is to act in the brain, a dosage of 50 mg/kg to 100 mg/kg
is usually appropriate. Generally, partially human antibodies and
fully human antibodies have a longer half-life within the human
body than other antibodies. Accordingly, lower dosages and less
frequent administration is often possible. Modifications such as
lipidation can be used to stabilize antibodies and to enhance
uptake and tissue penetration (e.g., into the brain). A method for
lipidation of antibodies is described by Cruikshank, et al., 1997,
J. Acquired Immune Deficiency Syndromes and Human Retrovirology
14:193).
[0086] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0087] The invention further provides a kit containing a peptide or
polypeptide of the invention of the present invention, or an
antibody or fragments thereof mimicking a polypeptide of the
invention, preferably with instructions for use, for example in the
treatment of sepsis, septic shock or sepsis-like conditions.
[0088] The invention provides a method for identifying (or
screening) modulators, i.e., candidate or test compounds or agents
(e.g., peptides, peptidomimetics, small molecules or other drugs)
which mimic a polypeptide of the invention or have a stimulatory or
inhibitory effect on, for example, activity of a polypeptide of the
invention. In particular, the invention provides a method of
screening compounds or compositions to treat sepsis, septic shock
or sepsis-like conditions, comprising: providing a TREM-1 peptide;
contacting an animal in a cecal ligation and puncture model (or
using other assay or model as described herein or known in the art)
with the TREM-1 peptide; determining if there was a modulation in
the sepsis, for example wherein an increase in survival indicates
that the TREM-1 peptide may be useful for treating sepsis, septic
shock or sepsis-like conditions.
[0089] The invention further pertains to novel agents identified by
the above-described screening assays and uses thereof for
treatments as described herein.
[0090] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as if each individual publication were
specifically and individually indicated to be incorporated by
reference herein as though fully set forth.
[0091] Preferred features of each aspect of the invention are
applicable to each other aspect, mutatis mutandis.
[0092] The present invention will now be described with reference
to the following non-limiting examples, with reference to the
figures, in which:
[0093] FIG. 1A. shows a sequence alignment of TREM-1 and TREM-2
family members. Human TREM-1 (SEQ ID NO:1) was aligned with mouse
TREM-1 (SEQ ID NO:2) and human and mouse TREM-2 (SEQ ID NO:26 and
27, respectively in order of appearance) using version 1.74 of
CLUSTAL W. Secondary structure assignments correspond to the
published human TREM-1 structure (arrows for .beta.-strands and
cylinder for a helices) (Radaev et al. (2003) Structure (Camb).
December; 11(12):1527-35). Residues involved in homo-heterodimer
formation are shown in white on black background. Cysteine making
disulfide bonds conserved for V-type Ig fold are in bold. Gaps are
indicated with (-), identical residues with (*), similar with (:
or.). An extended region of similarities between human and mouse
TREM1 sequences is shown in boxes on grey background. TREM-1
peptide sequences used in the Examples herein are indicated
underlined.
[0094] FIG. 1B. shows a ribbon diagram of the published TREM-1
homodimeric structure (Kelker, et al. (2004) J Mol Biol. September
24; 342(4): 1237-48). Postulated binding sites that comprise the
antibody equivalent Complementarity Determining Regions (CDRs) are
in red.
[0095] FIG. 2. shows that administration of TREM-1 peptides, 1 hour
before LPS, reduces death induced by endotoxaemia. BALB/c mice (10
per group) were injected intraperitoneally with 200 .mu.g LPS. The
TREM-1 peptides P1, P2, P3, or P5 (200 .mu.l of a 300 .mu.M
solution per mouse) were injected intraperitoneally 1 hour before
LPS. Viability of mice was monitored twice a day for 7 days.
Statistical analysis was performed by Logrank test. Data from
control mice represent cumulative survival curves from two
independent experiments performed under identical conditions.
[0096] FIG. 3. shows that TREM-1 peptide P1 is able to effectively
reduce death induced by endotoxaemia when injected at 4 hours after
LPS. BALB/c mice (10 per group) were injected intraperitoneally
with 200 .mu.g LPS. TREM-1 peptide P1, 200 .mu.l of a 300 .mu.M
solution per mouse was injected intraperitoneally 1 hour before or
4 hours after LPS. Viability of mice was monitored twice a day for
7 days. Statistical analysis was performed with the Logrank test.
Data from control mice represent cumulative survival curves from
two independent experiments performed under identical
conditions.
[0097] FIG. 4. shows that administration of TREM-1 peptides, 4
hours after LPS, reduces death induced by endotoxaemia. BALB/c mice
(10 per group) were injected intraperitoneally with 200 .mu.g LPS.
P1 peptide, 200 .mu.l of a 150, 300 and 600 .mu.M solution per
mouse (dots) or P3, 200 .mu.l of a 600 .mu.M solution per mouse
(filled squares) were injected intraperitoneally 4 hours after LPS.
Viability of mice was monitored twice a day for 7 days. Statistical
analysis was performed with the Logrank test.
[0098] FIG. 5. shows that TREM-1 peptide P1 protects against cecal
ligation and puncture (CLP). CLP was induced in C57BL/6 mice (15
per group) as described in Materials and Methods. P1 peptide (empty
dots) or P3 peptide (filled squares) (200 .mu.l of a 600 .mu.M
solution per mouse) were injected intraperitoneally 5 and 24 hours
after CLP induction. Viability of mice was monitored twice a day
for 10 days. Statistical analysis was performed with the Logrank
test.
[0099] FIG. 6. shows that P1, P2 and P5 peptides, but not P3
peptide, inhibit the binding of soluble TREM-1/IgG1 to TREM-1
Ligand positive peritoneal exudate cells. Cytofluorimetric analysis
of peritoneal exudate cells with 2 .mu.g/ml of mouse TREM-1/hIgG1
in the presence of a 500 .mu.M solution per mouse (thin line), 100
.mu.M solution per mouse (dotted line) or absence (thick line) of
the peptides is shown. The grey histogram represents immunostaining
with human IgG1 as a control.
[0100] FIG. 7A. shows the release of sTREM-1 from cultured
monocytes after stimulation with LPS with and without proteases
inhibitor. LPS stimulation induced the appearance of a 27-kD
protein that was specifically recognized by an anti-TREM-1 mAb
(inset). sTREM-1 levels in the conditioned culture medium were
measured by reflectance of immunodots. Data are shown as mean
.+-.SD (n=3).
[0101] FIG. 7B. shows expression of TREM-1 mRNA in monocytes.
Cultured monocytes were stimulated with LPS (1 .mu.g/mL) for 0, 1
and 16 hours as indicated. LPS induced TREM-1 mRNA production
within 1 hour.
[0102] FIG. 8A. shows the release of cytokines and sTREM-1 from
cultured monocytes. For cell activation, primary monocytes were
cultured in 24-well flat-bottom tissue culture plates in the
presence of LPS (1 .mu.g/mL). In some experiments this stimulus was
provided in combination with P5 (10 to 100 ng/mL), control peptide
(10 to 100 ng/mL) or rIL-10 (500 U/mL). To activate monocytes
through TREM-1, an agonist anti-TREM-1 mAb (10 .mu.g/mL) was added
as indicated. Cell-free supernatants were analysed for production
of TNF-.alpha., IL-1.beta. and sTREM-1 by ELISA or immunodot. All
experiments were performed in triplicate and data are expressed as
means (SEM). [0103] a: Media [0104] b: P5 10 ng/mL [0105] c:
Anti-TREM-1 [0106] d: LPS [0107] e: LPS+Anti-TREM-1 [0108] f:
LPS+P5 10 ng/mL [0109] g: LPS+P5 50 ng/mL [0110] h: LPS+P5 100
ng/mL [0111] is LPS+IL10
[0112] FIG. 8B. shows the effect of P5 on NF.kappa.B activation.
Monocytes were cultured for 24 hours in the presence of E. coli LPS
(O111:B4, 1 .mu.g/mL), anti-TREM-1 mAb (10 .mu.g/mL) and/or P5 (100
ng/mL) as indicated and the levels of NF.kappa.B p50 and p65 were
determined using an ELISA-based assay. Experiments were performed
in triplicate and data are expressed as means of optical densities
(SEM).
[0113] FIG. 9. shows accumulation of sTREM-1 in serum of
LPS-treated mice. Male Balb/C mice (20 to 23 g) were treated with
LPS (LD.sub.50, intraperitoneally). Serum was assayed for sTREM-1
by immunodot. Serum sTREM-1 was readily detectable 1 hour after LPS
administration and was maintained at a plateau level from 4 to 6
hours.
[0114] FIG. 10A. shows that P5 pre-treatment protects against LPS
lethality in mice. Male Balb/C mice (20 to 23 g) were randomly
grouped (10 mice per group) and treated with an LD.sub.100 of LPS.
P5 (50 .mu.g or 100 .mu.g ) or control vector was administered 60
min before LPS.
[0115] FIG. 10B. shows that delayed administration of P5 protects
LPS lethality in mice. Male Balb/C mice (20 to 23 g) were randomly
grouped (8 mice per group) and treated with an LD.sub.100 of LPS.
P5 (75 .mu.g) or control vector was administered 4 or 6 hours after
LPS as indicated.
[0116] FIG. 10C. shows that administration of agonist TREM-1 mAb is
lethal to mice. Male Balb/C mice (20 to 23 g) were randomly grouped
(8 mice per group) and treated with a combination of an LD.sub.50
of LPS+control vector, LD.sub.50 of LPS+anti-TREM-1 mAb (5 .mu.g)
or LD.sub.100 of LPS+control vector as indicated. Control vector
and anti-TREM-1 mAb were administered 1 hour after LPS
injection.
[0117] FIG. 11A. shows that P5 partially protects mice from
CLP-induced lethality. Male Balb/C mice (20 to 23 g) were randomly
grouped and treated with normal saline (n=14) or the control
peptide (n=14, 100 .mu.g) or with P5 (100 .mu.g) in a single
infection at HO (n=18), H+4 (n=18) or H+24 (n=18). The last group
of mice (n=18) was treated with repeated injections of P5 (100
.mu.g) at H+4, H+8 and H+24.
[0118] FIG. 11B. shows the does effect of P5 on survival. Mice
(n=15 per group) were treated with a single injection of normal
saline or 10 .mu.g, 20 .mu.g, 50 .mu.g, 100 .mu.g or 200 .mu.g of
P5 at HO after the CLP and monitored for survival.
[0119] FIG. 12. shows that P5 has no effect on bacterial counts
during CLP. Mice (5 per group) were killed under anaesthesia at 24
hours after CLP. Bacterial counts in peritoneal lavage fluid and
blood were determined and results are expressed as CFU per mL of
blood and CFU per mouse for the peritoneal lavage.
[0120] FIG. 13A and FIG. 13B show TNF-.alpha. and IL-1.beta. plasma
concentration evolution, respectively, after LPS (15 mb/kg)
administration in rats.
[0121] *p<0.05 P5-treated vs Control animals
[0122] .sctn.p<0.05 P5-treated vs P1-treated animals
[0123] FIG. 14 shows Nitrite/Nitrate concentrations evolution after
LPS (15 mg/kg) administration in rats. *p<0.05 P5-treated vs
Control and P1-treated animals.
[0124] FIG. 15 shows mean arterial pressure evolution during caecal
ligation and puncture-induced peritonitis in rats.
[0125] *p<0.05 vs Control animals
[0126] FIG. 16 shows TNF-.alpha. plasma concentration evolution
during caecal ligation and puncture-induced peritonitis in
rats.
[0127] *p<0.05 P5-treated vs Control animals
[0128] .sup..sctn.p<0.05 P1-treated vs Control animals
[0129] .sup.$p<0.05 P5 vs P1-treated animals
[0130] FIG. 17 shows Nitrite/Nitrate concentration evolution during
caecal ligation and puncture-induced peritonitis in rats.
[0131] *p<0.05 P5 and P1-treated vs Control animals
[0132] FIG. 18 shows that hP5 efficiently protects mice from LPS
induced septic shock (see Example 5). Septic shock was induced in
male Balb/c mice (n=15/group) with 200 mg of LPS as described in
Material and Methods. hP5 peptide or control were administered at
the following time points: -1 h, 0 h, +1 h +4 h. Mortality was
followed twice a day for 7 days. Data were analysed using GraphPad
Prism Survival curve analysis. Log-rank (Mantel-Cox) test showed a
statistically significant difference between the two curves
(p=0.0003).
[0133] FIG. 19. shows the results for male C57BL/6 mice
(n=15/group) that were subject to Cecal Ligation and Puncture as
described in Example 6 and treated with hP5 peptide or scrambled
peptide control at the following time points: -1 h, 0 h, +4 h and
+24 h. Mortality was followed twice a day for 10 days. Data were
analyzed using GraphPad Prism Survival curve analysis.
[0134] FIG. 20. Disease Activity Index (DAI). Colitis was induced
by oral administration of DSS (see Example 7). Upon colitis
induction, mice (n=8) were daily treated either with hP5 or its
scrambled control peptide. Animal weight, haemoccult or presence of
gross blood and stool consistency were used to determine the DAI
score as indicated in Example 7. *(grey)=DSS+hP5 versus
DSS+scrambled peptide; *(black)=DSS+hP5 vs DSS+vehicle;
***p.ltoreq.0.001, **p.ltoreq.0.01, *p.ltoreq.0.05.
[0135] FIG. 21. Body weight loss. Colitis was induced by oral
administration of DSS (see Example 7). Upon colitis induction, mice
were daily treated either with hP5 or its scrambled control
peptide. Weight was daily measured and percentage of weight loss
versus day 0 was calculated. *(grey)=DSS+hP5 versus DSS+scrambled
peptide; *(black)=DSS+hP5 vs DSS+vehicle; ***p.ltoreq.0.001,
**p.ltoreq.0.01.
[0136] FIG. 22. Haemoccult or presence of gross blood in the feces.
Colitis was induced by oral administration of DSS (see Example 7).
Upon colitis induction, mice were daily treated either with hP5 or
its scrambled control peptide. Haemoccult or presence of gross
blood was detected and score assigned as measured and score
assigned at the indicated time points as described in. Example 7.
*(grey)=DSS+hP5 versus DSS+scrambled peptide; *(black)=DSS+hP5 vs
DSS+vehicle; ***p.ltoreq.0.001, **p.ltoreq.0.01,
*p.ltoreq.0.05.
[0137] FIG. 23. Extent of diarrhea. Colitis was induced by oral
administration of DSS (see Example 7). Upon colitis induction, mice
were daily treated either with hP5 or its scrambled control
peptide. Stool consistency was measured and score assigned at the
indicated time points as described in Example 7. *(grey)=DSS+hP5
versus DSS+scrambled peptide; *(black)=DSS+hP5 vs DSS+vehicle;
***p.ltoreq.0.001, **p.ltoreq.0.01, *p.ltoreq.0.05.
[0138] FIG. 24. Shortening of the colon. Colitis was induced by
oral administration of DSS (see Example 7). Upon colitis induction,
mice were daily treated either with hP5 or its scrambled control
peptide. At day 11 mice were euthanized and the colon length of
each mouse measured from the anus to the end of the cecum.
***p.ltoreq.0.001, **p.ltoreq.0.01, *p.ltoreq.0.05.
EXAMPLE 1
TREM-1 Peptides Protect Mice From Death by Septic Shock
[0139] TREM-1 peptides matching the following criteria were
synthesized: i) highest homology between human and mouse TREM-1 and
lowest homology with TREM-2. ii) peptides spanning the
Complementarity Determining Regions (CDRs) of TREM-1. According to
the published crystal structure of TREM-1, and in analogy with
antibodies, these residues are likely to be involved in cognate
ligand recognition (Radaev et al. (2003) Structure (Camb).
December; 11(12):1527-35 & Kelker, et al. (2004) J Mol Biol.
September 24; 342(4):1237-48) (see FIG. 1A and FIG. 1B). One
peptide (P1) was designed in the CDR2 region and three peptides
(P2, P4 and P5) in the CDR3 region. A fourth peptide (P3) was
designed in the neck region connecting the V-type immunoglobulin
(Ig)-like domain (Ig-V) to the transmembrane domain. No peptide was
designed in the CDR1 region due to high sequence homology between
TREM-1 and TREM-2.
[0140] Thus, the following peptides of the TREM-1 protein were
ordered from and were synthesized and purified by the Protein and
Peptide Chemistry Facility, Institute of Biochemistry, University
of Lausanne:
TABLE-US-00004 P1 (CDR2 67-89) LVVTQRPFTRPSEVHMG [SEQ ID KFTLKH NO:
3] P2 (CDR3 114-136) VIYHPPNDPVVLFHPVR [SEQ ID LVVTKG NO: 4] P3
(neck region TTTRSLPKPTAVVSSPG [SEQ ID 168-184) NO: 5] P4 (CDR3
103-123) LQVTDSGLYRCVIYHPP [SEQ ID NDPV NO: 6] P5 (CDR3 103-119)
LQVTDSGLYRCVIYHPP [SEQ ID NO: 7] P1sc* (P1 scrambled
LTPKHGQRSTHVTKFRV [SEQ ID seq.) FEPVML NO: 8] P5sc* (P5 scrambled
TDSRCVIGLYHPPLQVY [SEQ ID seq.) NO: 9] *This is a control peptide
and indeed does not protect
[0141] In the experiments of this example, the peptides were
administered in a volume of 200 .mu.l of the solution molarity
indicated. To assess the ability of TREM1-peptides to protect mice
from LPS-induced endotoxaemia, the Inventors administered peptides
P1, P2, P3 and P5 (300 .mu.M) 1 hour before a lethal dose of
lipopolysaccharide (LPS) (FIG. 2). Lethality was monitored over
time and compared with animals that had received control injections
of vehicle alone. P5 injection confers maximal protection, with 90%
of the animals still alive 7 days after LPS injection, as compared
with 10% of control mice (p<0.001). 60% of the P I-treated mice
and 50% of the P2 treated mice survived endotoxaemia as compared
with 10% of control mice (p<0.01 and p<0.05 respectively).
Interestingly, all P3-treated mice died within 4 days after LPS
injection. These results indicate that peptides containing
sequences of the extracellular portion of TREM-1 corresponding to
the putative ligand binding site (CDR2 and CDR3) can protect mice
from lethal shock.
[0142] In order to investigate whether TREM-1 peptide treatment
could be delayed until after the administration of LPS, the
Inventors injected the peptides at 4 hours after LPS injection.
Only in the case of P1, this delayed treatment conferred
significant protection against a lethal dose of LPS (FIG. 3). 80%
of the mice injected with P1 4 hours after LPS survived
endotoxaemia compared to 60% of mice treated 1 h before LPS and 10%
of mice treated with vehicle alone (p<0.001 and p<0.01
respectively). Thus, P1 is effective even when injected after the
outbreak of endotoxaemia. No late death occurred over one week,
indicating that P1 did not merely delay the onset of LPS lethality,
but provided lasting protection. P1 administration conferred
maximal protection (80%) when administered at 600 .mu.M (p<0.01)
and the level of protection dropped to 50% at 300 .mu.M (p<0.05)
and further down to 30% at 150 .mu.M as compared to 20% of control
mice, indicating a dose dependent effect of P1 (FIG. 4). The
Inventors then investigated whether P1 protects against septic
shock in the "CLP" model (Cecal Ligation and Puncture is a widely
used experimental model of sepsis). Mice treated with two doses of
P1 at 5 and 24 hours after CLP were protected from death as
compared to control treated mice (p=0.0791) although the difference
was not statistically significant. 40% of the mice injected with P1
at 5 days after CLP survived compared to 5% of mice treated with P3
peptide. At 10 days after CLP, the treated mice were still alive,
indicating that P1 did not merely delay mortality, but provided
lasting protection (FIG. 5).
EXAMPLE 2
TREM-1 Peptide P1 Inhibits Binding of Soluble Mouse TREM-1/IgG to
TREM-1 Ligand Positive Cells
[0143] Among TREM-1 derived peptides tested in CLP, peptides P1, P2
and P5 demonstrate a protective activity. A possible mechanism of
action could be the ability of TREM-1 derived peptide to interfere
with TREM-1/TREM-1 ligand interaction. To address this question the
Inventors performed competition experiments on TREM-1 ligand
positive cells: PEC (Peritoneal Exudate Cells) from CLP treated
mice.
[0144] Peritoneal exudate cells (PEC) from mice suffering from a
caecal ligation and puncture (CLP)-induced peritonitis were
subjected to flow cytometry analysis after incubation with a
PE-conjugated anti-human IgG1 (Jackson Immunoresearch, Bar Harbor,
USA). Competition with TREM-1 peptides was performed by
pre-incubating cells with the indicated concentrations of peptides
for 45 minutes on ice before adding mTREM-1-IgG1.
[0145] As shown in FIG. 6, the P1 peptide, derived from the CDR2
region of mTREM-1, and the P2 and P5 peptides spanning the CDR3
region inhibit TREM-1 interaction with its ligand in a dose
dependent manner. Conversely the P3 peptide, derived from the neck
region of TREM-1 connecting the IgG like portion to the
transmembrane domain was ineffective.
EXAMPLE 3
[0146] Additional Studies on the Modulation of the Inflammatory
Response in Murine Sepsis by TREM-1 Peptide P5
[0147] Methods
Preparation of Monocytes from Peripheral Blood
[0148] Ten mL of peripheral blood samples were collected on EDTA-K
from 5 healthy volunteer donors originating from laboratory staff.
After dilution in RPMI (Life Technologies, Grand Island, N.Y.) v/v,
blood was centrifuged for 30 min at room temperature over a Ficoll
gradient (Amersham Pharmacia, Uppsala, Sweden) to isolate PBMC. The
cells recovered above the gradient were washed and counted. In
order to deplete the suspensions of lymphocytes, cells were then
plated in 24-well flat-bottom tissue culture plates (Corning,
Corning, N.Y.) at a concentration of 5.times.10.sup.6/mL and
allowed to adhere during 2 hours at 37.degree. C. The resulting
lymphocyte suspension was discarded and the adhering monocytic
cells were maintained in a 5% CO2 incubator at 37.degree. C. in
complete medium (RPMI 1640, 0.1 mM sodium pyruvate, 2 mM
Penicillin, 50 .mu.g/mL Streptomycin; Life Technologies)
supplemented with 10% FCS (Invitrogen, Cergy, France).
TREM-1 Peptide
[0149] Using the human TREM-1 sequence in Gen-Bank, accession #
AF287008 and the mouse TREM-1 sequence # AF241219, a peptide "P5"
(LQVTDSGLYRCVIYHPP; [SEQ ID NO:7]; was chemically synthesized as a
C-terminally amidated peptide (Pepscan Systems, Lelystad, The
Netherlands). The correct peptide was obtained in greater than 99%
yield and with measured mass of 1961 Da versus a calculated mass of
1962 Da and was homogeneous after preparative purification, as
confirmed by mass spectrometry and analytic reversed phase-high
performance liquid chromatography. A peptide "P5sc" containing the
same amino-acids than P5 but in a different sequence order
(TDSRCVIGLYHPPLQVY; [SEQ ID NO:9]) was similarly synthesized and
served as `control peptide`.
In Vitro Stimulation of Monocytes
[0150] For activation, monocytes were cultured in the presence of
E. coli LPS (O111:64, 1 .mu.g/mL, Sigma-Aldrich, La Verpilliere,
France). Cell viability was assessed by trypan blue exclusion and
by measuring lactate dehydrogenase release. In some experiments,
this stimulus was given in combination with TNF-.alpha. (5 to 100
ng/mL, R&D Systems, Lille, France), IL-1.beta. (5 to 100 ng/mL,
R&D Systems), rIFN-.gamma. (up to 100 U/mL, R&D Systems),
rIL-10 (500 U/mI, R&D Systems) or up to 100 ng/mL of P5 or
control peptide.
[0151] In order to activate monocytes through TREM-1, an
anti-TREM-1 agonist monoclonal antibody (R&D Systems) was added
as follows: flat-bottom plates were precoated with 10 .mu.g/mL
anti-TREM-1 per well. After thorough washing in phosphate buffered
saline (PBS), the monocyte suspensions were added at a similar
concentration as above. Some experiments were performed in the
presence of protease inhibitors (PMSF and Protease Cocktail
Inhibitor; Invitrogen). Cell-free supernatants were assayed for the
production of TNF-.alpha. and IL-1.beta. by ELISA according to the
recommendations of the manufacturer (BD Biosciences, San Diego,
USA). To address the effect of P5 on NF-.kappa.B activity in
monocytes, an ELISA-based assay was performed (BD Mercury.TM.
Transfactor Kit, BD Biosciences). Monocytes were cultured for 24
hours in the presence of E. coli LPS (O111:64, 1 .mu.g/mL), and/or
an agonist anti-TREM-1 monoclonal antibody (10 .mu.g/mL), and/or P5
(100 ng/mL). Whole-cell extracts were then prepared and levels of
NF-.kappa.B p50 and p65 were determined according to the
recommendations of the manufacturer. All experiments were performed
in triplicate and data are expressed as means (SEM).
Identification and Quantitation of sTREM-1 Release
[0152] Primary monocytes suspensions were cultured as described
above. The cells were treated with E. coli LPS (O111:64, 1
.mu.g/mL) for 24 hours at 37.degree. C. Cell-conditioned medium was
submitted to Western-blotting using an anti-TREM-1 monoclonal
antibody (R&D Systems) in order to confirm the presence of 27
kDa material recognized by anti-TREM-1. Soluble TREM-1 levels were
measured by assessing the optical intensity of bands on immunodots
by means of a reflectance scanner and the Quantity One Quantitation
Software (Bio-Rad, Cergy, France) as reported elsewhere (18).
Soluble TREM-1 concentration from each sample was determined by
comparing the optical densities of the samples with reference to
standard curves generated with purified TREM-1. All measurements
were performed in triplicate. The sensitivity of this technique
allows the detection of sTREM-1 levels as low as 5 pg/mL.
TREM-1 RT-PCR
[0153] Total mRNA was extracted from primary monocytes cultured in
the presence of LPS using a TRIzol reagent (Invitrogen), and
reverse transcribed using Superscript RT II (Invitrogen) to
generate cDNA. RT-PCR conditions then used for all reactions were
94.degree. C., 30 s/65.degree. C., 30 s/68.degree. C., 1 min for 30
cycles. Amplification was performed with 2.5 mM MgCl2, 0.2 mM dNTP,
2.0 U Taq polymerase, and 20 pM 5' and 3' oligonucleotide primers
(Proligos, Paris, France).
[0154] The sequences of the 5' and 3' primer pairs used were the
following:
TABLE-US-00005 for TREM-1 (17) [SEQ ID NO: 10]
TTGTCTCAGAACTCCGAGCTGC; and [SEQ ID NO: 11] GAGACATCGGCAGTTGACTTGG;
for TREM-1sv (19) [SEQ ID NO: 12] GGACGGAGAGATGCCCAAGACC; and [SEQ
ID NO: 13] ACCAGCCAGGAGAATGACAATG; for .beta.-actin (used as
housekeeping amplicon) [SEQ ID NO: 14] GGACGACATGGAGAAGATCTGG; and
[SEQ ID NO: 15] ATAGTAATGTCACGCACGATTTCC;
[0155] PCR products were run on agarose gels and visualized by
ethidium bromide staining.
LPS-Induced Endotoxinemia in Mice
[0156] After approval by the local ethical committee, male Balb/C
mice (20 to 23 g) were randomly grouped and treated with E. coli
LPS intraperitoneally (i.p.) in combination with P5 (in 500 .mu.l
normal saline) or control vector before or after LPS challenge. In
some experiments, 5 .mu.g of an anti TREM-1 monoclonal antibody was
administered i.p. one hour after LPS injection. The viability of
mice was examined every hour, or animals were sacrificed at regular
intervals. Serum samples were collected by cardiac puncture and
assayed for TNF-.alpha. and IL-1.beta. by ELISA (BD Biosciences),
and for sTREM-1 levels by immunodot.
CLP Polymicrobial Sepsis Model
[0157] Male Balb/C mice (7 to 9 weeks, 20 to 23 g) were
anaesthetized by i.p. administration of ketamine and xylazine in
0.2 mL sterile pyrogen-free saline. The caecum was exposed through
a 1.0 cm abdominal midline incision and subjected to a ligation of
the distal half followed by two punctures with a G21 needle. A
small amount of stool was expelled from the punctures to ensure
patency. The caecum was replaced into the peritoneal cavity and the
abdominal incision closed in two layers. After surgery all mice
were injected s.c. with 0.5 ml of physiologic saline solution for
fluid resuscitation and s.c. every 12 h with 1.25 mg (i.e. 50
.mu.g/g) of imipenem. The animals were randomly grouped and treated
with normal saline (n=14), the control peptide (n=14, 100 .mu.g) or
P5 (100 .mu.g) in a single injection at HO (n=18), H+4 (n=18) or
H+24 (n=18). The last group of mice (n=18) was treated with
repeated injections of P5 (100 .mu.g) at H+4, H+8 and H+24. All
treatments were diluted into 500 .mu.l of normal saline and
administered i.p. The Inventors next sought to determine the effect
of various doses of P5. For this purpose, mice (n=15 per group)
were treated with a single injection of normal saline or 10 .mu.g,
20 .mu.g, 50 .mu.g, 100 .mu.g or 200 .mu.g of P5 at HO after the
CLP and monitored for survival. Five additional animals per group
were killed under anaesthesia 24 hours after CLP for the
determination of bacterial count and cytokines levels. Peritoneal
lavage fluid was obtained using 2 mL RPMI 1640 (Life Technologies)
and blood was collected by cardiac puncture. Concentrations of
TNF-.alpha. and IL-1.beta. in the serum were determined by ELISA
(BD Biosciences). For the assessment of bacterial counts, blood and
peritoneal lavage fluid were plated in serial log dilutions on
tryptic soy supplemented with 5% sheep blood agar plates. After
plating, tryptic soy agar plates were incubated at 37.degree. C.
aerobically for 24 hours, and anaerobically for 48 hours. Results
are expressed as CFU per mL of blood and CFU per mouse for the
peritoneal lavage.
Statistical Analyses
[0158] Serum sTREM-1 and cytokines levels were expressed as mean
(.+-.SD).The protection against LPS lethality by P5 was assessed by
comparison of survival curves using the Log-Rank test. All
statistical analyses were completed with Statview software (Abacus
Concepts, Berkeley Calif.) and a two-tailed P<0.05 was
considered significant.
[0159] Results
[0160] A Soluble Form of TREM-1 is Released from Cultured Human
Monocytes After Stimulation with E. coli LPS
[0161] To identify the potential release of sTREM-1 in vitro, the
Inventors stimulated human monocytes with LPS and analyzed the
conditioned culture medium by SDS-PAGE. LPS stimulation induced the
appearance of a 27-kDa protein in a time-dependent manner (FIG.
7A). Western blotting analysis revealed that this protein was
specifically recognized by a monoclonal antibody directed against
the extra-cellular domain of TREM-1 (FIG. 7A). Cell viability was
unaffected at LPS concentrations that induced the presence of
sTREM-1 in conditioned medium, indicating that TREM-1 release was
not due to cell death. Similarly, treatment of monocytes with
protease inhibitors did not affect TREM-1 release (FIG. 7A). TREM-1
mRNA levels were increased upon LPS treatment (FIG. 7B) whereas
TREM-1sv mRNA levels remained undetectable. This suggests that
TREM-1 release is likely to be linked to an increased transcription
of the gene and unrelated to TREM-1sv expression. Stimulation of
monocytes for 16 hours with TNF-.alpha. (5 to 100 ng/mL) or
IL-1.beta. (5 to 100 ng/mL) induced very small TREM-1 release in a
cytokine dose-dependent manner. Stimulation with IFN-.gamma. did
not induce TREM-1 release, even at concentrations of up to 100
U/mL.
[0162] LPS Associated Release of Pro-Inflammatory Cytokines is
Attenuated by P5
[0163] Significant TNF-.alpha. and IL-1.beta. production was
observed in the supernatant of monocytes cultured with LPS.
TNF-.alpha. and IL-13 production was even higher for cells cultured
with both TREM-1 mAb and LPS as compared with those cultured with
mAb or LPS alone (FIG. 8A).
[0164] The inducible release of pro-inflammatory cytokines was
significantly lower after LPS stimulation when the medium was
supplemented with P5 or IL-10. P5 reduced, in a
concentration-dependent manner, the TNF-.alpha. and IL-1.beta.
production from cells cultured with LPS or with LPS and mAb and
simultaneously increased the release of sTREM-1 from cells cultured
with LPS. The control peptide displayed no action on cytokines or
sTREM-1 release (data not shown). In striking contrast, IL-10
totally inhibited the release of both TREM-1 and inflammatory
cytokines (FIG. 8A). Both LPS and TREM-1 mAb induced a strong
activation of monocytic NF-.kappa.B p50 and p65 and combined
administration of LPS and TREM-1 mAb lead to a synergistic effect.
P5 inhibited the NF-.kappa.B activation induced by the engagement
of TREM-1 but did not alter the effect of LPS (FIG. 8B).
[0165] Serum sTREM-1 Levels of LPS-Treated Mice are Increased
[0166] In order to determine whether sTREM-1 was released
systemically during endotoxemia in mice, the Inventors measured
serum sTREM-1 levels after LPS administration. Serum sTREM-1 was
readily detectable 1 hour after administration of an LD.sub.50 dose
of LPS and was maintained at peak plateau levels from 4 to 6 hours
after LPS treatment (FIG. 9).
[0167] TREM-1 Peptide "P5" Protects Endotoxemic Mice from
Lethality
[0168] Mice treated by a single dose of P5 60 min before a lethal
dose (LD.sub.100) of LPS were prevented from death in a
dose-dependent manner (FIG. 10A). In order to investigate whether
P5 treatment could be delayed until after the administration of
LPS, the Inventors injected P5 beginning 4 or 6 hours after LPS
injection. This delayed treatment up to 4 hours conferred
significant protection against a LD.sub.100 dose of LPS (FIG. 10B).
No late death occurred over one week, indicating that P5 did not
merely delay the onset of LPS lethality, but provided lasting
protection. Control mice all developed lethargy, piloerection, and
diarrhoea before death. By contrast, P5-treated mice remained well
groomed and active, had no diarrhoea, and were lively. To clarify
the mechanism by which P5 protected mice from LPS lethality, the
Inventors determined the serum levels of TNF-.alpha., IL-1.beta.
and sTREM-1 of endotoxemic mice at 2 and 4 hours. As compared to
controls, pre-treatment by 100 .mu.g of P5 reduced cytokines levels
by 30% and increased sTREM-1 levels by 2 fold as shown in Table
4:
TABLE-US-00006 TABLE 4 Serum concentrations of TNF-.alpha.,
IL-1.beta. and sTREM-1 in endotoxemic mice. TNF-.alpha. (ng/mL)
IL-1.beta. (ng/mL) sTREM-1 (ng/mL) H2 H4 H2 H4 H2 H4 Control 3.3
.+-. 1.0 0.4 .+-. 0.1 0.3 .+-. 0.1 1.5 .+-. 0.2 249 .+-. 48 139
.+-. 8 P5 (100 .mu.g) 2.4 .+-. 0.5 0.1 .+-. 0.1 0.2 .+-. 0.1 0.9
.+-. 0.2 475 .+-. 37 243 .+-. 28
[0169] Engagement of TREM-1 is Lethal to Mice
[0170] To further highlight the role of TREM-1 engagement in
LPS-mediated mortality, mice were treated with agonist anti-TREM-1
mAb in combination with the administration of an LD.sub.50 dose of
LPS. This induced a significant increase in mortality rate from 50%
to 100% (FIG. 10C).
[0171] P5 Protects Mice from CLP-Induced Lethality
[0172] To investigate the role of P5 in a more relevant model of
septic shock, the Inventors performed CLP experiments (FIG. 11A).
The control groups comprised mice injected with normal saline or
with the control peptide. In this model of polymicrobial sepsis, P5
still conferred a significant protection against lethality even
when administered as late as 24 hours after the onset of sepsis.
Interestingly, repeated injections of P5 had the more favourable
effect on survival (P<0.01). There was a dose response effect of
P5 on survival (FIG. 11B) and cytokine production (Table 5). P5 had
no effect on bacterial clearance (FIG. 12).
TABLE-US-00007 TABLE 5 Serum concentrations of TNF-.alpha.,
IL-1.beta. and sTREM-1 at 24 hours after CLP. TNF-.alpha. (pg/mL)
IL-1.beta. (pg/mL) sTREM-1 (ng/mL) Control peptide 105 .+-. 12 841
.+-. 204 52 .+-. 3 Control saline 118 .+-. 8 792 .+-. 198 35 .+-. 5
P5 10 .mu.g 110 .+-. 11 356 .+-. 62 43 .+-. 8 P5 20 .mu.g 89 .+-.
10 324 .+-. 58 58 .+-. 8 P5 50 .mu.g 24 .+-. 6 57 .+-. 11 93 .+-.
10 P5 100 .mu.g 20 .+-. 3 31 .+-. 3 118 .+-. 12 P5 200 .mu.g 21
.+-. 7 37 .+-. 8 158 .+-. 13
[0173] Sepsis exemplifies a complex clinical syndrome that results
from a harmful or damaging host response to severe infection.
Sepsis develops when the initial, appropriate host response to
systemic infection becomes amplified, and then dysregulated (4, 5).
Neutrophils and monocyte/macrophages exposed to LPS, for instance,
are activated and release such pro-inflammatory cytokines as
TNF-.alpha. and IL-1.beta.. Excessive production of these cytokines
is widely believed to contribute to the multi-organ failure that is
seen in septic patients (20-23).
[0174] TREM-1 is a recently identified molecule involved in
monocytic activation and inflammatory response (12, 14). It belongs
to a family related to NK cell receptors that activate downstream
signalling events. The expression of TREM-1 on PNNs and
monocytes/macrophages has been shown to be inducible by LPS (16,
17).
[0175] As described herein, the Inventors demonstrate that a
soluble form of TREM-1 was released from cultured human monocytes
after stimulation with E. coli LPS. Such a soluble form was also
detectable in the serum of endotoxemic mice as early as 1 hour
after LPS challenge. This is consistent with the implication of
TREM-1 in the very early phases of the innate immune response to
infection (14, 15, 24). The mechanism by which sTREM-1 is released
is not clearly elucidated but seems to be related to an increased
transcription of the TREM-1 gene. Nevertheless, although incubation
with a protease inhibitor cocktail does not alter the sTREM-1
release, cleavage of the surface TREM-1 from the membrane cannot be
totally excluded. Interestingly, stimulation of human monocytes
with such pro-inflammatory cytokines as TNF-.alpha., IL-1.beta. or
IFN-.gamma. induced very small sTREM-1 release unless LPS was added
as a co-stimulus. The expression of an alternative mRNA TREM-1
splice variant (TREM-1sv) has been detected in monocytes that might
translate into a soluble receptor (18) upon stimulation with cell
wall fraction of Mycobacterium bovis BCG but not LPS (25). This was
confirmed in this study as i) LPS did not increase the level of
mRNA TREM-1sv in monocytes and ii) only a 27-kDa protein was
released by monocytes upon LPS stimulation and not the 17.5-kDa
variant.
[0176] Although its natural ligand has not been identified (13,
14), engagement of TREM-1 on monocytes with an agonist monoclonal
antibody resulted in a further enhancement of pro-inflammatory
cytokines production, while P5 induced a decrease of these
syntheses in a concentration-dependent manner, and IL-10 completely
suppressed it.
[0177] Inflammatory cytokines, and especially TNF-.alpha., are
considered to be deleterious, yet they also possess beneficial
effects in sepsis (5) as shown by the fatal issue of peritonitis in
animals with impaired TNF-.alpha. responses (9-11). Moreover, in
clinical trials, the inhibition of TNF-.alpha. increased mortality
(8). Finally, the role of TNF-.alpha. in the clearance of infection
has been highlighted by the finding that sepsis is a frequent
complication in rheumatoid arthritis patients treated with
TNF-.alpha. antagonists (26).
[0178] The mechanism by which P5 modulates cytokine production is
not yet clear. P5 comprises the complementary determining region
(CDR)-3 and the `F` .beta. strand of the extracellular domain of
TREM-1. The latter contains a tyrosine residue mediating
dimerization. Radaev et al postulated that TREM-1 captures its
ligand with its CDR-equivalent loop regions (27). P5 could thus
impair TREM-1 dimerization and/or compete with the natural ligand
of TREM-1. Moreover, the increase of sTREM-1 release from monocytes
mediated by P5 could prevent the engagement of membrane TREM-1,
sTREM-1 acting as a decoy receptor, as in the TNF-.alpha. system
(28, 29).
[0179] Activation of the transcription factor NF-.kappa.B is a
critical step in monocyte inflammatory cytokine production after
exposure to bacterial stimuli such as LPS (30, 31). Among the
various NF-.kappa.B/Rel dimers, the p65/p50 heterodimer is the
prototypical form of LPS-inducible NF-.kappa.B in monocytes (32).
P5 abolishes the p65/p50 NF-.kappa.B over-activation induced by the
engagement of TREM-1. This might at least partially explain the
effects of P5 on cytokine production and the protection from
lethality shown here to occur when the peptide was injected one
hour before LPS-induced septic shock, or even up to 4 hours
after.
[0180] Endotoxemia is simple to achieve experimentally, but
imperfectly suited to reproduce human sepsis, while polymicrobial
sepsis induced by CLP is a more complex but better model, including
the use of fluid resuscitation and antibiotics. The latter was thus
also used in this study, and confirmed the dose-dependent
protection provided by P5, even when administered as late as 24
hours after the onset of sepsis. The favourable effect of P5 was
however unrelated to an enhanced bacterial clearance.
[0181] One difficulty in the use of immunomodulatory therapies is
that it is not possible to predict the development of sepsis, and,
thus, patients receiving those treatments frequently already have
well-established sepsis (6). Since P5 appeared to be effective even
when injected after the outbreak of sepsis, it could thus
constitute a realistic treatment (24, 33).
[0182] By contrast, engagement of TREM-1 by an agonist anti-TREM-1
monoclonal antibody mediated a dramatic increase of mortality rate
in LPS-challenged mice: this further underscores the detrimental
effect of TREM-1 engagement during septic shock.
[0183] Experimental septic shock reproduces human sepsis only in
part. Indeed, our group recently showed that significant levels of
sTREM-1 were released in the serum of critically ill patients with
sepsis patients (34), the highest levels being observed in patients
who survived. This is consistent with our experimental findings
indicating that the more important sTREM-1 release, the more
favourable is the outcome, and thus sustains, at least
theoretically, the potential value of soluble TREM peptides as
post-onset sepsis therapy.
[0184] TREM-1 appears to be a crucial player in the immediate
immune response triggered by infection. In the early phase of
infection, neutrophils and monocytes initiate the inflammatory
response owing to the engagement of pattern recognition receptors
by microbial products (3, 4). At the same time, bacterial products
induce the up-regulation and the release of sTREM-1. Upon
recognition of an unknown ligand, TREM-1 activates signalling
pathways which amplify these inflammatory responses, notably in
monocytes/macrophages. The modulation of TREM-1 signalling reduces,
although without complete inhibition, cytokine production and
protects septic animals from hyper-responsiveness and death.
Modulation of TREM-1 engagement with such a peptide as P5 might be
a suitable therapeutic tool for the treatment of sepsis,
particularly because it seems to be active even after the onset of
sepsis following infectious aggression.
EXAMPLE 4
Haemodynamic Studies in LPS Treated and Septic Rats Treated with P1
and P5
[0185] The role of TREM-1 peptides in further models of septic
shock, was investigated by performing LPS and CLP (caecal ligation
and puncture) experiments in rats.
[0186] Materials and Methods
[0187] LPS-Induced Endotoxinemia
[0188] Animals were randomly grouped (n=10-20) and treated with
Escherichia coli LPS (O111:64, Sigma-Aldrich, Lyon, France) i.p. in
combination with the TREM-1 or scrambled peptides.
[0189] CLP Polymicrobial Sepsis Model
[0190] The procedure has been described in details elsewhere (see
Mansart, A. et al. Shock 19:3844 (2003)). Briefly, rats (n=6-10 per
group) were anesthetized by i.p. administration of ketamine (150
mg/kg). The caecum was exposed through a 3.0-cm abdominal midline
incision and subjected to a ligation of the distal half followed by
two punctures with a G21 needle. A small amount of stool was
expelled from the punctures to ensure potency. The caecum was
replaced into the peritoneal cavity and the abdominal incision
closed in two layers. After surgery, all rats were injected s.c.
with 50 mL/kg of normal saline solution for fluid resuscitation.
TREM-1 or scrambled peptides were then administered as above.
[0191] Haemodynamic Measurements in Rats
[0192] Immediately after LPS administration as well as 16 hours
after CLP, arterial BP (systolic, diastolic, and mean), heart rate,
abdominal aortic blood flow, and mesenteric blood flow were
recorded using a procedure described elsewhere (see Mansart, A. et
al. Shock 19:3844 (2003)). Briefly, the left carotid artery and the
left jugular vein were cannulated with PE-50 tubing. Arterial BP
was continuously monitored by a pressure transducer and an
amplifier-recorder system (IOX EMKA Technologies, Paris, France).
Perivascular probes (Transonic Systems, Ithaca, N.Y.) wrapped up
the upper abdominal aorta and mesenteric artery, allowed to monitor
their respective flows by means of a flowmeter (Transonic Systems).
After the last measurement (4.sup.th hour during LPS experiments
and 24.sup.th hour after CLP), animals were sacrificed by an
overdose of sodium thiopental i.v.
[0193] Biological Measurements
[0194] Blood was sequentially withdrawn from the left carotid
artery. Arterial lactate concentrations and blood gases analyses
were performed on an automatic blood gas analyser (ABL 735,
Radiometer, Copenhagen, Denmark). Concentrations of TNF-.alpha. and
IL-1.beta. in the plasma were determined by an ELISA test
(Biosource, Nivelles, Belgium) according to the recommendations of
the manufacturer. Plasmatic concentrations of nitrates/nitrites
were measured using the Griess reaction (R&D Systems, Abingdon,
UK).
[0195] Statistical Analyses
[0196] Results are expressed as mean.+-.SD. Between-group
comparisons were performed using Student' t tests. All statistical
analyses were completed with Statview software (Abacus Concepts,
CA) and a two-tailed P<0.05 was considered significant.
[0197] Results
[0198] Endotoxinemia Model
[0199] Following LPS administration, arterial pressures, aortic and
mesenteric blood flows dropped rapidly in control animals
(scrambled peptides treated rats) while the heart rate remained
unchanged (Table 6). The decrease of arterial pressures and aortic
blood flow was delayed until the second hour in TREM-1 peptide
treated animals with significantly higher values by that time than
in control animals. There was no difference between P1 and P5
treated groups. By contrast, none of these two peptides had any
effect on the decrease of the mesenteric blood flow (Table 6).
[0200] Arterial pH remained constant over time until the fourth
hour after LPS injection where it severely dropped in the control
group only (Table 6). The significant arterial lactate level
elevation present in control animals after the third hour was
abolished by the TREM-1 peptides (Table 6). There was no difference
between P1 and P5 with regard to pH, arterial bicarbonate and
lactate concentrations.
[0201] As expected, a peak of TNF-.alpha. plasmatic concentration
was induced by LPS between 30 minutes and 1 hour after injection
followed by a progressive decline thereafter (FIG. 13A). P1 peptide
injection had no effect on this production, while P5 attenuated
TNF-.alpha. production by -30%.
[0202] P1 delayed the IL-1.beta. peak until the third hour after
LPS injection, but without attenuation. By contrast, P5 strongly
reduced IL-1.beta. release (FIG. 13B).
[0203] Nitrite/nitrate concentrations increased rapidly after LPS
administration in control and P1 treated animals but remained
stable upon P5 treatment (FIG. 14).
TABLE-US-00008 TABLE 6 Hemodynamic parameters during LPS-induced
endotoxinemia Heart Aortic Mesenteric Rate MAP blood flow blood
flow Lactate (bpm) (mmHg) (mL/min) (mL/min) pH (mmol/L) Control H0
486 .+-. 13 123 .+-. 21 45 .+-. 7 13.6 .+-. 3.4 7.31 .+-. 0.03 3.3
.+-. 0.8 H1 522 .+-. 16 103 .+-. 25 25 .+-. 8 .sup.a 9.6 .+-. 3.3
7.28 .+-. 0.03 4.2 .+-. 0.3 H2 516 .+-. 13 98 .+-. 23 12 .+-. 5
.sup.a,b 8.0 .+-. 3.7 7.29 .+-. 0.03 5.9 .+-. 0.6 H3 490 .+-. 20 78
.+-. 8 .sup.a,b 8 .+-. 3 .sup.a,b 5.8 .+-. 1.1 7.26 .+-. 0.01 7.9
.+-. 1.8 .sup.a,b H4 510 .+-. 18 67 .+-. 9 .sup.a,b 6 .+-. 1
.sup.a,b 4.1 .+-. 0.8 7.03 .+-. 0.10 .sup.a,b 11.5 .+-. 0.7
.sup.a,b P1 H0 464 .+-. 25 116 .+-. 10 49 .+-. 11 12.0 .+-. 3.7
7.32 .+-. 0.04 2.7 .+-. 0.1 H1 492 .+-. 26 119 .+-. 14 39 .+-. 12
.sup.a 10.5 .+-. 1.7 7.29 .+-. 0.04 4.9 .+-. 1.1 H2 492 .+-. 26 113
.+-. 21 26 .+-. 14 .sup.a 7.7 .+-. 2.7 7.30 .+-. 0.01 5.0 .+-. 0.9
H3 480 .+-. 30 97 .+-. 29 .sup.a 22 .+-. 8 .sup.a 5.0 .+-. 1.0 7.26
.+-. 0.06 5.7 .+-. 0.7 .sup.a H4 480 .+-. 20 92 .+-. 7 .sup.a 16
.+-. 6 .sup.a 4.8 .+-. 0.9 7.26 .+-. 0.08 .sup.a 7.9 .+-. 1.7
.sup.a P5 H0 474 .+-. 49 115 .+-. 16 48 .+-. 9 12.8 .+-. 6.4 7.33
.+-. 0.04 3.4 .+-. 1.5 H1 498 .+-. 26 99 .+-. 22 32 .+-. 8 11.4
.+-. 2.7 7.28 .+-. 0.06 5.4 .+-. 1.4 H2 510 .+-. 42 101 .+-. 18 23
.+-. 4 .sup.b 9.2 .+-. 1.9 7.32 .+-. 0.07 5.5 .+-. 1.6 H3 517 .+-.
62 93 .+-. 21 .sup.b 20 .+-. 7 .sup.b 6.0 .+-. 0.8 7.29 .+-. 0.11
5.9 .+-. 1.7 .sup.b H4 510 .+-. 26 89 .+-. 10 .sup.b 15 .+-. 6
.sup.b 5.0 .+-. 1 .0 7.28 .+-. 0.12 .sup.b 7.4 .+-. 1.8 .sup.b
.sup.a p < 0.05 P1 vs Controls .sup.b p < 0.05 P5 vs
Controls
CLP Model
[0204] As the severity of the Inventors' model was at its highest
16 to 20 hours after the completion of the CLP, the Inventors chose
to investigate animals by the 16.sup.th hour. Importantly, there
were no deaths before this time point. Although all animals were
fluid resuscitated, none received antibiotics in order to strictly
consider the role of the peptides.
[0205] There was a dramatic decline in arterial pressure in the
control animals over time, and by H24 systolic, diastolic and mean
arterial pressures were 58.+-.7 mmHg, 25.+-.4 mmHg and 38.+-.2 mmHg
respectively. This decrease was almost totally abolished with P1 or
P5 treatment with no significant difference between H16 and H24
(FIG. 15). There was no difference between P1 and P5 treated
rats.
[0206] TREM-1 peptides also prevented the aortic and mesenteric
blood flows decrease observed in control animals (Table 7). The
protective effect on mesenteric blood flow alterations was even
higher under P5 treatment. The relative preservation of blood flows
was not related to an increased heart rate, since the latter was
rather slower than in control animals (Table 7).
[0207] The progressive metabolic acidosis that developed in control
rats was attenuated by the P1 peptide, and almost abrogated by P5.
The same protective trend was observed for arterial lactate
elevation with a more pronounced effect of P5 (Table 7).
TABLE-US-00009 TABLE 7 Hemodynamic and selected biochemical
parameters during CLP polymicrobial sepsis Heart Aortic Mesenteric
Rate blood flow blood flow Bicarbonate Lactate (bpm) (mL/min)
(mL/min) pH (mmol/L) (mmol/L) Control H16 516 .+-. 44 .sup.a,b 38
.+-. 10 10.6 .+-. 3.0 .sup.b 7.31 .+-. 0.07 .sup.b 6.9 .+-. 2.7 4.7
.+-. 1.5 .sup.b H20 543 .+-. 35 .sup.a,b 9 .+-. 11 .sup.a,b 4.3
.+-. 1.5 .sup.b 7.23 .+-. 0.05 .sup.a,b 12.0 .+-. 5.6 .sup.a,b 8.5
.+-. 1.4 .sup.a,b H24 480 .+-. 20 14 .+-. 9 .sup.a,b 2.5 .+-. 0.7
.sup.b 7.17 .+-. 0.01 .sup.a,b 10.3 .+-. 3.3 .sup.a 10.8 .+-. 1.9
.sup.a,b P1 H16 462 .+-. 16 .sup.a 41 .+-. 12 13.5 .+-. 7.2 7.32
.+-. 0.04 16.8 .+-. 4.4 4.9 .+-. 0.4 H20 480 .+-. 30 .sup.a 28 .+-.
17 .sup.a 5.3 .+-. 3.0 .sup.c 7.31 .+-. 0.18 .sup.a 16.0 .+-. 5.4
.sup.b 5.3 .+-. 1.1 .sup.a,c H24 420 .+-. 30 22 .+-. 16 .sup.a 4.5
.+-. 2.1 .sup.c 7.24 .+-. 0.06 .sup.a,c 11.2 .+-. 0.8 .sup.c 6.8
.+-. 0.9 .sup.a,c P5 H16 460 .+-. 17 .sup.b 41 .+-. 14 15.3 .+-.
3.5 .sup.b 7.35 .+-. 0.01 .sup.b 18.6 .+-. 2.0 3.3 .+-. 0.4 .sup.b
H20 500 .+-. 17 .sup.b 31 .+-. 5 .sup.b 11.0 .+-. 6.9 .sup.b,c 7.34
.+-. 0.01 .sup.b 18.0 .+-. 0.9 .sup.a 3.6 .+-. 0.9 .sup.b,c H24 510
.+-. 20 28 .+-. 8 .sup.b 8.5 .+-. 3.5 .sup.b,c 7.36 .+-. 0.01
.sup.b,c 17.1 .+-. 0.9 .sup.a,c 4.9 .+-. 1.1 .sup.b,c .sup.a p <
0.05 P1 vs Controls .sup.b p < 0.05 P5 vs Controls .sup.c p <
0.05 P5 vs P1
[0208] Both P1 and P5 induced a decrease in TNF-.alpha. production,
again with a stronger effect of P5. By H20, plasmatic TNF-.alpha.
was almost undetectable under P5 treatment whereas it remained
elevated in the other groups of animals (FIG. 16).
[0209] Nitrite/nitrate concentrations were increased in control
animals but remained at a low level in both TREM-1 peptides treated
groups (FIG. 17).
[0210] A protective action of both P5 and P1 on hemodynamics was
thus observed in septic rats. Both arterial pressure and blood
flows were preserved, independently of heart rate. Moreover,
modulation of TREM-1 signalling reduced, although not completely,
cytokine production and protected septic animals from
hyper-responsiveness. The fact that the cytokine production was not
totally inhibited is a crucial point. Indeed, although inflammatory
cytokines such as TNF-.alpha. are considered deleterious, they also
display beneficial effects in sepsis as underlined by the fatal
issue of peritonitis models in animals with impaired TNF-.alpha.
responses.
[0211] The activation of iNOS observed during septic shock leads to
the production of large amount of NO that partly explains some of
the peripheral vascular disorders (notably vasodilation and
hypotension). On the myocardium itself, most of the action of NO is
mediated by an activation of the soluble guanylate-cyclase
responsible for the production of cGMP which impairs the effect of
cytosolic calcium on contraction. Cyclic GMP is also able to
stimulate the activity of some phosphodiesterases. The subsequent
decrease of intracellular cAMP levels could explain the ability of
NO to attenuate the effects of beta adrenergic stimulation. The
preservation of arterial pressure could therefore be partly
explained by a lessened production of NO, as reflected by the lower
concentrations of plasma nitrite/nitrate in TREM-1 peptides treated
animals.
[0212] The decrease in inflammatory cytokine production could
partly explain the effect noted on blood flows. Indeed, although
the list of potential cytokine mediators of myocardial depression
is long, TNF-.alpha. and IL-1.beta. have been shown to be good
candidates Both these latter cytokines depressed myocardial
contractility in vitro or ex vivo. Moreover, the neutralization or
removal of TNF-.alpha. or IL-1.beta. from human septic serum partly
abrogates the myocardial depressant effect in vitro and in vivo.
Although P1 and P5 had an identical action on blood flows and
arterial pressure during endotoxinemia, their action on cytokine
production differed with only a slight effect of P1 on plasma
TNF-.alpha. and IL-1.beta. concentrations. The protective role of
the TREM-1 peptides could therefore be only partly related to their
action on cytokine release, or involve redundant pathways.
[0213] The modulation of the TREM-1 pathway by the use of small
synthetic peptides had beneficial effects on haemodynamic
parameters during experimental septic shock in rats, along with an
attenuation of inflammatory cytokine production.
[0214] In summary, these data show that the TREM-1 peptides of the
invention 1) efficiently protect subject animals from
sepsis-related hemodynamic deterioration; 2) attenuate the
development of lactic acidosis; 3) modulate the production of such
pro-inflammatory cytokines as TNF-.alpha. and IL-1.beta. and 4)
decrease the generation of nitric oxide. Thus TREM-1 peptides are
potentially useful in the restoration of haemodynamic parameters in
patients with sepsis, septic shock or sepsis-like conditions and
therefore constitute a potential treatment for the aforesaid
conditions.
Example 5: hP5 Activity in a Murine Model of Sepsis:
Endotoxin-Induced Septic Shock.
[0215] The activity of human P5 (hP5) was investigated in a murine
model. hP5 differs from mP5 according to ClustalW comparison as set
our below:
TABLE-US-00010 Length Length SeqA Name (aa) SeqB Name (aa) Score 1
hP5 17 2 mP5 17 82 hP5 LQVEDSGLYQCVIYQPP 17 mP5 LQVTDSGLYRCVIYHPP
17 *** *****:****:**
[0216] For the experiment, male BALB/c mice (19-21 g) were randomly
grouped (15 mice per group) and injected intraperitoneally (i.p.)
with 200 microg of LPS from E. coli 0111:64 (Sigma). A blinded
investigator performed all injections. 200 microliters of TREM-1
peptides dissolved in water 10% DMSO, 9% Solutol were administered
intraperitoneally at -1 h, 0 h, +1 h, +4 h prior and after LPS
injection. Viability of treated mice was monitored twice a day for
7 days.
[0217] To then assess the ability of hP5 peptide to protect mice
from LPS-induced endotoxaemia, the inventors administered at -1 h,
0 h, +1 h, +4 h prior and after LPS injection a lethal dose of
lipopolysaccharide (LPS) (FIG. 18). Lethality was monitored
overtime and compared with animals that had received vehicle alone.
hP5 injection confers high protection, with 80% of the animals
still alive 7 days after LPS injection, as compared with no
survivors in the control group (p<0.0003). The human P5 peptide
shows >80% identity with mP5.
[0218] The results summarised in FIG. 18 clearly demonstrate that
the human P5 protects mice from lethal shock.
Example 6 hP5 Activity in a Murine Model of Sepsis: Cecal Ligation
and Puncture Model
[0219] For the experiment the mice underwent a standardized
preparation for laparotomy (anaesthesia with 2% inhaled isoflurane
in oxygen, shaving with animal clippers, alcohol scrub). A 1 cm
incision was made on the midline. The cecum was exposed and will be
tightly ligated at 50-80% over its base with a 4-0 silk suture
avoiding bowel obstruction. The cecum was then punctured once with
a 23 G needle. The cecum was gently squeezed until feces be just
visible through the puncture, and placed again in the abdominal
cavity. The incision was thereafter be closed with a 4-0 silk. 200
.mu.l of hP5 or its scrambled peptide control were freshly
dissolved in water 10% DMSO, 9% solutol and injected
intraperitoneally with a 22 G needle at the following time points:
-1, 0, +4, +24. No fluid resuscitation was administered. Survival
and Moribundity were observed twice a day for 10 days.
[0220] The Inventors then analysed whether hP5 protects against
septic shock in the "CLP" model. Mice treated with four doses of
hP5 at -1 h, 0 h, +4 h and +24 hours after CLP were protected from
death as compared to control treated mice. At 72 hours after CLP,
73.3% of the mice injected with hP5 survived compared to 60% of
mice treated with scrambled peptide. At 10 days after CLP, 66.6% of
the hP5 treated mice were still alive compared to 60% of the
control group, suggesting that hP5 could provide lasting protection
(FIG. 19).
Example 7 The Human TREM-1 Peptide P5 (hP5) Attenuates Established
Intestinal Inflammation in the DSS Colitis Model
[0221] The purpose of this example is to identify, characterize and
document the therapeutic potential of the TREM-1 derived peptide
hP5, in an experimental model of colitis in mice.
[0222] On day 0 of the study, the water supply was removed and
replaced with 3% Dextran Sulfate Sodium (DSS) From day 0 to day 6,
this solution was the only source of fluids. Water was administered
for the rest of the experiment (day 7-11). Healthy controls
received water only.
[0223] From day 3 to day 10 mice were treated with either hP5, or a
sequence-scrambled control peptide. Peptides were dissolved in DMSO
at 10 mg/ml and stored at +4.degree. C. Before administration, the
stock solution was diluted 1:10 in water 10% Solutol.RTM. HS 15
(BASF). Final vehicle concentrations: 1& DMSO, 10% solutol in
water. 200 .mu.l of these freshly prepared solutions of hP5, its
scrambled control or vehicle were injected intraperitoneally with a
25 G needle.
[0224] Animals were weighted daily and monitored on days 3, 4, 5,
6, 7 and 10 for rectal bleeding and stool consistency. For each
group, the disease activity index (DAI) was determined by
evaluating changes in weight, stool consistency and presence of
gross blood during the study, as described below:
TABLE-US-00011 Weight loss Blood in Score (%) Stool consistency
stool 0 <1 Normal Negative 1 1-4.9 Soft +/- 2 5-9.9 Mixed (soft
and liquid) + 3 10-15 Liquid ++ 4 >15 Diarrhea (liquid stools
that Gross adhere to the anus) bleeding
[0225] Scoring System for the Disease Activity Index (DAI).
Individual scores for each parameter are added and then divided by
three to give a DAI score for each mouse.
[0226] To determine the presence of occult blood in stool, a
pea-sized stool sample was placed on a slide. Then two drops of
reagent (Hemocult Sensa, Beckman Coulter) were placed onto the
stool sample on the slide and a change of colour was observed. The
presence of occult blood was graded using a score of 0 for no
colour; 1 for a very light blue (+/-) colour taking over 30 seconds
to appear, 2 for a blue colour developing in 30 seconds or more
(+); 3 for an immediate change in colour (++) and 4 for gross blood
observable on the slide.
[0227] On day 11 mice were euthanized by cervical dislocation to
allow colon length evaluation. An incision was done in the abdomen
to expose the colon. The stool in the colon was removed flushing
with saline. The entire colon from cecum to anus was removed and
the length was measured and reported as colon length (cm).
[0228] Data were analyzed using GraphPad Prism. Results are given
as means .+-. standard error of the mean. The BW score, stool score
and blood score were analyzed using two-way ANOVA test, Bonferroni
post test. Colon length was analysed using one-way ANOVA, Dunn's
post test. * P.ltoreq.0.05; **P.ltoreq.0.01; *** P.ltoreq.0.001
[0229] mP5 is a mTREM-1 derived peptide whose efficacy has been
proven in several models of sepsis. In this study, the
investigators have tested the efficacy of the human orthologue of
mP5, compared to its scrambled peptide control, in the above
described DSS-induced colitis model. All peptides were administered
in a therapeutic fashion, 3 days after initiation of DSS
treatment.
[0230] In all animals, body weight, haemoccult or presence of gross
blood and stool consistency were monitored. Bloody stools were
observed from day 3 onwards, loose stools and weight loss appeared
beginning from day 4-5. These comprehensive functional measures,
that were somewhat analogous to clinical symptoms observed in human
IBD, are summarized by the Disease Activity Index, as shown in FIG.
20. This scoring method, validated by repeated studies, showed
minimum variations and correlates well with more specific measures
of inflammation.
[0231] None of the control animals showed disease activity. DAI
peaked at day 5-6 and regressed upon DSS removal. As soon as
colitis was established (day 5), hP5 administration significantly
ameliorated stool consistency (FIG. 23) and colon bleeding (FIG.
22), as shown by approximately a 50% DAI inhibition, when compared
to mice treated with DSS+vehicle or DSS+scrambled peptide
(1.95.+-.0.170 vs. 2.92.+-.0.054, p.ltoreq.0.001 or 3.00.+-.0.088,
p.ltoreq.0.001, respectively).
[0232] Protection lasted during the whole treatment. Notably at day
10, in the hP5-treated group both stool score and hemoccult were
normal (FIGS. 23 and 22), while the groups treated with DSS+vehicle
and DSS+scrambled peptide still showed clinical signs of
inflammation (blood score: DSS+hP5=0.43.+-.0.202 vs
DSS+vehicle=1.25.+-.0.164; p.ltoreq.0.01; DSS+hP5=0.43.+-.0.202 vs
DSS+scrambled peptide=1.75.+-.0.250; p.ltoreq.0.001. Stool score:
DSS+hP5=0.71.+-.0.474 vs DSS+vehicle=1.75.+-.0.250 p.ltoreq.0.05;
DSS+hP5=0.71.+-.0.474 vs DSS+scrambled peptide=2.38.+-.0.324;
p.ltoreq.0.001).
[0233] We also monitored weight loss associated with the
development of colitis. FIG. 21 represents the percentage of weight
loss, expressed as a score. Mice started loosing weight at day 4
and the maximal weight loss was reached at day 8. hP5 treatment
significantly reduced weight loss in comparison with DSS+scrambled,
from day 8 on (day 10: hP5=2.00.+-.0.436 vs DSS+scrambled pep.
3.50.+-.0.189; p.ltoreq.0.001). At day 11 mice were euthanized and
the colon length of each mouse measured from the anus to the end of
the cecum (FIG. 24). DSS-induced colon inflammation caused a 30%
colon shortening when compared to naive animals. In the hP5-treated
group, colon shortening was significantly ameliorated when compared
to control group (hP5=7.41.+-.0.237 vs DSS+vehicle=5.96.+-.0.230;
p.ltoreq.0.05). In conclusion, blocking TREM-1 with a human TREM-1
derived peptide attenuates intestinal inflammation even when the
peptide is administered after the appearance of the clinical signs
of colitis. This finding indicates that the human TREM-1 -derived
peptide efficiently blocks interaction of the mouse TREM-1 receptor
with its endogenous ligand.
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Sequence CWU 1
1
271234PRTHomo sapiens 1Met Arg Lys Thr Arg Leu Trp Gly Leu Leu Trp
Met Leu Phe Val Ser1 5 10 15Glu Leu Arg Ala Ala Thr Lys Leu Thr Glu
Glu Lys Tyr Glu Leu Lys 20 25 30Glu Gly Gln Thr Leu Asp Val Lys Cys
Asp Tyr Thr Leu Glu Lys Phe 35 40 45Ala Ser Ser Gln Lys Ala Trp Gln
Ile Ile Arg Asp Gly Glu Met Pro 50 55 60Lys Thr Leu Ala Cys Thr Glu
Arg Pro Ser Lys Asn Ser His Pro Val65 70 75 80Gln Val Gly Arg Ile
Ile Leu Glu Asp Tyr His Asp His Gly Leu Leu 85 90 95Arg Val Arg Met
Val Asn Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln 100 105 110Cys Val
Ile Tyr Gln Pro Pro Lys Glu Pro His Met Leu Phe Asp Arg 115 120
125Ile Arg Leu Val Val Thr Lys Gly Phe Ser Gly Thr Pro Gly Ser Asn
130 135 140Glu Asn Ser Thr Gln Asn Val Tyr Lys Ile Pro Pro Thr Thr
Thr Lys145 150 155 160Ala Leu Cys Pro Leu Tyr Thr Ser Pro Arg Thr
Val Thr Gln Ala Pro 165 170 175Pro Lys Ser Thr Ala Asp Val Ser Thr
Pro Asp Ser Glu Ile Asn Leu 180 185 190Thr Asn Val Thr Asp Ile Ile
Arg Val Pro Val Phe Asn Ile Val Ile 195 200 205Leu Leu Ala Gly Gly
Phe Leu Ser Lys Ser Leu Val Phe Ser Val Leu 210 215 220Phe Ala Val
Thr Leu Arg Ser Phe Val Pro225 2302230PRTMus musculus 2Met Arg Lys
Ala Gly Leu Trp Gly Leu Leu Cys Val Phe Phe Val Ser1 5 10 15Glu Val
Lys Ala Ala Ile Val Leu Glu Glu Glu Arg Tyr Asp Leu Val 20 25 30Glu
Gly Gln Thr Leu Thr Val Lys Cys Pro Phe Asn Ile Met Lys Tyr 35 40
45Ala Asn Ser Gln Lys Ala Trp Gln Arg Leu Pro Asp Gly Lys Glu Pro
50 55 60Leu Thr Leu Val Val Thr Gln Arg Pro Phe Thr Arg Pro Ser Glu
Val65 70 75 80His Met Gly Lys Phe Thr Leu Lys His Asp Pro Ser Glu
Ala Met Leu 85 90 95Gln Val Gln Met Thr Asp Leu Gln Val Thr Asp Ser
Gly Leu Tyr Arg 100 105 110Cys Val Ile Tyr His Pro Pro Asn Asp Pro
Val Val Leu Phe His Pro 115 120 125Val Arg Leu Val Val Thr Lys Gly
Ser Ser Asp Val Phe Thr Pro Val 130 135 140Ile Ile Pro Ile Thr Arg
Leu Thr Glu Arg Pro Ile Leu Ile Thr Thr145 150 155 160Lys Tyr Ser
Pro Ser Asp Thr Thr Thr Thr Arg Ser Leu Pro Lys Pro 165 170 175Thr
Ala Val Val Ser Ser Pro Gly Leu Gly Val Thr Ile Ile Asn Gly 180 185
190Thr Asp Ala Asp Ser Val Ser Thr Ser Ser Val Thr Ile Ser Val Ile
195 200 205Cys Gly Leu Leu Ser Lys Ser Leu Val Phe Ile Ile Leu Phe
Ile Val 210 215 220Thr Lys Arg Thr Phe Gly225 230323PRTMus musculus
3Leu Val Val Thr Gln Arg Pro Phe Thr Arg Pro Ser Glu Val His Met1 5
10 15Gly Lys Phe Thr Leu Lys His 20423PRTMus musculus 4Val Ile Tyr
His Pro Pro Asn Asp Pro Val Val Leu Phe His Pro Val1 5 10 15Arg Leu
Val Val Thr Lys Gly 20517PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Thr Thr Thr Arg Ser Leu Pro
Lys Pro Thr Ala Val Val Ser Ser Pro1 5 10 15Gly621PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Leu
Gln Val Thr Asp Ser Gly Leu Tyr Arg Cys Val Ile Tyr His Pro1 5 10
15Pro Asn Asp Pro Val 20717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Leu Gln Val Thr Asp Ser Gly
Leu Tyr Arg Cys Val Ile Tyr His Pro1 5 10 15Pro823PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Leu
Thr Pro Lys His Gly Gln Arg Ser Thr His Val Thr Lys Phe Arg1 5 10
15Val Phe Glu Pro Val Met Leu 20917PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 9Thr
Asp Ser Arg Cys Val Ile Gly Leu Tyr His Pro Pro Leu Gln Val1 5 10
15Tyr1022DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 10ttgtctcaga actccgagct gc 221122DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
11gagacatcgg cagttgactt gg 221222DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 12ggacggagag atgcccaaga cc
221322DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 13accagccagg agaatgacaa tg 221422DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
14ggacgacatg gagaagatct gg 221524DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 15atagtaatgt cacgcacgat
ttcc 241623PRTHomo sapiens 16Leu Ala Cys Thr Glu Arg Pro Ser Lys
Asn Ser His Pro Val Gln Val1 5 10 15Gly Arg Ile Ile Leu Glu Asp
201723PRTHomo sapiens 17Val Ile Tyr Gln Pro Pro Lys Glu Pro His Met
Leu Phe Asp Arg Ile1 5 10 15Arg Leu Val Val Thr Lys Gly
201821PRTHomo sapiens 18Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln Cys
Val Ile Tyr Gln Pro1 5 10 15Pro Lys Glu Pro His 201917PRTHomo
sapiens 19Leu Gln Val Glu Asp Ser Gly Leu Tyr Gln Cys Val Ile Tyr
Gln Pro1 5 10 15Pro206PRTHomo sapiens 20Arg Pro Ser Lys Asn Ser1
5215PRTHomo sapiens 21Gln Pro Pro Lys Glu1 5226PRTMus musculus
22Arg Pro Phe Thr Arg Pro1 5235PRTMus musculus 23His Pro Pro Asn
Asp1 5244PRTHomo sapiens 24Gln Pro Pro Lys1254PRTMus musculus 25His
Pro Pro Asn126230PRTHomo sapiens 26Met Glu Pro Leu Arg Leu Leu Ile
Leu Leu Phe Val Thr Glu Leu Ser1 5 10 15Gly Ala His Asn Thr Thr Val
Phe Gln Gly Val Ala Gly Gln Ser Leu 20 25 30Gln Val Ser Cys Pro Tyr
Asp Ser Met Lys His Trp Gly Arg Arg Lys 35 40 45Ala Trp Cys Arg Gln
Leu Gly Glu Lys Gly Pro Cys Gln Arg Val Val 50 55 60Ser Thr His Asn
Leu Trp Leu Leu Ser Phe Leu Arg Arg Trp Asn Gly65 70 75 80Ser Thr
Ala Ile Thr Asp Asp Thr Leu Gly Gly Thr Leu Thr Ile Thr 85 90 95Leu
Arg Asn Leu Gln Pro His Asp Ala Gly Leu Tyr Gln Cys Gln Ser 100 105
110Leu His Gly Ser Glu Ala Asp Thr Leu Arg Lys Val Leu Val Glu Val
115 120 125Leu Ala Asp Pro Leu Asp His Arg Asp Ala Gly Asp Leu Trp
Phe Pro 130 135 140Gly Glu Ser Glu Ser Phe Glu Asp Ala His Val Glu
His Ser Ile Ser145 150 155 160Arg Ser Leu Leu Glu Gly Glu Ile Pro
Phe Pro Pro Thr Ser Ile Leu 165 170 175Leu Leu Leu Ala Cys Ile Phe
Leu Ile Lys Ile Leu Ala Ala Ser Ala 180 185 190Leu Trp Ala Ala Ala
Trp His Gly Gln Lys Pro Gly Thr His Pro Pro 195 200 205Ser Glu Leu
Asp Cys Gly His Asp Pro Gly Tyr Gln Leu Gln Thr Leu 210 215 220Pro
Gly Leu Arg Asp Thr225 23027227PRTMus musculus 27Met Gly Pro Leu
His Gln Phe Leu Leu Leu Leu Ile Thr Ala Leu Ser1 5 10 15Gln Ala Leu
Asn Thr Thr Val Leu Gln Gly Met Ala Gly Gln Ser Leu 20 25 30Arg Val
Ser Cys Thr Tyr Asp Ala Leu Lys His Trp Gly Arg Arg Lys 35 40 45Ala
Trp Cys Arg Gln Leu Gly Glu Glu Gly Pro Cys Gln Arg Val Val 50 55
60Ser Thr His Gly Val Trp Leu Leu Ala Phe Leu Lys Lys Arg Asn Gly65
70 75 80Ser Thr Val Ile Ala Asp Asp Thr Leu Ala Gly Thr Val Thr Ile
Thr 85 90 95Leu Lys Asn Leu Gln Ala Gly Asp Ala Gly Leu Tyr Gln Cys
Gln Ser 100 105 110Leu Arg Gly Arg Glu Ala Glu Val Leu Gln Lys Val
Leu Val Glu Val 115 120 125Leu Glu Asp Pro Leu Asp Asp Gln Asp Ala
Gly Asp Leu Trp Val Pro 130 135 140Glu Glu Ser Ser Ser Phe Glu Gly
Ala Gln Val Glu His Ser Thr Ser145 150 155 160Arg Asn Gln Glu Thr
Ser Phe Pro Pro Thr Ser Ile Leu Leu Leu Leu 165 170 175Ala Cys Val
Leu Leu Ser Lys Phe Leu Ala Ala Ser Ile Leu Trp Ala 180 185 190Val
Ala Arg Gly Arg Gln Lys Pro Gly Thr Pro Val Val Arg Gly Leu 195 200
205Asp Cys Gly Gln Asp Ala Gly His Gln Leu Gln Ile Leu Thr Gly Pro
210 215 220Gly Gly Thr225
* * * * *