U.S. patent application number 16/282469 was filed with the patent office on 2019-06-13 for use of cd31 peptides in the treatment of thrombotic and autoimmune disorders.
This patent application is currently assigned to INSERM (Institute National de la Sante et de la Recherche Medicale. The applicant listed for this patent is Giuseppina Caligiuri, Antonino Nicoletti. Invention is credited to Giuseppina Caligiuri, Antonino Nicoletti.
Application Number | 20190177396 16/282469 |
Document ID | / |
Family ID | 40342682 |
Filed Date | 2019-06-13 |
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United States Patent
Application |
20190177396 |
Kind Code |
A1 |
Caligiuri; Giuseppina ; et
al. |
June 13, 2019 |
USE OF CD31 PEPTIDES IN THE TREATMENT OF THROMBOTIC AND AUTOIMMUNE
DISORDERS
Abstract
The present invention stems from the finding that the
extracellular domain of CD31 proteins present on blood leukocytes
is shed and released in the circulation as a soluble form of CD31.
The invention relates to peptides corresponding to fragments of
CD31 that inhibit T-cell response, and to their use in the
treatment of thrombotic disorders such as atherothrombosis and
autoimmune disorders.
Inventors: |
Caligiuri; Giuseppina;
(Paris, FR) ; Nicoletti; Antonino; (Paris,
FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Caligiuri; Giuseppina
Nicoletti; Antonino |
Paris
Paris |
|
FR
FR |
|
|
Assignee: |
INSERM (Institute National de la
Sante et de la Recherche Medicale
Paris
FR
|
Family ID: |
40342682 |
Appl. No.: |
16/282469 |
Filed: |
February 22, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14809603 |
Jul 27, 2015 |
10253085 |
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16282469 |
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13001513 |
Mar 1, 2011 |
9127086 |
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14809603 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
7/02 20180101; G01N 2333/70596 20130101; A61P 19/04 20180101; A61P
43/00 20180101; G01N 33/505 20130101; C07K 14/70503 20130101; A61P
25/00 20180101; A61P 9/10 20180101; A61P 3/10 20180101; A61P 19/02
20180101; A61P 29/00 20180101; A61P 9/14 20180101; A61P 37/02
20180101; A61K 38/00 20130101; G01N 2500/10 20130101; A61P 5/14
20180101; A61P 1/00 20180101 |
International
Class: |
C07K 14/705 20060101
C07K014/705; G01N 33/50 20060101 G01N033/50 |
Claims
1-18. (canceled)
19. An isolated peptide consisting of: a) a fragment of at least 6
amino acids of the sequence defined by amino acids 579 to 601 of
SEQ ID NO: 1; b) a fragment of at least 6 amino acids of the
sequence corresponding to (a) in a non-human mammalian CD31; or c)
a fragment consisting of a sequence at least 80% identical to (a),
wherein said peptide exerts a dose-dependent inhibition of T-cell
proliferation in vitro and does not consist of an amino acid
sequence of SEQ ID NO: 5 or 6 and wherein said peptide optionally
comprises at least one chemical modification improving its
stability and/or its bioavailability selected from the group
consisting of: (a) modification to the N-terminal and/or C-terminal
end of the peptide by N-terminal acylation or deamination; (b)
modification of the C-terminal carboxyl group into an amide or an
alcohol group; (c) modification at the amide bond between two amino
acids by acylation or alkylation at the nitrogen atom or the alpha
carbon of the amide bond linking said two amino acids; (d)
modification at the alpha carbon of the amide bond linking two
amino acids by acylation or alkylation at the alpha carbon of the
amide bond linking said two amino acids; (e) replacement of one or
more naturally occurring L-enantiomeric amino acids with a
corresponding D-enantiomer; (f) retro-inversion, in which one or
more naturally occurring L-enantiomeric amino acids is replaced
with a corresponding D-enantiomer, together with inversion of the
amino acid chain; (g) replacement of one or more alpha carbons with
nitrogen atoms; and (h) binding of the amino group of one or more
amino acid to the .beta. carbon instead of the .alpha. carbon.
20. The peptide according to claim 19, wherein said peptide
consists of: a) a fragment of 6 to 15 amino acids of the sequence
defined by amino acids 579 to 601 of SEQ ID NO: 1; b) a fragment of
6 to 15 amino acids of the sequence corresponding to (a) in a
non-human mammalian CD31; c) a fragment consisting of a sequence at
least 80% identical to (a), or d) a peptide as defined in a), b) or
c) comprising at least one chemical modification improving its
stability and/or its bioavailability selected from the group
consisting of: (a) modification to the N-terminal and/or C-terminal
end of the peptide by N-terminal acylation or deamination; (b)
modification of the C-terminal carboxyl group into an amide or an
alcohol group; (c) modification at the amide bond between two amino
acids by acylation or alkylation at the nitrogen atom or the alpha
carbon of the amide bond linking said two amino acids; (d)
modification at the alpha carbon of the amide bond linking two
amino acids by acylation or alkylation at the alpha carbon of the
amide bond linking said two amino acids; (e) replacement of one or
more naturally occurring L-enantiomeric amino acids with a
corresponding D-enantiomer; (f) retro-inversion, in which one or
more naturally occurring L-enantiomeric amino acids is replaced
with a corresponding D-enantiomer, together with inversion of the
amino acid chain; (g) replacement of one or more alpha carbons with
nitrogen atoms; and (h) binding of the amino group of one or more
amino acid to the .beta. carbon instead of the .alpha. carbon;
wherein said peptide exerts a dose-dependent inhibition of T-cell
proliferation in vitro.
21. The peptide according to claim 19, wherein said peptide
comprises an amino acid sequence at least 80% identical to SEQ ID
NO: 2, 3 or 4.
22. A pharmaceutical composition comprising one or more of: a
peptide consisting of: a) a fragment of at least 6 amino acids of
the sequence defined by amino acids 579 to 601 of SEQ ID NO: 1; b)
a fragment of at least 6 amino acids of the sequence corresponding
to (a) in a non-human mammalian CD31; or c) a fragment consisting
of a sequence at least 80% identical to (a), wherein said peptide
exerts a dose-dependent inhibition of T-cell proliferation in vitro
and does not consist of an amino acid sequence of SEQ ID NO: 5 or 6
and wherein said peptide optionally comprises at least one chemical
modification improving its stability and/or its bioavailability
selected from the group consisting of: (a) modification to the
N-terminal and/or C-terminal end of the peptide by N-terminal
acylation or deamination; (b) modification of the C-terminal
carboxyl group into an amide or an alcohol group; (c) modification
at the amide bond between two amino acids by acylation or
alkylation at the nitrogen atom or the alpha carbon of the amide
bond linking said two amino acids; (d) modification at the alpha
carbon of the amide bond linking two amino acids by acylation or
alkylation at the alpha carbon of the amide bond linking said two
amino acids; (e) replacement of one or more naturally occurring
L-enantiomeric amino acids with a corresponding D-enantiomer; (f)
retro-inversion, in which one or more naturally occurring
L-enantiomeric amino acids is replaced with a corresponding
D-enantiomer, together with inversion of the amino acid chain; (g)
replacement of one or more alpha carbons with nitrogen atoms; and
(h) binding of the amino group of one or more amino acid to the
.beta. carbon instead of the .alpha. carbon, and a nucleic acid
encoding said peptide; and a physiologically acceptable
carrier.
23. The pharmaceutical composition according to claim 22, wherein
said peptide consists of: a) a fragment of 6 to 15 amino acids of
the sequence defined by amino acids 579 to 601 of SEQ ID NO: 1; b)
a fragment of 6 to 15 amino acids of the sequence corresponding to
(a) in a non-human mammalian CD31; c) a fragment consisting of a
sequence at least 80% identical to (a), or d) a peptide as defined
in a), b) or c) comprising at least one chemical modification
improving its stability and/or its bioavailability selected from
the group consisting of: (a) modification to the N-terminal and/or
C-terminal end of the peptide by N-terminal acylation or
deamination; (b) modification of the C-terminal carboxyl group into
an amide or an alcohol group; (c) modification at the amide bond
between two amino acids by acylation or alkylation at the nitrogen
atom or the alpha carbon of the amide bond linking said two amino
acids; (d) modification at the alpha carbon of the amide bond
linking two amino acids by acylation or alkylation at the alpha
carbon of the amide bond linking said two amino acids; (e)
replacement of one or more naturally occurring L-enantiomeric amino
acids with a corresponding D-enantiomer; (f) retro-inversion, in
which one or more naturally occurring L-enantiomeric amino acids is
replaced with a corresponding D-enantiomer, together with inversion
of the amino acid chain; (g) replacement of one or more alpha
carbons with nitrogen atoms; and (h) binding of the amino group of
one or more amino acid to the .beta. carbon instead of the .alpha.
carbon; wherein said peptide exerts a dose-dependent inhibition of
T-cell proliferation in vitro.
24. The pharmaceutical composition according to claim 22, wherein
said peptide comprises an amino acid sequence at least 80%
identical to SEQ ID NO: 2, 3 or 4.
25. A method of activating CD31-mediated signaling in an individual
in need thereof, comprising administering to the individual a
peptide comprising or consisting of: a) amino acids 579 to 601 of
SEQ ID NO: 1; b) the amino acids corresponding to (a) in a
non-human mammalian CD31; c) a fragment of at least 6 amino acids
of (a); d) a fragment of at least 6 amino acids of (b); or e) a
sequence at least 80% identical to (a) or (c), wherein said peptide
exerts a dose-dependent inhibition of T-cell proliferation in vitro
and wherein said peptide optionally comprises at least one chemical
modification improving its stability and/or its bioavailability
selected from the group consisting of: (a) modification to the
N-terminal and/or C-terminal end of the peptide by N-terminal
acylation or deamination; (b) modification of the C-terminal
carboxyl group into an amide or an alcohol group; (c) modification
at the amide bond between two amino acids by acylation or
alkylation at the nitrogen atom or the alpha carbon of the amide
bond linking said two amino acids; (d) modification at the alpha
carbon of the amide bond linking two amino acids by acylation or
alkylation at the alpha carbon of the amide bond linking said two
amino acids; (e) replacement of one or more naturally occurring
L-enantiomeric amino acids with a corresponding D-enantiomer; (f)
retro-inversion, in which one or more naturally occurring
L-enantiomeric amino acids is replaced with a corresponding
D-enantiomer, together with inversion of the amino acid chain; (g)
replacement of one or more alpha carbons with nitrogen atoms; and
(h) binding of the amino group of one or more amino acid to the
.beta. carbon instead of the .alpha. carbon.
26. The method according to claim 25, wherein said peptide has a
length of at most 30 amino acids.
27. The method according to claim 25, wherein said individual has a
CD31.sup.shed T lymphocytes phenotype.
28. The method according to claim 25, wherein said peptide consists
of: a) a peptide according to claim 19; b) a peptide comprising an
amino acid sequence at least 80% identical to SEQ ID NO: 5 or 6; or
c) a peptide comprising an amino acid sequence of SEQ ID NO: 5 or
6.
29. The method according to claim 25, wherein said peptide consists
of: a) a fragment of 6 to 15 amino acids of the sequence defined by
amino acids 579 to 601 of SEQ ID NO: 1; b) a fragment of 6 to 15
amino acids of the sequence corresponding to (a) in a non-human
mammalian CD31; c) a fragment consisting of a sequence at least 80%
identical to (a), or d) a peptide as defined in a), b) or c)
comprising at least one chemical modification improving its
stability and/or its bioavailability selected from the group
consisting of: (a) modification to the N-terminal and/or C-terminal
end of the peptide by N-terminal acylation or deamination; (b)
modification of the C-terminal carboxyl group into an amide or an
alcohol group; (c) modification at the amide bond between two amino
acids by acylation or alkylation at the nitrogen atom or the alpha
carbon of the amide bond linking said two amino acids; (d)
modification at the alpha carbon of the amide bond linking two
amino acids by acylation or alkylation at the alpha carbon of the
amide bond linking said two amino acids; (e) replacement of one or
more naturally occurring L-enantiomeric amino acids with a
corresponding D-enantiomer; (f) retro-inversion, in which one or
more naturally occurring L-enantiomeric amino acids is replaced
with a corresponding D-enantiomer, together with inversion of the
amino acid chain; (g) replacement of one or more alpha carbons with
nitrogen atoms; and (h) binding of the amino group of one or more
amino acid to the .beta. carbon instead of the .alpha. carbon;
wherein said peptide exerts a dose-dependent inhibition of T-cell
proliferation in vitro.
30. The method according to claim 25, wherein said peptide
comprises an amino acid sequence at least 80% identical to SEQ ID
NO: 2, 3 or 4.
31. The method according to claim 25, wherein said method is a
method for treating a thrombotic or an autoimmune disorder.
32. The method according to claim 25, wherein said method is a
method for treating a thrombotic disorder selected from the group
consisting of atherothrombosis, atherosclerosis, acute coronary
syndrome, ischemic stroke, peripheral arterial disease, abdominal
aortic aneurysm, deep vein thrombosis, myocardial infarction, and
pulmonary embolism.
33. The method according to claim 25, wherein said method is a
method for treating an autoimmune disorder selected from the group
consisting of rheumatoid arthritis, multiple sclerosis,
inflammatory bowel disease, systemic lupus erythematosus, Graves'
disease and diabetes mellitus.
Description
FIELD OF THE INVENTION
[0001] The present invention stems from the finding that the
extracellular domain of CD31 proteins present on blood leukocytes
is shed and released in the circulation as a soluble form of CD31.
The invention relates to peptides corresponding to fragments of
CD31 that inhibit T-cell response, and to their use in the
treatment of thrombotic and autoimmune disorders.
BACKGROUND
[0002] Thrombotic Disorders
[0003] In a healthy person, a homeostatic balance exists between
procoagulant (clotting) forces and anticoagulant and fibrinolytic
forces. Numerous genetic, acquired, and environmental factors can
tip the balance in favor of coagulation, leading to the pathologic
formation of thrombi in veins (e.g. deep vein thrombosis), arteries
(e.g. atherothrombosis, myocardial infarction, ischemic stroke), or
cardiac chambers. Thrombi can obstruct blood flow at the site of
formation or detach and embolize to block a distant blood vessel
(e.g. pulmonary embolism, stroke).
[0004] Accumulating evidences show that atherothrombosis, a
world-leading life-threatening disease, is linked to a defective
immunoregulation driving a pathologic activation of blood
leukocytes and a destructive inflammatory response within the
vascular wall. Consequently, a restoration of immunoregulation at
the blood-vessel interface would represent an innovative
therapeutic option to fight atherothrombosis.
[0005] Autoimmune Disorders
[0006] In autoimmune disorders, the immune system produces
antibodies to an endogenous antigen. Antibody-coated cells, like
any similarly coated foreign particle, activate the complement
system, resulting in tissue injury. Autoimmune disorders include
systemic lupus erythematodes (SLE), rheumatoid arthritis (RA),
multiple sclerosis (MS), inflammatory bowel disease (IBD), Graves'
disease and diabetes mellitus.
[0007] Several mechanisms may account for the body's attack on
itself. Autoantigens may become immunogenic because they are
altered chemically, physically, or biologically. Certain chemicals
couple with body proteins, making them immunogenic (as in contact
dermatitis). Drugs can produce several autoimmune reactions by
binding covalently to serum or tissue proteins (see below).
Photosensitivity exemplifies physically induced autoallergy:
Ultraviolet light alters skin protein, to which the patient becomes
allergic. In animal models, persistent infection with an RNA virus
that combines with host tissues alters autoantigens biologically,
resulting in an autoallergic disorder resembling SLE.
[0008] Most human autoimmune diseases are specific antigen-driven
T-cell diseases. T-cell clones responding to specific antigenic
epitopes are responsible for the initiation and/or the propagation
of these diseases. Similarly, specific antigen-driven T-cell
responses are responsible for the rejection of organ allografts and
the immune response to tumors. Activated T cells provide the
"engine" for the chronic inflammation that is associated with
autoimmune diseases, organ graft rejection and tumor immunity.
[0009] CD31 (PECAM-1)
[0010] Immune responses can be controlled by inhibitory immune
receptors among which CD31 (PECAM-1), which is expressed
exclusively and constitutively on cells at the blood-vessel
interface.
[0011] CD31 consists of a single chain molecule comprising six
Ig-like extracellular domains, a short transmembrane segment and a
cytoplasmic tail containing two ImmunoTyrosine-based Inhibitory
Motif (ITIM)s. The structure of CD31 is shown in the table
below.
TABLE-US-00001 Domain Position on SEQ ID No: 1 Signal peptide 1 to
27 Extracellular domain 28 to 601 First Ig-like extracellular
domain 34 to 121 Second Ig-like extracellular 145 to 233 domain
Third Ig-like extracellular domain 236 to 315 Fourth Ig-like
extracellular 328 to 401 domain Fifth Ig-like extracellular domain
424 to 493 Sixth Ig-like extracellular domain 499 to 591
Juxta-membrane domain 592 to 601 Transmembrane domain 602 to 620
Cytoplasmic domain 621 to 738
[0012] The immunoregulatory properties of CD31 are supported by the
fact that CD31 signalling drives mutual repulsion of blood
leukocytes and modulates the balance between inhibitory and
stimulatory signals of both innate and adaptive immune cells.
Mechanical engagement of the distal Ig-like extracellular domains
of CD31 induces outside-in inhibitory signalling triggered by the
phosphorylation of its ITIMs, and the recruitment and activation of
SH2-containing phosphatases.
[0013] Zehnder et al. (1995, Blood. 85(5):1282-8) identified a CD31
antibody that inhibited the mixed lymphocyte reaction (MLR) in a
specific and dose-dependent manner. They further found that a CD31
peptide corresponding to the epitope of this antibody, i.e. to the
23 membrane-proximal amino acids of CD31, strongly inhibited the
MLR. They hypothesized that the 23 membrane-proximal amino acids of
CD31 constitutes a functionally important region, and that the CD31
peptide interferes with lymphocyte activation by competing for
binding epitopes. However, Zehnder et al. failed to teach whether
CD31-mediated signaling is activated or inhibited by the CD31
peptide.
[0014] Chen et al. (1997, Blood. 89(4):1452-9) showed that this
peptide delayed onset of graft-versus-host disease (GVHD) and
increased long-term survival in a murine model of the disease. They
hypothesized that the CD31 peptide inhibits a common pathway in
T-cell activation. Again, Chen et al. failed to elucidate the role
played by the CD31 peptide in T-cell activation. In particular,
these previous works did not assess the putative effect of the
peptide on the CD31 signaling cascade and more precisely on the
phosporylation state of the CD31 ITIMs.
[0015] By a yet unknown mechanism, CD31 is "lost" on certain
circulating lymphocytes. Its loss is observed upon lymphocyte
activation and it has been recently shown that the absence of
lymphocyte CD31 signalling, in turn, heightens the pathologic
immune responses involved in the development of
atherothrombosis.
[0016] A soluble form of CD31, due to a variant transcript lacking
the transmembrane segment, has also been reported and therefore it
is currently thought that the individual amount of circulating CD31
is genetically determined. Consequently, a number of previous
studies have attempted to find a correlation between plasma levels
of soluble CD31 and the risk of atherothrombosis or other
autoimmune diseases. However, independently of the specific genetic
polymorphisms analyzed, data showed a broad range of plasma CD31
values and the results of these different studies were
contradicting.
[0017] There is therefore a need for better understanding the
biological function of CD31. This would allow the provision of more
efficient therapeutics for the treatment of diseases linked with
T-cell activation.
DESCRIPTION OF THE INVENTION
[0018] It has surprisingly been found that the assumed loss of CD31
on activated/memory T lymphocytes is actually incomplete and
results from shedding of CD31 between the 5.sup.th and the 6.sup.th
extracellular Ig-like domains. The shed extracellular domain of
CD31 (further referred to as "shed CD31") is then released into the
circulation, where it is present together with a soluble splice
variant of CD31.
[0019] In addition, it has been shown that a high risk of
atherothrombosis is linked with the increase in shed CD31 and
decrease in splice variant CD31 in the circulation, and not with
the total level of circulating CD31.
[0020] The finding that CD31 is not lost on blood lymphocytes but
only cleaved provides a unique opportunity to rescue its
physiological immunoregulatory function by targeting the residual
portion of the molecule. Specifically, the present invention
provides peptides corresponding to juxta-membrane amino acids of
the ectodomain of CD31 that are able to rescue the physiological
immunoregulatory function of CD31, even in patients having
apparently lost CD31 from the surface of their circulating T
lymphocytes.
[0021] It has been demonstrated that such peptides are capable of
preventing disease progression and aneurysm formation in a mouse
model for atherosclerosis. Shorter and more stable peptides
restricted to the last 10 or 6 COOH-terminal amino acids of the
known peptide of twenty-three amino acids display superior in vitro
immunosuppressive properties (lower ED and lower intra and
inter-assay variability) than the known peptide. These amino acids
correspond to a short extracellular fragment comprised between the
membrane and the 6.sup.th Ig-like domain of CD31.
[0022] The invention therefore provides peptides consisting of a
fragment comprising the membrane juxta-proximal part of
extracellular CD31 and part of the sixth Ig-like domain and the use
of such peptides in the treatment of a thrombotic or an autoimmune
as further described herein.
[0023] Such peptides have unique properties compared to soluble
forms of CD31 comprising all or most Ig-like domains of CD31.
Indeed, such peptides are highly homophilic since they have a Kd of
10.sup.-7 M, as assessed by BIAcore analysis. Hence they are able
to engage CD31 signaling by bridging the membrane juxta-proximal
part of extracellular CD31 that remains expressed after its
cleavage, via a strong homo-oligomerization. In contrast to this,
alternatively spliced soluble CD31 lacks the first 10 membrane
juxta-proximal amino acids and shows weak homophilic binding with
the 23-mer peptide (Kd of 17 .mu.M, as assessed by BIAcore
analysis). Furthermore, in vitro, only the peptides according to
the invention are capable of engaging the ITIM pathway downstream
of the truncated isoform of CD31, and are thus capable of restoring
CD31 signaling in T lymphocytes having apparently lost CD31.
[0024] CD31 Peptides
[0025] It has been found that the six-amino-acid-long CD31 peptide
of sequence SEQ ID NO: 2 and that a ten-amino-acid-long CD31
peptide of sequence SEQ ID NO: 3 are capable of inhibiting T-cell
activation. These two peptides correspond to fragments comprising
the membrane juxta-proximal part of extracellular CD31, which is
adjacent to the sixth extracellular Ig-like domain of CD31.
[0026] Therefore, the invention is directed to an isolated peptide
comprising or consisting of a fragment of CD31, wherein said
fragment is selected from the group consisting of: [0027] a) a
fragment of at least 3, 6 or 10 amino acids of the sequence defined
by amino acids 579 to 601 of SEQ ID NO: 1; [0028] b) a fragment of
at least 3, 6 or 10 amino acids of the sequence corresponding to
(a) in a non-human mammalian CD31; or [0029] c) a sequence at least
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to (a); with the
proviso that said peptide of (c) does not consist of an amino acid
sequence of SEQ ID NO: 5 or 6.
[0030] As used herein, the term "peptide" has the meaning usually
given in the art. More specifically, the dividing line between
proteins and peptides is usually set at a length of approximately
50 amino acids. Thus the peptides according to the invention
preferably have a length of at most 50, 40, 35, 30, 25, 20, 15 or
10 amino acids.
[0031] As used herein, the term "fragment" of a reference sequence
refers to a chain of contiguous nucleotides or amino acids that is
shorter than the reference sequence. More specifically, a fragment
of the sequence defined by amino acids 579 to 601 of SEQ ID NO: 1
is at most 22 amino acids long. Said fragment may have a length of
e.g. 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21 or 22 amino acids. Preferably, said fragment has a length
within the range of 3 to 18, 3 to 15, 3 to 10, 6 to 18, 6 to 15 or
6 to 10 amino acids.
[0032] The isolated peptide according to the invention may for
example comprise or consist of a fragment having a sequence
selected from the group consisting of SMRTSPRSSTLAVRVFLAPWKK (amino
acids 2 to 23 of SEQ ID NO: 5), MRTSPRSSTLAVRVFLAPWKK (amino acids
3 to 23 of SEQ ID NO: 5), RTSPRSSTLAVRVFLAPWKK (amino acids 4 to 23
of SEQ ID NO: 5), TSPRSSTLAVRVFLAPWKK (amino acids 5 to 23 of SEQ
ID NO: 5), SPRSSTLAVRVFLAPWKK (amino acids 6 to 23 of SEQ ID NO:
5), PRSSTLAVRVFLAPWKK (amino acids 7 to 23 of SEQ ID NO: 5),
RSSTLAVRVFLAPWKK (amino acids 8 to 23 of SEQ ID NO: 5),
SSTLAVRVFLAPWKK (amino acids 9 to 23 of SEQ ID NO: 5),
STLAVRVFLAPWKK (amino acids 10 to 23 of SEQ ID NO: 5),
TLAVRVFLAPWKK (amino acids 11 to 23 of SEQ ID NO: 5), LAVRVFLAPWKK
(amino acids 12 to 23 of SEQ ID NO: 5), AVRVFLAPWKK (amino acids 13
to 23 of SEQ ID NO: 5), VRVFLAPWKK (amino acids 14 to 23 of SEQ ID
NO: 5), RVFLAPWKK (amino acids 15 to 23 of SEQ ID NO: 5), VFLAPWKK
(amino acids 16 to 23 of SEQ ID NO: 5), FLAPWKK (amino acids 17 to
23 of SEQ ID NO: 5), LAPWKK (amino acids 18 to 23 of SEQ ID NO: 5),
APWKK (amino acids 19 to 23 of SEQ ID NO: 5), PWKK (amino acids 20
to 23 of SEQ ID NO: 5), WKK (amino acids 21 to 23 of SEQ ID NO: 5),
HASSVPRSKILTVRVILAPWKK (amino acids 2 to 23 of SEQ ID NO: 6),
ASSVPRSKILTVRVILAPWKK (amino acids 3 to 23 of SEQ ID NO: 6),
SSVPRSKILTVRVILAPWKK (amino acids 4 to 23 of SEQ ID NO: 6),
SVPRSKILTVRVILAPWKK (amino acids 5 to 23 of SEQ ID NO: 6),
VPRSKILTVRVILAPWKK (amino acids 6 to 23 of SEQ ID NO: 6),
PRSKILTVRVILAPWKK (amino acids 7 to 23 of SEQ ID NO: 6),
RSKILTVRVILAPWKK (amino acids 8 to 23 of SEQ ID NO: 6),
SKILTVRVILAPWKK (amino acids 9 to 23 of SEQ ID NO: 6),
KILTVRVILAPWKK (amino acids 10 to 23 of SEQ ID NO: 6),
ILTVRVILAPWKK (amino acids 11 to 23 of SEQ ID NO: 6), LTVRVILAPWKK
(amino acids 12 to 23 of SEQ ID NO: 6), TVRVILAPWKK (amino acids 13
to 23 of SEQ ID NO: 6), VRVILAPWKK (amino acids 14 to 23 of SEQ ID
NO: 6), RVILAPWKK (amino acids 15 to 23 of SEQ ID NO: 6), VILAPWKK
(amino acids 16 to 23 of SEQ ID NO: 6), ILAPWKK (amino acids 17 to
23 of SEQ ID NO: 6), SSMRTSPRSSTLAVRVFLAPWK (amino acids 1 to 22 of
SEQ ID NO: 5), SSMRTSPRSSTLAVRVFLAPW (amino acids 1 to 21 of SEQ ID
NO: 5), SSMRTSPRSSTLAVRVFLAP (amino acids 1 to 20 of SEQ ID NO: 5),
SSMRTSPRSSTLAVRVFLA (amino acids 1 to 19 of SEQ ID NO: 5),
SSMRTSPRSSTLAVRVFL (amino acids 1 to 18 of SEQ ID NO: 5),
SSMRTSPRSSTLAVRVF (amino acids 1 to 17 of SEQ ID NO: 5),
SSMRTSPRSSTLAVRV (amino acids 1 to 16 of SEQ ID NO: 5),
SSMRTSPRSSTLAVR (amino acids 1 to 15 of SEQ ID NO: 5),
SSMRTSPRSSTLAV (amino acids 1 to 14 of SEQ ID NO: 5), SSMRTSPRSSTLA
(amino acids 1 to 13 of SEQ ID NO: 5), SSMRTSPRSSTL (amino acids 1
to 12 of SEQ ID NO: 5), SSMRTSPRSST (amino acids 1 to 11 of SEQ ID
NO: 5), SSMRTSPRSS (amino acids 1 to 10 of SEQ ID NO: 5), SSMRTSPRS
(amino acids 1 to 9 of SEQ ID NO: 5), SSMRTSPR (amino acids 1 to 8
of SEQ ID NO: 5), SSMRTSP (amino acids 1 to 7 of SEQ ID NO: 5),
SSMRTS (amino acids 1 to 6 of SEQ ID NO: 5), SSMRT (amino acids 1
to 5 of SEQ ID NO: 5), SSMR (amino acids 1 to 4 of SEQ ID NO: 5),
SSM (amino acids 1 to 3 of SEQ ID NO: 5), NHASSVPRSKILTVRVILAPWK
(amino acids 1 to 22 of SEQ ID NO: 6), NHASSVPRSKILTVRVILAPW (amino
acids 1 to 21 of SEQ ID NO: 6), NHASSVPRSKILTVRVILAP (amino acids 1
to 20 of SEQ ID NO: 6), NHASSVPRSKILTVRVILA (amino acids 1 to 19 of
SEQ ID NO: 6), NHASSVPRSKILTVRVIL (amino acids 1 to 18 of SEQ ID
NO: 6), NHASSVPRSKILTVRVI (amino acids 1 to 17 of SEQ ID NO: 6),
NHASSVPRSKILTVRV (amino acids 1 to 16 of SEQ ID NO: 6),
NHASSVPRSKILTVR (amino acids 1 to 15 of SEQ ID NO: 6),
NHASSVPRSKILTV (amino acids 1 to 14 of SEQ ID NO: 6), NHASSVPRSKILT
(amino acids 1 to 13 of SEQ ID NO: 6), NHASSVPRSKIL (amino acids 1
to 12 of SEQ ID NO: 6), NHASSVPRSKI (amino acids 1 to 11 of SEQ ID
NO: 6), NHASSVPRSK (amino acids 1 to 10 of SEQ ID NO: 6), NHASSVPRS
(amino acids 1 to 9 of SEQ ID NO: 6), NHASSVPR (amino acids 1 to 8
of SEQ ID NO: 6), NHASSVP (amino acids 1 to 7 of SEQ ID NO: 6),
NHASSV (amino acids 1 to 6 of SEQ ID NO: 6), NHASS (amino acids 1
to 5 of SEQ ID NO: 6), NHAS (amino acids 1 to 4 of SEQ ID NO: 6),
NHA (amino acids 1 to 3 of SEQ ID NO: 6), SMRTSPRSSTLAVRVFLAPWK
(amino acids 2 to 22 of SEQ ID NO: 5), MRTSPRSSTLAVRVFLAPW (amino
acids 3 to 21 of SEQ ID NO: 5), RTSPRSSTLAVRVFLAP (amino acids 4 to
20 of SEQ ID NO: 5), TSPRSSTLAVRVFLA (amino acids 5 to 19 of SEQ ID
NO: 5), SPRSSTLAVRVFL (amino acids 6 to 18 of SEQ ID NO: 5),
PRSSTLAVRVF (amino acids 7 to 17 of SEQ ID NO: 5), RSSTLAVRV (amino
acids 8 to 16 of SEQ ID NO: 5), SSTLAVR (amino acids 9 to 15 of SEQ
ID NO: 5), STLAV (amino acids 10 to 14 of SEQ ID NO: 5), TLA (amino
acids 11 to 13 of SEQ ID NO: 5), HASSVPRSKILTVRVILAPWK (amino acids
2 to 22 of SEQ ID NO: 6), ASSVPRSKILTVRVILAPW (amino acids 3 to 21
of SEQ ID NO: 6), SSVPRSKILTVRVILAP (amino acids 4 to 20 of SEQ ID
NO: 6), SVPRSKILTVRVILA (amino acids 5 to 19 of SEQ ID NO: 6),
VPRSKILTVRVIL (amino acids 6 to 18 of SEQ ID NO: 6), PRSKILTVRVI
(amino acids 7 to 17 of SEQ ID NO: 6), RSKILTVRV (amino acids 8 to
16 of SEQ ID NO: 6), SKILTVR (amino acids 9 to 15 of SEQ ID NO: 6),
KILTV (amino acids 10 to 14 of SEQ ID NO: 6) and ILT (amino acids
11 to 13 of SEQ ID NO: 6).
[0033] In a preferred embodiment, said fragment corresponds to a
juxta-membrane fragment, i.e. to a fragment immediately adjacent to
the transmembrane domain of CD31. In other words, said fragment
preferably corresponds to a fragment including the C-terminal
extremity of the sequence consisting of amino acids 579 to 601 of
SEQ ID NO: 1 or of the corresponding sequence in a non-human
mammalian CD31. For example, a peptide consisting of a
juxta-membrane fragment of 15 amino acids of the sequence defined
by amino acids 579 to 601 of SEQ ID NO: 1 consists of amino acids
587 to 601 of SEQ ID NO: 1.
[0034] In a specific embodiment, peptides comprising the
transmembrane domain of CD31 or part thereof are excluded from the
scope of the present invention.
[0035] The sequence of CD31 peptides according to the invention is
preferably derived from the sequence of human or murine CD31.
However, the sequence of CD31 may be derived from any non-human
mammalian CD31 sequence. FIG. 1 shows an alignment between the
human, murine, bovine and pig CD31 sequences. The sequence
corresponding to the sequence defined by amino acids 579 to 601 of
SEQ ID NO: 1 is highlighted by a box. The skilled in the art can
easily identify the corresponding sequence in another non-human
mammalian CD31 protein by performing a sequence alignment with the
sequences shown in FIG. 1.
[0036] In addition to the CD31 fragment, the peptide may optionally
comprise sequences heterologous to CD31. These heterologous
sequences may e.g. correspond to a carrier molecule such as the
Keyhole Limpet Hemocyanin (KLH), bovine serum albumine (BSA),
ovoalbumin (OVA), thyroglobulin (THY) or the multiple antigenic
peptide (MAP).
[0037] In a preferred embodiment, the peptide comprises or consists
of a peptide selected from the group consisting of: [0038] the
six-amino-acid-long peptide shown as SEQ ID NO: 2, which is present
both in human and murine CD31 sequences; [0039] the
ten-amino-acid-long peptide shown as SEQ ID NO: 3, the sequence of
which is derived from the mouse CD31 sequence; and [0040] the
ten-amino-acid-long peptide shown as SEQ ID NO: 4, the sequence of
which is derived from the human CD31 sequence.
[0041] In another preferred embodiment, the peptide comprises or
consists of an amino acid sequence at least 80%, 85%, 90%, 95%,
96%, 97%, 98% or 99% identical to any one of SEQ ID NOs: 2, 3 or
4.
[0042] CD31 peptides according to the invention have the biological
activity of exerting a dose-dependent inhibition of T-cell
proliferation in vitro and/or of inhibiting the mixed-lymphocyte
reaction (MLR). Their biological activity may for example be
measured as described in Example 3, Example 6 or in Zehnder et al.
(1995, Blood. 85(5):1282-8).
[0043] The T-cell proliferation assay may comprise comparing the
radioactivity incorporated into T-cells cultured either in the
presence or in the absence of the compound to be tested. This assay
may for example be performed as follows: [0044] providing a
multi-well plate comprising complete medium supplemented with
anti-CD3 antibodies; [0045] supplementing the wells with increasing
concentrations of the compound to be tested; [0046] plating
peripheral blood mononuclear cells (e.g. of spleen cells); [0047]
culturing the cells for about 72 hours; [0048] adding (.sup.3H)
thymidine and culturing the cells for about 16 hours; [0049]
measuring the radioactivity; and [0050] comparing the radioactivity
measured in the presence of the compound to be tested with the
radioactivity measured in the absence of said compound, and/or in
the presence of a reference compound, and/or in the presence of a
negative control.
[0051] Alternatively, the T-cell proliferation assay may comprise
comparing expression levels of the early activation marker CD69 in
T-cells cultured either in the presence or in the absence of the
compound to be tested. This assay may for example be performed as
follows: [0052] providing purified CD4+ cells (e.g. purified from
C571316 mice); [0053] stimulating said CD4+ cells by addition of
anti-CD3 purified antibodies and bone marrow derived dendritic
cells; [0054] culturing the cells for about 18 hours; and [0055]
analyzing said cells for the expression of the early activation
marker CD69, e.g. by flow cytometry; and [0056] comparing CD69
expression in the presence of the compound to be tested.
[0057] By a polypeptide having an amino acid sequence at least, for
example, 95% "identical" to a query amino acid sequence of the
present invention, it is intended that the amino acid sequence of
the subject polypeptide is identical to the query sequence except
that the subject polypeptide sequence may include up to five amino
acid alterations per each 100 amino acids of the query amino acid
sequence. In other words, to obtain a polypeptide having an amino
acid sequence at least 95% identical to a query amino acid
sequence, up to 5% (5 of 100) of the amino acid residues in the
subject sequence may be inserted, deleted, or substituted with
another amino acid.
[0058] Methods for comparing the identity and homology of two or
more sequences are well known in the art. Thus for instance,
programs available in the Wisconsin Sequence Analysis Package,
version 9.1, for example the programs BESTFIT and GAP, may be used
to determine the % identity between two polynucleotides and the %
identity and the % homology between two polypeptide sequences.
BESTFIT uses the "local homology" algorithm of Smith and Waterman
and finds the best single region of similarity between two
sequences. Other programs for determining identity and/or
similarity between sequences are also known in the art, for
instance the BLAST family of programs, accessible through the home
page of the NCBI at world wide web site ncbi.nim.nih.gov) and
FASTA.
[0059] Peptides consisting of an amino acid sequence "at least 80%,
85%, 90%, 95%, 96%, 97%, 98% or 99% identical" to a reference
sequence may comprise mutations such as deletions, insertions
and/or substitutions compared to the reference sequence. In case of
substitutions, the substitution preferably corresponds to a
conservative substitution as indicated in the table below. In a
preferred embodiment, the peptide consisting of an amino acid
sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to a reference sequence only differs from the reference
sequence by conservative substitutions. In another preferred
embodiment, the peptide consisting of an amino acid sequence at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a
reference sequence corresponds to a naturally-occurring allelic
variant of the reference sequence. In still another preferred
embodiment, the peptide consisting of an amino acid sequence at
least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a
reference sequence corresponds to a homologous sequence derived
from another non-human mammalian species than the reference
sequence.
TABLE-US-00002 Conservative substitutions Type of Amino Acid Ala,
Val, Leu, Ile, Met, Pro, Amino acids with aliphatic hydrophobic
Phe, Trp side chains Ser, Tyr, Asn, Gln, Cys Amino acids with
uncharged but polar side chains Asp, Glu Amino acids with acidic
side chains Lys, Arg, His Amino acids with basic side chains Gly
Neutral side chain
[0060] In a preferred embodiment, the peptide according to the
invention consists of any one of the sequences shown in the table
below:
TABLE-US-00003 Swissprot accession SEQ species number position
sequence ID NO: Human P16284 592-601 VRVILAPWKK 4 Mouse Q08481
581-590 VRVFLAPWKK 3 Rat Q3SWT0 580-589 VRVFLAPWKK 10 Pig Q95242
593-602 VRVYLAPWKK 11 Bovin P51866 591-600 VRVYL-PLEK 12
[0061] CD31 peptides according to the invention may be prepared by
any well-known procedure in the art, such as solid phase synthesis,
liquid phase synthesis or genetic engineering. As a solid phase
synthesis, for example, the amino acid corresponding to the
C-terminus of the peptide to be synthesized is bound to a support
which is insoluble in organic solvents, and by alternate repetition
of reactions, one wherein amino acids with their amino groups and
side chain functional groups protected with appropriate protective
groups are condensed one by one in order from the C-terminus to the
N-terminus, and one where the amino acids bound to the resin or the
protective group of the amino groups of the peptides are released,
the peptide chain is thus extended in this manner. After synthesis
of the desired peptide, it is subjected to the deprotection
reaction and cut out from the solid support.
[0062] The CD31 peptides of the invention may optionally comprise
chemical modifications improving their stability and/or their
biodisponibility. Such chemical modifications aim at obtaining
peptides with increased protection of the peptides against
enzymatic degradation in vivo, and/or increased capacity to cross
membrane barriers, thus increasing its half-life and maintaining or
improving its biological activity. Any chemical modification known
in the art can be employed according to the present invention. Such
chemical modifications include but are not limited to: [0063]
modifications to the N-terminal and/or C-terminal ends of the
peptides such as e.g. N-terminal acylation (preferably acetylation)
or desamination, or modification of the C-terminal carboxyl group
into an amide or an alcohol group; [0064] modifications at the
amide bond between two amino acids: acylation (preferably
acetylation) or alkylation (preferably methylation) at the nitrogen
atom or the alpha carbon of the amide bond linking two amino acids;
[0065] modifications at the alpha carbon of the amide bond linking
two amino acids such as e.g. acylation (preferably acetylation) or
alkylation (preferably methylation) at the alpha carbon of the
amide bond linking two amino acids. [0066] chirality changes such
as e.g. replacement of one or more naturally occurring amino acids
(L enantiomer) with the corresponding D-enantiomers; [0067]
retro-inversions in which one or more naturally-occurring amino
acids (L-enantiomer) are replaced with the corresponding
D-enantiomers, together with an inversion of the amino acid chain
(from the C-terminal end to the N-terminal end); [0068]
azapeptides, in which one or more alpha carbons are replaced with
nitrogen atoms; and/or [0069] betapeptides, in which the amino
group of one or more amino acid is bonded to the .beta. carbon
rather than the .alpha. carbon.
[0070] By an "isolated" peptide, it is intended that the peptide is
not present within a living organism, e.g. within human body.
However, the isolated peptide may be part of a composition or a
kit. The isolated peptide is preferably purified.
[0071] The compounds of the invention may be produced by any
well-known procedure in the art, including chemical synthesis
technologies and recombinant technologies.
[0072] Examples of chemical synthesis technologies are solid phase
synthesis and liquid phase synthesis. As a solid phase synthesis,
for example, the amino acid corresponding to the C-terminus of the
peptide to be synthesized is bound to a support which is insoluble
in organic solvents, and by alternate repetition of reactions, one
wherein amino acids with their amino groups and side chain
functional groups protected with appropriate protective groups are
condensed one by one in order from the C-terminus to the
N-terminus, and one where the amino acids bound to the resin or the
protective group of the amino groups of the peptides are released,
the peptide chain is thus extended in this manner. Solid phase
synthesis methods are largely classified by the tBoc method and the
Fmoc method, depending on the type of protective group used.
Typically used protective groups include tBoe (t-butoxycarbonyl),
Cl-Z (2-chlorobenzyloxycarbonyl), Br-Z (2-bromobenzyloyycarbonyl),
Bzl (benzyl), Fmoc (9-fluorenylmcthoxycarbonyl), Mbh
(4,4'-dimethoxydibenzhydryl), Mtr
(4-methoxy-2,3,6-trimethylbenzenesulphonyl), Trt (trityl), Tos
(tosyl), Z (benzyloxycarbonyl) and Clz-Bzl (2,6-dichlorobenzyl) for
the amino groups; NO2 (nitro) and Pmc
(2,2,5,7,8-pentamethylchromane-6-sulphonyl) for the guanidino
groups); and tBu (t-butyl) for the hydroxyl groups). After
synthesis of the desired peptide, it is subjected to the
de-protection reaction and cut out from the solid support. Such
peptide cutting reaction may be carried with hydrogen fluoride or
tri-fluoromethane sulfonic acid for the Boc method, and with TFA
for the Fmoc method.
[0073] Alternatively, the peptide may be synthesized using
recombinant techniques. In this case, a nucleic acid encoding a
peptide according to the invention (further referred to as "a
nucleic acid according to the invention") is cloned into an
expression vector. The nucleic acid of the invention is preferably
placed under the control of expression signals (e.g. a promoter, a
terminator and/or an enhancer) allowing its expression. The
expression vector is then transfected into a host cell (e.g. a
human, CHO, mouse, monkey, fungal or bacterial host cell), and the
transfected host cell is cultivated under conditions suitable for
the expression of the peptide.
[0074] The method of producing the peptide may optionally comprise
the steps of purifying said peptide, chemically modifying said
peptide, and/or formulating said peptide into a pharmaceutical
composition.
[0075] The invention also encompasses a method for identifying a
peptidomimetic of a peptide according to the invention, said
peptidomimetic being a candidate compound for the treatment and/or
prevention of a thrombotic or an autoimmune disorder, comprising
the steps of:
[0076] a) providing a peptidomimetic; and
[0077] b) determining whether said peptidomimetic exerts: [0078] i.
a dose-dependent inhibition of T-cell proliferation in vitro and/or
of the mixed-lymphocyte reaction (MLR); [0079] ii. an
immunosuppressant activity; and/or [0080] iii. an antiplatelet
activity. wherein, if said peptidomimetic exerts a dose-dependent
inhibition of T-cell proliferation in vitro and/or of the
mixed-lymphocyte reaction (MLR), an immunosuppressant activity
and/or an antiplatelet activity, said peptidomimetic is a candidate
compound for the treatment and/or prevention of a thrombotic or an
autoimmune disorder.
[0081] Step (a) of the above method may comprise designing and
synthesizing said peptidomimetic.
[0082] Step (b) of the above method for identifying a
peptidomimetic of a peptide according to the invention may comprise
or consist of (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii)
and (iii); or (i) and (ii) and (iii).
[0083] Methods for determining whether said peptidomimetic exerts a
dose-dependent inhibition of T-cell proliferation in vitro and/or
of the mixed-lymphocyte reaction (MLR), an immunosuppressant
activity and/or an antiplatelet activity are well known to the
skilled in the art.
[0084] The determination whether said peptidomimetic exerts a
dose-dependent inhibition of T-cell proliferation in vitro and/or
of the mixed-lymphocyte reaction (MLR) may for example be performed
as described in Example 3.
[0085] Immunosuppressant and antiplatelet activity can for example
be the evaluated by analyzing the expression of activation surface
markers (e.g. CD62P for platelets and/or CD69 for leukocytes) or of
soluble activation markers (e.g. TXA2 for platelets and/or IL-2 for
Lymphocytes).
[0086] As used herein, the term peptidomimetic refers to a compound
containing non-peptidic structural elements that mimics the
biological action of a CD31 peptide according to the invention.
Methods for designing and synthesizing peptidomimetics of a given
peptide are well-known in the art and include e.g. those described
in Ripka and Rich (Curr Opin Chem Biol. 1998; 2(4):441-52) and in
Patch and Barron (Curr Opin Chem Biol. 2002; 6(6):872-7).
[0087] Use of CD31 Peptides for the Treatment of Thrombotic and
Autoimmune Disorders
[0088] It has been found that CD31 peptides corresponding to
fragments comprising part of the sixth extracellular Ig-like domain
of CD31 are capable of activating CD31-mediated signaling, even in
CD31.sup.- (i.e. CD31.sup.shed) T lymphocytes. In addition, such
peptides are capable of preventing disease progression and aneurysm
formation in a mouse model for atherosclerosis.
[0089] Therefore, the invention is directed to an isolated peptide
comprising or consisting of
[0090] a) amino acids 579 to 601 of SEQ ID NO: 1;
[0091] b) the amino acids corresponding to (a) in a non-human
mammalian CD31;
[0092] c) a fragment of at least 6 amino acids of (a);
[0093] d) a fragment of at least 6 amino acids of (b); or
[0094] e) a sequence at least 80% identical to (a) or (c);
for use in activating CD31-mediated signaling. These peptides
preferably exert a dose-dependent inhibition of T-cell
proliferation in vitro. The activation of CD31-mediated signaling
may be an in vitro or an in vivo activation.
[0095] As used throughout the present specification, the term
"CD31-mediated signaling" refers to a signaling pathway in which
CD31 is involved. Such pathways are well known in the art and
include those described e.g. in Newman and Newman (2003
Arterioscler Thromb Vasc Biol 23:953-964) and in Newton-Nash and
Newman (1999. J Immunol 163:682-688).
[0096] The invention is further directed to an isolated peptide
comprising or consisting of:
[0097] a) amino acids 579 to 601 of SEQ ID NO: 1;
[0098] b) the amino acids corresponding to (a) in a non-human
mammalian CD31;
[0099] c) a fragment of at least 6 amino acids of (a);
[0100] d) a fragment of at least 6 amino acids of (b); or
[0101] e) a sequence at least 80% identical to (a) or (c);
for use in the treatment of a thrombotic or an autoimmune disorder.
These peptides preferably exert a dose-dependent inhibition of
T-cell proliferation in vitro.
[0102] As used throughout the present specification, the term
"thrombotic disorder" includes but is not limited to
atherothrombosis, atherosclerosis, acute coronary syndrome,
ischemic stroke, peripheral arterial disease and abdominal aortic
aneurysm.
[0103] As used throughout the present specification, the term
"autoimmune disorder" includes but is not limited to rheumatoid
arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease
(IBD), systemic lupus erythematodes (SLE), Graves' disease and
diabetes mellitus.
[0104] In a preferred embodiment of the invention, said thrombotic
or autoimmune disorder is associated with a loss of CD31.sup.+ T
lymphocytes phenotype. Indeed, it has been surprisingly found that
CD31 peptides restore CD31 signaling even in individuals with a
CD31.sup.- T lymphocytes phenotype. Therefore, in the context of
the present invention, CD31 peptides are preferably used to treat a
subgroup of individuals and/or patients having a CD31.sup.- T
lymphocytes phenotype.
[0105] As used herein, the term "CD31.sup.- T lymphocytes
phenotype" is used interchangeably with the term "CD31.sup.shed T
lymphocytes phenotype". These terms refer to the phenotype of an
individual having apparently lost CD31 on its circulating T cells
when conventional prior art methods for detecting CD31, e.g. such
as those described in Stockinger et al. (Immunology, 1992,
75(1):53-8), Demeure et al. (Immunology, 1996, 88(1):110-5),
Caligiuri et al. (Arterioscler Thromb Vasc Biol, 2005,
25(8):1659-64) or Caligiuri et al. (Arterioscler Thromb Vasc Biol,
2006, 26(3):618-23) are used. In such methods, the antibody used
for detecting CD31 binds to an epitope located on any one of the
1.sup.St to the 5.sup.th extracellular Ig-like domains.
[0106] Preferably, individuals having a CD31.sup.- T lymphocytes
phenotype have lost at least 50%, 60%, 65%, 70%, 75%, 80%, 90% or
95% of their circulating CD4.sup.+/CD31.sup.+ T lymphocytes. In
other words, at least 50%, 60%, 65%, 70%, 75%, 80%, 90% or 95% of
their circulating T lymphocytes are CD31.sup.shed lymphocytes.
Either the plasmatic rate or the cellular rate of CD31.sup.- T
lymphocytes, compared to CD31.sup.+ T lymphocytes, may be
measured.
[0107] The CD31 peptide used for activating CD31-mediated signaling
and/or treating a thrombotic or an autoimmune disorder may be any
one of the peptides described in the above paragraph entitled "CD31
peptides".
[0108] Alternatively, the CD31 peptide used for activating
CD31-mediated signaling and/or treating a thrombotic or an
autoimmune disorder comprises or consists of: [0109] a) amino acids
579 to 601 of SEQ ID NO: 1; [0110] b) the amino acids corresponding
to (a) in a non-human mammalian CD31; [0111] c) a sequence at least
80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to (a) or
(b);
[0112] The peptides used herein may have a length comprised between
e.g. 10-100, 15-80, 20-60, 25-50 and 20-40 amino acids. These
peptides preferably comprise or consist of fragments immediately
adjacent to the transmembrane domain of CD31. The peptide may for
example correspond to a peptide consisting of an amino acid
sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%
identical to SEQ ID NO: 5 or 6. The peptide preferably consists of
SEQ ID NO: 5 or 6. These peptides may optionally comprise chemical
modifications improving their stability and/or their
biodisponibility.
[0113] The invention is also directed to a method of treating or
preventing a thrombotic or an autoimmune disorder comprising the
step of administering an effective amount of a peptide as described
herein, or a nucleic coding therefore, to an individual in need
thereof. Said individual in need thereof preferably suffers from or
is at risk of suffering from a thrombotic or an autoimmune
disorder. Most preferably, said individual has a CD31.sup.- T
lymphocytes phenotype.
[0114] By "effective amount", is meant an amount sufficient to
achieve a concentration of peptide which is capable of preventing,
treating or slowing down the disease to be treated. Such
concentrations can be routinely determined by those of skilled in
the art. The amount of the compound actually administered will
typically be determined by a physician, in the light of the
relevant circumstances, including the condition to be treated, the
chosen route of administration, the actual compound administered,
the age, weight, and response of the individual patient, the
severity of the patient's symptoms, and the like. It will also be
appreciated by those of stalled in the art that the dosage may be
dependent on the stability of the administered peptide.
[0115] The individuals to be treated in the frame of the invention
are preferably human individuals. However, the veterinary use of
CD31 peptides for treating other mammals is also contemplated by
the present invention.
[0116] Pharmaceutical Compositions
[0117] The CD31 peptides described herein may be formulated into a
pharmaceutical composition. Thus the invention contemplates a
pharmaceutical composition comprising any one of the above CD31
peptides and a physiologically acceptable carrier. Physiologically
acceptable carriers can be prepared by any method known by those
skilled in the art.
[0118] Pharmaceutical compositions comprising at least one peptide
of the invention include all compositions wherein the peptide(s)
are contained in an amount effective to achieve the intended
purpose. In addition, the pharmaceutical compositions may contain
suitable pharmaceutically acceptable carriers comprising excipients
and auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Suitable
pharmaceutically acceptable vehicles are well known in the art and
are described for example in Remington's Pharmaceutical Sciences
(Mack Publishing Company, Easton, USA, 1985), which is a standard
reference text in this field. Pharmaceutically acceptable vehicles
can be routinely selected in accordance with the mode of
administration, solubility and stability of the peptides. For
example, formulations for intravenous administration may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives. The use of biomaterials and other
polymers for drug delivery, as well the different techniques and
models to validate a specific mode of administration, are disclosed
in literature.
[0119] The peptides of the present invention may be administered by
any means that achieve the intended purpose. For example,
administration may be achieved by a number of different routes
including, but not limited to subcutaneous, intravenous,
intradermal, intramuscular, intraperitoneal, intracerebral,
intrathecal, intranasal, oral, rectal, transdermal, buccal,
topical, local, inhalant or subcutaneous use.
[0120] Dosages to be administered depend on individual needs, on
the desired effect and the chosen route of administration. It is
understood that the dosage administered will be dependent upon the
age, sex, health, and weight of the recipient, concurrent
treatment, if any, frequency of treatment, and the nature of the
effect desired. The total dose required for each treatment may be
administered by multiple doses or in a single dose.
[0121] Depending on the intended route of delivery, the compounds
may be formulated as liquid (e.g., solutions, suspensions), solid
(e.g., pills, tablets, suppositories) or semisolid (e.g., creams,
gels) forms.
[0122] In a preferred embodiment, the compositions are presented in
unit dosage forms to facilitate accurate dosing. The term "unit
dosage forms" refers to physically discrete units suitable as
unitary dosages for human subjects and other mammals, each unit
containing a pre-determined quantity of active material calculated
to produce the desired therapeutic effect, in association with a
suitable pharmaceutical excipient. Typical unit dosage forms
include pre-filled, pre-measured ampoules or syringes of the liquid
compositions or pills, tablets, capsules or the like in the case of
solid compositions. In such compositions, the compound of the
invention is usually a minor component (from about 0.1 to about 50%
by weight or preferably from about 1 to about 40% by weight) with
the remainder being various vehicles or carriers and processing
aids helpful for forming the desired dosing form.
[0123] The compounds of this invention can also be administered in
sustained release forms or from sustained release drug delivery
systems.
[0124] The expression "physiologically acceptable" is meant to
encompass any carrier, which does not interfere with the
effectiveness of the biological activity of the active ingredient
and that is not toxic to the host to which is administered. For
example, for parenteral administration, the above active
ingredients may be formulated in unit dosage form for injection in
vehicles such as saline, dextrose solution, serum albumin and
Ringer's solution.
[0125] Besides the pharmaceutically acceptable carrier, the
compositions of the invention can also comprise minor amounts of
additives, such as stabilizers, excipients, buffers and
preservatives.
[0126] The invention also contemplates a pharmaceutical composition
comprising a nucleic acid encoding the peptide of the invention in
the frame of e.g. a treatment by gene therapy. In this case, the
nucleic acid is preferably present on a vector, on which the
sequence coding for the peptide is placed under the control of
expression signals (e.g. a promoter, a terminator and/or an
enhancer) allowing its expression. The vector may for example
correspond to a viral vector such as an adenoviral or a lentiviral
vector.
[0127] The invention further provides kits comprising a
pharmaceutical composition comprising a CD31 peptide of the
invention and instructions regarding the mode of administration.
These instructions may e.g. indicate the medical indication, and/or
the route of administration, and/or the dosage, and/or the group of
patients to be treated.
[0128] All references cited herein, including journal articles or
abstracts, published or unpublished patent application, issued
patents or any other references, are entirely incorporated by
reference herein, including all data, tables, figures and text
presented in the cited references.
[0129] Although having distinct meanings, the terms "comprising",
"having", "containing" and "consisting of" have been used
interchangeably throughout this specification and may be replaced
with one another.
[0130] The invention will be further evaluated in view of the
following examples and figures.
BRIEF DESCRIPTION OF THE FIGURES
[0131] FIG. 1A-B shows an alignment between the sequences of human,
mouse, pig and bovine CD31. The sequence defined by amino acids 579
to 601 of SEQ ID NO: 4 (human CD31) and the corresponding sequence
in mouse, pig and bovine CD31 is highlighted by a box.
[0132] FIG. 2 shows a representative example of 10-color
flow-cytometry analysis of human peripheral blood cells from a
healthy donor. Isotype controls of antibodies anti-CD31 dom1 and
anti-CD31 dom6 are shown in the insets. Lymphocytes, Monocytes and
Granulocytes were gated within the FSC/SSC scatter. B (CD20 A700+)
and T (CD3 PE-TR+) lymphocytes were identified and gated within the
"Lymphocytes" and CD8+ (PerCP) and CD4+ (APC) subpopulations were
gated within T lymphocytes. CD8+ and CD4+ T cells were further
analyzed for the expression of HLA-DR and CD45RA and accordingly
subdivided in activated (1), memory (2) and naive (3) cells. All
leukocytes were positive for CD31 dom6. Lack of dom1 increased from
naive (3) to memory (2) to activated (1) T cells.
[0133] FIG. 3A-B shows that the apparent loss of CD31 on
lymphocytes is due to its extracellular shedding. a. Solubilized
cell membrane-bound CD31 molecules were extracted from cultured
Jurkat CD4+ T cells and coupled to fluorescent beads. The
percentage of dom1- bead-bound molecules is <6% in resting
conditions and >99% 5' after TCR engagement. b. Most soluble
CD31 in culture supernatant (.quadrature.) of TCR-activated T cells
and in human plasma (.box-solid.) consists of a single truncated
fragment comprising dom1-dom5 and lacking dom6. Negligible levels
of truncated CD31 lacking both dom5 and dom6 could be detected only
in plasma.
[0134] FIG. 4A-B shows that a peptide homotypic of the residual
extracellular fragment on CD31.sup.shed T induces CD31-ITIM
phosphorylation. a. Proliferative response to TCR engagement of
human peripheral blood mononuclear cells in the presence of
increasing doses of CD31 peptide 551-574. *p<0.05 vs dose "0".
b. Flow cytometry assessment of 686ITIM phosphorylation on
solubilized membrane-bound CD31 from cultured Jurkat CD4+ T cells.
Solubilized proteins were captured by E9-PECAM-1.2 (dom6)
functional CBA beads and detection was carried out by anti-pY686
rabbit sera followed by AlexaFluor.RTM.488-anti-rabbit secondary
antibody. The histogram shows the Median Fluorescent Intensity
(MFI).+-.the % of the variability coefficient (CV %) of
Alexafluor.RTM.488 (pY686) over 2000 E9-PECAM-1.2 acquired beads.
Pervan=positive control (pervanadate); CD3/CD28=anti-CD3 and
anti-CD28 antibodies (1 .mu.g/ml each); peptide=CD31 peptide
551-574 (100 .mu.M).
[0135] FIG. 5A-D shows that CD31 homotypic peptide 551-574 inhibits
T-cell responses. a. BIAcore.RTM. analysis of mouse CD31 peptide
551-574 homophilic binding at two-fold stepwise dilutions of the
analyte (12.5, 25, 50 and 100 .mu.M). Data are normalised against
the control channels and expressed as ARU (Resonance Units). b.
TCR-induced intracellular calcium mobilisation determined by flow
cytometry in Fluo-3AM-loaded spleen cells. Data are expressed as
MFI (530/30 nm). Grey arrow=addition of either anti-CD3/CD28
antibodies and crosslinker alone (.box-solid.=control) or with
peptide 551-554 at 100 .mu.M (.circle-solid.) or with anti-mouse
CD31 antibodies (.largecircle.). c. Proliferation in response to
TCR-stimulation in CD31+/+(black columns) and CD31-/- (white
columns) spleen cells. A dose-dependent inhibition is observed in
CD31+/+ cells while only the highest dose of the peptide affect
proliferation of CD31-/- splenocytes. No effect was observed with
100 .mu.M dose of the scramble peptide on CD31+/+ cells (crisscross
column). *p<0.05 vs previous peptide dose. d. Immunosuppressive
effect of the peptide in the DTH model. *p<0.01 vs scramble
(crisscross column) and 10 .mu.M dose of peptide 551-574. Data are
expressed as mean.+-.SEM.
[0136] FIG. 6A-C shows that CD31 peptide biotherapy prevents
acceleration of atherosclerosis and aneurysm formation. a.
Mean.+-.SEM of Atherosclerotic lesion surface area in serial
cross-sections of the aortic root at 200, 400, 600, 800 .mu.m from
the appearance of the first cusp in control (.box-solid.) and
peptide 551-574 (.quadrature.) treated mice. *p<0.05 vs control,
ANOVA, repeated measure. b. The presence of an abdominal aortic
aneurysm (AAA) was macroscopically, blindly evaluated by A.G. and
A. N. after careful dissection of the adventitial tissue. An
aneurysm was present in 8/10 (Exp #1) or 6/8 (Exp #2) control mice
as opposed to only 1/8 peptide-treated mice in both experiments
(p<0.001 by Chi squared test). The images below show an example
of aneurysm (AAA; arrows) as compared to the absence of aneurysm
(no AAA) of the abdominal aorta. c. Flow cytometry analysis of the
effect of the peptide (50 .mu.M) on CD8+ T cell (% cytolysis) and
macrophage (MMP-2/9 activity) functions. Data represent mean.+-.SEM
of cultures from 3-4 mous/group. *p<0.05 vs control.
[0137] FIG. 7 shows the clinical score of EAE activity (paralysis
level). "pepREG" refers to the peptide of SEQ ID NO: 3. "pepSCRA"
refers to the peptide of SEQ ID NO: 14.
BRIEF DESCRIPTION OF THE SEQUENCES
[0138] SEQ ID NO: 1 corresponds to the sequence of human CD31.
[0139] SEQ ID NOs: 2, 3, 4, 10, 11 and 12 correspond to CD31
peptides according to the invention.
[0140] SEQ ID NOs: 5 and 6 correspond to CD31 peptides for use in
the methods according to the invention.
[0141] SEQ ID NO: 7 corresponds to the sequence of murine CD31.
[0142] SEQ ID NO: 8 corresponds to the sequence of bovine CD31.
[0143] SEQ ID NO: 9 corresponds to the sequence of pig CD31.
[0144] SEQ ID NOs: 13 and 14 correspond to scramble peptides used
as controls.
EXAMPLES
Example 1: Material and Methods
[0145] Assessment of CD31.sup.+ and CD31.sup.shed Blood
Leukocytes.
[0146] Ten-color flow cytometry was performed on peripheral blood
leukocytes from 5 healthy individuals either in basal conditions or
after overnight stimulation with soluble 1 .mu.g/ml of purified
anti-CD3 antibody (R&D Systems). Ten-color flow cytometry was
performed after erythrocyte hypotonic lysis (10 minutes at
37.degree. C. 1:10 v:v in Tris 10 mM, NH.sub.4Cl 155 mM, KHCO.sub.3
10 mM, pH 7.4) on heparinized peripheral blood leukocytes from 5
healthy individuals, fixed in PBS/Formaldehyde 1%/FCS 1% for 4
minutes at 37.degree. C. prior to processing. All experiments on
human blood were approved by the International Ethical committee
(see world wide web page clinicaltrials.gov; Identifier:
NCT00430820). Pelleted cells were incubated for 30 minutes at room
temperature and protected from light with a cocktail of fluorescent
monoclonal antibodies directed to CD3 (PE-Texas Red), CD4 (PE-Cy7),
CD8 (PerCP), HLA-DR (APC-Cy7), CD45RA (Pacific Blue), and CD31
(WM59, PE) from BD Biosciences and anti-CD20 (AlexaFluor.RTM.700)
and anti-CD31 (PECAM 1.2, FITC) from Invitrogen (1 .mu.l of each).
At least 50,000 events were acquired in the lymphocyte gate using a
BD LSRII.RTM. equipped with 3 lasers (405, 488 and 633 nm) and
analysed with BD DIVA.RTM. 6.0 software.
[0147] Subtractive Measurement of Soluble CD31.
[0148] To detect the splice variant and truncated CD31 in plasma
and the culture supernatant, a cytokine bead array (CBA.RTM., BD)
has been customized. Three differently functional CBA beads (A9, D5
and E9) were coupled with either one of the following purified
monoclonal anti-CD31 antibodies JC70A (domain 1, DAKO), MEM-05
(domain 5, Zymed) and PECAM 1.2 (domain 6, Invitrogen). The coupled
beads were then incubated with the plasma of the same 5 healthy
controls (FIG. 2) or the culture supernatant and positive binding
of circulating CD31 was detected by a fourth anti-CD31 monoclonal
antibody, WM-59 (domains 1-2) coupled to PE (BD). The concentration
of plasma CD31 including at least domain 1 (JC70A), or domains 1 to
5 (MEM-05) or all the extracellular domains 1 to 6 of CD31 (PECAM
1.2) was determined by analysing the median fluorescent intensity
of the detecting antibody on .gtoreq.1000 gated beads on samples
and serial dilutions of the same standard (recombinant, full length
extracellular CD31, R&D Systems). The standard curve was
obtained for each of the beads using the same known concentrations
of the recombinant CD31 in order to overcome any bias due to
differences in binding affinity of the diverse antibodies. The
concentration in ng/ml of CD31 determined with PECAM 1.2 coupled
beads (dom 1-6) was subtracted from the one obtained using MEM-05
coupled beads to obtain the amount of circulating CD31 lacking dom6
(dom 1-5). The latter was subtracted from the concentration of CD31
obtained using the JC70A-coupled beads to calculate the value of
soluble CD31 lacking both dom 5 and 6 but containing at least
domains 1 and 2 (dom 1-2).
[0149] Assessment of CD31-ITIM Phosphorylation.
[0150] Log-phase Jurkat cells (10.sup.7 cells/condition) were
either left unstimulated (negative control) or incubated with
pervanadate (positive control) or stimulated with anti-CD3 and
anti-CD28 antibodies (R&D Systems, 1 .mu.g/ml each) in the
presence or absence of peptide 551-574 (100 .mu.M), or incubated
with the peptide alone during 20 minutes. Cells were then lysed
with 1 ml of RIPA buffer on ice for 30 minutes, ultracentrifuged
and 16 .mu.l of the supernatant was incubated with PECAM 1.2-coated
Functional E9 CBA.RTM. beads (BD) for 2 hours at room temperature.
Beads were subsequently washed with CBA washing buffer and
incubated with 2 .mu.l of undiluted rabbit anti-CD31
phospho-tyrosine 686 (pY686) sera followed by two washings and
incubation with AlexaFluor.RTM.488-conjugated (Fab).sub.2 fragments
(1:100 in CBA whashing buffer) of goat-anti-rabbit IgG
(Invitrogen). The beads (2000/condition) were finally analysed by
flow cytometry in the FITC channel (530/30 nm) and data are
expressed as Median fluorescence intensity (MFI).+-.the percentage
of the coefficient of variability (% CV) calculated with the DIVA
6.0.RTM. software (BD). Duplicate lysate aliquots and serial
dilutions of recombinant CD31 were incubated with the PECAM
1.2-coated beads and the amount of dom1+ cell-bound CD31 was
revealed using anti-CD31 WM59-R-PE (dom1) and PECAM 1.2-FITC (dom6)
antibodies (data shown in FIG. 3a).
[0151] Fluorescent Peptide Binding.
[0152] For visualisation of peptide binding to CD31.sup.+ and
CD31.sup.shed CD4.sup.+ T cells, freshly purified peripheral blood
leukocytes prepared as above were washed with a buffered solution
containing 2 mM EDTA (to avoid endocytosis of the peptide) and
incubated overnight at room temperature in a dark humidified
chamber with 50 .mu.M FITC-labelled CD31 peptide 551-574 and 1:10
dilution of fluorescent monoclonal anti-CD31 (PE) and anti-CD4
(APC) antibodies (BD Biosciences) in a poly-D-Lysine coated
Ibidi.RTM. 8-well culture chamber (Biovalley). Cells were then
washed twice, nuclei counterstained with DAPI and digital images of
a 0.3 .mu.m intracellular section were acquired on a Zeiss Axiovert
M200 microscope (.times.63 immersion objective) equipped with the
ApoTome.RTM. and a cooled monochromatic digital camera (Zeiss).
[0153] Calcium Mobilisation Assay.
[0154] Spleen cells from C57Bl/6 mice were prepared as described in
Caligiuri et al. (2005 Arterioscler Thromb Vasc Biol 25:1659-1664).
Cells were incubated with Fluo-3AM (Invitrogen, # F1242) as per the
instructions of the manufacturer. Fluorescence of calcium-bound
tracer was measured in the FITC channel on an LSRII.RTM. cytometer
(BD Biosciences) prior to and during 60 seconds following the
addition of hamster anti-mouse CD3/CD28 monoclonal antibodies (40
.mu.g/ml each) and rat/hamster compBead.RTM. (1:50) either alone or
in the presence of rat anti-mouse CD31 antibody (clone 390, 10
.mu.g/ml) or in the presence of CD31 peptide 551-574 (100 .mu.M).
Negative controls included rat IgG isotype control and scramble
peptide. Antibodies and compBeads.RTM. were from BD
Biosciences.
[0155] Plasmon Surface Resonance. Homophilic binding association
and dissociation constants were calculated by surface plasmon
resonance (BIAcore.RTM. 2000, GE). In brief, peptide 551-574 was
coated at 3400 resonance units (RU) on CM5 chips according to the
manufacturer's instructions. Soluble peptide 551-574 (12.5, 25, 50
and 100 .mu.M in 200 .mu.l of 10 mM HEPES pH 7.4, 150 mM NaCl,
0.005% Tween 20) was injected at 20 .mu.l/min at 25.degree. C., on
the peptide-coated channel and on an uncoated channel. Dissociation
was monitored for 300 seconds. Association (kon) and dissociation
(koff) constants were calculated using the BIAevaluation 3.0
Software (GE). Injection of peptide 551-574 on a channel coated
with the scramble peptide yielded negligible signal.
[0156] Evaluation of Immunoregulation In Vitro.
[0157] CD8.sup.+ T cell-mediated cytolytic activity against
allogeneic mouse aortic smooth muscle cells and measurement of
macrophage gelatinase (MMP-2/9) activity were performed as
previously described for human cells in Caligiuri et al. (2006
Arterioscler Thromb Vasc Biol 26:618-623) using kits and reagents
from Invitrogen. Briefly, primary cultures of FVB/N mouse aorta
smooth muscle cells were labelled with the lipophylic tracer DIO
(green) and co-cultured for 3 hours with CD8+ T cell-enriched
spleen cells from C57131/6 mice (n=3 scramble peptide and n=3
peptide 551-574, 50 .mu.M). Cytolysis was evaluated by
intracellular accumulation of propidium iodide (PI). Cells were
analysed by flow cytometry and the % of cytolysis was calculated by
expressing the number of dead (PI+) cells among the target (DIO+)
cells. Intracellular MMP-2/9 (gelatinase) activity was measured by
flow cytometry in 7-day bone-marrow derived macrophages from
C57Bl/6 mice (n=3 scramble peptide and n=3 peptide 551-574, 50
.mu.M) three hours after the incorporation of OregonGreen.RTM.
gelatine (MFI). T-cell proliferation was performed using either
human peripheral blood mononuclear cells of spleen cells from
C57Bl/6 (CD31.sup.+/+) and CD31.sup.-/- mice (Charles River France)
as previously described (Caligiuri et al. 2005 Arterioscler Thromb
Vasc Biol 25:1659-1664). Briefly, cells were plated in triplicates
at 0.2.times.10.sup.6 cells/well in a U bottom 96-well plate in
complete medium (RPMI 1640, 1% pyruvate, 1% glutamine, 1%
penicillin-streptomicyne-fungizone, 10% decomplemented fetal calf
serum, all from Invitrogen) containing 1 .mu.g/ml anti-mouse
CD3/CD28 or 5 .mu.g/ml anti-human CD3 antibodies (BD) as
appropriate. CD31 (551-574) and scramble peptide at 25, 50 and 100
.mu.M final concentration were deposited in the wells just before
cell plating. Plated cells were cultured for 72 hours in 5% CO2 at
37.degree. C. (.sup.3H) thymidine (0.5 .mu.Ci/well) was added for
the last 16 hours and proliferation evaluated using a Tomtec
harvester and analysis on a Wallac micro beta counter. Data are
expressed as mean.+-.SEM of cpm in triplicates.
[0158] Evaluation of Immunoregulation In Vivo.
[0159] Delayed type hypersensitivity (DTH) suppression was
evaluated as described in the "Current Protocols in Immunology
(2001) 4.0.1-4.0.2 Unit 4.2". Briefly, TNCB
(2-chloro-1,3,5-trinitrobenzene, Fluka #79874) was dissolved in
acetone/olive oil (1:1 v/v) at a concentration of 10 mg/ml. BALB/c
mice (n=6/group) were primed by painting the shaved regions of the
abdomen a with a total 0.2 ml of the preparation (n=6/group). The
experiment included 3 groups for peptide 551-574 (10, 50, 100
.mu.M) and 1 group treated with scramble peptide at 100 .mu.M).
Five days after priming, 10 .mu.l of the TNCB-solvent mixture was
painted on the right pinna, 30 minutes after subcutaneous
(interscapolar) administration of the peptide 551-574 or the
scramble peptide. Ear thickness increases were calculated by
subtracting the thickness of the right and the left pinna of each
mouse (right-left/left.times.100), measured at 24 h with a dial
caliper ("Pocotest", Kroeplin Langenmesstechnick). The measure was
performed 5 times on each ear and averaged for further analysis.
The immunosuppressive effect of the peptide was calculated as %
suppression=(1-.DELTA.TE/.DELTA.TS).times.100, where .DELTA.T=(ear
thickness 24 hr after elicitation)-(baseline ear thickness),
E=sensitised animals, S=treated animals. Data are expressed as
mean.+-.SEM.
[0160] Detection of Atherosclerotic Lesion Size and Aneurysm
Formation.
[0161] Male 28-week old apolipoprotein E mice (n=8-10 mice/group)
from our breeding facility were maintained on a regular chow diet
and kept under standard conditions. Acceleration of atherosclerosis
and aneurysm formation was induced by subcutaneous angiotensin II
(Sigma, #A9525) infusion (1 mg/kg/d) for 28 days using osmotic
minipumps (Alzet, #2004) as previously described (Daugherty et al.
J Clin Invest 105:1605-1612). All experiments were approved by our
institutional Ethical committee. Atherosclerotic lesion size was
measured as previously described (Caligiuri et al. 2005
Arterioscler Thromb Vasc Biol 25:1659-1664). These experiments were
repeated twice with similar results.
[0162] Peptides.
[0163] All experiments on human material were carried out using the
human peptide sequence while the mouse equivalent was used in all
mouse experiments. The sequences of the peptides are shown in the
table below
TABLE-US-00004 Human NH2-NHASSVPRSKIL SEQ ID NO: 6 TVRVILAPWKK-COOH
Mouse NH2-SSMRTSPRSSTL SEQ ID NO: 5 AVRVFLAPWKK-COOH Scramble
NH2-SMPAVRSRFSAT SEQ ID NO: 13 SLVTLKSRWPK-COOH
Example 2: The Apparent Loss of CD31 at the Surface of Blood
Lymphocytes is Due to its Shedding Between the 5th and 6th
Extracellular Ig-Like Domains
[0164] In order to establish whether the loss CD31 was restricted
to part or extended to the totality of its 6 extracellular Ig-like
domains, a multicolor flow cytometry analysis of whole blood
leukocytes from 5 healthy donors using two different antibodies
specifically recognizing the membrane-distal and membrane-proximal
Ig-like domains of the molecule was performed. To be able to
discriminate between the different leukocyte populations and assess
their state of maturation and activation, a panel of lineage
markers as well as the expression of CD45RA and HLA-DR were
simultaneously used. While the expression of CD31, as detected by a
monoclonal antibody specific for the first domains of CD31 (clone
WM-59, dom1-2) was recognized on naive but not on activated/memory
blood T cells, all cells expressed the membrane-proximal
extracellular fragment of the molecule detected by another
monoclonal antibody specific for the 6.sup.th Ig-like domain of
CD31 (clone PECAM 1.2, dom6), irrespective of their state of
maturation/activation (FIG. 2).
[0165] Flow cell cytometry showed that T-cell receptor (TCR)
engagement induces a shift of >80% of blood resting T cells from
a CD31 dom1.sup.+/dom6.sup.+ to a dom1.sup.-/dom6.sup.+
(CD31.sup.shed) phenotype. Molecular analysis of the membrane
proteins from cultured T-cell lysates demonstrated that >99% of
the T cell-bound CD31 molecules drop the distal portion containing
dom1 already 5' minutes after TCR stimulation in vitro (FIG. 3a).
Analysis of the supernatant showed that, simultaneously, a single
truncated soluble protein limited to the first 5 Ig-like domains of
CD31, accumulates in the culture supernatant (FIG. 3b).
Furthermore, the analysis of the plasma of the same healthy donors
showed that major part of soluble CD31 in plasma was constituted of
a truncated molecule comprising Ig-like domains 1 to 5 and
specifically lacking the membrane-proximal 6.sup.th domain (FIG.
3b) that always remains anchored to the apparently CD31-negative
(CD31 dom1.sup.-) lymphocytes both in vitro and in vivo. Only a
minimal fraction of soluble CD31 contained all 6 extracellular
domains predicted in the previously reported (Goldberger et al. J
Biol Chem 269:17183-17191) variant spliced form both in culture
supernatant and in plasma (FIG. 3b). No significant other cleavage
of the molecule occurs upstream of the 5.sup.th domain since the
latter was virtually always present concomitantly with the first
domain in the truncated soluble CD31 proteins (FIG. 3b).
Example 3: A Peptide Contained in the Residual Extracellular CD31
Fragment on CD31.sup.shed T Cells Enhances Phosphorylation of
CD31-ITIM
[0166] A CD31 dom6-derived synthetic peptide corresponding to the
juxta-membrane 23 aminoacids (551-574) of the ectodomain of the
human molecule binds both to CD31 dom1.sup.+ and to CD31 dom1.sup.-
(CD31.sup.shed) CD4.sup.+ T lymphocytes ex vivo. Importantly, the
binding of this peptide on T cells has functional consequences on
immune cell control since it exerted dose-dependent inhibition of
human peripheral blood T-cell proliferation in vitro (FIG. 4a). To
assess whether the inhibitory effect of the peptide could be
mediated by homophilic binding and engagement of the CD31
signaling, the level of phosphorylation of the CD31 ITIM tyrosine
at position 686 (.sub.686ITIM) in cultured T cells was evaluated.
Stimulation of the TCR by anti-CD3 and anti-CD28 antibodies alone
or the sole presence of the peptide induced a minor increase of
CD31 pY686 (FIG. 4b) but concomitant TCR-stimulation in the
presence of the peptide boosted the phosphorylation the CD31
.sub.686ITIM by a factor of >23 (FIG. 4b).
Example 4: CD31 Homotypic Peptide 551-574 Inhibits a Large Array of
Cell-Mediated Immune Responses In Vitro and In Vivo
[0167] To further test our hypothesis, in vivo experiments were
performed, and the murine equivalent of CD31 (551-574) peptide was
therefore synthesized. Its properties were evaluated in vitro and
in vivo. Its homophilic interaction by surface plasmon resonance
analysis was first established (FIG. 5a). The association and
dissociation constants were of 134.+-.120 M.sup.-1s.sup.-1 and
1.58E-03.+-.1.07E-03 s.sup.-1, respectively and the affinity at
equilibrium was 17.8.+-.9.7 .mu.M, in agreement with previous
measurement done on recombinant human CD31 homo-dimerization using
other systems. It was next assessed whether the mouse CD31
(551-574) peptide was able to inhibit calcium mobilization in
response to the co-engagement of the CD3 and of the co-stimulatory
molecule CD28 in spleen lymphocytes. The data showed that
homophilic targeting of the juxta-membrane portion of CD31 whit
this small peptide is as an efficient immunoregulatory strategy as
it is the co-ligation of CD3 and CD28 with the distal portion of
CD31 by cross-linking the molecules using specific monoclonal
antibodies (FIG. 5b). The engagement of the CD31 molecular pathway
by this peptide could attain effective suppression of
antigen-driven lymphocyte responses in vitro and in vivo. It was
found that peptide 551-574 inhibits in a dose-dependent manner the
proliferation of spleen cells in response to stimulation of the T
cell-receptor in vitro (FIG. 5c). At low dose, the effect of the
peptide was exclusively due to its homophilic binding with CD31
molecules since no effect was observed on spleen cells from
CD31.sup.-/- mice (FIG. 3c). However, at high concentrations the
peptide can also bind to other (low affinity) heterophilic ligands
as suggested by its effect at 100 .mu.M dose also on cells lacking
CD31 (FIG. 5c). The scramble peptide, used at the highest dose, was
ineffective (FIG. 5c). Finally, the therapeutic immunosuppressive
potential of this peptide in vivo in the context of matched,
histocompatible antigen-driven immune responses was evaluated using
a model of delayed type of hypersensitivity. Distal recall of an
hapten-elicited specific immune response was suppressed in a
dose-dependent manner by peptide 551-574 in this model (FIG. 5d).
Effective immunosuppression was achieved by a single subcutaneous
administration of 50 .mu.M of the peptide 551-574 while the
scramble peptide was ineffective (FIG. 5d).
Example 5: CD31.sup.shed Cell-Targeting Peptide Biotherapy Prevents
Disease Progression and Aneurysm Formation in Atherosclerotic
Mice
[0168] The model of angiotensin II infusion into aged
apolipoprotein E-/- mice (25) was used, this model closely
mimicking atherothrombosis in humans. Daily subcutaneous
administration of 50 .mu.M of the peptide prevented both the
acceleration of plaque growth in the aortic root (FIG. 6a) and the
formation of atherothrombotic abdominal aortic aneurysms (AAA) in
this model (FIG. 6b). in vitro, the CD31-derived peptide 551-574
inhibited both the activity of matrix degrading enzymes in
bone-marrow derived macrophages and CD8.sup.+ T cell-dependent
cytolysis of murine aortic smooth muscle cells (FIG. 6c).
Example 6: In Silico and In Vitro Validation of the 10 Amino
Acid-Long Peptide Candidate Peptide "PepReg"
[0169] A shorter nested peptide corresponding to the ten COOH
terminal amino acids was tested. This peptide is shown as SEQ ID
NO: 3 and referred to as "PepReg". As shown in the table below,
this peptide was expected to be more stable than the CD31 551-574
peptide of 23 amino acids.
TABLE-US-00005 TABLE Characteristics of PepReg vs the 23aa CD31
peptide in silico PepReg CD31 564-574 (10aa) CD31 551-574 (23aa)
sequence VRVFLAPWKK SSMRTSPRSSTLAVRV (SEQ ID NO: 3) FLAPWKK (SEQ ID
NO: 5) Number of amino 10 23 acids Molecular weight 1243.5 2606.0
Theoretical pI 11.17 12.31 Formula C62H98N16O11 C116H193N35O31S1
-(Asp, Glu)/+(Arg, 0/3 0/5 Lys) charges Instability index 25.38
65.09 Estimated half-life 100 hours 1.9 hours (mammalian
reticulocytes, in vitro) Aliphatic index 107.00 68.83
[0170] The immunosuppressive properties of PepReg (10aa) vs the 23
aa parent peptide were evaluated in vitro. Negatively purified CD4+
cells from C57Bl6 mice were stimulated by soluble anti-CD3 purified
antibodies and bone marrow derived dendritic cells. Cells from
triplicate wells were analyzed for the expression of the early
activation marker CD69 after 18 hours culture in complete RPMI
medium supplemented with 10% fetal calf serum. Flow cytometry
showed that PepReg was at least as efficient as the 23aa peptide in
suppressing T cell activation as determined by the percentage of
CD4 cells expressing CD69. Interestingly, the effect was more
reproducible (smaller standard deviation) and was observed with
lower doses (50 .mu.g/ml vs 100 .mu.g/ml) of PepReg (10aa) as
compared to the parent (23aa) peptide. The suppression of T cell
proliferation by PepReg was analyzed by [H3] thymidin incorporation
and persisted up to 7 days of culture at 37.degree. C. in 10% serum
demonstrating that the data regarding the stability of the peptide
obtained in silico were validated in vitro.
Example 7: In Vivo Validation of the 10 Amino Acid-Long Peptide
Candidate Peptide "PepReg" in the EAE Model
[0171] Experimental Autoimmune Encephalomyelitis (EAE), also called
Experimental Allergic Encephalomyelitis, is an animal model of
Multiple Sclerosis.
[0172] Twelve-week old female C57BL/6J mice were immunized with 300
.mu.g of MOG35-55 peptide emulsified in Complete Freund's Adjuvant
1:1 by volume containing 800 .mu.g of nonviable desiccated
Mycobacterium tuberculosis H37RA. A final volume of 200 .mu.l of
the emulsion was injected subcutaneously at 4 sites (50 .mu.l/site)
over the flanks. In addition, 300 ng of Pertussis toxin was
injected intravenously (retro-orbital plexus) on the same day and 2
days later. Clinical signs of EAE ware assessed daily by the
following scoring system: 0, no signs; 1, hindlimb weakness; 2,
hindlimb weakness and tail paralysis; 3, hindlimb and tail
paralysis; 4, hindlimb and tail paralysis and forelimb weakness; 5,
moribund state; and 6, death. The peak (waxing phase) occured
around day 21. In this C57BL/6J mouse model, there was no waning
phase as assessed in our laboratory up to day 41.
[0173] The experiment was carried out with ten mice per group. The
mice of each group were treating with either of: [0174] PBS; [0175]
PepReg (SEQ ID NO: 3); or [0176] PepScra (SEQ ID NO: 14).
[0177] The dosing was of 50 .mu.g of peptide per mice and per day
(i.e. about 2 mg/Kg per day). The peptide was administered by a
subcutaneous injection.
[0178] Disease protection was associated with reduced infiltration
of IL17+ and IFNg+ T helper CD4+ cells and increased proportion of
regulatory CD25+/foxP3 CD4+ cells in the central nervous system of
the mice.
[0179] As shown on FIG. 7, PepReg is capable of arresting disease
development in the waxing phase (Day 15) and reducing disease
extension in the plateau phase (Days 21 through to 35).
[0180] The scrambled peptide was also beneficial, although less
than PepReg. This result suggests that the amino acid composition
rather than the sequence per se is important for the beneficial
effect. This result has important implications for the development
of peptidomimetics.
Example 8: Detection of Shed CD31 in Plasma from Patients Suffering
from Atherothrombosis and in Unaffected Individuals
[0181] The total amount of CD31, the amount of shed CD31 and the
amount of spliced CD31 has been measured both in eleven individuals
suffering from atherothrombosis and in twenty-three unaffected
individuals.
[0182] The group "Atherothrombosis" comprised eleven individuals
suffering from chest pain even at rest and presenting an abnormal
coronarography.
[0183] The group "No Atherothrombosis" comprised twenty-three
individuals. A sub-analysis was carried out on the group "No
Atherothrombosis", which was found to comprise: [0184] eight
individuals presenting a normal coronarography and a normal carotid
echodoppler in spite of chest pain; [0185] four individuals
presenting a normal coronarography in spite of chest pain, but in
whom atherosclerosis was detected by carotid echodoppler; and
[0186] eleven individuals suffering from chest pain only on effort
and presenting an abnormal coronarography (i.e. suffering from
coronary atherosclerosis without thrombosis).
[0187] The total amount of CD31, the amount of shed CD31 and the
amount of spliced CD31 was determined as follows.
[0188] 1. The total amount (1 .mu.l/test) of beads (E9, coupled
with clone JC70A, DAKO) was transferred to a conical tube and
centrifuged at 200 g for 5 minutes. The supernatant was carefully
discarded and replaced with same amount of serum enhancement buffer
(BD #51-9002150), and incubated at room temperature for 15
minutes.
[0189] 2. The fluorescently-labeled antibody antibody mix (PE-WM59;
FITC-HCl/6; PB-PECAM1.2) was prepared, each at 1 .mu.g/ml, 1 .mu.l
each/condition.
[0190] 3. 1 tube precondition was prepared, each containing 3 .mu.l
of a standard dilution or a plasma sample. The reconstituted beads
were centrifuged at 200 g for 5 minutes, the supernatant was
discarded and the serum enhancement buffer was replaced with the
fluorescently-labeled antibody mix. 3 .mu.l of this solution was
distributed in each of the tubes containing the standard dilution
and samples, and the solution incubated for 1 hour at 4.degree. C.
in the dark.
[0191] 4. 150 .mu.l of Washing buffer (BD #51-9003797) were added
to each tube, and the signal was acquired.
[0192] As shown in the table below, the percentage of shed CD31 was
higher in individuals suffering from atherothrombosis than in
unaffected individuals, in spite of the fact that all individuals
were suffering from chest pain.
TABLE-US-00006 CD31 Plasma Level (ng/ml) total splice shed
Atherothrombosis 11.55 .+-. 0.7 -7.02 .+-. 2.69 18.57 .+-. 2.67 (n
= 11) No Atherothrombosis 11.58 .+-. 0.49 5.26 .+-. 1.850 6.31 .+-.
1.85 (N = 23) T-test Prob > F 0.9756 0.0007 0.0006
[0193] Total CD31 amounts were similar in the four groups, while
the amount of shed CD31 and the amount of spliced CD31 were
significantly different in each paired group comparison. Shed CD31
was increased in individuals with abnormal coronarography, with
highest values in those suffering from atherothrombosis. Splice
CD31 was still present in patients suffering from atherosclerosis
without atherothrombosis, while it was almost undetectable in
patients suffering from atherothrombosis.
[0194] These results demonstrate that high levels of CD31 soluble
splice variants associated with low levels of shed CD31 indicates
that the patient suffers from non specific chest pain, eventually
associated with carotid plaques. A slight increase of shed CD31
levels associated with normal or reduced levels of CD31 soluble
splice variants indicates that the patient suffers from
atherosclerosis. An important increase of shed CD31 levels
associated with undetectable amounts of CD31 soluble splice
variants indicates that the patient suffers from
atherothrombosis.
Example 9: Discussion of the Results
[0195] Dysimmune diseases are linked to lack of appropriate control
of immune responses. Atherosclerosis and its complications are not
only due to metabolic disturbances but are increasingly recognized
as a dysimmune disease and an important current issue is the
identification of interventional tools able to restore
immunoregulation. It has been previously shown that
atherothrombotic manifestations such as plaque rupture and
thrombosis (Caligiuri et al. 2005 Arterioscler Thromb Vasc Biol
25:1659-1664) or aneurysm complication (Caligiuri et al. 2006
Arterioscler Thromb Vasc Biol 26:618-623) are associated with a
significant reduction of CD31+ T lymphocytes in the peripheral
blood. In these previous works, we documented that lack of CD31
signaling on lymphocytes elicited pro-atherothrombotic immune
responses whilst the presence of CD31 on T cells was able to
inhibit them.
[0196] Here it is demonstrated that the assumed loss of the
molecule on activated/memory T lymphocytes is actually incomplete
and results from shedding of CD31 between the 5th and 6th
extracellular Ig-like domains. CD31 shedding occurred immediately
after cell activation on T lymphocytes and was accompanied by the
accumulation of the truncated molecule in the supernatant together
with trace levels of the spliced variant produced by the cells.
This finding was unsuspected because all commercially available
tests to detect plasma CD31 use antibodies directed to CD31 domains
1 to 5, and therefore cannot discriminate between the spliced
variant (containing all the 6 extracellular domains) and the
truncated (domains 1 to 5) forms of CD31. The subtractive
immunosorbent assay described herein is able to discriminate
between the two forms of soluble CD31 and precisely quantify the
proportion of each of them in the plasma. This assay showed that
the major part of plasma CD31 comprises domains 1 to 5 but lacks
the membrane-proximal 6th domain, which remains anchored to blood
CD31 dom1- lymphocytes. Therefore, it is proposed to refer to these
lymphocytes as CD31shed rather that CD31 "negative" cells. Previous
work in vitro had indicated that CD31 shedding at an unidentified
position N-terminal from the transmembrane segment of the molecule
can occur in endothelial cells undergoing apoptosis (Ilan et al.
2001. Faseb J 15:362-372). For the first time, it is shown herein
that shedding is responsible for the CD31 (incomplete) loss on
blood lymphocytes and that the circulating CD31 consists mainly of
a truncated form derived from its cleavage between the Ig-like
domains 5 and 6, rather than of the secreted spliced variant form.
Genetic polymorphisms for CD31 have been described, but the
predictive value of soluble CD31 levels was conflicting either in
atherothrombosis or other dysimmune diseases. In fact, while the
amount of the spliced form can be predicted by different genetic
variants, the proportion of the form resulting from protein
shedding is not determined by CD31 gene polymorphism. It is
proposed that the disparity between the different studies was due
to fact that circulating CD31 is a mixture of the genetic variant
and of the truncated form and discrimination between the two forms
of CD31 was not possible. The subtractive method described herein
will allow the differentiation of the prognostic value determined
by genetic variants of CD31 independently of that linked to CD31
shedding.
[0197] The fact that CD31 is not completely lost on blood
lymphocytes offers a unique opportunity to rescue its physiological
immunoregulatory function by targeting the residual extracellular
portion of the molecule. Indeed, it has been documented herein that
this can be achieved by a homotypic peptide-based therapy, both in
vitro and in vivo. Homophilic binding of this peptide dramatically
enhanced the phosphorylation the CD31 686ITIM and inhibited their
TCR-induced proliferation. Induction of CD31 ITIM phosphorylation
by antibody-mediated cross-linking of CD31 and CD3 surface
molecules was previously known to inhibit calcium mobilization
induced by anti-CD3 antibodies in human T-cell lines. Remarkably,
it has been found that targeting the juxta-membrane portion of CD31
whit the small homotypic peptide is as an efficient
immunoregulatory strategy as it is the co-ligation of CD3 and CD28
with the distal portion of CD31 by cross-linking the molecules with
antibodies. A selective small synthetic peptide strategy is
obviously simpler and might also be safer than using large
proteins, such as monoclonal antibodies and cross-linkers, for the
biotherapy of immunological disorders (Isaacs 2007. Curr Opin
Pharmacol 7:418-425).
[0198] With this idea in mind, it was assessed whether the
engagement of the CD31 molecular pathway by this peptide could
attain effective suppression of antigen-driven lymphocyte responses
in vitro and in vivo in the context of matched, histocompatible
antigen-driven immune responses. Distal recall of an
hapten-elicited specific immune response was suppressed in a
dose-dependent manner by a single subcutaneous administration of
the peptide. A similar protective effect of a single peptide shot
was also observed in experimental autoimmune encephalomyelitis (a
mouse model of multiple sclerosis) and lasted for up to 5 days.
[0199] Consequently, it was evaluated whether rescuing of
CD31-mediated immunoregulation by the synthetic peptide 551-574
could be employed in a biotherapy to fight atherothrombosis since
CD31-mediated immunoregulation is typically lost in this disease.
It was chosen to use the angiotensin-induced model of
atherothrombosis because the abrupt acceleration of atherosclerotic
plaque growth and the development of abdominal aortic aneurysms
complicated by a thrombus in this model are produced simply by
excess bioavailability of a physiological peptide--angiotensin
II--and does not require the use of surgery, gene transfer or high
fat diet, each of which could considerably bias the interpretation
of the results. The CD31-peptide prevented both the acceleration of
plaque growth in the aortic root and the formation of
atherothrombotic abdominal aortic aneurysms in this model. Such a
dramatic protective effect was superior to any therapeutic molecule
ever tested and equivalent to that achieved by genetic
manipulation.
[0200] Macrophages and lymphocytes represent the most important
immune cells involved in the development of atherothrombosis. The
local function of these cells injure the cellular and extracellular
components of the arterial layers resulting in either plaque
rupture and luminal thrombosis, when occurring in the fibrous cap
of atherosclerotic plaques, or aneurysm formation and eventually
rupture, when happening in the outer layers of the artery.
Degradation of the extracellular matrix is essentially due to the
activity of macrophage-derived matrix metalloproteases while death
of arterial smooth muscle cells is putatively caused by T
cell-mediated cytolysis. Remarkably, CD31+ T lymphocytes exert an
important immunosuppressive function on both these phenomena which
are conversely aggravated by CD31shed T cells. An aberrant
reduction of CD31+ cells in patients with atherothrombosis
underlies the defective immunoregulation observed in the disease.
Here it is shown that the CD31-derived peptide 551-574 inhibits
both the activity of matrix degrading enzymes and T cell-dependent
cytolysis of the arterial wall cells. The immunoregulation conveyed
by this peptide is as efficient as that exerted by immunoregulatory
CD31+ T cells and hence may counterweight the loss of the
physiologic CD31-dependent immunoregulation in human
atherothrombosis.
[0201] This is the first time that a peptide-based biotherapy is
envisaged to correct the defective immunoregulation characteristic
of atherothrombosis and prevent development of the disease in
patients. In addition, such biotherapy may broaden over to other
debilitating dysimmune diseases. In particular, experimental
studies have suggested that CD31-signalling might play a protective
role in rheumatoid arthritis (Wong et al. 2005. J Clin Immunol
25:19-28), multiple sclerosis (Graesser et al. 2002. J Clin Invest
109:383-392) and non-alchoolic fatty liver disease (Goel et al.
2007. Am J Physiol Gastrointest Liver Physiol 293:G1205-1214).
Example 10: Evaluation of Ninety Six Peptides According to the
Invention
[0202] The ninety six peptides consisting of a fragment having a
sequence selected from the group consisting of: [0203] amino acids
2 to 23, 3 to 23, 4 to 23, 5 to 23, 6 to 23, 7 to 23, 8 to 23, 9 to
23, 10 to 23, 11 to 23, 12 to 23, 13 to 23, 14 to 23, 15 to 23, 16
to 23, 17 to 23, 18 to 23, 19 to 23, 20 to 23 and 21 to 23 of SEQ
ID NO: 5; and [0204] amino acids 2 to 23, 3 to 23, 4 to 23, 5 to
23, 6 to 23, 7 to 23, 8 to 23, 9 to 23, 10 to 23, 11 to 23, 12 to
23, 13 to 23, 14 to 23, 15 to 23, 16 to 23 and 17 to 23 of SEQ ID
NO: 6; and [0205] amino acids 1 to 22, 1 to 21, 1 to 20, 1 to 19, 1
to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to
11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4 and 1
to 3 of SEQ ID NO: 5; and [0206] amino acids 1 to 22, 1 to 21, 1 to
20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13,
1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5,
1 to 4 and 1 to 3 of SEQ ID NO: 6; and [0207] amino acids 2 to 22,
3 to 21, 4 to 20, 5 to 19, 6 to 18, 7 to 17, 8 to 16, 9 to 15, 10
to 14 and 11 to 13 of SEQ ID NO: 5; and [0208] amino acids 2 to 22,
3 to 21, 4 to 20, 5 to 19, 6 to 18, 7 to 17, 8 to 16, 9 to 15, 10
to 14 and 11 to 13 of SEQ ID NO: 6; are tested for confirming that
these peptides induce CD31-ITIM phosphorylation. CD31-ITIM
phosphorylation is assessed by studying the effect of increasing
doses of the peptides according to the invention on CD31-ITIM
phosphorylation of cultured Jurkat cells stimulated with anti-CD3
and/or anti-CD28 antibodies, as described in Example 3.
[0209] It is confirmed that the above peptides inhibit T-cell
proliferation using the protocol described by Caligiuri et al.
(2005 Arterioscler Thromb Vasc Biol 25:1659-1664).
[0210] It is confirmed that the peptides can achieve effective
immunosuppression using the protocol described in the Current
Protocols in Immunology (2001, 4.0.1-4.0.2 Unit 4.2).
[0211] The efficiency of the peptides for treating a thrombotic
disorder is confirmed in the model of angiotensin II infusion into
aged apolipoprotein E-/- mice, which closely mimicks
atherothrombosis in humans.
[0212] The efficiency of the peptides for treating an autoimmune
disorder is confirmed in the Experimental Autoimmune
Encephalomyelitis (EAE) model, which is an animal model of Multiple
Sclerosis, and in a model for rheumatoid arthritis.
Example 11: Evaluation of the Peptides According to the Invention
in an Animal Model of the Rheumatoid Arthritis (RA)
[0213] It is confirmed that the peptides according to the invention
(e.g. PepReg and/or the peptides described in Example 10) are
capable of arresting disease development and/or reducing disease
extension in an animal model of Rheumatoid arthritis (RA).
[0214] Rheumatoid arthritis (RA) is a chronic and systemic
inflammatory autoimmune disorder that causes the immune system to
attack the joints. The disease is characterized by aggressive
synovial hyperplasia (pannus formation) and inflammation
(synovitis), which, if left untreated, lead to progressive
destruction of joint cartilage and bone. The destructive lesions
result from both immune responses and non-antigen-specific innate
inflammatory processes. Several studies have shown that CD31 plays
a critical role in this disease since the disease onset and
progression is accelerated in its absence.
[0215] DBA/1 mice are used in this experiment. Induction of RA is
initiated on 12 week-old mice. On day 0, mice are immunized
intradermally at the base of the tail with 150 .mu.g of bovine type
II collagen (CII) emulsified with an equal volume of Freund's
complete adjuvant containing 200 .mu.g of H37RA Mycobacterium
tuberculosis. On day 21, mice are given a booster (intradermal
injection of 150 .mu.g of bovine CII in Freund's incomplete
adjuvant). Simultaneously, mice receive an intravenous injection of
50 .mu.g of LPS. Mice are followed up for two months. Following
immunization, the animals develop an autoimmune polyarthritis that
is characterized by severe cartilage and bone erosions. Mouse
collagen-induced RA shares several clinical, histopathological and
immunological features with human RA.
[0216] The peptides according to the invention are administered
following one of the below treatment schemes. [0217] Scheme 1:
Preventive administration. Two doses (50 and 100 mg/kg) of the
peptide is administered to the mice one day before the CII
immunization and than either daily, or twice a week, or weekly, for
the study period (2 months). A peptide with a scrambled sequence
(i.e. a peptide comprising the same amino acids as the peptide
according to the invention, but not the same sequence) is
administered to a control group of mice. Treatment is pursued for
one month. [0218] Scheme 2: Curative administration. The peptide
(50 and 100 mg/kg) and the scramble peptide are administered to
mice after the beginning of the symptoms, either daily, or twice a
week or weekly until the end of the study. Equivalent groups of
mice are kept in conventional housing facilities and are bled
weekly from a tail vein to monitor bleeding time and specific
pathogen antibody raise in sera (CDTA, Orleans).
[0219] Arthritis development is monitored by physical examination 3
times per week. Inflammation in each of the 4 paws is graded on a
scale of 0 to 3, and the scores for the 4 paws will be cumulated
(yielding a maximum score of 12 per mouse). The arthritis index is
calculated by dividing the total score in the experimental mice by
the number of arthritic mice.
[0220] Lesions in the joints are also followed. Ankle joints of
mice are excised 6 weeks after immunization and fixed in 10%
buffered formalin, decalcified in 10% EDTA, embedded in paraffin,
sectioned, and stained with hematoxylin and eosin. The intensity of
synovial hyperplasia, cellular inflammation, and pannus formation
is evaluated, and arthritis is graded in a blinded manner on a
scale of 0 to 4.
[0221] Immunohistochemistry is further used to track and to
phenotype inflammatory cells infiltrated in the joints.
[0222] The immunoregulation status is evaluated by measuring levels
of serum IgG1 and IgG2a to CII. The measurement is performed by
enzyme-linked immunosorbent assay (ELISA). The proliferation of T
cells isolated from draining lymph nodes and the spleen is tested
by the incorporation of .sup.3H thymidine in response to CII-loaded
dendritic cells.
[0223] Cell populations in the lymphoid organs and in the synovia
is analyzed by flow cytometry.
[0224] This experiment allows confirming that continuous
administration of the peptides according to the invention prevents
onset of RA. The curative phase allows evaluating the therapeutic
potential of the peptides for treating RA in patients that do not
respond to the current biologicals (i.e. 40% of the patients). Dose
and frequency of the administrations able to drive a regression of
the inflammatory cells in the joints, and regression of the
clinical score, are also determined.
Sequence CWU 1
1
1011738PRTHomo sapiensSIGNAL(1)..(27)DOMAIN(28)..(601)extracellular
domainDOMAIN(34)..(121)First Ig-like domainDOMAIN(145)..(233)Second
Ig-like domainDOMAIN(236)..(315)Third Ig-like
domainDOMAIN(328)..(401)Fourth Ig-like
domainDOMAIN(424)..(493)Fifth Ig-like domainDOMAIN(499)..(591)Sixth
Ig-like domainDOMAIN(602)..(620)transmembrane
domainDOMAIN(621)..(738)cytoplasmic 1Met Gln Pro Arg Trp Ala Gln
Gly Ala Thr Met Trp Leu Gly Val Leu1 5 10 15Leu Thr Leu Leu Leu Cys
Ser Ser Leu Glu Gly Gln Glu Asn Ser Phe 20 25 30Thr Ile Asn Ser Val
Asp Met Lys Ser Leu Pro Asp Trp Thr Val Gln 35 40 45Asn Gly Lys Asn
Leu Thr Leu Gln Cys Phe Ala Asp Val Ser Thr Thr 50 55 60Ser His Val
Lys Pro Gln His Gln Met Leu Phe Tyr Lys Asp Asp Val65 70 75 80Leu
Phe Tyr Asn Ile Ser Ser Met Lys Ser Thr Glu Ser Tyr Phe Ile 85 90
95Pro Glu Val Arg Ile Tyr Asp Ser Gly Thr Tyr Lys Cys Thr Val Ile
100 105 110Val Asn Asn Lys Glu Lys Thr Thr Ala Glu Tyr Gln Leu Leu
Val Glu 115 120 125Gly Val Pro Ser Pro Arg Val Thr Leu Asp Lys Lys
Glu Ala Ile Gln 130 135 140Gly Gly Ile Val Arg Val Asn Cys Ser Val
Pro Glu Glu Lys Ala Pro145 150 155 160Ile His Phe Thr Ile Glu Lys
Leu Glu Leu Asn Glu Lys Met Val Lys 165 170 175Leu Lys Arg Glu Lys
Asn Ser Arg Asp Gln Asn Phe Val Ile Leu Glu 180 185 190Phe Pro Val
Glu Glu Gln Asp Arg Val Leu Ser Phe Arg Cys Gln Ala 195 200 205Arg
Ile Ile Ser Gly Ile His Met Gln Thr Ser Glu Ser Thr Lys Ser 210 215
220Glu Leu Val Thr Val Thr Glu Ser Phe Ser Thr Pro Lys Phe His
Ile225 230 235 240Ser Pro Thr Gly Met Ile Met Glu Gly Ala Gln Leu
His Ile Lys Cys 245 250 255Thr Ile Gln Val Thr His Leu Ala Gln Glu
Phe Pro Glu Ile Ile Ile 260 265 270Gln Lys Asp Lys Ala Ile Val Ala
His Asn Arg His Gly Asn Lys Ala 275 280 285Val Tyr Ser Val Met Ala
Met Val Glu His Ser Gly Asn Tyr Thr Cys 290 295 300Lys Val Glu Ser
Ser Arg Ile Ser Lys Val Ser Ser Ile Val Val Asn305 310 315 320Ile
Thr Glu Leu Phe Ser Lys Pro Glu Leu Glu Ser Ser Phe Thr His 325 330
335Leu Asp Gln Gly Glu Arg Leu Asn Leu Ser Cys Ser Ile Pro Gly Ala
340 345 350Pro Pro Ala Asn Phe Thr Ile Gln Lys Glu Asp Thr Ile Val
Ser Gln 355 360 365Thr Gln Asp Phe Thr Lys Ile Ala Ser Lys Ser Asp
Ser Gly Thr Tyr 370 375 380Ile Cys Thr Ala Gly Ile Asp Lys Val Val
Lys Lys Ser Asn Thr Val385 390 395 400Gln Ile Val Val Cys Glu Met
Leu Ser Gln Pro Arg Ile Ser Tyr Asp 405 410 415Ala Gln Phe Glu Val
Ile Lys Gly Gln Thr Ile Glu Val Arg Cys Glu 420 425 430Ser Ile Ser
Gly Thr Leu Pro Ile Ser Tyr Gln Leu Leu Lys Thr Ser 435 440 445Lys
Val Leu Glu Asn Ser Thr Lys Asn Ser Asn Asp Pro Ala Val Phe 450 455
460Lys Asp Asn Pro Thr Glu Asp Val Glu Tyr Gln Cys Val Ala Asp
Asn465 470 475 480Cys His Ser His Ala Lys Met Leu Ser Glu Val Leu
Arg Val Lys Val 485 490 495Ile Ala Pro Val Asp Glu Val Gln Ile Ser
Ile Leu Ser Ser Lys Val 500 505 510Val Glu Ser Gly Glu Asp Ile Val
Leu Gln Cys Ala Val Asn Glu Gly 515 520 525Ser Gly Pro Ile Thr Tyr
Lys Phe Tyr Arg Glu Lys Glu Gly Lys Pro 530 535 540Phe Tyr Gln Met
Thr Ser Asn Ala Thr Gln Ala Phe Trp Thr Lys Gln545 550 555 560Lys
Ala Ser Lys Glu Gln Glu Gly Glu Tyr Tyr Cys Thr Ala Phe Asn 565 570
575Arg Ala Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val
580 585 590Arg Val Ile Leu Ala Pro Trp Lys Lys Gly Leu Ile Ala Val
Val Ile 595 600 605Ile Gly Val Ile Ile Ala Leu Leu Ile Ile Ala Ala
Lys Cys Tyr Phe 610 615 620Leu Arg Lys Ala Lys Ala Lys Gln Met Pro
Val Glu Met Ser Arg Pro625 630 635 640Ala Val Pro Leu Leu Asn Ser
Asn Asn Glu Lys Met Ser Asp Pro Asn 645 650 655Met Glu Ala Asn Ser
His Tyr Gly His Asn Asp Asp Val Arg Asn His 660 665 670Ala Met Lys
Pro Ile Asn Asp Asn Lys Glu Pro Leu Asn Ser Asp Val 675 680 685Gln
Tyr Thr Glu Val Gln Val Ser Ser Ala Glu Ser His Lys Asp Leu 690 695
700Gly Lys Lys Asp Thr Glu Thr Val Tyr Ser Glu Val Arg Lys Ala
Val705 710 715 720Pro Asp Ala Val Glu Ser Arg Tyr Ser Arg Thr Glu
Gly Ser Leu Asp 725 730 735Gly Thr26PRTArtificialmouse or
human-derived CD31 peptide 2Leu Ala Pro Trp Lys Lys1
5310PRTArtificialmouse-derived CD31 peptide 3Val Arg Val Phe Leu
Ala Pro Trp Lys Lys1 5 10410PRTArtificialhuman-derived CD31 peptide
4Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5
10523PRTArtificialmouse-derived CD31 peptide 5Ser Ser Met Arg Thr
Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10 15Phe Leu Ala Pro
Trp Lys Lys 20623PRTArtificialhuman-derived CD31 peptide 6Asn His
Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile
Leu Ala Pro Trp Lys Lys 207727PRTMus musculus 7Met Leu Leu Ala Leu
Gly Leu Thr Leu Val Leu Tyr Ala Ser Leu Gln1 5 10 15Ala Glu Glu Asn
Ser Phe Thr Ile Asn Ser Ile His Met Glu Ser Leu 20 25 30Pro Ser Trp
Glu Val Met Asn Gly Gln Gln Leu Thr Leu Glu Cys Leu 35 40 45Val Asp
Ile Ser Thr Thr Ser Lys Ser Arg Ser Gln His Arg Val Leu 50 55 60Phe
Tyr Lys Asp Asp Ala Met Val Tyr Asn Val Thr Ser Arg Glu His65 70 75
80Thr Glu Ser Tyr Val Ile Pro Gln Ala Arg Val Phe His Ser Gly Lys
85 90 95Tyr Lys Cys Thr Val Met Leu Asn Asn Lys Glu Lys Thr Thr Ile
Glu 100 105 110Tyr Glu Val Lys Val His Gly Val Ser Lys Pro Lys Val
Thr Leu Asp 115 120 125Lys Lys Glu Val Thr Glu Gly Gly Val Val Thr
Val Asn Cys Ser Leu 130 135 140Gln Glu Glu Lys Pro Pro Ile Phe Phe
Lys Ile Glu Lys Leu Glu Val145 150 155 160Gly Thr Lys Phe Val Lys
Arg Arg Ile Asp Lys Thr Ser Asn Glu Asn 165 170 175Phe Val Leu Met
Glu Phe Pro Ile Glu Ala Gln Asp His Val Leu Val 180 185 190Phe Arg
Cys Gln Ala Gly Ile Leu Ser Gly Phe Lys Leu Gln Glu Ser 195 200
205Glu Pro Ile Arg Ser Glu Tyr Val Thr Val Gln Glu Ser Phe Ser Thr
210 215 220Pro Lys Phe Glu Ile Lys Pro Pro Gly Met Ile Ile Glu Gly
Asp Gln225 230 235 240Leu His Ile Arg Cys Ile Val Gln Val Thr His
Leu Val Gln Glu Phe 245 250 255Thr Glu Ile Ile Ile Gln Lys Asp Lys
Ala Ile Val Ala Thr Ser Lys 260 265 270Gln Ser Ser Glu Ala Val Tyr
Ser Val Met Ala Met Val Glu Tyr Ser 275 280 285Gly His Tyr Thr Cys
Lys Val Glu Ser Asn Arg Ile Ser Lys Ala Ser 290 295 300Ser Ile Met
Val Asn Ile Thr Glu Leu Phe Pro Lys Pro Lys Leu Glu305 310 315
320Phe Ser Ser Ser Arg Leu Asp Gln Gly Glu Leu Leu Asp Leu Ser Cys
325 330 335Ser Val Ser Gly Thr Pro Val Ala Asn Phe Thr Ile Gln Lys
Glu Glu 340 345 350Thr Val Leu Ser Gln Tyr Gln Asn Phe Ser Lys Ile
Ala Glu Glu Ser 355 360 365Asp Ser Gly Glu Tyr Ser Cys Thr Ala Gly
Ile Gly Lys Val Val Lys 370 375 380Arg Ser Gly Leu Val Pro Ile Gln
Val Cys Glu Met Leu Ser Lys Pro385 390 395 400Ser Ile Phe His Asp
Ala Lys Ser Glu Ile Ile Lys Gly His Ala Ile 405 410 415Gly Ile Ser
Cys Gln Ser Glu Asn Gly Thr Ala Pro Ile Thr Tyr His 420 425 430Leu
Met Lys Ala Lys Ser Asp Phe Gln Thr Leu Glu Val Thr Ser Asn 435 440
445Asp Pro Ala Thr Phe Thr Asp Lys Pro Thr Arg Asp Met Glu Tyr Gln
450 455 460Cys Arg Ala Asp Asn Cys His Ser His Pro Ala Val Phe Ser
Glu Ile465 470 475 480Leu Arg Val Arg Val Ile Ala Pro Val Asp Glu
Val Val Ile Ser Ile 485 490 495Leu Ser Ser Asn Glu Val Gln Ser Gly
Ser Glu Met Val Leu Arg Cys 500 505 510Ser Val Lys Glu Gly Thr Ser
Pro Ile Thr Phe Gln Phe Tyr Lys Glu 515 520 525Lys Glu Asp Arg Pro
Phe His Gln Ala Val Val Asn Asp Thr Gln Ala 530 535 540Phe Trp His
Asn Lys Gln Ala Ser Lys Lys Gln Glu Gly Gln Tyr Tyr545 550 555
560Cys Thr Ala Ser Asn Arg Ala Ser Ser Met Arg Thr Ser Pro Arg Ser
565 570 575Ser Thr Leu Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys
Gly Leu 580 585 590Ile Ala Val Val Val Ile Gly Val Val Ile Ala Thr
Leu Ile Val Ala 595 600 605Ala Lys Cys Tyr Phe Leu Arg Lys Ala Lys
Ala Lys Gln Lys Pro Val 610 615 620Glu Met Ser Arg Pro Ala Ala Pro
Leu Leu Asn Ser Asn Ser Glu Lys625 630 635 640Ile Ser Glu Pro Ser
Val Glu Ala Asn Ser His Tyr Gly Tyr Asp Asp 645 650 655Val Ser Gly
Asn Asp Ala Val Lys Pro Ile Asn Gln Asn Lys Asp Pro 660 665 670Gln
Asn Met Asp Val Glu Tyr Thr Glu Val Glu Val Ser Ser Leu Glu 675 680
685Pro His Gln Ala Leu Gly Thr Arg Ala Thr Glu Thr Val Tyr Ser Glu
690 695 700Ile Arg Lys Val Asp Pro Asn Leu Met Glu Asn Arg Tyr Ser
Arg Thr705 710 715 720Glu Gly Ser Leu Asn Gly Thr 7258739PRTBos
taurus 8Met Gln Leu Arg Trp Thr Gln Arg Gly Met Met Trp Leu Gly Ala
Leu1 5 10 15Leu Thr Leu Leu Leu Cys Ser Ser Leu Lys Gly Gln Glu Asn
Ser Phe 20 25 30Thr Ile Asn Ser Ile His Met Gln Ile Leu Pro His Ser
Thr Val Gln 35 40 45Asn Gly Glu Asn Leu Thr Leu Gln Cys Leu Val Asp
Val Ser Thr Thr 50 55 60Ser Arg Val Lys Pro Leu His Gln Val Leu Phe
Tyr Lys Asp Asp Val65 70 75 80Leu Leu His Asn Val Ser Ser Arg Arg
Asn Thr Glu Ser Tyr Leu Ile 85 90 95Pro His Val Arg Val Cys Asp Ser
Gly Arg Tyr Lys Cys Asn Val Ile 100 105 110Leu Asn Asn Lys Glu Lys
Thr Thr Pro Glu Tyr Glu Val Trp Val Lys 115 120 125Gly Val Ser Asp
Pro Arg Val Thr Leu Asp Lys Lys Glu Val Ile Glu 130 135 140Gly Gly
Val Val Val Val Asn Cys Ser Val Pro Glu Glu Lys Ala Pro145 150 155
160Val His Phe Thr Ile Glu Lys Phe Glu Leu Asn Ile Arg Gly Ala Lys
165 170 175Lys Lys Arg Glu Lys Thr Ser Gln Asn Gln Asn Phe Val Thr
Leu Glu 180 185 190Phe Thr Val Glu Glu Gln Asp Arg Thr Ile Arg Phe
Gln Cys Gln Ala 195 200 205Lys Ile Phe Ser Gly Ser Asn Val Glu Ser
Ser Arg Pro Ile Gln Ser 210 215 220Asp Leu Val Thr Val Arg Glu Ser
Phe Ser Asn Pro Lys Phe His Ile225 230 235 240Ile Pro Glu Gly Lys
Val Met Glu Gly Asp Asp Leu Gln Val Lys Cys 245 250 255Thr Val Gln
Val Thr His Gln Ala Gln Ser Phe Pro Glu Ile Ile Ile 260 265 270Gln
Lys Asp Arg Glu Ile Val Ala His Asn Ser Leu Ser Ser Glu Ala 275 280
285Val Tyr Ser Val Met Ala Thr Thr Glu His Asn Gly Asn Tyr Thr Cys
290 295 300Lys Val Glu Ala Ser Arg Ile Ser Lys Val Ser Ser Val Val
Val Asn305 310 315 320Val Thr Glu Leu Phe Ser Lys Pro Lys Leu Glu
Ser Ser Ala Thr His 325 330 335Leu Asp Gln Gly Glu Asp Leu Asn Leu
Leu Cys Ser Ile Pro Gly Ala 340 345 350Pro Pro Ala Asn Phe Thr Ile
Gln Lys Gly Gly Met Thr Val Ser Gln 355 360 365Thr Gln Asn Phe Thr
Lys Arg Val Ser Glu Trp Asp Ser Gly Leu Tyr 370 375 380Thr Cys Val
Ala Gly Val Gly Arg Val Phe Lys Arg Ser Asn Thr Val385 390 395
400Gln Ile Thr Val Cys Glu Met Leu Ser Lys Pro Ser Ile Phe His Asp
405 410 415Ser Arg Ser Glu Val Ile Lys Gly Gln Thr Ile Glu Val Ser
Cys Gln 420 425 430Ser Val Asn Gly Thr Ala Pro Ile Phe Tyr Gln Leu
Ser Asn Thr Ser 435 440 445Lys Pro Val Ala Asn Gln Ser Val Gly Ser
Asn Lys Pro Ala Ile Phe 450 455 460Arg Val Lys Pro Thr Lys Asp Val
Glu Tyr Cys Cys Ser Ala Asp Asn465 470 475 480Cys His Ser His Ser
Lys Met Phe Ser Glu Val Leu Arg Val Lys Val 485 490 495Ile Ala Pro
Val Asp Glu Ala Gln Leu Val Val Leu Lys Gly Glu Val 500 505 510Glu
Pro Gly Glu Pro Ile Val Phe Tyr Cys Ser Val Asn Glu Gly Ser 515 520
525Phe Pro Ile Thr Tyr Lys Phe Tyr Lys Glu Lys Glu Ser Lys Pro Phe
530 535 540Tyr Gln Asp Thr Ile Asn Ala Thr Gln Ile Met Trp His Lys
Thr Thr545 550 555 560Ala Ser Lys Glu Tyr Glu Gly Gln Tyr Tyr Cys
Thr Ala Ser Asn Arg 565 570 575Ala Asn Leu Ser Lys His Val Ile Gln
Ser Asn Thr Leu Thr Val Arg 580 585 590Val Tyr Leu Pro Leu Glu Lys
Gly Leu Ile Ala Val Val Val Ile Gly 595 600 605Val Ile Ile Val Thr
Leu Val Leu Gly Ala Lys Cys Tyr Phe Leu Lys 610 615 620Lys Ala Lys
Ala Lys Gln Met Pro Val Glu Met Ser Arg Pro Ala Val625 630 635
640Pro Leu Leu Asn Ser Asn Asn Glu Lys Thr Leu Ser Asp Ala Gly Thr
645 650 655Glu Ala Asp Arg His Tyr Gly Tyr Asn Glu Asp Val Gly Asn
His Ala 660 665 670Met Lys Pro Leu Asn Glu Asn Lys Glu Pro Leu Thr
Leu Asp Val Glu 675 680 685Tyr Thr Glu Val Glu Val Thr Ser Pro Glu
Pro His Gln Gly Leu Gly 690 695 700Thr Lys Gly Thr Glu Thr Glu Thr
Val Tyr Ser Glu Ile Arg Lys Ala705 710 715 720Asp Pro Asp Phe Val
Glu Asn Arg Tyr Ser Arg Thr Glu Gly Ser Leu 725 730 735Asp Gly
Ser9740PRTSus scrofa 9Met Arg Leu Arg Trp Thr Gln Gly Gly Asn Met
Trp Leu Gly Val Leu1 5 10 15Leu Thr Leu Gln Leu Cys Ser Ser Leu Glu
Gly Gln Glu Asn Ser Phe 20 25 30Thr Ile Asn Ser Ile His Met Glu Met
Leu Pro Gly Gln Glu Val His 35 40 45Asn Gly Glu Asn Leu Thr Leu Gln
Cys Ile Val Asp Val Ser Thr Thr 50 55 60Ser Ser Val Lys Pro Gln His
Gln Val Leu Phe Tyr Lys Asp Asp Val65 70 75 80Leu Phe His Asn Val
Ser Ser Thr Lys Asn Thr Glu Ser Tyr Phe Ile 85 90 95Ser
Glu Ala Arg Val Tyr Asn Ser Gly Arg Tyr Lys Cys Thr Val Ile 100 105
110Leu Asn Asn Lys Glu Lys Thr Thr Ala Glu Tyr Lys Val Val Val Glu
115 120 125Gly Val Ser Asn Pro Arg Val Thr Leu Asp Lys Lys Glu Val
Ile Glu 130 135 140Gly Gly Val Val Lys Val Thr Cys Ser Val Pro Glu
Glu Lys Pro Pro145 150 155 160Val His Phe Ile Ile Glu Lys Phe Glu
Leu Asn Val Arg Asp Val Lys 165 170 175Gln Arg Arg Glu Lys Thr Ala
Asn Asn Gln Asn Ser Val Thr Leu Glu 180 185 190Phe Thr Val Glu Glu
Gln Asp Arg Val Ile Leu Phe Ser Cys Gln Ala 195 200 205Asn Val Ile
Phe Gly Thr Arg Val Glu Ile Ser Asp Ser Val Arg Ser 210 215 220Asp
Leu Val Thr Val Arg Glu Ser Phe Ser Asn Pro Lys Phe His Ile225 230
235 240Ser Pro Lys Gly Val Ile Ile Glu Gly Asp Gln Leu Leu Ile Lys
Cys 245 250 255Thr Ile Gln Val Thr His Gln Ala Gln Ser Phe Pro Glu
Ile Ile Ile 260 265 270Gln Lys Asp Lys Glu Ile Val Ala His Ser Arg
Asn Gly Ser Glu Ala 275 280 285Val Tyr Ser Val Met Ala Thr Val Glu
His Asn Ser Asn Tyr Thr Cys 290 295 300Lys Val Glu Ala Ser Arg Ile
Ser Lys Val Ser Ser Ile Met Val Asn305 310 315 320Ile Thr Glu Leu
Phe Ser Arg Pro Lys Leu Lys Ser Ser Ala Thr Arg 325 330 335Leu Asp
Gln Gly Glu Ser Leu Arg Leu Trp Cys Ser Ile Pro Gly Ala 340 345
350Pro Pro Glu Ala Asn Phe Thr Ile Gln Lys Gly Gly Met Met Met Leu
355 360 365Gln Asp Gln Asn Leu Thr Lys Val Ala Ser Glu Arg Asp Ser
Gly Thr 370 375 380Tyr Thr Cys Val Ala Gly Ile Gly Lys Val Val Lys
Arg Ser Asn Glu385 390 395 400Val Gln Ile Ala Val Cys Glu Met Leu
Ser Lys Pro Ser Ile Phe His 405 410 415Asp Ser Gly Ser Glu Val Ile
Lys Gly Gln Thr Ile Glu Val Ser Cys 420 425 430Gln Ser Ile Asn Gly
Thr Ser Pro Ile Ser Tyr Gln Leu Leu Lys Gly 435 440 445Ser Asp Leu
Leu Ala Ser Gln Asn Val Ser Ser Asn Glu Pro Ala Val 450 455 460Phe
Lys Asp Asn Pro Thr Lys Asp Val Glu Tyr Gln Cys Ile Ala Asp465 470
475 480Asn Cys His Ser His Ala Gly Met Pro Ser Lys Val Leu Arg Val
Lys 485 490 495Val Ile Ala Pro Val Glu Glu Val Lys Leu Ser Ile Leu
Leu Ser Glu 500 505 510Glu Val Glu Ser Gly Gln Ala Ile Val Leu Gln
Cys Ser Val Lys Glu 515 520 525Gly Ser Gly Pro Ile Thr Tyr Lys Phe
Tyr Lys Glu Lys Glu Asn Lys 530 535 540Pro Phe His Gln Val Thr Leu
Asn Asp Thr Gln Ala Ile Trp His Lys545 550 555 560Pro Lys Ala Ser
Lys Asp Gln Glu Gly Gln Tyr Tyr Cys Leu Ala Ser 565 570 575Asn Arg
Ala Thr Pro Ser Lys Asn Phe Leu Gln Ser Asn Ile Leu Ala 580 585
590Val Arg Val Tyr Leu Ala Pro Trp Lys Lys Gly Leu Ile Ala Val Val
595 600 605Val Ile Ala Val Ile Ile Ala Val Leu Leu Leu Gly Ala Arg
Phe Tyr 610 615 620Phe Leu Lys Lys Ser Lys Ala Lys Gln Met Pro Val
Glu Met Cys Arg625 630 635 640Pro Ala Ala Pro Leu Leu Asn Ser Asn
Asn Glu Lys Thr Leu Ser Asp 645 650 655Pro Asn Thr Glu Ala Asn Arg
His Tyr Gly Tyr Asn Glu Asp Val Gly 660 665 670Asn His Ala Met Lys
Pro Leu Asn Glu Asn Lys Glu Pro Leu Thr Leu 675 680 685Asp Val Glu
Tyr Thr Glu Val Glu Val Thr Ser Pro Glu Pro His Arg 690 695 700Gly
Leu Gly Thr Lys Gly Thr Glu Thr Val Tyr Ser Glu Ile Arg Lys705 710
715 720Ala Asp Pro Asp Leu Val Glu Asn Arg Tyr Ser Arg Thr Glu Gly
Ser 725 730 735Leu Asp Gly Thr 7401010PRTArtificialrat-derived CD31
peptide 10Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5
101110PRTArtificialpig-derived CD31 peptide 11Val Arg Val Tyr Leu
Ala Pro Trp Lys Lys1 5 10129PRTArtificialbovin-derived CD31 peptide
12Val Arg Val Tyr Leu Pro Leu Glu Lys1 51323PRTArtificialscramble
peptide 13Ser Met Pro Ala Val Arg Ser Arg Phe Ser Ala Thr Ser Leu
Val Thr1 5 10 15Leu Lys Ser Arg Trp Pro Lys
201410PRTArtificialscramble peptide 14Trp Pro Lys Leu Arg Lys Phe
Val Ala Val1 5 101522PRTArtificial SequenceCD31 peptide 15Ser Met
Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe1 5 10 15Leu
Ala Pro Trp Lys Lys 201621PRTArtificial SequenceCD31 peptide 16Met
Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu1 5 10
15Ala Pro Trp Lys Lys 201720PRTArtificial SequenceCD31 peptide
17Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu Ala1
5 10 15Pro Trp Lys Lys 201819PRTArtificial SequenceCD31 peptide
18Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu Ala Pro1
5 10 15Trp Lys Lys1918PRTArtificial SequenceCD31 peptide 19Ser Pro
Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu Ala Pro Trp1 5 10 15Lys
Lys2017PRTArtificial SequenceCD31 peptide 20Pro Arg Ser Ser Thr Leu
Ala Val Arg Val Phe Leu Ala Pro Trp Lys1 5 10
15Lys2116PRTArtificial SequenceCD31 peptide 21Arg Ser Ser Thr Leu
Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 10
152215PRTArtificial SequenceCD31 peptide 22Ser Ser Thr Leu Ala Val
Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 10 152314PRTArtificial
SequenceCD31 peptide 23Ser Thr Leu Ala Val Arg Val Phe Leu Ala Pro
Trp Lys Lys1 5 102413PRTArtificial SequenceCD31 peptide 24Thr Leu
Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 102512PRTArtificial
SequenceCD31 peptide 25Leu Ala Val Arg Val Phe Leu Ala Pro Trp Lys
Lys1 5 102611PRTArtificial SequenceCD31 peptide 26Ala Val Arg Val
Phe Leu Ala Pro Trp Lys Lys1 5 10279PRTArtificial SequenceCD31
peptide 27Arg Val Phe Leu Ala Pro Trp Lys Lys1 5288PRTArtificial
SequenceCD31 peptide 28Val Phe Leu Ala Pro Trp Lys Lys1
5297PRTArtificial SequenceCD31 peptide 29Phe Leu Ala Pro Trp Lys
Lys1 5305PRTArtificial SequenceCD31 peptide 30Ala Pro Trp Lys Lys1
5314PRTArtificial SequenceCD31 peptide 31Pro Trp Lys
Lys13222PRTArtificial SequenceCD31 peptide 32His Ala Ser Ser Val
Pro Arg Ser Lys Ile Leu Thr Val Arg Val Ile1 5 10 15Leu Ala Pro Trp
Lys Lys 203321PRTArtificial SequenceCD31 peptide 33Ala Ser Ser Val
Pro Arg Ser Lys Ile Leu Thr Val Arg Val Ile Leu1 5 10 15Ala Pro Trp
Lys Lys 203420PRTArtificial SequenceCD31 peptide 34Ser Ser Val Pro
Arg Ser Lys Ile Leu Thr Val Arg Val Ile Leu Ala1 5 10 15Pro Trp Lys
Lys 203519PRTArtificial SequenceCD31 peptide 35Ser Val Pro Arg Ser
Lys Ile Leu Thr Val Arg Val Ile Leu Ala Pro1 5 10 15Trp Lys
Lys3618PRTArtificial SequenceCD31 peptide 36Val Pro Arg Ser Lys Ile
Leu Thr Val Arg Val Ile Leu Ala Pro Trp1 5 10 15Lys
Lys3717PRTArtificial SequenceCD31 peptide 37Pro Arg Ser Lys Ile Leu
Thr Val Arg Val Ile Leu Ala Pro Trp Lys1 5 10
15Lys3816PRTArtificial SequenceCD31 peptide 38Arg Ser Lys Ile Leu
Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 10
153915PRTArtificial SequenceCD31 peptide 39Ser Lys Ile Leu Thr Val
Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 10 154014PRTArtificial
SequenceCD31 peptide 40Lys Ile Leu Thr Val Arg Val Ile Leu Ala Pro
Trp Lys Lys1 5 104113PRTArtificial SequenceCD31 peptide 41Ile Leu
Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 104212PRTArtificial
SequenceCD31 peptide 42Leu Thr Val Arg Val Ile Leu Ala Pro Trp Lys
Lys1 5 104311PRTArtificial SequenceCD31 peptide 43Thr Val Arg Val
Ile Leu Ala Pro Trp Lys Lys1 5 10449PRTArtificial SequenceCD31
peptide 44Arg Val Ile Leu Ala Pro Trp Lys Lys1 5458PRTArtificial
SequenceCD31 peptide 45Val Ile Leu Ala Pro Trp Lys Lys1
5467PRTArtificial SequenceCD31 peptide 46Ile Leu Ala Pro Trp Lys
Lys1 54722PRTArtificial SequenceCD31 peptide 47Ser Ser Met Arg Thr
Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10 15Phe Leu Ala Pro
Trp Lys 204821PRTArtificial SequenceCD31 peptide 48Ser Ser Met Arg
Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10 15Phe Leu Ala
Pro Trp 204920PRTArtificial SequenceCD31 peptide 49Ser Ser Met Arg
Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10 15Phe Leu Ala
Pro 205019PRTArtificial SequenceCD31 peptide 50Ser Ser Met Arg Thr
Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10 15Phe Leu
Ala5118PRTArtificial SequenceCD31 peptide 51Ser Ser Met Arg Thr Ser
Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10 15Phe
Leu5217PRTArtificial SequenceCD31 peptide 52Ser Ser Met Arg Thr Ser
Pro Arg Ser Ser Thr Leu Ala Val Arg Val1 5 10
15Phe5315PRTArtificial SequenceCD31 peptide 53Ser Ser Met Arg Thr
Ser Pro Arg Ser Ser Thr Leu Ala Val Arg1 5 10 155414PRTArtificial
SequenceCD31 peptide 54Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr
Leu Ala Val1 5 105513PRTArtificial SequenceCD31 peptide 55Ser Ser
Met Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala1 5 105612PRTArtificial
SequenceCD31 peptide 56Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr
Leu1 5 105711PRTArtificial SequenceCD31 peptide 57Ser Ser Met Arg
Thr Ser Pro Arg Ser Ser Thr1 5 105810PRTArtificial SequenceCD31
peptide 58Ser Ser Met Arg Thr Ser Pro Arg Ser Ser1 5
10599PRTArtificial SequenceCD31 peptide 59Ser Ser Met Arg Thr Ser
Pro Arg Ser1 5608PRTArtificial SequenceCD31 peptide 60Ser Ser Met
Arg Thr Ser Pro Arg1 5617PRTArtificial SequenceCD31 peptide 61Ser
Ser Met Arg Thr Ser Pro1 5626PRTArtificial SequenceCD31 peptide
62Ser Ser Met Arg Thr Ser1 5635PRTArtificial SequenceCD31 peptide
63Ser Ser Met Arg Thr1 5644PRTArtificial SequenceCD31 peptide 64Ser
Ser Met Arg16522PRTArtificial SequenceCD31 peptide 65Asn His Ala
Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile Leu
Ala Pro Trp Lys 206621PRTArtificial SequenceCD31 peptide 66Asn His
Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile
Leu Ala Pro Trp 206720PRTArtificial SequenceCD31 peptide 67Asn His
Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile
Leu Ala Pro 206819PRTArtificial SequenceCD31 peptide 68Asn His Ala
Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile Leu
Ala6918PRTArtificial SequenceCD31 peptide 69Asn His Ala Ser Ser Val
Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile
Leu7017PRTArtificial SequenceCD31 peptide 70Asn His Ala Ser Ser Val
Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10
15Ile7116PRTArtificial SequenceCD31 peptide 71Asn His Ala Ser Ser
Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10
157215PRTArtificial SequenceCD31 peptide 72Asn His Ala Ser Ser Val
Pro Arg Ser Lys Ile Leu Thr Val Arg1 5 10 157314PRTArtificial
SequenceCD31 peptide 73Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile
Leu Thr Val1 5 107413PRTArtificial SequenceCD31 peptide 74Asn His
Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr1 5 107512PRTArtificial
SequenceCD31 peptide 75Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile
Leu1 5 107611PRTArtificial SequenceCD31 peptide 76Asn His Ala Ser
Ser Val Pro Arg Ser Lys Ile1 5 107710PRTArtificial SequenceCD31
peptide 77Asn His Ala Ser Ser Val Pro Arg Ser Lys1 5
10789PRTArtificial SequenceCD31 peptide 78Asn His Ala Ser Ser Val
Pro Arg Ser1 5798PRTArtificial SequenceCD31 peptide 79Asn His Ala
Ser Ser Val Pro Arg1 5807PRTArtificial SequenceCD31 peptide 80Asn
His Ala Ser Ser Val Pro1 5816PRTArtificial SequenceCD31 peptide
81Asn His Ala Ser Ser Val1 5825PRTArtificial SequenceCD31 peptide
82Asn His Ala Ser Ser1 5834PRTArtificial SequenceCD31 peptide 83Asn
His Ala Ser18421PRTArtificial SequenceCD31 peptide 84Ser Met Arg
Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe1 5 10 15Leu Ala
Pro Trp Lys 208519PRTArtificial SequenceCD31 peptide 85Met Arg Thr
Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu1 5 10 15Ala Pro
Trp8617PRTArtificial SequenceCD31 peptide 86Arg Thr Ser Pro Arg Ser
Ser Thr Leu Ala Val Arg Val Phe Leu Ala1 5 10
15Pro8715PRTArtificial SequenceCD31 peptide 87Thr Ser Pro Arg Ser
Ser Thr Leu Ala Val Arg Val Phe Leu Ala1 5 10 158813PRTArtificial
SequenceCD31 peptide 88Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val
Phe Leu1 5 108911PRTArtificial SequenceCD31 peptide 89Pro Arg Ser
Ser Thr Leu Ala Val Arg Val Phe1 5 10909PRTArtificial SequenceCD31
peptide 90Arg Ser Ser Thr Leu Ala Val Arg Val1 5917PRTArtificial
SequenceCD31 peptide 91Ser Ser Thr Leu Ala Val Arg1
5925PRTArtificial SequenceCD31 peptide 92Ser Thr Leu Ala Val1
59321PRTArtificial SequenceCD31 peptide 93His Ala Ser Ser Val Pro
Arg Ser Lys Ile Leu Thr Val Arg Val Ile1 5 10 15Leu Ala Pro Trp Lys
209419PRTArtificial SequenceCD31 peptide 94Ala Ser Ser Val Pro Arg
Ser Lys Ile Leu Thr Val Arg Val Ile Leu1 5 10 15Ala Pro
Trp9517PRTArtificial SequenceCD31 peptide 95Ser Ser Val Pro Arg Ser
Lys Ile Leu Thr Val Arg Val Ile Leu Ala1 5 10
15Pro9615PRTArtificial SequenceCD31 peptide 96Ser Val Pro Arg Ser
Lys Ile Leu Thr Val Arg Val Ile Leu Ala1 5 10 159713PRTArtificial
SequenceCD31 peptide 97Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val
Ile Leu1 5 109811PRTArtificial SequenceCD31 peptide 98Pro Arg Ser
Lys Ile Leu Thr Val Arg Val Ile1 5 10999PRTArtificial SequenceCD31
peptide 99Arg Ser Lys Ile Leu Thr Val Arg Val1 51007PRTArtificial
SequenceCD31 peptide 100Ser Lys Ile Leu Thr Val Arg1
51015PRTArtificial SequenceCD31 peptide 101Lys Ile Leu Thr Val1
5
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