WO2012172343A2 - Traitement de l'angiocholite sclérosante primitive - Google Patents

Traitement de l'angiocholite sclérosante primitive Download PDF

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Publication number
WO2012172343A2
WO2012172343A2 PCT/GB2012/051353 GB2012051353W WO2012172343A2 WO 2012172343 A2 WO2012172343 A2 WO 2012172343A2 GB 2012051353 W GB2012051353 W GB 2012051353W WO 2012172343 A2 WO2012172343 A2 WO 2012172343A2
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WIPO (PCT)
Prior art keywords
ccr9
ccr7
cxcr4
ccr5
chemokine
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PCT/GB2012/051353
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English (en)
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WO2012172343A3 (fr
Inventor
Graham Cotton
Ola Winqvist
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Ith Immune Therapy Holdings
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Publication of WO2012172343A2 publication Critical patent/WO2012172343A2/fr
Publication of WO2012172343A3 publication Critical patent/WO2012172343A3/fr
Priority to US14/105,628 priority Critical patent/US9726666B2/en
Priority to US15/629,691 priority patent/US10451620B2/en
Priority to US15/629,705 priority patent/US10429385B2/en
Priority to US15/629,697 priority patent/US10502736B2/en
Priority to US15/629,713 priority patent/US10408832B2/en
Priority to US15/629,700 priority patent/US10422800B2/en
Priority to US15/629,708 priority patent/US10401357B2/en
Priority to US16/537,224 priority patent/US20190361020A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4, KC
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3679—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits by absorption
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7158—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/08—Hepato-biliairy disorders other than hepatitis

Definitions

  • the various embodiments of the present invention relates to products for and methods of treating inflammatory conditions, such as primary sclerosing cholangitis. Companion diagnostics are also described.
  • PSC Primary sclerosing cholangitis
  • PSC is associated with an increased incidence of other forms of cancer, in particular cholangiosarcoma, cancer of the biliary tree, and colorectal cancer.
  • cholangiosarcoma cancer of the biliary tree
  • colorectal cancer The majority of patients with PSC also suffer with inflammatory bowel disease, in particular the form of this disease known as ulcerative colitis.
  • Apheresis is a treatment used for depletion of blood components, such as antibodies, low- density lipoproteins (LDL) and blood cells.
  • LDL low- density lipoproteins
  • Leukapheresis is the apheresis treatment used for removal of white blood cells, leukocytes.
  • the patient is connected to an extracorporeal blood circulating system; the blood is drawn from a vein in one arm, passed through a column device and returned into the other arm of the patient.
  • WO2010/029317 describes apheresis columns useful for treating inflammatory conditions including a chemokine immobilised on a solid support.
  • Chemokines are a class of cytokine molecules involved in cell recruitment and activation in inflammation. Chemokines cause chemotaxis and activation of various subpopulations of cells in the immune system. The activity of chemokines is mediated primarily through tight binding to their receptors on the surface of leukocytes. In certain embodiments the present invention is based on the realisation that the interaction between chemokines and cells expressing their receptors may be exploited for the treatment of primary sclerosing cholangitis (PSC). The inventors have determined that targeting increased recruitment of specific chemokine receptor-expressing cells to the site of inflammation presents a new therapeutic approach to treat such conditions.
  • PSC primary sclerosing cholangitis
  • chemokine receptor expression on each cell may be increased again providing a therapeutic approach to treat such conditions.
  • PSC patients include higher levels of CCR9 expressing monocytes in the blood.
  • the disease relevant monocytes may also have high expression of HLA-DR (referred to herein as HLA-DR hl ).
  • HLA-DR is a MHC class II cell surface receptor encoded by the human leukocyte antigen complex on chromosome 6 region 6p21 .31 .
  • the invention serves to reduce the recruitment of inflammatory leukocytes, which express characteristic chemokine receptors, and possibly express characteristic chemokine receptors at increased levels, to sites of inflammation linked to disorders such as primary sclerosing cholangitis. This is achieved using specific binding reagents to capture specific chemokine receptor-expressing inflammatory leukocytes from the patient.
  • the invention provides in a first aspect a method for treating primary sclerosing cholangitis comprising applying peripheral blood from a patient to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to one or more chemokine receptors, in particular the chemokine receptor CCR9, CXCR4, CCR7 and/or CCR5, immobilized directly or indirectly on the support thus removing one or more chemokine receptors, in particular CCR9, CXCR4, CCR7 and/or CCR5, expressing cells from the peripheral blood of the patient.
  • the peripheral blood from which the chemokine receptor expressing cells have been removed may then be returned to the patient in order to complete the treatment.
  • the invention may thus rely on a continuous extracorporeal circuit in some embodiments.
  • in certain chemokine receptors in particular the chemokine receptor CCR9, CXCR4, CCR7 and/or CCR5
  • the invention may comprise steps of obtaining peripheral blood from the patient, applying the peripheral blood to the column and subsequently returning the peripheral blood from which the chemokine receptor expressing cells have been removed to the patient.
  • suitable binding reagents can be immobilized onto a solid support, either directly or indirectly, to generate an apheresis column suitable for capturing relevant chemokine receptor-expressing cells. Where increased levels of chemokine receptor expression are observed, such cells may be preferably removed from the peripheral blood using the columns of the various embodiments of the invention.
  • the methods of the various embodiments of the invention may preferably target one or more of CCR9 hl ,
  • CXCR4 hi , CCR7 hi and CCR5 hi cells as defined herein for removal from the peripheral blood.
  • "High" expression may be determined according to standard flow cytometry techniques. The level is measured relative to levels of expression of the chemokine receptor in cells taken from a healthy subject.
  • the attached Figure 12 provides an example of a gating strategy.
  • the CCR9 hi cells may be monocytes and may also be HLA-DR hi .
  • the invention further provides a binding reagent capable of specifically binding to one or more chemokine receptors, in particular to a chemokine receptor/the chemokine receptor CCR9, CXCR4, CCR7 and/or CCR5, for use in the treatment of primary sclerosing cholangitis, wherein the one or more binding reagents is immobilized, directly or indirectly on a solid support contained within an apheresis column, to which is applied peripheral blood from a patient thus removing one or more chemokine receptor/CCR9, CXCR4, CCR7 and CCR5 expressing cells from the peripheral blood of the patient.
  • the invention also provides for use of one or more binding reagents capable of specifically binding to a chemokine receptor/the chemokine receptor CCR9, CXCR4, CCR7 and/or CCR5 for use in the manufacture of an apheresis column for treatment of primary sclerosing cholangitis, wherein the one or more binding reagents is immobilized on a solid support contained within the apheresis column, to which is applied peripheral blood from a patient thus removing chemokine receptor/CCR9, CXCR4, CCR7 and/or CCR5 expressing cells from the peripheral blood of the patient.
  • the invention aims to treat primary sclerosing cholangitis.
  • treatment is meant a reduction in the specific chemokine receptor expressing cells in the peripheral blood of the patient.
  • the reduction may comprise a reduction in cells that express chemokine receptors, in particular one or more of CCR9, CXCR4, CCR7 and CCR5, at increased levels in diseased patients.
  • the patient is typically a human patient but the term patient may include both human and non-human animal subjects in some embodiments.
  • this typically involves a reduction in one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells, such as "CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi expressing cells, in the peripheral blood of the patient.
  • the CCR9, CXCR4, CCR7 and/or CCR5 expressing cells comprise, consist essentially of or consist of monocytes, macrophages and/or lymphocytes, in particular T- lymphocytes, in certain embodiments.
  • the cells may be monocytes and in particular may comprise HLA-DR hl monocytes. Monocytes are produced by the bone marrow from haematopoietic stem cell precursors called monoblasts.
  • Monocytes may differentiate into macrophages or dendritic cells. Monocytes and their macrophage and dendritic cell progeny serve a number of functions in the immune system including phagocytosis, antigen presentation and cytokine production. Monocytes may be characterized with reference to expression of the cell surface marker CD14, optionally together with CD16.
  • Classical monocytes may be characterized by high level expression of the CD14 cell surface receptor (CD14++ CD16- monocyte).
  • Non-classical monocytes may be characterized by low level expression of CD14 and with additional co-expression of the CD16 receptor (CD14+CD16++ monocyte).
  • Intermediate monocytes may be characterized by high level expression of CD14 and low level expression of CD16 (CD14++CD16+ monocytes).
  • Macrophages are derived from monocytes and are responsible for protecting tissues from foreign substances. They are cells that possess a large smooth nucleus, a large area of cytoplasm and internal vesicles for processing foreign material.
  • the term "macrophage” may refer to a monocyte-derived cell expressing one or more of the following cell surface markers CD14, CD1 1 b, Lysozyme M, MAC-1 /MAC-3 and CD68.
  • the term macrophage includes both activated and un-activated macrophages. Activated macrophages may be characterized by expression of CD69, ENG, FCER2 and IL2RA, HLA-DR, CD86.
  • M1 macrophages may be characterized by expression of one or more of CD1 6 + CD32 + CD64 + and secrete mainly IL-23 and IL-1 , TNF, IL-6 and high levels of IL-12 and in addition effector molecules such as iNOS and ROI.
  • M1 macrophages have cytotoxic features as opposed to M2 macrophages.
  • M2 macrophages may be characterized by expression of one or more of SRA/B + CD163 + MR + CD14 + and express TGF , IL-10 and IL-1 Ra.
  • Tumour associated macrophages share many characteristics with the M2 macrophages and are considered as M2 polarised macrophages.
  • the M1 /M2 paradigm can also be found in monocyte subsets where CD14 + CD1 6 CXC3R1 low monocytes are considered the
  • T-lymphocyte includes CD4 + T cells such as T helper cells (Th1 cells and Th2 cells), and CD8 + T cells such as cytotoxic T cells.
  • Th1 cells may be characterized by expression of CCR5 and/or by production of IFN- ⁇ .
  • Th2 cells may be characterized by expression of CCR3 and/or by production of IL-4.
  • CCR9, CXCR4, CCR7 and/or CCR5 expressed on these aforementioned cells binds to chemokines such as CCL25 (TECK), CXCL12, CCL19, CCL21 or CCL5 (RANTES).
  • CCR9 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C- C motif) receptor 9. The HGNC ID for this gene is 1610. The gene is located at
  • chromosome position 3p22 The previous symbol and name for the gene is GPR28.
  • CXCR4 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-X-C motif) receptor 4.
  • the HGNC ID for this gene is 2561 .
  • the gene is located at chromosome position 2q21 .
  • the previous symbol and name for the gene is "chemokine (C-X-C motif), receptor 4 (fusin)".
  • Synonyms for this gene include CD1 84, D2S201 E, fusin, HM89, HSY3RR, LESTR, NPY3R, NPYR, NPYY3R.
  • the Genbank reference sequence for CXCR4 is AF005058.1 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CCR7 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) receptor 7.
  • the HGNC ID for this gene is 1 608.
  • the gene is located at chromosome position 17q12-q21 .2.
  • the previous symbol and name for the gene is CMKBR7, EBI1 .
  • Synonyms for this gene include BLR2, CD197 and CDw197.
  • the RefSeq reference sequence for CCR7 is NM 001838.3 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CCR5 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) receptor 5.
  • the HGNC ID for this gene is 1606.
  • the gene is located at chromosome position 3p21 .
  • the previous symbol and name for the gene is CMKBR5.
  • Synonyms for this gene include CC-CKR-5, CD195 CKR-5, IDDM22 and CKR5.
  • the RefSeq reference sequence for CCR1 is NM 000579.3 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • the various embodiments of the methods of the invention may involve specific binding interactions with any these further cell-surface markers in addition to the removal based upon binding to CCR9, CXCR4, CCR7 and/or CCR5.
  • Suitable binding reagents can be prepared to specifically bind to these cell-surface markers.
  • the discussion of CCR9, CXCR4, CCR7 and/or CCR5 specific binding reagents thus applies mutatis mutandis.
  • Treatment according to the various embodiments of the invention may result in alleviation or amelioration of symptoms, prevention of progression, regression of the condition, or complete recovery.
  • Measurable parameters of successful treatment include one or more, up to all, of a liver values AST, ALT, ALP, bilirubin, PK and ERCP, MRI.
  • a single treatment is sufficient to cause a depletion of around 10%, 20%, 30%, 40%, 50%, 60% or 70%, or higher up to 80%, 90%, 95% or more, or any range of values between and including these amounts, of a specific chemokine receptor, in particular one or more of CCR9, CXCR4, CCR7 and CCR5, expressing cells from the peripheral blood of the patient.
  • At least around 50% depletion is achieved in a single treatment.
  • successful treatment may be defined with reference to depletion of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells.
  • Treatment may lead to depletion of between approximately 100 and 500 million CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, such as monocytes, in certain embodiments, and more particularly to about 100, 150, 200, 250, 300, 350, 400, 450, or 500 million CCR9, CXCR4, CCR7 and/or CCR5 expressing cells.
  • chemokine receptor expressing cells are immobilized.
  • chemokine receptors which may be of the same or different type to those used for capture.
  • additional chemokine receptors may permit circulating chemokines which contribute to the inflammatory condition to be captured from the peripheral blood.
  • a reduction in circulating (specific) chemokine levels may provide a measure of successful treatment.
  • the duration of treatment may be readily determined by one skilled in the art and will depend upon factors such as the flow rate of the peripheral blood. Duration of treatment may be tied into monitoring of the treatment itself, with the treatment considered complete once a measurable parameter of treatment has reached a defined threshold. Any suitable parameter may be employed as discussed herein.
  • treatment may be considered complete when a reduction in one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells, such as a 50% reduction in one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells, has been achieved.
  • the apheresis system may be operated at a flow rate of around 10-80 imL/min, or more specifically between around 20-70 imL/min, or between around 30-60 imL/min.
  • the treatment is performed for a period of around 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10,120 etc., or any range of values between and including these amounts, minutes.
  • the treatment is typically not aimed to remove all of the cells expressing the chemokine receptor in the peripheral blood, as a basal level of those cells is required in healthy subjects.
  • around 10-80% or more specifically around 20, 30, 40 or 50%, or any range of values between and including these amounts, of the patient's blood is applied to the column in a single treatment.
  • the volume of blood circulated through the apheresis column or system may be in the region of around 1000-3000ml, such as around 1000, 1200, 1400, 1600, 1800 or 2000ml or any range of values between and including these amounts.
  • the treatment may be considered complete once this volume of blood has been circulated.
  • the patient may be administered anticoagulants prior to each treatment session.
  • a suitable solution such as a sterile saline solution, optionally including an anticoagulant such as Heparin, may be used for priming the apheresis (extracorporeal) system.
  • An additional volume of anticoagulant may be added to the circuit at the start of each treatment session, for example as a bolus injection.
  • the invention relies upon a binding reagent which is capable of specifically binding to a chemokine receptor.
  • This specific binding reaction permits cells expressing the chemokine receptor to be removed from the peripheral blood of the patient when the blood is passed over the solid support upon or within which the binding reagent is immobilized.
  • Specific chemokine receptors of interest include CCR9, CXCR4, CCR7 and CCR5, particularly CCR9.
  • the binding reagent can be any binding reagent capable of specifically binding to the receptor in question.
  • specific binding is meant that the binding reagent displays sufficient specificity of binding and appropriate binding affinity/kinetics to permit removal of cells expressing one or more of CCR9, CXCR4, CCR7 and/or CCR5 from the peripheral blood.
  • the binding reagent is capable of binding to other molecules, such as other chemokine receptors
  • the binding reagent will preferentially bind to cells expressing one or more of CCR9, CXCR4, CCR7 and CCR5 and in particular to cells expressing increased levels of CCR9, CXCR4, CCR7 and/or CCR5 (as defined further herein).
  • the binding reagent capable of specifically binding to CCR9, CXCR4, CCR7 and/or CCR5 may be either an agonist or an antagonist of CCR9, CXCR4, CCR7 and/or CCR5, respectively.
  • the binding reagent capable of specifically binding to CCR9, CXCR4, CCR7 and/or CCR5 is an antagonist of CCR9, CXCR4, CCR7 and/or CCR5, respectively.
  • Chemokines are typically, although not necessarily exclusively (particularly in the case of truncated or modified forms) agonists of their cognate receptor and serve to activate the cells expressing the relevant receptor, as would be appreciated by one skilled in the art.
  • Antibodies against the relevant chemokine receptor are generally considered to be antagonists, as would be appreciated by one skilled in the art.
  • binding reagents include proteins or polypeptides, such as antibodies and receptor ligands, in particular chemokines.
  • the binding reagent may be a nucleic acid molecule in certain embodiments.
  • the nucleic acid is an aptamer.
  • Nucleic acid aptamers are polynucleotides of approximately 15-40 nucleotides long. Nucleic acid aptamers can be made using the SELEX process (systemic evolution of ligands by exponential enrichment) or any other process known to those of skill in the art.
  • the binding reagent may be a peptide, and in certain instances, a peptide aptamer.
  • Peptide aptamers are artificial recognition molecules that consist of a variable peptide sequence inserted into a constant scaffold protein (Baines IC, Colas P. Peptide aptamers as guides for small molecule drug discovery. Drug Discov Today.
  • a number of methodologies such as phage display, ribosome display and yeast two-hybrid screening systems are available for screening a library of potential peptide-based binding agents.
  • protein scaffolds based on domains such as fibronectins, ankyrin repeats, protein A, SH3 domains, lipocalins and ubiquitin can be used as the binding agent.
  • technologies such as phage display and ribosome display are available for screening a library of protein - based binding agents.
  • libraries of candidate chemical compounds can be screened for specific binding to the relevant chemokine receptor using suitable screening techniques known in the art, which may be high throughput screens in certain embodiments.
  • the candidate binding agent may be immobilized on a solid support and the ability of the agent to specifically retain cells expressing the chemokine receptor of interest or labelled chemokine receptors determined.
  • a range of cell types may be applied to the solid supports to confirm specificity of binding, or alternatively a mixed sample (such as peripheral blood) may be applied to the solid support. Retention of the cell type of interest (expressing the appropriate chemokine receptor) can be confirmed to identify suitable binding agents.
  • chemokine also comprises biotinylated or otherwise labelled chemokines.
  • chemokine also comprises modified and truncated versions of the chemokine and chemokine fragments with the proviso that the modified or truncated form retains its ability to bind to its cognate receptor (and thus remains functional in the context of the various embodiments of the invention).
  • the chemokine does not necessarily need to retain biological activity as it is specific binding affinity for CCR9, CXCR4, CCR7 and/or CCR5 that is required.
  • the chemokine lacks biological activity, for example in terms of activation of the (CCR9, CXCR4, CCR7 and/or CCR5) receptor.
  • Modifications may be made to improve protein synthesis, for example uniformity of product and yield.
  • exemplary modifications may comprise amino acid additions, substitutions, deletions or other modifications to one or more amino acids in the chemokine.
  • Modifications may comprise substitution of the wild type amino acid with non-natural amino acids such as norleucine
  • NLeu NLeu
  • pyroGlu derivatized amino acids
  • Such modifications may be made to minimize side-product formation during storage and use of the columns of the various embodiments of the invention.
  • Modifications may be made to improve labelling, for example inclusion of a polyethylene glycol (PEG) spacer to facilitate biotinylation.
  • PEG polyethylene glycol
  • the biotinylation and/or conjugation with fluorochromes or other labelling groups of the chemokine is performed in a manner which does not substantially affect the receptor binding capacity. Site specific biotinylation or other labelling is preferred as non-selective labelling of chemokines may compromise receptor binding activity.
  • Biotinylation or other labelling is generally preferred at or towards the C-terminus of the protein as the inventors have found that modifications in this area are generally well tolerated (in terms of a minimal effect on receptor binding capability).
  • Biotinylation may be carried out site-specifically at any suitable amino acid.
  • suitable amino acids include lysine, diaminopropionic acid and ornithine.
  • suitable amino acids include lysine, diaminopropionic acid and ornithine.
  • suitable amino acids include lysine, diaminopropionic acid and ornithine.
  • suitable amino acids include lysine, diaminopropionic acid and ornithine.
  • suitable amino acids include lysine, diaminopropionic acid and ornithine.
  • Truncations may involve deletion of either N or C terminal amino acids as appropriate, or both.
  • the truncated version will retain the residues required for the chemokine to fold correctly, for example to retain a chemokine fold structure, consistent with the requirement that a truncated version must retain the ability to bind to the relevant receptor (expressed by (on the surface of) a leukocyte).
  • Chemokine molecules typically include disulphide bonds between the 1 st and 3 rd and 2 nd and 4 th cysteine residues respectively, as would be understood by one skilled in the art. Where sequences are presented herein, it is assumed that these disulphide bonds will form in the folded protein (unless otherwise stated).
  • Truncated versions may comprise anywhere between 1 and 100 less amino acids, such as 1 , 2, 3, 4, 5 etc amino acids, than the wild type amino acid sequence in certain embodiments.
  • truncated versions may comprise further modification as detailed herein.
  • the modified or truncated version may have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more overall amino acid sequence identity with the full length wild type chemokine (where a deletion is counted as a difference in amino acid sequence) in certain embodiments.
  • Sequence identity may be determined using known algorithms, such as BLAST or GAP analysis (GCG).
  • Chemokines may lack the N- terminal signal peptide which is cleaved off during synthesis in vivo.
  • Specific chemokines useful in the various embodiments of the present invention for binding to CCR9, CXCR4, CCR7 and/or CCR5 include CCL25 (TECK), CXCL12, CCL19, CCL21 or CCL5 (RANTES).
  • CCL25, CXCL12, CCL21 or CCL5 are able to bind to chemokine receptors implicated in primary sclerosing cholangitis.
  • CCL25, CXCL12, CCL19, CCL21 or CCL5 are useful for removing CCR9, CXCR4, CCR7 and/or CCR5 expressing cells from the blood of a patient.
  • the chemokines described in greater detail herein (with reference to the relevant figures and amino acid sequences, as set forth in the SEQ ID NOs) may each be applied according to the various embodiments of the present invention.
  • chemokines may each be applied according to the present invention. Such modified forms may instruct the skilled person regarding additional modified forms of the same and other chemokines which may be suitable for use in the invention.
  • Chemokines show variable sequence homology varying from less than 20% to over 90% but all share very similar tertiary structures consisting of a disordered N-terminus, followed by a long loop (the N-loop) that ends in a 3 10 helix, a 3-stranded ⁇ -sheet and a C-terminal helix. The overlall topology is stabilsed by disulphide bonds.
  • Truncations within this N-terminal region can maintain binding to the receptor but can lead to a change or loss of function (for examples Zhang YJ et al, J. Biol. Chem., 1994, 269, 15918, ; Gong J-H and Clark-Lewis I., J. Exp. Med., 1995, 181 , 631 -640; Fernandez EJ annd Lolis E., Annu. Rev. Pharmacol. Toxicol., 202, 42, 469-99; Allen SJ et al, Annu. Rev. Immunol., 2007, 25, 787-820, each of which is incorporated herein by reference). Truncations at the C- terminus of the chemokine can also be made and maintain receptor binding activity (Treating Inflammatory Disorders, Ola Winqvist and Graham Cotton, WO2010/029317, incorporated herein by reference in its entirety).
  • fragments and variants of chemokines are used in the devices and methods as disclosed herein. More particularly, such fragments and variants retain the ability to specifically bind to their cognate chemokine receptor.
  • Chemokines are known by those skilled in the art to share specific receptor binding domains, including a similar monomeric fold, characterized, for example, by a disordered amino-terminal domain, followed by a conserved core region, consisting of the so called "N-loop," three anti-parallel ⁇ -strands, and a carboxyl-terminal a-helix.
  • chemokine-chemokine receptor interaction is a two-step mechanism, in which the core of the chemokine interacts first with a binding site formed by the extracellular domains of the receptor, while another interaction is formed between the chemokine N terminus and a second binding site on the receptor in order to trigger receptor activation.
  • a "fragment,” such as a functional fragment of a chemokine is intended to mean a portion of the amino acid sequence of the protein that retains binding for its cognate receptor.
  • the fragment may include, for example, the monomeric fold region, or portions thereof such as the amino- terminal domain, the conserved core region and/or the "N-loop," the anti-parallel ⁇ -strands, and/or the carboxyl-terminal a-helix or combinations and portions thereof.
  • a polypeptide can be considerably mutated without materially altering one or more of the polypeptide's functions, for example, without altering specific binding and/or the folding of the protein.
  • the genetic code is well known to be degenerate, and thus different codons encode the same amino acids. Even where an amino acid substitution is introduced, the mutation can be conservative and have no material impact on the essential functions of a protein (see for example, Stryer, Biochemistry 4th Ed., W.
  • part of a polypeptide chain can be deleted without impairing or eliminating all of its functions.
  • the deletion of between about 1 and about 20 amino acids on the C- and/or N-terminus such as deletions of about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids at the C- and/or N-terminus, can result in a chemokine that retains function, such as specific binding of its cognate receptor.
  • Such truncations can retain the full function of an entire protein, and/or can allow for retained functions such as protein-protein interactions as in the case of ligand-receptor interactions.
  • Chemokines having deletions of a small number of amino acids for example, less than about 20% (such as less than about 18%, less than about 15%, less than about 10%, less than about 8%, less than about 5%, less than about 2%, or less than about 1 %) of the total number of amino acids in the wild type chemokine can also be used in the methods and devices disclosed herein.
  • insertions or additions can be made in the polypeptide chain for example, adding epitope tags, without impairing or eliminating its functions (Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-lntersciences, 1998).
  • a functional fragment of a chemokine may consist of about 10 or more, about 25 or more, about 50 or more, about 75 or more, about 100 or more, about 125 or more, about 150, about 175 or more, or about more or 200 or more amino acid residues of a chemokine amino acid sequence.
  • the chemokine or a functional fragment thereof has an amino acid that has at least about 60% or 65% sequence identity, about 70% or 75% sequence identity, about 80% or 85% sequence identity, about 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity over its full length as compared to a reference sequence, such as those detailed herein, for example using the NCBI Blast 2.0 gapped BLAST set to default parameters. Alignment may also be performed manually by inspection.
  • One or more conservative amino acid modifications can also be made in the chemokine amino acid sequence, whether an addition, deletion or modification, that does not substantially alter the 3-dimensional structure of the polypeptide or its ability to bind to the cognate receptor.
  • a conservative amino acid substitution does not affect the ability of the chemokine to specifically bind its cognate receptor.
  • Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Peptides such as chemokines and fragments thereof, can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity or function— such as binding to a cognate receptor— as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein may be provided in the form of a salt of a pharmaceutically- acceptable cation or esterif ied to form a C1 -C16 ester, or converted to an amide of formula NR1 R2 wherein R1 and R2 are each independently H or C1 -C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring.
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be modified to C1 -C16 alkyl or dialkyl amino or further converted to an amide.
  • a pharmaceutically-acceptable acid addition salt such as the HCI, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts
  • Hydroxyl groups of the peptide side chains can be converted to C1 -C16 alkoxy or to a C1 - C16 ester using well-recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains can be substituted with one or more halogen atoms, such as F, CI, Br or I, or with C1 - C16 alkyl, C1 -C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • Methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups.
  • cyclic structures into the peptides of this disclosure to select and provide conformational constraints to the structure that result in enhanced stability.
  • a C- or N-terminal cysteine can be added to the peptide, so that when oxidized the peptide will contain a disulfide bond, generating a cyclic peptide.
  • Other peptide cyclizing methods include the formation of thioethers and carboxyl- and amino-terminal amides and esters.
  • Peptidomimetic and organomimetic embodiments are also within the scope of the present disclosure, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three-dimensional arrangement of the peptide backbone and component amino acid side chains, resulting in such peptido- and
  • organomimetics of the proteins of this disclosure For computer modeling applications, a pharmacophore is an idealized, three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). See Walters, "Computer-Assisted Modeling of Drugs", in Klegerman & Groves, eds., 1993, Pharmaceutical Biotechnology, Interpharm Press: Buffalo Grove, IL, pp. 165 174 and
  • amino acids in a peptide, polypeptide, or protein generally are chemically bound together via amide linkages (CONH). Additionally, amino acids may be bound together by other chemical bonds.
  • CCL3, CCL5, CCL8 all bind to CCR5 and may thus be useful in the invention.
  • CCL25 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) ligand 25.
  • the HGNC ID for this gene is 10624.
  • the gene is located at chromosome position 19p13.2.
  • the previous symbol and name for the gene is SCYA25, "small inducible cytokine subfamily A (Cys-Cys), member 25".
  • Synonyms for this gene include "Ck beta-15", Ckb15, TECK, "TECKvar", "thymus expressed chemokine”.
  • Genbank reference sequence for CCL25 is U86358.1 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CXCL12 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-X-C motif) ligand 12.
  • the HGNC ID for this gene is 10672.
  • the gene is located at chromosome position 10q1 1 .1 .
  • the previous symbol and name for the gene is SDF1 , SDF1 A, SDF1 B, "stromal cell-derived factor 1 ".
  • Synonyms for this gene include PBSF, SCYB12, SDF-1 a, SDF-1 b, TLSF-a, TLSF-b, TPAR1 .
  • the Genbank reference sequence for CXCL12 is L36033.1 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CCL21 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) ligand 21 .
  • the HGNC ID for this gene is 10620.
  • the gene is located at chromosome position 9p13.
  • the previous symbol and name for the gene is SCYA21 , "small inducible cytokine subfamily A (Cys-Cys), member 21 ".
  • Synonyms for this gene include 6Ckine, "beta chemokine exodus-2", CKb9, ECL, "Efficient Chemoattractant for Lymphocytes", exodus-2, "secondary lymphoid tissue chemokine", SLC, TCA4.
  • Genbank reference sequence for CCL21 is AB002409.1 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CCL5 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) ligand 5, also known as RANTES.
  • the HGNC ID for this gene is 10632.
  • the gene is located at chromosome position 17q1 1 .2-q12.
  • the previous symbol and name for the gene is D17S136E, SCYA5, "small inducible cytokine A5 (RANTES)”.
  • CCL5 "regulated upon activation, normally T-expressed, and presumably secreted", "SIS-delta”, SISd, "small inducible cytokine subfamily A (Cys-Cys), member 5", "T-cell specific protein p288", “T-cell specific RANTES protein”, TCP228.
  • Genbank reference sequence for CCL5 is AF043341 .1 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CCL19 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) ligand 19, also known as MIP-3b.
  • the HGNC ID for this gene is 10617.
  • the gene is located at chromosome position 9p13.
  • the previous symbol and name for the gene is SCYA19, "small inducible cytokine subfamily A (Cys-Cys), member 19".
  • CCL8 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) ligand 8, also known as MCP-2.
  • the HGNC ID for this gene is 10635.
  • the gene is located at chromosome position 17q1 1 .2.
  • the previous symbol and name for the gene is SCYA8, "small inducible cytokine subfamily A (Cys-Cys), member 8 (monocyte chemotactic protein 2)".
  • Another synonym for this gene is HC14.
  • the Genbank reference sequence for CCL8 is X99886.1 as available on 13 June 201 1 , which is incorporated herein by reference in its entirety.
  • CCL3 is the gene symbol approved by the HUGO Gene Nomenclature Committee for chemokine (C-C motif) ligand 3, also known as MIP-1 a.
  • the HGNC ID for this gene is
  • the gene is located at chromosome position 17q12.
  • the previous symbol and name for the gene is SCYA3, "small inducible cytokine A3 (homologous to mouse Mip-1 a)".
  • a chemokine of the various embodiments of the invention containing both modifications and a truncation and specifically adapted for use in the invention is described in detail herein.
  • the truncated CCL25 corresponds to residues 1 to 74 of the full length mature protein (and thus lacks amino acids 75 to 127 and the N-terminal signal peptide of 23 amino acids) and thus retains the chemokine fold.
  • a methionine to Norleucine substitution is incorporated, to prevent oxidation of the residue during chain assembly.
  • the N terminal glutamine residue is substituted with pyroglutamine to permit uniformity of product during synthesis.
  • Biotinylation is achieved via a PEG spacer at the ⁇ -functionality of the lysine residue found at position 72.
  • the amino acid sequence of the linear molecule (i.e. without the PEG spacer and biotin molecule at amino acid 72 shown) comprises, consists essentially of or consists of the amino acid sequence presented as SEQ ID NO: 1 .
  • Chemokines of the invention may be synthesised through any suitable means.
  • the final protein may thus comprise, consist essentially of or consist of the amino acid sequence of SEQ ID NO: 4 (see Example 6 below).
  • chemokine of the various embodiments of the invention containing modifications and specifically adapted for use in the invention is described in detail herein (see Example 3 below).
  • the modified CCL8 (MCP-2) corresponds to residues 1 to 76 of the full length mature protein (and lacks the N-terminal signal peptide of 23 amino acids, which is cleaved off) and thus retains the chemokine fold.
  • the Gin at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions.
  • Gln1 of the sequence is substituted with pyroglutamine to prevent mixed species of N-terminal Gin and pyroGlu being generated (SEQ ID NO: 5).
  • FmocLys(ivDde)-OH is incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 6).
  • the naturally occurring lysine at position 75 is modified through biotinylation.
  • a suitable spacer such as a PEG spacer, may be incorporated between the ⁇ -amino functionality and the biotin (SEQ ID NO: 7):
  • the invention also relates to a modified chemokine comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7:
  • X1 pyroGlu (but may remain as Gin in some embodiments)
  • X75 an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • diaminopropionic acid and optionally is biotinylated, optionally via a spacer
  • chemokine of the various embodiments of the invention containing modifications and specifically adapted for use in the invention is described in detail herein (see Example 4 below).
  • the modified CCL5 corresponds to residues 1 to 68 of the full length mature protein (and lacks the N-terminal signal peptide of 23 amino acids, which is cleaved off) and thus retains the chemokine fold.
  • the single methionine (Met67) within the sequence is mutated to lysine, to mitigate against oxidation of this residue during the chain assembly.
  • SEQ ID NO: 8 presents the amino acid sequence prior to mutation.
  • This Met to Lys substitution provides a lysine at position 67 which can be modified through biotinylation.
  • Fmocl_ys(ivDde)-OH is incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 9).
  • the biotinylated version comprises, consists essentially of or consists of the amino acid sequence of SEQ ID NO: 10.
  • the invention also relates to a modified chemokine comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 10:
  • X is an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG (e.g. K(Biotin))
  • a chemokine of the various embodiments of the invention containing truncation and modifications and specifically adapted for use in the invention is described in detail herein (see Example 6 below).
  • the truncated CXCL12 (SDF-1 a) corresponds to residues 1 to 67 of the full length mature protein (and lacks the N-terminal signal peptide of 21 amino acids, which is cleaved off from an immature protein of total length 93 amino acids) and thus retains the chemokine fold (SEQ ID NO: 1 1 ).
  • FmocLys(ivDde)-OH is incorporated as residue 64 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 12).
  • the naturally occurring lysine at position 64 is modified through biotinylation.
  • the final protein may thus comprise, consist essentially of or consist of the amino acid sequence of SEQ ID NO: 13.
  • the invention also relates to a modified chemokine comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 13:
  • X is an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • chemokine of the various embodiments of the invention containing modifications and specifically adapted for use in the invention is described in detail herein (see Example 9 below).
  • the modified CCL19 ( ⁇ -3 ⁇ ) corresponds to residues 1 to 77 of the full length mature protein (and lacks the N-terminal signal peptide of 21 amino acids, which is cleaved off) and thus retains the chemokine fold.
  • An additional lysine is inserted at the C-terminus, at position 78.
  • the chemokine may thus comprise, consist essentially of or consist of the amino acid sequence of SEQ ID NO: 14.
  • FmocLys(ivDde)-OH is incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 15).
  • the ⁇ -amino side chain functionality of Lys(78) is modified through biotinylation.
  • the final protein may thus comprise, consist essentially of or consist of the amino acid sequence of SEQ ID NO: 16.
  • the invention also relates to a modified chemokine comprising, consisting essentially of or consisting of the amino acid sequence of SEQ ID NO: 16:
  • X is an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin)
  • Chemokines useful in the various embodiments of the invention may be synthesised through any suitable means known in the art.
  • the chemokines are chemically synthesised as this facilitates modification and labelling etc.
  • recombinant DNA based approaches may also be employed in combination with appropriate labelling and modification technologies as required.
  • the invention also provides a nucleic acid molecule encoding the chemokines of the various embodiments of the invention.
  • the invention also relates to a vector containing such a nucleic acid molecule and a host cell containing the vector.
  • the vector may additionally comprise a suitable promoter operably linked to the nucleic acid molecule, to facilitate transcription of the corresponding imRNA molecule.
  • the host cell may be capable of expressing the protein by transcription and translation of the nucleic acid molecule encoding a chemokine of the various embodiments of the invention.
  • the chemokines useful in the various embodiments of the invention can be biotinylated by methods known in the art such as described in WO 00/50088 A2, which is incorporated herein by reference.
  • site-specific labelling of the chemokines of the various embodiments of the invention is preferable, although any labelling technique which does not significantly affect the receptor-binding capacity of the chemokine may be employed.
  • Various site-specifically biotinylated chemokines and native chemokines are available commercially, for instance from Almac, Craigavon, UK.
  • the one or more chemokines are biotinylated via a spacer group.
  • the spacer may be employed to prevent the biotin group from impacting on the activity of the chemokine, in particular binding of the chemokine to its cognate receptor. Any suitable spacer that facilitates retention of receptor binding properties of the chemokine may be employed in the various embodiments of the invention. Thus, in the specific embodiments described above, spacers other than PEG spacers may be employed as appropriate. In specific embodiments, the spacer is a polyethylene glycol (PEG) spacer.
  • PEG polyethylene glycol
  • the term "antibody” includes all immunoglobulins or immunoglobulin-like molecules with specific binding affinity for the relevant chemokine receptor (including by way of example and without limitation, IgA, IgD, IgE, IgG and IgM, combinations thereof, and similar molecules produced during an immune response in any vertebrate, for example, in mammals such as humans, goats, rabbits and mice).
  • Specific immunoglobulins useful in the various embodiments of the invention include IgG isotypes.
  • the antibodies useful in the various embodiments of the invention may be monoclonal or polyclonal in origin, but are typically monoclonal antibodies.
  • Antibodies may be human antibodies, non-human antibodies, or humanized versions of non- human antibodies, or chimeric antibodies. Various techniques for antibody humanization are well established and any suitable technique may be employed.
  • the term "antibody” also refers to a polypeptide ligand comprising at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, and it extends to all antibody derivatives and fragments that retain the ability to specifically bind to the relevant chemokine receptor.
  • antibody encompasses antibodies comprised of both heavy and light chains, but also heavy chain (only) antibodies.
  • the antibodies may be engineered so as to be specific for more than one chemokine receptor, for example bi-specific to permit binding to two different chemokine receptors. Suitable commercially available antibodies which bind to the chemokine receptors of interest are listed in table 1 below. They may or may not be labelled. General reference may be made to "Antibodies a laboratory manual: By E Harlow and D Lane, pp 726. Cold Spring Harbor Laboratory. 1988", which reference is incorporated herein in its entirety.
  • Anti-CCR2 antibodies are described for example in WO 2010/021697, incorporated herein by reference. Further examples of potentially useful antibodies include MLN-1202, an anti-CCR2 monoclonal antibody currently undergoing clinical trials (Millennium Pharmaceuticals).
  • the chemokine receptor expressing cells may thus be targeted using alternative binding agents, such as antibodies or other chemical compounds, as defined herein, rather than the natural chemokine binding partner.
  • alternative binding agents such as antibodies or other chemical compounds, as defined herein, rather than the natural chemokine binding partner. This approach is a new approach to treating
  • the various embodiments of the invention also provide an apheresis column loaded with a solid support comprising a binding reagent capable of specifically binding to a chemokine receptor immobilized directly or indirectly on the support to permit removal of a cell expressing the chemokine receptor from the peripheral blood of a patient, wherein the binding reagent is not a chemokine.
  • the binding reagent capable of specifically binding to the chemokine receptor may be an agonist or an antagonist of the chemokine receptor.
  • the binding reagent capable of specifically binding to the chemokine receptor is selected from an antibody and a chemical compound.
  • the invention thus also provides a method for treating an inflammatory condition comprising applying peripheral blood from a patient/subject to an apheresis column as defined above (an apheresis column loaded with a solid support comprising a binding reagent capable of specifically binding to a chemokine receptor immobilized directly or indirectly on the support to permit removal of a cell expressing the chemokine receptor from the peripheral blood of a patient, wherein the binding reagent is not a chemokine) thus removing chemokine receptor expressing cells from the peripheral blood of the
  • the method may comprise returning the blood depleted of the chemokine receptor expressing cells to the patient/subject.
  • the invention provides a binding reagent capable of specifically binding to a chemokine receptor for use in the treatment of an inflammatory condition, wherein the binding reagent is immobilized on a solid support contained within an apheresis column as defined above (an apheresis column loaded with a solid support comprising a binding reagent capable of specifically binding to a chemokine receptor immobilized directly or indirectly on the support to permit removal of a cell expressing the chemokine receptor from the peripheral blood of a patient/subject, wherein the binding reagent is not a chemokine), to which is applied peripheral blood from a patient thus removing chemokine receptor expressing cells from the peripheral blood of the patient.
  • Solid support materials for immobilizing the binding reagents of the various embodiments of the invention are known in the art.
  • Solid support refers to, for example, materials having a rigid or semi-rigid surface or surfaces, and may take the form of beads, resins, gels, microspheres, or other geometric configurations.
  • a useful support material is one that does not activate blood cells so as to make them coagulate or adhere to the support as peripheral whole blood is applied to the device.
  • a support treated with an agent to provide it with anti-coagulation properties in particular a heparinized support is employed.
  • the blood of the patient may be treated with an anti-coagulant such as heparin prior to application to the support.
  • Useful support materials comprise high molecular weight carbohydrates, in particular carbohydrates having a molecular weight of 100 kDa or more, such as agarose, in particulate form, optionally cross-linked, and cellulose.
  • Other preferred support materials are polymers, such as carboxylated polystyrene, and glass.
  • the support of the various embodiments of the invention may be provided in the form of particles or fibres.
  • the support particles may have regular form, such as spheres or beads, or irregular form.
  • SepharoseTM a cross linked, beaded-form of agarose, is used as column matrix. It is chosen for its optimal distribution capacity and can provide a large available area for affinity binding.
  • Solid supports may be provided in the form of magnetic beads, with the specific binding reagent immobilized on the magnetic beads. Following capture of the (CCR9, CXCR4, CCR7 and/or CCR5) chemokine receptor expressing cells from the blood, the beads can be removed from the blood with the aid of an appropriate magnetic separator.
  • a binding reagent such as a chemokine, antibody, peptide, nucleic acid or chemical compound
  • a binding reagent can be immobilized on the support in a direct or indirect manner. Immobilization can be by means of a suitable linker in some embodiments.
  • “Avidin” or “avidin molecule” refers to any type of protein that specifically binds biotin to the substantial exclusion of other (small) molecules that might be present in a biological sample.
  • avidin examples include avidins that are naturally present in egg white, oilseed protein (e.g., soybean meal), and grain (e.g., corn/maize), and streptavidin, which is a protein of bacterial origin.
  • oilseed protein e.g., soybean meal
  • grain e.g., corn/maize
  • streptavidin which is a protein of bacterial origin.
  • such a method may comprise providing the binding reagent in biotinylated form, providing a solid support having streptavidin immobilized on its surface, contacting the support with an aqueous solution of the biotinylated binding reagent, and rinsing the support with an aqueous solvent.
  • binding pair interactions such as antibody - antigen interactions, may also be utilised for indirect immobilisation of binding reagent onto a support.
  • the support may be derivatised with one member of a binding pair, such as an antibody or fragment or derivative thereof, as defined herein, which has known affinity for a particular peptide sequence or small molecule hapten.
  • the binding reagent may be modified to include the peptide sequence or hapten into the linear molecule or may be added as a side chain or label. Any suitable antibody-antigen pair may be employed.
  • the antibody fragment or derivative may be any fragment or derivative that retains specific binding affinity for the appropriate antigen. Examples include Fab, F(ab') 2 fragments, scFV, VH domains, single domain antibodies (such as nanobodies), heavy chain antibodies and humanized version of non-human antibodies etc.
  • binding reagents can be synthesised or derivatised with one of the interacting partners and the solid support synthesised or derivatised with the other interacting partner without loss of binding activity (i.e. binding of the binding reagent to the appropriate chemokine receptor). Immobilization may occur via essentially the same interaction in reverse in some embodiments.
  • the binding reagent which may be a chemokine for example, may be attached to an antibody as defined herein, and the solid support derivatised with the antigen.
  • the chemokine may be produced as a fusion protein with the antibody.
  • binding reagents such as chemokines and antibodies
  • binding reagents can be immobilised directly onto a solid support using bioconjugation techniques established in the field. For example direct immobilisation of proteins onto cyanogen bromide activated solid supports via amino functionalities within the primary sequence of the protein.
  • sulphydryl functionalities within proteins can be used to directly immobilise the protein to alkyl halide derivatised supports or supports containing free thiol functionalities.
  • proteins containing a-thioester functionalities can be directly immobilised on supports containing 1 ,2 amino thiol moieties (eg N-terminal cysteine) using the native chemical ligation reaction.
  • proteins modified with ketones and aldehydes can be immobilised on solid supports derivatised with hydrazinyl, hydrazide and aminoxy functionalities using hydrazone / oxime bond forming ligation reactions (and vice versa).
  • 'Click' chemistry can be used to immobilise proteins onto solid supports, whereby the protein and the support are derivatised with the appropriate mutually reactive chemical functionalities (azides and alkynes).
  • Staudinger ligation chemistry can be used to immobilise appropriately derivatised proteins onto the appropriately derivatised solid supports.
  • the solid support is contained within or carried by the apheresis column.
  • loaded is meant that the column carries or contains the solid support in a manner such that
  • the solid support provides a matrix within the column through which blood flows, in continuous fashion in certain embodiments. This permits cells expressing the specific chemokine receptor to be removed from the blood passing through the column, such that blood exiting the column is depleted of the specific chemokine receptor-expressing cells.
  • the apheresis column is loaded with a support comprising streptavidin immobilized on the support and one or more biotinylated binding reagents, such as chemokines, bound to the streptavidin on the support.
  • the solid support may be comprised of a high-molecular weight carbohydrate, optionally cross-linked, such as agarose.
  • the binding reagent is coupled to the solid support.
  • the relative amounts of binding reagent may be controlled to ensure that coupling between the solid support and the binding reagent will be immediate, minimising the risk of binding reagent decoupling from the solid support. Thus, it may be ensured that there is a relative excess of immobilization sites for the binding reagent on the solid support. Alternatively, or additionally, following immobilization of the binding reagent on the solid support, the solid support may be washed to remove any unbound binding reagent.
  • the apheresis column utilised in the various embodiments of the present invention acts as a leukapheresis treatment for primary sclerosing cholangitis.
  • the column acts to specifically remove one or more of CCR9, CXCR4, CCR7 and CCR5-expressing monocytes or leukocytes by exploiting the interaction between CCR9, CXCR4, CCR7 and/or CCR5 expressed on the cell surface and a specific binding reagent immobilized on a solid support contained within or carried by the column.
  • the overall column typically comprises, consists of, or consists essentially of three combined components; 1 ) a housing which contains or carries 2) the solid support and 3) one or more binding reagents (immobilized thereon) which specifically bind one or more chemokine receptors.
  • the housing may be manufactured from any suitable material for clinical use. In certain embodiments the housing is composed of a plastic material.
  • the housing includes an in flow site for entry of blood and an out flow site for blood (depleted of target cells) to exit the column.
  • the housing may be designed to maintain a continuous blood flow through the solid support matrix.
  • the housing (as shown for example in Figure 3) may include a top portion which comprises a distribution plate (2) at the inflow site (1 ) to spread the blood evenly over the entire matrix area.
  • the distribution plate may act as a first safety barrier preventing larger particles flowing through the column and into the patient. However, the distribution plate is not essential and may be removed in some embodiments to decrease the overall resistance in the system.
  • the column may contain one or more safety filter units (3 and 4) placed at the inflow (1 ) and/or outflow (5) sites of the plastic housing. Such filter units may act to prevent particles larger than blood cells passing in and/or out of the column.
  • the safety filter units may contain a plurality of filters, such as two, three or four filters designed to be a robust barrier and stop all particles larger than blood cells passing through the column.
  • the safety filters may comprise of any suitable pore size to prevent particles larger than blood cells from passing through the column, as would be readily understood by one skilled in the art. Suitable filters are commercially available. In specific embodiments, the pore size of the filter(s) is between approximately 60 ⁇ and ⁇ ⁇ , more specifically approximately 80 ⁇ .
  • the solid support and binding reagent components are discussed in further detail herein.
  • the volume of the housing may be varied depending upon the blood volumes intended to pass through the column. Typically, the volume of the housing is between approximately 40ml and 200ml, more specifically 50ml to 150ml or 60ml to 120ml.
  • the column is generally applied in the form of an apheresis circuit.
  • the overall system includes the apheresis column, tubing and an appropriate pump to pump the blood around the circuit.
  • the system is illustrated in figure 4.
  • the patient (1 ) is connected to the extracorporeal circuit via sterile needles to veins in the right and the left arms.
  • a saline bag (3) is also connected and the saline solution is pumped with a suitable pump (2).
  • Blood is drawn from one arm of the patient through the sterile tubing system by the blood pump (4) and passed through the column (6) and back to the patient.
  • the tubing system may be connected to the column via any suitable coupling, such as standard dialysis luer-lock couplings.
  • the couplings on the column may be colour-coded for correct assembly. For example, red tubing for inflow to the red column top and blue tubing for outflow back to the patient.
  • An air detector (8) may be present in the circuit.
  • Inlet pressure (5) and/or Pven sensors (7) may additionally be employed to monitor the pressure in the circuit.
  • An apheresis pump such as the 4008 ADS pump manufactured by Fresenius Medical Care or the Adamonitor pump, may monitor the patient's inflow and outflow.
  • the pump may also monitor the pressure in the extracorporeal circulation.
  • the pump may be able to discriminate air by a bubble catcher and air detector.
  • a clot catcher filter may be positioned inside the bubble catcher.
  • the pump may also incorporate an optical detector to distinguish between light, e.g. saline solution or air present in the tubing system and dark e.g. blood present in the tubing system.
  • FIG. 5 A schematic diagram of a suitable pump, showing the air detector and optical filter is shown in figure 5. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump may stop immediately. Alternatively or additionally a visual/ audible alarm may be emitted.
  • the treatment methods of the various embodiments of the invention may thus rely upon an extracorporeal circuit.
  • the methods may be considered as ex vivo or in vitro methods and be defined solely with reference to steps performed outside of the patient.
  • the method further comprises, prior to application of the blood to the column, collecting peripheral blood from the patient.
  • the method further comprises, following the application of the blood to the column, infusing the blood depleted of (CCR9, CXCR4, CCR7 and/or CCR5) chemokine receptor expressing cells to the patient. This is then a complete leukapheresis treatment method.
  • a leukaphereis method for treating primary sclerosing cholangitis, comprises collecting peripheral blood from the patient; applying the peripheral blood to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to one or more chemokine receptors, in particular the chemokine receptor CCR9, CXCR4, CCR7 and/or CCR5, immobilized directly or indirectly on the support thus removing one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells from the peripheral blood of the patient; and infusing the depleted blood (of chemokine receptor expressing cells) to the patient.
  • the peripheral blood may be continuously collected from the patient.
  • the depleted blood may be continuously infused to the patient, through use of an appropriate circuit as described herein.
  • the support may be disposed in a column through which the blood is made to flow. This may be achieved using a suitable pump for example, as also described herein. Blood flow through the column enables the binding reagent(s) immobilized on the solid support to capture the cells expressing the chemokine receptor, thus depleting them from the blood and preventing their contribution to the inflammatory primary sclerosing cholangitis.
  • the methods of the various embodiments of the invention and binding reagents for use in the methods of the various embodiments of the invention may require that the patient has been selected for treatment on the basis of detecting an increase in the level of chemokine receptor, in particular, one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells in a sample obtained from the patient.
  • Such companion diagnostic methods are described in greater detail herein and are based, for example, on the observation that CCR9, CXCR4, CCR7 and/or CCR5 expression on monocytes is elevated in patients with primary sclerosing cholangitis.
  • the invention also provides a method of diagnosing, monitoring progression of, or monitoring treatment of primary sclerosing cholangitis comprising determining:
  • CCR7 and CCR5 expressing cells high levels of expression of one or more of CCR9, CXCR4, CCR7 and CCR5 or high levels of cells with high expression of CCR9, CXCR4, CCR7 or CCR5 or increased levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells compared to control, increased levels of expression of one or more of CCR9, CXCR4, CCR7 and CCR5 compared to a control or increased levels of cells with high expression of one or more of CCR9, CXCR4, CCR7 and CCR5 compared to a control indicate the presence or progression of primary sclerosing cholangitis.
  • chemokine receptor expression may also be investigated as increased levels of chemokine receptor expression per cell may also be diagnostically relevant.
  • the cells may be monocytes, in particular CCR9 expressing and HLA-DR hl monocytes as described herein.
  • “Diagnosing” is defined herein to include screening for a disease/condition or pre-indication of a disease/condition, identifying a disease/condition or pre-indication of a disease/condition and checking for recurrence of disease/condition following treatment.
  • the methods of the various embodiments of the invention may also have prognostic value, and this is included within the definition of the term "diagnosis”.
  • the prognostic value of the methods of the various embodiments of the invention may be used as a marker of potential susceptibility to primary sclerosing cholangitis by identifying levels of CCR9, CXCR4, CCR7 and/or CCR5 expression linked to conditions associated with that syndrome.
  • CCR9, CXCR4, CCR7 and/or CCR5 expression linked to conditions associated with that syndrome Thus patients at risk may be identified before the disease has a chance to manifest itself in terms of symptoms identifiable in the patient.
  • diagnosis may be made in conjunction with other objective indicators of PSC.
  • diagnosis is made in conjunction with the following indicators:
  • Diagnosis is made clinically with associated ALT, AST, ALP, bilirubin and patognomonic ERCP picture.
  • identification of circulating biliary tract homing proinflammatory cells cam be monitored for chemokine receptor expression.
  • Monitoring progression of includes performing the methods to monitor the stage and/or the state and progression of the primary sclerosing cholangitis. Monitoring progression may involve performing the diagnostic methods multiple times on the same patient to determine whether the levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells are increasing, decreasing or remaining stable over a certain time period. This may be in the context of a treatment regime.
  • “Monitoring the success of a particular treatment” is defined to include determining the levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells before and after a treatment.
  • the cells may be monocytes, in particular CCR9 expressing and HLA-DR hl monocytes as described herein.
  • the treatment is generally one aimed at treating primary sclerosing cholangitis and may be a treatment according to one of the methods of the various embodiments of the invention as defined herein.
  • Successful treatment may be determined with reference to a decrease in one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells as a result of, or following, the treatment.
  • a level of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells is determined prior to treatment. This level is recorded and a further assessment made at a predetermined time following the treatment. The comparison of levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells permits the success of the treatment to be monitored.
  • a single treatment is sufficient to cause a depletion of around 10%, 20%, 30%, 40%, 50%, 60% or 70%, or higher, up to 80%, 90%, 95% or more, or any range of values between and including these amounts, of one or more specific chemokine receptors, in particular CCR9, CXCR4, CCR7 and/or CCR5, expressing cells from the peripheral blood of the patient.
  • one or more specific chemokine receptors in particular CCR9, CXCR4, CCR7 and/or CCR5, expressing cells from the peripheral blood of the patient.
  • at least around 50% depletion is achieved in a single treatment.
  • successful treatment may be defined with reference to depletion of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells.
  • Treatment may lead to depletion of between approximately 100 and 500 million of one of more of CCR9, CXCR4, CCR7 and CCR5 expressing cells, such as monocytes, in certain embodiments. Additional factors may be included to determine successful treatment. For example, a lack of increase in one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells following treatment may indicate successful treatment in terms of preventing further progression of the condition, optionally combined with an improvement in other markers or staging of the primary sclerosing cholangitis.
  • chemokine receptor expressing cells By binding to the column through the binding reagent-chemokine receptor interaction, chemokine receptor expressing cells are immobilized. These immobilized cells express further unoccupied chemokine receptors, which may be of the same or different type to those used for capture. These additional chemokine receptors may permit circulating chemokines which contribute to the inflammatory condition to be captured from the peripheral blood. Thus, a reduction in circulating (specific) chemokine levels may provide a measure of successful treatment.
  • the sample in which one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cell levels, levels of expression of one or more of CCR9, CXCR4, CCR7 and CCR5 and/or levels of cells with high expression of one or more of CCR9, CXCR4, CCR7 and CCR5 may comprise any suitable tissue sample or body fluid sample.
  • the test sample is obtained from a human subject.
  • the sample is a blood sample, in particular a peripheral blood sample.
  • the sample may comprise a liver sample, such as a liver biopsy in certain embodiments.
  • the methods may involve determining levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing monocytes, macrophages or lymphocytes in certain embodiments.
  • Levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 may be determined according to any suitable method.
  • flow cytometry may be employed in order to determine the number of cells expressing CCR9, CXCR4, CCR7 and/or CCR5 in the sample, to determine levels of CCR9, CXCR4, CCR7 and/or CCR5 expression and/or to identify levels of CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi cells.
  • Flow cytometric techniques are described herein and examples of commercially available antibodies suitably labelled for use in flow cytometry are set out in Table 1 for example.
  • the method may involve steps of collecting and fixing the cells in the sample, followed by incubation with a suitable binding reagent that binds specifically to the CCR9, CXCR4, CCR7 and/or CCR5 chemokine receptor expressing cells in the sample.
  • a suitable binding reagent as defined herein, may be employed.
  • a CCR9, CXCR4, CCR7 and/or CCR5 specific antibody may be employed.
  • a wash step may be adopted following an incubation period to remove any unbound reagent. Suitable wash steps and incubation conditions would be well known to one skilled in the art.
  • the binding reagent may be directly labeled in order to permit antibody binding to be directly determined.
  • a secondary binding reagent such as an antibody
  • the primary and secondary binding reagents may form two halves of a binding pair.
  • the binding interaction should not prevent the primary binding reagent binding to the CCR9, CXCR4, CCR7 and/or CCR5 receptor expressing cells, unless a competition assay is being employed.
  • the two halves of a binding pair may comprise an antigen-antibody, antibody- antibody, receptor-ligand, biotin-streptavidin pair etc. in certain embodiments.
  • CCR9, CXCR4, CCR7 and/or CCR5 Other techniques used to quantify chemokine (CCR9, CXCR4, CCR7 and/or CCR5) receptor expressing cell levels, to quantify levels of CCR9, CXCR4, CCR7 and/or CCR5 expression and/or to quantify levels of CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi cells include PCR- based techniques such as QT-PCR and protein based methods such as western blot.
  • Quantitation may be achieved with reference to fixed cell lines carrying known numbers of various receptor expressing cells and/or known levels of receptor expression per cell.
  • Such fixed cell lines are available commercially (for example ChemiScreenTM cell lines from Millipore). Methods analogous to the treatment methods of the various embodiments of the invention may also be employed, with binding of CCR expressing cells to the solid support being determined following peripheral blood being passed through the column.
  • the levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 may be determined relative to a suitable control.
  • a threshold level of cells, level of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or level of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 may be set at or over which a positive diagnosis is made.
  • This threshold may be determined based upon measuring levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 (defined as CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi ) in samples obtained from diseased patients and comparing these levels with levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 (defined as CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hl ) in samples obtained from healthy subjects.
  • primary sclerosing cholangitis is diagnosed on the basis of levels of chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells.
  • a positive diagnosis may be made in subjects based upon the presence of greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or more chemokine receptor expressing cells in the sample, as a percentage of total cells in the sample.
  • primary sclerosing cholangitis is diagnosed on the basis of the presence of a about a 1 .2 fold or greater increase, such as about a 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative to healthy controls.
  • a 1 .2 fold or greater increase such as about a 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative to healthy controls.
  • primary sclerosing cholangitis is diagnosed on the basis of levels of CCR9 expressing cells, in particular monocytes.
  • a positive diagnosis may be made in subjects based upon the presence of greater than about 8%, greater than about 9%, greater than about 10%, greater than about 12% or greater than about 15% CCR9 expressing cells, in particular monocytes in the sample, as a percentage of total cells in the sample.
  • Primary sclerosing cholangitis may also be diagnosed on the basis of the presence of a about a 1 .2 fold or greater increase, such as about a 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in the CCR9 expressing cells, in particular monocytes, relative to healthy controls.
  • progression of primary sclerosing cholangitis is monitored on the basis of levels of chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells at different time points.
  • chemokine receptor expressing cells such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells at different time points.
  • Progression of primary sclerosing cholangitis may be indicated in subjects based upon an increase of greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, or greater than about 10%, such as an increase of greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75% or more chemokine receptor expressing cells in the sample, as a percentage of total cells in the sample, compared to a sample taken from the same subject at an earlier time point.
  • progression of primary sclerosing cholangitis is confirmed on the basis of the presence of a about a 1 .2 fold or greater increase, such as about a 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in chemokine receptor expressing cells , such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative to a sample taken from the same subject at an earlier time point.
  • chemokine receptor expressing cells such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells
  • primary sclerosing cholangitis is monitored on the basis of levels of CCR9 expressing cells, in particular monocytes.
  • Progression of the disease which may be in the context of a treatment regime, may be indicated in subjects based upon the presence of an increase of greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, or greater than about 10% or more chemokine receptor expressing cells in the sample, as a percentage of total cells in the sample, compared to a sample taken from the same subject at an earlier time point.
  • progression of primary sclerosing cholangitis is confirmed on the basis of the presence of a about a 1 .2 fold or greater increase, such as about a 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in CCR9 expressing cells, in particular monocytes, relative to a sample taken from the same subject at an earlier time point.
  • Regression or successful treatment may be monitored based upon similar decreases over various time points.
  • regression or successful treatment may be indicated in subjects based upon a decrease of greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, or about 10%, such as a decrease of about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more chemokine receptor expressing cells in the sample, as a percentage of total cells in the sample, compared to a sample taken from the same subject at an earlier time point.
  • regression of primary sclerosing cholangitis is confirmed on the basis of the presence of a about a 1 .2 fold or greater decrease, such as about a 1 .5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold decrease in chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative to a sample taken from the same subject at an earlier time point.
  • chemokine receptor expressing cells such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells
  • primary sclerosing cholangitis is monitored on the basis of levels of CCR9 expressing cells, in particular monocytes.
  • Regression or successful treatment of the disease may be made in subjects based upon a decrease of greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, or about 10%, such as a decrease of about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75% or more CCR9 expressing cells, in particular monocytes in the sample, as a percentage of total cells in the sample or by a decrease of about 2%, such as a decrease of about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35% or more CCR9 expressing cells, in particular monocytes in the sample, as
  • Suitable software is freely available (such as the R project for statistical computing) to perform the necessary statistical analysis of the data obtained to calculate a useful threshold.
  • the threshold may be set to maximize sensitivity and/or specificity of the test. Performance of the test in these respects may be measured by plotting a receiver operating characteristics (ROC) curve (sensitivity versus specificity). The area under the curve provides an indication of the overall performance of the test.
  • ROC receiver operating characteristics
  • the test sample is generally tested in parallel with the control sample.
  • the test sample level of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 (defined as CCR9 hi , CXCR4 hl , CCR7 hl and/or CCR5 hl ) can then be compared with that of the control sample to make the diagnosis.
  • a control sample from a disease patient may also be tested in certain embodiments.
  • Reference to controls permits relative levels ("high”, “low” etc.) of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells in the test sample to be readily identified and the significance thereof interpreted. Reference to controls also permits relative levels of CCR9, CXCR4, CCR7 and/or CCR5 expression ("high”, “low” etc.) within the cell population to be determined and the significance thereof interpreted. Such determination may, for example, indicate the average levels of CCR9, CXCR4, CCR7 and/or CCR5 expression per cell in the test sample.
  • high or higher levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells or high or higher levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expression for example average CCR9, CXCR4, CCR7 and/or CCR5 expression per cell, or high or higher levels of one or more of CCR9 hi , CXCR4 hi , CCR7 hi and CCR5 hi cells correlate with active disease or more active primary sclerosing cholangitis.
  • lower or low levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells, or low or lower levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expression, for example average CCR9, CXCR4, CCR7 and/or CCR5 expression per cell, or low or lower levels of one or more of CCR9 hi , CXCR4 hi , CCR7 hi and hi CCR5 hi cells may correlate with a lack of active inflammation or primary sclerosing cholangitis. This may be defined as "less active disease”.
  • control samples may be assessed and levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 (defined as CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi ) determined across the range of severities of primary sclerosing cholangitis.
  • This may assist in correlating the levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 (defined as CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi ) in the test sample with the relative severity of the condition.
  • control samples may be taken from the subject at an earlier time point. They may, however, be based upon known reference values as discussed above. Thus, relative levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative levels of CCR9, CXCR4, CCR7 and/or CCR5 expression including relative levels of average CCR9, CXCR4, CCR7 and/or CCR5 expression per cell or relative levels of CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hl cells may be with reference to samples taken from the same subject at a different point in time.
  • decreased levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells decreased relative levels of CCR9, CXCR4, CCR7 and/or CCR5 expression including decreased relative levels of average CCR9, CXCR4, CCR7 and/or
  • CCR5 expression per cell or decreased relative levels of CCR9 hi , CXCR4 hi , CCR7 hi and/or hl CCR5 hl cells correlate with successful treatment.
  • the treatment may be any suitable treatment, but in specific embodiments is a treatment according to the various embodiments of the invention.
  • increased levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells increased relative levels of CCR9, CXCR4, CCR7 and/or CCR5 expression including increased relative levels of average CCR9, CXCR4, CCR7 and/or CCR5 expression per cell or increased relative levels of CCR9, CXCR4, CCR7 and/or CCR5hi cells may indicate the progression of condition or disease.
  • levels of CCR9, CXCR4, CCR7 and/or CCR5 expressing cells levels of expression of CCR9, CXCR4, CCR7 and/or CCR5 and/or levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 (defined as CCR9 hi , CXCR4 hi , CCR7 hi and/or CCR5 hi ) are increased in a sample taken later than a sample from the same patient this may indicate progression of the condition.
  • the invention provides a method of selecting a suitable treatment for primary sclerosing cholangitis comprising determining:
  • CCR7 and CCR5 expressing cells high levels of expression of one or more of CCR9, CXCR4, CCR7 and CCR5 or high levels of cells with high expression of CCR9, CXCR4, CCR7 and/or CCR5 or increased levels of one or more of CCR9, CXCR4, CCR7 and CCR5 expressing cells compared to control, increased levels of expression of one or more of CCR9, CXCR4, CCR7 and CCR5 compared to a control or increased levels of cells with high expression of one or more of CCR9, CXCR4, CCR7 and CCR5 compared to a control, result in selection of a treatment as defined herein for treatment of the primary sclerosing cholangitis.
  • the chemokine receptor expressing cells are high chemokine receptor expressing cells, in particular, high CCR9, CXCR4, CCR7 and/or CCR5 expressing cells.
  • primary sclerosing cholangitis is treated on the basis of measuring levels of chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells.
  • chemokine receptor expressing cells such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells.
  • a treatment according to the various embodiments of the invention may be applied based upon the presence of greater than about 10%, greater than about
  • chemokine receptor expressing cells in the sample, as a percentage of total cells in the sample.
  • primary sclerosing cholangitis is treated according to the various embodiments of the invention on the basis of the presence of a about a 1 .5 fold or greater increase, such as about a 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative to healthy controls.
  • a about a 1 .5 fold or greater increase such as about a 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in chemokine receptor expressing cells, such as CCR9, CXCR4, CCR7 and/or CCR5 expressing cells, relative to healthy controls.
  • primary sclerosing cholangitis is treated on the basis of measuring levels of CCR9 expressing cells, in particular monocytes.
  • a treatment according to the various embodiments of the invention may be applied based upon the presence of greater than about 2%, greater than about 3%, greater than about 4%, greater than about 5%, greater than about 6%, greater than about 7%, greater than about 10%, greater than about 12% or greater than about 15% or more CCR9 expressing cells, in particular monocytes in the sample, as a percentage of total cells in the sample or on the basis of the presence of a about a 1 .5 fold or greater increase, such as about a 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50 or 100 or greater fold increase in CCR9 expressing cells, in particular monocytes, relative to healthy controls.
  • Diagnosis is made clinically with associated ALT, AST, ALP, bilirubin and patognomonic ERCP picture.
  • identification of circulating biliary tract homing proinflammatory cells cam be monitored for chemokine receptor expression
  • CCR9, CXCR4, CCR7 and CCR5 expressing cells such as monocytes, macrophages and lymphocytes, in particular monocytes, upregulated in various inflammatory conditions associated with primary sclerosing cholangitis, an important factor in the inflammatory process of primary sclerosing cholangitis can be controlled.
  • FIG. 1 a, 1 b & 1 c the binding of biotinylated CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor;
  • FIG. 2a, 2b & 2c the binding of biotinylized CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a patient with CD;
  • FIG. 3 The plastic house and top showing the distribution plate (2) and safety filter units (3 and 4).
  • FIG. 4 The overall leukapheresis system.
  • FIG. 5 - The pump with air detector and optical detector (4).
  • FIG. 6 Depletion of CCR9 -expressing cell populations in one blood donor. Total cell populations are unaffected after the column passage.
  • FIG. 7 Depletion of CCR9-expressing cell populations in one IBD patient. Total cell populations are unaffected after the column passage
  • FIG. 8 Sequence and biotinylation of RANTES derivative
  • FIG. 9 Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.
  • FIG. 10 HPLC of purified folded Biotin-TECK (Nleu)
  • FIG. 1 Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu).
  • FIG. 12 Example of gating criteria for CCR2 expressing monocytes
  • FIG. 13a Expression of CCR9 expressing monocytes in 4 patients with Primary sclerosing cholangitis (PSC) and in 20 healthy controls (HC).
  • PSC Primary sclerosing cholangitis
  • HC healthy controls
  • FIG.13b Expression of CCR9 in activated (HLADR hl ) monocytes in 4 patients with Primary sclerosing cholangitis (PSC) and in 20 healthy controls (HC) .
  • PSC Primary sclerosing cholangitis
  • HC healthy controls
  • FIG. 14 Binding of the chemokine bTECK (CCL25) to monocytes from a patient with Primary sclerosing cholangitis. Blood from a patient with PSC was incubated with bTECK and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.
  • FIG. 15a Depletion of CCR9 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bTECK.
  • FIG. 15b Depletion of activated CCR9 expressing monocytes with Sepharose Streptavidin- matrix conjugated with bTECK.
  • Blood cells from a patient with PSC were incubated with bTECK- Sepharose Streptavidin- matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bTECK-matrix (Before Depletion).
  • CCL25, CXCL12, CCL21 or CCL5 mediates recruitment of CCR9, CXCR4, CCR7 and/or CCR5+ lymphocytes to the liver.
  • CCR9, CXCR4, CCR7 and/or CCR5+ lymphocytes have been activated in the gut and homing to the liver might contribute to the strong correlation between IBD and PSC. (Eksteen B 2004)
  • Neolymphoid tissue develops in the liver in patients with PSC, contributing to the
  • the inventors have also determined that PSC patients include higher levels of CCR9 expressing monocytes in the blood.
  • the disease relevant monocytes may also have high expression of HLA-DR (referred to herein as HLA-DR hl ).
  • the treatments and diagnostic methods herein may accordingly be applicable in particular to monocytes and to HLA-DR hl monocytes.
  • peripheral blood leukocytes Heparinized peripheral blood from healthy blood donors or IBD patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83 % ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.
  • Chemokines The leukocytes were incubated for 30 min in the dark at 4 Q C with the following biotinylated and Alexa647 Fluor® labeled chemokines: CCL25 (in concentrations of
  • the flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San Jose, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.
  • EXAMPLE 1 Affinity of blood cells to CCL25.
  • T-cells CD4+ lymphocytes; CD8+ lymphocytes
  • monocytes CD14+ monocytes
  • EXAMPLE 2 Preparation of a chemokine column for blood cell apheresis.
  • streptavidin cross-linked agarose ProZyme, San Leandro, CA, U.S.A.
  • beads in the range from 75 ⁇ to 300 ⁇ suspended (200 ml, -50 %, v/v) in an aqueous solution of 25 mM sodium phosphate (pH 7.0) and 150 mM NaCI was added a solution of 75 ⁇ g biotinylated MIP-1 a (Almac Sciences) in the same buffer at 22 °C and slowly stirred by hand for 3 min. After standing for another 20 min, the support was filtered off, washed thrice with neutral aqueous sodium phosphate/sodium chloride and filled into a glass column (i.d. 25 mm, length 12 cm).
  • EXAMPLE 3 Separation of monocytes from peripheral blood of a healthy donor with the chemokine column of Example 2. Heparinized peripheral blood from a healthy male donor was analyzed by flow cytometry for CD4+ lymphocytes, CD8+ lymphocytes and CD14 monocytes. 100 ml of the blood was filtered through the column at a rate of about 8 ml per min and washed with FACS buffer. The filtered blood was analyzed for the same cells. It was found that about 95 % of the monocytes had been retained by the column whereas more than 90 % each of CD4+ and CD8+ lymphocytes had been recovered. EXAMPLE 4 - Tailored leukapheresis
  • Apheresis is an established treatment used for depletion of blood components, such as antibodies, low-density lipoproteins (LDL) and blood cells.
  • LDL low-density lipoproteins
  • Leukapheresis is the apheresis treatment used for removal of white blood cells, leukocytes.
  • the patient is connected to an extracorporeal blood circulating system; the blood is drawn from a vein in one arm, passed through a column device and returned into the other arm of the patient.
  • Side effects of leukapheresis treatments are varying from mild events like headache, dizziness, hypotension, palpitation and flush seen in 0.1 to 5% of treated patients.
  • the column is intended to be used as a leukapheresis treatment for primary sclerosing cholangitis. It will specifically remove CCR9 -expressing gut-homing leukocytes through the use of a bTECK containing resin, exploiting the CCR9 -TECK interaction.
  • the column consists of three combined components, the plastic house, the streptavidin (SA)
  • SepharoseTM BigBeads matrix and bTECK bound to the matrix The treatment is conducted using the same techniques as a standard apheresis procedure.
  • the plastic house (FIG. 3)
  • the plastic house designed to keep a continuous blood flow through the matrix, consists of a transparent body and red-coloured top.
  • the top has a distribution plate (2) at the inflow site (1 ) to spread the blood evenly over the entire matrix area.
  • the plate is the first safety barrier preventing larger particles flowing through the column and into the patient.
  • Safety filter units (3 and 4) are placed at the inflow (1 ) and outflow (5) sites of the plastic housing.
  • the safety filter unit contains three filters designed to be a robust barrier and stop all particles larger than blood cells passing through the column.
  • the plastic housing design is shown in Figure 3.
  • the design with safety filters (3 and 4) at both ends of the column device will minimize the risk of leakage of particles into the patient, including in the event that the device is placed up side down with the blood flow in the opposite direction to that anticipated.
  • the second component in the device is the affinity matrix called streptavidin SepharoseTM BigBeads (SepharoseTM GE Healthcare, Sweden).
  • SepharoseTM is a cross linked, beaded- form of agarose, which is a polysaccharide extracted from seaweed.
  • SepharoseTM and agarose are commonly used as column matrices in biomedical affinity techniques. It is chosen for its optimal distribution capacity and can provide a large available area for affinity binding.
  • the bTECK Coupled to the matrix is the third component of the device, the bTECK.
  • This bTECK peptide is a synthetic, engineered version of the human chemokine TECK, which is truncated and biotinylated, but retains its binding activity to the TECK receptor CCR9.
  • biotinylating the engineered TECK it is able to bind to the streptavidin molecules in the SepharoseTM matrix.
  • the biotin-streptavidin binding is known be one of the strongest biological interactions with a Kd in the order of 4 x 10 "14 M.
  • the calculated ratio of streptavidin :biotin binding sites in the column is 10:1 . Therefore, the coupling between the matrix and bTECK will be immediate, minimising the risk of bTECK decoupling from the matrix.
  • the apheresis system To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).
  • the system is illustrated in Figure 4.
  • the patient (1 ) is connected to the extracorporeal circuit via sterile Venflon needles to veins in the right and the left arms.
  • a saline bag (3) is also connected and the saline solution is pumped with an ACD pump (2).
  • Blood is drawn from one arm of the patient through the sterile tubing system by the blood pump (4) and passed through the column (6) and back to the patient.
  • the tubing system is connected to the column via standard dialysis luer-lock couplings.
  • the couplings on the column are colour- coded for correct assembly; red tubing for inflow to the red column top and blue tubing for outflow back to the patient.
  • An air detector (8) is present.
  • Inlet pressure(5) and Pven sensors (7) are employed to monitor the pressure in the circuit.
  • An apheresis pump monitors the patient's inflow and outflow, the pressure in the extracorporeal circulation and can discriminate air by a bubble catcher and air detector.
  • a clot catcher filter is placed inside the bubble catcher.
  • the pump also has an optical detector to distinguish between light, e.g. saline solution or air present in the tubing system and dark e.g. blood present in the tubing system.
  • FIG. 5 A schematic diagram of the pump, showing the air detector and optical filter is shown in Figure 5. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/ audible alarm are emitted.
  • Modules (left to right - Blood pump, ACD pump, Air detector)
  • the patient will be administered anticoagulants prior to each treatment session.
  • a sterile saline solution with 5000 IE Heparin will be used for priming the extracorporeal system, thereafter a bolus injection with 4000 IE Heparin will be added into the circuit at the start of each treatment session.
  • Leukapheresis time and flow rate 5000 IE Heparin will be used for priming the extracorporeal system, thereafter a bolus injection with 4000 IE Heparin will be added into the circuit at the start of each treatment session.
  • the apheresis system should be operated at a flow rate of 30-60 imL/min. A treatment is finalised after 1800mL of blood has been circulated. Storage conditions
  • the column devices should be stored between 1 and 25 °C avoiding freezing and more elevated temperatures. Stability data > 3 months indicate no difference in functionality over time or by temperature (room temperature and refrigerated). The columns will be kept in refrigerated conditions until use. Mechanical damage as those resulting from violent vibrations and trauma should be avoided. Column stored outside of these recommendations should not be used.
  • the column devices will be transported under refrigerated condition, avoiding freezing and more elevated temperatures. Mechanical damage such as those resulting from violent vibrations and trauma should be avoided.
  • This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.
  • the side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in figure 8.
  • the protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.
  • the results demonstrate significant depletion of the target population chemokine receptor- expressing cells post matrix perfusion. Depletion tests were performed on blood from a healthy donor. The results are shown in Figure 9.
  • TECK Metal to Nleu substitution
  • Truncated form of human TECK corresponding to residues 1 -74 of the mature protein, which encompasses the sequence corresponding to the chemokine fold.
  • the full length mature protein is 127 amino acids (the signal peptide is 23 amino acids in a 150 amino acid immature protein).
  • the single methionine within the sequence was altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly, which was observed during the synthesis of the natural sequence derivative.
  • the Gin at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gin and pyroGlu being generated.
  • the linear amino acid sequence (SEQ ID NO: 1 ) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):
  • X1 pyroGlu or Gin
  • the engineered TECK sequence was assembled on a solid support, using Fmoc protocols for solid-phase peptide synthesis (SEQ ID NO: 3):
  • XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRHRKVCGNPKSREVQ RAXKLLDARNXVF-RESIN X1 pyroGlu or Gin
  • FmocLys(Dde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein.
  • the Dde protecting group was removed by treatment of all resin (2.5g) with a solution of 2% hydrazine in DMF (100ml) over 1 hour period to afford 2.0g resin.
  • Resin (1 .5g) was swollen in DMF (2ml) and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38g, 1 mmol), DIC solution (2ml, 0.5M in DMF) and HOCt solution (2ml, 0.5M in DMF) was added. The mixture was sonicated for 2 hours and then washed with DMF.
  • the resin was capped with 0.5M acetic anhydride/DMF solution (20ml) for 5 minutes and then washed with DMF.
  • Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2 x 50ml) for 15 minutes each. The resin was washed with DMF.
  • the folded protein was purified by reverse phase HPLC using a Jupiter C18, 250x21 mm column, 9ml/min, 10-60%B over 50 minutes. 1 1 .1 mg of pure folded Nle-TECK-Biotin was afforded.
  • Figure 10 shows HPLC of purified folded Biotin-TECK(Nleu). The protein eluted in a single peak at 21 .6 mins.
  • Figure 1 1 shows Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu). The expected mass was 8959.4 Da.
  • TECK-Biotin-Nle was tested for agonist activity in an Aequorin assay against hCCR9, (Euroscreen) and an EC50 value of 63.6nM was reported, c.f. EC50 for native TECK is 67.87nM.
  • the final active chemokine thus has the following sequence (SEQ ID NO: 4):
  • X1 pyroGlu or Gin
  • X72 an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, such as K(PEG-Biotin)
  • chemokines Chemical synthesis of chemokines was performed using standard Fmoc solid phase peptides synthesis (SPPS) techniques on an ABI 433 peptide synthesiser. DIC (0.5 M in DMF) and OxymaPure (0.5 M in DMF) were used for activation, acetic anhydride (0.5 M in DMF) for capping, and 20 % piperidine in DMF for Fmoc deprotection. Rink Amide resin was utilised for the generation of C-terminal amide chemokines and Wang resin for C-terminal acid chemokines. After assembly, the resin was washed with DMF and DCM and then dried in vacuo.
  • SPPS Fmoc solid phase peptides synthesis
  • the Dde protecting group was removed by treatment of resin with a solution of 2.5% hydrazine in DMF (200ml) over a 2 hour period. The resin was then washed with DMF.
  • Couple Fmoc-8-amino-3,6-dioctanoic acid (PEG) Resin was swollen in DMF and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38g, 1 mmol), DIC solution (2ml, 0.5 M in DMF) and OxymaPure solution (2ml, 0.5 M in DMF) was added. The mixture was sonicated for 3 hours and then washed with DMF.
  • the resin was capped with acetic anhydride solution (0.5 M in DMF, 10ml) for 5 minutes and then washed with DMF.
  • Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2 x 50ml) for 15 minutes each. The resin was washed with DMF.
  • Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ⁇ ), thioanisole (500 ⁇ ), water (500 ⁇ ), DMS (500 ⁇ ), EDT (250 ⁇ ), NH 4 I (500 ⁇ g) and phenol (500 ⁇ g) and the mixture was stirred at room temperature for 5 hours.
  • the solution was filtered into cold ether and the resin rinsed with TFA.
  • the precipitated peptide was centrifuged, washed with ether, centrifuged and lyophilised.
  • the crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250 x 21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1 % TFA, Buffer B: acetonitrile containing 0.1 % TFA].
  • Pure peptide (10mg) was dissolved into 6M GnHCI (16 ml) and then rapidly diluted to 2M GnHCI concentration by the addition of 50mM TRIS pH 8.5 (84 ml) containing 0.3mM GSSG and 3mM GSH. The mixture was stirred at room temperature for 24 hours and then analysed by RP-HPLC (Jupiter C18, 250 x 4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.
  • Target Molecule MCP-2 derivatised at the ⁇ -amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)
  • Human MCP-2 corresponding to residues 1 -76 is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off.
  • the Gin at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions.
  • Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gin and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use.
  • the naturally occurring lysine at position 75 was modified through biotinylation on the resin.
  • a PEG spacer was incorporated between the ⁇ -amino functionality and the biotin.
  • the linear amino acid sequence (SEQ ID NO: 5) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):
  • the engineered MCP-2 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:
  • X1 pyroGlu or Gin
  • FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 6). Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine (SEQ ID NO: 7):
  • X1 pyroGlu or Gin
  • X75 an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer
  • PEG e.g. PEG-Biotin
  • Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained 9263.6 Da; expected 9263.8 Da.
  • biotinMCP-2 was tested for activity in an Aequorin assay against hCCR2b, (Euroscreen) and was shown to be a partial agonist with an EC50 value of 50.9nM. c.f . EC50 for recombinant native MCP-2 is 23.5nM (partial agonist).
  • Example 8 biotinRANTES (CCL5)
  • Target Molecule RANTES derivatised at the ⁇ -amino side chain functionality of Lys(67) with Biotin (TFA salt)
  • Human RANTES corresponding to residues 1 -68 is initially expressed as 91 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off.
  • the single methionine (Met67) within the sequence was mutated to lysine, to mitigate against oxidation of this residue during the chain assembly, which was observed during the synthesis of the natural sequence derivative.
  • This Met to Lys substitution provided a lysine at position 67 which was modified through biotinylation on the resin.
  • the linear amino acid sequence (SEQ ID NO: 8) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):
  • the engineered RANTES sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:
  • X is K(ivDde) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 9). Subsequent removal of the ivDde protecting group, followed by coupling of the Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine (SEQ ID NO: 10):
  • X is an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG (e.g. K(Biotin))
  • Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained 8068.9 Da; expected 8070.2 Da. Functional Assay Data:
  • BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5nM was reported.
  • Target Molecule SDF-1 a derivatised at the ⁇ -amino side chain functionality of Lys(64) with Biotin (TFA salt) Modifications: Truncated form of human SDF-1 a corresponding to residues 1 -67 of the mature protein, which encompasses the sequence corresponding to the chemokine fold. The full length mature protein is 72 amino acids (the signal peptide is 21 amino acids in a 93 amino acid immature protein). The naturally occurring lysine at position 64 was modified through biotinylation on the resin.
  • the linear amino acid sequence (SEQ ID NO: 1 1 ) is shown, prior to attachment of the biotin molecule at amino acid 64 (K):
  • X K(ivDde) FmocLys(ivDde)-OH was incorporated as residue 64 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 12). Subsequent removal of the ivDde protecting group, followed by coupling of the Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine (SEQ ID NO: 13):
  • X is an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin)
  • Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinSDF-1 a: obtained 8055.5 Da; expected 8057.5 Da.
  • biotinSDF-1 a was tested for agonist activity in an Aequorin assay against hCXCR4, (Euroscreen) and an EC50 value of 17.3nM was reported, c.f. EC50 for recombinant native SDF-1 a is 12.0nM.
  • Target Molecule ⁇ -3 ⁇ derivatised at the ⁇ -amino side chain functionality of Lys(78) with Biotin (TFA salt)
  • the linear amino acid sequence (SEQ ID NO: 14) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):
  • the engineered ⁇ -3 ⁇ sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:
  • X is K(ivDde) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 15). Subsequent removal of the ivDde protecting group, followed by coupling of the Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine (SEQ ID NO: 16):
  • X is an amino acid residue that can be biotinylated, such as lysine, ornithine or
  • diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, e.g. K(PEG-Biotin)
  • Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMIP-3 : obtained 9148.8 Da; expected 9149.7 Da. Functional Assay Data:
  • biotinMip-3 was tested for agonist activity in an Aequorin assay against hCCR7,
  • Peripheral blood from patients and healthy controls was collected in heparin tubes.
  • the red blood cells were lysed using Fix Buffer (Phosphate Buffer Saline (PBS) citrate with 4% paraformaldehyde) for four minutes at 37°C and Lysing buffer (PBS with 10mM Tris and 160mM NH 4 CI, pH 7.5) for 15 min at 37 ⁇ ⁇ .
  • the cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum for 15min at room temperature (RT) and stained with antibodies (Table 2) at 4 ⁇ ⁇ for 30min.
  • the cells were analysed by flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences).
  • Peripheral blood from patients and healthy controls was collected in heparin tubes.
  • the red blood cells were lysed using Fix Buffer (Phosphate Buffer Saline (PBS) citrate with 4% paraformaldehyde) for four minutes at 37°C and Lysing buffer (PBS with 10mM Tris and 160mM NH4CI, pH 7.5) for 15 min at 37 ⁇ ⁇ .
  • the cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum 15min at room temperature (RT) and stained with cell specific antibodies together with biotinylated chemokine (1 ⁇ ) or the corresponding chemokine receptor antibody at 4°C for 30min (Table 2).
  • biotinylated chemokine was detected via the interaction between biotin and a fluorophore conjugated Streptavidin.
  • the samples were analysed by flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences). 3. Cell depletion by matrix conjugated with biotinylated chemokine
  • Cells were prepared from peripheral blood (section 1 ). 1 imL Sepharose BigBeads matrix conjugated with 0.4mg/ml_ Streptavidin (GE Healthcare) was washed in 50 imL PBS and added to a 5 imL polystyrene tube (BD FalconTM). Biotinylated chemokine (1 ⁇ ) was added to the tube and incubated for 20 min at RT to enable immobilization of the chemokine on the matrix via the biotin-streptavidin interaction. Next, the cells were added to the chemokine- matrix and incubated for 20 min at RT. The cells that did not bind to the matrix were removed by washing the matrix with PBS in a sterile 40um nylon filter (BD FalconTM Cell Strainer). The flow through cells were stained with antibodies (Table 2), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine- matrix.
  • Table 2 The flow through cells were stained with antibodies (Table 2), analysed by
  • CCR9 binds to the chemokine TECK (CCL25) which is mainly expressed in the gut but is also reported to be upregulated in PSC liver. 27% of the monocytes bind to the biotinylated TECK (bTECK) (Fig 14). 3. Cell depletion by matrix conjugated with biotinylated chemokine
  • monocytes in PSC express CCR9 and bind the ligand bTECK.
  • CCR9 expressing monocytes can be removed with Sepharose Streptavidin matrix conjugated with bTECK.

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Abstract

L'invention concerne une méthode de traitement de l'angiocholite sclérosante primitive consistant à appliquer du sang périphérique d'un patient ou sujet sur une colonne d'aphérèse chargée d'un support solide comprenant un ou plusieurs réactifs de liaison pouvant se lier spécifiquement à un récepteur de chimiokines, éventuellement le récepteur de chimiokines CCR9, CXCR4, CCR7 ou CCR5, immobilisé directement ou indirectement sur le support, éliminant ainsi au moins un récepteur de chimiokines, éventuellement des cellules exprimant CCR9, CXCR4, CCR7 et CCR5 à partir du sang périphérique du patient ou sujet. L'invention concerne en outre diverses méthodes thérapeutiques associées, ainsi que des réactifs de liaison utiles.
PCT/GB2012/051353 2011-06-13 2012-06-13 Traitement de l'angiocholite sclérosante primitive WO2012172343A2 (fr)

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