CA2676962A1 - Mcam modulation and uses thereof in the management of neuroinflammatory conditions - Google Patents
Mcam modulation and uses thereof in the management of neuroinflammatory conditions Download PDFInfo
- Publication number
- CA2676962A1 CA2676962A1 CA 2676962 CA2676962A CA2676962A1 CA 2676962 A1 CA2676962 A1 CA 2676962A1 CA 2676962 CA2676962 CA 2676962 CA 2676962 A CA2676962 A CA 2676962A CA 2676962 A1 CA2676962 A1 CA 2676962A1
- Authority
- CA
- Canada
- Prior art keywords
- cell
- mcam
- ninjurin
- secreting
- condition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Methods, uses, agents and compositions useful for the prevention, treatment and/or diagnosis of inflammatory conditions, including neuroinflammatory conditions such as multiple sclerosis and spinal cord injury based on the modulation of nerve injury-induced protein-1 (Ninjurin-1) and/or melanoma cell adhesion molecule (MCAM) are disclosed. Methods, and kits useful for the identification and selection of inflammatory cytokine-secreting T cells or precursors thereof, are also disclosed.
Description
FIELD OF THE INVENTION
[0001] The present invention generally relates to inflammation, and more particularly to the prevention, treatment and/or diagnosis of diseases and conditions associated with inflammation such as multiple sclerosis (MS) and spinal cord injury (SCI).
BACKGROUND OF THE INVENTION
[0001] The present invention generally relates to inflammation, and more particularly to the prevention, treatment and/or diagnosis of diseases and conditions associated with inflammation such as multiple sclerosis (MS) and spinal cord injury (SCI).
BACKGROUND OF THE INVENTION
[0002] A number of disorders are associated with inflammation. For example, multiple sclerosis (MS) is an immune-mediated inflammatory disorder of the central nervous system (CNS) characterized by multifocal areas of leukocyte infiltration, demyelination and axonal damage. Typically, demyelination is centered around pericapillary and periveinular accumulation of CD4+ and CD8+ memory T lymphocytes, macrophages and dendritic cells (DCs). These cells arise from migration of peripheral blood (PB) immune cells across the CNS microvascular endothelium.
[0003] There are few treatment regimens currently used in MS. Corticosteroids have anti-inflammatory and immunosuppressive effects, which also transiently restores the blood-brain barrier (Noseworthy et al., (2000) Neurology 54(9): 1726-33). They shorten the duration of the relapse and accelerate recovery. Since they are only effective as a short-term treatment, they are most commonly used to treat an acute relapse (Andersson and Goodkin, (1998) J Neurol Sci. 160(1): 16-25; Bansil et al., (1995) Ann Neurol. 37 Suppl 1: S87-101).
Further, the responsiveness to corticosteroids declines over time, and extended use may lead to adrenal suppression, cardiovascular collapse and arrhythmias (C. F.
Lacy et al., Drug information handbook 8th Edition, 2001, pp. 549-551).
Further, the responsiveness to corticosteroids declines over time, and extended use may lead to adrenal suppression, cardiovascular collapse and arrhythmias (C. F.
Lacy et al., Drug information handbook 8th Edition, 2001, pp. 549-551).
[0004] Interferon (IFN)-(3 has been used as a therapy for patients with active Relapsing/Remitting Multiple Sclerosis (RRMS) since the 1980's. It is recently being used for secondary progressive patients as well. Recombinant IFN is available in 3 drugs: IFN-13-lb (BetaseronTM) and two IFN(3-la preparations (AvonexTM and RebifTM) (Polman and Uitedehaag, supra). These drugs reduce the rate of clinical relapse. However, neutralizing antibodies develop against these drugs rendering them ineffective with time.
Also, flu-like symptoms are a prominent side effect early on in the treatment.
Also, flu-like symptoms are a prominent side effect early on in the treatment.
[0005] Glatiramer acetate (CopaxoneTM) is a synthetic co-polymer of tyrosine, glutamate, alanine and lysine, thought to mimic myelin basic protein (MBP) and thus, block T cell recognition of MBP (Karin N. et al., (1994) J Exp Med. 180(6): 2227-37). This drug is therefore beneficial in RRMS but not progressive MS. This drug also decreases the rate of relapse and appears to be better tolerated by patients than interferon therapy. Further, treatment with this drug may cause cardiovascular problems such as chest pain, flushing and tachycardia, and respiratory problems such as dyspnea (C. F. Lacy et al., supra).
[0006] Another drug that has been approved for the use in RRMS and secondary progressive MS is mitroxantrone. This drug is used to arrest the cell cycle and prevent cellular division, and it is primarily used in the treatment of leukemia (Rolak L.A., (2001) Neurol Clin. 19(1): 107-18). In MS, it reduces relapse rate and increases the length between exacerbations. This drug however has long-term side effects causing cardiac toxicity and chronic myeloid leukemias.
[0007] Therefore, there are a few moderately effective treatments for RRMS and secondary progressive MS that have shown to reduce both the frequency of the disease and severity of exacerbations.
[0008] Spinal cord injury (SCI) occurs due to traumatic injuries resulting from for example traffic accidents, sport accidents, or falls and drops from heights, and to spinal cord compression, or the like. It also occurs due to other disorders, for example, when stroke is accompanied by pyramidal tract transection. Spinal cord injury results in permanent loss of motor, sensory and autonomic functions. Following the initial injury, presumably as part of the inflammatory/immune response to the injury, a series of degenerative processes which promote tissue damage beyond the original site of injury are initiated. After the initial mechanical disruption of nerves and nerve fibers at the time of injury, hemorrhaging is usually observed within 30 minutes at the area of damage and may expand over the next few hours. Within several hours following the injury, immune/inflammatory cells, e.g., neutrophils and macrophages, infiltrate the area and cause further damage to the nerve tissue, i.e., cell-mediated damage. These post-traumatic events are referred to as "secondary injury" (or "secondary spinal cord injury"). Therefore, a significant aspect of the tissue damage and functional loss may be preventable as it is the result of secondary events triggered by the trauma. It is important to treat as promptly as possible when the spinal cord is damaged, in order to promote recovery from or to prevent progress, of neurologic function deficit. It would be advantageous to prevent further damage to the spinal cord and surrounding tissue following a spinal cord injury by treatment as soon as possible after the initial trauma to prevent secondary injury effects.
[0009] Currently, the conventional treatment for reducing or minimizing the damage resulting from secondary injury is intravenous injection of the glucocorticoid, methylprednisolone (Bracken et al., JAMA, 277(20): 1597-1604 (1997)).
Unfortunately, prolonged administration of glucocorticoids has adverse systemic side effects, e.g., increased incidence of sepsis and pneumonia, and a limited therapeutic window.
Furthermore, recent studies have raised doubts about the beneficial effects of high doses methylprednisolone after SCI (Schroter et al., Neuroscience 2009 161(3): 753-63. Epub 2009 Apr 11).
Unfortunately, prolonged administration of glucocorticoids has adverse systemic side effects, e.g., increased incidence of sepsis and pneumonia, and a limited therapeutic window.
Furthermore, recent studies have raised doubts about the beneficial effects of high doses methylprednisolone after SCI (Schroter et al., Neuroscience 2009 161(3): 753-63. Epub 2009 Apr 11).
[0010] There is therefore a continued need for improved materials and methods for the treatment of conditions/diseases associated with inflammation, such as neuroinflammatory conditions like MS and SCI.
[0011] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
SUMMARY OF THE INVENTION
[0012] The present invention generally relates to inflammation such as neuroinflammation, and more particularly to the prevention, treatment and/or diagnosis of diseases and conditions associated with neuroinflammation, such as multiple sclerosis (MS) and spinal cord injury (SCI).
[0013] In an aspect, the present invention provides a method of preventing or treating an inflammatory condition in a subject, said method comprising administering to said subject an effective amount of a nerve injurin-induced protein-1 (Ninjurin-1) and/or melanoma cell adhesion molecule (MCAM) inhibitor.
[0014] In another aspect, the present invention provides a method of inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium comprising contacting said cell and/or said CNS
endothelium with an effective amount of a Ninjurin-1 and/or MCAM inhibitor.
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium comprising contacting said cell and/or said CNS
endothelium with an effective amount of a Ninjurin-1 and/or MCAM inhibitor.
[0015] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for preventing or treating an inflammatory condition in a subject.
[0016] In another aspect, the present invention provides a Use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition in a subject.
[0017] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium.
[0018] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium.
[0019] In another aspect, the present invention provides a Ninjurin-1 and/or MCAM
inhibitor for preventing or treating an inflammatory condition in a subject.
inhibitor for preventing or treating an inflammatory condition in a subject.
[0020] In another aspect, the present invention provides a Ninjurin-1 and/or MCAM
inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition in a subject.
inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition in a subject.
[0021] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating an inflammatory condition in a subject, said composition comprising a Ninjurin-1 and/or MCAM inhibitor and a pharmaceutically acceptable carrier.
[0022] A method of identifying a compound for preventing or treating an inflammatory condition, said method comprising determining whether:
(a) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(b) a level of Ninjurin-1 and/or MCAM activity; or (c) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for preventing or treating said inflammatory condition.
(a) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(b) a level of Ninjurin-1 and/or MCAM activity; or (c) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for preventing or treating said inflammatory condition.
[0023] In another aspect, the present invention provides a method of identifying or characterizing a compound for preventing or treating a neuroinflammatory condition, said method comprising:
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene 5 capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for preventing or treating said inflammatory condition.
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene 5 capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for preventing or treating said inflammatory condition.
[0024] In another aspect, the present invention provides a method of identifying a compound for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii) across the CNS endothelium, said method comprising determining whether:
(a) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(b) a level of Ninjurin-1 and/MCAM activity; or (c) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
(a) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(b) a level of Ninjurin-1 and/MCAM activity; or (c) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
[0025] In another aspect, the present invention provides a method of identifying or characterizing a compound for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii) across the CNS endothelium, said method comprising:
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
[0026] In another aspect, the present invention provides a method for diagnosing an inflammatory condition in a first subject, said method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said first subject (b) comparing said expression and/or activity to a corresponding reference expression and/or activity; and (c) diagnosing said inflammatory condition based on said comparison.
[0027] In another aspect, the present invention provides a method for monitoring the course of treatment of a patient suffering from an inflammatory condition, the method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein a decrease in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at an earlier time is indicative that said patient is responsive to said treatment.
[0028] In another aspect, the present invention provides a method of determining whether a subject is suffering from a relapse of an inflammatory condition, said method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein an increase in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at a time point of remission is indicative that said patient is suffering from a relapse of said inflammatory condition.
[0029] In another aspect, the present invention provides a method of identifying an inflammatory cytokine-secreting T cell or precursor thereof in a sample, said method comprising (i) contacting said cell with an MCAM ligand and (ii) identifying said inflammatory cytokine-secreting T cell or precursor thereof based on the binding to said MCAM ligand.
[0030] In another aspect, the present invention provides a method of purifying an inflammatory cytokine-secreting T cell or precursor thereof from a population of cells in a sample, said method comprising contacting said sample with an MCAM ligand and (ii) purifying said inflammatory cytokine-secreting T cell or precursor thereof on the basis of binding to said MCAM ligand.
[0031] In another aspect, the present invention provides a kit for identifying and/or purifying an inflammatory cytokine-secreting T cell or a precursor thereof in a cell sample, said kit comprising (i) an MCAM ligand and (ii) instructions for identifying and/or purifying said inflammatory cytokine-secreting T cell or precursor thereof from said sample.
[0032] In an embodiment, the above-mentioned Ninjurin-1 inhibitor blocks Ninjurin-1/Ninjurin-1 homotypic interaction.
[0033] In an embodiment, the above-mentioned Ninjurin-1 inhibitor binds to an extracellular domain of a Ninjurin-1 polypeptide.
[0034] In an embodiment, the above-mentioned Ninjurin-1 inhibitor binds to a domain comprising a motif corresponding to residues 28 to 35 of a Ninjurin-1 polypeptide.
[0035] In an embodiment, the above-mentioned Ninjurin-1 polypeptide is a human Ninjurin-1 polypeptide.
[0036] In an embodiment, the above-mentioned Ninjurin-1 inhibitor is a peptide comprising a domain of formula I:
[0037] Xaa'-Xaa2-Arg-Trp-Xaa3-Xaa4-Arg-Xaa5-Arg-Xaa6-Xaa7-Xaa8 (I),
[0038] wherein
[0039] Xaa', Xaa2, Xaa6, Xaa7 and Xaa6 is each independently any amino acid or is absent;
[0040] Xaa3, Xaa4 and Xaa5 is each independently any amino acid;
(0041] or a functional analog thereof.
[0042] In an embodiment, Xaa2 is Pro. In an embodiment, Xaa' is Pro. In an embodiment, Xaa6 is Pro. In an embodiment, Xaa7 is Ile. In an embodiment, Xaa8 is Asn. In an embodiment, Xaa3 is Gly. In an embodiment, Xaa4 is Leu. In an embodiment, Xaa5 is Asn or Leu.
[0043] In an embodiment, the above-mentioned domain is Pro-Pro-Arg-Trp-Gly-Leu-Arg-Asn-Arg-Pro-Ile-Asn.
[0044] In an embodiment, the above-mentioned Ninjurin-1 inhibitor is a peptide consisting of the domain of formula I defined in any one of claims 6 to 15.
[0045] In an embodiment, the above-mentioned MCAM inhibitor blocks MCAM/MCAM
homotypic interaction.
homotypic interaction.
[0046] In an embodiment, the above-mentioned inhibitor is an antibody or a fragment thereof.
[0047] In an embodiment, the above-mentioned MCAM inhibitor is an siRNA
molecule.
molecule.
[0048] In an embodiment, the above-mentioned MCAM inhibitor blocks MCAM-induced cell signalling.
[0049] In an embodiment, the above-mentioned inflammatory condition is a neuroinflammatory condition.
[0050] In an embodiment, the above-mentioned neuroinflammatory condition is associated with recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), to the central nervous system (CNS).
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), to the central nervous system (CNS).
[0051] In an embodiment, the above-mentioned myeloid cell is a monocyte, a macrophage and/or a dendritic cell.
[0052] In an embodiment, the above-mentioned inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
[0053] In an embodiment, the above-mentioned inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-y), (iii) Tumor Necrosis Factor-alpha (TNF-a), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-y), (iii) Tumor Necrosis Factor-alpha (TNF-a), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
[0054] In an embodiment, the above-mentioned neuroinflammatory condition is associated with a CNS trauma. In a further embodiment, the above-mentioned CNS
trauma is spinal cord injury (SCI).
trauma is spinal cord injury (SCI).
[0055] In an embodiment, the above-mentioned neuroinflammatory condition is an autoimmune CNS condition. In a further embodiment, the above-mentioned autoimmune CNS condition is multiple sclerosis (MS).
[0056] In an embodiment, the above-mentioned method of any one of claims 21 to 29, wherein said method further comprises (i) reducing the neurological signs, symptoms and/or clinical scores of the neuroinflammatory condition; (ii) reducing tissue damage; and/or (iii) reducing infiltration of myeloid cells into the CNS.
[0057] In an embodiment, the above-mentioned CNS endothelium is exposed to an inflammatory environment. In a further embodiment, the above-mentioned inflammatory environment comprises Interferon-gamma (IFN-y) and/or Tumor Necrosis Factor-alpha (TNF-a). In a further embodiment, the above-mentioned inflammatory environment is associated with a neuroinflammatory condition.
[0058] In an embodiment, the above-mentioned reference expression and/or activity corresponds to an expression and/or activity determined in a sample from a control subject known to not having an inflammatory condition, and wherein a higher expression and/or activity in said sample from said first subject is indicative that said first subject has an inflammatory condition.
[0059] In an embodiment, the above-mentioned expression and/or activity of Ninjurin-1 and/or MCAM is determined by measuring the amount of Ninjurin-1- and/or MCAM-expressing cells in said sample. In a further embodiment, the above-mentioned amount is a relative amount.
[0060] In an embodiment, the above-mentioned MCAM ligand is (i) MCAM, (ii) an MCAM
binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii).
binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii).
[0061] In an embodiment, the above-mentioned MCAM ligand is bound to a solid support.
[0062] In an embodiment, the above-mentioned inflammatory condition is a neuroinflammatory condition. In an embodiment, the above-mentioned neuroinflammatory condition is associated with a CNS trauma. In a further embodiment, the above-mentioned CNS trauma is spinal cord injury (SCI).
[0063] In another embodiment, the above-mentioned neuroinflammatory condition is an autoimmune CNS condition. In a further embodiment, the above-mentioned autoimmune CNS condition is multiple sclerosis (MS).
[0064] Other objects, advantages and features of the present invention will become 5 more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
BRIEF DESCRIPTION OF THE DRAWINGS
[0065] In the appended drawings:
10 [0066] Figure 1 shows the expression of nerve injury-induced protein-1 (Ninjurin-1) in vitro in human blood-brain barrier (BBB)-endothelial cells. (A) FACS analyses of the expression of Ninjurin-1 in resting human primary BBB-ECs (left panel) treated with astrocyte conditioned media (ACM) (40%, 24h) (center panel) or treated with pro-inflammatory cytokines TNF + IFN-y (100 U/ml, 24h) in the presence of ACM
(right panel).
Data shown are representative of ten independent experiments. (B) Ninjurin-1 immunostaining of resting or activated BBB-ECs. Confocal microscopy confirmed Ninjurin-1 (green) expression at the surface of BBB-ECs and up-regulation following treatment with TNF and IFN-y (100 U/ml, 16 h). TO-PRO-3 was used for nuclear staining. Scale bar: 10 pm.
Data shown are representative of three independent experiments;
[0067] Figure 2 shows the expression of Ninjurin-1 on human CD14+ monocytes and CD83+ dendritic cells (DCs). (A) FACS analysis of Ninjurin-1 expression on ex vivo human peripheral blood lymphocytes (CD4+, CD8+, CD19+) demonstrates low levels on T
and B
lymphocytes. (B) FACS analysis of Ninjurin-1 expression on human ex vivo CD14+
monocytes, and in vitro CD68+ macrophages and CD83+ dendritic cells (DCs) demonstrates high expression of Ninjurin-1 on myeloid antigen-presenting cells. (C) Western blot (WB) analysis of the expression of Ninjurin-1 in ex vivo human leukocytes (CD8+, CD4+, CD14+
and CD19+). R-actin was used as a loading control. (D) Expression of Ninjurin-1 on human CD68+ microglia grown in primary cultures. Data shown are representative of seven (A), five (C), and two (B and D) independent experiments using an equivalent number of preparations and donors. Isotype controls are shown in clear histograms and Ninjurin-1 immunostaining in gray;
[0068] Figure 3 shows the in situ detection of Ninjurin-1 in multiple sclerosis (MS) lesions. (A) Ninjurin-1 immunostaining in normal-appearing white matter (NAWM) blood vessels of human CNS post-mortem sections (left panel). Ninjurin-1 expression on infiltrating immune cells and on ECs in MS lesions (right panel). Arrowheads show Ninjurin-expressing ECs. Scale bars, 50 pm. (B) Percentage of CD14+ and CD11c+ cells co-expressing Ninjurin-1 in the cerebrospinal fluid (CSF) and the peripheral blood of MS
patients (n = 5). Mean SEM from five independent experiments. (C) Co-expression of Ninjurin-1 and Caveolin-1 on ECs within NAWM from MS patients. Scale bar, 30 pm. (D) Immunofluorescent staining and confocal microscopy analysis of active MS
lesions expressing Ninjurin-1 and either MHC II+ (top panels), CD68+ (middle panels) or DC-SIGN+
(bottom panels). Co-localization is seen in right panels with TO-PRO-3 nuclear staining.
Scale bars, 30 pm. Photomicrographs shown are representative of immunostainings performed on 12 active plaques and 15 NAWM obtained from frozen CNS material of MS
patients (n = 3).;
[0069] Figure 4 shows the expression of Ninjurin-1 in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) and spinal cord injury (SCI).
(A) Western blot for Ninjurin-1 in spinal cord homogenates of myelin oligodendrocyte glycoprotein (MOG)35-55-immunized EAE mice (C57BL/6) revealed an upregulation of Ninjurin-1 compared to the control (unimmunized mice). Levels of Ninjurin-1 correlated with EAE
scores (center lane: score 1; right lane: score 3.0). (B) Flow cytometry analysis of Ninjurin-1 expression on MHC ll+ (left panel) CD11c+ DCs (middle panel) and F4/80+ macrophages (right panel) obtained from the CNS (brain and spinal cord) of MOG35-55-immunized EAE mice (C57BU6).
Data shown are representative of two experiments, gated on CD3ne9 CD45hand either MHC
II-, CD11c- or F4/80-expressing cells. (C) Spinal cords from EAE mice immunostained for Ninjurin-1, MHC II (left panels), CD11c (middle panels), F4/80 (right panels) and nuclear stain TO-PRO-3 (day 14 post-induction, n = 6). High power view of areas marked confirmed co-localization of Ninjurin-1 in CNS myeloid antigen-presenting cells (red).
Scale bars, 30 pm. (D) Western blot for Ninjurin-1 in spinal cord homogenates after SCI in C57BL/6 mice.
Spinal cord mechanical injury was performed in mice using the Infinite HorizonsTM impactor device. Samples were extracted from uninjured (UI) and from injured mice 1, 3, 7, 14, 21 and 28 days after injury. The expression of Ninjurin-1 increases in the spinal cord of injured mice;
[0070] Figure 5 shows the effect of Ninjurin-1 blockade on CD14+ monocyte migration across human BBB-endothelial cells. The adhesion motif of human Ninjurin-1 is used as a blocking peptide, called Ninj26_37 (sequence: NH2-PPRWGLRNRPIN-COOH, SEQ ID
NO: 5).
(A) In vitro model of the BBB. Human BBB-endothelial cells are grown in Boyden chambers and treated with the Ninjurin-1 blocking peptide Ninj26-37 or control peptide (top chamber) 1 hour prior to the addition of immune cells. Human ex vivo CD4+ and CD8+ T
lymphocytes (B) and CD14+ monocytes (C) were allowed to migrate across human BBB-endothelial cells for 24 h, in the presence or absence of the human Ninj26_37 blocking peptide.
Ninjurin-1 blockade (0.4 mM) significantly restricts the migration of CD14+ monocyte, but not that of CD4+ or CD8+ T cells, across human BBB-endothelial cells. Data shown are representative of seven independent experiments (n = 7 blood donors) on six distinct BBB-EC
preparations performed in triplicate. (D) CFSE-labeled human CD14+ monocytes (green) were seeded on a confluent monolayer of human TNF/IFN-y-activated BBB-ECs, fixed and immunostained for Ninjurin-1 (left panels). A 15 pm z-stack reconstruction (x-z and y-z) shows Ninjurin-1 (arrows) around the CFSE-loaded migrating monocyte and in the transmigratory cup (arrowheads). Photomicrograph shown is representative of >20 fields obtained from four independent immunostainings performed using four blood donors and two distinct BBB-ECs preparations. Scale bar, 20 pm;
[0071] Figure 6 shows the effects of Ninjurin-1 blocking peptide (Ninj26_37) in EAE mice.
EAE was induced by active immunization with MOG35-55/CFA in C57BL/6 mice. Some animals received intraperitoneal (i.p.) injections of 200 pg twice a day (b.i.d.) of mouse Ninj26_ 37 from day 3 to day 23 post-immunization (=, n = 24 mice); others received PBS (o, n = 22 mice) in the same manner. (A) Mice treated with Ninj26_37 blocking peptide show a significant reduction of the neurological signs, symptoms and clinical scores of the disease. (clinical score: 0 = no clinical symptoms; 0.5 = partial floppy tail; 1 = floppy tail; 2 = ataxia; 2.5 =
weakness of hind limbs; 3 = paralysis of one hind limb; 4 = paralysis of both hind limbs). (B) FACS analysis of CNS infiltrating leukocytes 14 days postimmunization, comparing the number of CD3+ lymphocytes, CD11c+ DCs (top panels) and F4/80+ macrophages (lower panels) in Ninj26-37-treated mice vs. control animals. Data shown are representative of two independent experiments obtained from four mice, gated on CD45hi cells. (C) Luxol fast blue-hematoxylin and eosin stainings of EAE spinal cords from Ninj26-37-treated mice show a reduction in immune cell infiltration and demyelination, as compared to the control group.
Photomicrographs shown are representative of >20 stainings performed on four animals.
Doted lines delineate areas of demyelination. Scale bar, 50 pm. (D) Immunofluorescent analyses of spinal cords (14 days post-immunization) confirmed reduction of infiltrating MHC
ll+ (top panels), F4/80+ (middle panels) and CD11 c+ (bottom panels) cells in Ninj26_37-treated mice vs. control animals. Nuclei were stained with TO-PRO-3. Photomicrographs shown are representative of >20 immunostainings performed on post-mortem material from 4 animals.
Scale bar, 30 pm;
[0072] Figure 7 shows the clinical effect of Ninjurin-1 blocking peptide after spinal cord injury. Spinal cord mechanical injury was performed in C57BL/6 mice using the Infinite HorizonsTM impactor device. (A) WB of spinal cords samples showing Ninjurin-1 expression at day 1, 3, 7, 14, 21 and 28 post-injury. Ctl represents baseline (uninjured) control. Data shown are representative of two experiments performed with three animals. (B) and (C) Immunostainings and cell counts of Iba1+ and CD11c+ myeloid cell infiltrates in the spinal cord of SCI animals treated with Ninj26_37 blocking peptide (200 pg i.p., twice a day) vs.
controls. Photomicrographs shown are representative of immunostainings obtained from control and Ninj26-37-treated animals mice (n = 4 per group, day 7 post-injury). Scale bars, 500 pm (D) The clinical scores (Basso mouse scale, BMS, Basso D.M. et al., (2006). J
Neurotrauma. 23(5): 635-59) of mice treated with Ninj26-37 blocking peptide (=, 200 pg i.p., twice a day) were reduced when compared to control animals (o). Data shown represent two independent experiments using 11 mice per group;
[0073] Figure 8 shows the nucleotide and amino acid sequences of Ninjurin-1.
(A) Nucleotide sequence of human Ninjurin-1 (SEQ ID NO: 1, NCBI Reference Sequence:
NM_004148.3). The coding sequence (residues 72 to 530) is indicated in bold.
(B) Amino acid sequence of human Ninjurin-1 (SEQ ID NO: 2, NCBI Reference Sequence:
NP_004139.2). (C) Nucleotide sequence of mouse Ninjurin-1 (SEQ ID NO: 3, NCBI
Reference Sequence: NM_013610.2). The coding sequence (residues 17 to 475) is indicated in bold. (D) Amino acid sequence of mouse Ninjurin-1 (SEQ ID NO: 4, NCBI
Reference Sequence: NP_038638.1);
[0074] Figure 9 shows the effect of Ninjurin-1 blockade on monocyte adhesion to a BBB-ECs monolayer in an in vitro flow system. BBB-ECs were grown to confluence on flow capillary slides (Ibidi micro-slide I Luer 0.6). Primary cultures of human BBB-ECs were then submitted to flow conditions (0.25 ml/min, Ibidi pump system) and CFSE-labelled ex vivo isolated monocytes were added in the absence or presence of the Ninjurin blocking peptide (4 mM). Image acquisition took place in the next 40 minutes, pictures and movies (10 images per second) were acquired and analysed on VolocityTM software (Improvision). The number of adherent monocytes was evaluated on at least 15 random fields; two representative images are shown in (A). Quantification of the number of adherent monocytes under the different flow conditions (presence or absence of Ninjurin blocking peptides, BBB-ECs stimulated or not with TNF/IFN-y) is shown in the right panel (B);
[0075] Figure 10 shows the effect of Ninjurin-1 blockade on the velocity of monocyte on a BBB-ECs monolayer in an in vitro flow system. BBB-ECs were grown to confluence on flow capillary slides (Ibidi micro-slide Luer 0.6). Primary cultures of human BBB-ECs were then submitted to flow conditions (0.25 ml/min, Ibidi pump system) and CFSE
labelled ex vivo isolated monocytes were added in the absence or presence of the Ninjurin blocking peptide (4 mM). Image acquisition took place in the next 40 minutes, movies (10 images per second) were acquired and analysed on VolocityTM software (Improvision).
Flowing monocyte velocities were calculated from the movies using the software. The mean velocity of all the moving monocytes was compared under the different conditions (presence or absence of Ninjurin blocking peptides, BBB-ECs stimulated or not with TNF/IFN-y);
[0076] Figure 11 shows the expression of melanoma cell adhesion molecule (MCAM) on endothelial cells of the blood-brain barrier (BBB-ECs). (A) Expression of MCAM protein (105-115 kDa) in primary human cultures of BBB-ECs, as compared to umbilical vein ECs (HUVECs). n = 3 different preparations, from 3 distinct donors. (B) MCAM
protein can be detected, by Western blot analysis, in detergent resistant membrane microdomains and lipid rafts of BBB-ECs (fractions 4 to 6). GM1 is shown as a marker of the lipid raft fractions (fractions 4 to 6). (C) Expression of MCAM on the surface of BBB-ECs (dark histogram) is up-regulated in the presence of TNF-a and IFN-y (100 U/ml, open histogram), n = 3 as measured by flow cytometry;
[0077] Figure 12 shows the expression of MCAM in MS lesions. Paraffin-embedded material from control human CNS (top panels, A) and human active MS lesions (bottom panels, B) are shown. Immunostaining for Caveolin-1 (Cav-1, endothelial cell marker, middle panels) and MCAM (left panels) were acquired by fluorescent microscopy. Data shown are representative of 5 active MS lesions from 4 distinct MS-affected donors and 4 controls.
Merged images are presented on the right panels and demonstrate co-localization of MCAM
with Cav-1 on infiltrated blood vessels in MS and control tissue. Scale bar is 75 pm and nuclei were stained with TOPRO-3. These results indicate that MCAM is expressed by BBB-ECs in MS lesions and in control CNS tissue. The expression of MCAM does not seem to be significantly modulated in MS lesions;
[0078] Figure 13 shows an immunocytofluorescent staining of the expression of MCAM
during leukocyte migration. Ex vivo CD4+ T lymphocytes isolated from healthy donors were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then allowed to migrate across a monolayer of BBB-ECs for 2 hours. The monolayer was then washed extensively with PBS to remove non adherent cells and fixed with 4% paraformaldehyde.
Cells were 5 stained with anti-MCAM antibody. Scale bar is 25 pm. MCAM enrichment is seen surrounding the lymphocytes at points of EC contact and entry in a structure reminiscent of the transmigratory cup;
[0079] Figure 14 shows the expression of MCAM as assessed by flow cytometry on freshly isolated peripheral blood cells. Cells were labeled with anti-MCAM
antibody 10 concurrently with specific cell markers: (A) CD4+ T lymphocytes, (B) CD8+ T
lymphocytes, (C) CD14+ monocytes and (D) CD19+ B lymphocytes. (E) MCAM+ CD3+ T lymphocytes are mainly CD45RO+ and CD45RA", indicative of a memory T cell phenotype. (F) Overview of the markers associated with MCAM expression in ex vivo peripheral blood cells;
[0080] Figure 15 shows a representative quantification of selected lymphocyte markers 15 as assessed by flow cytometry on ex vivo MCAM+ human peripheral blood lymphocytes obtained from healthy donors. Quantification is shown for CD69 early activation marker (A), CD107a degranulation marker (B), the chemokine receptors CCR7 (C) and CCR6 (D) and the inflammatory cytokine IL-17 (E). Cells were labeled with anti-MCAM
antibody concurrently with cell specific marker (n > 4). * p < 0.05, ** p < 0.01, *** p < 0.001. The data demonstrates that MCAM+ cells more frequently express CD161, CCR6 and IL-17, and express lower levels of CCR7;
[0081] Figure 16 shows a flow cytometry analysis of MCAM expression on ex vivo and in vitro activated (with OKT3 + CD14 monocytes + IL-2) memory lymphocytes. One million CD4+ CD45RO+ lymphocytes were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and IL-2 (20 units/ml) for 3 days. (A) Representative FACS histogram. (B) Quantification of MCAM expression after in vitro activation (n > 8, *** p < 0.001) demonstrates a significant increase in the number of MCAM+ cells upon non-antigen specific TCR-mediated activation;
[0082] Figure 17 shows the expression of selected cell surface markers on isolated CD4+ CD45RO+ MCAM+ and MCAM" lymphocytes after in vitro activation with OKT3 +
monocytes + selected cytokines. One million lymphocytes sorted according to expression of MCAM were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and either IL-2 (n > 7, 20 units/ml) or IL-23 (n > 3, 10 ng/ml) with anti-IFN-y (5 p/ml) and anti-IL-4 (5 pg/ml). Following activation, CD4+ CD45RO+ cells were assessed for the Th17 marker CD161 (A), CD107a (B), CD25 (C) and CD69 (D). * p < 0.05, ** p <
0.01, *** p < 0.001. The data demonstrates that MCAM expressing cells more frequently co-express CD161, CD107a and CD69 when stimulated with IL-2 or with IL-23;
[0083] Figure 18 shows a representative flow cytometry analysis of the expression of the cytokines Interferon-gamma (A) and IL-17 (A and B) on in vitro activated CD4+
CD45RO+ T cells (A) and (B) on CD4+ CD45RO+ lymphocytes sorted for MCAM
expression (CD4+ CD45RO+ MCAM+ vs. CD4+ CD45RO+ MCAM"). One million lymphocytes sorted according to expression of MCAM were cultured with 500 000 autologous CD14+
monocytes in the presence of anti-CD3 (2.5 pg/ml) and either IL-2 (20 units/ml). The data demonstrates that IL-17 expression and the number of IL-17 expressing cells are significantly increased in MCAM expressing CD4 memory lymphocytes;
[0084] Figure 19 shows the quantification of flow cytometry analysis of the expression of selected cytokines by isolated CD4+ CD45RO+ MCAM+ and MCAM- lymphocytes after days of in vitro culture. One million lymphocytes sorted according to expression of MCAM
were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and either IL-2 (n > 7, 20 units/ml) or IL-23 (n > 3, 10 ng/ml) with anti-IFN-y (5 p/ml) and anti-IL-4 (5 pg/ml). Following activation, CD4+ CD45RO+ cells were assessed for expression of IL-17 (A), GM-CSF (B), IFN-gamma (C) and IL-4 (D). * p < 0.05, ** p < 0.01, *** p < 0.001. The data demonstrates that MCAM expression on CD4+ CD45RO+
lymphocytes predicts the expression of IL-17, GM-CSF and IFN-y;
[0085] Figure 20 shows an overview of the markers associated with in vitro activated MCAM+ lymphocytes;
[0086] Figure 21 shows MCAM expression on MCAM+-sorted lymphocytes after 5 days in culture in a Th17 polarization environment. Memory T cells expressing CD45RO, CD4 and MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions). (A) Representative histogram showing expression of MCAM in memory T cells expressing CD45RO and CD4 (black line) compared with isotype (solid grey) after 5 days in culture. (B) Quantification of the proportion of cells that are MCAM+ or MCAM" after 5 days in Th17 polarization conditions and originate from memory T cells expressing CD45RO and CD4 sorted for MCAM expression (MCAM+
fraction). n = 3. Statistical analysis were obtained with Paired T test;
[0087] Figure 22 shows MCAM expression on MCAM-negative-sorted lymphocytes after days in culture in Th17 polarization environment. Memory T cells expressing CD45RO and CD4, but negative for MCAM were sorted with a purity of >99% and cultured for 5 days in 5 presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions). (A) Representative histogram showing expression of MCAM expression in memory T
cells expressing CD45RO and CD4 (grey line) compared with isotype (solid grey) after 5 days in culture. (B) Quantification of the proportion of cells that are MCAM+ or MCAM-after 5 days in Th17 polarization conditions and originate from memory T cells expressing CD45RO and CD4 negatively sorted for MCAM expression (MCAM- fraction) (n = 3).
Statistical analysis were obtained with Paired T test, ns = not significant;
[0088] Figure 23 shows the expression of IL-17 and IFN-y in MCAM+ memory T
cells originating from MCAM+ sorted lymphocytes. Memory T cells expressing CD45RO, CD4 and MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions). Intracellular stainings for IL-17 (left panel), IFN-y (middle panel) and IL-17 + IFN-y (right panel) were performed on cultured CD45RO+ CD4+ MCAM+ cells and CD45RO+ CD4+ MCAM- cells stimulated with PMA/ionomycin/Brefeldin A for 4 hours (n = 3). Statistical analysis were obtained with Paired T test, ns = not significant;
[0089] Figure 24 shows the expression of IL-17 and IFN-y in MCAM+ memory T
cells originating from MCAM" sorted lymphocytes. Memory T cells expressing CD45RO
and CD4 but negative for MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions).
Intracellular stainings for IL-17 (left panel), IFN-y (middle panel) and IL-17 + IFN-y (right panel) were performed on cultured CD45RO+ CD4+ MCAM+ cells and CD45RO+ CD4+
MCAM" cells stimulated with PMA/ionomycin/Brefeldin A for 4 hours (n = 3).
Statistical analysis were obtained with Paired T test, ns = not significant;
[0090] Figure 25 shows the expression of IL-8 on MCAM+ memory T cells originating from MCAM+ and MCAM sorted lymphocytes. Memory T cells expressing CD45RO and CD4, and positive or negative for MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions).
Intracellular staining for IL-8 was performed on cultured CD45RO+ CD4+ MCAM+
cells and CD45RO+ CD4+ MCAM- stimulated with PMA/ionomycin/ Brefeldin A for 4 hours.
n=3.
Statistical analysis were obtained with Paired T test;
[0091] Figure 26 shows the expression of MCAM on CD4+ and CD8+ T lymphocytes obtained from healthy donors (controls) and MS patients. Human ex vivo peripheral blood collected from healthy donors and MS patients was directly subjected to whole blood FAGS
staining with anti-CD3, anti-CD4, anti-CD8, anti-MCAM antibodies. FAGS
acquisition was performed the same day. (A) Proportion of CD3+ CD4+ MCAM+ lymphocytes in 35 healthy donors and in 42 MS patients in relapse (** p < 0.0001). (B) Proportion of CD3+ CD8+
MCAM+ lymphocytes in 35 healthy donors and in 42 MS patients in relapse (* p <
0.005). (C) Enrichment of CD3+ CD4+ MCAM+ and CD3+ CD8+ MCAM+ lymphocytes in the cerebro-spinal fluid (CSF) as compared to the peripheral blood in MS patients (n = 5, * p < 0.005);
[0092] Figure 27 shows CD4+ CD45RO+ human peripheral blood lymphocytes from MS
patients and healthy controls cultured under Th17 skewing conditions and assessed for MCAM association with IL-17 secretion (n = 3 MS patients and 7 controls). One million CD4+
CD45RO+ lymphocytes were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and IL-23 (10 ng/ml) with anti-IFN-y (5 p/ml) and anti-IL-4 (5 pg/ml). Following activation CD4+ CD45RO+ cells were assessed for IL-17 and MCAM co-expression. (A) Shows representative FACS plots from one control and one MS
patient.
Panel (B) is the quantification of MCAM+ IL-17+ cells compared to MCAM- IL-17+
cells. CD4+
CD45RO+ MCAM+ from MS patients grown under Th17 polarizing conditions express significantly more IL-17 compared to healthy controls (* p < 0.05), presumably due to the increase proportion of MCAM+ CD4+ seen in MS peripheral blood;
[0093] Figure 28 shows the presence of CD4+ and CD8+ MCAM+ T lymphocytes in various organs of mice affected by myelin-oligodendrocyte (MOG) peptide-induced experimental allergic encephalomyelitis (EAE). EAE was induced in C57BL6 mice by injection of MOG35-55 in complete Freunds adjuvant. At the peak of the disease (day 14), two animals were sacrificed and the presence of CD4+ and CD8+ MCAM+ lymphocytes was assessed in the spleen, the lymph nodes (LN) and the CNS of the animals. (A) Representative FAGS analysis of the presence of MCAM+ lymphocytes in the spleen, LN
and brain. Panel (B) shows the quantification of the percentage of MCAM+ CD4+
and CD8+
lymphocytes in the spleen, the LN and the brain of EAE animals. These data show that MCAM+ lymphocytes are recruited to the brain in the course of EAE, the animal model of MS; and [0094] Figure 29 shows the (A) nucleotide (NCBI Reference Sequence:
NM_006500.2, SEQ ID NO: 6) and (B) amino acid (NCBI Reference Sequence: NM_006491.2, SEQ ID
NO:
7) sequences of human MCAM. The coding sequence (residues 30-1970) is indicated in bold in the nucleotide sequence.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0095] The present inventors have shown that two molecules expressed on endothelial cells of the blood-brain barrier, namely nerve injury-induced protein-1 (Ninjurin-1) and melanoma cell adhesion molecule (MCAM), are associated with inflammatory conditions such as neuroinflammatory conditions, and that the expression and/or modulation of these molecules may be used in therapeutic, diagnostic and/or prognostic applications.
[0096] More specifically, in the studies described herein, the expression of nerve injury-induced protein-1 (Ninjurin-1) on brain and spinal cord endothelial cells (ECs) and on peripheral blood monocytes, as well as its ability to promote myeloid cell recruitment across the BBB, was investigated. The instant inventors have determined that while Ninjurin-1 is expressed at low levels in healthy human and mouse CNS, its immunoreactivity is localized to CNS microvascular endothelium, as well as on microglia, infiltrating macrophages and dendritic cells during neuroinflammatory events. In the peripheral blood, Ninjurin-1 expression was found predominantly on human CD14+ monocytes, but not on the surface of CD4+ or CD8+ T lymphocytes. Using an oligopeptide corresponding to residue 26 to 37 of Ninjurin-1 (Ninj26.37), it was further demonstrated that Ninjurin-1 significantly contributes to monocyte migration into the CNS in vitro and in vivo during EAE and SCI, and that Ninjurin-1 neutralization protects against EAE and promotes repair following SCI.
[0097] It is also demonstrated that the proportion of T cells expressing melanoma cell adhesion molecule (MCAM) is higher in the peripheral blood (PB) and cerebro-spinal fluid (CSF) of MS patients as compared to healthy controls, and that MCAM+ T cells are enriched in the CSF compared to the PB. Furthermore, it is also shown that MCAM is expressed on human T lymphocytes that are preferentially programmed to secrete the inflammatory cytokines IL-17, IFN-y, GM-CSF and TNF-a following activation.
[0098] Accordingly, in an aspect, the present invention provides a method of preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject, said method comprising administering to said subject an effective amount of a Ninjurin-1 and/or MCAM inhibitor.
[0099] In another aspect, the present invention provides a method of inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., the CNS endothelium), said method comprising contacting said immune 5 cell and/or said endothelium with an effective amount of a Ninjurin-1 and/or MCAM inhibitor.
[00100] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject. The present invention also relates to a use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for preventing or 10 treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject.
[00101] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., a CNS endothelium). The present invention also relates to a use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a 15 medicament for inhibiting the recruitment of an immune cell across the endothelium of a tissue/organ.
[00102] In another aspect, the present invention provides a Ninjurin-1 and/or MCAM
inhibitor for preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject. The present invention further relates to a Ninjurin-1 and/or MCAM
20 inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject.
[00103] In another aspect, the present invention provides a Ninjurin-1 and/or MCAM
inhibitor for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the CNS endothelium. The present invention further relates to a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for inhibiting the recruitment of an immune cell across the CNS endothelium.
[00104] Ninjurin-1 (also known as NIN1 or NINJ1) is a type 3b membrane protein known to interact in a homophilic manner through an extracellular binding motif. The nucleotide and amino acid sequences of human and mouse Ninjurin-1 are illustrated in Fig. 8.
While Ninjurin-1 is expressed during embryogenesis and is thought to contribute to CNS and peripheral nervous system (PNS) development, its expression is strikingly up-regulated on neurons and Schwann cells during experimental peripheral nerve injury. In rats, functional inhibition of Ninjurin-1/Ninjurin-1 homotypic interaction using the blocking oligopeptide (Ninj26_37) was shown to reduce post-lesional neurite outgrowth, suggesting a positive influence of Ninjurin-1 on nerve regeneration.
[00105] MCAM (also known as CD146 and MUC18) is a cell surface glycoprotein belonging to the immunoglobulin superfamily involved in cell adhesion, and in cohesion of the endothelial monolayer at intercellular junctions in vascular tissue. It also promotes tumor progression of many cancers including melanoma and prostate cancer. It is known to interact in a homotypic/homophilic manner and may also bind to other ligands.
The nucleotide and amino acid sequences of human MCAM are shown in Fig. 29.
[00106] "Inflammatory condition" or "autoimmune condition" as used herein refers to a condition associated with inflammation of an organ or tissue and/or a dysregulated immune response, and which generally involves cell, organ or tissue damage.
Inflammatory conditions are typically characterized by various events including dilatation of arterioles, capillaries, and venules, increased permeability and blood flow; exudation of fluids, including plasma proteins; and/or immune cell recruitment/migration to the site of inflammation. In an embodiment, the above-mentioned Inflammatory condition is associated with the recruitment of immune cells to the site of inflammation. In a further embodiment, the above-mentioned immune cells are myeloid cells, such as monocytes, macrophages and/or dendritic cells. In another embodiment, the above-mentioned immune cells are T cells (or T
lymphocytes), for example CD4+ and/or CD8+ T cells. In a further embodiment, the above-mentioned T cells are inflammatory cytokine-secreting T cells or inflammatory cytokine-secreting T cell precursors. As used herein, "inflammatory cytokine-secreting T cell" refers to a T cell having the potential/capacity to perform effector functions such as to secrete inflammatory cytokines (e.g., IL-17, IFN-y) and/or mediate cytotoxic killing, for example following activation/stimulation (e.g., T-cell receptor(TCR)-mediated activation/stimulation). In an embodiment, the above-mentioned inflammatory cytokine-secreting T cell express one or more of the markers depicted in Figs. 14 to 20 (e.g., CD161, CD107a and CD69).
In a further embodiment, the above-mentioned inflammatory cytokine-secreting T cell is a Th17 cell (Dong C., Microbes Infect. 2009 11(5): 584-8. Epub 2009 Apr 14). The term "inflammatory cytokine-secreting T cell precursor" refers to a T cell which has the potential/capacity to differentiate into an inflammatory cytokine-secreting T
cell following exposure to specific conditions (polarizing conditions), such as Th17-polarizing conditions.
[00107] "Neuroinflammatory condition" as used herein refers to a condition associated with inflammation of the nervous system, and more particularly the central nervous system (CNS), and which is associated with cell/tissue damage. It is typically characterized by, for example, increased glial activation, increased pro-inflammatory cytokine/chemokine levels (e.g., TNF-a, IFN-y, IL-1p), increased blood-brain-barrier permeability, and/or increased immune cell (e.g., leukocyte) recruitment/invasion to the CNS. It includes chronic neuroinflammation, such as an inflammation associated with chronic activation of cells of the immune system (i.e., autoimmune-associated neuroinflammation). Such chronic neuroinflammation is observed, for example, in multiple sclerosis. It also includes acute neuroinflammation, such as inflammation resulting from an initial trauma to the CNS. Acute neuroinflammation is observed, for example, following CNS injury (e.g., spinal cord injury), and is associated with CNS tissue damage beyond the original site of injury.
[00108] In an embodiment, the above-mentioned neuroinflammatory condition is multiple sclerosis (MS). In a further embodiment, the above-mentioned MS is of one of the following subtypes: clinically isolated syndromes (CIS) suggestive of MS, relapsing-remitting MS, primary progressive MS, secondary progressive MS, progressive relapsing MS, or borderline forms of MS (e.g., Devic's disease, Balo concentric sclerosis, Schilder's diffuse sclerosis, Marburg multiple sclerosis), as well as any neurological diseases with signs and symptoms suggestive of MS.
[00109] In another embodiment, the above-mentioned neuroinflammatory condition is a neural injury, in a further embodiment spinal cord injury, and in a further embodiment secondary spinal cord injury.
[00110] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic or therapeutic result. An effective amount refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:
[00111] (A) Preventing the disease; for example, preventing a neuroinflammatory disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, [00112] (B) Inhibiting the disease; for example, inhibiting a neuroinflammatory disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and [00113] (C) Ameliorating the disease; for example, ameliorating a neuroinflammatory disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
[00114] The amount of the Ninjurin-1 and/or MCAM inhibitor which is effective in the prevention and/or treatment of a particular disease, disorder or condition (e.g., a neuroinflammatory disease, disorder or condition) will depend on the nature and severity of the disease, the chosen prophylactic/therapeutic regimen, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient.
Typically, 0.001 to 1000 mg/kg of body weight/day will be administered to the subject. In an embodiment, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in a further embodiment of about 1 mg/kg to about 100 mg/kg, in a further embodiment of about 10 mg/kg to about 50 mg/kg, may be used. The dose administered to a patient, in the context of the present invention should be sufficient to effect/induce a beneficial prophylactic and/or therapeutic response in the patient over time.
The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems.
For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.
[00115] In an embodiment, the above-mentioned treatment comprises the use/administration of more than one (i.e., a combination of) active/therapeutic agent, at least one of which being the above-mentioned Ninjurin-1 and/or MCAM inhibitor. The combination of prophylactic/therapeutic agents and/or compositions of the present invention may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent may be administered to a patient before, concomitantly, before and after, or after a second active agent is administered. The agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time. In an embodiment, the one or more active agent(s) is used/administered in combination with one or more agent(s) currently used to prevent or treat the disorder in question (e.g., a neuroinflammatory condition such as MS or SCI). In an embodiment, a Ninjurin-1 inhibitor is co-administered with an MCAM inhibitor.
[00116] As used herein, the term "Ninjurin and/or MCAM inhibitor" includes any compound able to directly or indirectly affect the regulation of Ninjurin-1 and/or MCAM by reducing for example the expression of Ninjurin-1 and/or MCAM (i.e., at the transcription and/or translation level), or a Ninjurin-1 and/or MCAM activity. It includes intracellular as well as extracellular Ninjurin-1 and/or MCAM inhibitors. Without being so limited, such inhibitors include siRNA, antisense molecules, proteins, peptides, small molecules, antibodies, etc.
[00117] As used herein the terms "Ninjurin-1 and/or MCAM activity" and "Ninjurin-1 and/or MCAM function" refer to detectable enzymatic, biochemical or cellular activity attributable to Ninjurin-1 and/or MCAM. Ninjurin-1 activity may also be measured by protein-protein binding assay using purified Ninjurin-1 and a purified Ninjurin-1 ligand (e.g., Ninjurin-1). As such, in an embodiment, determining whether a compound decreases Ninjurin-1 activity comprises determining whether the compound inhibits or decreases Ninjurin-1 - Ninjurin-1 homotypic binding. Similarly, MCAM activity may also be measured by protein-protein binding assay using purified MCAM and a purified MCAM ligand (e.g., MCAM). As such, in an embodiment, determining whether a compound decreases MCAM activity comprises determining whether the compound inhibits or decreases MCAM - MCAM homotypic binding.
[00118] In an embodiment, such a binding assay may be performed using cells expressing Ninjurin-1 and/or MCAM on their surface, thus via measurement of cell-cell binding of such Ninjurin-1- and/or MCAM-positive cells.
[00119] Ninjurin-1 activity may also be measured in a cell recruitment/migration assay, such as the assay described in Examples 4 and 6 below, or using a Ninjurin-mediated adhesion assay, as described for example in Araki et al., J. Biol Chem. (1997) 272(34):
21373-21380 and in U.S. Patent No. 6,559,288).
[00120] Ninjurin-1 and/or MCAM activity could also be indirectly measured by evaluating the level of expression of Ninjurin-1 and/or MCAM, or a fragment thereof, in cells as well as in a biological sample (tissue, organ, fluid). Ninjurin-1 and/or MCAM
expression levels could 5 be determined at the polypeptide and/or nucleic acid levels using any standard methods known in the art (see below). Ninjurin-1 and/or MCAM activity could also be indirectly measured by evaluating the level of expression or activity of a reporter gene (e.g., luciferase, R-galactosidase, alkaline phosphatase, GFP) operably linked to a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene.
10 [00121] In an embodiment, the above-mentioned Ninjurin-1 and/or MCAM
inhibitor is an antisense or RNAi-based inhibitory molecule.
[00122] Generally, the principle behind antisense technology is that an antisense molecule hybridizes to a target nucleic acid and effects modulation of gene expression such as transcription, splicing, translocation of the RNA to the site of protein translation, 15 translation of protein from the RNA. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi is a form of antisense-20 mediated gene silencing involving the introduction of dsRNA (typically of less than 30 nucleotides in length, and generally about 19 to 24 nucleotides in length) leading to the sequence-specific reduction of targeted endogenous mRNA levels, here the RNA
transcript of the Ninjurin-1 gene. Such dsRNA are generally substantially complementary to at least part of an RNA transcript of the Ninjurin-1 gene. Another example of modulation of gene 25 expression is the RNA analogue Locked Nucleic Acid (LNA). Other examples relate to double stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), micro-RNA (miRNA). The use of single stranded antisense oligonucleotides (ASO) is also encompassed by the method of the present invention. Sequence-specificity makes antisense compounds extremely attractive as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of any one of a variety of diseases.
[00123] Chemically modified nucleosides, such as 2'-substituted arabinonucleosides (e.g., 2'F-ANA) and 2'-substituted RNA (e.g., 2'F-RNA), may be used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
[00124] As used herein "antisense molecule" is meant to refer to an oligomeric molecule, particularly an antisense oligonucleotide for use in modulating the activity or function of nucleic acid molecules encoding a Ninjurin-1 polypeptide (e.g., the polypeptide of SEQ ID
NO: 2) and/or MCAM polypeptide (e.g., the polypeptide of SEQ ID NO: 7), ultimately modulating the amount of Ninjurin-1 and/or MCAM produced in cells (e.g., CNS
cells, immune cells). This is accomplished by providing oligonucleotide molecules which specifically hybridize with one or more nucleic acids encoding Ninjurin-1 and/or MCAM. As used herein, the term "nucleic acid encoding a Ninjurin-1 polypeptide"
encompasses DNA
encoding said polypeptide, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA (e.g., a nucleic acid comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO: 1). As used herein, the term "nucleic acid encoding an MCAM polypeptide" encompasses DNA encoding said polypeptide, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA (e.g., a nucleic acid comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO: 6). The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
The overall effect of such interference with target nucleic acid function is the decrease of the expression of Ninjurin-1 and/or MCAM.
[00125] In the context of this invention, "hybridization" means hydrogen bonding between complementary nucleoside or nucleotide bases. Terms "specifically hybridizable" and "complementary" are the terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. Such conditions may comprise, for example, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 to 70 C for 12 to 16 hours, followed by washing. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
[00126] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. Examples of modified nucleotides include a 2'-0- methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate and a non-natural base comprising nucleotide.
[00127] Methods to produce antisense molecules directed against a nucleic acid are well known in the art. The antisense molecules of the invention may be synthesized in vitro or in vivo.
[00128] Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, TX, USA), New England Biolabs Inc. (Beverly, MA, USA) and Invitrogen (Carlsbad, CA, USA).
[00129] The antisense molecule may be expressed from recombinant viral vectors, such as vectors derived from adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, and the like. Such vectors typically comprises a sequence encoding an antisense molecule of interest (e.g., a dsRNA specific for Ninjurin-1) and a suitable promoter operatively linked to the antisense molecule for expressing the antisense molecule. The vector may also comprise other sequences, such as regulatory sequences, to allow, for example, expression in a specific cell/tissue/organ, or in a particular intracellular environment/compartment. Methods for generating, selecting and using viral vectors are well known in the art.
[00130] Antisense molecules (siRNA and shRNA) inhibiting the expression of human Ninjurin-1 are commercially available, for example from Santa Cruz Biotechnology Inc. (Cat.
Nos sc-75915, sc-75915-SH and sc-75915-V) and from Invitrogen (NINJ1 Stealth RNAiTM
siRNA, Cat. Nos. HSS107188, HSS107189 and HSS107190). Antisense molecules (siRNA
and shRNA) inhibiting the expression of human MCAM are commercially available, for example from Invitrogen (MCAM Stealth RNAiTM siRNA, Cat. Nos. HSS106378, and HSS106380) and Applied Biosystems (siRNA ID Nos. s8571, s8572 and s8573).
Also, several providers (e.g., InvivoGen, Qiagen, Ambion, Inc.) offer custom-made antisense synthesis services.
[00131] In an embodiment, the above-mentioned Ninjurin-1 and/or MCAM inhibitor is a Ninjurin-1 and/or MCAM antibody.
[00132] By "Ninjurin-1 antibody" or "anti-Ninjurin-1" in the present context is meant an antibody capable of detecting (i.e. binding to) a Ninjurin-1 protein or a Ninjurin-1 protein fragment. In an embodiment, the above-mentioned antibody inhibits the biological activity of Ninjurin-1, such as Ninjurin-1/Ninjurin-1 homotypic interaction or Ninjurin-1-mediated cell recruitment. In another embodiment, the Ninjurin-1 protein fragment is an extracellular domain of Ninjurin-1.
[00133] By "MCAM antibody" or "anti-MCAM" in the present context is meant an antibody capable of detecting (i.e. binding to) a MCAM protein or a MCAM protein fragment. In an embodiment, the above-mentioned antibody inhibits the biological activity of MCAM, such as MCAM/MCAM homotypic interaction or MCAM-mediated cell recruitment.
[00134] In an embodiment, the antibody specifically binds to (interacts with) a Ninjurin-1 polypeptide (e.g., the polypeptide of SEQ ID NO: 2) and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as a Ninjurin-1 polypeptide. In another embodiment, the antibody specifically binds to (interacts with) an MCAM polypeptide (e.g., the polypeptide of SEQ ID
NO: 7) and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as an MCAM polypeptide.
[00135] The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.
[00136] In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH
Laboratories). The term antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g., Fab and Fab' fragments) which inhibit or neutralize their respective interaction domains and/or are specific thereto.
[00137] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c.), intravenous (i.v.) or intraperitoneal (i.p.) injections of the relevant antigen (e.g., Ninjurin-1 or MCAM polypeptide or a fragment thereof) with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R'N=C=NR, where R and R1 are different alkyl groups.
[00138] Animals may be immunized against the antigen (e.g., a Ninjurin-1 or MCAM
polypeptide or a fragment thereof, such as a fragment comprising residues 26 to 37 of a Ninjurin-1 polypeptide), immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 pg for rabbits or 5 pg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with 1/5 to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at 5 multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as 10 alum are suitably used to enhance the immune response.
[00139] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA
methods (e.g., U.S. Patent No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 as well as U.S. Patent 15 Application Publication Nos. 2002/0160970 and 2003/0083293.
[00140] In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes 20 then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
[00141] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the 25 enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[00142] In an embodiment, the above-mentioned antibody is raised against an extracellular domain of a Ninjurin-1 or MCAM polypeptide (i.e. an extracellular domain of a 30 Ninjurin-1 polypeptide is used for immunization). In a further embodiment, the above-mentioned antibody is raised against a Ninjurin-1 polypeptide fragment comprising a domain corresponding to residues 28 to 35 of a Ninjurin-1 polypeptide. In a further embodiment, the above-mentioned antibody is raised against a Ninjurin-1 polypeptide fragment comprising a domain corresponding to residues 26 to 37 of a Ninjurin-1 polypeptide.
[00143] Ninjurin-1 and/or MCAM inhibitors may also be in the form of non-antibody-based scaffolds, such as avimers (Avidia); DARPins (Molecular Partners); Adnectins (Adnexus), Anticalins (Pieris) and Affibodies (Affibody). The use of alternative scaffolds for protein binding is well known in the art (see, for example, Binz and PlOckthun, 2005, Curr. Opin.
Biotech. 16: 1-11).
[00144] In an embodiment, the Ninjurin-1 inhibitor (e.g., anti-Ninjurin-1 antibody) blocks Ninjurin-1/Ninjurin-1 homotypic interaction, for example by competing for the Ninjurin-1 binding domain or by sterically hindering the Ninjurin-1 binding domain. In a further embodiment, the above-mentioned Ninjurin-1 inhibitor binds to an extracellular domain of a Ninjurin-1 polypeptide. In another embodiment, the above-mentioned inhibitor binds to a domain corresponding to or comprised within residues 28 to 35 of a Ninjurin-1 polypeptide.
In a further embodiment, the above-mentioned antibody binds to a domain corresponding to or comprised within residues 26 to 37 of a Ninjurin-1 polypeptide (e.g., the mouse or human Ninjurin-1 polypeptide depicted in Fig. 8).
[00145] In an embodiment, the MCAM inhibitor (e.g., anti-MCAM antibody) blocks MCAM/MCAM homotypic interaction, for example by competing for the MCAM binding domain or by sterically hindering the MCAM binding domain. In a further embodiment, the above-mentioned MCAM inhibitor binds to an extracellular domain of a MCAM
polypeptide.
[00146] In an embodiment, the above-mentioned Ninjurin-1 inhibitor is a peptide comprising a domain of formula I:
[00147] Xaa'-Xaa2-Arg-Trp-Xaa3-Xaa4-Arg-Xaa5-Arg-Xaa6-Xaa7-Xaa8 (I), [00148] wherein [00149] Xaa', Xaa2, Xaa6, Xaa7 and Xaa6 is any amino acid or is absent;
[00150] Xaa3, Xaa4 and Xaa5 is any amino acid;
[00151] or a functional analog thereof.
[00152] In an embodiment, Xaa2 is Pro. In an embodiment, Xaa' is Pro. In an embodiment, Xaa6 is Pro. In an embodiment, Xaa7 is Ile. In an embodiment, Xaa8 is Asn. In an embodiment, Xaa3 is Gly. In an embodiment, Xaa4 is Leu. In an embodiment, Xaa5 is Asn or Leu.
[00153] In an embodiment, the above-mentioned domain is Pro-Pro-Arg-Trp-Gly-Leu-Arg-Asn-Arg-Pro-lle-Asn (SEQ ID NO: 5).
[00154] In another embodiment, the above-mentioned Ninjurin-1 inhibitor is a peptide consisting of the domain of formula I defined above.
[00155] The term "amino acid" as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of peptides.
Examples of naturally-occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. Other amino acids include for example norleucine, norvaline, cyclohexyl alanine, biphenyl alanine, homophenyl alanine, naphthyl alanine, pyridyl alanine, phenyl alanines substituted at the ortho, para and meta positions with alkoxy, halogen or nitro groups etc. These amino acids are well known in the art of biochemistry/peptide chemistry.
[00156] The term "functional analog" (or "functional variant/derivative") refers to a peptide/domain having at least one modification as compared to the peptide/domain defined above, and which retain the activity of inhibiting Ninjurin-1 and/or MCAM
(e.g., blocking Ninjurin-1/Ninjurin-1 and/or MCAM/MCAM homotypic interaction).
[00157] In embodiments, the modification is a deletion, an insertion, a substitution or a chemical modification of one or more amino acids. The modification may be, for example, a deletion of (e.g., one to five) consecutive or non-consecutive amino acids, a substitution of (e.g., one to five) amino acids, one or more substitution(s) of a naturally occurring amino acid (L-amino acid) by a corresponding D-amino acid, an extension of the sequence by e.g., one, two, three or more amino acids.
[00158] In an embodiment, the above-mentioned substitution(s) are conserved amino acid substitutions.
[00159] As used herein, the term "conserved amino acid substitutions" (or sometimes "conservative amino acid substitutions") refers to the substitution of one amino acid for another at a given location in the peptide/domain, where the substitution can be made without substantial loss of the relevant Ninjurin-1 inhibitory activity. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide/domain by routine testing.
[00160] In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6) are assigned to amino acid residues (as detailed in U.S. Patent. No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); GIn (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5);
Leu (-1.8); Ile (-1.8);
Tyr (-2.3); Phe (-2.5); and Trp (-3.4).
[00161] In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8);
Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
[00162] In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral:
Gly, Ser, Thr, Cys, Asn, GIn, Tyr.
[00163] Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylgiycine, N-methylisoleucine, phenyiglycine, cyclohexylalanine, norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chiorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid.
[00164] In other embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. (J.
Mol. Biol.
179: 125-142, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, lie, Pro, Met and Trp, and genetically, encoded hydrophilic amino acids include Thr, His, Glu, Gin, Asp, Arg, Ser, and Lys.
[00165] Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents.
[00166] An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held, equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain.
Genetically encoded aliphatic amino acids include Ala, Leu, Val, and lie.
[00167] A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and GIn.
[00168] An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and 5 Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH
due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.
[00169] The above classifications are not absolute and an amino acid may be classified in 10 more than one category. In addition, amino acids can be classified based on known behavior and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains.
[00170] Conservative changes can also include the substitution of a chemically 15 derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid.
[00171] In addition to the substitutions outlined above, synthetic amino acids providing similar side chain functionality can also be introduced into the peptide/domain. For example, aromatic amino acids may be replaced with D- or L-naphthylalanine, D- or L-phenylglycine, 20 D- or L-2-thienylalanine, D- or L-1-, 2-, 3-, or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L-p-biphenylalanine D-or L-p-methoxybiphenylalanine, D- or L-2-indole(alkyl)alanines, and D- or L-alkylalanines wherein the alkyl group is selected from 25 the group consisting of substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, and iso-pentyl.
[00172] Non-carboxylate amino acids can be made to possess a negative charge, as provided by phosphono- or sulfated (e.g., -SO3H) amino acids, which are to be considered as non-limiting examples.
30 [00173] Other substitutions may include unnatural alkylated amino acids, made by combining an alkyl group with any natural amino acid. Basic natural amino acids such as lysine and arginine may be substituted with alkyl groups at the amine (NH2) functionality. Yet other substitutions include nitrile derivatives (e.g., containing a CN-moiety in place of the CONH2 functionality) of asparagine or glutamine, and sulfoxide derivative of methionine. In addition, any amide linkage in the peptide/domain may be replaced by a ketomethylene, hydroxyethyl, ethyl/reduced amide, thioamide or reversed amide moieties, (e.g., (-C=O)-CH2-), (-CHOH)-CH2-), (CH2-CH2-), (-C=S)-NH-), or (-NH-(-C=O) for (-C=O)-NH-)).
[00174] Other modifications are also included within the definition of functional analog of the peptide/domain of the present invention. For example, the size of the peptide/domain can be reduced by deleting one or more amino acids, and/or amino acid mimetics or dipeptide mimics containing non-peptide bonds may be used. Examples of using molecular scaffolds such as benzodiazepine, azepine, substituted gamma lactam rings, keto-methylene pseudopeptides, P-turn dipeptide cores and R-aminoalcohols for these purposes are known to peptide chemists and are described in for example Peptidomimetic protocols (Methods in molecular medicine Vol. 23) W. M. Kazmierski (ed.), Humana Press and Advances in Amino Acid Mimetics and Peptidomimetics, Vols. 1 & 2, A. Abell (Ed).
[00175] Covalent modifications of the peptide/domain are thus included within the scope of the present invention. Such modifications may be introduced into the peptide/domain for example by reacting targeted amino acid residues of the peptide/domain with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
The following examples of chemical derivatives are provided by way of illustration and not by way of limitation.
[00176] Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH
6Ø
[00177] Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters, e.g., methyl picolinimidate; pyridoxal phosphate; pyridoxal; chIoroborohydride;
trinitrobenzenesulfonic acid; O-methyl isourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
[00178] Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues is typically performed in alkaline conditions because of the high pKa of the guanidine functional group.
Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosinyl residues per se is well-known, such as for introducing spectral labels into tyrosinyl residues by reaction with aromatic diazonium compounds or tetranitromethane. N-acetylimidazol and tetranitromethane may be used to form O-acetyl tyrosinyl species and 3-nitro derivatives, respectively.
[00179] Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'-N=C=N-R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethyl pentyl) carbodiimide.
Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Other modifications of the peptides in the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N-methyl amino acids) and, in some instances, amidation of the C-terminal carboxyl groups, according to known method steps.
[00180] Covalent attachment of fatty acids (e.g., C6-C16) to the peptide/domain may confer additional biological properties such as protease resistance, plasma protein binding, increased plasma half-life, intracellular penetration, etc. The above description of modification of a peptide/domain does not limit the scope of the approaches nor the possible modifications that can be engineered.
[00181] In embodiments, the N- and/or C-terminal amino acids of the above-mentioned peptide may be modified by amidation, acetylation, acylation or other modifications known in the art. In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end of the peptide) of the peptide is modified (e.g., for protection against degradation). In an embodiment, the modification is acylation with a C2-C16 acyl group, in a further embodiment, the modification is acetylation.
[00182] In an embodiment, the carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the peptide) of said peptide is modified (e.g., for protection against degradation). In an embodiment, the modification is an amidation.
[00183] In an embodiment, the above-mentioned peptide contains about 100 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 90 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 80 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 70 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 60 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 50 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 40 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 30 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 20 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 15 amino acids or less. In a further embodiment, the above-mentioned peptide contains between about 5 to about 15 amino acids. In a further embodiment, the above-mentioned peptide contains 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
[00184] The above-mentioned peptide may be produced by expression in a host cell comprising a nucleic acid encoding the peptide (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be readily synthesized by automated solid phase procedures well known in the art. Suitable syntheses can be performed by utilizing "T-boc" or "Fmoc" procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A
Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the peptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37: 933-936, 1996;
Baca et al., J. Am.
Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45:
209-216, 1995; Schnolzer and Kent, Science 256: 221-225, 1992; Liu and Tam, J. Am.
Chem. Soc.
116: 4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91: 6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31: 322-334, 1988). Other methods useful for synthesizing the peptides are described in Nakagawa et al., J. Am. Chem. Soc.
107: 7087-7092, 1985. Commercial providers of peptide synthetic services may also be used to prepare synthetic peptides in the the D- or L-configuration. Such providers include, for example, Advanced ChemTech (Louisville, Ky.), Applied Biosystems (Foster City, Calif.), Anaspec (San Jose, Calif.), and Cell Essentials (Boston, Mass.).
[00185] Peptides and peptide analogues comprising naturally occurring amino acids encoded by the genetic code may also be prepared using recombinant DNA
technology using standard methods. Peptides produced by recombinant technology may be modified (e.g., N-terminal acylation [e.g., acetylation], C-terminal amidation, cyclization/formation of a loop within the peptide [e.g., via formation of a disulphide bridge between Cys residues]) using methods well known in the art. Therefore, in embodiments, in cases where a peptide described herein contains naturally occurring amino acids encoded by the genetic code, the peptide may be produced using recombinant methods, and may in embodiments be subjected to for example the just-noted modifications (e.g., acylation, amidation, cyclization).
[00186] "Recombinant technology" refers to the production of a peptide or polypeptide by recombinant techniques, wherein generally, a nucleic acid encoding peptide is inserted into a suitable expression vector which is in turn used to transform/transfect a host cell to produce the protein. The term "recombinant" when made in reference to a protein or a polypeptide refers to a peptide, polypeptide or protein molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.
Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as "recombinant" therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention.
Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation/transfection. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species.
Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.
[00187] The peptides of the invention can be purified by many techniques well known in the art, such as reverse phase chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like. The actual conditions used to purify a particular peptide or peptide analog will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art. For affinity chromatography purification, any antibody which specifically binds the peptide or peptide analog may for example be used.
5 [00188] In an embodiment, the above-mentioned peptide is substantially pure.
A
compound is "substantially pure" when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or 10 produced by recombinant technology will generally be substantially free from its naturally associated components. A substantially pure peptide can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
15 [00189] In another aspect, the present invention provides a composition comprising the above-mentioned Ninjurin-1 and/or MCAM inhibitor and a pharmaceutically acceptable carrier or excipient. In an embodiment, the above-mentioned composition is used for the prevention or treatment of an inflammatory condition, such as a neuroinflammatory condition.
20 [00190] Such compositions may be prepared in a manner well known in the pharmaceutical art. Supplementary active compounds can also be incorporated into the compositions. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The 25 carrier can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration (see Remington: The Science and Practice of Pharmacy by Alfonso R.
Gennaro, 2003, 21th edition, Mack Publishing Company).
30 [00191] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of active agent(s)/composition(s) suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
[00192] Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds/compositions of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain excipients, (e.g., lactose) or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
[00193] For preparing pharmaceutical compositions from the compound(s)/composition(s) of the present invention, pharmaceutically acceptable carriers are either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substance, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
[00194] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may typically contain from 5% or 10% to 70% of the active compound/composition. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
[00195] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
[00196] Aqueous solutions suitable for oral use are prepared by dissolving the Ninjurin-1 and/or MCAM inhibitor in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
[00197] In an embodiment, the Ninjurin-1 and/or MCAM inhibitor is formulated/administered such that it comes into contact with neural cells or neural tissue, such as central nervous system (CNS) cells or tissue. Such tissue includes brain and spinal cord (e.g., cervical, thoracic, or lumbar) tissue. As such, in embodiments, the Ninjurin-1 and/or MCAM inhibitor can be administered to treat neural cells/tissue in vivo via direct intracranial injection or injection into the cerebrospinal fluid (e.g., intrathecal injection).
Alternatively, the Ninjurin-1 and/or MCAM inhibitor can be administered systemically (e.g.
intravenously) and may come into contact with the affected neural tissue via lesions (where the blood-brain barrier is compromised), or, in a further embodiment, may be in a form capable of crossing the blood-brain barrier and entering the neural system (e.g., CNS).
Further, in an embodiment, a composition of the invention may be formulated for such administration to neural cells/tissue.
[00198] The composition may also contain more than one active compound for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. It may be desirable to use the above-mentioned Ninjurin-1 and/or MCAM inhibitor or composition in addition to one or more agents currently used to prevent or treat the disorder in question. The above-mentioned Ninjurin-1 and/or MCAM
inhibitor may be formulated in a single composition or in several individual compositions which may be co-administered in the course of the treatment.
[00199] The invention further provides a kit or package comprising the above-mentioned Ninjurin-1 and/or MCAM inhibitor or the above-mentioned composition, together with instructions for (i) the prevention and/or treatment of an inflammatory (e.g., neuroinflammatory) condition in a subject and/or (ii) for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ, such as the CNS endothelium. The kit may further comprise, for example, containers, buffers, a device (e.g., syringe) for administering the Ninjurin-1 and/or MCAM inhibitor or a composition comprising same.
[00200] Given the correlation between Ninjurin-1 and MCAM expression/activity and (i) inflammation (e.g., neuroinflammation) as well as (ii) immune cell recruitment to the CNS
endothelium, compounds which are capable of decreasing Ninjurin-1 and/or MCAM
expression/activity may be used for the prevention and/or treatment of inflammatory conditions and/or for inhibiting immune cell recruitment to the endothelium of a tissue/organ, such as the CNS endothelium. Therefore, the invention further relates to screening methods (e.g. in vitro methods) for the identification and characterization of compounds capable of decreasing/inhibiting Ninjurin-1 and/or MCAM expression and/or activity, which may be used for the prevention and/or treatment of inflammatory conditions and/or for inhibiting immune cell recruitment to the endothelium of a tissue/organ, such as the CNS
endothelium. In an embodiment, the above-mentioned endothelium is exposed to an inflammatory environment (e.g., the endothelium of an inflamed tissue/organ).
[00201] In another aspect, the present invention provides a method of identifying a compound for preventing or treating an inflammatory condition, said method comprising determining whether: (a) a level of expression of a Ninjurin-1 and/or MCAM
nucleic acid or encoded polypeptide; (b) a level of Ninjurin-1 and/or MCAM activity (e.g., Ninjurin-1 and/or MCAM homotypic binding activity); or (c) a combination of (a) and (b), is decreased in the presence of a test compound relative to in the absence of said test compound;
wherein said decrease is indicative that said test compound may be used for preventing or treating said inflammatory condition.
[00202] In another aspect, the present invention provides a method of identifying or characterizing a compound for preventing or treating an inflammatory condition, said method comprising: (a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for preventing or treating said inflammatory condition.
[00203] In another aspect, the present invention provides a method of identifying a compound for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., CNS endothelium), said method comprising determining whether: (a) a level of expression of a Ninjurin-1 and/or MCAM
nucleic acid or encoded polypeptide; (b) a level of Ninjurin-1 and/or MCAM activity (e.g., Ninjurin-1 and/or MCAM homotypic binding activity); or (c) a combination of (a) and (b), is decreased in the presence of a test compound relative to in the absence of said test compound;
wherein said decrease is indicative that said test compound may be used for inhibiting the recruitment of an immune cell across the endothelium of the tissue/organ.
[00204] In another aspect, the present invention provides a method of identifying a compound for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., CNS endothelium), said method comprising:
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM
gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound:
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for inhibiting the recruitment of an immune cell across the endothelium of the tissue/organ.
[00205] In an embodiment, the above-mentioned Ninjurin-1 and/or MCAM activity is a binding activity. Methods to measure the binding of a compound to Ninjurin-1 are well known in the art (see, for example, U.S. Patent No. 6,559,288).
[00206] The above-noted screening method or assay may be applied to a single test compound or to a plurality or "library" of such compounds (e.g., a combinatorial library). Any such compounds may be utilized as lead compounds and further modified to improve their therapeutic, prophylactic and/or pharmacological properties for preventing and/or treating a neuroinflammatory condition.
[00207] Test compounds (drug candidates) may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are 5 available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides.
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
10 [00208] Screening assay systems may comprise a variety of means to enable and optimize useful assay conditions. Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g., protease inhibitors), temperature control means for optimal activity and/or stability, of Ninjurin-1, and detection 15 means to enable the detection of its activity. A variety of such detection means may be used, including but not limited to one or a combination of the following:
radiolabelling, antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., 20 biotin/(strept)avidin), and others.
[00209] As noted above, the invention further relates to methods for the identification and characterization of compounds capable of decreasing Ninjurin-1 and/or MCAM
gene expression. Such a method may comprise assaying Ninjurin-1 and/or MCAM gene expression in the presence versus the absence of a test compound. Such gene expression 25 may be measured by detection of the corresponding RNA or protein, or via the use of a suitable reporter construct comprising one or more transcriptional regulatory element(s) normally associated with a Ninjurin-1 gene, operably-linked to a reporter gene.
[00210] A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the 30 second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
[00211] Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since, for example, enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory element" is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked. The expression of such a reporter gene may be measured on the transcriptional or translational level, e.g., by the amount of RNA or protein produced. RNA may be detected by for example Northern analysis or by the reverse transcriptase-polymerase chain reaction (RT-PCR) method (see for example Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA).
[00212] Protein levels may be detected either directly using affinity reagents (e.g., an antibody or fragment thereof (for methods, see for example Harlow, E. and Lane, D (1988) Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); a ligand which binds the protein) or by other properties (e.g., fluorescence in the case of green fluorescent protein) or by measurement of the protein's activity, which may entail enzymatic activity to produce a detectable product (e.g., with altered spectroscopic properties) or a detectable phenotype (e.g., alterations in cell growth/function). Suitable reporter genes include but are not limited to chloramphenicol acetyltransferase, beta-D
galactosidase, luciferase, or green fluorescent protein (GFP).
[00213] Ninjurin-1 and/or MCAM protein expression levels could be determined using any standard methods known in the art. Non-limiting examples of such methods include Western blot, tissue microarray, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and assays based on a property of the protein including but not limited to DNA
binding, ligand binding, or interaction with other protein partners.
[00214] Methods to determine Ninjurin-1 and/or MCAM nucleic acid (mRNA) levels are known in the art, and include for example polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR) (e.g., as in Example 3 below), in situ PCR, SAGE, quantitative PCR (q-PCR), in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA
hybridization platforms.
For RNA expression, preferred methods include, but are not limited to:
extraction of cellular mRNA and Northern blotting using labeled probes that hybridize to transcripts encoding all or part of one or more of the genes of this invention; amplification of mRNA
expressed from one or more of the genes of this invention using gene-specific primers, polymerase chain reaction (PCR), quantitative PCR (q-PCR), and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the cells, which is then labeled and used to probe cDNAs or oligonucleotides encoding all or part of the genes of this invention, arrayed on any of a variety of surfaces; in situ hybridization.
[00215] In embodiments, competitive screening assays may be done by combining a Ninjurin-1 and/or MCAM polypeptide, or a fragment thereof (a Ninjurin-1 and/or MCAM
binding domain) and a probe to form a probe : Ninjurin-1 and/or MCAM binding domain complex in a first sample followed by adding a test compound. The binding of the test compound is determined, and a change, or difference in binding of the probe in the presence of the test compound indicates that the test compound is capable of binding to the Ninjurin-1 and/or MCAM binding domain and potentially modulating Ninjurin-1 and/or MCAM
activity.
[00216] The binding of the test compound may be determined through the use of competitive binding assays. In this embodiment, the probe is labeled with an affinity label such as biotin. Under certain circumstances, there may be competitive binding between the test compound and the probe, with the probe displacing the candidate agent. In one case, the test compound may be labeled. Either the test compound, or a compound of the present invention, or both, is added first to the Ninjurin-1 binding domain for a time sufficient to allow binding to form a complex.
[00217] The assay may be carried out in vitro utilizing a source of Ninjurin-1 and/or MCAM which may comprise a naturally isolated or recombinantly produced Ninjurin-1 and/or MCAM (or a variant/fragment thereof), in preparations ranging from crude to pure. Such assays may be performed in an array format. In certain embodiments, one or a plurality of the assay steps are automated.
[00218] In embodiments, the assays described herein may be performed in a cell or cell-free format.
[00219] A homolog, variant and/or fragment of Ninjurin-1 and/or MCAM which retains activity (e.g., a binding activity) may also be used in the methods of the invention.
[00220] "Homology", "homologous" and "homolog" refer to sequence similarity between two polypeptide molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences. Two nucleotide or amino acid sequences are considered "substantially identical"
if, when optimally aligned (with gaps permitted), they share at least about 50%
sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%, e.g., with any of the sequences described herein.
As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An "unrelated" or "non-homologous"
sequence shares less than 40% identity, though preferably less than about 25 %
identity, with any of the sequences described herein.
[00221] Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.).
Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings).
Software for performing BLAST analysis may be available through the National Center for Biotechnology Information.
The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl.
Acad. Sci. USA
89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[00222] An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M
NaHPO4, 7%
sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 C, and washing in 0.2 x SSC/0.19% SIDS at 42 C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
2.10.3).
Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4i 7% SDS, 1 mM EDTA at 65 C, and washing in 0.1 x SSC/0.1% SDS at 68 C (see Ausubel, et al. (eds), 1989, supra).
Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, stringent conditions are selected to be about 5 C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
[00223] In an embodiment, the above-mentioned homolog, variant and/or fragment of Ninjurin-1 comprises a region corresponding to residues 28 to 35 (Arg28-Pro35) of the human Ninjurin-1 polypeptide (Fig. 8B). In a further embodiment, the above-mentioned Ninjurin-1 polypeptide or fragment thereof comprises a region corresponding to residues 26 to 37 (Pro26-Asn37) of the human Ninjurin-1 polypeptide.
[00224] The present inventors have shown in human samples and neuroinflammatory mouse models (EAE and SCI) that Ninjurin-1 is expressed or overexpressed on CNS cells 5 and peripheral immune cells (myeloid cells) in neuroinflammatory conditions, and thus that Ninjurin-1 may be used as a biological marker for the detection and characterization of neuroinflammatory conditions. They have also demonstrated that the proportion of T cells expressing MCAM is higher in the peripheral blood (PB) and cerebro-spinal fluid (CSF) of MS patients as compared to healthy controls.
10 [00225] Therefore, in another aspect, the invention relates to the diagnosis and prognosis of an inflammatory condition, such as a neuroinflammatory condition. The invention thus provides a method for diagnosing or prognosing an inflammatory condition in a subject based on the expression and/or activity of Ninjurin-1 and/or MCAM determined in a sample (e.g., a CNS sample or a blood/blood cell sample) from the subject. The expression and 15 activity of Ninjurin-1 and/or MCAM in the sample may be determined using the assays/methods described above.
[00226] In an embodiment, the method may comprise determining whether Ninjurin-and/or MCAM activity and/or expression is modulated, e.g., upregulated or increased, relative to a control/reference activity or expression. In yet another embodiment, the control 20 Ninjurin-1 and/or MCAM expression or activity can be selected from an established standard, a corresponding Ninjurin-1 and/or MCAM expression or activity determined in the subject (in a sample from the subject) at an earlier time; a corresponding Ninjurin-1 and/or MCAM expression or activity determined in a control subject known to not having an inflammatory condition (e.g., a healthy subject). In such cases, an increased or higher 25 expression and/or activity in the sample from the subject relative to the control activity or expression is indicative that the subject has an inflammatory condition.
"Higher expression"
as used herein refers to (i) higher expression of Ninjurin-1 and/or MCAM on one or more given cells present in the sample and/or (ii) increased amount (absolute or relative amount) of Ninjurin-1- and/or MCAM-expressing/positive cells in the sample.
30 [00227] In another embodiment, the control Ninjurin-1 and/or MCAM
expression or activity is a corresponding expression or activity in a control subject known to have an inflammatory condition. In such a case, a comparable or higher Ninjurin-1 and/or MCAM
expression and/or activity in a sample from the subject relative to the control expression or activity is indicative that the subject has an inflammatory condition.
[00228] Methods for normalizing the level of expression of a gene are well known in the art. For example, the expression level of a gene of the present invention can be normalized on the basis of the relative ratio of the mRNA level of this gene to the mRNA
level of a housekeeping gene or the relative ratio of the protein level of the protein encoded by this gene to the protein level of the housekeeping protein, so that variations in the sample extraction efficiency among cells or tissues are reduced in the evaluation of the gene expression level. A "housekeeping gene" is a gene the expression of which is substantially the same from sample to sample or from tissue to tissue, or one that is relatively refractory to change in response to external stimuli. A housekeeping gene can be any RNA
molecule other than that encoded by the gene of interest that will allow normalization of sample RNA
or any other marker that can be used to normalize for the amount of total RNA
added to each reaction. For example, the GAPDH gene, the G6PD gene, the actin gene, ribosomal RNA, 36B4 RNA, PGK1, RPLPO, or the like, may be used as a housekeeping gene.
[00229] Methods for calibrating the level of expression of a gene are well known in the art.
For example, the expression of a gene can be calibrated using reference samples, which are commercially available. Examples of reference samples include, but are not limited to:
StratageneTM QPCR Human Reference Total RNA, ClontechTM Universal Reference Total RNA, and XpressRefTM Universal Reference Total RNA.
[00230] In an embodiment, the above-mentioned method comprises determining the level of a Ninjurin-1 nucleic acid (e.g., the nucleic acid of SEQ ID NO: 1 or a nucleic acid which encodes the polypeptide of SEQ ID NO: 2) in the sample. In another embodiment, the above-mentioned method comprises determining the level of a Ninjurin-1 polypeptide (e.g., the polypeptide of SEQ ID NO: 2) in the sample. In an embodiment, the level of a Ninjurin-1 polypeptide is determined using an anti-Ninjurin-1 antibody. In another embodiment, the above-mentioned method comprises determining the level of an MCAM nucleic acid (e.g., the nucleic acid of SEQ ID NO: 6 or a nucleic acid which encodes the polypeptide of SEQ ID
NO: 7) in the sample. In another embodiment, the above-mentioned method comprises determining the level of an MCAM polypeptide (e.g., the polypeptide of SEQ ID
NO: 7) in the sample. In an embodiment, the level of an MCAM polypeptide is determined using an anti-an MCAM antibody.
[00231] "Sample" or "biological sample" refers to any solid or liquid sample isolated from a live being. In a particular embodiment, it refers to any solid or liquid sample isolated from a human, such as a biopsy material, blood, saliva, synovial fluid, urine, amniotic fluid and cerebrospinal fluid. In an embodiment, the above-mentioned sample is obtained from the central nervous system (e.g., a CNS cell, tissue or fluid). In a further embodiment, the CNS
cell is obtained by a biopsy. In a further embodiment, the CNS cell is a CNS
endothelial cell, such as an endothelial cell of the blood-brain barrier.
[00232] In another embodiment, the above-mentioned sample is a blood sample or a blood cell sample. In a further embodiment, the above-mentioned blood cell sample is a peripheral blood mononuclear cell (PBMC) sample. In an embodiment, the above-mentioned blood or blood cell sample comprises myeloid cells, such as monocytes and/or dendritic cells. In another embodiment, the above-mentioned blood or blood cell sample comprises lymphoid cells, and more particularly T cells (CD4+ and/or CD8+ T cells). In an embodiment, the above-mentioned blood or blood cell sample comprises memory T cells.
[00233] In an embodiment, the above-mentioned blood or blood cell sample may be submitted to one or more cell depletion or enrichment steps, so as to enrich the sample in one or more cell types of interest (e.g., myeloid cells, such as monocytes and/or dendritic cells, or T cell, such as memory T cells). In an embodiment, the above-mentioned method comprises determining the proportion or relative amount of Ninjurin-1+- and/or MCAM+-cell in said sample and comparing it to a corresponding proportion or relative amount in a control/reference sample. Accordingly, in another aspect, the present invention provides a method for diagnosing an inflammatory condition, such as a neuroinflammatory condition, in a first subject, said method comprising (a) determining the extent/amount of Ninjurin-1 and/or MCAM expression (e.g., the number of Ninjurin-1- and/or MCAM-expressing cells and/or the intensity of Ninjurin-1 and/or MCAM expression per cell) in a sample said first subject (b) comparing said amount to a corresponding reference amount; and (c) diagnosing an inflammatory condition, such as a neuroinflammatory condition, based on said comparison.
[00234] The proportion of Ninjurin-1- and/or MCAM-expressing/positive cells in a sample may be measured using methods well known in the art, for example by flow cytometry, as described below.
[00235] In another aspect, the present invention provides a method for monitoring the course of treatment of a patient suffering from an inflammatory condition, such as a neuroinflammatory condition (e.g., MS, SCI), the method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein a decrease in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at an earlier time is indicative that said patient is responsive to said treatment. In an embodiment, a similar or an increased expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at an earlier time is indicative that said patient is not responsive to said treatment.
[00236] In another aspect, the present invention provides a method of determining whether a subject is suffering from a relapse (exacerbation) of an inflammatory condition, said method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein an increase in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at a time point of remission is indicative that said patient is suffering from a relapse of said inflammatory condition. The treatment regimen (e.g., drug, dose) may thus be adapted accordingly.
[00237] In an embodiment, the above-mentioned expression and/or activity of Ninjurin-1 and/or MCAM is determined based on the amount of Ninjurin-1- and/or MCAM-expressing cells in said sample. In a further embodiment, the above-mentioned amount is a relative amount (e.g., the proportion of Ninjurin-1- and/or MCAM-expressing cells relative to the total cells present in the sample, or relative to another cell type present in the sample).
[00238] In an embodiment, the methods of diagnosis/prognostication noted above may be performed in conjunction with the therapeutic/prophylactic methods noted above, for preventing and/or treating an inflammatory condition in a subject. Such a method thus comprises the diagnosis or prognostication of an inflammatory condition in a subject and, in accordance with the diagnosis/prognosis, decreasing Ninjurin-1 and/or MCAM
levels in the subject (e.g., in a cell, tissue or organ of the subject) thereby to prevent or treat the inflammatory condition.
[00239] The present invention also provides a kit or package comprising a reagent useful for determining Ninjurin-1 and/or MCAM expression and activity (e.g., a ligand that specifically binds a Ninjurin-1 and/or MCAM polypeptide such as an antibody, or a ligand that specifically binds a Ninjurin-1 and/or MCAM nucleic acid such as an oligonucleotide).
Such kit may further comprise, for example, instructions for the prognosis and/or diagnosis of an inflammatory condition, control samples, containers, reagents useful for performing the methods (e.g., buffers, enzymes), etc.
[00240] As used herein the term "subject" is meant to refer to any animal, such as a mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In an embodiment, the above-mentioned subject is a mammal, in a further embodiment a human.
[00241] The present inventors have further determined that MCAM expression permits the identification of T cells having the capacity or the potential to perform effector functions, for example to produce various inflammatory cytokines (e.g., IL-17 and IFN-y), upon activation (e.g., TCR-mediated activation).
[00242] Accordingly, in another aspect, the present invention provides a method of identifying an inflammatory cytokine-secreting T cell or a precursor thereof in a sample, said method comprising identifying MCAM-expressing cells in the sample, for example by contacting said cell with an MCAM ligand (e.g., (i) MCAM, (ii) an MCAM binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii)) and identifying said inflammatory cytokine-secreting T cell precursor based on the binding to said MCAM ligand.
[00243] In another aspect, the present invention provides a method of purifying an inflammatory cytokine-secreting T cell or a precursor thereof from a cell sample, or enriching a sample comprising a cell sample in inflammatory cytokine-secreting T cells or a precursor thereof, said method comprising contacting said sample with an MCAM ligand (e.g., (i) MCAM, (ii) an MCAM binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii)) and isolating or purifying said inflammatory cytokine-secreting T cell or precursor thereof on the basis of its binding to the MCAM
ligand.
[00244] In another aspect, the present invention provides a kit for identifying and/or purifying an inflammatory cytokine-secreting T cell or a precursor thereof from a cell sample, or enriching a sample comprising a cell sample in inflammatory cytokine-secreting T cell precursors, said kit comprising an MCAM ligand (e.g., (i) MCAM, (ii) an MCAM
binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii)) and instructions for identifying and/or purifying the inflammatory cytokine-secreting T cell or a precursor thereof from the sample.
[00245] In an embodiment, the above-mentioned MCAM ligand is labeled (using a fluorescent moiety, for example).
5 [00246] In an embodiment, the above-mentioned MCAM ligand is an MCAM-specific antibody or MCAM-binding fragment thereof.
[00247] In an embodiment, the above-mentioned MCAM ligand is bound to a solid support, such a bead (e.g., a magnetic bead) such as to allow easy separation/isolating of the cell bound to the MCAM ligand from the other cells in the sample (using a column, for 10 example), and thus to obtain a sample enriched in inflammatory cytokine-secreting T cells and/or precursors thereof.
[00248] In an embodiment, the above-mentioned inflammatory cytokine-secreting T cell or precursor thereof has the capacity to secrete preferentially: (i) IL-17, (ii) IFN-y, (iii) TNF-a, (iv) GM-CSF, (v) IL-8, or (vi) any combination of (i) to (v), upon activation/stimulation, such 15 as antigen-specific or non-antigen specific TCR-mediated stimulation.
[00249] In an embodiment, the above-mentioned inflammatory cytokine-secreting T cell or precursor thereof is a memory T cell. In another embodiment, the above-mentioned inflammatory cytokine-secreting T cell or precursor thereof is a CD4+ T cell.
In a further embodiment, the above-mentioned inflammatory cytokine-secreting T cell precursor is a 20 CD4+ T cell having the capacity to differentiate into an IL-17-secreting cell (typically referred to as a "Th17 cell") following exposure to specific polarizing condition.
[00250] The present invention is illustrated in further details by the following non-limiting examples.
Example 1: Materials and Methods 25 [00251] Patients and sample collection. All MS patients were observed at the CHUM-Notre-Dame Hosptial MS clinic and diagnosed according to the McDonald criteria. Human peripheral blood (PB) was collected from MS patients and from healthy donors.
PB
mononuclear cells were obtained from heparinized whole blood using FicollTM
density gradient separation (Amersham Biosciences, Baie D'Urfe, Quebec, Canada). CD14+
30 monocytes, CD4+ CD45RO+ lymphocytes and MCAM+ T cells were isolated using the magnetic cell sorting (MACSTM) isolation columns, according to manufacturer's protocol (Miltenyi, Auburn, CA).
[00252] BBB-endothelial cell isolation and culture. BBB-endothelial cells were isolated from non-epileptic material according to a previously published protocol (Prat et al., J
Neuropathol Exp Neurol. 2000 59(10):896-906; Biernacki et al., J Neuropathol Exp Neurol.
2001 60(12): 1127-36; Prat et al., Arch Neurol. 2002 59(3): 391-7). BBB-endothelial cells were grown in primary cultures in media composed of Medium 199 (Gibco Invitrogen, Burlington, ON, Canada) supplemented with 20% clone M3 conditioned media, 10%
fetal bovine serum (FBS), 5% normal human serum (HS), 0.13% endothelial cell growth supplement (ECGS) and 0.2% insulin-transferrin-selenium on 0.5% gelatin-coated tissue culture plastic plates (all reagents from Sigma, Oakville, ON, Canada). For treatments, the BBB-endothelial cells were grown in culture media in the presence of 40%
astrocyte conditioned media (ACM), until they reach confluency. When indicated BBB-endothelial cells were activated for 24 hours with 100 U/ml of Tumor Necrosis Factor (TNF) and 100 U/ml of Interferon (IFN)-y (Biosource-Invitrogen, Carlsbad, CA, USA) in the presence of 40% ACM
and the absence of ECGS. As previously demonstrated, these cells express factor VIII, von Willebrand factor, Ulex Agglutenens Europaensis-1-binding sites, endothelial antigen HT-7;
and are susceptible to tumor necrosis factor (TNF)-induced CD54 and CD106 up-regulation.
Immunoreactivity for glial fibrillary acidic protein and a-myosin could not be detected, confirming the absence of contaminating astrocytes and smooth muscle cells, respectively.
We also confirmed the absence of monocytes and macrophages by immunostaining with anti-CD14 and anti-CD11c antibodies.
[00253] Astrocyte isolation and culture. Astrocytes were cultured as previously described (Jack et al., J Immunol. 2005 175(7): 4320-4330; Wosik et al., J Neurosci.
2007 27(34):
9032-422007). Astrocytes were grown in primary cultures in complete Dulbecco's Modified Eagle Media (DMEM) (Invitrogen) supplemented with 10% FBS on plastic plates.
Astrocyte-conditioned media (ACM) was harvested once a week from confluent flasks and used in the culture media of the BBB-endothelial cells when mentioned.
[00254] Leukocyte isolation and culture. Venous blood samples were obtained from consenting healthy donors or Multiple Sclerosis (MS) patients in accordance with institutional guidelines. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by a density gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare, Bio-Sciences AB, Sweden). Ex vivo CD4+, CD8+, CD14+ and CD19' cells were isolated from PBMCs using CD4, CD8, CD14 and CD19 mouse anti-human MicroBeadsTM respectively (Miltenyi Biotec Inc., Auburn, CA, USA). To generate mature DCs, PBMCs were first cultured in (Wisent Inc., St-Bruno, Qc, Canada), supplemented with 5% HS (Sigma), 2mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycine (Sigma) for 2 hours at 37 C to enable the cells to adhere. The media was removed and the cells were washed with PBS to remove the non adherent cells. Fresh culture media containing 20 ng/ml of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, BD Biosciences) and 10 ng/ml of Interleukin (IL)-4 (R&D Systems, Minneapolis, MN, USA). Culture media was replaced every 2 days with fresh media, containing GM-CSF and IL-4, to remove the non adherent cells. After 6 days, 100 ng/ml of Iipopolysaccharide (LPS, from Escherichia coli Serotype 0111:B4, Sigma) was added. Mature DCs were harvested 2 days later for analysis.
[00255] Flow Cytometric analysis of Ninjurin-1 expression. Cells were harvested and resuspended in FACS buffer composed of PBS supplemented with 1% FBS (Biosource-Invitrogen) and 0.1% NaN3 (Sigma). The cells were incubated with HS (Biosource-Invitrogen) to prevent unspecific binding. BBB-endothelial cells and PBMCs were incubated for 1 h at 4 C with the primary unconjugated monoclonal mouse anti-Ninjurin-1 antibody (20 pg/ml, BD Biosciences), or with the Functional Grade Purified Mouse IgG2a Isotype Control (20 pg/ml, ebioscience, Inc., San Diego, CA, USA). Cells were then incubated for 30 min at 4 C with an allophycocyanin (APC)-conjugated goat anti-mouse Ig (2 pg/ml, BD
Biosciences) to detect specific binding. Cells were then counter-stained for 30 min at 4 C
with mouse anti-human conjugated antibodies: Human Leukocyte Antigen (HLA)-ABC, Intracellular cell adhesion molecule (ICAM)-1, CD3, CD4, CD8, CD19, HLA-DR, CD14, CD83, CD123, CD209, CD11c or with corresponding isotype controls (all from BD
Biosciences). To study the expression of Ninjurin-1 on murine immune cells, the mouse monoclonal anti-Ninjurin-1 antibody was biotinylated, to prevent unspecific binding of the secondary antibody. A buffer exchange was performed with a Slide-A-Lyzer Dialysis Cassette (3.5-20K Cassettes, Thermo Scientific, Rockford, IL, USA) to remove glycerol and bovine serum albumin (BSA) from the antibody solution using MelonTM Gel IgG
Spin Purification Kit. Biotin was added using the EZ-Link Micro NHS-PEO4-Biotinylation kit according to the manufacturer's instructions (Thermo Scientific). Murine peripheral blood or CNS mononuclear cells were incubated for 1h at 4 C with the primary biotinylated monoclonal mouse anti-Ninjurin-1 antibody (5 pg/ml, BD Biosciences), or with the Biotin-conjugated IgG2a, K isotype control (5 pg/ml, ebioscience). Cells were then incubated for 30 min at 4 C with secondary antibody goat anti-peroxidase-APC (2 pg/ml, BD
Biosciences).
Counter-staining with anti-mouse CD3, CD4, CD8, CD11 c, F4/80 or with corresponding isotype controls (BD Biosciences and BioLegend, San Diego, CA, USA) were performed.
Cells were acquired on a BD LSRTM II Flow cytometer and analyzed using the BD
FACSDivaTM software (BD Biosciences).
[00256] Western blot. Ex vivo CD4+, CD8+, CD14+ and CD19+ cells (isolated using MACSTM beads, Miltenyi Biotec), mature dendritic cells, primary cultures of BBB endothelial and CNS material from EAE or SCI animals were lysed in denaturing buffer (0.1%
SDS in 50mM Tris-HCI pH 8.5) with Proteinase Inhibitor Cocktail (BD BaculoGoldTM, BD
Biosciences), and sonicated using the Vibra CeIITM ultrasonic processor (Sonics & Materials, Inc., Newtown, CT, USA). Proteins were quantified using the BCATM Protein Assay kit (Thermo Scientific). Thirty micrograms of proteins were separated on a 12% SDS-PAGE gel and the proteins were transfered on Immun-BlotTM PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked (1 h at room temperature) with 5%
donkey serum (Sigma), or with 5% non-fat dry milk, in Tris Buffered Saline-0.1 % Tween 20 (Sigma-Aldrich, St-Louis, MO, USA) and then incubated overnight at 4 C with the primary antibody: polyclonal sheep anti-Human Ninjurin-1 (1:100 dilution; R&D Systems) or monoclonal mouse anti-Ninjurin-1 (1:150 dilution; BD Biosciences). Secondary antibody Peroxidase-conjugated affiniPureTM Donkey Anti-Sheep IgG (H+L) (1:10,000 dilution;
Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or secondary antibody rabbit anti-mouse immunoglobulins/HRP (1:1000 dilution; DakoCytomation, Glostrup, Denmark), and ECLTM Westernblotting Analysis System (AmershamTM GE Healthcare, Buckinghamshire, UK) were used to detect specific binding. Mouse anti-13-Actin (1:20,000 dilution; Sigma) was used as a loading control (rabbit anti-mouse immunoglobulins/HRP;
1:1000 dilution; DakoCytomation). HepG2 cell lysate was used as a positive control for the expression of Ninjurin-1 (BD Bioscience). For spinal cord injury material, proteins were extracted from 5 mm length of spinal cord tissue containing the lesion site.
[00257] Immunocytofluorescent stainings. BBB-endothelial cells were grown in chambers slides (Lab TekTM, NuncTM, by Thermoscientific) and were either un-treated or treated for 24 h with TNF and IFN-y (100 U/ml). Cells were fixed for 10 minutes at room temperature with 70% ethanol and incubated overnight at 4 C with mouse monoclonal anti-Ninjurin-1 (1:50 dilution; BD Biosciences). Counter-stainings with F-actin (Sigma) were performed. Slides were mounted using Gelvatol containing either Hoechst 33258 pentahydrate (10 pg/ml, Molecular Probes, Eugene, OR, USA) or TO-PRO-3 (Invitrogen), as nuclear stains.
[00258] Immunofluorescent stainings of human and mouse CNS material. Luxol Fast Blue (LFB) and H&E stainings (Wosik et al., 2007, supra) were performed on human and mouse brain tissue specimens obtained from four MS patients (autopsy) and EAE/SCI
animals.
Sections showing acute demyelinating lesions and active perivascular mononuclear cell infiltration were selected (8 to 12 blocks per MS donor), and compared to normal-appearing white matter from the same donors (8 blocks per donor) and to non-neurological disease controls (3 donors; 9-11 blocks per donor). Mean age was 49 6 years and disease duration ranged from 3 to 23 years. The cause of death was pneumonia (2), urosepsis (1) and barbiturate intoxication (1). Sections (n=40) from MS patients (n= 5) and disease controls (n=5) were fixed in -20 C acetone for 10 minutes, hydrated in PBS and blocked with the avidin/biotin blocking kit (Invitrogen). CNS material from EAE (n=6) and SCI
(n=6) animals were collected following rapid intra-cardiac PBS perfusion and snap-frozen in liquid nitrogen.
Non-specific immunoglobulin binding was blocked with 10% goat serum for 30 minutes at room temperature (rt). Sections were incubated for 40 minutes with Biotin-labelled mouse anti-Ninjurin-1 (1/50, R&D Systems) diluted in 3% goat serum and washed 7 times with PBS
and 0.05% Tween 20 after each incubation. Ninjurin-1 immunostaining was revealed using Cy3- or HRP-labelled streptavidin (DakoCytomation). Sections were mounted using Gelvatol containing either Hoechst 33258 pentahydrate (10 pg/ml, Molecular Probes) or (Invitrogen), as nuclear stains. Additional immunostainings were performed using monoclonal Abs raised against human or mouse CD3, -CD4, CD11c, F4/80, Mac-2 and -IBA-1. Negative controls were performed omitting the primary antibody.
Fluorescence was visualized on a LeicaTM DM6000 B epifluorescent microscope equipped with a digital camera (Leica Microsystems, Wetzlar Germany) or on a LeicaTM SP5 confocal microscope. Images were acquired using OpenlabTM 4Ø4 (Improvision, Waltham, MA) and processed and analyzed with Adobe PhotoshopTM CS2 (Adobe, Mountain View, CA).
[00259] Ninjurin-1 blocking peptides. Peptides corresponding to the adhesion motif of Ninjurin-1, located between amino acids 26 and 37 of Ninjurin-1 [human sequence:
PARWGWRHGPIN (SEQ ID NO: 5); mouse sequence: PPRWGLRNRPIN (SEQ ID NO: 5)], were used as a blocking peptides (referred to as Ninj26_37). Custom Ninj26_37 blocking peptides were synthesized by Alpha Diagnostic International (ADI, San Antonio, TX, USA).
[00260] In vitro model of the BBB. BBB-ECs grown in primary cultures were used to generate an in vitro model of the human BBB. Human BBB-endothelial cells (25 x cells/chamber) were grown in primary culture on 3 pm porous membrane (Becton Dickinson Labware, Franklin Lakes, NJ, USA), coated with 0.5% gelatin (Sigma), in endothelial cell culture media supplemented with 40% ACM, for 4 days to reach confluency. The BBB-endothelial cells were treated with 0.4 mM of human Ninj26_37 (PARWGWRHGPIN, SEQ ID
NO: 5) one hour prior to the addition of the leukocytes. Human ex vivo CD14+
monocytes, or 5 CD4, or CD8+ lymphocytes were isolated, from consenting healthy donors, as described above. The leukocytes were added to the upper chamber (1 X 106 cells /
chamber) and were allowed to migrate across human BBB-endothelial cells for 24h in the presence or absence of the human Ninj26_37 blocking peptide. PBS was used as a vehicle control.
The cells that migrated through the BBB-endothelial cells, were recovered from the lower chamber and 10 counted manually. All migration data shown represent at least 3 independent experiments performed in triplicate.
[00261] EAE mice. Six to eight week-old female wild-type C57BL/6 mice were purchased from Charles River Laboratories (Montreal, Qc, Canada). Experimental autoimmune encephalomyelitis (EAE) was induced by active immunization in female C57BL/6 mice 15 (Jackson Laboratory, Bar Habor, Maine, USA). Seven to eight week old mice were injected subcutaneously with 200 pg of myelin oligodendrocyte glycoprotein (MOG)35-55 peptide [Sequence: MEVGWYRSPFSRWHLYRNGK (SEQ ID NO: 8)] emulsified in complete Freund's Adjuvant supplemented with 600 pg of Mycobacterium tuberculosis H37RA
(DIFCO
Laboratories, Detroit, MI, USA). On day 0 and 2, mice were injected intraperitoneally with 20 500 ng of Pertussis toxin (List Biological Laboratories, INC., Campbell, CA, USA). Starting on day 3, mice were injected intraperitoneally, twice a day, with 200 pg of murine Ninj26-37 blocking peptide. PBS was used as the vehicle control. Animals were monitored daily for signs of EAE and the scoring system was as follows: 0 = no clinical symptoms;
0.5 = partial floppy tail, 1 = floppy tail; 2 = ataxia; 2.5 = weakness in hind limbs, 3 =
paralysis of one hind 25 limb; 4 = paralysis of both hind limbs, 5 = moribund.
[00262] Spinal cord injury. Adult (8-10 weeks old) female C57BL/6 mice were anesthetized with ketamine:xylazine:acepromazine (50:5:1 mg/kg). After performing a laminectomy at the 11th thoracic vertebrae, the exposed spinal cord was contused using the Infinite HorizonsTM Impactor device (Precision Scientific Instrumentation, Lexington, KY).
30 Injuries were made using a force of 60 kDynes, and only animals that had tissue displacements ranging between 500-700 pm were used for experiments. Mice were injected intraperitoneally with 100 pg of the murine Ninj26-37 blocking peptide or PBS
(in 200 pl) every 12 hours, starting one hour after injury and for 7 days, or with PBS as a vehicle control.
Locomotor recovery was evaluated in an open-field test using the Basso Mouse Scale (BMS) (Basso D.M. et al., (2006). J Neurotrauma. 23(5): 635-59). The BMS
analysis of hind limb movements and coordination was carried out by two trained independent technicians and the consensus score taken. The final score is presented as mean SEM.
[00263] Quantitative Real Time PCR (qRT-PCR). RNA from 5 mm length of the uninjured and injured spinal cord containing the lesion site was harvested at 1, 3, 7, 14, 21 and 28 dpi and extracted using RNeasyTM Lipid Tissue kit (Qiagen, Mississauga, Ontario, Canada).
Three individual spinal cords per time point were used. 1 pl of the RT product was added to 24 pi of BrilliantTM SYBRTM Green quantitative PCR Master Mix (Stratagene), and qRT-PCR
was performed to analyze the expression of Ninjurin-1 (MX4000 apparatus, Stratagene). The primers used for Ninjurin-1 amplification were: 5'-AGG GCC ATG AAG ATC AGA ACT
GGA
-3' (sense, SEQ ID NO: 9) and 5'- ATG GAT TTG CTG CAT GTC CTT GGG -3' (antisense, SEQ ID NO: 10). GAPDH was used as a housekeeping gene for control, and the primers used for GAPDH amplification were: 5'- CAA AGT TGT CAT GGA TGA CC -3' (sense, SEQ
ID NO: 11) and 5'- CCA TGG AGA AGG CTG GGG -3' (antisense, SEQ ID NO: 12) (Cayrol et al., Nat Immunol. 2008 9(2): 137-145). The amount of cDNA was calculated based on the threshold cycle (CT) value, and was standardized by the amount of housekeeping gene using the 2- MOCT method (Livak and Schmittgen, Methods 2001 25(4): 402-408).
MCT
were calculated as follow: MMCT= (CT. Target - CT, GAPDH) - (CT. Target - CT, GADPH).
[00264] Western blot analysis of MCAM expression in whole cell lysates and lipid rafts of BBB-ECs. Specific protein detection was performed by Western blot. Forty (40) pg of BBB-EC proteins were analyzed by standard SDS-PAGE using anti-MCAM antibody (mouse, 1/100 ebioscience). Lipid raft were isolated from confluent BBB-ECs using a previously published protocol (Wosik et al., 2007, supra). Cholesterol, phospholipid and protein concentrations in each fraction were assayed using commercially available kits: Cholesterol assay kit (Molecular Probes), Phospholipids B colorimetric method kit (Wako) and BCA
Protein assay kit (Pierce). Lipid raft markers were assessed by SDS-PAGE (GM1, horse radish peroxidase (HRP) -cholera toxin B subunit, Molecular Probes; goat anti-CD59, 1/200, R&D Systems). Specific binding was revealed with HRP-conjugated anti-mouse and anti-goat antibody using the ECLTM system and anti-actin was used as a loading control.
[00265] Immunostaining analysis of MCAM expression on CNS vessel. Formaldehyde-fixed CNS specimens from five MS-affected individuals were examined, (2 males, both 42 years of age and 3 females, 54, 62 and 66 years of age); evolution of disease was between 2 and 20 years. Five control brains from patients who died of non-neurological traumatic injury (34, 46, 51, 69 years of age and unknown, 3 males and 2 females) were used as controls. Autopsies were performed within 6-12 h from time of death. Active MS
lesions were identified by LFB and haematoxilin and eosin (H&E) staining and defined as areas of demyelination associated with intense perivascular immune cell infiltration (48.2 3 nuclei/field centered on a vessel). Normal appearing white matter (NAWM) from MS cases and control brains (non-neurological traumatic death) were defined as areas of normal myelin staining in the absence of immune cell infiltration (21.6 4 nuclei/vessel and 23.2 5 nuclei/vessel, respectively). For immunohistofluorescence, 5 m thick sections were deparaffinized and stained with antibodies specific for MCAM (mouse monoclonal, 1/50, ebioscience), Cav-1 (polyclonal rabbit, 1/75, Santa Cruz Biotechnology), or a with donkey anti mouse-specific antibody (1:400, biotin-conjugated, followed by streptavidin-FITC; Dako and Jackson ImmunoResearch Laboratories, respectively) or a Cy3-conjugated goat rabbit-specific antibody (1:400, 30 min; Jackson ImmunoResearch). Nuclei were stained with TOPRO-3 (1:300 in PBS; Molecular Probes). Control staining was performed with an isotype control antibody. For immunocytochemistry human BBB-ECs were treated, or not, with IFN-y and TNF (100 U/ml) for 16 h. Isolated peripheral blood CD4+ T lymphocytes were stained with CFSE (green) and allowed to migrated for 2 h and then fixed with 4%
paraformaldehyde. MCAM was detected with anti-MCAM (mouse monoclonal, 1/50, ebioscience) followed by a goat anti-mouse antibody conjugated to FITC (green, 1:400, biotin-conjugated, Dako. All images were acquired using a LeicaTM SP5 confocal microscope and analyzed using the LeicaTM LAS AF software (Leica Wetzlar).
[00266] MCAM and Ninjurin-1 expression in peripheral blood and cerobro-spinal fluid samples. Venous blood samples were obtained from consenting healthy donors and MS
patients. PB mononuclear cells were obtained from heparinized whole blood using FicolITM
density gradient separation (Amersham Biosciences, Baie D'Urfe, Quebec, Canada).
Mononuclear cells were assessed by flow cytometry using anti-CD14-FITC
(fluorescein isothiocyanate), anti-CD8-R-PE (R-Phycoerythrin), anti-CD19-PE-Cy5 and anti CD4-pacific blue and other cell specific markers (all antibodies from BD PharMingen). All cell staining were acquired on a BD LSRTM II (Becton Dickinson) and analyzed using the BD
FACSDivaTM software (BD Bioscience).
[00267] T cell stimulation. CD14+ monocytes, CD4+ CD45RO+ lymphocytes and MCAM+ T
cells were isolated from PB mononuclear cells using the magnetic cell sorting (MACSTM) isolation columns, according to manufacturer's protocol (Miltenyi, Auburn, CA). Isolated CD4 T cells (one million cells/ml) were cultured with autologous CD14+ monocytes (0.5 million/ml). CD4+ lymphocytes were labeled with the vital dye 5,6-carboxyfluorescein diacetate succinimidyl ester as required (CFSE), were cultured for 5 days in the presence of autologous CD14+ monocytes, anti-CD3 (OKT3, 2.5 pg/ml) and either IL-2 (20 units/ml) or IL-23 (10 ng/ml) in the presence of anti-IFN-gamma (clone K3.53) and anti-IL-4 (clone 3007, both at 5 pg/ml) in RPMI 1640 supplemented with 5% human serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin (Sigma). After 5 days, the proliferation of the CD4+ T lymphocyte, the presence of the different activation marker (CD45RO, CD161, CD25, CD69, CD107a, etc.) and the capacity to produce cytokines (IFN-y, IL-17, GM-CSF, IL-4, TNF, etc.) were assessed by flow cytometry. All cell staining were acquired on a BD
LSRTM II (Becton Dickinson) and analyzed using the BD FACSDivaTM software (BD
Bioscience).
[00268] Intracellular cytokine staining. For intracellular cytokine staining, CD4+
lymphocyte and the corresponding APC co-cultures were activated for 18 h with 1 pg/mL
ionomycin and 20 ng/mL phorbol 12-myristate 13-acetate (PMA) in the presence of 2 pg/mL
brefeldin A (Sigma) for the last 6 h of co-culture. Recombinant cytokines and antibodies were purchased from R&D Systems. Cells were stained for surface markers and were then fixed and permeabilized in 4% (w/v) paraformaldehyde with 0.1% (w/v) saponin in Hank's Balanced Salt Solution for 10 minutes at room temperature. Intracellular staining was performed by incubating cells with antibodies against cytokines (eBioscience, San Diego, CA) (1 mg/mL) for 30 m on ice in PBS buffer containing 0.1 % (w/v) saponin, 1 % FBS, 0.1 %
(w/v) NaN3, followed by two washes and resuspended in staining buffer (1%
(v/v) FBS, 0.1%
(w/v) NaN3 in PBS). All cell staining were acquired on a BD LSRTM II (Becton Dickinson) and analyzed using the BD FACSDivaTM software (BD Bioscience).
[00269] Statistical analysis. Statistical analyses were performed using PRISM
GraphpadTM Software (San Diego, CA) and data are presented as the mean the standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed followed by Bonferroni multiple comparison post-test for all experiments except for the migration across the BBB, which was done using two-way ANOVA. Only p values < 0.05 were considered significant. The data reported are either from either one representative experiment out of 3 independent experiments or pooled from 3 to 10 experiments. Statistical comparison of IL-17 and IFN-y staining was done using non-parametric student T-test. Differences between groups were considered significant when p < 0.05.
Example 2: Expression of Ninjurin-1 in human BBB-endothelial cells and blood cells [00270] Ninjurin-1 is expressed on the surface of human brain endothelial cells in vitro and in situ, and its expression is upregulated in the context of inflammation.
The expression of Ninjurin-1 on BBB-endothelial cells was shown to increase following treatment with proinflammatory cytokines, such as TNF-a and IFN-y, as demonstrated by flow cytometry and immunocytofluorescence (Figs. 1A and 113). In addition to being present on endothelial cells, Ninjurin-1 is also expressed by different subtypes of human leukocytes, preferentially on cells of the myeloid lineage such as CD14+ monocytes, CD68+ macrophages and CD83+
dendritic cells (DCs) (Figs. 2B and 2C). In contrast, human peripheral blood T
and B
lymphocytes (CD4+ T cells, CD8+ T cells, as well as CD19+ B cells) only show weak or modest expression of Ninjurin-1 (Figs. 2A and 2C). Thus, Ninjurin-1, the ligand involved in the interaction with endothelial Ninjurin-1 through homotypic interaction, is primarily expressed by human peripheral blood myeloid cells (myeloid antigen-presenting cells).
Example 3: Expression of Ninjurin-1 in neuroinflammatory conditions [00271] Although Ninjurin-1 signal is weak on vessels in human control non-inflamed CNS
specimens in situ, its expression significantly increases on the cerebral vascular endothelium in MS lesions (Fig. 3A). Ninjurin-1 is also detected in situ in infiltrating immune cells within MS lesions, and particularly in MHC II+, CD68+ and DC-SIGN+ cells (Fig. 3D).
Fig. 3B shows that the proportion of CD14+ and CD11 c+ cells co-expressing Ninjurin-1 is increased in the cerebrospinal fluid (CSF) as compared to the peripheral blood of MS patients.
These results suggest a role for Ninjurin-1 in the histopathology of MS. Similarly, Ninjurin-1 is expressed in spinal cord homogenates of mice affected with experimental autoimmune encephalomyelitis (EAE) and on CD11c+ DCs and F4/80+ macrophages infiltrating the CNS. EAE was induced in C57BL/6 mice by active immunization with myelin oligodendrocytes glycoprotein (MOG)35_ 55 peptide (myelin oligodendrocyte glycoprotein emulsified in complete Freund's adjuvant) as previously described (Cayrol et al., Nat Immunol. 2008 9(2):137-145). An increase in the expression of Ninjurin-1 protein in the spinal cord of mice affected with EAE
was observed (Fig. 4A). Upregulation of Ninjurin-1 expression correlated with higher disease score (Fig.
4A). Furthermore, MHC II+, CD11c+ and F4/80+ myeloid cells that have reached the CNS
express high levels of Ninjurin-1, and their proportion significantly increases in the CNS of mice affected with EAE as compared to the spleen of the same animals, as observed by flow cytometry (Fig. 4B) and immunostaining of spinal cords (Fig. 4C).
Example 4: Effect of Ninjurin-1 blockade on immune cell migration across the human BBB endothelium [00272] Using an in vitro model of the human BBB (Fig. 5A), it was demonstrated that 5 addition of the human blocking peptide Ninj26_37 to BBB-endothelial cells decreases the migration of freshly isolated (ex vivo) peripheral blood human CD14+ monocytes across the endothelium (Fig. 5C). The blocking Ninj26-37 peptide did not any effect on the migration of CD4+ and CD8+ T lymphocytes in this model (Fig. 5B). Fig. 5D shows that Ninjurin-1 staining is observed around CFSE-loaded migrating CD14+ monocytes and in the transmigratory cup 10 (arrowheads).
Example 5: Effect of Ninjurin-1 blockade in murine models of neuroinflammation [00273] Two distinct murine models of CNS inflammation were used: EAE
(autoimmune-based inflammation, mimicking MS in humans) and SCI (traumatic- or injury-induced inflammation). As shown above, Ninjurin-1 is expressed in the CNS of mice affected with 15 EAE, and on infiltrating F4/80+ macrophages and CD11 c+ dendritic cells (Fig. 4). EAE was induced by active immunization of C57BL/6 mice with MOG35-55 peptide, as described above.
Some animals received i.p. injections of 200 g b.i.d. of Ninj26_37 from day 3 to day 23 post-immunization; while control animals received a physiological saline solution.
The data depicted in Fig. 6A shows a significant reduction of the neurological signs, symptoms and 20 clinical scores of the disease in animals treated with the Ninj26_37 blocking peptide as compared to the control group. The histopathological analysis shows an important reduction in tissue damage (demyelination) in animals treated with Ninj26_37 (Fig. 6D).
Furthermore, a reduction of infiltrating immune cells (more particularly F4/80+ macrophages and CD11c+
dendritic cells) was also observed by flow cytometry (Fig. 6B) and immunofluorescence (Fig.
25 6D).
[00274] The infiltration of myeloid cells in the injured spinal cord significantly contributes to tissue damage and delays the clinical and pathological recovery in the murine model of SCI. SCI experiments were performed with the Infinite HorizonsTM impactor device (Precision Scientific Instrumentation). Fig. 7A shows that there is a significant increase in the 30 expression of Ninjurin-1 in spinal cord homogenates of injured mice.
Neutralization of Ninjurin-1 was performed with the murine Ninj26-37 blocking peptide after SCI.
Some animals
10 [0066] Figure 1 shows the expression of nerve injury-induced protein-1 (Ninjurin-1) in vitro in human blood-brain barrier (BBB)-endothelial cells. (A) FACS analyses of the expression of Ninjurin-1 in resting human primary BBB-ECs (left panel) treated with astrocyte conditioned media (ACM) (40%, 24h) (center panel) or treated with pro-inflammatory cytokines TNF + IFN-y (100 U/ml, 24h) in the presence of ACM
(right panel).
Data shown are representative of ten independent experiments. (B) Ninjurin-1 immunostaining of resting or activated BBB-ECs. Confocal microscopy confirmed Ninjurin-1 (green) expression at the surface of BBB-ECs and up-regulation following treatment with TNF and IFN-y (100 U/ml, 16 h). TO-PRO-3 was used for nuclear staining. Scale bar: 10 pm.
Data shown are representative of three independent experiments;
[0067] Figure 2 shows the expression of Ninjurin-1 on human CD14+ monocytes and CD83+ dendritic cells (DCs). (A) FACS analysis of Ninjurin-1 expression on ex vivo human peripheral blood lymphocytes (CD4+, CD8+, CD19+) demonstrates low levels on T
and B
lymphocytes. (B) FACS analysis of Ninjurin-1 expression on human ex vivo CD14+
monocytes, and in vitro CD68+ macrophages and CD83+ dendritic cells (DCs) demonstrates high expression of Ninjurin-1 on myeloid antigen-presenting cells. (C) Western blot (WB) analysis of the expression of Ninjurin-1 in ex vivo human leukocytes (CD8+, CD4+, CD14+
and CD19+). R-actin was used as a loading control. (D) Expression of Ninjurin-1 on human CD68+ microglia grown in primary cultures. Data shown are representative of seven (A), five (C), and two (B and D) independent experiments using an equivalent number of preparations and donors. Isotype controls are shown in clear histograms and Ninjurin-1 immunostaining in gray;
[0068] Figure 3 shows the in situ detection of Ninjurin-1 in multiple sclerosis (MS) lesions. (A) Ninjurin-1 immunostaining in normal-appearing white matter (NAWM) blood vessels of human CNS post-mortem sections (left panel). Ninjurin-1 expression on infiltrating immune cells and on ECs in MS lesions (right panel). Arrowheads show Ninjurin-expressing ECs. Scale bars, 50 pm. (B) Percentage of CD14+ and CD11c+ cells co-expressing Ninjurin-1 in the cerebrospinal fluid (CSF) and the peripheral blood of MS
patients (n = 5). Mean SEM from five independent experiments. (C) Co-expression of Ninjurin-1 and Caveolin-1 on ECs within NAWM from MS patients. Scale bar, 30 pm. (D) Immunofluorescent staining and confocal microscopy analysis of active MS
lesions expressing Ninjurin-1 and either MHC II+ (top panels), CD68+ (middle panels) or DC-SIGN+
(bottom panels). Co-localization is seen in right panels with TO-PRO-3 nuclear staining.
Scale bars, 30 pm. Photomicrographs shown are representative of immunostainings performed on 12 active plaques and 15 NAWM obtained from frozen CNS material of MS
patients (n = 3).;
[0069] Figure 4 shows the expression of Ninjurin-1 in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) and spinal cord injury (SCI).
(A) Western blot for Ninjurin-1 in spinal cord homogenates of myelin oligodendrocyte glycoprotein (MOG)35-55-immunized EAE mice (C57BL/6) revealed an upregulation of Ninjurin-1 compared to the control (unimmunized mice). Levels of Ninjurin-1 correlated with EAE
scores (center lane: score 1; right lane: score 3.0). (B) Flow cytometry analysis of Ninjurin-1 expression on MHC ll+ (left panel) CD11c+ DCs (middle panel) and F4/80+ macrophages (right panel) obtained from the CNS (brain and spinal cord) of MOG35-55-immunized EAE mice (C57BU6).
Data shown are representative of two experiments, gated on CD3ne9 CD45hand either MHC
II-, CD11c- or F4/80-expressing cells. (C) Spinal cords from EAE mice immunostained for Ninjurin-1, MHC II (left panels), CD11c (middle panels), F4/80 (right panels) and nuclear stain TO-PRO-3 (day 14 post-induction, n = 6). High power view of areas marked confirmed co-localization of Ninjurin-1 in CNS myeloid antigen-presenting cells (red).
Scale bars, 30 pm. (D) Western blot for Ninjurin-1 in spinal cord homogenates after SCI in C57BL/6 mice.
Spinal cord mechanical injury was performed in mice using the Infinite HorizonsTM impactor device. Samples were extracted from uninjured (UI) and from injured mice 1, 3, 7, 14, 21 and 28 days after injury. The expression of Ninjurin-1 increases in the spinal cord of injured mice;
[0070] Figure 5 shows the effect of Ninjurin-1 blockade on CD14+ monocyte migration across human BBB-endothelial cells. The adhesion motif of human Ninjurin-1 is used as a blocking peptide, called Ninj26_37 (sequence: NH2-PPRWGLRNRPIN-COOH, SEQ ID
NO: 5).
(A) In vitro model of the BBB. Human BBB-endothelial cells are grown in Boyden chambers and treated with the Ninjurin-1 blocking peptide Ninj26-37 or control peptide (top chamber) 1 hour prior to the addition of immune cells. Human ex vivo CD4+ and CD8+ T
lymphocytes (B) and CD14+ monocytes (C) were allowed to migrate across human BBB-endothelial cells for 24 h, in the presence or absence of the human Ninj26_37 blocking peptide.
Ninjurin-1 blockade (0.4 mM) significantly restricts the migration of CD14+ monocyte, but not that of CD4+ or CD8+ T cells, across human BBB-endothelial cells. Data shown are representative of seven independent experiments (n = 7 blood donors) on six distinct BBB-EC
preparations performed in triplicate. (D) CFSE-labeled human CD14+ monocytes (green) were seeded on a confluent monolayer of human TNF/IFN-y-activated BBB-ECs, fixed and immunostained for Ninjurin-1 (left panels). A 15 pm z-stack reconstruction (x-z and y-z) shows Ninjurin-1 (arrows) around the CFSE-loaded migrating monocyte and in the transmigratory cup (arrowheads). Photomicrograph shown is representative of >20 fields obtained from four independent immunostainings performed using four blood donors and two distinct BBB-ECs preparations. Scale bar, 20 pm;
[0071] Figure 6 shows the effects of Ninjurin-1 blocking peptide (Ninj26_37) in EAE mice.
EAE was induced by active immunization with MOG35-55/CFA in C57BL/6 mice. Some animals received intraperitoneal (i.p.) injections of 200 pg twice a day (b.i.d.) of mouse Ninj26_ 37 from day 3 to day 23 post-immunization (=, n = 24 mice); others received PBS (o, n = 22 mice) in the same manner. (A) Mice treated with Ninj26_37 blocking peptide show a significant reduction of the neurological signs, symptoms and clinical scores of the disease. (clinical score: 0 = no clinical symptoms; 0.5 = partial floppy tail; 1 = floppy tail; 2 = ataxia; 2.5 =
weakness of hind limbs; 3 = paralysis of one hind limb; 4 = paralysis of both hind limbs). (B) FACS analysis of CNS infiltrating leukocytes 14 days postimmunization, comparing the number of CD3+ lymphocytes, CD11c+ DCs (top panels) and F4/80+ macrophages (lower panels) in Ninj26-37-treated mice vs. control animals. Data shown are representative of two independent experiments obtained from four mice, gated on CD45hi cells. (C) Luxol fast blue-hematoxylin and eosin stainings of EAE spinal cords from Ninj26-37-treated mice show a reduction in immune cell infiltration and demyelination, as compared to the control group.
Photomicrographs shown are representative of >20 stainings performed on four animals.
Doted lines delineate areas of demyelination. Scale bar, 50 pm. (D) Immunofluorescent analyses of spinal cords (14 days post-immunization) confirmed reduction of infiltrating MHC
ll+ (top panels), F4/80+ (middle panels) and CD11 c+ (bottom panels) cells in Ninj26_37-treated mice vs. control animals. Nuclei were stained with TO-PRO-3. Photomicrographs shown are representative of >20 immunostainings performed on post-mortem material from 4 animals.
Scale bar, 30 pm;
[0072] Figure 7 shows the clinical effect of Ninjurin-1 blocking peptide after spinal cord injury. Spinal cord mechanical injury was performed in C57BL/6 mice using the Infinite HorizonsTM impactor device. (A) WB of spinal cords samples showing Ninjurin-1 expression at day 1, 3, 7, 14, 21 and 28 post-injury. Ctl represents baseline (uninjured) control. Data shown are representative of two experiments performed with three animals. (B) and (C) Immunostainings and cell counts of Iba1+ and CD11c+ myeloid cell infiltrates in the spinal cord of SCI animals treated with Ninj26_37 blocking peptide (200 pg i.p., twice a day) vs.
controls. Photomicrographs shown are representative of immunostainings obtained from control and Ninj26-37-treated animals mice (n = 4 per group, day 7 post-injury). Scale bars, 500 pm (D) The clinical scores (Basso mouse scale, BMS, Basso D.M. et al., (2006). J
Neurotrauma. 23(5): 635-59) of mice treated with Ninj26-37 blocking peptide (=, 200 pg i.p., twice a day) were reduced when compared to control animals (o). Data shown represent two independent experiments using 11 mice per group;
[0073] Figure 8 shows the nucleotide and amino acid sequences of Ninjurin-1.
(A) Nucleotide sequence of human Ninjurin-1 (SEQ ID NO: 1, NCBI Reference Sequence:
NM_004148.3). The coding sequence (residues 72 to 530) is indicated in bold.
(B) Amino acid sequence of human Ninjurin-1 (SEQ ID NO: 2, NCBI Reference Sequence:
NP_004139.2). (C) Nucleotide sequence of mouse Ninjurin-1 (SEQ ID NO: 3, NCBI
Reference Sequence: NM_013610.2). The coding sequence (residues 17 to 475) is indicated in bold. (D) Amino acid sequence of mouse Ninjurin-1 (SEQ ID NO: 4, NCBI
Reference Sequence: NP_038638.1);
[0074] Figure 9 shows the effect of Ninjurin-1 blockade on monocyte adhesion to a BBB-ECs monolayer in an in vitro flow system. BBB-ECs were grown to confluence on flow capillary slides (Ibidi micro-slide I Luer 0.6). Primary cultures of human BBB-ECs were then submitted to flow conditions (0.25 ml/min, Ibidi pump system) and CFSE-labelled ex vivo isolated monocytes were added in the absence or presence of the Ninjurin blocking peptide (4 mM). Image acquisition took place in the next 40 minutes, pictures and movies (10 images per second) were acquired and analysed on VolocityTM software (Improvision). The number of adherent monocytes was evaluated on at least 15 random fields; two representative images are shown in (A). Quantification of the number of adherent monocytes under the different flow conditions (presence or absence of Ninjurin blocking peptides, BBB-ECs stimulated or not with TNF/IFN-y) is shown in the right panel (B);
[0075] Figure 10 shows the effect of Ninjurin-1 blockade on the velocity of monocyte on a BBB-ECs monolayer in an in vitro flow system. BBB-ECs were grown to confluence on flow capillary slides (Ibidi micro-slide Luer 0.6). Primary cultures of human BBB-ECs were then submitted to flow conditions (0.25 ml/min, Ibidi pump system) and CFSE
labelled ex vivo isolated monocytes were added in the absence or presence of the Ninjurin blocking peptide (4 mM). Image acquisition took place in the next 40 minutes, movies (10 images per second) were acquired and analysed on VolocityTM software (Improvision).
Flowing monocyte velocities were calculated from the movies using the software. The mean velocity of all the moving monocytes was compared under the different conditions (presence or absence of Ninjurin blocking peptides, BBB-ECs stimulated or not with TNF/IFN-y);
[0076] Figure 11 shows the expression of melanoma cell adhesion molecule (MCAM) on endothelial cells of the blood-brain barrier (BBB-ECs). (A) Expression of MCAM protein (105-115 kDa) in primary human cultures of BBB-ECs, as compared to umbilical vein ECs (HUVECs). n = 3 different preparations, from 3 distinct donors. (B) MCAM
protein can be detected, by Western blot analysis, in detergent resistant membrane microdomains and lipid rafts of BBB-ECs (fractions 4 to 6). GM1 is shown as a marker of the lipid raft fractions (fractions 4 to 6). (C) Expression of MCAM on the surface of BBB-ECs (dark histogram) is up-regulated in the presence of TNF-a and IFN-y (100 U/ml, open histogram), n = 3 as measured by flow cytometry;
[0077] Figure 12 shows the expression of MCAM in MS lesions. Paraffin-embedded material from control human CNS (top panels, A) and human active MS lesions (bottom panels, B) are shown. Immunostaining for Caveolin-1 (Cav-1, endothelial cell marker, middle panels) and MCAM (left panels) were acquired by fluorescent microscopy. Data shown are representative of 5 active MS lesions from 4 distinct MS-affected donors and 4 controls.
Merged images are presented on the right panels and demonstrate co-localization of MCAM
with Cav-1 on infiltrated blood vessels in MS and control tissue. Scale bar is 75 pm and nuclei were stained with TOPRO-3. These results indicate that MCAM is expressed by BBB-ECs in MS lesions and in control CNS tissue. The expression of MCAM does not seem to be significantly modulated in MS lesions;
[0078] Figure 13 shows an immunocytofluorescent staining of the expression of MCAM
during leukocyte migration. Ex vivo CD4+ T lymphocytes isolated from healthy donors were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then allowed to migrate across a monolayer of BBB-ECs for 2 hours. The monolayer was then washed extensively with PBS to remove non adherent cells and fixed with 4% paraformaldehyde.
Cells were 5 stained with anti-MCAM antibody. Scale bar is 25 pm. MCAM enrichment is seen surrounding the lymphocytes at points of EC contact and entry in a structure reminiscent of the transmigratory cup;
[0079] Figure 14 shows the expression of MCAM as assessed by flow cytometry on freshly isolated peripheral blood cells. Cells were labeled with anti-MCAM
antibody 10 concurrently with specific cell markers: (A) CD4+ T lymphocytes, (B) CD8+ T
lymphocytes, (C) CD14+ monocytes and (D) CD19+ B lymphocytes. (E) MCAM+ CD3+ T lymphocytes are mainly CD45RO+ and CD45RA", indicative of a memory T cell phenotype. (F) Overview of the markers associated with MCAM expression in ex vivo peripheral blood cells;
[0080] Figure 15 shows a representative quantification of selected lymphocyte markers 15 as assessed by flow cytometry on ex vivo MCAM+ human peripheral blood lymphocytes obtained from healthy donors. Quantification is shown for CD69 early activation marker (A), CD107a degranulation marker (B), the chemokine receptors CCR7 (C) and CCR6 (D) and the inflammatory cytokine IL-17 (E). Cells were labeled with anti-MCAM
antibody concurrently with cell specific marker (n > 4). * p < 0.05, ** p < 0.01, *** p < 0.001. The data demonstrates that MCAM+ cells more frequently express CD161, CCR6 and IL-17, and express lower levels of CCR7;
[0081] Figure 16 shows a flow cytometry analysis of MCAM expression on ex vivo and in vitro activated (with OKT3 + CD14 monocytes + IL-2) memory lymphocytes. One million CD4+ CD45RO+ lymphocytes were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and IL-2 (20 units/ml) for 3 days. (A) Representative FACS histogram. (B) Quantification of MCAM expression after in vitro activation (n > 8, *** p < 0.001) demonstrates a significant increase in the number of MCAM+ cells upon non-antigen specific TCR-mediated activation;
[0082] Figure 17 shows the expression of selected cell surface markers on isolated CD4+ CD45RO+ MCAM+ and MCAM" lymphocytes after in vitro activation with OKT3 +
monocytes + selected cytokines. One million lymphocytes sorted according to expression of MCAM were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and either IL-2 (n > 7, 20 units/ml) or IL-23 (n > 3, 10 ng/ml) with anti-IFN-y (5 p/ml) and anti-IL-4 (5 pg/ml). Following activation, CD4+ CD45RO+ cells were assessed for the Th17 marker CD161 (A), CD107a (B), CD25 (C) and CD69 (D). * p < 0.05, ** p <
0.01, *** p < 0.001. The data demonstrates that MCAM expressing cells more frequently co-express CD161, CD107a and CD69 when stimulated with IL-2 or with IL-23;
[0083] Figure 18 shows a representative flow cytometry analysis of the expression of the cytokines Interferon-gamma (A) and IL-17 (A and B) on in vitro activated CD4+
CD45RO+ T cells (A) and (B) on CD4+ CD45RO+ lymphocytes sorted for MCAM
expression (CD4+ CD45RO+ MCAM+ vs. CD4+ CD45RO+ MCAM"). One million lymphocytes sorted according to expression of MCAM were cultured with 500 000 autologous CD14+
monocytes in the presence of anti-CD3 (2.5 pg/ml) and either IL-2 (20 units/ml). The data demonstrates that IL-17 expression and the number of IL-17 expressing cells are significantly increased in MCAM expressing CD4 memory lymphocytes;
[0084] Figure 19 shows the quantification of flow cytometry analysis of the expression of selected cytokines by isolated CD4+ CD45RO+ MCAM+ and MCAM- lymphocytes after days of in vitro culture. One million lymphocytes sorted according to expression of MCAM
were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and either IL-2 (n > 7, 20 units/ml) or IL-23 (n > 3, 10 ng/ml) with anti-IFN-y (5 p/ml) and anti-IL-4 (5 pg/ml). Following activation, CD4+ CD45RO+ cells were assessed for expression of IL-17 (A), GM-CSF (B), IFN-gamma (C) and IL-4 (D). * p < 0.05, ** p < 0.01, *** p < 0.001. The data demonstrates that MCAM expression on CD4+ CD45RO+
lymphocytes predicts the expression of IL-17, GM-CSF and IFN-y;
[0085] Figure 20 shows an overview of the markers associated with in vitro activated MCAM+ lymphocytes;
[0086] Figure 21 shows MCAM expression on MCAM+-sorted lymphocytes after 5 days in culture in a Th17 polarization environment. Memory T cells expressing CD45RO, CD4 and MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions). (A) Representative histogram showing expression of MCAM in memory T cells expressing CD45RO and CD4 (black line) compared with isotype (solid grey) after 5 days in culture. (B) Quantification of the proportion of cells that are MCAM+ or MCAM" after 5 days in Th17 polarization conditions and originate from memory T cells expressing CD45RO and CD4 sorted for MCAM expression (MCAM+
fraction). n = 3. Statistical analysis were obtained with Paired T test;
[0087] Figure 22 shows MCAM expression on MCAM-negative-sorted lymphocytes after days in culture in Th17 polarization environment. Memory T cells expressing CD45RO and CD4, but negative for MCAM were sorted with a purity of >99% and cultured for 5 days in 5 presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions). (A) Representative histogram showing expression of MCAM expression in memory T
cells expressing CD45RO and CD4 (grey line) compared with isotype (solid grey) after 5 days in culture. (B) Quantification of the proportion of cells that are MCAM+ or MCAM-after 5 days in Th17 polarization conditions and originate from memory T cells expressing CD45RO and CD4 negatively sorted for MCAM expression (MCAM- fraction) (n = 3).
Statistical analysis were obtained with Paired T test, ns = not significant;
[0088] Figure 23 shows the expression of IL-17 and IFN-y in MCAM+ memory T
cells originating from MCAM+ sorted lymphocytes. Memory T cells expressing CD45RO, CD4 and MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions). Intracellular stainings for IL-17 (left panel), IFN-y (middle panel) and IL-17 + IFN-y (right panel) were performed on cultured CD45RO+ CD4+ MCAM+ cells and CD45RO+ CD4+ MCAM- cells stimulated with PMA/ionomycin/Brefeldin A for 4 hours (n = 3). Statistical analysis were obtained with Paired T test, ns = not significant;
[0089] Figure 24 shows the expression of IL-17 and IFN-y in MCAM+ memory T
cells originating from MCAM" sorted lymphocytes. Memory T cells expressing CD45RO
and CD4 but negative for MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions).
Intracellular stainings for IL-17 (left panel), IFN-y (middle panel) and IL-17 + IFN-y (right panel) were performed on cultured CD45RO+ CD4+ MCAM+ cells and CD45RO+ CD4+
MCAM" cells stimulated with PMA/ionomycin/Brefeldin A for 4 hours (n = 3).
Statistical analysis were obtained with Paired T test, ns = not significant;
[0090] Figure 25 shows the expression of IL-8 on MCAM+ memory T cells originating from MCAM+ and MCAM sorted lymphocytes. Memory T cells expressing CD45RO and CD4, and positive or negative for MCAM were sorted with a purity of >99% and cultured for 5 days in presence of IL-23, anti-CD3, anti-IL-4 and anti-IFN-y (Th17 polarization conditions).
Intracellular staining for IL-8 was performed on cultured CD45RO+ CD4+ MCAM+
cells and CD45RO+ CD4+ MCAM- stimulated with PMA/ionomycin/ Brefeldin A for 4 hours.
n=3.
Statistical analysis were obtained with Paired T test;
[0091] Figure 26 shows the expression of MCAM on CD4+ and CD8+ T lymphocytes obtained from healthy donors (controls) and MS patients. Human ex vivo peripheral blood collected from healthy donors and MS patients was directly subjected to whole blood FAGS
staining with anti-CD3, anti-CD4, anti-CD8, anti-MCAM antibodies. FAGS
acquisition was performed the same day. (A) Proportion of CD3+ CD4+ MCAM+ lymphocytes in 35 healthy donors and in 42 MS patients in relapse (** p < 0.0001). (B) Proportion of CD3+ CD8+
MCAM+ lymphocytes in 35 healthy donors and in 42 MS patients in relapse (* p <
0.005). (C) Enrichment of CD3+ CD4+ MCAM+ and CD3+ CD8+ MCAM+ lymphocytes in the cerebro-spinal fluid (CSF) as compared to the peripheral blood in MS patients (n = 5, * p < 0.005);
[0092] Figure 27 shows CD4+ CD45RO+ human peripheral blood lymphocytes from MS
patients and healthy controls cultured under Th17 skewing conditions and assessed for MCAM association with IL-17 secretion (n = 3 MS patients and 7 controls). One million CD4+
CD45RO+ lymphocytes were cultured with 500 000 autologous CD14+ monocytes in the presence of anti-CD3 (2.5 pg/ml) and IL-23 (10 ng/ml) with anti-IFN-y (5 p/ml) and anti-IL-4 (5 pg/ml). Following activation CD4+ CD45RO+ cells were assessed for IL-17 and MCAM co-expression. (A) Shows representative FACS plots from one control and one MS
patient.
Panel (B) is the quantification of MCAM+ IL-17+ cells compared to MCAM- IL-17+
cells. CD4+
CD45RO+ MCAM+ from MS patients grown under Th17 polarizing conditions express significantly more IL-17 compared to healthy controls (* p < 0.05), presumably due to the increase proportion of MCAM+ CD4+ seen in MS peripheral blood;
[0093] Figure 28 shows the presence of CD4+ and CD8+ MCAM+ T lymphocytes in various organs of mice affected by myelin-oligodendrocyte (MOG) peptide-induced experimental allergic encephalomyelitis (EAE). EAE was induced in C57BL6 mice by injection of MOG35-55 in complete Freunds adjuvant. At the peak of the disease (day 14), two animals were sacrificed and the presence of CD4+ and CD8+ MCAM+ lymphocytes was assessed in the spleen, the lymph nodes (LN) and the CNS of the animals. (A) Representative FAGS analysis of the presence of MCAM+ lymphocytes in the spleen, LN
and brain. Panel (B) shows the quantification of the percentage of MCAM+ CD4+
and CD8+
lymphocytes in the spleen, the LN and the brain of EAE animals. These data show that MCAM+ lymphocytes are recruited to the brain in the course of EAE, the animal model of MS; and [0094] Figure 29 shows the (A) nucleotide (NCBI Reference Sequence:
NM_006500.2, SEQ ID NO: 6) and (B) amino acid (NCBI Reference Sequence: NM_006491.2, SEQ ID
NO:
7) sequences of human MCAM. The coding sequence (residues 30-1970) is indicated in bold in the nucleotide sequence.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0095] The present inventors have shown that two molecules expressed on endothelial cells of the blood-brain barrier, namely nerve injury-induced protein-1 (Ninjurin-1) and melanoma cell adhesion molecule (MCAM), are associated with inflammatory conditions such as neuroinflammatory conditions, and that the expression and/or modulation of these molecules may be used in therapeutic, diagnostic and/or prognostic applications.
[0096] More specifically, in the studies described herein, the expression of nerve injury-induced protein-1 (Ninjurin-1) on brain and spinal cord endothelial cells (ECs) and on peripheral blood monocytes, as well as its ability to promote myeloid cell recruitment across the BBB, was investigated. The instant inventors have determined that while Ninjurin-1 is expressed at low levels in healthy human and mouse CNS, its immunoreactivity is localized to CNS microvascular endothelium, as well as on microglia, infiltrating macrophages and dendritic cells during neuroinflammatory events. In the peripheral blood, Ninjurin-1 expression was found predominantly on human CD14+ monocytes, but not on the surface of CD4+ or CD8+ T lymphocytes. Using an oligopeptide corresponding to residue 26 to 37 of Ninjurin-1 (Ninj26.37), it was further demonstrated that Ninjurin-1 significantly contributes to monocyte migration into the CNS in vitro and in vivo during EAE and SCI, and that Ninjurin-1 neutralization protects against EAE and promotes repair following SCI.
[0097] It is also demonstrated that the proportion of T cells expressing melanoma cell adhesion molecule (MCAM) is higher in the peripheral blood (PB) and cerebro-spinal fluid (CSF) of MS patients as compared to healthy controls, and that MCAM+ T cells are enriched in the CSF compared to the PB. Furthermore, it is also shown that MCAM is expressed on human T lymphocytes that are preferentially programmed to secrete the inflammatory cytokines IL-17, IFN-y, GM-CSF and TNF-a following activation.
[0098] Accordingly, in an aspect, the present invention provides a method of preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject, said method comprising administering to said subject an effective amount of a Ninjurin-1 and/or MCAM inhibitor.
[0099] In another aspect, the present invention provides a method of inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., the CNS endothelium), said method comprising contacting said immune 5 cell and/or said endothelium with an effective amount of a Ninjurin-1 and/or MCAM inhibitor.
[00100] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject. The present invention also relates to a use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for preventing or 10 treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject.
[00101] In another aspect, the present invention provides a use of a Ninjurin-1 and/or MCAM inhibitor for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., a CNS endothelium). The present invention also relates to a use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a 15 medicament for inhibiting the recruitment of an immune cell across the endothelium of a tissue/organ.
[00102] In another aspect, the present invention provides a Ninjurin-1 and/or MCAM
inhibitor for preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject. The present invention further relates to a Ninjurin-1 and/or MCAM
20 inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition (e.g., a neuroinflammatory condition) in a subject.
[00103] In another aspect, the present invention provides a Ninjurin-1 and/or MCAM
inhibitor for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the CNS endothelium. The present invention further relates to a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for inhibiting the recruitment of an immune cell across the CNS endothelium.
[00104] Ninjurin-1 (also known as NIN1 or NINJ1) is a type 3b membrane protein known to interact in a homophilic manner through an extracellular binding motif. The nucleotide and amino acid sequences of human and mouse Ninjurin-1 are illustrated in Fig. 8.
While Ninjurin-1 is expressed during embryogenesis and is thought to contribute to CNS and peripheral nervous system (PNS) development, its expression is strikingly up-regulated on neurons and Schwann cells during experimental peripheral nerve injury. In rats, functional inhibition of Ninjurin-1/Ninjurin-1 homotypic interaction using the blocking oligopeptide (Ninj26_37) was shown to reduce post-lesional neurite outgrowth, suggesting a positive influence of Ninjurin-1 on nerve regeneration.
[00105] MCAM (also known as CD146 and MUC18) is a cell surface glycoprotein belonging to the immunoglobulin superfamily involved in cell adhesion, and in cohesion of the endothelial monolayer at intercellular junctions in vascular tissue. It also promotes tumor progression of many cancers including melanoma and prostate cancer. It is known to interact in a homotypic/homophilic manner and may also bind to other ligands.
The nucleotide and amino acid sequences of human MCAM are shown in Fig. 29.
[00106] "Inflammatory condition" or "autoimmune condition" as used herein refers to a condition associated with inflammation of an organ or tissue and/or a dysregulated immune response, and which generally involves cell, organ or tissue damage.
Inflammatory conditions are typically characterized by various events including dilatation of arterioles, capillaries, and venules, increased permeability and blood flow; exudation of fluids, including plasma proteins; and/or immune cell recruitment/migration to the site of inflammation. In an embodiment, the above-mentioned Inflammatory condition is associated with the recruitment of immune cells to the site of inflammation. In a further embodiment, the above-mentioned immune cells are myeloid cells, such as monocytes, macrophages and/or dendritic cells. In another embodiment, the above-mentioned immune cells are T cells (or T
lymphocytes), for example CD4+ and/or CD8+ T cells. In a further embodiment, the above-mentioned T cells are inflammatory cytokine-secreting T cells or inflammatory cytokine-secreting T cell precursors. As used herein, "inflammatory cytokine-secreting T cell" refers to a T cell having the potential/capacity to perform effector functions such as to secrete inflammatory cytokines (e.g., IL-17, IFN-y) and/or mediate cytotoxic killing, for example following activation/stimulation (e.g., T-cell receptor(TCR)-mediated activation/stimulation). In an embodiment, the above-mentioned inflammatory cytokine-secreting T cell express one or more of the markers depicted in Figs. 14 to 20 (e.g., CD161, CD107a and CD69).
In a further embodiment, the above-mentioned inflammatory cytokine-secreting T cell is a Th17 cell (Dong C., Microbes Infect. 2009 11(5): 584-8. Epub 2009 Apr 14). The term "inflammatory cytokine-secreting T cell precursor" refers to a T cell which has the potential/capacity to differentiate into an inflammatory cytokine-secreting T
cell following exposure to specific conditions (polarizing conditions), such as Th17-polarizing conditions.
[00107] "Neuroinflammatory condition" as used herein refers to a condition associated with inflammation of the nervous system, and more particularly the central nervous system (CNS), and which is associated with cell/tissue damage. It is typically characterized by, for example, increased glial activation, increased pro-inflammatory cytokine/chemokine levels (e.g., TNF-a, IFN-y, IL-1p), increased blood-brain-barrier permeability, and/or increased immune cell (e.g., leukocyte) recruitment/invasion to the CNS. It includes chronic neuroinflammation, such as an inflammation associated with chronic activation of cells of the immune system (i.e., autoimmune-associated neuroinflammation). Such chronic neuroinflammation is observed, for example, in multiple sclerosis. It also includes acute neuroinflammation, such as inflammation resulting from an initial trauma to the CNS. Acute neuroinflammation is observed, for example, following CNS injury (e.g., spinal cord injury), and is associated with CNS tissue damage beyond the original site of injury.
[00108] In an embodiment, the above-mentioned neuroinflammatory condition is multiple sclerosis (MS). In a further embodiment, the above-mentioned MS is of one of the following subtypes: clinically isolated syndromes (CIS) suggestive of MS, relapsing-remitting MS, primary progressive MS, secondary progressive MS, progressive relapsing MS, or borderline forms of MS (e.g., Devic's disease, Balo concentric sclerosis, Schilder's diffuse sclerosis, Marburg multiple sclerosis), as well as any neurological diseases with signs and symptoms suggestive of MS.
[00109] In another embodiment, the above-mentioned neuroinflammatory condition is a neural injury, in a further embodiment spinal cord injury, and in a further embodiment secondary spinal cord injury.
[00110] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic or therapeutic result. An effective amount refers to the amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinician, which includes one or more of the following:
[00111] (A) Preventing the disease; for example, preventing a neuroinflammatory disease, condition or disorder in an individual that may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease, [00112] (B) Inhibiting the disease; for example, inhibiting a neuroinflammatory disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology), and [00113] (C) Ameliorating the disease; for example, ameliorating a neuroinflammatory disease, condition or disorder in an individual that is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology).
[00114] The amount of the Ninjurin-1 and/or MCAM inhibitor which is effective in the prevention and/or treatment of a particular disease, disorder or condition (e.g., a neuroinflammatory disease, disorder or condition) will depend on the nature and severity of the disease, the chosen prophylactic/therapeutic regimen, the target site of action, the patient's weight, special diets being followed by the patient, concurrent medications being used, the administration route and other factors that will be recognized by those skilled in the art. The dosage will be adapted by the clinician in accordance with conventional factors such as the extent of the disease and different parameters from the patient.
Typically, 0.001 to 1000 mg/kg of body weight/day will be administered to the subject. In an embodiment, a daily dose range of about 0.01 mg/kg to about 500 mg/kg, in a further embodiment of about 0.1 mg/kg to about 200 mg/kg, in a further embodiment of about 1 mg/kg to about 100 mg/kg, in a further embodiment of about 10 mg/kg to about 50 mg/kg, may be used. The dose administered to a patient, in the context of the present invention should be sufficient to effect/induce a beneficial prophylactic and/or therapeutic response in the patient over time.
The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration. Effective doses may be extrapolated from dose response curves derived from in vitro or animal model test systems.
For example, in order to obtain an effective mg/kg dose for humans based on data generated from rat studies, the effective mg/kg dosage in rat may be divided by six.
[00115] In an embodiment, the above-mentioned treatment comprises the use/administration of more than one (i.e., a combination of) active/therapeutic agent, at least one of which being the above-mentioned Ninjurin-1 and/or MCAM inhibitor. The combination of prophylactic/therapeutic agents and/or compositions of the present invention may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent may be administered to a patient before, concomitantly, before and after, or after a second active agent is administered. The agents may in an embodiment be combined/formulated in a single composition and thus administered at the same time. In an embodiment, the one or more active agent(s) is used/administered in combination with one or more agent(s) currently used to prevent or treat the disorder in question (e.g., a neuroinflammatory condition such as MS or SCI). In an embodiment, a Ninjurin-1 inhibitor is co-administered with an MCAM inhibitor.
[00116] As used herein, the term "Ninjurin and/or MCAM inhibitor" includes any compound able to directly or indirectly affect the regulation of Ninjurin-1 and/or MCAM by reducing for example the expression of Ninjurin-1 and/or MCAM (i.e., at the transcription and/or translation level), or a Ninjurin-1 and/or MCAM activity. It includes intracellular as well as extracellular Ninjurin-1 and/or MCAM inhibitors. Without being so limited, such inhibitors include siRNA, antisense molecules, proteins, peptides, small molecules, antibodies, etc.
[00117] As used herein the terms "Ninjurin-1 and/or MCAM activity" and "Ninjurin-1 and/or MCAM function" refer to detectable enzymatic, biochemical or cellular activity attributable to Ninjurin-1 and/or MCAM. Ninjurin-1 activity may also be measured by protein-protein binding assay using purified Ninjurin-1 and a purified Ninjurin-1 ligand (e.g., Ninjurin-1). As such, in an embodiment, determining whether a compound decreases Ninjurin-1 activity comprises determining whether the compound inhibits or decreases Ninjurin-1 - Ninjurin-1 homotypic binding. Similarly, MCAM activity may also be measured by protein-protein binding assay using purified MCAM and a purified MCAM ligand (e.g., MCAM). As such, in an embodiment, determining whether a compound decreases MCAM activity comprises determining whether the compound inhibits or decreases MCAM - MCAM homotypic binding.
[00118] In an embodiment, such a binding assay may be performed using cells expressing Ninjurin-1 and/or MCAM on their surface, thus via measurement of cell-cell binding of such Ninjurin-1- and/or MCAM-positive cells.
[00119] Ninjurin-1 activity may also be measured in a cell recruitment/migration assay, such as the assay described in Examples 4 and 6 below, or using a Ninjurin-mediated adhesion assay, as described for example in Araki et al., J. Biol Chem. (1997) 272(34):
21373-21380 and in U.S. Patent No. 6,559,288).
[00120] Ninjurin-1 and/or MCAM activity could also be indirectly measured by evaluating the level of expression of Ninjurin-1 and/or MCAM, or a fragment thereof, in cells as well as in a biological sample (tissue, organ, fluid). Ninjurin-1 and/or MCAM
expression levels could 5 be determined at the polypeptide and/or nucleic acid levels using any standard methods known in the art (see below). Ninjurin-1 and/or MCAM activity could also be indirectly measured by evaluating the level of expression or activity of a reporter gene (e.g., luciferase, R-galactosidase, alkaline phosphatase, GFP) operably linked to a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene.
10 [00121] In an embodiment, the above-mentioned Ninjurin-1 and/or MCAM
inhibitor is an antisense or RNAi-based inhibitory molecule.
[00122] Generally, the principle behind antisense technology is that an antisense molecule hybridizes to a target nucleic acid and effects modulation of gene expression such as transcription, splicing, translocation of the RNA to the site of protein translation, 15 translation of protein from the RNA. The modulation of gene expression can be achieved by, for example, target degradation or occupancy-based inhibition. An example of modulation of RNA target function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi). RNAi is a form of antisense-20 mediated gene silencing involving the introduction of dsRNA (typically of less than 30 nucleotides in length, and generally about 19 to 24 nucleotides in length) leading to the sequence-specific reduction of targeted endogenous mRNA levels, here the RNA
transcript of the Ninjurin-1 gene. Such dsRNA are generally substantially complementary to at least part of an RNA transcript of the Ninjurin-1 gene. Another example of modulation of gene 25 expression is the RNA analogue Locked Nucleic Acid (LNA). Other examples relate to double stranded nucleic acid molecules including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), micro-RNA (miRNA). The use of single stranded antisense oligonucleotides (ASO) is also encompassed by the method of the present invention. Sequence-specificity makes antisense compounds extremely attractive as therapeutics to selectively modulate the expression of genes involved in the pathogenesis of any one of a variety of diseases.
[00123] Chemically modified nucleosides, such as 2'-substituted arabinonucleosides (e.g., 2'F-ANA) and 2'-substituted RNA (e.g., 2'F-RNA), may be used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics or affinity for a target RNA.
[00124] As used herein "antisense molecule" is meant to refer to an oligomeric molecule, particularly an antisense oligonucleotide for use in modulating the activity or function of nucleic acid molecules encoding a Ninjurin-1 polypeptide (e.g., the polypeptide of SEQ ID
NO: 2) and/or MCAM polypeptide (e.g., the polypeptide of SEQ ID NO: 7), ultimately modulating the amount of Ninjurin-1 and/or MCAM produced in cells (e.g., CNS
cells, immune cells). This is accomplished by providing oligonucleotide molecules which specifically hybridize with one or more nucleic acids encoding Ninjurin-1 and/or MCAM. As used herein, the term "nucleic acid encoding a Ninjurin-1 polypeptide"
encompasses DNA
encoding said polypeptide, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA (e.g., a nucleic acid comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO: 1). As used herein, the term "nucleic acid encoding an MCAM polypeptide" encompasses DNA encoding said polypeptide, RNA (including pre-mRNA and mRNA) transcribed from such DNA, and also cDNA derived from such RNA (e.g., a nucleic acid comprising the coding sequence of the nucleotide sequence set forth in SEQ ID NO: 6). The specific hybridization of an oligomeric compound with its target nucleic acid interferes with the normal function of the nucleic acid.
The overall effect of such interference with target nucleic acid function is the decrease of the expression of Ninjurin-1 and/or MCAM.
[00125] In the context of this invention, "hybridization" means hydrogen bonding between complementary nucleoside or nucleotide bases. Terms "specifically hybridizable" and "complementary" are the terms which are used to indicate a sufficient degree of complementarity or precise pairing such that stable and specific binding occurs between the oligonucleotide and the DNA or RNA target. It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. An antisense compound is specifically hybridizable when binding of the compound to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and in the case of in vitro assays, under conditions in which the assays are performed. Such conditions may comprise, for example, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, at 50 to 70 C for 12 to 16 hours, followed by washing. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.
[00126] In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target and increased stability in the presence of nucleases. Examples of modified nucleotides include a 2'-0- methyl modified nucleotide, a nucleotide comprising a 5'-phosphorothioate group, a terminal nucleotide linked to a cholesteryl derivative, a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate and a non-natural base comprising nucleotide.
[00127] Methods to produce antisense molecules directed against a nucleic acid are well known in the art. The antisense molecules of the invention may be synthesized in vitro or in vivo.
[00128] Reagents and kits for performing RNAi are available commercially from for example Ambion Inc. (Austin, TX, USA), New England Biolabs Inc. (Beverly, MA, USA) and Invitrogen (Carlsbad, CA, USA).
[00129] The antisense molecule may be expressed from recombinant viral vectors, such as vectors derived from adenoviruses, adeno-associated viruses, retroviruses, herpesviruses, and the like. Such vectors typically comprises a sequence encoding an antisense molecule of interest (e.g., a dsRNA specific for Ninjurin-1) and a suitable promoter operatively linked to the antisense molecule for expressing the antisense molecule. The vector may also comprise other sequences, such as regulatory sequences, to allow, for example, expression in a specific cell/tissue/organ, or in a particular intracellular environment/compartment. Methods for generating, selecting and using viral vectors are well known in the art.
[00130] Antisense molecules (siRNA and shRNA) inhibiting the expression of human Ninjurin-1 are commercially available, for example from Santa Cruz Biotechnology Inc. (Cat.
Nos sc-75915, sc-75915-SH and sc-75915-V) and from Invitrogen (NINJ1 Stealth RNAiTM
siRNA, Cat. Nos. HSS107188, HSS107189 and HSS107190). Antisense molecules (siRNA
and shRNA) inhibiting the expression of human MCAM are commercially available, for example from Invitrogen (MCAM Stealth RNAiTM siRNA, Cat. Nos. HSS106378, and HSS106380) and Applied Biosystems (siRNA ID Nos. s8571, s8572 and s8573).
Also, several providers (e.g., InvivoGen, Qiagen, Ambion, Inc.) offer custom-made antisense synthesis services.
[00131] In an embodiment, the above-mentioned Ninjurin-1 and/or MCAM inhibitor is a Ninjurin-1 and/or MCAM antibody.
[00132] By "Ninjurin-1 antibody" or "anti-Ninjurin-1" in the present context is meant an antibody capable of detecting (i.e. binding to) a Ninjurin-1 protein or a Ninjurin-1 protein fragment. In an embodiment, the above-mentioned antibody inhibits the biological activity of Ninjurin-1, such as Ninjurin-1/Ninjurin-1 homotypic interaction or Ninjurin-1-mediated cell recruitment. In another embodiment, the Ninjurin-1 protein fragment is an extracellular domain of Ninjurin-1.
[00133] By "MCAM antibody" or "anti-MCAM" in the present context is meant an antibody capable of detecting (i.e. binding to) a MCAM protein or a MCAM protein fragment. In an embodiment, the above-mentioned antibody inhibits the biological activity of MCAM, such as MCAM/MCAM homotypic interaction or MCAM-mediated cell recruitment.
[00134] In an embodiment, the antibody specifically binds to (interacts with) a Ninjurin-1 polypeptide (e.g., the polypeptide of SEQ ID NO: 2) and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as a Ninjurin-1 polypeptide. In another embodiment, the antibody specifically binds to (interacts with) an MCAM polypeptide (e.g., the polypeptide of SEQ ID
NO: 7) and displays no substantial binding to other naturally occurring proteins other than the ones sharing the same antigenic determinants as an MCAM polypeptide.
[00135] The term antibody or immunoglobulin is used in the broadest sense, and covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies, and antibody fragments so long as they exhibit the desired biological activity. Antibody fragments comprise a portion of a full length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments.
Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies. Additionally, any secondary antibodies, either monoclonal or polyclonal, directed to the first antibodies would also be included within the scope of this invention.
[00136] In general, techniques for preparing antibodies (including monoclonal antibodies and hybridomas) and for detecting antigens using antibodies are well known in the art (Campbell, 1984, In "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology", Elsevier Science Publisher, Amsterdam, The Netherlands) and in Harlow et al., 1988 (in: Antibody A Laboratory Manual, CSH
Laboratories). The term antibody encompasses herein polyclonal, monoclonal antibodies and antibody variants such as single-chain antibodies, humanized antibodies, chimeric antibodies and immunologically active fragments of antibodies (e.g., Fab and Fab' fragments) which inhibit or neutralize their respective interaction domains and/or are specific thereto.
[00137] Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (s.c.), intravenous (i.v.) or intraperitoneal (i.p.) injections of the relevant antigen (e.g., Ninjurin-1 or MCAM polypeptide or a fragment thereof) with or without an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCI2, or R'N=C=NR, where R and R1 are different alkyl groups.
[00138] Animals may be immunized against the antigen (e.g., a Ninjurin-1 or MCAM
polypeptide or a fragment thereof, such as a fragment comprising residues 26 to 37 of a Ninjurin-1 polypeptide), immunogenic conjugates, or derivatives by combining the antigen or conjugate (e.g., 100 pg for rabbits or 5 pg for mice) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with the antigen or conjugate (e.g., with 1/5 to 1/10 of the original amount used to immunize) in Freund's complete adjuvant by subcutaneous injection at 5 multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, for conjugate immunizations, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as 10 alum are suitably used to enhance the immune response.
[00139] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA
methods (e.g., U.S. Patent No. 6,204,023). Monoclonal antibodies may also be made using the techniques described in U.S. Patent Nos. 6,025,155 and 6,077,677 as well as U.S. Patent 15 Application Publication Nos. 2002/0160970 and 2003/0083293.
[00140] In the hybridoma method, a mouse or other appropriate host animal, such as a rat, hamster or monkey, is immunized (e.g., as hereinabove described) to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen used for immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes 20 then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.
[00141] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the 25 enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.
[00142] In an embodiment, the above-mentioned antibody is raised against an extracellular domain of a Ninjurin-1 or MCAM polypeptide (i.e. an extracellular domain of a 30 Ninjurin-1 polypeptide is used for immunization). In a further embodiment, the above-mentioned antibody is raised against a Ninjurin-1 polypeptide fragment comprising a domain corresponding to residues 28 to 35 of a Ninjurin-1 polypeptide. In a further embodiment, the above-mentioned antibody is raised against a Ninjurin-1 polypeptide fragment comprising a domain corresponding to residues 26 to 37 of a Ninjurin-1 polypeptide.
[00143] Ninjurin-1 and/or MCAM inhibitors may also be in the form of non-antibody-based scaffolds, such as avimers (Avidia); DARPins (Molecular Partners); Adnectins (Adnexus), Anticalins (Pieris) and Affibodies (Affibody). The use of alternative scaffolds for protein binding is well known in the art (see, for example, Binz and PlOckthun, 2005, Curr. Opin.
Biotech. 16: 1-11).
[00144] In an embodiment, the Ninjurin-1 inhibitor (e.g., anti-Ninjurin-1 antibody) blocks Ninjurin-1/Ninjurin-1 homotypic interaction, for example by competing for the Ninjurin-1 binding domain or by sterically hindering the Ninjurin-1 binding domain. In a further embodiment, the above-mentioned Ninjurin-1 inhibitor binds to an extracellular domain of a Ninjurin-1 polypeptide. In another embodiment, the above-mentioned inhibitor binds to a domain corresponding to or comprised within residues 28 to 35 of a Ninjurin-1 polypeptide.
In a further embodiment, the above-mentioned antibody binds to a domain corresponding to or comprised within residues 26 to 37 of a Ninjurin-1 polypeptide (e.g., the mouse or human Ninjurin-1 polypeptide depicted in Fig. 8).
[00145] In an embodiment, the MCAM inhibitor (e.g., anti-MCAM antibody) blocks MCAM/MCAM homotypic interaction, for example by competing for the MCAM binding domain or by sterically hindering the MCAM binding domain. In a further embodiment, the above-mentioned MCAM inhibitor binds to an extracellular domain of a MCAM
polypeptide.
[00146] In an embodiment, the above-mentioned Ninjurin-1 inhibitor is a peptide comprising a domain of formula I:
[00147] Xaa'-Xaa2-Arg-Trp-Xaa3-Xaa4-Arg-Xaa5-Arg-Xaa6-Xaa7-Xaa8 (I), [00148] wherein [00149] Xaa', Xaa2, Xaa6, Xaa7 and Xaa6 is any amino acid or is absent;
[00150] Xaa3, Xaa4 and Xaa5 is any amino acid;
[00151] or a functional analog thereof.
[00152] In an embodiment, Xaa2 is Pro. In an embodiment, Xaa' is Pro. In an embodiment, Xaa6 is Pro. In an embodiment, Xaa7 is Ile. In an embodiment, Xaa8 is Asn. In an embodiment, Xaa3 is Gly. In an embodiment, Xaa4 is Leu. In an embodiment, Xaa5 is Asn or Leu.
[00153] In an embodiment, the above-mentioned domain is Pro-Pro-Arg-Trp-Gly-Leu-Arg-Asn-Arg-Pro-lle-Asn (SEQ ID NO: 5).
[00154] In another embodiment, the above-mentioned Ninjurin-1 inhibitor is a peptide consisting of the domain of formula I defined above.
[00155] The term "amino acid" as used herein includes both L- and D-isomers of the naturally occurring amino acids as well as other amino acids (e.g., naturally-occurring amino acids, non-naturally-occurring amino acids, amino acids which are not encoded by nucleic acid sequences, etc.) used in peptide chemistry to prepare synthetic analogs of peptides.
Examples of naturally-occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, threonine, etc. Other amino acids include for example norleucine, norvaline, cyclohexyl alanine, biphenyl alanine, homophenyl alanine, naphthyl alanine, pyridyl alanine, phenyl alanines substituted at the ortho, para and meta positions with alkoxy, halogen or nitro groups etc. These amino acids are well known in the art of biochemistry/peptide chemistry.
[00156] The term "functional analog" (or "functional variant/derivative") refers to a peptide/domain having at least one modification as compared to the peptide/domain defined above, and which retain the activity of inhibiting Ninjurin-1 and/or MCAM
(e.g., blocking Ninjurin-1/Ninjurin-1 and/or MCAM/MCAM homotypic interaction).
[00157] In embodiments, the modification is a deletion, an insertion, a substitution or a chemical modification of one or more amino acids. The modification may be, for example, a deletion of (e.g., one to five) consecutive or non-consecutive amino acids, a substitution of (e.g., one to five) amino acids, one or more substitution(s) of a naturally occurring amino acid (L-amino acid) by a corresponding D-amino acid, an extension of the sequence by e.g., one, two, three or more amino acids.
[00158] In an embodiment, the above-mentioned substitution(s) are conserved amino acid substitutions.
[00159] As used herein, the term "conserved amino acid substitutions" (or sometimes "conservative amino acid substitutions") refers to the substitution of one amino acid for another at a given location in the peptide/domain, where the substitution can be made without substantial loss of the relevant Ninjurin-1 inhibitory activity. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide/domain by routine testing.
[00160] In some embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0), where the following may be an amino acid having a hydropathic index of about -1.6 such as Tyr (-1.3) or Pro (-1.6) are assigned to amino acid residues (as detailed in U.S. Patent. No. 4,554,101, incorporated herein by reference): Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); GIn (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5);
Leu (-1.8); Ile (-1.8);
Tyr (-2.3); Phe (-2.5); and Trp (-3.4).
[00161] In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another having a similar hydropathic index (e.g., within a value of plus or minus 2.0). In such embodiments, each amino acid residue may be assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics, as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8);
Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).
[00162] In other embodiments, conserved amino acid substitutions may be made where an amino acid residue is substituted for another in the same class, where the amino acids are divided into non-polar, acidic, basic and neutral classes, as follows: non-polar: Ala, Val, Leu, Ile, Phe, Trp, Pro, Met; acidic: Asp, Glu; basic: Lys, Arg, His; neutral:
Gly, Ser, Thr, Cys, Asn, GIn, Tyr.
[00163] Conservative amino acid changes can include the substitution of an L-amino acid by the corresponding D-amino acid, by a conservative D-amino acid, or by a naturally-occurring, non-genetically encoded form of amino acid, as well as a conservative substitution of an L-amino acid. Naturally-occurring non-genetically encoded amino acids include beta-alanine, 3-amino-propionic acid, 2,3-diamino propionic acid, alpha-aminoisobutyric acid, 4-amino-butyric acid, N-methylglycine (sarcosine), hydroxyproline, ornithine, citrulline, t-butylalanine, t-butylgiycine, N-methylisoleucine, phenyiglycine, cyclohexylalanine, norleucine, norvaline, 2-napthylalanine, pyridylalanine, 3-benzothienyl alanine, 4-chiorophenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 1,2,3,4-tetrahydro-isoquinoline-3-carboxylix acid, beta-2-thienylalanine, methionine sulfoxide, homoarginine, N-acetyl lysine, 2-amino butyric acid, 2-amino butyric acid, 2,4,-diamino butyric acid, p-aminophenylalanine, N-methylvaline, homocysteine, homoserine, cysteic acid, epsilon-amino hexanoic acid, delta-amino valeric acid, or 2,3-diaminobutyric acid.
[00164] In other embodiments, conservative amino acid changes include changes based on considerations of hydrophilicity or hydrophobicity, size or volume, or charge. Amino acids can be generally characterized as hydrophobic or hydrophilic, depending primarily on the properties of the amino acid side chain. A hydrophobic amino acid exhibits a hydrophobicity of greater than zero, and a hydrophilic amino acid exhibits a hydrophilicity of less than zero, based on the normalized consensus hydrophobicity scale of Eisenberg et al. (J.
Mol. Biol.
179: 125-142, 1984). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, lie, Pro, Met and Trp, and genetically, encoded hydrophilic amino acids include Thr, His, Glu, Gin, Asp, Arg, Ser, and Lys.
[00165] Hydrophobic or hydrophilic amino acids can be further subdivided based on the characteristics of their side chains. For example, an aromatic amino acid is a hydrophobic amino acid with a side chain containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents.
[00166] An apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at physiological pH and which has bonds in which a pair of electrons shared in common by two atoms is generally held, equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded apolar amino acids include Gly, Leu, Val, Ile, Ala, and Met. Apolar amino acids can be further subdivided to include aliphatic amino acids, which is a hydrophobic amino acid having an aliphatic hydrocarbon side chain.
Genetically encoded aliphatic amino acids include Ala, Leu, Val, and lie.
[00167] A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at physiological pH, but which has one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and GIn.
[00168] An acidic amino acid is a hydrophilic amino acid with a side chain pKa value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include Asp and 5 Glu. A basic amino acid is a hydrophilic amino acid with a side chain pKa value of greater than 7. Basic amino acids typically have positively charged side chains at physiological pH
due to association with hydronium ion. Genetically encoded basic amino acids include Arg, Lys, and His.
[00169] The above classifications are not absolute and an amino acid may be classified in 10 more than one category. In addition, amino acids can be classified based on known behavior and or characteristic chemical, physical, or biological properties based on specified assays or as compared with previously identified amino acids. Amino acids can also include bifunctional moieties having amino acid-like side chains.
[00170] Conservative changes can also include the substitution of a chemically 15 derivatised moiety for a non-derivatised residue, by for example, reaction of a functional side group of an amino acid.
[00171] In addition to the substitutions outlined above, synthetic amino acids providing similar side chain functionality can also be introduced into the peptide/domain. For example, aromatic amino acids may be replaced with D- or L-naphthylalanine, D- or L-phenylglycine, 20 D- or L-2-thienylalanine, D- or L-1-, 2-, 3-, or 4-pyrenylalanine, D- or L-3-thienylalanine, D- or L-(2-pyridinyl)-alanine, D- or L-(3-pyridinyl)-alanine, D- or L-(2-pyrazinyl)-alanine, D- or L-(4-isopropyl)-phenylglycine, D-(trifluoromethyl)-phenylglycine, D-(trifluoromethyl)-phenylalanine, D-p-fluorophenylalanine, D- or L-p-biphenylalanine D-or L-p-methoxybiphenylalanine, D- or L-2-indole(alkyl)alanines, and D- or L-alkylalanines wherein the alkyl group is selected from 25 the group consisting of substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, and iso-pentyl.
[00172] Non-carboxylate amino acids can be made to possess a negative charge, as provided by phosphono- or sulfated (e.g., -SO3H) amino acids, which are to be considered as non-limiting examples.
30 [00173] Other substitutions may include unnatural alkylated amino acids, made by combining an alkyl group with any natural amino acid. Basic natural amino acids such as lysine and arginine may be substituted with alkyl groups at the amine (NH2) functionality. Yet other substitutions include nitrile derivatives (e.g., containing a CN-moiety in place of the CONH2 functionality) of asparagine or glutamine, and sulfoxide derivative of methionine. In addition, any amide linkage in the peptide/domain may be replaced by a ketomethylene, hydroxyethyl, ethyl/reduced amide, thioamide or reversed amide moieties, (e.g., (-C=O)-CH2-), (-CHOH)-CH2-), (CH2-CH2-), (-C=S)-NH-), or (-NH-(-C=O) for (-C=O)-NH-)).
[00174] Other modifications are also included within the definition of functional analog of the peptide/domain of the present invention. For example, the size of the peptide/domain can be reduced by deleting one or more amino acids, and/or amino acid mimetics or dipeptide mimics containing non-peptide bonds may be used. Examples of using molecular scaffolds such as benzodiazepine, azepine, substituted gamma lactam rings, keto-methylene pseudopeptides, P-turn dipeptide cores and R-aminoalcohols for these purposes are known to peptide chemists and are described in for example Peptidomimetic protocols (Methods in molecular medicine Vol. 23) W. M. Kazmierski (ed.), Humana Press and Advances in Amino Acid Mimetics and Peptidomimetics, Vols. 1 & 2, A. Abell (Ed).
[00175] Covalent modifications of the peptide/domain are thus included within the scope of the present invention. Such modifications may be introduced into the peptide/domain for example by reacting targeted amino acid residues of the peptide/domain with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues.
The following examples of chemical derivatives are provided by way of illustration and not by way of limitation.
[00176] Cysteinyl residues may be reacted with alpha-haloacetates (and corresponding amines), such as 2-chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Histidyl residues may be derivatized by reaction with compounds such as diethylprocarbonate e.g., at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain, and para-bromophenacyl bromide may also be used; e.g., where the reaction is preferably performed in 0.1 M sodium cacodylate at pH
6Ø
[00177] Lysinyl and amino terminal residues may be reacted with compounds such as succinic or other carboxylic acid anhydrides. Other suitable reagents for derivatizing alpha-amino-containing residues include compounds such as imidoesters, e.g., methyl picolinimidate; pyridoxal phosphate; pyridoxal; chIoroborohydride;
trinitrobenzenesulfonic acid; O-methyl isourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
[00178] Arginyl residues may be modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin according to known method steps. Derivatization of arginine residues is typically performed in alkaline conditions because of the high pKa of the guanidine functional group.
Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. The specific modification of tyrosinyl residues per se is well-known, such as for introducing spectral labels into tyrosinyl residues by reaction with aromatic diazonium compounds or tetranitromethane. N-acetylimidazol and tetranitromethane may be used to form O-acetyl tyrosinyl species and 3-nitro derivatives, respectively.
[00179] Carboxyl side groups (aspartyl or glutamyl) may be selectively modified by reaction with carbodiimides (R'-N=C=N-R') such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethyl pentyl) carbodiimide.
Furthermore aspartyl and glutamyl residues may be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions. Glutaminyl and asparaginyl residues may be frequently deamidated to the corresponding glutamyl and aspartyl residues. Other modifications of the peptides in the present invention may include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains acetylation of the N-terminal amine, methylation of main chain amide residues (or substitution with N-methyl amino acids) and, in some instances, amidation of the C-terminal carboxyl groups, according to known method steps.
[00180] Covalent attachment of fatty acids (e.g., C6-C16) to the peptide/domain may confer additional biological properties such as protease resistance, plasma protein binding, increased plasma half-life, intracellular penetration, etc. The above description of modification of a peptide/domain does not limit the scope of the approaches nor the possible modifications that can be engineered.
[00181] In embodiments, the N- and/or C-terminal amino acids of the above-mentioned peptide may be modified by amidation, acetylation, acylation or other modifications known in the art. In an embodiment, the amino terminal residue (i.e., the free amino group at the N-terminal end of the peptide) of the peptide is modified (e.g., for protection against degradation). In an embodiment, the modification is acylation with a C2-C16 acyl group, in a further embodiment, the modification is acetylation.
[00182] In an embodiment, the carboxy terminal residue (i.e., the free carboxy group at the C-terminal end of the peptide) of said peptide is modified (e.g., for protection against degradation). In an embodiment, the modification is an amidation.
[00183] In an embodiment, the above-mentioned peptide contains about 100 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 90 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 80 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 70 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 60 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 50 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 40 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 30 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 20 amino acids or less. In a further embodiment, the above-mentioned peptide contains about 15 amino acids or less. In a further embodiment, the above-mentioned peptide contains between about 5 to about 15 amino acids. In a further embodiment, the above-mentioned peptide contains 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids.
[00184] The above-mentioned peptide may be produced by expression in a host cell comprising a nucleic acid encoding the peptide (recombinant expression) or by chemical synthesis (e.g., solid-phase peptide synthesis). Peptides can be readily synthesized by automated solid phase procedures well known in the art. Suitable syntheses can be performed by utilizing "T-boc" or "Fmoc" procedures. Techniques and procedures for solid phase synthesis are described in for example Solid Phase Peptide Synthesis: A
Practical Approach, by E. Atherton and R. C. Sheppard, published by IRL, Oxford University Press, 1989. Alternatively, the peptides may be prepared by way of segment condensation, as described, for example, in Liu et al., Tetrahedron Lett. 37: 933-936, 1996;
Baca et al., J. Am.
Chem. Soc. 117: 1881-1887, 1995; Tam et al., Int. J. Peptide Protein Res. 45:
209-216, 1995; Schnolzer and Kent, Science 256: 221-225, 1992; Liu and Tam, J. Am.
Chem. Soc.
116: 4149-4153, 1994; Liu and Tam, Proc. Natl. Acad. Sci. USA 91: 6584-6588, 1994; and Yamashiro and Li, Int. J. Peptide Protein Res. 31: 322-334, 1988). Other methods useful for synthesizing the peptides are described in Nakagawa et al., J. Am. Chem. Soc.
107: 7087-7092, 1985. Commercial providers of peptide synthetic services may also be used to prepare synthetic peptides in the the D- or L-configuration. Such providers include, for example, Advanced ChemTech (Louisville, Ky.), Applied Biosystems (Foster City, Calif.), Anaspec (San Jose, Calif.), and Cell Essentials (Boston, Mass.).
[00185] Peptides and peptide analogues comprising naturally occurring amino acids encoded by the genetic code may also be prepared using recombinant DNA
technology using standard methods. Peptides produced by recombinant technology may be modified (e.g., N-terminal acylation [e.g., acetylation], C-terminal amidation, cyclization/formation of a loop within the peptide [e.g., via formation of a disulphide bridge between Cys residues]) using methods well known in the art. Therefore, in embodiments, in cases where a peptide described herein contains naturally occurring amino acids encoded by the genetic code, the peptide may be produced using recombinant methods, and may in embodiments be subjected to for example the just-noted modifications (e.g., acylation, amidation, cyclization).
[00186] "Recombinant technology" refers to the production of a peptide or polypeptide by recombinant techniques, wherein generally, a nucleic acid encoding peptide is inserted into a suitable expression vector which is in turn used to transform/transfect a host cell to produce the protein. The term "recombinant" when made in reference to a protein or a polypeptide refers to a peptide, polypeptide or protein molecule which is expressed using a recombinant nucleic acid construct created by means of molecular biological techniques.
Recombinant nucleic acid constructs may include a nucleotide sequence which is ligated to, or is manipulated to become ligated to, a nucleic acid sequence to which it is not ligated in nature, or to which it is ligated at a different location in nature. Referring to a nucleic acid construct as "recombinant" therefore indicates that the nucleic acid molecule has been manipulated using genetic engineering, i.e., by human intervention.
Recombinant nucleic acid constructs may for example be introduced into a host cell by transformation/transfection. Such recombinant nucleic acid constructs may include sequences derived from the same host cell species or from different host cell species, which have been isolated and reintroduced into cells of the host species.
Recombinant nucleic acid construct sequences may become integrated into a host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and/or repair events.
[00187] The peptides of the invention can be purified by many techniques well known in the art, such as reverse phase chromatography, high performance liquid chromatography (HPLC), ion exchange chromatography, size exclusion chromatography, affinity chromatography, gel electrophoresis, and the like. The actual conditions used to purify a particular peptide or peptide analog will depend, in part, on synthesis strategy and on factors such as net charge, hydrophobicity, hydrophilicity, and the like, and will be apparent to those of ordinary skill in the art. For affinity chromatography purification, any antibody which specifically binds the peptide or peptide analog may for example be used.
5 [00188] In an embodiment, the above-mentioned peptide is substantially pure.
A
compound is "substantially pure" when it is separated from the components that naturally accompany it. Typically, a compound is substantially pure when it is at least 60%, more generally 75%, preferably over 90% and more preferably over 95%, by weight, of the total material in a sample. Thus, for example, a polypeptide that is chemically synthesized or 10 produced by recombinant technology will generally be substantially free from its naturally associated components. A substantially pure peptide can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid molecule encoding a peptide compound; or by chemical synthesis. Purity can be measured using any appropriate method such as column chromatography, gel electrophoresis, HPLC, etc.
15 [00189] In another aspect, the present invention provides a composition comprising the above-mentioned Ninjurin-1 and/or MCAM inhibitor and a pharmaceutically acceptable carrier or excipient. In an embodiment, the above-mentioned composition is used for the prevention or treatment of an inflammatory condition, such as a neuroinflammatory condition.
20 [00190] Such compositions may be prepared in a manner well known in the pharmaceutical art. Supplementary active compounds can also be incorporated into the compositions. As used herein "pharmaceutically acceptable carrier" or "excipient" includes any and all solvents, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The 25 carrier can be suitable, for example, for intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration (see Remington: The Science and Practice of Pharmacy by Alfonso R.
Gennaro, 2003, 21th edition, Mack Publishing Company).
30 [00191] Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of active agent(s)/composition(s) suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
[00192] Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for compounds/compositions of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
Formulations for inhalation may contain excipients, (e.g., lactose) or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
[00193] For preparing pharmaceutical compositions from the compound(s)/composition(s) of the present invention, pharmaceutically acceptable carriers are either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substance, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
[00194] In powders, the carrier is a finely divided solid, which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. The powders and tablets may typically contain from 5% or 10% to 70% of the active compound/composition. Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
The term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it.
Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
[00195] Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
[00196] Aqueous solutions suitable for oral use are prepared by dissolving the Ninjurin-1 and/or MCAM inhibitor in water and adding suitable colorants, flavors, stabilizers, and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well-known suspending agents.
[00197] In an embodiment, the Ninjurin-1 and/or MCAM inhibitor is formulated/administered such that it comes into contact with neural cells or neural tissue, such as central nervous system (CNS) cells or tissue. Such tissue includes brain and spinal cord (e.g., cervical, thoracic, or lumbar) tissue. As such, in embodiments, the Ninjurin-1 and/or MCAM inhibitor can be administered to treat neural cells/tissue in vivo via direct intracranial injection or injection into the cerebrospinal fluid (e.g., intrathecal injection).
Alternatively, the Ninjurin-1 and/or MCAM inhibitor can be administered systemically (e.g.
intravenously) and may come into contact with the affected neural tissue via lesions (where the blood-brain barrier is compromised), or, in a further embodiment, may be in a form capable of crossing the blood-brain barrier and entering the neural system (e.g., CNS).
Further, in an embodiment, a composition of the invention may be formulated for such administration to neural cells/tissue.
[00198] The composition may also contain more than one active compound for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. It may be desirable to use the above-mentioned Ninjurin-1 and/or MCAM inhibitor or composition in addition to one or more agents currently used to prevent or treat the disorder in question. The above-mentioned Ninjurin-1 and/or MCAM
inhibitor may be formulated in a single composition or in several individual compositions which may be co-administered in the course of the treatment.
[00199] The invention further provides a kit or package comprising the above-mentioned Ninjurin-1 and/or MCAM inhibitor or the above-mentioned composition, together with instructions for (i) the prevention and/or treatment of an inflammatory (e.g., neuroinflammatory) condition in a subject and/or (ii) for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ, such as the CNS endothelium. The kit may further comprise, for example, containers, buffers, a device (e.g., syringe) for administering the Ninjurin-1 and/or MCAM inhibitor or a composition comprising same.
[00200] Given the correlation between Ninjurin-1 and MCAM expression/activity and (i) inflammation (e.g., neuroinflammation) as well as (ii) immune cell recruitment to the CNS
endothelium, compounds which are capable of decreasing Ninjurin-1 and/or MCAM
expression/activity may be used for the prevention and/or treatment of inflammatory conditions and/or for inhibiting immune cell recruitment to the endothelium of a tissue/organ, such as the CNS endothelium. Therefore, the invention further relates to screening methods (e.g. in vitro methods) for the identification and characterization of compounds capable of decreasing/inhibiting Ninjurin-1 and/or MCAM expression and/or activity, which may be used for the prevention and/or treatment of inflammatory conditions and/or for inhibiting immune cell recruitment to the endothelium of a tissue/organ, such as the CNS
endothelium. In an embodiment, the above-mentioned endothelium is exposed to an inflammatory environment (e.g., the endothelium of an inflamed tissue/organ).
[00201] In another aspect, the present invention provides a method of identifying a compound for preventing or treating an inflammatory condition, said method comprising determining whether: (a) a level of expression of a Ninjurin-1 and/or MCAM
nucleic acid or encoded polypeptide; (b) a level of Ninjurin-1 and/or MCAM activity (e.g., Ninjurin-1 and/or MCAM homotypic binding activity); or (c) a combination of (a) and (b), is decreased in the presence of a test compound relative to in the absence of said test compound;
wherein said decrease is indicative that said test compound may be used for preventing or treating said inflammatory condition.
[00202] In another aspect, the present invention provides a method of identifying or characterizing a compound for preventing or treating an inflammatory condition, said method comprising: (a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for preventing or treating said inflammatory condition.
[00203] In another aspect, the present invention provides a method of identifying a compound for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., CNS endothelium), said method comprising determining whether: (a) a level of expression of a Ninjurin-1 and/or MCAM
nucleic acid or encoded polypeptide; (b) a level of Ninjurin-1 and/or MCAM activity (e.g., Ninjurin-1 and/or MCAM homotypic binding activity); or (c) a combination of (a) and (b), is decreased in the presence of a test compound relative to in the absence of said test compound;
wherein said decrease is indicative that said test compound may be used for inhibiting the recruitment of an immune cell across the endothelium of the tissue/organ.
[00204] In another aspect, the present invention provides a method of identifying a compound for inhibiting the recruitment of an immune cell (e.g., a myeloid or lymphoid cell) across the endothelium of a tissue/organ (e.g., CNS endothelium), said method comprising:
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM
gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound:
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for inhibiting the recruitment of an immune cell across the endothelium of the tissue/organ.
[00205] In an embodiment, the above-mentioned Ninjurin-1 and/or MCAM activity is a binding activity. Methods to measure the binding of a compound to Ninjurin-1 are well known in the art (see, for example, U.S. Patent No. 6,559,288).
[00206] The above-noted screening method or assay may be applied to a single test compound or to a plurality or "library" of such compounds (e.g., a combinatorial library). Any such compounds may be utilized as lead compounds and further modified to improve their therapeutic, prophylactic and/or pharmacological properties for preventing and/or treating a neuroinflammatory condition.
[00207] Test compounds (drug candidates) may be obtained from any number of sources including libraries of synthetic or natural compounds. For example, numerous means are 5 available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides.
Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means.
10 [00208] Screening assay systems may comprise a variety of means to enable and optimize useful assay conditions. Such means may include but are not limited to: suitable buffer solutions, for example, for the control of pH and ionic strength and to provide any necessary components for optimal activity and stability (e.g., protease inhibitors), temperature control means for optimal activity and/or stability, of Ninjurin-1, and detection 15 means to enable the detection of its activity. A variety of such detection means may be used, including but not limited to one or a combination of the following:
radiolabelling, antibody-based detection, fluorescence, chemiluminescence, spectroscopic methods (e.g., generation of a product with altered spectroscopic properties), various reporter enzymes or proteins (e.g., horseradish peroxidase, green fluorescent protein), specific binding reagents (e.g., 20 biotin/(strept)avidin), and others.
[00209] As noted above, the invention further relates to methods for the identification and characterization of compounds capable of decreasing Ninjurin-1 and/or MCAM
gene expression. Such a method may comprise assaying Ninjurin-1 and/or MCAM gene expression in the presence versus the absence of a test compound. Such gene expression 25 may be measured by detection of the corresponding RNA or protein, or via the use of a suitable reporter construct comprising one or more transcriptional regulatory element(s) normally associated with a Ninjurin-1 gene, operably-linked to a reporter gene.
[00210] A first nucleic acid sequence is "operably-linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the 30 second nucleic acid sequence. For instance, a promoter is operably-linked to a coding sequence if the promoter affects the transcription or expression of the coding sequences.
[00211] Generally, operably-linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since, for example, enhancers generally function when separated from the promoters by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably-linked but not contiguous. "Transcriptional regulatory element" is a generic term that refers to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals which induce or control transcription of protein coding sequences with which they are operably-linked. The expression of such a reporter gene may be measured on the transcriptional or translational level, e.g., by the amount of RNA or protein produced. RNA may be detected by for example Northern analysis or by the reverse transcriptase-polymerase chain reaction (RT-PCR) method (see for example Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA).
[00212] Protein levels may be detected either directly using affinity reagents (e.g., an antibody or fragment thereof (for methods, see for example Harlow, E. and Lane, D (1988) Antibodies : A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); a ligand which binds the protein) or by other properties (e.g., fluorescence in the case of green fluorescent protein) or by measurement of the protein's activity, which may entail enzymatic activity to produce a detectable product (e.g., with altered spectroscopic properties) or a detectable phenotype (e.g., alterations in cell growth/function). Suitable reporter genes include but are not limited to chloramphenicol acetyltransferase, beta-D
galactosidase, luciferase, or green fluorescent protein (GFP).
[00213] Ninjurin-1 and/or MCAM protein expression levels could be determined using any standard methods known in the art. Non-limiting examples of such methods include Western blot, tissue microarray, immunoblot, enzyme-linked immunosorbant assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), flow cytometry, and assays based on a property of the protein including but not limited to DNA
binding, ligand binding, or interaction with other protein partners.
[00214] Methods to determine Ninjurin-1 and/or MCAM nucleic acid (mRNA) levels are known in the art, and include for example polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR) (e.g., as in Example 3 below), in situ PCR, SAGE, quantitative PCR (q-PCR), in situ hybridization, Southern blot, Northern blot, sequence analysis, microarray analysis, detection of a reporter gene, or other DNA/RNA
hybridization platforms.
For RNA expression, preferred methods include, but are not limited to:
extraction of cellular mRNA and Northern blotting using labeled probes that hybridize to transcripts encoding all or part of one or more of the genes of this invention; amplification of mRNA
expressed from one or more of the genes of this invention using gene-specific primers, polymerase chain reaction (PCR), quantitative PCR (q-PCR), and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantitative detection of the product by any of a variety of means; extraction of total RNA from the cells, which is then labeled and used to probe cDNAs or oligonucleotides encoding all or part of the genes of this invention, arrayed on any of a variety of surfaces; in situ hybridization.
[00215] In embodiments, competitive screening assays may be done by combining a Ninjurin-1 and/or MCAM polypeptide, or a fragment thereof (a Ninjurin-1 and/or MCAM
binding domain) and a probe to form a probe : Ninjurin-1 and/or MCAM binding domain complex in a first sample followed by adding a test compound. The binding of the test compound is determined, and a change, or difference in binding of the probe in the presence of the test compound indicates that the test compound is capable of binding to the Ninjurin-1 and/or MCAM binding domain and potentially modulating Ninjurin-1 and/or MCAM
activity.
[00216] The binding of the test compound may be determined through the use of competitive binding assays. In this embodiment, the probe is labeled with an affinity label such as biotin. Under certain circumstances, there may be competitive binding between the test compound and the probe, with the probe displacing the candidate agent. In one case, the test compound may be labeled. Either the test compound, or a compound of the present invention, or both, is added first to the Ninjurin-1 binding domain for a time sufficient to allow binding to form a complex.
[00217] The assay may be carried out in vitro utilizing a source of Ninjurin-1 and/or MCAM which may comprise a naturally isolated or recombinantly produced Ninjurin-1 and/or MCAM (or a variant/fragment thereof), in preparations ranging from crude to pure. Such assays may be performed in an array format. In certain embodiments, one or a plurality of the assay steps are automated.
[00218] In embodiments, the assays described herein may be performed in a cell or cell-free format.
[00219] A homolog, variant and/or fragment of Ninjurin-1 and/or MCAM which retains activity (e.g., a binding activity) may also be used in the methods of the invention.
[00220] "Homology", "homologous" and "homolog" refer to sequence similarity between two polypeptide molecules. Homology can be determined by comparing each position in the aligned sequences. A degree of homology between amino acid sequences is a function of the number of identical or matching amino acids at positions shared by the sequences. Two nucleotide or amino acid sequences are considered "substantially identical"
if, when optimally aligned (with gaps permitted), they share at least about 50%
sequence similarity or identity, or if the sequences share defined functional motifs. In alternative embodiments, sequence similarity in optimally aligned substantially identical sequences may be at least 60%, 70%, 75%, 80%, 85%, 90% or 95%, e.g., with any of the sequences described herein.
As used herein, a given percentage of homology between sequences denotes the degree of sequence identity in optimally aligned sequences. An "unrelated" or "non-homologous"
sequence shares less than 40% identity, though preferably less than about 25 %
identity, with any of the sequences described herein.
[00221] Optimal alignment of sequences for comparisons of identity may be conducted using a variety of algorithms, such as the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math 2: 482, the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444, and the computerised implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI, U.S.A.).
Sequence identity may also be determined using the BLAST algorithm, described in Altschul et al., 1990, J. Mol. Biol. 215:403-10 (using the published default settings).
Software for performing BLAST analysis may be available through the National Center for Biotechnology Information.
The BLAST algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold. Initial neighbourhood word hits act as seeds for initiating searches to find longer HSPs. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction is halted when the following parameters are met: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program may use as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1992, Proc. Natl.
Acad. Sci. USA
89: 10915-10919) alignments (B) of 50, expectation (E) of 10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a comparison of both strands. One measure of the statistical similarity between two sequences using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. In alternative embodiments of the invention, nucleotide or amino acid sequences are considered substantially identical if the smallest sum probability in a comparison of the test sequences is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
[00222] An alternative indication that two nucleic acid sequences are substantially complementary is that the two sequences hybridize to each other under moderately stringent, or preferably stringent, conditions. Hybridization to filter-bound sequences under moderately stringent conditions may, for example, be performed in 0.5 M
NaHPO4, 7%
sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 C, and washing in 0.2 x SSC/0.19% SIDS at 42 C (see Ausubel, et al. (eds), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
2.10.3).
Alternatively, hybridization to filter-bound sequences under stringent conditions may, for example, be performed in 0.5 M NaHPO4i 7% SDS, 1 mM EDTA at 65 C, and washing in 0.1 x SSC/0.1% SDS at 68 C (see Ausubel, et al. (eds), 1989, supra).
Hybridization conditions may be modified in accordance with known methods depending on the sequence of interest (see Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology -- Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York). Generally, stringent conditions are selected to be about 5 C lower than the thermal melting point for the specific sequence at a defined ionic strength and pH.
[00223] In an embodiment, the above-mentioned homolog, variant and/or fragment of Ninjurin-1 comprises a region corresponding to residues 28 to 35 (Arg28-Pro35) of the human Ninjurin-1 polypeptide (Fig. 8B). In a further embodiment, the above-mentioned Ninjurin-1 polypeptide or fragment thereof comprises a region corresponding to residues 26 to 37 (Pro26-Asn37) of the human Ninjurin-1 polypeptide.
[00224] The present inventors have shown in human samples and neuroinflammatory mouse models (EAE and SCI) that Ninjurin-1 is expressed or overexpressed on CNS cells 5 and peripheral immune cells (myeloid cells) in neuroinflammatory conditions, and thus that Ninjurin-1 may be used as a biological marker for the detection and characterization of neuroinflammatory conditions. They have also demonstrated that the proportion of T cells expressing MCAM is higher in the peripheral blood (PB) and cerebro-spinal fluid (CSF) of MS patients as compared to healthy controls.
10 [00225] Therefore, in another aspect, the invention relates to the diagnosis and prognosis of an inflammatory condition, such as a neuroinflammatory condition. The invention thus provides a method for diagnosing or prognosing an inflammatory condition in a subject based on the expression and/or activity of Ninjurin-1 and/or MCAM determined in a sample (e.g., a CNS sample or a blood/blood cell sample) from the subject. The expression and 15 activity of Ninjurin-1 and/or MCAM in the sample may be determined using the assays/methods described above.
[00226] In an embodiment, the method may comprise determining whether Ninjurin-and/or MCAM activity and/or expression is modulated, e.g., upregulated or increased, relative to a control/reference activity or expression. In yet another embodiment, the control 20 Ninjurin-1 and/or MCAM expression or activity can be selected from an established standard, a corresponding Ninjurin-1 and/or MCAM expression or activity determined in the subject (in a sample from the subject) at an earlier time; a corresponding Ninjurin-1 and/or MCAM expression or activity determined in a control subject known to not having an inflammatory condition (e.g., a healthy subject). In such cases, an increased or higher 25 expression and/or activity in the sample from the subject relative to the control activity or expression is indicative that the subject has an inflammatory condition.
"Higher expression"
as used herein refers to (i) higher expression of Ninjurin-1 and/or MCAM on one or more given cells present in the sample and/or (ii) increased amount (absolute or relative amount) of Ninjurin-1- and/or MCAM-expressing/positive cells in the sample.
30 [00227] In another embodiment, the control Ninjurin-1 and/or MCAM
expression or activity is a corresponding expression or activity in a control subject known to have an inflammatory condition. In such a case, a comparable or higher Ninjurin-1 and/or MCAM
expression and/or activity in a sample from the subject relative to the control expression or activity is indicative that the subject has an inflammatory condition.
[00228] Methods for normalizing the level of expression of a gene are well known in the art. For example, the expression level of a gene of the present invention can be normalized on the basis of the relative ratio of the mRNA level of this gene to the mRNA
level of a housekeeping gene or the relative ratio of the protein level of the protein encoded by this gene to the protein level of the housekeeping protein, so that variations in the sample extraction efficiency among cells or tissues are reduced in the evaluation of the gene expression level. A "housekeeping gene" is a gene the expression of which is substantially the same from sample to sample or from tissue to tissue, or one that is relatively refractory to change in response to external stimuli. A housekeeping gene can be any RNA
molecule other than that encoded by the gene of interest that will allow normalization of sample RNA
or any other marker that can be used to normalize for the amount of total RNA
added to each reaction. For example, the GAPDH gene, the G6PD gene, the actin gene, ribosomal RNA, 36B4 RNA, PGK1, RPLPO, or the like, may be used as a housekeeping gene.
[00229] Methods for calibrating the level of expression of a gene are well known in the art.
For example, the expression of a gene can be calibrated using reference samples, which are commercially available. Examples of reference samples include, but are not limited to:
StratageneTM QPCR Human Reference Total RNA, ClontechTM Universal Reference Total RNA, and XpressRefTM Universal Reference Total RNA.
[00230] In an embodiment, the above-mentioned method comprises determining the level of a Ninjurin-1 nucleic acid (e.g., the nucleic acid of SEQ ID NO: 1 or a nucleic acid which encodes the polypeptide of SEQ ID NO: 2) in the sample. In another embodiment, the above-mentioned method comprises determining the level of a Ninjurin-1 polypeptide (e.g., the polypeptide of SEQ ID NO: 2) in the sample. In an embodiment, the level of a Ninjurin-1 polypeptide is determined using an anti-Ninjurin-1 antibody. In another embodiment, the above-mentioned method comprises determining the level of an MCAM nucleic acid (e.g., the nucleic acid of SEQ ID NO: 6 or a nucleic acid which encodes the polypeptide of SEQ ID
NO: 7) in the sample. In another embodiment, the above-mentioned method comprises determining the level of an MCAM polypeptide (e.g., the polypeptide of SEQ ID
NO: 7) in the sample. In an embodiment, the level of an MCAM polypeptide is determined using an anti-an MCAM antibody.
[00231] "Sample" or "biological sample" refers to any solid or liquid sample isolated from a live being. In a particular embodiment, it refers to any solid or liquid sample isolated from a human, such as a biopsy material, blood, saliva, synovial fluid, urine, amniotic fluid and cerebrospinal fluid. In an embodiment, the above-mentioned sample is obtained from the central nervous system (e.g., a CNS cell, tissue or fluid). In a further embodiment, the CNS
cell is obtained by a biopsy. In a further embodiment, the CNS cell is a CNS
endothelial cell, such as an endothelial cell of the blood-brain barrier.
[00232] In another embodiment, the above-mentioned sample is a blood sample or a blood cell sample. In a further embodiment, the above-mentioned blood cell sample is a peripheral blood mononuclear cell (PBMC) sample. In an embodiment, the above-mentioned blood or blood cell sample comprises myeloid cells, such as monocytes and/or dendritic cells. In another embodiment, the above-mentioned blood or blood cell sample comprises lymphoid cells, and more particularly T cells (CD4+ and/or CD8+ T cells). In an embodiment, the above-mentioned blood or blood cell sample comprises memory T cells.
[00233] In an embodiment, the above-mentioned blood or blood cell sample may be submitted to one or more cell depletion or enrichment steps, so as to enrich the sample in one or more cell types of interest (e.g., myeloid cells, such as monocytes and/or dendritic cells, or T cell, such as memory T cells). In an embodiment, the above-mentioned method comprises determining the proportion or relative amount of Ninjurin-1+- and/or MCAM+-cell in said sample and comparing it to a corresponding proportion or relative amount in a control/reference sample. Accordingly, in another aspect, the present invention provides a method for diagnosing an inflammatory condition, such as a neuroinflammatory condition, in a first subject, said method comprising (a) determining the extent/amount of Ninjurin-1 and/or MCAM expression (e.g., the number of Ninjurin-1- and/or MCAM-expressing cells and/or the intensity of Ninjurin-1 and/or MCAM expression per cell) in a sample said first subject (b) comparing said amount to a corresponding reference amount; and (c) diagnosing an inflammatory condition, such as a neuroinflammatory condition, based on said comparison.
[00234] The proportion of Ninjurin-1- and/or MCAM-expressing/positive cells in a sample may be measured using methods well known in the art, for example by flow cytometry, as described below.
[00235] In another aspect, the present invention provides a method for monitoring the course of treatment of a patient suffering from an inflammatory condition, such as a neuroinflammatory condition (e.g., MS, SCI), the method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein a decrease in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at an earlier time is indicative that said patient is responsive to said treatment. In an embodiment, a similar or an increased expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at an earlier time is indicative that said patient is not responsive to said treatment.
[00236] In another aspect, the present invention provides a method of determining whether a subject is suffering from a relapse (exacerbation) of an inflammatory condition, said method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein an increase in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at a time point of remission is indicative that said patient is suffering from a relapse of said inflammatory condition. The treatment regimen (e.g., drug, dose) may thus be adapted accordingly.
[00237] In an embodiment, the above-mentioned expression and/or activity of Ninjurin-1 and/or MCAM is determined based on the amount of Ninjurin-1- and/or MCAM-expressing cells in said sample. In a further embodiment, the above-mentioned amount is a relative amount (e.g., the proportion of Ninjurin-1- and/or MCAM-expressing cells relative to the total cells present in the sample, or relative to another cell type present in the sample).
[00238] In an embodiment, the methods of diagnosis/prognostication noted above may be performed in conjunction with the therapeutic/prophylactic methods noted above, for preventing and/or treating an inflammatory condition in a subject. Such a method thus comprises the diagnosis or prognostication of an inflammatory condition in a subject and, in accordance with the diagnosis/prognosis, decreasing Ninjurin-1 and/or MCAM
levels in the subject (e.g., in a cell, tissue or organ of the subject) thereby to prevent or treat the inflammatory condition.
[00239] The present invention also provides a kit or package comprising a reagent useful for determining Ninjurin-1 and/or MCAM expression and activity (e.g., a ligand that specifically binds a Ninjurin-1 and/or MCAM polypeptide such as an antibody, or a ligand that specifically binds a Ninjurin-1 and/or MCAM nucleic acid such as an oligonucleotide).
Such kit may further comprise, for example, instructions for the prognosis and/or diagnosis of an inflammatory condition, control samples, containers, reagents useful for performing the methods (e.g., buffers, enzymes), etc.
[00240] As used herein the term "subject" is meant to refer to any animal, such as a mammal including human, mice, rat, dog, cat, pig, cow, monkey, horse, etc. In an embodiment, the above-mentioned subject is a mammal, in a further embodiment a human.
[00241] The present inventors have further determined that MCAM expression permits the identification of T cells having the capacity or the potential to perform effector functions, for example to produce various inflammatory cytokines (e.g., IL-17 and IFN-y), upon activation (e.g., TCR-mediated activation).
[00242] Accordingly, in another aspect, the present invention provides a method of identifying an inflammatory cytokine-secreting T cell or a precursor thereof in a sample, said method comprising identifying MCAM-expressing cells in the sample, for example by contacting said cell with an MCAM ligand (e.g., (i) MCAM, (ii) an MCAM binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii)) and identifying said inflammatory cytokine-secreting T cell precursor based on the binding to said MCAM ligand.
[00243] In another aspect, the present invention provides a method of purifying an inflammatory cytokine-secreting T cell or a precursor thereof from a cell sample, or enriching a sample comprising a cell sample in inflammatory cytokine-secreting T cells or a precursor thereof, said method comprising contacting said sample with an MCAM ligand (e.g., (i) MCAM, (ii) an MCAM binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii)) and isolating or purifying said inflammatory cytokine-secreting T cell or precursor thereof on the basis of its binding to the MCAM
ligand.
[00244] In another aspect, the present invention provides a kit for identifying and/or purifying an inflammatory cytokine-secreting T cell or a precursor thereof from a cell sample, or enriching a sample comprising a cell sample in inflammatory cytokine-secreting T cell precursors, said kit comprising an MCAM ligand (e.g., (i) MCAM, (ii) an MCAM
binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii)) and instructions for identifying and/or purifying the inflammatory cytokine-secreting T cell or a precursor thereof from the sample.
[00245] In an embodiment, the above-mentioned MCAM ligand is labeled (using a fluorescent moiety, for example).
5 [00246] In an embodiment, the above-mentioned MCAM ligand is an MCAM-specific antibody or MCAM-binding fragment thereof.
[00247] In an embodiment, the above-mentioned MCAM ligand is bound to a solid support, such a bead (e.g., a magnetic bead) such as to allow easy separation/isolating of the cell bound to the MCAM ligand from the other cells in the sample (using a column, for 10 example), and thus to obtain a sample enriched in inflammatory cytokine-secreting T cells and/or precursors thereof.
[00248] In an embodiment, the above-mentioned inflammatory cytokine-secreting T cell or precursor thereof has the capacity to secrete preferentially: (i) IL-17, (ii) IFN-y, (iii) TNF-a, (iv) GM-CSF, (v) IL-8, or (vi) any combination of (i) to (v), upon activation/stimulation, such 15 as antigen-specific or non-antigen specific TCR-mediated stimulation.
[00249] In an embodiment, the above-mentioned inflammatory cytokine-secreting T cell or precursor thereof is a memory T cell. In another embodiment, the above-mentioned inflammatory cytokine-secreting T cell or precursor thereof is a CD4+ T cell.
In a further embodiment, the above-mentioned inflammatory cytokine-secreting T cell precursor is a 20 CD4+ T cell having the capacity to differentiate into an IL-17-secreting cell (typically referred to as a "Th17 cell") following exposure to specific polarizing condition.
[00250] The present invention is illustrated in further details by the following non-limiting examples.
Example 1: Materials and Methods 25 [00251] Patients and sample collection. All MS patients were observed at the CHUM-Notre-Dame Hosptial MS clinic and diagnosed according to the McDonald criteria. Human peripheral blood (PB) was collected from MS patients and from healthy donors.
PB
mononuclear cells were obtained from heparinized whole blood using FicollTM
density gradient separation (Amersham Biosciences, Baie D'Urfe, Quebec, Canada). CD14+
30 monocytes, CD4+ CD45RO+ lymphocytes and MCAM+ T cells were isolated using the magnetic cell sorting (MACSTM) isolation columns, according to manufacturer's protocol (Miltenyi, Auburn, CA).
[00252] BBB-endothelial cell isolation and culture. BBB-endothelial cells were isolated from non-epileptic material according to a previously published protocol (Prat et al., J
Neuropathol Exp Neurol. 2000 59(10):896-906; Biernacki et al., J Neuropathol Exp Neurol.
2001 60(12): 1127-36; Prat et al., Arch Neurol. 2002 59(3): 391-7). BBB-endothelial cells were grown in primary cultures in media composed of Medium 199 (Gibco Invitrogen, Burlington, ON, Canada) supplemented with 20% clone M3 conditioned media, 10%
fetal bovine serum (FBS), 5% normal human serum (HS), 0.13% endothelial cell growth supplement (ECGS) and 0.2% insulin-transferrin-selenium on 0.5% gelatin-coated tissue culture plastic plates (all reagents from Sigma, Oakville, ON, Canada). For treatments, the BBB-endothelial cells were grown in culture media in the presence of 40%
astrocyte conditioned media (ACM), until they reach confluency. When indicated BBB-endothelial cells were activated for 24 hours with 100 U/ml of Tumor Necrosis Factor (TNF) and 100 U/ml of Interferon (IFN)-y (Biosource-Invitrogen, Carlsbad, CA, USA) in the presence of 40% ACM
and the absence of ECGS. As previously demonstrated, these cells express factor VIII, von Willebrand factor, Ulex Agglutenens Europaensis-1-binding sites, endothelial antigen HT-7;
and are susceptible to tumor necrosis factor (TNF)-induced CD54 and CD106 up-regulation.
Immunoreactivity for glial fibrillary acidic protein and a-myosin could not be detected, confirming the absence of contaminating astrocytes and smooth muscle cells, respectively.
We also confirmed the absence of monocytes and macrophages by immunostaining with anti-CD14 and anti-CD11c antibodies.
[00253] Astrocyte isolation and culture. Astrocytes were cultured as previously described (Jack et al., J Immunol. 2005 175(7): 4320-4330; Wosik et al., J Neurosci.
2007 27(34):
9032-422007). Astrocytes were grown in primary cultures in complete Dulbecco's Modified Eagle Media (DMEM) (Invitrogen) supplemented with 10% FBS on plastic plates.
Astrocyte-conditioned media (ACM) was harvested once a week from confluent flasks and used in the culture media of the BBB-endothelial cells when mentioned.
[00254] Leukocyte isolation and culture. Venous blood samples were obtained from consenting healthy donors or Multiple Sclerosis (MS) patients in accordance with institutional guidelines. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by a density gradient centrifugation using Ficoll-PaqueTM PLUS (GE Healthcare, Bio-Sciences AB, Sweden). Ex vivo CD4+, CD8+, CD14+ and CD19' cells were isolated from PBMCs using CD4, CD8, CD14 and CD19 mouse anti-human MicroBeadsTM respectively (Miltenyi Biotec Inc., Auburn, CA, USA). To generate mature DCs, PBMCs were first cultured in (Wisent Inc., St-Bruno, Qc, Canada), supplemented with 5% HS (Sigma), 2mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycine (Sigma) for 2 hours at 37 C to enable the cells to adhere. The media was removed and the cells were washed with PBS to remove the non adherent cells. Fresh culture media containing 20 ng/ml of Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, BD Biosciences) and 10 ng/ml of Interleukin (IL)-4 (R&D Systems, Minneapolis, MN, USA). Culture media was replaced every 2 days with fresh media, containing GM-CSF and IL-4, to remove the non adherent cells. After 6 days, 100 ng/ml of Iipopolysaccharide (LPS, from Escherichia coli Serotype 0111:B4, Sigma) was added. Mature DCs were harvested 2 days later for analysis.
[00255] Flow Cytometric analysis of Ninjurin-1 expression. Cells were harvested and resuspended in FACS buffer composed of PBS supplemented with 1% FBS (Biosource-Invitrogen) and 0.1% NaN3 (Sigma). The cells were incubated with HS (Biosource-Invitrogen) to prevent unspecific binding. BBB-endothelial cells and PBMCs were incubated for 1 h at 4 C with the primary unconjugated monoclonal mouse anti-Ninjurin-1 antibody (20 pg/ml, BD Biosciences), or with the Functional Grade Purified Mouse IgG2a Isotype Control (20 pg/ml, ebioscience, Inc., San Diego, CA, USA). Cells were then incubated for 30 min at 4 C with an allophycocyanin (APC)-conjugated goat anti-mouse Ig (2 pg/ml, BD
Biosciences) to detect specific binding. Cells were then counter-stained for 30 min at 4 C
with mouse anti-human conjugated antibodies: Human Leukocyte Antigen (HLA)-ABC, Intracellular cell adhesion molecule (ICAM)-1, CD3, CD4, CD8, CD19, HLA-DR, CD14, CD83, CD123, CD209, CD11c or with corresponding isotype controls (all from BD
Biosciences). To study the expression of Ninjurin-1 on murine immune cells, the mouse monoclonal anti-Ninjurin-1 antibody was biotinylated, to prevent unspecific binding of the secondary antibody. A buffer exchange was performed with a Slide-A-Lyzer Dialysis Cassette (3.5-20K Cassettes, Thermo Scientific, Rockford, IL, USA) to remove glycerol and bovine serum albumin (BSA) from the antibody solution using MelonTM Gel IgG
Spin Purification Kit. Biotin was added using the EZ-Link Micro NHS-PEO4-Biotinylation kit according to the manufacturer's instructions (Thermo Scientific). Murine peripheral blood or CNS mononuclear cells were incubated for 1h at 4 C with the primary biotinylated monoclonal mouse anti-Ninjurin-1 antibody (5 pg/ml, BD Biosciences), or with the Biotin-conjugated IgG2a, K isotype control (5 pg/ml, ebioscience). Cells were then incubated for 30 min at 4 C with secondary antibody goat anti-peroxidase-APC (2 pg/ml, BD
Biosciences).
Counter-staining with anti-mouse CD3, CD4, CD8, CD11 c, F4/80 or with corresponding isotype controls (BD Biosciences and BioLegend, San Diego, CA, USA) were performed.
Cells were acquired on a BD LSRTM II Flow cytometer and analyzed using the BD
FACSDivaTM software (BD Biosciences).
[00256] Western blot. Ex vivo CD4+, CD8+, CD14+ and CD19+ cells (isolated using MACSTM beads, Miltenyi Biotec), mature dendritic cells, primary cultures of BBB endothelial and CNS material from EAE or SCI animals were lysed in denaturing buffer (0.1%
SDS in 50mM Tris-HCI pH 8.5) with Proteinase Inhibitor Cocktail (BD BaculoGoldTM, BD
Biosciences), and sonicated using the Vibra CeIITM ultrasonic processor (Sonics & Materials, Inc., Newtown, CT, USA). Proteins were quantified using the BCATM Protein Assay kit (Thermo Scientific). Thirty micrograms of proteins were separated on a 12% SDS-PAGE gel and the proteins were transfered on Immun-BlotTM PVDF Membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were blocked (1 h at room temperature) with 5%
donkey serum (Sigma), or with 5% non-fat dry milk, in Tris Buffered Saline-0.1 % Tween 20 (Sigma-Aldrich, St-Louis, MO, USA) and then incubated overnight at 4 C with the primary antibody: polyclonal sheep anti-Human Ninjurin-1 (1:100 dilution; R&D Systems) or monoclonal mouse anti-Ninjurin-1 (1:150 dilution; BD Biosciences). Secondary antibody Peroxidase-conjugated affiniPureTM Donkey Anti-Sheep IgG (H+L) (1:10,000 dilution;
Jackson ImmunoResearch Laboratories, West Grove, PA, USA) or secondary antibody rabbit anti-mouse immunoglobulins/HRP (1:1000 dilution; DakoCytomation, Glostrup, Denmark), and ECLTM Westernblotting Analysis System (AmershamTM GE Healthcare, Buckinghamshire, UK) were used to detect specific binding. Mouse anti-13-Actin (1:20,000 dilution; Sigma) was used as a loading control (rabbit anti-mouse immunoglobulins/HRP;
1:1000 dilution; DakoCytomation). HepG2 cell lysate was used as a positive control for the expression of Ninjurin-1 (BD Bioscience). For spinal cord injury material, proteins were extracted from 5 mm length of spinal cord tissue containing the lesion site.
[00257] Immunocytofluorescent stainings. BBB-endothelial cells were grown in chambers slides (Lab TekTM, NuncTM, by Thermoscientific) and were either un-treated or treated for 24 h with TNF and IFN-y (100 U/ml). Cells were fixed for 10 minutes at room temperature with 70% ethanol and incubated overnight at 4 C with mouse monoclonal anti-Ninjurin-1 (1:50 dilution; BD Biosciences). Counter-stainings with F-actin (Sigma) were performed. Slides were mounted using Gelvatol containing either Hoechst 33258 pentahydrate (10 pg/ml, Molecular Probes, Eugene, OR, USA) or TO-PRO-3 (Invitrogen), as nuclear stains.
[00258] Immunofluorescent stainings of human and mouse CNS material. Luxol Fast Blue (LFB) and H&E stainings (Wosik et al., 2007, supra) were performed on human and mouse brain tissue specimens obtained from four MS patients (autopsy) and EAE/SCI
animals.
Sections showing acute demyelinating lesions and active perivascular mononuclear cell infiltration were selected (8 to 12 blocks per MS donor), and compared to normal-appearing white matter from the same donors (8 blocks per donor) and to non-neurological disease controls (3 donors; 9-11 blocks per donor). Mean age was 49 6 years and disease duration ranged from 3 to 23 years. The cause of death was pneumonia (2), urosepsis (1) and barbiturate intoxication (1). Sections (n=40) from MS patients (n= 5) and disease controls (n=5) were fixed in -20 C acetone for 10 minutes, hydrated in PBS and blocked with the avidin/biotin blocking kit (Invitrogen). CNS material from EAE (n=6) and SCI
(n=6) animals were collected following rapid intra-cardiac PBS perfusion and snap-frozen in liquid nitrogen.
Non-specific immunoglobulin binding was blocked with 10% goat serum for 30 minutes at room temperature (rt). Sections were incubated for 40 minutes with Biotin-labelled mouse anti-Ninjurin-1 (1/50, R&D Systems) diluted in 3% goat serum and washed 7 times with PBS
and 0.05% Tween 20 after each incubation. Ninjurin-1 immunostaining was revealed using Cy3- or HRP-labelled streptavidin (DakoCytomation). Sections were mounted using Gelvatol containing either Hoechst 33258 pentahydrate (10 pg/ml, Molecular Probes) or (Invitrogen), as nuclear stains. Additional immunostainings were performed using monoclonal Abs raised against human or mouse CD3, -CD4, CD11c, F4/80, Mac-2 and -IBA-1. Negative controls were performed omitting the primary antibody.
Fluorescence was visualized on a LeicaTM DM6000 B epifluorescent microscope equipped with a digital camera (Leica Microsystems, Wetzlar Germany) or on a LeicaTM SP5 confocal microscope. Images were acquired using OpenlabTM 4Ø4 (Improvision, Waltham, MA) and processed and analyzed with Adobe PhotoshopTM CS2 (Adobe, Mountain View, CA).
[00259] Ninjurin-1 blocking peptides. Peptides corresponding to the adhesion motif of Ninjurin-1, located between amino acids 26 and 37 of Ninjurin-1 [human sequence:
PARWGWRHGPIN (SEQ ID NO: 5); mouse sequence: PPRWGLRNRPIN (SEQ ID NO: 5)], were used as a blocking peptides (referred to as Ninj26_37). Custom Ninj26_37 blocking peptides were synthesized by Alpha Diagnostic International (ADI, San Antonio, TX, USA).
[00260] In vitro model of the BBB. BBB-ECs grown in primary cultures were used to generate an in vitro model of the human BBB. Human BBB-endothelial cells (25 x cells/chamber) were grown in primary culture on 3 pm porous membrane (Becton Dickinson Labware, Franklin Lakes, NJ, USA), coated with 0.5% gelatin (Sigma), in endothelial cell culture media supplemented with 40% ACM, for 4 days to reach confluency. The BBB-endothelial cells were treated with 0.4 mM of human Ninj26_37 (PARWGWRHGPIN, SEQ ID
NO: 5) one hour prior to the addition of the leukocytes. Human ex vivo CD14+
monocytes, or 5 CD4, or CD8+ lymphocytes were isolated, from consenting healthy donors, as described above. The leukocytes were added to the upper chamber (1 X 106 cells /
chamber) and were allowed to migrate across human BBB-endothelial cells for 24h in the presence or absence of the human Ninj26_37 blocking peptide. PBS was used as a vehicle control.
The cells that migrated through the BBB-endothelial cells, were recovered from the lower chamber and 10 counted manually. All migration data shown represent at least 3 independent experiments performed in triplicate.
[00261] EAE mice. Six to eight week-old female wild-type C57BL/6 mice were purchased from Charles River Laboratories (Montreal, Qc, Canada). Experimental autoimmune encephalomyelitis (EAE) was induced by active immunization in female C57BL/6 mice 15 (Jackson Laboratory, Bar Habor, Maine, USA). Seven to eight week old mice were injected subcutaneously with 200 pg of myelin oligodendrocyte glycoprotein (MOG)35-55 peptide [Sequence: MEVGWYRSPFSRWHLYRNGK (SEQ ID NO: 8)] emulsified in complete Freund's Adjuvant supplemented with 600 pg of Mycobacterium tuberculosis H37RA
(DIFCO
Laboratories, Detroit, MI, USA). On day 0 and 2, mice were injected intraperitoneally with 20 500 ng of Pertussis toxin (List Biological Laboratories, INC., Campbell, CA, USA). Starting on day 3, mice were injected intraperitoneally, twice a day, with 200 pg of murine Ninj26-37 blocking peptide. PBS was used as the vehicle control. Animals were monitored daily for signs of EAE and the scoring system was as follows: 0 = no clinical symptoms;
0.5 = partial floppy tail, 1 = floppy tail; 2 = ataxia; 2.5 = weakness in hind limbs, 3 =
paralysis of one hind 25 limb; 4 = paralysis of both hind limbs, 5 = moribund.
[00262] Spinal cord injury. Adult (8-10 weeks old) female C57BL/6 mice were anesthetized with ketamine:xylazine:acepromazine (50:5:1 mg/kg). After performing a laminectomy at the 11th thoracic vertebrae, the exposed spinal cord was contused using the Infinite HorizonsTM Impactor device (Precision Scientific Instrumentation, Lexington, KY).
30 Injuries were made using a force of 60 kDynes, and only animals that had tissue displacements ranging between 500-700 pm were used for experiments. Mice were injected intraperitoneally with 100 pg of the murine Ninj26-37 blocking peptide or PBS
(in 200 pl) every 12 hours, starting one hour after injury and for 7 days, or with PBS as a vehicle control.
Locomotor recovery was evaluated in an open-field test using the Basso Mouse Scale (BMS) (Basso D.M. et al., (2006). J Neurotrauma. 23(5): 635-59). The BMS
analysis of hind limb movements and coordination was carried out by two trained independent technicians and the consensus score taken. The final score is presented as mean SEM.
[00263] Quantitative Real Time PCR (qRT-PCR). RNA from 5 mm length of the uninjured and injured spinal cord containing the lesion site was harvested at 1, 3, 7, 14, 21 and 28 dpi and extracted using RNeasyTM Lipid Tissue kit (Qiagen, Mississauga, Ontario, Canada).
Three individual spinal cords per time point were used. 1 pl of the RT product was added to 24 pi of BrilliantTM SYBRTM Green quantitative PCR Master Mix (Stratagene), and qRT-PCR
was performed to analyze the expression of Ninjurin-1 (MX4000 apparatus, Stratagene). The primers used for Ninjurin-1 amplification were: 5'-AGG GCC ATG AAG ATC AGA ACT
GGA
-3' (sense, SEQ ID NO: 9) and 5'- ATG GAT TTG CTG CAT GTC CTT GGG -3' (antisense, SEQ ID NO: 10). GAPDH was used as a housekeeping gene for control, and the primers used for GAPDH amplification were: 5'- CAA AGT TGT CAT GGA TGA CC -3' (sense, SEQ
ID NO: 11) and 5'- CCA TGG AGA AGG CTG GGG -3' (antisense, SEQ ID NO: 12) (Cayrol et al., Nat Immunol. 2008 9(2): 137-145). The amount of cDNA was calculated based on the threshold cycle (CT) value, and was standardized by the amount of housekeeping gene using the 2- MOCT method (Livak and Schmittgen, Methods 2001 25(4): 402-408).
MCT
were calculated as follow: MMCT= (CT. Target - CT, GAPDH) - (CT. Target - CT, GADPH).
[00264] Western blot analysis of MCAM expression in whole cell lysates and lipid rafts of BBB-ECs. Specific protein detection was performed by Western blot. Forty (40) pg of BBB-EC proteins were analyzed by standard SDS-PAGE using anti-MCAM antibody (mouse, 1/100 ebioscience). Lipid raft were isolated from confluent BBB-ECs using a previously published protocol (Wosik et al., 2007, supra). Cholesterol, phospholipid and protein concentrations in each fraction were assayed using commercially available kits: Cholesterol assay kit (Molecular Probes), Phospholipids B colorimetric method kit (Wako) and BCA
Protein assay kit (Pierce). Lipid raft markers were assessed by SDS-PAGE (GM1, horse radish peroxidase (HRP) -cholera toxin B subunit, Molecular Probes; goat anti-CD59, 1/200, R&D Systems). Specific binding was revealed with HRP-conjugated anti-mouse and anti-goat antibody using the ECLTM system and anti-actin was used as a loading control.
[00265] Immunostaining analysis of MCAM expression on CNS vessel. Formaldehyde-fixed CNS specimens from five MS-affected individuals were examined, (2 males, both 42 years of age and 3 females, 54, 62 and 66 years of age); evolution of disease was between 2 and 20 years. Five control brains from patients who died of non-neurological traumatic injury (34, 46, 51, 69 years of age and unknown, 3 males and 2 females) were used as controls. Autopsies were performed within 6-12 h from time of death. Active MS
lesions were identified by LFB and haematoxilin and eosin (H&E) staining and defined as areas of demyelination associated with intense perivascular immune cell infiltration (48.2 3 nuclei/field centered on a vessel). Normal appearing white matter (NAWM) from MS cases and control brains (non-neurological traumatic death) were defined as areas of normal myelin staining in the absence of immune cell infiltration (21.6 4 nuclei/vessel and 23.2 5 nuclei/vessel, respectively). For immunohistofluorescence, 5 m thick sections were deparaffinized and stained with antibodies specific for MCAM (mouse monoclonal, 1/50, ebioscience), Cav-1 (polyclonal rabbit, 1/75, Santa Cruz Biotechnology), or a with donkey anti mouse-specific antibody (1:400, biotin-conjugated, followed by streptavidin-FITC; Dako and Jackson ImmunoResearch Laboratories, respectively) or a Cy3-conjugated goat rabbit-specific antibody (1:400, 30 min; Jackson ImmunoResearch). Nuclei were stained with TOPRO-3 (1:300 in PBS; Molecular Probes). Control staining was performed with an isotype control antibody. For immunocytochemistry human BBB-ECs were treated, or not, with IFN-y and TNF (100 U/ml) for 16 h. Isolated peripheral blood CD4+ T lymphocytes were stained with CFSE (green) and allowed to migrated for 2 h and then fixed with 4%
paraformaldehyde. MCAM was detected with anti-MCAM (mouse monoclonal, 1/50, ebioscience) followed by a goat anti-mouse antibody conjugated to FITC (green, 1:400, biotin-conjugated, Dako. All images were acquired using a LeicaTM SP5 confocal microscope and analyzed using the LeicaTM LAS AF software (Leica Wetzlar).
[00266] MCAM and Ninjurin-1 expression in peripheral blood and cerobro-spinal fluid samples. Venous blood samples were obtained from consenting healthy donors and MS
patients. PB mononuclear cells were obtained from heparinized whole blood using FicolITM
density gradient separation (Amersham Biosciences, Baie D'Urfe, Quebec, Canada).
Mononuclear cells were assessed by flow cytometry using anti-CD14-FITC
(fluorescein isothiocyanate), anti-CD8-R-PE (R-Phycoerythrin), anti-CD19-PE-Cy5 and anti CD4-pacific blue and other cell specific markers (all antibodies from BD PharMingen). All cell staining were acquired on a BD LSRTM II (Becton Dickinson) and analyzed using the BD
FACSDivaTM software (BD Bioscience).
[00267] T cell stimulation. CD14+ monocytes, CD4+ CD45RO+ lymphocytes and MCAM+ T
cells were isolated from PB mononuclear cells using the magnetic cell sorting (MACSTM) isolation columns, according to manufacturer's protocol (Miltenyi, Auburn, CA). Isolated CD4 T cells (one million cells/ml) were cultured with autologous CD14+ monocytes (0.5 million/ml). CD4+ lymphocytes were labeled with the vital dye 5,6-carboxyfluorescein diacetate succinimidyl ester as required (CFSE), were cultured for 5 days in the presence of autologous CD14+ monocytes, anti-CD3 (OKT3, 2.5 pg/ml) and either IL-2 (20 units/ml) or IL-23 (10 ng/ml) in the presence of anti-IFN-gamma (clone K3.53) and anti-IL-4 (clone 3007, both at 5 pg/ml) in RPMI 1640 supplemented with 5% human serum, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin (Sigma). After 5 days, the proliferation of the CD4+ T lymphocyte, the presence of the different activation marker (CD45RO, CD161, CD25, CD69, CD107a, etc.) and the capacity to produce cytokines (IFN-y, IL-17, GM-CSF, IL-4, TNF, etc.) were assessed by flow cytometry. All cell staining were acquired on a BD
LSRTM II (Becton Dickinson) and analyzed using the BD FACSDivaTM software (BD
Bioscience).
[00268] Intracellular cytokine staining. For intracellular cytokine staining, CD4+
lymphocyte and the corresponding APC co-cultures were activated for 18 h with 1 pg/mL
ionomycin and 20 ng/mL phorbol 12-myristate 13-acetate (PMA) in the presence of 2 pg/mL
brefeldin A (Sigma) for the last 6 h of co-culture. Recombinant cytokines and antibodies were purchased from R&D Systems. Cells were stained for surface markers and were then fixed and permeabilized in 4% (w/v) paraformaldehyde with 0.1% (w/v) saponin in Hank's Balanced Salt Solution for 10 minutes at room temperature. Intracellular staining was performed by incubating cells with antibodies against cytokines (eBioscience, San Diego, CA) (1 mg/mL) for 30 m on ice in PBS buffer containing 0.1 % (w/v) saponin, 1 % FBS, 0.1 %
(w/v) NaN3, followed by two washes and resuspended in staining buffer (1%
(v/v) FBS, 0.1%
(w/v) NaN3 in PBS). All cell staining were acquired on a BD LSRTM II (Becton Dickinson) and analyzed using the BD FACSDivaTM software (BD Bioscience).
[00269] Statistical analysis. Statistical analyses were performed using PRISM
GraphpadTM Software (San Diego, CA) and data are presented as the mean the standard error of the mean (SEM). One-way analysis of variance (ANOVA) was performed followed by Bonferroni multiple comparison post-test for all experiments except for the migration across the BBB, which was done using two-way ANOVA. Only p values < 0.05 were considered significant. The data reported are either from either one representative experiment out of 3 independent experiments or pooled from 3 to 10 experiments. Statistical comparison of IL-17 and IFN-y staining was done using non-parametric student T-test. Differences between groups were considered significant when p < 0.05.
Example 2: Expression of Ninjurin-1 in human BBB-endothelial cells and blood cells [00270] Ninjurin-1 is expressed on the surface of human brain endothelial cells in vitro and in situ, and its expression is upregulated in the context of inflammation.
The expression of Ninjurin-1 on BBB-endothelial cells was shown to increase following treatment with proinflammatory cytokines, such as TNF-a and IFN-y, as demonstrated by flow cytometry and immunocytofluorescence (Figs. 1A and 113). In addition to being present on endothelial cells, Ninjurin-1 is also expressed by different subtypes of human leukocytes, preferentially on cells of the myeloid lineage such as CD14+ monocytes, CD68+ macrophages and CD83+
dendritic cells (DCs) (Figs. 2B and 2C). In contrast, human peripheral blood T
and B
lymphocytes (CD4+ T cells, CD8+ T cells, as well as CD19+ B cells) only show weak or modest expression of Ninjurin-1 (Figs. 2A and 2C). Thus, Ninjurin-1, the ligand involved in the interaction with endothelial Ninjurin-1 through homotypic interaction, is primarily expressed by human peripheral blood myeloid cells (myeloid antigen-presenting cells).
Example 3: Expression of Ninjurin-1 in neuroinflammatory conditions [00271] Although Ninjurin-1 signal is weak on vessels in human control non-inflamed CNS
specimens in situ, its expression significantly increases on the cerebral vascular endothelium in MS lesions (Fig. 3A). Ninjurin-1 is also detected in situ in infiltrating immune cells within MS lesions, and particularly in MHC II+, CD68+ and DC-SIGN+ cells (Fig. 3D).
Fig. 3B shows that the proportion of CD14+ and CD11 c+ cells co-expressing Ninjurin-1 is increased in the cerebrospinal fluid (CSF) as compared to the peripheral blood of MS patients.
These results suggest a role for Ninjurin-1 in the histopathology of MS. Similarly, Ninjurin-1 is expressed in spinal cord homogenates of mice affected with experimental autoimmune encephalomyelitis (EAE) and on CD11c+ DCs and F4/80+ macrophages infiltrating the CNS. EAE was induced in C57BL/6 mice by active immunization with myelin oligodendrocytes glycoprotein (MOG)35_ 55 peptide (myelin oligodendrocyte glycoprotein emulsified in complete Freund's adjuvant) as previously described (Cayrol et al., Nat Immunol. 2008 9(2):137-145). An increase in the expression of Ninjurin-1 protein in the spinal cord of mice affected with EAE
was observed (Fig. 4A). Upregulation of Ninjurin-1 expression correlated with higher disease score (Fig.
4A). Furthermore, MHC II+, CD11c+ and F4/80+ myeloid cells that have reached the CNS
express high levels of Ninjurin-1, and their proportion significantly increases in the CNS of mice affected with EAE as compared to the spleen of the same animals, as observed by flow cytometry (Fig. 4B) and immunostaining of spinal cords (Fig. 4C).
Example 4: Effect of Ninjurin-1 blockade on immune cell migration across the human BBB endothelium [00272] Using an in vitro model of the human BBB (Fig. 5A), it was demonstrated that 5 addition of the human blocking peptide Ninj26_37 to BBB-endothelial cells decreases the migration of freshly isolated (ex vivo) peripheral blood human CD14+ monocytes across the endothelium (Fig. 5C). The blocking Ninj26-37 peptide did not any effect on the migration of CD4+ and CD8+ T lymphocytes in this model (Fig. 5B). Fig. 5D shows that Ninjurin-1 staining is observed around CFSE-loaded migrating CD14+ monocytes and in the transmigratory cup 10 (arrowheads).
Example 5: Effect of Ninjurin-1 blockade in murine models of neuroinflammation [00273] Two distinct murine models of CNS inflammation were used: EAE
(autoimmune-based inflammation, mimicking MS in humans) and SCI (traumatic- or injury-induced inflammation). As shown above, Ninjurin-1 is expressed in the CNS of mice affected with 15 EAE, and on infiltrating F4/80+ macrophages and CD11 c+ dendritic cells (Fig. 4). EAE was induced by active immunization of C57BL/6 mice with MOG35-55 peptide, as described above.
Some animals received i.p. injections of 200 g b.i.d. of Ninj26_37 from day 3 to day 23 post-immunization; while control animals received a physiological saline solution.
The data depicted in Fig. 6A shows a significant reduction of the neurological signs, symptoms and 20 clinical scores of the disease in animals treated with the Ninj26_37 blocking peptide as compared to the control group. The histopathological analysis shows an important reduction in tissue damage (demyelination) in animals treated with Ninj26_37 (Fig. 6D).
Furthermore, a reduction of infiltrating immune cells (more particularly F4/80+ macrophages and CD11c+
dendritic cells) was also observed by flow cytometry (Fig. 6B) and immunofluorescence (Fig.
25 6D).
[00274] The infiltration of myeloid cells in the injured spinal cord significantly contributes to tissue damage and delays the clinical and pathological recovery in the murine model of SCI. SCI experiments were performed with the Infinite HorizonsTM impactor device (Precision Scientific Instrumentation). Fig. 7A shows that there is a significant increase in the 30 expression of Ninjurin-1 in spinal cord homogenates of injured mice.
Neutralization of Ninjurin-1 was performed with the murine Ninj26-37 blocking peptide after SCI.
Some animals
66 received i.p. injections of 200 g b.i.d. of Ninj26_37 from day 1 to 7 following injury, and the control group was injected in the same manner with an irrelevant peptide (sequence:
WRGNPGIRWAPH, SEQ ID NO: 13). The infiltration of lba1+ (Fig. 7B) and CD11c+
(Fig. 7C) myeloid cells in the spinal cord of SCI animals treated with Ninj26-37 blocking peptide was reduced as compared to animals treated with the control peptide. Also, the clinical scores (Basso mouse scale, BMS) of mice treated with 200 g of the Ninj26-37 blocking peptide were reduced as compared to control animals (Fig. 7D).
Example 6: Effect of Ninjurin-1 blockade on monocyte adhesion to a BBB-ECs monolayer in an in vitro flow system [00275] The effect of Ninjurin-1 blockade on monocyte adhesion to a BBB-ECs monolayer was tested in an in vitro flow system. The results presented in Fig. 9 shows that the Ninjurin-1 blocking peptide significantly reduces the number of adherent monocytes to BBB-ECs under flow conditions. Furthermore, Ninjurin-1 blockade by the peptide significantly increases the monocyte mean velocity (Fig. 10), demonstrating a reduced adhesion/interaction of the monocytes to the BBB-ECs monolayer. The reduced adhesion/interaction of monocytes is observed in non-inflammatory conditions (no treatment) and inflammatory conditions (TNF-a + IFN-7 treatment).
Example 7: Expression of MCAM in human BBB-endothelial cells and blood cells [00276] The expression of MCAM (melanoma cell adhesion molecule/CD146) on BBB-ECs was studied in vitro and in situ. MCAM is expressed by BBB-ECs in vitro (primary cultures of human BBB-ECs) and in situ (in control and MS patient archival autopsy central nervous system samples) (Figs. 11 and 12). In vitro MCAM expression was shown to be up-regulated with inflammatory cytokine treatment (TNF-a and IFN-y, 100 U/ml for 16 hours), as shown in Fig. 11 B. In situ expression of MCAM on CNS vessels is strong and did not vary significantly between control vessels, MS normal appearing white matter, and active MS
lesions (Fig. 12). Using BBB-ECs in culture as a monolayer with isolated human lymphocytes from the peripheral blood (PB) of healthy controls, it was shown that adherent lymphocytes are surrounded by a MCAM+ ring-like structure that is reminiscent of a transmigratory cup, a structure involved in leukocyte diapedesis across vascular beds (Fig.
13).
Example 8: Expression of MCAM on human T lymphocytes
WRGNPGIRWAPH, SEQ ID NO: 13). The infiltration of lba1+ (Fig. 7B) and CD11c+
(Fig. 7C) myeloid cells in the spinal cord of SCI animals treated with Ninj26-37 blocking peptide was reduced as compared to animals treated with the control peptide. Also, the clinical scores (Basso mouse scale, BMS) of mice treated with 200 g of the Ninj26-37 blocking peptide were reduced as compared to control animals (Fig. 7D).
Example 6: Effect of Ninjurin-1 blockade on monocyte adhesion to a BBB-ECs monolayer in an in vitro flow system [00275] The effect of Ninjurin-1 blockade on monocyte adhesion to a BBB-ECs monolayer was tested in an in vitro flow system. The results presented in Fig. 9 shows that the Ninjurin-1 blocking peptide significantly reduces the number of adherent monocytes to BBB-ECs under flow conditions. Furthermore, Ninjurin-1 blockade by the peptide significantly increases the monocyte mean velocity (Fig. 10), demonstrating a reduced adhesion/interaction of the monocytes to the BBB-ECs monolayer. The reduced adhesion/interaction of monocytes is observed in non-inflammatory conditions (no treatment) and inflammatory conditions (TNF-a + IFN-7 treatment).
Example 7: Expression of MCAM in human BBB-endothelial cells and blood cells [00276] The expression of MCAM (melanoma cell adhesion molecule/CD146) on BBB-ECs was studied in vitro and in situ. MCAM is expressed by BBB-ECs in vitro (primary cultures of human BBB-ECs) and in situ (in control and MS patient archival autopsy central nervous system samples) (Figs. 11 and 12). In vitro MCAM expression was shown to be up-regulated with inflammatory cytokine treatment (TNF-a and IFN-y, 100 U/ml for 16 hours), as shown in Fig. 11 B. In situ expression of MCAM on CNS vessels is strong and did not vary significantly between control vessels, MS normal appearing white matter, and active MS
lesions (Fig. 12). Using BBB-ECs in culture as a monolayer with isolated human lymphocytes from the peripheral blood (PB) of healthy controls, it was shown that adherent lymphocytes are surrounded by a MCAM+ ring-like structure that is reminiscent of a transmigratory cup, a structure involved in leukocyte diapedesis across vascular beds (Fig.
13).
Example 8: Expression of MCAM on human T lymphocytes
67 [00277] The expression of MCAM on human peripheral blood immune cells was assessed (Fig. 14). A sub-group of human PB CD4+ and CD8 T lymphocytes express MCAM.
The different leukocyte markers that were associated with the MCAM+ lymphocytes was assessed and it was demonstrated that MCAM+ lymphocytes express certain markers (CD2, CD3, CD6, CD45RO, CD161, CCR6 and CD28, for example) and were less associated, if they express at all, with other markers (CD45RA, CCD56, CD62L, and CCR7, for example) (Fig. 14). These studies demonstrate MCAM+ is expressed by T lymphocytes having a memory phenotype. These cells express very little cytokines ex vivo (as assessed by intra-cellular FACS staining), comparable to MCAM" T lymphocytes of the human PB.
Example 9: Functional characterization of MCAM+ lymphocytes [00278] The function of MCAM+ lymphocytes was studied using different activation regimen and looking at different activation markers (proliferation, CD161, CD107a, CD69) and by studying the cytokine profile associated with activated MCAM+
lymphocytes compared to MCAM- lymphocytes. A polyclonal non-antigen specific TCR-mediated activation strategy was used (anti-CD3, autologous CD14+ cells, in presence of either IL-2 or IL-23) on whole PB mononuclear cells or on isolated leukocytes. As shown in Figs. 15 to 20, activated MCAM+ lymphocytes preferentially express certain markers and certain cytokines.
CD4+ MCAM+ lymphocytes behave similarly to MCAM memory lymphocytes concerning lymphocyte proliferation and certain activation markers (such as CD25). In contrast, CD4+
MCAM+ lymphocytes are significantly associated with CD161, CD107a and CD69.
Furthermore CD4+ MCAM+ lymphocytes express significantly higher levels of the cytokines IL-17, IFN-y, GM-CSF and TNF, as compared to memory CD4+ MCAM" lymphocytes from the same donors. The expression of MCAM by human Th17 cells (human CD4+ T
cells activated under conditions that favor the production of IL-17) was further shown by a proteomic screen that demonstrated that human Th17 cells express the protein MCAM. The association of MCAM on lymphocytes and the secretion of the cytokine IL-17 following T cell activation is the strongest and indicates that MCAM may be used as a marker of lymphocytes that have the potential to secrete IL-17 following re-activation.
[00279] The results presented at Fig. 21 show that the expression of MCAM is maintained on MCAM positive sorted lymphocytes after 5 days in culture in a Th17 polarization environment (more than 90% of the cells remains MCAM positive after 5 days).
Also, a significant proportion of MCAM-negative sorted cells expresses MCAM after 5 days of in vitro culture under Th17 polarization conditions (Fig. 22), demonstrating that MCAM is a
The different leukocyte markers that were associated with the MCAM+ lymphocytes was assessed and it was demonstrated that MCAM+ lymphocytes express certain markers (CD2, CD3, CD6, CD45RO, CD161, CCR6 and CD28, for example) and were less associated, if they express at all, with other markers (CD45RA, CCD56, CD62L, and CCR7, for example) (Fig. 14). These studies demonstrate MCAM+ is expressed by T lymphocytes having a memory phenotype. These cells express very little cytokines ex vivo (as assessed by intra-cellular FACS staining), comparable to MCAM" T lymphocytes of the human PB.
Example 9: Functional characterization of MCAM+ lymphocytes [00278] The function of MCAM+ lymphocytes was studied using different activation regimen and looking at different activation markers (proliferation, CD161, CD107a, CD69) and by studying the cytokine profile associated with activated MCAM+
lymphocytes compared to MCAM- lymphocytes. A polyclonal non-antigen specific TCR-mediated activation strategy was used (anti-CD3, autologous CD14+ cells, in presence of either IL-2 or IL-23) on whole PB mononuclear cells or on isolated leukocytes. As shown in Figs. 15 to 20, activated MCAM+ lymphocytes preferentially express certain markers and certain cytokines.
CD4+ MCAM+ lymphocytes behave similarly to MCAM memory lymphocytes concerning lymphocyte proliferation and certain activation markers (such as CD25). In contrast, CD4+
MCAM+ lymphocytes are significantly associated with CD161, CD107a and CD69.
Furthermore CD4+ MCAM+ lymphocytes express significantly higher levels of the cytokines IL-17, IFN-y, GM-CSF and TNF, as compared to memory CD4+ MCAM" lymphocytes from the same donors. The expression of MCAM by human Th17 cells (human CD4+ T
cells activated under conditions that favor the production of IL-17) was further shown by a proteomic screen that demonstrated that human Th17 cells express the protein MCAM. The association of MCAM on lymphocytes and the secretion of the cytokine IL-17 following T cell activation is the strongest and indicates that MCAM may be used as a marker of lymphocytes that have the potential to secrete IL-17 following re-activation.
[00279] The results presented at Fig. 21 show that the expression of MCAM is maintained on MCAM positive sorted lymphocytes after 5 days in culture in a Th17 polarization environment (more than 90% of the cells remains MCAM positive after 5 days).
Also, a significant proportion of MCAM-negative sorted cells expresses MCAM after 5 days of in vitro culture under Th17 polarization conditions (Fig. 22), demonstrating that MCAM is a
68 marker of Th17 differentiated T cells. The results depicted in Fig. 23 show that MCAM+ cells derived from MCAM positive sorted lymphocytes and cultured for 5 days in Th17 polarization conditions express IL-17 and IFN-y in a higher proportion as compared to MCAM-cells.
Similarly, MCAM+ cells derived from MCAM negative sorted lymphocytes and cultured for 5 days in Th17 polarization conditions express IL-17 and IL-8 in a higher proportion as compared to MCAM- cells (Fig. 24, left panel and Fig. 25). A higher proportion of MCAM+
derived from MCAM negative sorted cells appear to express IFN-7 (Fig. 24, middle panel) and both IL-17 and IFN-y (Fig. 24, right panel), although it did not reach statistical significance.
Example 10: Expression of MCAM on samples from MS patients [00280] The proportion of MCAM+ lymphocytes in the PB of MS patients in relapse was determined and compared to that of healthy donor PB. As shown in Fig. 26A and 26B, the PB of MS patients contains a significantly higher proportion of CD4+ and CD8+
T
lymphocytes that express MCAM, as compared to healthy controls. Fig. 26C shows that MCAM+ lymphocytes (both CD4 and CD8) are enriched in the cerebro-spinal fluid (CSF) of MS patients, as compared to the PB. These data indicate that the proportion of MCAM+
lymphocytes in biological samples may be used to diagnose the presence of neuroinflammation, such as MS.
Example 11: Expression of MCAM on Th17 cells derived from controls and MS
patients [00281] The data presented in Fig. 27 demonstrate that the proportion of MCAM+
IL17+ T
cells is significantly higher in MS patients as compared to control.
Example 12: Expression of MCAM in samples from EAE mice [00282] As shown in Fig. 28, MCAM+ T cells are enriched in the CNS and the lymph nodes of EAE mice suffering from neurological symptoms, suggestive of a role for MCAM in the migration of IL-17-secreting cells to the CNS.
[00283] The studies presented herein highlight the role of Ninjurin-1 and MCAM
in the recruitment of immune cells within the CNS, and in the pathophysiology of neuroinflammatory diseases/conditions. The selective blockade of Ninjurin-1 -mediated recruitment of myeloid cells to the CNS reduces tissue destruction observed in inflammatory
Similarly, MCAM+ cells derived from MCAM negative sorted lymphocytes and cultured for 5 days in Th17 polarization conditions express IL-17 and IL-8 in a higher proportion as compared to MCAM- cells (Fig. 24, left panel and Fig. 25). A higher proportion of MCAM+
derived from MCAM negative sorted cells appear to express IFN-7 (Fig. 24, middle panel) and both IL-17 and IFN-y (Fig. 24, right panel), although it did not reach statistical significance.
Example 10: Expression of MCAM on samples from MS patients [00280] The proportion of MCAM+ lymphocytes in the PB of MS patients in relapse was determined and compared to that of healthy donor PB. As shown in Fig. 26A and 26B, the PB of MS patients contains a significantly higher proportion of CD4+ and CD8+
T
lymphocytes that express MCAM, as compared to healthy controls. Fig. 26C shows that MCAM+ lymphocytes (both CD4 and CD8) are enriched in the cerebro-spinal fluid (CSF) of MS patients, as compared to the PB. These data indicate that the proportion of MCAM+
lymphocytes in biological samples may be used to diagnose the presence of neuroinflammation, such as MS.
Example 11: Expression of MCAM on Th17 cells derived from controls and MS
patients [00281] The data presented in Fig. 27 demonstrate that the proportion of MCAM+
IL17+ T
cells is significantly higher in MS patients as compared to control.
Example 12: Expression of MCAM in samples from EAE mice [00282] As shown in Fig. 28, MCAM+ T cells are enriched in the CNS and the lymph nodes of EAE mice suffering from neurological symptoms, suggestive of a role for MCAM in the migration of IL-17-secreting cells to the CNS.
[00283] The studies presented herein highlight the role of Ninjurin-1 and MCAM
in the recruitment of immune cells within the CNS, and in the pathophysiology of neuroinflammatory diseases/conditions. The selective blockade of Ninjurin-1 -mediated recruitment of myeloid cells to the CNS reduces tissue destruction observed in inflammatory
69 cerebral lesions, including the lesions induced by traumatic stress (e.g., SCI) as well as those associated with autoimmune inflammatory processes, as observed in MS.
These studies also show that Ninjurin-1 and MCAM expression constitute markers of ongoing inflammation, and thus may be used for diagnostic and/or prognostic purposes.
[00284] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
These studies also show that Ninjurin-1 and MCAM expression constitute markers of ongoing inflammation, and thus may be used for diagnostic and/or prognostic purposes.
[00284] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.
Claims (110)
1. A method of preventing or treating an inflammatory condition in a subject, said method comprising administering to said subject an effective amount of a nerve injurin-induced protein-1 (Ninjurin-1) and/or melanoma cell adhesion molecule (MCAM) inhibitor.
2. The method of claim 1, wherein said Ninjurin-1 inhibitor blocks Ninjurin-1/Ninjurin-1 homotypic interaction.
3. The method of claim 2, wherein said Ninjurin-1 inhibitor binds to an extracellular domain of a Ninjurin-1 polypeptide.
4. The method of claim 3, wherein said Ninjurin-1 inhibitor binds to a domain comprising a motif corresponding to residues 28 to 35 of a Ninjurin-1 polypeptide.
5. The method of claim 3 or 4, wherein said Ninjurin-1 polypeptide is a human Ninjurin-1 polypeptide.
6. The method of any one of claims 1 to 5, wherein said Ninjurin-1 inhibitor is a peptide comprising a domain of formula I:
Xaa1-Xaa2-Arg-Trp-Xaa3-Xaa4-Arg-Xaa5-Arg-Xaa6-Xaa7-Xaa8 (I), wherein Xaa1, Xaa2, Xaa6, Xaa7 and Xaa8 is each independently any amino acid or is absent;
Xaa3, Xaa4 and Xaa5 is each independently any amino acid;
or a functional analog thereof.
Xaa1-Xaa2-Arg-Trp-Xaa3-Xaa4-Arg-Xaa5-Arg-Xaa6-Xaa7-Xaa8 (I), wherein Xaa1, Xaa2, Xaa6, Xaa7 and Xaa8 is each independently any amino acid or is absent;
Xaa3, Xaa4 and Xaa5 is each independently any amino acid;
or a functional analog thereof.
7. The method of claim 6, wherein Xaa 2 is Pro.
8. The method of claim 6 or 7, wherein Xaa1 is Pro.
9. The method of any one of claims 6 to 8, wherein Xaa6 is Pro.
10. The method of any one of claims 6 to 9, wherein Xaa7 is lie.
11. The method of any one of claims 6 to 10, wherein Xaa8 is Asn.
12. The method of any one of claims 6 to 11, wherein Xaa3 is Gly.
13. The method of any one of claims 6 to 12, wherein Xaa4 is Leu.
14. The method of any one of claims 6 to 13, wherein Xaa5 is Asn or Leu.
15. The method of claim 14, wherein said domain is Pro-Pro-Arg-Trp-Gly-Leu-Arg-Asn-Arg-Pro-Ile-Asn.
16. The method of any one of claims 1 to 5, wherein said Ninjurin-1 inhibitor is a peptide consisting of the domain of formula I defined in any one of claims 6 to 15.
17. The method of claim 1, wherein said MCAM inhibitor blocks MCAM/MCAM
homotypic interaction.
homotypic interaction.
18. The method of claim 1 or 17, wherein said inhibitor is an antibody or a fragment thereof.
19. The method of claim 1, wherein said MCAM inhibitor is an siRNA molecule
20. The method of claim 1, wherein said MCAM inhibitor blocks MCAM-induced cell signalling.
21. The method of any one of claims 1 to 20, wherein said inflammatory condition is a neuroinflammatory condition.
22. The method of claim 21, wherein said neuroinflammatory condition is associated with recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), to the central nervous system (CNS).
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), to the central nervous system (CNS).
23. The method of claim 22, wherein said myeloid cell is a monocyte, a macrophage and/or a dendritic cell.
24. The method of claim 22, wherein said inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
25. The method of claim 22 or 24, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
26. The method of any one of claims 21 to 25, wherein said neuroinflammatory condition is associated with a CNS trauma.
27. The method of claim 26, wherein said CNS trauma is spinal cord injury (SCI).
28. The method of any one of claims 21 to 25, wherein said neuroinflammatory condition is an autoimmune CNS condition.
29. The method of claim 28, wherein said autoimmune CNS condition is multiple sclerosis (MS).
30. The method of any one of claims 21 to 29, wherein said method further comprises (i) reducing the neurological signs, symptoms and/or clinical scores of the neuroinflammatory condition; (ii) reducing tissue damage; and/or (iii) reducing infiltration of myeloid cells into the CNS.
31. A method of inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium comprising contacting said cell and/or said CNS endothelium with an effective amount of a Ninjurin-1 and/or MCAM
inhibitor.
inhibitor.
32. The method of claim 31, wherein said Ninjurin-1 inhibitor is the Ninjurin-1 inhibitor defined in any one of claims 2 to 16.
33. The method of claim 31, wherein said MCAM inhibitor is the MCAM inhibitor defined in any one of claims 17 to 20.
34. The method of claim 31, wherein said myeloid cell is a monocyte, a macrophage and/or a dendritic cell.
35. The method of claim 31, wherein said inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
36. The method of claim 31 or 35, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
37. The method of any one of claims 31 to 36, wherein said CNS endothelium is exposed to an inflammatory environment.
38. The method of claim 37, wherein said inflammatory environment comprises Interferon-gamma (IFN-.gamma.) and/or Tumor Necrosis Factor-alpha (TNF-.alpha.).
39. The method of claim 37, wherein said inflammatory environment is associated with a neuroinflammatory condition.
40. The method of claim 39, wherein said neuroinflammatory condition is a CNS
trauma.
trauma.
41. The method of claim 40, wherein said CNS trauma is spinal cord injury (SCI).
42. The method of claim 39, wherein said neuroinflammatory condition is an autoimmune CNS condition.
43. The method of claim 42, wherein said autoimmune CNS condition is multiple sclerosis (MS).
44. Use of a Ninjurin-1 and/or MCAM inhibitor for preventing or treating an inflammatory condition in a subject.
45. Use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition in a subject.
46. The use of claim 44 or 45, wherein said Ninjurin-1 inhibitor is the Ninjurin-1 inhibitor defined in any one of claims 2 to 16.
47. The use of claim 44 or 45, wherein said MCAM inhibitor is the MCAM
inhibitor defined in any one of claims 17 to 20.
inhibitor defined in any one of claims 17 to 20.
48. The use of any one of claims 44 to 47, wherein said inflammatory condition is a neuroinflammatory condition.
49. The use of claim 48, wherein said neuroinflammatory condition is associated with the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii) to the central nervous system (CNS).
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii) to the central nervous system (CNS).
50. The use of claim 49, wherein said myeloid cell is a monocyte, a macrophage and/or a dendritic cell.
51. The use of claim 49, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
52. The use of claim 48 or 51, wherein said inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
53. The use of any one of claims 48 to 52, wherein said neuroinflammatory condition is associated with a CNS trauma.
54. The use of claim 53, wherein said CNS trauma is spinal cord injury (SCI).
55. The use of any one of claims 48 to 52, wherein said neuroinflammatory condition is an autoimmune CNS condition.
56. The use of claim 55, wherein said autoimmune CNS condition is multiple sclerosis (MS).
57. Use of a Ninjurin-1 and/or MCAM inhibitor for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium.
58. Use of a Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), across the CNS endothelium.
59. The use of claim 57 or 58, wherein said Ninjurin-1 inhibitor is the Ninjurin-1 inhibitor defined in any one of claims 2 to 16.
60. The use of claim 57 or 58, wherein said MCAM inhibitor is the MCAM
inhibitor defined in any one of claims 17 to 20.
inhibitor defined in any one of claims 17 to 20.
61. The use of any one of claims 57 to 60, wherein said myeloid cell is a monocyte, a macrophage and/or a dendritic cell.
62. The use of any one of claims 57 to 60, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
63. The use of claim any one of claims 57 to 60 and 62, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete: (i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
64. The use of any one of claims 57 to 63, wherein said CNS endothelium is exposed to an inflammatory environment.
65. The use of claim 64, wherein said inflammatory environment comprises Interferon-gamma (IFN-.gamma.) and/or Tumor Necrosis Factor-alpha (TNF-.alpha.).
66. The use of claim 64 or 65, wherein said inflammatory environment is associated with a neuroinflammatory condition.
67. The use of claim 66, wherein said neuroinflammatory condition is associated with a CNS trauma.
68. The use of claim 67, wherein said CNS trauma is spinal cord injury (SCI).
69. The use of claim 66, wherein said neuroinflammatory condition is an autoimmune CNS condition.
70. The use of claim 69, wherein said autoimmune CNS condition is multiple sclerosis (MS).
71. A Ninjurin-1 and/or MCAM inhibitor for preventing or treating an inflammatory condition in a subject.
72. A Ninjurin-1 and/or MCAM inhibitor for the preparation of a medicament for preventing or treating an inflammatory condition in a subject.
73. The inhibitor of claim 71 or 72, wherein said inflammatory condition is a neuroinflammatory condition.
74. A pharmaceutical composition for preventing or treating an inflammatory condition in a subject, said composition comprising a Ninjurin-1 and/or MCAM inhibitor and a pharmaceutically acceptable carrier.
75. The pharmaceutical composition of claim 74, wherein said inflammatory condition is a neuroinflammatory condition.
76. A method of identifying a compound for preventing or treating an inflammatory condition, said method comprising determining whether:
(a) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(b) a level of Ninjurin-1 and/or MCAM activity; or (c) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for preventing or treating said inflammatory condition.
(a) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(b) a level of Ninjurin-1 and/or MCAM activity; or (c) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for preventing or treating said inflammatory condition.
77. A method of identifying or characterizing a compound for preventing or treating a neuroinflammatory condition, said method comprising:
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for preventing or treating said inflammatory condition.
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for preventing or treating said inflammatory condition.
78. A method of identifying a compound for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii) across the CNS endothelium, said method comprising determining whether:
(d) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(e) a level of Ninjurin-1 and/MCAM activity; or (f) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
(d) a level of expression of a Ninjurin-1 and/or MCAM nucleic acid or encoded polypeptide;
(e) a level of Ninjurin-1 and/MCAM activity; or (f) a combination of (a) and (b);
is decreased in the presence of a test compound relative to in the absence of said test compound; wherein said decrease is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
79. A method of identifying or characterizing a compound for inhibiting the recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii) across the CNS endothelium, said method comprising:
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
(a) contacting a test compound with a cell comprising a first nucleic acid comprising a transcriptionally regulatory element normally associated with a Ninjurin-1 and/or MCAM gene, operably linked to a second nucleic acid comprising a reporter gene capable of encoding a reporter protein; and (b) determining whether reporter gene expression or reporter protein activity is decreased in the presence of said test compound relative to in the absence of said test compound;
wherein a decrease in said reporter gene expression or reporter protein activity is indicative that said test compound may be used for inhibiting the recruitment of said cell across the CNS endothelium.
80. The method of claim 76 or 77, wherein said inflammatory condition is a neuroinflammatory condition.
81. The method of claim 80, wherein said neuroinflammatory condition is associated with recruitment of (i) a myeloid cell, (ii) an inflammatory cytokine-secreting T
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), to the central nervous system (CNS).
cell, (iii) an inflammatory cytokine-secreting T cell precursor or (iv) any combination of (i) to (iii), to the central nervous system (CNS).
82. The method of claim 81, wherein said myeloid cell is a monocyte, a macrophage and/or a dendritic cell.
83. The method of claim 81, wherein said inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
84. The method of claim 81 or 83, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete:
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.UPSILON.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
(i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.UPSILON.), (iii) Tumor Necrosis Factor-alpha (TNF-.alpha.), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
85. The method of any one of claims 80 to 84, wherein said neuroinflammatory condition is associated with a CNS trauma.
86. The method of claim 85, wherein said CNS trauma is spinal cord injury (SCI).
87. The method of any one of claims 80 to 84, wherein said neuroinflammatory condition is an autoimmune CNS condition.
88. The method of claim 87, wherein said autoimmune CNS condition is multiple sclerosis (MS).
89. A method for diagnosing an inflammatory condition in a first subject, said method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said first subject (b) comparing said expression and/or activity to a corresponding reference expression and/or activity; and (c) diagnosing said inflammatory condition based on said comparison.
90. The method of claim 89, wherein said reference expression and/or activity corresponds to an expression and/or activity determined in a sample from a control subject known to not having an inflammatory condition, and wherein a higher expression and/or activity in said sample from said first subject is indicative that said first subject has an inflammatory condition.
91. The method of claim 89, wherein said reference expression and/or activity corresponds to an expression and/or activity determined in a sample from a control subject known to have an inflammatory condition, and wherein a comparable or higher expression and/or activity in said sample from said first subject is indicative that said first subject has an inflammatory condition.
92. A method for monitoring the course of treatment of a patient suffering from an inflammatory condition, the method comprising (a) determining the expression and/or activity of Ninjurin-1 and/or MCAM in a sample from said patient; wherein a decrease in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at an earlier time is indicative that said patient is responsive to said treatment.
93. A method of determining whether a subject is suffering from a relapse of an inflammatory condition, said method comprising (a) determining the expression and/or activity of Ninjurin and/or MCAM in a sample from said patient; wherein an increase in said expression and/or activity relative to a corresponding expression and/or activity of Ninjurin-1 and/or MCAM determined in a biological sample obtained from said patient at a time point of remission is indicative that said patient is suffering from a relapse of said inflammatory condition.
94. The method of any one of claims 89 to 93, wherein said inflammatory condition is a neuroinflammatory condition.
95. The method of claim 94, wherein said sample is a CNS cell, tissue or fluid.
96. The method of claim 95, wherein said CNS fluid is cerebro-spinal fluid (CSF).
97. The method of any one of claims 89 to 94, wherein said sample is a blood cell sample.
98. The method of claim 97, wherein said blood cell sample is a peripheral blood mononuclear cell (PBMC) sample.
99. The method of any one of claims 89 to 98, wherein said sample comprises (i) myeloid cells, (ii) an inflammatory cytokine-secreting T cells, (iii) an inflammatory cytokine-secreting T cell precursors or (iv) any combination of (i) to (iii).
100. The method of claim 99, wherein said myeloid cells are (i) monocytes;
(ii) macrophages, (iii) dendritic cells or (iv) any combination of (i) to (iii).
(ii) macrophages, (iii) dendritic cells or (iv) any combination of (i) to (iii).
101. The method of claim 99, wherein said inflammatory cytokine-secreting T
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
cell or inflammatory cytokine-secreting T cell precursor is a memory T cell.
102. The method of claim 99 or 101, wherein said inflammatory cytokine-secreting T cell or inflammatory cytokine-secreting T cell precursor has the capacity to secrete: (i) interleukin-17 (IL-17), (ii) Interferon-gamma (IFN-.gamma.), (iii) Tumor Necrosis Factor-alpha (TNF-a), (iv) Granulocyte-macrophage colony-stimulating factor (GM-CSF), (v) IL-8, or (vi) any combination of (i) to (v), upon activation.
103. The method of any one of claims 89 to 102, wherein said expression and/or activity of Ninjurin-1 and/or MCAM is determined by measuring the amount of Ninjurin-1-and/or MCAM-expressing cells in said sample.
104. The method of claim 103, wherein said amount is a relative amount.
105. The method of any one of claims 89 to 104, wherein said inflammatory condition is multiple sclerosis.
106. A method of identifying an inflammatory cytokine-secreting T cell or precursor thereof in a sample, said method comprising (i) contacting said cell with an MCAM
ligand and (ii) identifying said inflammatory cytokine-secreting T cell or precursor thereof based on the binding to said MCAM ligand.
ligand and (ii) identifying said inflammatory cytokine-secreting T cell or precursor thereof based on the binding to said MCAM ligand.
107. A method of purifying an inflammatory cytokine-secreting T cell or precursor thereof from a population of cells in a sample, said method comprising contacting said sample with an MCAM ligand and (ii) purifying said inflammatory cytokine-secreting T cell or precursor thereof on the basis of binding to said MCAM ligand.
108. The method of claim 106 or 107, wherein said MCAM ligand is (i) MCAM, (ii) an MCAM binding partner, (iii) an MCAM-specific antibody, or (iv) an MCAM-binding fragment of any of (i) to (iii).
109. The method of any one of claims 106 to 108, wherein said MCAM ligand is bound to a solid support.
110. A kit for identifying and/or purifying an inflammatory cytokine-secreting T cell or a precursor thereof in a cell sample, said kit comprising (i) an MCAM ligand and (ii) instructions for identifying and/or purifying said inflammatory cytokine-secreting T cell or precursor thereof from said sample.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US12/796,221 US8703711B2 (en) | 2009-06-09 | 2010-06-08 | Ninjurin-1 modulation and uses thereof |
US12/797,055 US8293468B2 (en) | 2009-06-10 | 2010-06-09 | MCAM modulation and uses thereof |
US13/617,617 US9017682B2 (en) | 2009-06-10 | 2012-09-14 | Treatment of neuroinflammatory conditions using antibodies directed against MCAM |
US14/694,328 US20150218266A1 (en) | 2009-06-10 | 2015-04-23 | Inhibition of th17 cells migration to inflamed tissues using antibodies directed against mcam |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US18542009P | 2009-06-09 | 2009-06-09 | |
US61/185,420 | 2009-06-09 | ||
US18570709P | 2009-06-10 | 2009-06-10 | |
US61/185,707 | 2009-06-10 |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2676962A1 true CA2676962A1 (en) | 2010-12-09 |
CA2676962C CA2676962C (en) | 2020-11-10 |
Family
ID=43332953
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2676962A Active CA2676962C (en) | 2009-06-09 | 2009-08-25 | Mcam modulation and uses thereof in the management of neuroinflammatory conditions |
Country Status (2)
Country | Link |
---|---|
AU (1) | AU2009212789B2 (en) |
CA (1) | CA2676962C (en) |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20140314744A1 (en) * | 2011-06-06 | 2014-10-23 | Neotope Biosciences Limited | Mcam antagonists and methods of treatment |
CN102861331B (en) * | 2011-07-06 | 2015-08-12 | 中国科学院生物物理研究所 | The application of CD146 and the diseases associated with inflammation such as antibody diagnosis and treatment autoimmune disease thereof |
-
2009
- 2009-08-25 AU AU2009212789A patent/AU2009212789B2/en active Active
- 2009-08-25 CA CA2676962A patent/CA2676962C/en active Active
Also Published As
Publication number | Publication date |
---|---|
CA2676962C (en) | 2020-11-10 |
AU2009212789B2 (en) | 2014-05-15 |
AU2009212789A1 (en) | 2010-12-23 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11787846B2 (en) | EphA2 T-cell epitope agonists and uses therefore | |
US8703711B2 (en) | Ninjurin-1 modulation and uses thereof | |
US9017682B2 (en) | Treatment of neuroinflammatory conditions using antibodies directed against MCAM | |
JP4538464B2 (en) | Optic myelitis marker | |
JP6886816B2 (en) | Immune system regulator | |
US20210077577A1 (en) | Blockade of ccl18 signaling via ccr6 as a therapeutic option in fibrotic diseases and cancer | |
EP2100140B1 (en) | Methods for diagnosing fibrotic lung diseases | |
CA2676962C (en) | Mcam modulation and uses thereof in the management of neuroinflammatory conditions | |
UA110119C2 (en) | TOMM34 peptides and vaccines containing them | |
KR102281512B1 (en) | Composition for diagnosing inflammatory diseases comprising agents detecting the level of acetylation of transforming growth factor β-induced protein |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20140428 |