CD31SHED AS A MOLECULAR TARGET FOR IMAGING OF INFLAMMATION

Abstract

Disclosed is CD31.sup.shed for use as a molecular imaging target in the molecular imaging of an inflammatory condition. Administering the radiolabeled peptide P8RI as CD31.sup.shed ligand in different rat models of inflammation indeed showed that CD31.sup.shed is present on activated cells in a quantity allowing a detectable signal, whereas the noise signal corresponding to CD31.sup.shed present on activated circulating cells and on other organs or cells not involved in inflammation was little. Also disclosed is a labeled CD31.sup.shed ligand and the use thereof as a molecular imaging agent in the molecular imaging of an inflammatory condition. The molecular imaging of inflammatory sites particularly allows determining whether a subject suffers from or is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to the molecular imaging of inflammatory sites, in particular for determining whether a subject suffers or is at risk of having or developing an inflammatory condition and for monitoring the changes in the extent of an inflammatory condition after an anti-inflammatory treatment.

BACKGROUND

[0002] Imaging of inflammation sites would be a useful tool for the diagnosis of an inflammation condition. The high level of glucose metabolism in inflammation has prompted to the use of .sup.18F-FDG (2-deoxy-2-.sup.18F-fluoro-D-glucose) PET. .sup.18F-FDG however suffers from poor specificity and its high physiologic uptake in the heart, lung and brain limits its use. More specific targets for imaging inflamed tissues are therefore needed.

[0003] CD31 is a transmembrane glycoprotein receptor constitutively expressed by leukocytes, platelets and endothelial cells. It consists of a single chain molecule comprising six Ig-like extracellular domains, a short transmembrane segment and a cytoplasmic tail comprising two important tyrosine-based motifs (around Y663 and Y686) that act as Immunoreceptor Tyrosine-based Inhibitory Motifs (ITIMs). The structure of CD31 is shown in the Table 1 below.

TABLE-US-00001 TABLE 1 Structure of CD31 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 domain 145 to 233 Third Ig-like extracellular domain 236 to 315 Fourth Ig-like extracellular domain 328 to 401 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

[0004] Due to its homophilic and inhibitory functions, CD31 exerts a crucial role in the homeostasis of the circulation. In pro-inflammatory conditions, the homophilic portion of the receptor is lost due to a cleavage and activated cells express a truncated form of CD31. WO2010/000756 indeed discloses that CD31 is shed on activated/memory T lymphocytes between the 5th and the 6th extracellular Ig-like domains whereas WO2013/152919 discloses that activated platelets also express a CD31 that is truncated between the 6.sup.th extracellular domain and the juxtamembrane sequence. Both types of shed extracellular sequences of CD31 (referred to as "soluble CD31") are then released into the circulation, where they are present together with a soluble splice variant of CD31, produced by healthy endothelial cells. The remaining small CD31 ectodomain which remains anchored to the membrane after shedding is referred to as "CD31.sup.shed". WO2010/000756 and WO2013/152919 discloses a method for diagnosing a thrombotic or an autoimmune disorder based on the detection of said soluble CD31 in a biological sample of an individual.

[0005] WO2010/000741 discloses peptides corresponding to juxta-membrane amino acids of the ectodomain of CD31 that are able to rescue the physiological immunoregulatory function of CD31, by specifically targeting CD31.sup.shed on activated leukocytes and platelets. Such peptides are useful for the treatment of thrombotic or autoimmune disorders. WO2013/190014 further discloses specific peptides of 8 amino acids, within the membrane juxta-proximal part of extracellular CD31, which hold useful for the treatment of thrombotic or inflammatory disorders and display physic-chemical properties that are more suitable for drug development.

[0006] There is still a need to provide reliable solutions for the imaging and diagnostic of inflammatory conditions.

DESCRIPTION OF THE INVENTION

[0007] The Inventors have unexpectedly found that CD31.sup.shed itself can be used as a molecular target for the diagnosis of an inflammatory condition, in particular by allowing the molecular imaging of inflammatory sites. CD31.sup.shed was indeed surprisingly found to be present on activated cells in a quantity allowing to obtain a detectable signal when using a labeled CD31.sup.shed ligand, whereas the noise signal corresponding to CD31.sup.shed present on activated circulating cells and on other organs or cells not involved in inflammation was little, thereby leading to a good signal to noise ratio. They have indeed shown in vivo in an animal model of inflammation that administering to said animal a radiolabeled CD31.sup.shed ligand specific for CD31.sup.shed allows localizing the inflammation site, due to the specific binding of the radiolabeled CD31.sup.shed ligand to the activated cells expressing CD31.sup.shed and its subsequent concentration in the inflammation site, whereas the remaining radiolabeled CD31.sup.shed ligand is rapidly cleared from the body of the animal.

[0008] In this context, the Inventors used peptide P8RI of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids coupled to HYNIC (6-Hydrazinopyridine-3-carboxylic acid) via a PEG spacer and radiolabeled with 99mTc. Said radiolabeled CD31.sup.shed ligand was found stable both in vitro and in vivo and specific to CD31.sup.shed, with very low binding to plasma proteins.

[0009] Therefore, labelled CD31.sup.shed ligands, in particular small peptides, able to specifically bind to CD31.sup.shed are useful as a tracer for molecular imaging of inflammation.

[0010] One object of the present invention is thus a labeled CD31.sup.shed ligand comprising a CD31.sup.shed ligand and at least one imaging label, preferably at least one radionuclide.

[0011] Said CD31.sup.shed ligand is preferably:

[0012] a) a peptide selected in the group consisting of:

[0013] (i) a peptide consisting of a fragment of 3 to 15 amino acids of the sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1,

[0014] (ii) a peptide consisting of a fragment of 3 to 15 amino acids of a sequence corresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1 in a non-human mammalian CD31,

[0015] (iii) a peptide of 3 to 15 amino acids consisting of a sequence at least 70% identical to the sequence of peptide (i),

[0016] (iv) a peptide consisting of a retro-inverso sequence of peptide (i), (ii) or (iii), and

[0017] (v) the peptide (i), (ii), (iii) or (iv) comprising at least one or at least one further chemical modification,

[0018] or

[0019] b) a peptidomimetic of peptide a).

[0020] The CD31.sup.shed ligand of (v) preferably comprises at least one amino acid in the D-enantiomer form.

[0021] The CD31.sup.shed ligand is preferably selected in the group consisting of a peptide of sequence SEQ ID NO: 2, a peptide of sequence SEQ ID NO: 3, a peptide of sequence SEQ ID NO: 4, a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids, a peptide of sequence SEQ ID NO: 7 and a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomer amino acids.

[0022] A preferred CD31.sup.shed ligand is a peptide of sequence SEQ ID NO: 5, or a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids.

[0023] The radionuclide of the labeled CD31.sup.shed ligand is preferably detectable by molecular imaging technique(s), such as Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), an hybrid of SPECT and/or PET, or their combinations.

[0024] For example, the radionuclide is technetium-99m (99mTc or .sup.99mTc), gallium-67 (.sup.67Ga), gallium-68 (.sup.68Ga), yttrium-90 (.sup.90Y), indium-111 (.sup.111In), rhenium-186 (.sub.186Re), fluorine-18 (.sup.18F), copper-64 (.sup.64Cu) or thallium-201 (.sup.201TI).

[0025] A preferred labeled CD31.sup.shed ligand comprises a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids as CD31.sup.shed ligand and 99mTc as radionuclide.

[0026] In one preferred embodiment, the CD31.sup.shed ligand is coupled with HYNIC (6-Hydrazinopyridine-3-carboxylic acid), optionally via at least one spacer.

[0027] Another object of the present invention relates to the labeled CD31.sup.shed ligand as defined above for use as a molecular imaging agent for imaging of inflammatory sites, in particular for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition. The present invention also relates to the use of the labeled CD31.sup.shed ligand as defined above as a molecular imaging agent, in particular for imaging inflammation sites.

[0028] Another object of the present invention relates to a method, preferably an in vitro method, for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition, wherein said method comprises:

detecting the presence of CD31.sup.shed on the surface of cells with a labeled CD31.sup.shed ligand as defined above in a biological sample obtained from the subject.

[0029] Another object of the present invention relates to CD31.sup.shed for use as a molecular imaging target for imaging of inflammatory sites, more particularly for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition, is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition. The present invention also relates to the use of CD31.sup.shed for use as a molecular imaging target, in particular for imaging inflammation sites.

[0030] Inflammatory Condition

[0031] An inflammatory condition underlies a large number of diseases. For example, the immune system is often involved with inflammatory conditions, as demonstrated in both allergic reactions and some myopathies, with many immune system disorders resulting in abnormal inflammation.

[0032] As used throughout the present specification, the term "inflammatory condition" includes but is not limited to a chronic inflammatory condition, an immune disorder, an autoimmune disorder, an acute and chronic grant alloimmune conditions, an acute and chronic infectious-driven inflammatory condition, a non-immune disease with etiological origins in inflammatory processes or their combinations.

[0033] The chronic inflammatory condition is for example inflammatory bowel disease, psoriasis, atopic dermatitis, cerebral amyloid angiopathy an/or vasculitis.

[0034] An immune disorder is for example allergies and/or myopathy.

[0035] The autoimmune disorder is for example rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel disease (IBD), systemic lupus erythematodes (SLE), Graves' disease and/or diabetes mellitus.

[0036] Acute and chronic alloimmune conditions are for example allograft rejection or graft versus host disease (GVHD).

[0037] Acute and chronic infectious-driven inflammatory conditions include the formation of septic granulomas (abscess) and/or septic shock.

[0038] The non-immune disease with etiological origins in inflammatory processes is for example cancer, thrombosis, ischaemic and/or ischemia-reperfusion organ damage (for example heart and/or brain infarction) arterial inflammatory condition (such as atherothrombosis, arterial dissection and/or unhealed/thromboses arterial aneurysm) and/or neurodegenerative disease.

[0039] The inflammatory condition is for example selected from the group consisting of rheumatoid arthritis, multiple sclerosis, allergies, myopathy, inflammatory bowel disease, psoriasis, atopic dermatitis, cerebral amyloid angiopathy, vasculitis, systemic lupus erythematosus, Graves' disease, diabetes mellitus, acute or chronic graft rejection, cancer, thrombosis, atherothrombosis, ischaemic heart and/or brain infarction, and/or neurodegenerative disease.

[0040] Subject for Imaging, to be Diagnosed and/or Treated

[0041] A "subject" in the context of the present invention is a human being or a non-human mammal.

[0042] The terms "human being", "individual" or "patient" are herein synonymous and may be used interchangeably.

[0043] Said human being may be of any sex, for example male or female and of any age, for example an infant, child, adolescent, adult, elderly people.

[0044] A non-human mammal is preferably a mouse, rat, cat, dog, rabbit, hamster, swine, sheep, horse or primate.

[0045] The subject may suffer from an inflammatory condition, be suspected to suffer from an inflammatory condition, be at risk of having an inflammatory condition or be at risk of recurrence of an inflammatory condition.

[0046] The expressions "at risk of having an inflammatory condition", "at risk of suffering from an inflammatory condition" and "at risks of developing an inflammatory condition" are herein synonymous.

[0047] "Risk" in the context of the present invention, relates to the probability that an event will occur over a specific time period, for example the conversion to an inflammatory condition.

[0048] Imaging of Inflammation

[0049] The expressions "imaging of inflammation sites" and "imaging of inflammation" are herein synonymous.

[0050] Imaging of inflammation sites allows to localize the inflamed tissues and/or inflamed organs in the body of a subject.

[0051] Imaging of inflammation sites presents many advantages.

[0052] For example, imaging of inflammation sites allows diagnosing an inflammation condition, confirming an inflammation condition, localizing the inflamed tissues or organs, monitoring the response to a treatment, for example to an anti-inflammatory treatment, monitoring the inflammatory side-effects of a treatment, predicting the risk of developing an inflammatory condition and/or determining the risk of recurrence of an inflammatory condition after an anti-inflammatory treatment.

[0053] Imaging of inflammation sites may thus be used for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition, is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition.

[0054] A molecular imaging target and/or a molecular imaging agent are needed to carry out imaging of inflammation.

[0055] As used herein, the term "molecular imaging target" refers to a compound that can be detected by using molecular imaging techniques.

[0056] As used herein, the term "molecular imaging agent" refers to a compound that can be used to detect specific biological elements, in particular a molecular imaging target, by using molecular imaging techniques.

[0057] The molecular imaging agent is preferably an agent coupled to an imaging label, covalently or non-covalently.

[0058] In the context of the invention, the molecular imaging agent is used to detect CD31.sup.shed in vivo or in vitro, for example in a blood sample of a subject.

[0059] CD31.sup.shed for Use as a Molecular Imaging Target

[0060] The present invention thus relates to CD31.sup.shed for use as a molecular imaging target for imaging of inflammation sites.

[0061] The present invention also relates to the use of CD31.sup.shed as a molecular imaging target for imaging of inflammation sites.

[0062] The present invention particularly relates to CD31.sup.shed for use as a molecular imaging target in a method for imaging inflammation sites, particularly in vivo.

[0063] As used herein "CD31.sup.shed" refers to the remaining small CD31 ectodomain which remains anchored to the membrane of endothelial cells, platelets or leukocytes, after shedding of the CD31 transmembrane glycoprotein receptor.

[0064] CD31.sup.shed on the surface of activated endothelial cells, platelets and leukocytes lacks at least the 1.sup.st to 5.sup.th extracellular Ig-like domains of CD31.

[0065] CD31.sup.shed on the surface of activated platelets and activated endothelial cells lacks the 1.sup.st to 5.sup.th extracellular Ig-like domains of CD31 and at least one part of the 6th extracellular Ig-like domain.

[0066] CD31.sup.shed on the surface of activated leukocytes lacks the 1.sup.st to 5.sup.th extracellular Ig-like domains, but comprises the 6th extracellular Ig-like domain.

[0067] The present invention also relates to CD31.sup.shed for use as defined above, in a method, preferably an in vivo method, for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition, is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition.

[0068] The present invention also relates to CD31.sup.shed for use for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition, is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition, in particular in vivo.

[0069] The expression "inflammatory condition" and "imaging of inflammation sites" are as defined above.

[0070] CD31.sup.shed Ligand

[0071] The term "CD31.sup.shed ligand" refers to any compound that is able to specifically bind with CD31.sup.shed.

[0072] The specific binding of a compound to CD31.sup.shed present on a cell surface may be assessed by any method well-known by the skilled person.

[0073] For example, the specific binding may be measured by plasmon surface resonance, flow cytometry or beta-imager.

[0074] In a preferred embodiment, the binding of a compound to be tested to CD31.sup.shed present on a cell surface is assessed as follows: the compound to be tested, or an irrelevant analogue as negative control, is bound to a fluorescent probe (e.g. fluoresceine). The compound is incubated at consecutive dilutions (for example 1, 10, 100 .mu.mol) with CD31+ cells (for example from a cell line, such as Jurkat T cells, or primary cells, such as peripheral blood T cells), at a density of 10.sup.6 cells/ml in a saline buffer comprising Ca++ and Mg++ (HBSS, culture medium). Parallel conditions are incubated in the presence of a cell activator (e.g. TCR crosslinking, such as 1 .mu.g/ml anti-CD3e antibodies+20 .mu.g/ml secondary F(ab')2 fragment if the cells are T lymphocytes). The reaction is stopped after 5 or 20 minutes by repeated washing steps with cold buffer. Cells are fixed with paraformaldehyde and washed again. Binding of the compound to be tested on individual cells is detected by the relative fluorescent signal using a flow cytometer. A greater signal for the specific compound as compared to the control and in the conditions comprising the cell activator as compared to resting cells indicates appropriate binding to CD31.sup.shed.

[0075] Alternatively, the compound to be tested, or an irrelevant analogue as negative control, is tested for its binding to activated endothelial cells (for example primary cells from human blood vessels of immortalized cell lines). The cells are cultured on collagen type I thin layer to confluence. For the activation, the cells are incubated with 20 ng/ml Human recombinant TNFa for 30 minutes in culture medium. The reaction is stopped by rinsing the cells with cold saline buffer and the cells are fixed (for instance with a Zinc-fixative solution for 10 minutes at room temperature). After extensive rinsing in a saline buffer comprising, activated endothelial cells are labeled by the compound to be tested, or an irrelevant analogue as negative control, is bound to a fluorescent probe (e.g. fluoresceine) and counterstained by a plasma membrane dye (e g Cell Mask.TM.) and a nuclear staining (eg Hoechst 33342). Binding of the compound to be tested on individual cells is detected by the relative fluorescent signal using a fluorescence microscope. A greater signal for the specific compound as compared to the control and in the conditions comprising the cell activator as compared to resting cells indicates appropriate binding to CD31.sup.shed.

[0076] The CD31.sup.shed ligand may be a peptide, a peptidomimetic, a chemical compound, an antibody or an aptamer.

[0077] The term "antibody" is thus used to refer to any antibody-like molecule that has an antigen binding region, and this term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs or VHH), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is non-internalizing. As used herein the term "non-internalizing antibody" refers to an antibody, respectively, that has the property of to bind to a target antigen present on a cell surface, and that, when bound to its target antigen, does not enter the cell and become degraded in the lysosome. In some embodiments, the heterologous polypeptide is a light immunoglobulin chain. In some embodiments, the heterologous polypeptide is a heavy immunoglobulin chain. In some embodiments, the heterologous polypeptide is a heavy single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains.

[0078] Particularly, in the context of the invention, the antibody is a single domain antibody. The term "single domain antibody" has its general meaning in the art and refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such single domain antibody are also called VHH or "nanobody.RTM.". For a general description of (single) domain antibodies, reference is also made to the prior art cited above, as well as to EP 0 368 684, Ward et al. (Nature 1989 Oct 12; 341 (6242): 544-6), Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and WO 06/030220, WO 06/003388. The amino acid sequence and structure of a single domain antibody can be considered to be comprised of four framework regions or "FRs" which are referred to in the art and herein as "Framework region 1" or "FRI"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4" respectively; which framework regions are interrupted by three complementary determining regions or "CDRs", which are referred to in the art as "Complementarity Determining Region for "CDRI"; as "Complementarity Determining Region 1" or "CDR2" and as "Complementarity Determining Region 2" or "CDR3" and as "Complementarity Determining Region 2", respectively. Accordingly, the single domain antibody can be defined as an amino acid sequence with the general structure: FRI--CDRI--FR2--CDR2--FR3--CDR3--FR4 in which FRI to FR4 refer to framework regions 1 to 4 respectively, and in which CDRI to CDR3 refer to the complementarity determining regions 1 to 3. In the context of the invention, the amino acid residues of the single domain antibody are numbered according to the general numbering for VH domains given by the International ImMunoGeneTics information system aminoacid numbering (http://imgt.cines.fr/).

[0079] Particularly, in the context of the invention, the antibody is a single chain variable fragment. The term "single chain variable fragment" or "scFv fragment" refers to a single folded polypeptide comprising the VH and VL domains of an antibody linked through a linker molecule. In such a scFv fragment, the VH and VL domains can be either in the VH--linker--VL or VL--linker--VH order. In addition to facilitate its production, a scFv fragment may contain a tag molecule linked to the scFv via a spacer. A scFv fragment thus comprises the VH and VL domains implicated into antigen recognizing but not the immunogenic constant domains of corresponding antibody.

[0080] In one embodiment of the invention, the CD31.sup.shed ligand is a peptide.

[0081] The peptide is preferably a peptide as disclosed in WO2010/000741 or WO2013/190014.

[0082] Preferably, the CD31.sup.shed ligand is a synthetic peptide.

[0083] By a "synthetic peptide", it is intended that the peptide is not present within a living organism, e.g. within human body.

[0084] The synthetic peptide is preferably purified.

[0085] The synthetic peptide may be part of a composition or a kit.

[0086] The peptide may be selected in the group consisting of:

[0087] (i) a peptide consisting of a fragment of 3 to 15 amino acids of the sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1,

[0088] (ii) a peptide consisting of a fragment of 3 to 15 amino acids of a sequence corresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1 in a non-human mammalian CD31,

[0089] (iii) a peptide of 3 to 15 amino acids consisting of a sequence at least 70% identical to the sequence of peptide (i),

[0090] (iv) a peptide consisting of a retro-inverso sequence of peptide (i), (ii) or (iii), and

[0091] (v) the peptide (i), (ii), (iii) or (iv) comprising at least one or at least one further chemical modification, preferably at least one amino acid in the D-enantiomer form.

[0092] A "fragment" refers herein to a sequence of consecutive amino acids. For example, a fragment may be a fragment of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.

[0093] The sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1 is sequence SEQ ID NO: 12.

[0094] Thus, the peptide may consist of a fragment of 3 to 15 amino acids of sequence SEQ ID NO: 12.

[0095] The peptide may also consist of a fragment of 3 to 15 amino acids of a sequence corresponding to sequence SEQ ID NO: 12 in a non-human mammalian CD31.

[0096] Non-limiting examples of non-human mammalian CD31 are the murine CD31 of sequence SEQ ID NO: 9, the bovine CD31 of sequence SEQ ID NO: 10 and the pig CD31 of sequence SEQ ID NO: 11.

[0097] The person skilled in the art can easily identify a sequence corresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1, i.e. to sequence SEQ ID NO: 12, in a non-human mammalian CD31 protein, for example by performing a sequence alignment between sequence SEQ ID NO: 1 and the sequence of said non-human mammalian CD31 protein, for example with one of sequences SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11.

[0098] Methods for sequence alignment and determination of sequence identity are well known in the art, for example using publicly available computer software such as BioPerl, BLAST, BLAST-2, CS-BLAST, FASTA, ALIGN, ALIGN-2, LALIGN, Jaligner, matcher or Megalign (DNASTAR) software and alignment algorithms such as the Needleman-Wunsch and Smith-Waterman algorithms.

[0099] The sequence of the CD31 peptide according to the invention is preferably derived from the sequence of human CD31 or murine CD31.

[0100] The peptide may also be a peptide of 3 to 15 amino acids consisting of a sequence at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical to the sequence of peptide (i), i.e. to the sequence of a fragment of 3 to 15 amino acids of the sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1.

[0101] A peptide sequence at least 70% identical to a given sequence of 4 to 6 amino acids differs from said given sequence of at most one amino acid. A peptide sequence at least 70% identical to a given sequence of 7 to 9 amino acids differs from said given sequence of at most two amino acids.

[0102] A peptide sequence at least 70% identical to a given sequence of 10 to 13 amino acids differs from said given sequence of at most three amino acids.

[0103] A peptide sequence at least 70% identical to a given sequence of 14 or 15 amino acids differs from said given sequence of at most four amino acids.

[0104] By "a sequence at least x % identical to a reference sequence", it is intended that the amino acid sequence of the subject peptide is identical to the reference sequence or differ from the reference sequence by up to 100-x amino acid alterations per each 100 amino acids of the reference sequence. In other words, to obtain a polypeptide having an amino acid sequence at least x % identical to a reference amino acid sequence, up to 100-x % of the amino acid residues in the subject sequence may be inserted, deleted or substituted with another amino acid.

[0105] Methods for comparing the identity of two or more sequences are well known in the art. 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 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 between sequences are also known in the art, for instance the Needle program, which is based on the Needleman and Wunsch algorithm, described in Needleman and Wunsch (1970) J. Mol Biol. 48:443-453, with for example the following parameters for polypeptide sequence comparison: comparison matrix: BLOSUM62, gap open penalty: 10 and gap extend penalty: 0.5, end gap penalty: false, end gap open penalty=10, end gap extend penalty=0.5; and the following parameters for polynucleotide sequence comparison: comparison matrix: DNAFULL; gap open penalty=10, gap extend penalty=0.5, end gap penalty: false, end gap open penalty=10, end gap extend penalty=0.5.

[0106] Peptides consisting of an amino acid sequence "at least 70%, 75%, 80%, 85%, or 90% identical" to a reference sequence may comprise mutations, such as deletions, insertions and/or substitutions compared to the reference sequence.

[0107] In case of substitutions, the substitution preferably corresponds to a conservative substitution as indicated in the Table 1 below. In a preferred embodiment, the peptide consisting of an amino acid sequence at least 70%, 75%, 80%, 85% or 90% identical to a reference sequence only differs from the reference sequence by conservative substitutions.

TABLE-US-00002 TABLE 1 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

[0108] In another preferred embodiment, the peptide consisting of an amino acid sequence at least 70%, 75%, 80%, 85% or 90% identical to a reference sequence corresponds to a naturally-occurring allelic variant of the reference sequence.

[0109] In still another preferred embodiment, the peptide consisting of an amino acid sequence at least 70%, 75%, 80%, 85%or 90% identical to a reference sequence corresponds to a homologous sequence derived from another non-human mammalian species than the reference sequence.

[0110] In a preferred embodiment, the peptide consisting of an amino acid sequence at least 70%, 75%, 80%, 85% or 90% identical to a reference sequence differs from the reference sequence by conservative substitutions and/or corresponds to a homologous sequence derived from another non-human mammalian species than the reference sequence.

[0111] By the expression "a peptide consisting of a retro-inverso sequence of peptide (i), (ii) or (iii)", it is herein meant a peptide that differs from the peptide (i), (ii) or (iii) in that its amino acids are in the reverse order by comparison to the sequence of peptide (i), (ii) or (iii), respectively, and consist of D-amino acids instead of the naturally-occurring L-amino acids.

[0112] D-enantiomers of amino acids (also called D-amino acids) are referred to by the same letter as their corresponding L-enantiomer (also called L-amino acid), but in lower case. Thus, for example, the L-enantiomer of arginine is referred to as `R`, while the D-enantiomer is referred to as `r`.

[0113] Preferably, the peptide is soluble in an organic or nonorganic solvent.

[0114] In a preferred embodiment, the peptide is soluble in water. More particularly, the peptide is preferably soluble in water and/or in aqueous buffer such as trifluoroacetate (for example in a 0.1% trifluoroacetate solution), NaCl 9 g/L, PBS, Tris or Tris-phosphate. The solubility in water and aqueous buffers is particularly advantageous on the pharmacological point of view. Thanks to such solubility, the peptide may be dissolved in an aqueous solution, for example at a concentration equal to, at least of or at most of 1 micromolar, 10 micromolar, 50 micromolar, 100 micromolar, 500 micromolar, 1 mM, 50 mM or 100 mM.

[0115] A CD31.sup.shed ligand of the invention that is readily soluble in water may be obtained by the presence of at least one charged amino acid (preferably arginine (R) and/or lysine (K)), wherein said charged amino acid is not comprised between two hydrophobic residues.

[0116] Thus, in a preferred embodiment, the CD31.sup.shed ligand according to the invention comprises at least one charged amino acid, preferably arginine and/or lysine, wherein said charged amino acid is not comprised between two hydrophobic residues.

[0117] In a more preferred embodiment, said charged amino acid is located either at the N- or C-terminal end of the sequence.

[0118] For example, the sequence of a preferred CD31.sup.shed ligand according to the invention begins with the motif RV (for example instead of VRV).

[0119] In a preferred embodiment, the peptide is resistant to peptidase, in particular to eukaryote peptidase.

[0120] By "resistant to peptidase", it is herein meant that the CD31.sup.shed ligand remains undigested, as determined by reverse phase--high-performance liquid chromatography (RP-HPLC) and mass spectroscopy (MS), upon incubation at 37.degree. C. with mammalian serum or injection in a living laboratory animal. Laboratory tests aimed at evaluating serum stability of the peptides are well standardized (see for example Jenssen and Aspmo, 2008, Methods Mol Biol 494, 177-186). Highly peptidase-resistant peptides are those that remains undigested for up to 70% of their original mass and/or displaying a half life longer than 240 minutes in the presence of proteolytic enzymes (see for example Kumarasinghe and Hruby, 2015, In Peptide Chemistry and Drug Design, B. M. Dunn, ed. (Hoboken, N.J.: Wiley), pp. 247-266).

[0121] The CD31.sup.shed ligand is preferably resistant to peptidases present in blood, such as soluble peptidases or peptidases present on cell surface.

[0122] The CD31.sup.shed ligand may also comprise at least one or at least one further chemical modification, preferably to improve its stability and/or bioavailability.

[0123] Such chemical modifications generally 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/or maintaining or improving its biological activity. Any chemical modification known in the art can be employed according to the present invention.

[0124] The CD31.sup.shed ligand may comprise at least one artificial amino acid, said artificial amino acid being preferably selected from the group consisting of a D-enantiomer amino acid, a beta-methyl amino acid, a alpha-substituted alpha-amino acid and an amino acid analog.

[0125] By "beta-methyl amino acid", it is herein meant a derivative of the amino acid alanine with an aminomethyl group on the side chain. This non-proteinogenic amino acid is classified as a polar base.

[0126] By "alpha-substituted alpha-amino acid", it is herein meant that the group on the alpha carbon of an L-amino acid (NH2) has been changed to another, non proteinaceous group, such as a methyl-, aryl- or acyl- group.

[0127] By "amino acid analog", it is herein meant any other artificial analog of a natural amino acid.

[0128] The CD31.sup.shed ligand may thus comprise at least one amino acid in the D-enantiomer form. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 of the amino acids of the CD31.sup.shed ligand defined above may be in the D-enantiomer form.

[0129] In one embodiment, the CD31.sup.shed ligand consists of D-amino acids.

[0130] The CD31.sup.shed ligand may also comprise an inverted sequence, namely an inversion of the amino acid chain (from the C-terminal end to the N-terminal end). The entire amino acid sequence of the peptide may be inverted, or a portion of the amino acid sequence may be inverted. For example, a consecutive sequence of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids may be inverted. Reference herein to `inverted` amino acids refers to inversion of the sequence of consecutive amino acids in the sequence.

[0131] Other chemical modifications include, but are not limited to:

[0132] modifications to the N-terminal and/or C-terminal ends of the peptides such as e.g. N-terminal methylation, N-terminal acylation (preferably acetylation) or deamination, or modification of the C-terminal carboxyl group into an amide or an alcohol group;

[0133] 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;

[0134] 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;

[0135] 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);

[0136] azapeptides, in which one or more alpha carbons are replaced with nitrogen atoms; betapeptides, in which the amino group of one or more amino acid is bonded to the .beta. carbon rather than the a carbon,

[0137] ester linkage(s) (such as .alpha.-hydroxy acid(s)),

[0138] insertion of extra methylene group(s) (for example .beta.- and .gamma.-amino acid(s), and/or

[0139] Peptoid(s), oligourea(s), arylamide(s) and/or oligohydrazide(s).

[0140] The CD31.sup.shed ligand includes amino acids modified either by natural processes, such as post-translational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modification(s) can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini, it will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given polypeptide. Also, a given polypeptide may contain many types of modifications. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from natural post-translational processes or may be made by synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, araidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidyl inositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cystine, formation of pyroglutarnate, formylation, gamma-carboxylation, glycosylation, GP6'I anchor formation, hydroxylation, iodination, mefhylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoyiation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0141] In a preferred embodiment of the invention, the CD31.sup.shed ligand is selected in the group consisting of:

[0142] (i) a peptide consisting of a fragment of 3 to 15 amino acids of the sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1, said fragment comprising the amino acids 579 to 581, the amino acids 589 to 591, the amino acids 599 to 601 and/or the amino acids 593 to 595 of SEQ ID NO: 1,

[0143] (ii) a peptide consisting of a fragment of 3 to 15 amino acids of a sequence corresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1 in a non-human mammalian CD31, for example a fragment of 3 to 15 amino acids of the sequence defined by amino acids 568 to 590 of sequence SEQ ID NO: 9, said fragment preferably comprising the amino acids 568 to 570, the amino acids 578 to 580, the amino acids 588 to 590 and/or the amino acids 582 to 584 of SEQ ID NO: 9,

[0144] (iii) a peptide of 3 to 15 amino acids consisting of a sequence at least 70% identical, preferably at least 75% identical, preferably at least 80% identical, more preferably at least 85% identical, still more preferably at least 90% identical to the sequence of peptide (i),

[0145] (iv) a peptide consisting of a retro-inverso sequence of peptide (i), (ii) or (iii), and

[0146] (v) the peptide (i), (ii), (iii) or (iv) comprising at least one or at least one further chemical modification.

[0147] Such peptide has, for example, a sequence selected from the group consisting of: SSTLAVRVFLAPWKK (SEQ ID NO: 13, amino acids 576 to 590 of SEQ ID NO: 9), STLAVRVFLAPWKK (SEQ ID NO: 14, amino acids 577 to 590 of SEQ ID NO: 9), TLAVRVFLAPWKK (SEQ ID NO: 15, amino acids 578 to 590 of SEQ ID NO: 9), LAVRVFLAPWKK (SEQ ID NO: 16, amino acids 579 to 590 of SEQ ID NO: 9), AVRVFLAPWKK (SEQ ID NO: 17, amino acids 580 to 590 of SEQ ID NO: 9), VRVFLAPWKK (SEQ ID NO: 3, amino acids 581 to 590 of SEQ ID NO: 9), RVFLAPWKK (SEQ ID NO: 18, amino acids 582 to 590 of SEQ ID NO: 9), VFLAPWKK (SEQ ID NO: 19, amino acids 583 to 590 of SEQ ID NO: 9), FLAPWKK (SEQ ID NO: 20, amino acids 584 to 590 of SEQ ID NO: 9), LAPWKK (SEQ ID NO: 2, amino acids 585 to 590 of SEQ ID NO: 9), APWKK (SEQ ID NO: 21, amino acids 586 to 590 of SEQ ID NO: 9), PWKK SEQ ID NO: 22, amino acids 587 to 590 of SEQ ID NO: 9), WKK (amino acids 588 to 590 of SEQ ID NO: 9), SKILTVRVILAPWKK (SEQ ID NO: 23, amino acids 587 to 601 of SEQ ID NO: 1), KILTVRVILAPWKK (SEQ ID NO: 24, amino acids 588 to 601 of SEQ ID NO: 1), ILTVRVILAPWKK (SEQ ID NO: 25, amino acids 589 to 601 of SEQ ID NO: 1), LTVRVILAPWKK (SEQ ID NO: 26, amino acids 590 to 601 of SEQ ID NO: 1), TVRVILAPWKK (SEQ ID NO: 27, amino acids 591 to 601 of SEQ ID NO: 1), VRVILAPWKK (SEQ ID NO: 4, amino acids 592 to 601 of SEQ ID NO: 1), RVILAPWKK (SEQ ID NO: 28, amino acids 593 to 601 of SEQ ID NO: 1), VILAPWKK (SEQ ID NO: 29, amino acids 594 to 601 of SEQ ID NO: 1), ILAPWKK (SEQ ID NO: 30, amino acids 595 to 601 of SEQ ID NO: 1), SSMRTSPRSSTLAVR (SEQ ID NO: 31, amino acids 568 to 582 of SEQ ID NO: 9), SSMRTSPRSSTLAV (SEQ ID NO: 32, amino acids 568 to 581 of SEQ ID NO: 9), SSMRTSPRSSTLA (SEQ ID NO: 33, amino acids 568 to 580 of SEQ ID NO: 9), SSMRTSPRSSTL (SEQ ID NO: 34, amino acids 568 to 579 of SEQ ID NO: 9), SSMRTSPRSST (SEQ ID NO: 35, amino acids 568 to 578 of SEQ ID NO: 9), SSMRTSPRSS (SEQ ID NO: 36, amino acids 568 to 577 of SEQ ID NO: 9), SSMRTSPRS (SEQ ID NO: 37, amino acids 568 to 576 of SEQ ID NO: 9), SSMRTSPR (SEQ ID NO: 38, amino acids 568 to 575 of SEQ ID NO: 9), SSMRTSP (SEQ ID NO: 39, amino acids 568 to 574 of SEQ ID NO: 9), SSMRTS (SEQ ID NO: 40, amino acids 568 to 573 of SEQ ID NO: 9), SSMRT (SEQ ID NO: 41, amino acids 568 to 572 of SEQ ID NO: 9), SSMR (SEQ ID NO: 42, amino acids 568 to 571 of SEQ ID NO: 9), SSM (amino acids 568 to 570 of SEQ ID NO: 9), NHASSVPRSKILTVR (SEQ ID NO: 43, amino acids 579 to 593 of SEQ ID NO: 1), NHASSVPRSKILTV (SEQ ID NO: 44, amino acids 579 to 592 of SEQ ID NO: 1), NHASSVPRSKILT (SEQ ID NO: 45, amino acids 579 to 591 of SEQ ID NO: 1), NHASSVPRSKIL (SEQ ID NO: 46, amino acids 579 to 590 of SEQ ID NO: 1), NHASSVPRSKI (SEQ ID NO: 47, amino acids 579 to 589 of SEQ ID NO: 1), NHASSVPRSK (SEQ ID NO: 48, amino acids 579 to 588 of SEQ ID NO: 1), NHASSVPRS (SEQ ID NO: 49, amino acids 579 to 587 of SEQ ID NO: 1), NHASSVPR (SEQ ID NO: 50, amino acids 579 to 586 of SEQ ID NO: 1), NHASSVP (SEQ ID NO: 51, amino acids 579 to 585 of SEQ ID NO: 1), NHASSV (SEQ ID NO: 52, amino acids 579 to 584 of SEQ ID NO: 1), NHASS (SEQ ID NO: 53, amino acids 579 to 583 of SEQ ID NO: 1), NHAS (SEQ ID NO: 54, amino acids 579 to 582 of SEQ ID NO: 1), NHA (amino acids 579 to 581 of SEQ ID NO: 1), TSPRSSTLAVRVFLA (SEQ ID NO: 55, amino acids 572 to 586 of SEQ ID NO: 9), SPRSSTLAVRVFL (SEQ ID NO: 56, amino acids 573 to 585 of SEQ ID NO: 9), PRSSTLAVRVF (SEQ ID NO: 57, amino acids 574 to 584 of SEQ ID NO: 9), RSSTLAVRV (SEQ ID NO: 58, amino acids 575 to 583 of SEQ ID NO: 9), SSTLAVR (SEQ ID NO: 59, amino acids 576 to 582 of SEQ ID NO: 9), STLAV (SEQ ID NO: 60, amino acids 577 to 581 of SEQ ID NO: 9), TLA (amino acids 578 to 580 of SEQ ID NO: 9), SVPRSKILTVRVILA (SEQ ID NO: 61, amino acids 583 to 597 of SEQ ID NO: 1), VPRSKILTVRVIL (SEQ ID NO: 62, amino acids 584 to 596 of SEQ ID NO: 1), PRSKILTVRVI (SEQ ID NO: 63, amino acids 585 to 595 of SEQ ID NO: 1), RSKILTVRV (SEQ ID NO: 64, amino acids 586 to 594 of SEQ ID NO: 1), SKILTVR (SEQ ID NO: 65, amino acids 587 to 593 of SEQ ID NO: 1), KILTV (SEQ ID NO: 66, amino acids 588 to 562 of SEQ ID NO: 1), ILT (amino acids 589 to 591 of SEQ ID NO: 1), RVF (amino acids 582 to 584 of SEQ ID NO: 9), RVFL (SEQ ID NO: 67, amino acids 582 to 585 of SEQ ID NO: 9), RVFLA (SEQ ID NO: 68, amino acids 582 to 586 of SEQ ID NO: 9), RVFLAP (SEQ ID NO: 69, amino acids 582 to 587 of SEQ ID NO: 9), RVFLAPW (SEQ ID NO: 70, amino acids 582 to 588 of SEQ ID NO: 9), RVFLAPWK (SEQ ID NO: 5, amino acids 582 to 589 of SEQ ID NO: 9), RVI (amino acids 593 to 595 of SEQ ID NO: 1), RVIL (SEQ ID NO: 71, amino acids 593 to 596 of SEQ ID NO: 1), RVILA (SEQ ID NO: 72, amino acids 593 to 597 of SEQ ID NO: 1), RVILAP (SEQ ID NO: 73, amino acids 593 to 598 of SEQ ID NO: 1), RVILAPW (SEQ ID NO: 74, amino acids 593 to 599 of SEQ ID NO: 1), RVILAPWK (SEQ ID NO: 7, amino acids 593 to 600 of SEQ ID NO: 1).

[0148] In a more preferred embodiment of the invention, the CD31.sup.shed ligand is selected in the group consisting of:

[0149] (i) a peptide consisting of a fragment of 3 to 15 amino acids of the sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1, said fragment comprising the amino acids 579 to 582, the amino acids 588 to 592, the amino acids 598 to 601 and/or the amino acids 593 to 595 of SEQ ID NO: 1,

[0150] (ii) a peptide consisting of a fragment of 3 to 15 amino acids of a sequence corresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1 in a non-human mammalian CD31, for example a fragment of 3 to 15 amino acids of the sequence defined by amino acids 568 to 590 of sequence SEQ ID NO: 9, said fragment preferably comprising the amino acids 568 to 571, the amino acids 578 to 580 the amino acids 587 to 590 and/or the amino acids 582 to 584 of SEQ ID NO: 9,

[0151] (iii) a peptide of 3 to 15 amino acids consisting of a sequence at least 70% identical, preferably at least 75% identical, preferably at least 80% identical, more preferably at least 85% identical, still more preferably at least 90% identical to the sequence of peptide (i),

[0152] (iv) a peptide consisting of a retro-inverso sequence of peptide (i), (ii) or (iii), and

[0153] (v) the peptide (i), (ii), (iii) or (iv) comprising at least one or at least one further chemical modification.

[0154] In a preferred embodiment, the CD31.sup.shed ligand is an 8 amino-acid fragment comprising inversions and/or at least one unnatural amino acid, such as at least one D-amino acids. Such peptides indeed retain the activity of the original peptide or even demonstrate improved activity. Incorporation of unnatural amino acids in peptides intended for therapeutic use is of utility in increasing the stability of the peptide, in particular in vivo stability.

[0155] In another preferred embodiment of the invention, the CD31.sup.shed ligand is selected in the group consisting of a peptide of sequence SEQ ID NO: 2, a peptide of sequence SEQ ID NO: 3, a peptide of sequence SEQ ID NO: 4, a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids, a peptide of sequence SEQ ID NO: 7 and a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomer amino acids.

[0156] A more preferred CD31.sup.shed ligand is a peptide of sequence SEQ ID NO: 5 or a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids.

[0157] The CD31.sup.shed ligand may additionally comprise at least one chelating agent.

[0158] A chelating agent is a molecule covalently bound to the ligand, which allows complexing radiometal(s).

[0159] Typically, chelating agents could be: 6-Hydrazinopyridine-3-carboxylic acid (HYNIC), chelating peptide such as Gly-Gly-Cys or His-based sequence (Francesconi 2004, Waibel 1999, Ali 2011), MAG3 en N-ter (Okarvi 2012, 2004), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA), diethylene triamine penta-acetic acid (DTPA), 1,4,7-tris(carboxymethylaza)cyclododecane-10-azaacetylamide (DO3A), nitrilotriacetic acid (NTA), D-penicillamine, 2,3-dimercaptosuccinic acid,2,3-dimercapto-1-propanesulfonic acid, 2,3-dimercaptopropanol (BAL), triethylenetetramine, ammonium tetrathiomolybdate anion, ethylenediaminetetraacetic acid (EDTA), 2-(p-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (IB4M) or hydroxypyridinone (HOPO).

[0160] The CD31.sup.shed ligand may be linked to the chelating agent directly (for example the chelating agent being attached to a lateral amino acid long chain) or indirectly, for example via at least one spacer.

[0161] A preferred chelating agent is 6-Hydrazinopyridine-3-carboxylic acid (HYNIC).

[0162] In such case, the peptide of the CD31.sup.shed ligand may be linked to HYNIC directly (for example HYNIC being attached to a lateral amino acid long chain) or indirectly, for example via at least one spacer.

[0163] The spacer for example comprises or consists of at least one PEG (polyethylene glycol), for example one, two or at least three PEG, more preferably three PEG and/or at least one aliphatic spacer.

[0164] A preferred CD31.sup.shed ligand is a compound of the following formula:

##STR00001##

[0165] This compound is also called HYNIC-P8RI in the following.

[0166] The CD31.sup.shed ligand may be prepared by any well-known procedure in the art, such as chemical synthesis, for example solid phase synthesis or 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. 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.

[0167] 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), CI-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).

[0168] Alternatively, the CD31.sup.shed ligand may be synthesized using recombinant techniques.

[0169] The method of producing the CD31.sup.shed ligand may optionally comprise the steps of purifying said CD31.sup.shed ligand, chemically modifying said CD31.sup.shed ligand, and/or formulating said CD31.sup.shed ligand into a pharmaceutical composition.

[0170] In an embodiment, the CD31.sup.shed ligand is a peptidomimetic of a peptide as defined above, i.e. a compound that mimics said peptide.

[0171] A peptidomimetic is a compound consisting of or essentially consisting of non-peptidic structural elements that mimics a given peptide, thereby conferring to said compound a biological activity equal to or similar to said peptide.

[0172] The peptidomimetic is preferably soluble in an organic or nonorganic solvent.

[0173] As the peptide previously, the peptidomimetic is preferably soluble in water.

[0174] 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).

[0175] Imaging Label

[0176] The labeled CD31.sup.shed ligand according to the invention comprises at least one imaging label.

[0177] Said imaging label may be a radionucleide or a contrastophor.

[0178] The term "contrastophor" as used herein refers to a contrast agent, for example comprising a chelating agent.

[0179] Examples of chelating agent that may be used in a contrastophor are given below in the section "CD31.sup.shed ligand".

[0180] Non-limitative examples of contrastophor are Gd-DTPA (complex of gadolinium and DTPA) or Gd-DOTA (complex of gadolinium and DOTA).

[0181] A contrastophor is preferably used in MRI (also called thereafter "IRM" or "Magnetic Resonance Imaging").

[0182] The term "radionuclide" as used herein has the same meaning as radioactive nuclide, radioisotope or radioactive isotope.

[0183] The radionuclide is preferably detectable by nuclear medicine molecular imaging technique(s), such as, Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), an hybrid of SPECT and/or PET or their combinations.

[0184] Single Photon Emission Computed Tomography (SPECT) herein includes planar scintigraphy (PS).

[0185] An hybrid of SPECT and/or PET is for example SPECT/CT, PET/CT, PET/IRM or SPECT/IRM.

[0186] SPECT and PET acquire information on the concentration (or uptake) of radionuclides introduced into a subject's body. PET generates images by detecting pairs of gamma rays emitted indirectly by a positron-emitting radionuclide. A PET analysis results in a series of thin slice images of the body over the region of interest (e.g., brain, breast, liver, . . . ). These thin slice images can be assembled into a three dimensional representation of the examined area. SPECT is similar to PET, but the radioactive substances used in SPECT have longer decay times than those used in PET and emit single instead of double gamma rays. Although SPECT images exhibit less sensitivity and are less detailed than PET images, the SPECT technique is much less expensive than PET and offers the advantage of not requiring the proximity of a particle accelerator. Actual clinical PET presents higher sensitivity and better spatial resolution than SPECT, and presents the advantage of an accurate attenuation correction due to the high energy of photons; so PET provides more accurate quantitative data than SPECT. Planar scintigraphy (PS) is similar to SPECT in that it uses the same radionuclides. However, PS only generates 2D-information.

[0187] SPECT produces computer-generated images of local radiotracer uptake, while CT produces 3-D anatomic images of X ray density of the human body. Combined SPECT/CT imaging provides sequentially functional information from SPECT and the anatomic information from CT, obtained during a single examination. CT data are also used for rapid and optimal attenuation correction of the single photon emission data. By precisely localizing areas of abnormal and/or physiological tracer uptake, SPECT/CT improves sensitivity and specificity, but can also aid in achieving accurate dosimetric estimates as well as in guiding interventional procedures or in better defining the target volume for external beam radiation therapy. Gamma camera imaging with single photon emitting radiotracers represents the majority of procedures.

[0188] In a preferred embodiment, the radionuclide is detectable by SPECT or an hybrid SPECT/CT.

[0189] The radionuclide may be selected in the group consisting of technetium-99m (.sup.99mTc), gallium-67 (.sup.67Ga), gallium-68 (.sup.68Ga) yttrium-90 (.sup.90Y), indium-111 (.sup.111In), rhenium-186 (.sup.186RE), flourine-18 (.sup.18F), copper-64 (.sup.64Cu) or thallium-201 (.sup.201TI).

[0190] A preferred radionuclide is technetium-99m (99mTc).

[0191] Labeled CD31.sup.shed Ligand

[0192] The present invention thus particularly relates to a labeled CD31.sup.shed ligand comprising a CD31.sup.shed ligand and at least one imaging label.

[0193] The CD31.sup.shed ligand and the imaging label are as defined above in the sections of the same name.

[0194] The radiochemical specific activity of labeled CD31.sup.shed ligand is preferably greater than 70 GBq/pmol, more preferably greater than 80 GBq/pmol, more preferably greater than 90 GBq/pmol, for example greater than 100 GBq/pmol.

[0195] The labeled CD31.sup.shed ligand is preferably stable.

[0196] The stability of the labeled CD31.sup.shed ligand may be assessed in vitro and/or in vivo by any method well known by the skilled person, for example by determining radiochemical purity (RCP).

[0197] The radiochemical purity (RCP) may be assessed by high performance liquid chromatography (HPLC) and/or instant thin layer chromatography (ITLC), preferably using both methods.

[0198] For example, the in vitro stability may be assessed after incubation of the labeled CD31.sup.shed ligand in vitro in plasma, preferably in human plasma at 37.degree. C., for example for at least 1 hour, at least 2 hours, at least 3 hours or at least 4 hours.

[0199] For example, the in vivo stability may be assessed in a sample of human or non-human mammal urine, for example of rat urine, after administration of the labeled CD31.sup.shed ligand in said human or non-human mammal.

[0200] The in vitro and/or in vivo RCP of the labeled CD31.sup.shed ligand is preferably greater than 80%, preferably greater than 85%, for example greater than 89%.

[0201] The biodistribution and specific uptake of the labeled CD31.sup.shed ligand by activated platelets, endothelial cells and leukocytes may be assessed in an inflammation model, for example in a rat model of heart inflammation, brain inflammation or vascular inflammation, such as the experimental abdominal aorta aneurysm (AAA) induced by local infusion of elastase and followed by intravenous injection of a periodontal bacterium present in human AAA and known to induce AAA inflammation. For example, immediately after intravenous injection of the labeled CD31.sup.shed ligand, sequential whole-body acquisitions (for example every 10 minutes for the first hour) are performed, for example using a hybrid SPECT/CT camera (NanoSPECT/CT, Bioscan Inc.).

[0202] The binding of the labeled CD31.sup.shed ligand to plasma proteins is preferably low, for example lower than 10%, more preferably lower than 5% after 4 hours of incubation in human plasma at 37.degree. C.

[0203] The low binding to plasma proteins may be assessed by any method well known by the skilled person, such as size exclusion chromatography, for example after incubation of the labeled CD31.sup.shed ligand in vitro in plasma, preferably in human plasma, at 37.degree. C., for example for at least 1 hour, at least 2 hours, at least 3 hours or at least 4 hours.

[0204] The CD31.sup.shed ligand and the imaging label may be linked covalently or non-covalently.

[0205] The CD31.sup.shed ligand and the imaging label are preferably linked non-covalently.

[0206] When the CD31.sup.shed ligand comprises a chelating agent, the CD31.sup.shed ligand is preferably linked to the imaging label via said chelating agent, and preferably non covalently.

[0207] The labeled CD31.sup.shed ligand is preferably a radiolabeled CD31.sup.shed ligand.

[0208] A preferred labeled CD31.sup.shed ligand thus comprises a CD31.sup.shed ligand and at least one radionuclide.

[0209] In one preferred embodiment of the invention, the labeled CD31.sup.shed ligand comprises a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids as CD31.sup.shed ligand and .sup.99mTc as a radionuclide.

[0210] The labeled CD31.sup.shed ligand may additionally comprise at least one co-ligand, i.e. one or more ligands coupled to the CD31.sup.shed ligand,

[0211] The co-ligand(s) is/are preferably used to stabilize the imaging label binding to the CD31.sup.shed ligand.

[0212] For example, the co-ligand may be tricine, EDDA (ethylendiaminediacetic acid) (EDDA), aminocarboxylate, phosphine or pyridine.

[0213] In on embodiment, the labeled CD31.sup.shed ligand comprises both tricine and EDDA as co-ligands, for example when the radionuclide is 99mTc.

[0214] When at least one co-ligand is used, the labeled CD31.sup.shed ligand comprises or consists of a complex comprising said co-ligand(s), said CD31.sup.shed ligand and said radionuclide.

[0215] In one embodiment of the invention, the CD31.sup.shed ligand is the peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids coupled with HYNIC via a spacer as defined above.

[0216] A preferred labeled CD31.sup.shed ligand, referred to as 99mTc-HYNIC-P8RI, comprises:

[0217] the compound of formula:

##STR00002##

[0218] and

[0219] the radionuclide 99mTc.

[0220] HYNIC forms a 99mTc-N bond between technetium and the hydrazine moiety of HYNIC.

[0221] The labeled CD31.sup.shed ligand can be prepared by conventional methods perfectly known by a man skilled in the art.

[0222] In the methods and uses described herein, the labeled CD31.sup.shed ligand may be used per se or as a pharmaceutical composition as defined below.

[0223] The methods and uses described herein may be performed by using a kit as defined below.

[0224] Labeled CD31.sup.shed Ligand for Use as a Molecular Imaging Agent

[0225] The present invention also relates to the use of a labeled CD31.sup.shed ligand as defined above as a molecular imaging agent for imaging inflammation sites.

[0226] The present invention thus also relates to a labeled CD31.sup.shed ligand as defined above for use as a molecular imaging agent for imaging of inflammatory sites.

[0227] The present invention particularly relates to a labeled CD31.sup.shed ligand as defined above for use as a molecular imaging agent in an in vivo method of detecting, more particularly imaging, inflammatory sites.

[0228] In particular, the present invention relates to a labeled CD31.sup.shed ligand for use as defined above, in a method, preferably an in vivo method, for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition.

[0229] The invention also relates to a labeled CD31.sup.shed ligand for use as defined above, wherein the presence, localization and/or amount of CD31.sup.shed is determined. The labeled CD31.sup.shed ligand indeed binds to CD31.sup.shed.

[0230] The presence, localization and/or amount of CD31.sup.shed is determined by the presence, localization and/or amount of the labeled CD31.sup.shed ligand, i.e. of the signal detected corresponding to the imaging label of said labeled CD31.sup.shed ligand.

[0231] The presence, localization and/or amount of the labeled CD31.sup.shed ligand may be determined by planar scintigraphy (PS), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), an hybrid SPECT/CT or their combinations.

[0232] The labeled CD31.sup.shed ligand is preferably used or administered intravenously.

[0233] The labeled CD31.sup.shed ligand is preferably used or administered in an amount sufficient to obtain a detectable signal, in particular one injected intravenously.

[0234] The signal corresponding to the imaging label of the labeled CD31.sup.shed ligand is preferably detected immediately after the administration of the labeled CD31.sup.shed ligand.

[0235] The signal may be detected in the entire body of the subject or in only one part of the subject (particularly in only one part of the body of the subject).

[0236] The present invention thus particularly relates to a labeled CD31.sup.shed ligand as defined above for use in a method for detecting, more particularly imaging, inflammation sites, wherein said method comprises:

[0237] administering said labeled CD31.sup.shed ligand to the subject, and

[0238] imaging said subject or at least one part of the body of said subject, thereby detecting the binding of the labeled CD31.sup.shed ligand to CD31.sup.shed in said subject or in said part of the body of the subject.

[0239] The present invention thus particularly relates to a labeled CD31.sup.shed ligand as defined above for use in a method for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition, wherein said method comprises:

[0240] administering said labeled CD31.sup.shed ligand to the subject, and

[0241] imaging said subject or at least one part of the body of said subject, thereby detecting the binding of the labeled CD31.sup.shed ligand to CD31.sup.shed in said subject or in said part of the body of the subject.

[0242] The molecular imaging agent of the invention represents a powerful tool for diagnosing or assessing an inflammatory condition associated with CD31.sup.shed.

[0243] a) Determining if a subject suffers from an inflammatory or is at risk of suffering from an inflammatory condition

[0244] In a method for determining whether a subject suffers from an inflammatory condition or is at risk of suffering from an inflammatory condition, the subject to be diagnosed is suspected to suffer or be likely to suffer from an inflammatory condition or suffers from an inflammatory condition. The method can indeed be performed to confirm that the subject suffers from an inflammatory condition and/or to specify the kind of inflammatory condition.

[0245] The presence, localization and/or amount of CD31.sup.shed may indicate that the subject suffers from an inflammatory condition.

[0246] The localization of CD31.sup.shed may further indicate the kind of inflammatory condition.

[0247] The amount of CD31.sup.shed may further indicate the severity of the inflammation condition.

[0248] The presence, localization and/or amount of CD31.sup.shed may be compared to a reference value or a control biological image. When the reference value or the control biological sample corresponds to a healthy subject, in particular a subject that does not suffer from an inflammatory condition or a panel of healthy subjects, a presence, localization and/or amount of CD31.sup.shed detected in the biological sample that is greater than those of reference value or of the control image indicates that the subject suffers or, is at risk of having an inflammatory condition.

[0249] b) Determining the risk of recurrence of an inflammatory condition after an anti-inflammatory treatment

[0250] The methods and uses according to the invention can also be used to follow the risk of recurrence of an inflammatory condition after an anti-inflammatory treatment. In particular, they can be used to monitor the subject after an anti-inflammatory treatment. For example, this can be achieved by repeating the method at least one time, preferably two times after the end of said treatment, in order to determine the presence, localization and/or amount of CD31.sup.shed. An increased presence, localization and/or amount of CD31.sup.shed may indicate a high risk of recurrence of an inflammatory condition after an anti-inflammatory treatment. A stable or a decreased presence, localization and/or amount of CD31.sup.shed may indicate a low risk of recurrence of an inflammatory condition after an anti-inflammatory treatment. For example, an image of a biological sample and/or of the subject or at least one part of the subject is generated at the end of the treatment and at least one time after treatment. Comparison of the images at the end and after treatment allows the risk of recurrence of an inflammatory condition after an anti-inflammatory treatment to be monitored.

[0251] c) Monitoring the response to a treatment of a subject suffering from an inflammatory condition

[0252] The methods and uses according to the invention can also be used to monitor the response to a treatment of a subject suffering from an inflammatory condition. For example, this can be achieved by repeating the method at least two times, for example one time before treatment and at least two times during the treatment, or at least two times during the treatment. For example, an image of a biological sample and/or of the subject or at least one part of the subject is generated before treatment and at least one time during the treatment, for example with an anti-inflammatory treatment. Comparison of the images before and during treatment allows the response of the subject to that particular treatment to be monitored.

[0253] An increased presence, localization and/or amount of CD31.sup.shed may indicate that the treatment is not or not any more efficient. A stable or a decreased presence, localization and/or amount of CD31.sup.shed may indicate that the treatment is efficient.

[0254] The expression "monitoring the response to a treatment" and "monitoring the efficacy of a treatment" are herein synonymous.

[0255] Said treatment may be a curative or preventive treatment.

[0256] Said treatment may comprise or consist in administering at least one anti-inflammatory agent, at least one immunosuppressant, at least one probiotic (i.e. live microorganisms that may confer a health benefit on the subject), at least one antibiotic or their combinations.

[0257] Method Using a Labeled CD31.sup.shed Ligand as a Molecular Imaging Agent

[0258] The present invention also relates to a method for imaging of inflammation sites using a labeled CD31.sup.shed ligand as defined above.

[0259] The present invention also relates to a method, preferably an in vitro method, for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition in a subject, wherein said method comprises detecting the presence of CD31.sup.shed on the surface of cells with a labeled CD31.sup.shed ligand as defined above in a biological sample of the subject.

[0260] The cells are preferably platelets, leukocytes and/or endothelial cells.

[0261] By "biological sample", it is herein meant any sample able to contain endothelial cells, platelets or leukocytes, such as a blood sample or fraction thereof (for example a thrombus) or a tissue sample, for example a biopsy.

[0262] The method may comprise a first step of providing a biological sample from the subject.

[0263] The present invention also relates to a method as defined above, wherein the detection of CD31.sup.shed on the surface of the cells in said biological sample indicates that the subject suffers, is at risk of having or developing an inflammatory condition or is at risk of recurrence of an inflammatory condition.

[0264] The present invention also relates to a method as defined above, wherein the amount of CD31.sup.shed detected on the surface of the cells in said biological sample is compared to a reference value or a control biological sample. When the reference value or the control biological sample corresponds to a healthy subject, in particular a subject that does not suffer from an inflammatory condition or a panel of healthy subjects, an amount of CD31.sup.shed detected in the biological sample that is greater than those of reference value or of the control biological sample indicates that the subject suffers, is at risk of having or developing an inflammatory condition or is at risk of recurrence of an inflammatory condition.

[0265] The present invention also relates to a method as defined above, wherein an increased amount of CD31.sup.shed detected on the surface of the cells in said biological sample by comparison to the amount detected in a biological sample before treatment or earlier during treatment indicates that said treatment is not efficient and/or wherein a stable or a decreased amount of CD31.sup.shed detected on the surface of the cells in said biological sample by comparison to the amount detected in a biological sample before treatment or earlier during treatment indicates that said treatment is efficient.

[0266] Said method may further comprise determining the amount of CD31.sup.shed on the surface of cells.

[0267] The detection of CD31.sup.shed on the surface of the cells in said biological sample is preferably carried out by imaging techniques.

[0268] The step of detection as mentioned above comprises contacting the biological sample of the subject with a labeled CD31.sup.shed ligand, preferably an effective amount of a labeled CD31.sup.shed ligand, as a molecular imaging agent.

[0269] The present invention also relates to a method as defined above, wherein said method comprises:

[0270] contacting a biological sample of the subject with a labeled CD31.sup.shed ligand, and

[0271] detecting the labeled CD31.sup.shed ligand bound to CD31.sup.shed, thereby detecting the presence of CD31.sup.shed on the surface of cells in the biological sample.

[0272] The contact is preferably carried out under conditions that allow the molecular imaging agent (1) to reach the cells of the subject that may express CD31.sup.shed (preferably endothelial cells, platelets and/or leukocyte) and (2) to interact with such CD31.sup.shed, so that the interaction results in the binding of the molecular imaging agent to the CD31.sup.shed. After contact with the labeled CD31.sup.shed ligand and after sufficient time has elapsed for the interaction to take place, the molecular imaging agent bound to CD31.sup.shed present in the subject sample is detected by a molecular imaging technique as disclosed above.

[0273] An "effective amount" is an amount sufficient to allow the molecular imaging agent to complete these three conditions of (1) reaching the cells, (2) interacting with CD31.sup.shed and (3) being detected.

[0274] Method of Treatment of an Inflammation Condition

[0275] The present invention also relates to a method for preventing and/or treating an inflammatory condition in a subject in need thereof, wherein said method comprises,

[0276] performing a method as defined above for determining whether a subject suffers from an inflammatory condition, is at risk of having an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or for monitoring the efficacy of a treatment of an inflammatory condition in a subject,

[0277] when the subject is determined to suffer from an inflammatory condition, to be at risk of having an inflammatory condition or to be at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment or if the treatment is not efficient, administering to said subject a suitable treatment.

[0278] Said suitable treatment may comprise or consist in administering at least one anti-inflammatory agent, at least one immunosuppressant, at least one probiotic (i.e. live microorganisms that may confer a health benefit on the subject), at least one antibiotic, at least one CD31.sup.shed ligand linked to an active ingredient or their combinations.

[0279] The present invention also relates to a method for preventing and/or treating an inflammatory condition in a subject in need thereof, wherein said method comprises administering to said subject a CD31.sup.shed ligand linked to an active ingredient.

[0280] The active ingredient is for example a drug, such as an anti-inflammatory agent. The CD31.sup.shed ligand is thereby used as a drug targeting agent and allows improving the effect of said drug.

[0281] Pharmaceutical Composition Comprising the Molecular Imaging Agent

[0282] The pharmaceutical composition according to the invention comprises at least one labeled CD31.sup.shed ligand and at least one pharmaceutically acceptable carrier.

[0283] As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredient, i.e. the molecular imaging agent, and which is not excessively toxic to the subject at the concentration(s) at which it is administered. This term includes solvent(s), dispersion medium/media, coating(s), antibacterial and/or antifungal agent(s), isotonic agent(s), adsorption delaying agent(s) and their combinations. The use of such medium/media and/or agent(s) for pharmaceutically active substance(s) is well known in the art.

[0284] The pharmaceutical composition may be administered by injection. For administration by injection, the pharmaceutical composition comprising the molecular imaging agent may be formulated as sterile aqueous or non-aqueous solution or alternatively as sterile powder for the extemporaneous preparation of a sterile injectable solution. The pharmaceutical composition should be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0285] Pharmaceutically acceptable carrier(s) for administration by injection are solvent(s) or dispersion medium/media, such as aqueous solution(s) (e.g., Hank's solution, alcoholic/aqueous solution or saline solution), and non-aqueous carrier(s) (e.g. propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate). The injectable pharmaceutical composition may also contain parenteral vehicle(s) (such as sodium chloride and Ringer's dextrose) and/or intravenous vehicle(s) (such as fluid and nutrient replenishers); as well as other conventional, pharmaceutically acceptable, non-toxic excipient(s) and additive(s) including salt(s), buffer(s) and preservative(s) such as antibacterial and/or antifungal agent(s) (e.g. parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like). Prolonged absorption of the injectable composition can be brought about by adding agents that can delay absorption (e.g. aluminum monostearate and/or gelatin). The pH and concentration of the various components can readily be determined by those skilled in the art.

[0286] The sterile injectable solution may be prepared by incorporating the active compound(s), i.e. the molecular imaging agent, and other ingredient(s) in the required amount of an appropriate solvent and then by sterilizing the resulting mixture, for example, by filtration and/or irradiation.

[0287] In general, the dosage of the molecular imaging agent (or pharmaceutical composition comprising thereof) will vary depending on considerations such as age, sex and weight of the subject, as well as the particular inflammatory condition suspected to affect the patient, the extent of the disease, the tissue(s) of the body to be examined and/or the sensitivity of the imaging label. Factors such as contraindications, therapies, and other variables are also to be taken into account to adjust the dosage of molecular imaging agent to be administered. This, however, can be readily achieved by a trained physician.

[0288] In general, one suitable dose of molecular imaging agent, i.e. of labeled CD31.sup.shed ligand, or a pharmaceutical composition comprising thereof, corresponds to the lowest amount of molecular imaging agent or pharmaceutical composition that is sufficient to allow molecular imaging of any relevant CD31.sup.shed present in the subject. To minimize this dose, it is preferred that administration be intravenous, intramuscular, intraperitoneal or subcutaneous, and preferably proximal to the site to be examined.

[0289] Kits

[0290] The present invention also provides a kit comprising material(s) useful for carrying out the methods and uses of the invention.

[0291] In certain embodiments, the kit comprises at least one CD31.sup.shed ligand as above described and at least one imaging label, preferably at least one radionuclide, and, optionally, instructions for associating said CD31.sup.shed ligand and said imaging label, preferably radionuclide, to form a labeled CD31.sup.shed ligand according to the invention.

[0292] The radionuclide is preferably a short-lived radionuclide, such as technetium-99m (99mTc), gallium-67 (67Ga), yttrium-90 (90Y), indium-111 (111In), rhenium-186 (186Re), and thallium-201 (201TI), more preferably 99mTc.

[0293] In addition, the kit may further comprise at least one co-ligand, such as tricine and/or ethylendiaminediacetic acid (EDDA).

[0294] In addition, the kit may further comprise one or more of: labelling buffer, labelling reagent, purification buffer, purification reagent, purification means, injection medium, and/or injection reagent. Protocols for using these buffer(s), reagent(s) and/or means for performing different steps of the preparation procedure and/or administration may be included in the kit.

[0295] The different components included in the kit may be supplied in a solid (e.g. lyophilized) or liquid form.

[0296] The kit may optionally comprise different containers (e.g. vial, ampoule, test tube, flask or bottle) for each individual component. Each component will generally be suitable as aliquoted in its respective container or provided in a concentrated form. Other containers suitable for conducting certain steps of the preparation methods may also be provided. The individual containers of the kit are preferably maintained in close confinement for commercial sale.

[0297] In certain embodiments, the kit further comprises instructions for using its components for the imaging of an inflammatory condition as described herein, and in particular for determining whether a subject suffers from, is at risk of having or developing an inflammatory condition or is at risk of recurrence of an inflammatory condition after an anti-inflammatory treatment, as described herein.

[0298] Instructions for using the kit according to the invention may comprise instructions for preparing labeled CD31.sup.shed ligand from the CD31.sup.shed ligand and the imaging label, instructions concerning dosage and mode of administration of the molecular imaging agent thereby obtained, instructions for performing the detection of CD31.sup.shed, and/or instructions for interpreting the results obtained. A kit may also contain a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products.

[0299] The present invention will be further illustrated in view of the following examples and figures.

[0300] 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.

BRIEF DESCRIPTION OF THE FIGURES

[0301] FIG. 1: Radio high performance liquid chromatography (HPLC) of 99mTc-HYNIC-P8RI with tricine (A) and tricine/EDDA as coligands (B). HPLC analysis of the acetonitrile fraction shows a major specie (Tr=10.1 min) with tricine/EDDA contrary to the multiple species profile with tricine.

[0302] FIG. 2: Radio-HPLC stability study of 99mTc-HYNIC-P8RI with tricine/EDDA as coligands. A: plasma stability after 4 h of incubation in human plasma. B: Radio-HPLC chromatogram of urine collected in a rat bladder 1 hour after injection of 74 MBq of 99mTc-HYNIC-P8RI with tricine/EDDA as coligands. For A and B, peak in the acetonitrile fraction (Tr=10.1 min) corresponds to the initial specie with no signs of degradation.

[0303] FIG. 3: SPECT/CT images of [99mTc]EDDA/HYNIC-P8RI in a male wistar rat with an abdominal aortic aneurysm (AAA) receiving injection of Porphyromonas gingivalis weekly. Images were acquired 30 min after a tracer injection of 74 MBq and 21 days after AAA surgery. The axial (A), coronal (B) and sagittal (C) planes of the abdominal region are shown. It is observed the tracer elimination via kidneys (white arrows in B) and the bladder (white arrow in C). Focal uptake of [99mTc]EDDA/HYNIC-P8RI can be observed at the location of the AAA (black arrows in A, B, C).

[0304] FIG. 4: Comparison of average counts ratio between right hindlimb and left hindlimb measured in the same region of the thigh for technetium-99m radiolabeled mertiatide (n=3) and P8RI injected rats (n=3), in a model of right hindlimb inflammation. Briefly, turpentine oil was injected intramuscularly in Wistar rats right hindlimb and the contralateral thight was injected similarly and simultaneously with saline. SPECT/CT acquisitions were performed 48 hours later and 30 min after injection of 80 MBq of [99mTc]-HYNIC-PEGS-P8RI or [99mTc]-mertiatide. Comparison between the two groups was realized using a Mann-Withney U test, a p value of less than 0.05 was considered significant.

BRIEF DESCRIPTION OF THE SEQUENCES

[0305] SEQ ID NO: 1 corresponds to the sequence of human CD31.

[0306] SEQ ID NO: 2 corresponds to the sequence LAPWKK of a 6 amino acid peptide derived from human or murine CD31.

[0307] SEQ ID NO: 3 corresponds to the sequence VRVFLAPWKK of a 10 amino acid peptide derived from murine CD31, also called PepReg CD31.

[0308] SEQ ID NO: 4 corresponds to the sequence VRVILAPWKK of a 10 amino acid peptide derived from human CD31.

[0309] SEQ ID NO: 5 corresponds to the sequence RVFLAPWK of a 8 amino acid peptide derived from murine CD31, also called P8F.

[0310] SEQ ID NO: 6 corresponds to the sequence kwpalfvr of a 8 amino acid peptide, also called P8RI, having the inverted sequence of SEQ ID NO: 5 and consisting of D-amino acids.

[0311] SEQ ID NO: 7 corresponds to the sequence RVILAPWK of a 8 amino acid peptide derived from human CD31.

[0312] SEQ ID NO: 8 corresponds to the sequence kwpalivr of a 8 amino acid peptide having the inverted sequence of SEQ ID NO: 7 and consisting of D-amino acids.

[0313] SEQ ID NO: 9 corresponds to the sequence of murine CD31.

[0314] SEQ ID NO: 10 corresponds to the sequence of bovine CD31.

[0315] SEQ ID NO: 11 corresponds to the sequence of pig CD31.

[0316] SEQ ID NO: 12 corresponds to the amino acids 579 to 601 of sequence SEQ ID NO: 1.

[0317] Sequences SEQ ID NO: 13 to 74 are as defined above.

EXAMPLES

Materials and Methods

1. Materials

[0318] Reagents were purchased from Sigma-Aldrich Corporation except when otherwise stated and used as received. P8RI (H-kwpalfvr-OH) was synthetized on solid phase with a purity>85% analyzed by RP-HPLC/MS. HYNIC- P8RI was prepared by solid phase peptide synthesis using Fmoc chemistry (yield after RP-HPLC purification 52%, purity by RP-HPLC 96%).

Na.sup.99mTcO.sub.4.sup.- was obtained from a commercial .sup.99Mo/.sup.99mTc generator (TEKCIS.RTM., Iba molecular, France).

2. Analytical Methods

2.1 HPLC

[0319] A Dionex Ultimate 3000 system coupled to a Berthold radiometric detector was used for RP-HPLC analysis. A ACE 3 C18, 3 .mu., 100 .ANG., 150.times.4.6 mm column, at a flow rate of 1 mUmin and with UV detection at 220 nm, was employed with the following mobile phases, A: 0.1% TFA/water), B Acetonitrile (ACN). Gradient was: 0-2 min 23% B, 2-20 min 23-50% B, 20-23 min 50-100% B, 23-25 min 100-23% B, 25-28 min 23% B.

2.2 Thin Layer Chromatography

[0320] TLC was performed using a radiochromatogaph (MiniGita, Raytest, Germany). Stationary phase was silica gel (ITLC-SG, Agilent technologies, USA) and different mobile phases were employed. MEK was used to determine the amount of free .sup.99mTcO.sub.4.sup.- (Rf=1), Anticoagulant Citrate Dextrose Solution (ACD-A, Baxter International, USA) to determine non-peptide-bound .sup.99mTc-coligands and .sup.99mTcO.sub.4.sup.- (Rf=1), 60% ACN for .sup.99mTc-colloid (Rf =0)

3. .sup.99mTc Radiolabeling

3.1 Tricine as Coligand

[0321] In a rubber-sealed N2 purged vial 20 .mu.g of HYNIC- P8RI were incubated with 500 .mu.L of a tricine solution (40 mg/mL in PBS 1.times. buffer pH 7.2), 80 .mu.L of a tin(II) solution (1 mg/mL in HCl 0.1N), 1 GBq .sup.99mTcO4- eluate and PBS qs a 3 mL total volume for 30 min at room temperature (RT).

3.2 EDDA as Coligand

[0322] In a rubber-sealed N2 purged vial 20 .mu.g of HYNIC- P8RI were incubated with 500 .mu.L of a EDDA solution (20 mg/mL in NaOH 0.1N), 80 .mu.L of a tin(II) solution (1 mg/mL in HCl 0.1N), 1 GBq .sup.99mTcO4.sup.- eluate and PBS 1.times. qs a 3 mL total volume for 30 min at RT.

3.3 Tricine/EDDA Exchange Labeling

[0323] Same procedure than tricine as coligand except that 500 .mu.L of a EDDA solution (20 mg/mL in NaOH 0.1N) was added in the reaction vial and heated for 10 min at 100.degree. C.

4. Purification Procedure

[0324] Purification was realized using C18 Sep-Pak cartridge (Sep-Pak C18 Plus Light Cartridge, Waters, USA) preactivated with 10 mL of ethanol followed by 10 mL of water and 5 mL of air. After passing the radiolabeling preparation through the cartridge and washing it with 8 mL of water, the radiolabeled peptide was eluted with 80% ACN which was then evaporated under vacuum.

5. In Vitro Stability Study

[0325] Stability of .sup.99mTc complexes was assessed in fresh human plasma at 37.degree. C. after 0 min, 30 min, 1 hour, 2 hours and 4 hours of incubation at a concentration of 100 pmol/mL. Next, plasma samples were precipitated with methanol and centrifuged (20000 g, 10 min). Supernatants were collected and filtered (Millex-GV 0.22 .mu.m PVDF, Merck Millipore, Germany) then assessed by radio-HPLC.

6. In Vivo Stability Study

[0326] 74 MBq of 99mTc-HYNIC-P8RI (obtained with tricine/EDDA as coligands) was injected to a male wistar rat. After one hour, rat was sacrificed and urine was directly collected from the bladder using a syringe and analyzed by radio-HPLC after a 0.22 .mu.m filtration (Millex-GV 0.22 .mu.m PVDF, Merck Millipore, Germany).

7. Protein Binding

[0327] Protein binding of the purified radiolabeled peptide was determined after 0 min, 30 min, 1 hour, 2 hours and 4 hours of incubation in fresh human plasma at 37.degree. C. and analyzed after size exclusion chromatography (illustra Microspin G-50 Columns, Sephadex G-50, GE Healthcare, UK). G-50 columns were prespun at 2000.times.g for 1 min then 20 .mu.L of mixture was added and the column was centrifuged at 2000.times.g for 2 min. Protein binding of the radiolabeled peptide was estimated by measuring columns and eluates in a gamma-counter (Cobra II, Packard Bioscience). In the same time, radiolabeled peptide was incubated for 1 h in PBS 1.times. at 37.degree. C. as a control.

8. Experimental Models

[0328] 8.1. Abdominal Aorta Aneurysm (AAA) Model

[0329] Rat model of vascular inflammation that has been set up at the laboratory: the experimental abdominal aorta aneurysm (AAA) induced by local infusion of elastase and followed by intravenous injection of a periodontal bacterium present in human AAA (Porphyromonas gingivalis) and known to induce AAA inflammation (Delbosc et al., 2011, PLoS ONE 6(4): e18679. doi:10.1371/journal.pone.0018679).

[0330] 8.2. Hindlimb Inflammation Model

[0331] Rat model of hindlimb inflammation was induced by intramuscular injection of turpentine oil (150 .mu.l) in the right hindlimb, whereas a saline solution (150 .mu.l) was injected in the left hindlimb (contralateral control).

9. SPECT/CT Imaging--AAA Model

[0332] Immediately after intravenous injection via the penis vein of 74 MBq of radiolabelled HYNIC-P8RI (obtained using Tricine/EDDA), sequential whole-body acquisitions (every 10 minutes for the first hour) were performed with a hybrid SPECT/CT camera (NanoSPECT/CT, Bioscan Inc.) dedicated to small animals.

10. X ray CT Scanner, SPECT and SPECT/CT Acquisitions--Right Hindlimb Inflammation

[0333] Acquisitions were performed 30 min after injection of 80 MBq of 99mTc-HYNIC-P8RI (obtained with tricine/EDDA as coligands) and .sup.99mTc-mertiatide, respectively.

Results

1. Radiolabeling

[0334] 6-Hydrazinopyridine-3-carboxylic acid (HYNIC) was coupled to the N-terminal amino-acid of the P8RI peptide via a 3(PEG) spacer to obtain HYNIC-P8RI.

[0335] HYNIC-P8RI was then labeled at high specific activities (>71 GBq/.mu.mol) using tricine, EDDA or Tricine/EDDA as coligands. Labelling yields varied from 65.5% to 98.3% as shown in Table 2. The tricine/EDDA exchange labeling strategy was chosen because of high labeling yield and resulting in a single major specie as analyzed by HPLC (see FIG. 1).

TABLE-US-00003 TABLE 2 Labelling yields of 99mTc-HYNIC-P8RI using different coligands Co-ligand N yield(%) SD (%) Tricine 3 98.3 0.5 EDDA 3 65.5 7.3 Tricine/EDDA 3 93.8 2.8

2. Stability Study

[0336] In vitro stability in human plasma revealed a high stability of the 99mTc complex with no significant release of radiolabeled impurities or radiolabeled peptide degradation. The RCP (radiochemical purity) was superior to 89% after 4 h of incubation (see FIG. 2 A). In vivo stability study on rat urine by radio-HPLC analysis showed one major species excreted with a retention time corresponding to the injected radiotracer. This result indicates that 99mTc-HYNIC-P8RI may be excreted unchanged (see FIG. 28).

3. Protein Binding

[0337] Very low levels of protein binding as determined by size exclusion chromatography (<5% after 4 h incubation) were found using the Tricine/EDDA coligands exchange labelling strategy. This finding suggests that 99mTc-HYNIC-P8RI may be a hydrophilic compound.

[0338] 4. SPECT/CT Imaging--AAA Model

[0339] Representative SPECT and CT images were obtained after an acquisition performed 30 min after injection of 99mTc-HYNIC-P8RI (obtained with tricine/EDDA as coligands) in AAA rat as shown in FIG. 3. The pattern of biodistribution indicated almost exclusive renal uptake and excretion. Interestingly, there was a focal uptake of the radiotracer on the aorta pathway corresponding to the location of the AAA.

5. X ray CT Scanner, SPECT and SPECT/CT Acquisitions--Right Hindlimb Inflammation model

[0340] .sup.99mTc- HYNIC-P8RI was further assessed in a rat model of right hindlimb inflammation using turpentine oil.

[0341] Mertiatide is a non-specific marker of renal function and was used as a negative control, because of its biodistribution pattern close to peptide P8RI (in particular, low molecular weight and quick clearance).

[0342] In this model, the technetium-99m radiolabeled P8RI uptake ratio between the turpentine oil injected hindlimb and the saline injected hindlimb was higher compared to the control group injected with technetium-99m radiolabeled mertiatide, thereby confirming the specific binding of P8RI to inflammatory sites (see FIG. 4).

Conclusion

[0343] The RCP of [99mTc]EDDA/HYNIC-P8RI was >93% (HPLC and ITLC) without any purification and the specific activity was >71 GBq/pmol. There was no significant release of degraded radiolabelled peptide (RCP>89%) and the radiotracer binding to plasma proteins was very low (<5% after 4 h incubation). In vivo, blood clearance of the tracer was almost exclusively renal with a peak activity in kidneys and bladder 1 h after injection, corresponding to the unaltered form of the peptide on HPLC. In addition, .sup.99mTc-HYNIC-P8RI uptake by AAA was detectable from 30 min after injection in animals and associated with activated platelets and leukocytes on immunohistochemistry. .sup.99mTc-HYNIC-P8RI was also specifically detected in inflammatory sites in a model of right hindlimb inflammation. By specifically targeting activated cells involved in inflammation expressing truncated CD31 (CD31.sup.shed) and with a rapid blood clearance, radiolabelled P8RI constitutes a useful novel approach in inflammation imaging.

Sequence CWU 1

1

741738PRTHomo 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(592)..(601)Juxta-membrane domainDOMAIN(602)..(620)Transmembrane domainDOMAIN(621)..(738)Cytoplasmic domain 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 Thr26PRTArtificial Sequencemuse or human-derived CD31 peptide 2Leu Ala Pro Trp Lys Lys1 5310PRTArtificial Sequencemouse-derived CD31 peptide 3Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 10410PRTArtificial Sequencehuman-derived CD31 peptide 4Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 1058PRTArtificial Sequencemurine-derived CD31 peptide 5Arg Val Phe Leu Ala Pro Trp Lys1 568PRTArtificial Sequencemurine-derived CD31 peptide 6Lys Trp Pro Ala Leu Phe Val Arg1 578PRTArtificial Sequencehuman-derived CD31 peptide 7Arg Val Ile Leu Ala Pro Trp Lys1 588PRTArtificial Sequencehuman-derived CD31 sequence 8Lys Trp Pro Ala Leu Ile Val Arg1 59727PRTMus musculus 9Met 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 72510739PRTBos taurus 10Met 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 Ser11740PRTSus scrofa 11Met 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 7401223PRTArtificial Sequencehuman-derived CD31 peptide 12Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val1 5 10 15Ile Leu Ala Pro Trp Lys Lys 201315PRTArtificial Sequencepeptide 13Ser Ser Thr Leu Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 10 151414PRTArtificial Sequencepeptide 14Ser Thr Leu Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 101513PRTArtificial Sequencepeptide 15Thr Leu Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 101612PRTArtificial Sequencepeptide 16Leu Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 101711PRTArtificial Sequencepeptide 17Ala Val Arg Val Phe Leu Ala Pro Trp Lys Lys1 5 10189PRTArtificial Sequencepeptide 18Arg Val Phe Leu Ala Pro Trp Lys Lys1 5198PRTArtificial Sequencepeptide 19Val Phe Leu Ala Pro Trp Lys Lys1 5207PRTArtificial Sequencepeptide 20Phe Leu Ala Pro Trp Lys Lys1 5215PRTArtificial Sequencepeptide 21Ala Pro Trp Lys Lys1 5224PRTArtificial Sequencepeptide 22Pro Trp Lys Lys12315PRTArtificial Sequencepeptide 23Ser Lys Ile Leu Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 10 152414PRTArtificial Sequencepeptide 24Lys Ile Leu Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 102513PRTArtificial Sequencepeptide 25Ile Leu Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 102612PRTArtificial Sequencepeptide 26Leu Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 102711PRTArtificial Sequencepeptide 27Thr Val Arg Val Ile Leu Ala Pro Trp Lys Lys1 5 10289PRTArtificial Sequencepeptide 28Arg Val Ile Leu Ala Pro Trp Lys Lys1 5298PRTArtificial Sequencepeptide 29Val Ile Leu Ala Pro Trp Lys Lys1 5307PRTArtificial Sequencepeptide 30Ile Leu Ala Pro Trp Lys Lys1 53115PRTArtificial Sequencepeptide 31Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg1 5 10 153214PRTArtificial Sequencepeptide 32Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala Val1 5 103313PRTArtificial Sequencepeptide 33Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr Leu Ala1 5 103412PRTArtificial Sequencepeptide 34Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr Leu1 5 103511PRTArtificial Sequencepeptide 35Ser Ser Met Arg Thr Ser Pro Arg Ser Ser Thr1 5 103610PRTArtificial Sequencepeptide 36Ser Ser Met Arg Thr Ser Pro Arg Ser Ser1 5 10379PRTArtificial Sequencepeptide 37Ser Ser Met Arg Thr Ser Pro Arg Ser1 5388PRTArtificial Sequencepeptide 38Ser Ser Met Arg Thr Ser Pro Arg1 5397PRTArtificial Sequencepeptide 39Ser Ser Met Arg Thr Ser Pro1 5406PRTArtificial Sequencepeptide 40Ser Ser Met Arg Thr Ser1 5415PRTArtificial Sequencepeptide 41Ser Ser Met Arg Thr1 5424PRTArtificial Sequencepeptide 42Ser Ser Met Arg14315PRTArtificial Sequencepeptide 43Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg1 5 10 154414PRTArtificial Sequencepeptide 44Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr Val1 5 104513PRTArtificial Sequencepeptide 45Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu Thr1 5 104612PRTArtificial Sequencepeptide 46Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile Leu1 5 104711PRTArtificial Sequencepeptide 47Asn His Ala Ser Ser Val Pro Arg Ser Lys Ile1 5 104810PRTArtificial Sequencepeptide 48Asn His Ala Ser Ser Val Pro Arg Ser Lys1 5 10499PRTArtificial Sequencepeptide 49Asn His Ala Ser Ser Val Pro Arg Ser1 5508PRTArtificial Sequencepeptide 50Asn His Ala Ser Ser Val Pro Arg1 5517PRTArtificial Sequencepeptide 51Asn His Ala Ser Ser Val Pro1 5526PRTArtificial Sequencepeptide 52Asn His Ala Ser Ser Val1 5535PRTArtificial Sequencepeptide 53Asn His Ala Ser Ser1 5544PRTArtificial Sequencepeptide 54Asn His Ala Ser15515PRTArtificial Sequencepeptide 55Thr Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu Ala1 5 10 155613PRTArtificial Sequencepeptide 56Ser Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe Leu1 5 105711PRTArtificial Sequencepeptide 57Pro Arg Ser Ser Thr Leu Ala Val Arg Val Phe1 5 10589PRTArtificial Sequencepeptide 58Arg Ser Ser Thr Leu Ala Val Arg Val1 5597PRTArtificial Sequencepeptide 59Ser Ser Thr Leu Ala Val Arg1 5605PRTArtificial Sequencepeptide 60Ser Thr Leu Ala Val1 56115PRTArtificial Sequencepeptide 61Ser Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val Ile Leu Ala1 5 10 156213PRTArtificial Sequencepeptide 62Val Pro Arg Ser Lys Ile Leu Thr Val Arg Val Ile Leu1 5 106311PRTArtificial Sequencepeptide 63Pro Arg Ser Lys Ile Leu Thr Val Arg Val Ile1 5 10649PRTArtificial Sequencepeptide 64Arg Ser Lys Ile Leu Thr Val Arg Val1 5657PRTArtificial Sequencepeptide 65Ser Lys Ile Leu Thr Val Arg1 5665PRTArtificial Sequencepeptide 66Lys Ile Leu Thr Val1 5674PRTArtificial Sequencepeptide 67Arg Val Phe Leu1685PRTArtificial Sequencepeptide 68Arg Val Phe Leu Ala1 5696PRTArtificial Sequencepeptide 69Arg Val Phe Leu Ala Pro1 5707PRTArtificial Sequencepeptide 70Arg Val Phe Leu Ala Pro Trp1 5714PRTArtificial Sequencepeptide 71Arg Val Ile Leu1725PRTArtificial Sequencepeptide 72Arg Val Ile Leu Ala1 5736PRTArtificial Sequencepeptide 73Arg Val Ile Leu Ala Pro1 5747PRTArtificial Sequencepeptide 74Arg Val Ile Leu Ala Pro Trp1 5

Claims

1. A labeled CD31.sup.shed ligand comprising a CD31.sup.shed ligand and at least one imaging label.

2. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand is a peptide, a peptidomimetic, a chemical compound, an antibody, or an aptamer.

3. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand is a single domain antibody or a scFv fragment.

4. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand is a) a peptide selected in the group consisting of: (i) a peptide consisting of a fragment of 3 to 15 amino acids of the sequence defined by amino acids 579 to 601 of sequence SEQ ID NO: 1, (ii) a peptide consisting of a fragment of 3 to 15 amino acids of a sequence corresponding to the amino acids 579 to 601 of sequence SEQ ID NO: 1 in a non-human mammalian CD31, (iii) a peptide of 3 to 15 amino acids consisting of a sequence at least 70% identical to the sequence of peptide (i), (iv) a peptide consisting of a retro-inverso sequence of peptide (i), (ii), or (iii), and (v) the peptide (i), (ii), (iii), or (iv) comprising at least one chemical modification, or b) a peptidomimetic of peptide a).

5. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand is a peptide selected in the group consisting of a peptide of sequence SEQ ID NO: 2, a peptide of sequence SEQ ID NO: 3, a peptide of sequence SEQ ID NO: 4, a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids, a peptide of sequence SEQ ID NO: 7, and a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomer amino acids.

6. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shedl ligand is a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids, a peptide of sequence SEQ ID NO: 7, or a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomer amino acids.

7. The labeled CD31.sup.shed ligand according to claim 1, wherein said imaging label is a radionuclide.

8. The labeled CD31.sup.shed ligand according to claim 1, wherein said imaging label is a radionuclide detectable by Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT), a hybrid of SPECT and/or PET or their combinations.

9. The labeled CD31.sup.shed ligand according to claim 1, wherein said imaging label is a radionuclide selected from the group consisting of technetium-99m (99mTc), gallium-67 (67Ga), gallium-68 (68Ga) yttrium-90 (90Y), indium-111 (111In), rhenium-186 (186Re), fluorine-18 (18F), copper-64 (64Cu), and thallium-201 (201Tl).

10. The labeled CD31.sup.shed ligand according to claim 1, wherein said imaging label is 99mTc.

11. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand is a peptide selected in the group consisting of a peptide of sequence SEQ ID NO: 2, a peptide of sequence SEQ ID NO: 3, a peptide of sequence SEQ ID NO: 4, a peptide of sequence SEQ ID NO: 5, a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids, a peptide of sequence SEQ ID NO: 7, and a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomer amino acids; and wherein said imaging label is a radionuclide.

12. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand is a peptide of sequence SEQ ID NO: 6 consisting of D-enantiomer amino acids or a peptide of sequence SEQ ID NO: 8 consisting of D-enantiomer amino acids; and wherein said imaging label is 99mTc.

13. The labeled CD31.sup.shed ligand according to claim 1, wherein said CD31.sup.shed ligand comprises 6-hydrazinopyridine-3-carboxylic acid (HYNIC).