Treating conditions associated with metabolic syndrome

Abstract

A method for treating a condition associated with metabolic syndrome or for treating adiposis dolorosa (AD) comprises applying peripheral blood from a patient or subject to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to a chemokine receptor, optionally the chemokine receptor CCR2, CCR1, CCR3, CCR4 or CCR5 immobilized directly or indirectly on the support thus removing one or more chemokine receptor, optionally CCR2, CCR1, CCR3, CCR4 and CCR5 expressing cells from the peripheral blood of the patient or subject. Various companion diagnostic methods and useful binding reagents are also described.

Claims

1. A method for treating a condition associated with metabolic syndrome or for treating adiposis dolorosa (AD) in a subject in need thereof, the method comprising applying peripheral blood from the subject to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to a chemokine receptor CCR2 immobilized directly or indirectly on the support, whereby one or more cells expressing chemokine receptor CCR2 are removed from the peripheral blood of the subject, wherein the applied blood is recirculated back into the systemic circulation of the subject, whereby the condition associated with metabolic syndrome or adiposis dolorosa (AD) is treated.

2. The method of claim 1 wherein the condition associated with metabolic syndrome is selected from diabetes, obesity, insulin resistance, increased serum triacylglycerol concentrations, and hypertension.

3. The method of claim 1, wherein the binding reagent is an agonist or an antagonist of CCR2.

4. The method of claim 1, wherein the binding reagent is an antibody or a chemokine.

5. The method of claim 4, wherein the chemokine is selected from MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5.

6. The method of claim 5, wherein the chemokine is MCP-1 or MCP-5.

7. The method of claim 1, wherein the one or more cells are CCR2-expressing monocytes, or CCR2-expressing B lymphocytes.

8. The method of claim 1, wherein the subject has increased levels of expression of CCR2 as compared to a subject that does not have a condition associated with metabolic syndrome or adiposis dolorosa (AD).

9. The method of claim 1, wherein 20-50% of the subject's blood is applied to the column in a single treatment.

Description

DESCRIPTION OF THE FIGURES

(1) FIGS. 1a, 1b & 1cthe binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(2) FIG. 2abinding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(3) FIG. 2bbinding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(4) FIG. 3The plastic house and top showing the distribution plate (2) and safety filter units (3 and 4).

(5) FIG. 4The overall leukapheresis system

(6) FIG. 5The pump with air detector and optical detector (4).

(7) FIG. 6aResults of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(8) FIG. 6bResults 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.

(9) FIG. 7Sequence and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification

(10) FIG. 8Sequence and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution

(11) FIG. 9Sequence and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution

(12) FIG. 10Alignment of MCP-1 and MCP-5 amino acid sequences

(13) FIG. 11Sequence and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification

(14) FIG. 12Sequence and biotinylation, of RANTES derivative

(15) FIG. 13Example of gating criteria for CCR2-expressing monocytes

(16) FIG. 14Expression of CCR2 expressing monocytes in 9 patients with diabetes mellitus (DM) and in 20 healthy controls (HC). Blood from patients with DM and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells.

(17) FIG. 15Binding of the chemokine bMCP1 (CCL2) to monocytes from 3 patients with Diabetes Mellitus and in 20 healthy controls (HC). Blood from 3 patient with DM was incubated with bMCP1 and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(18) FIG. 16Depletion of CCR2 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bMCP1. Blood cells from a patient with DM were incubated with bMCP1-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 bMCP1-matrix (Before Depletion).

(19) FIG. 17Expression of CCR2 expressing B cells in seven patients with adiposis dolorosa (AD) and in 20 healthy controls (HC). Blood from patients with AD and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The B cells were characterized as CD19 positive cells.

(20) FIG. 18Binding of the chemokine bMCP1 (CCL2) to B cells from seven patient with Adiposis Dolorosa. Blood from a patient with AD was incubated with bMCP1 and analysed with flow cytometry. The B cells were characterized as CD19 positive cells.

(21) FIG. 19Depletion of CCR2 expressing B cells with Sepharose Streptavidin-matrix conjugated with bMCP1. Blood cells from a patient with AD were incubated with bMCP1-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 bMCP1-matrix (Before Depletion).

(22) FIG. 20Increased frequency of CCR1 expressing monocytes in 4 patients with Adiposis dolorosa compared to healthy controls.

(23) FIG. 21Depletion of CCR1 expressing moncoytes with SepharoseStrepavidin-matrix-bRANTES. Blood cells from a healthy control were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion).

(24) FIG. 22Increased frequency of CCR4 expressing T cells in four patients with type 2 Diabetes.

(25) FIG. 23Increased frequency of CCR5 expressing T cells in four patients with type 2 Diabetes.

DESCRIPTION OF PREFERRED EMBODIMENTS

(26) It has been shown (Jianli Niu and Pappachan E. Kolattakudy. Clinical Science (2009) 117, 95-109) that CCR2 expression on monocytes is elevated in diabetic patients. Transgenic mice with an adipose-tissue-specific expression of MCP-1 have macrophage infiltration into adipose tissue, increased hepatic triacylglycerol content and insulin resistance. MCP-1-knockout mice fed a high fat diet have a drastically reduced macrophage accumulation into adipose tissue and hepatic steatosis when compared with high-fatfed wildtype mice. Inhibition of MCP1 function by the acute expression of a dominant negative mutant of MCP-1 ameliorated insulin resistance in db/db mice and in high-fat-fed wild-type mice. Moreover, increased level of serum MCP-1 correlates with markers of the metabolic syndrome, including obesity, insulin resistance, Type 2 diabetes, hypertension and increased serum triacylglyerol concentrations. Monocyte chemoattractant protein-1 (MCP-1) is a major chemoattractant for monocytes and memory T cells by means of their binding to its specific cell-surface receptor, CC-chemokine receptor-2 (CCR2). On this basis, the inventors have selected CCR2 expressing cells as a target for treatment of conditions associated with metabolic syndrome. Serum levels of RANTES are increased in patients with obesity, impaired oral glucose tolerance test and in patients with Type 2 diabetes. RANTES is produced by adipocytes and it recruits and activates T cells, monocytes, basophils, eosinophils and mast cells by binding to CCR1, CCR3 and CCR5 chemokine receptors, which causes an inflammation leading to increased insulin resistance. Polymorphisms of the RANTES gene are associated with the Type 2 diabetes, again linking RANTES to diabetes development. In a diabetes prevention study with lifestyle intervention of owerweight patients with impaired oral glucose tolerance test the patients with the highest serum levels of RANTES were more prone to develop Type 2 diabetes as compared to controls, implying RANTES in the disease development of Type 2 diabetes.

(27) It is shown herein that MCP-1 can be used to reduce CCR2-expressing monocyte levels in diabetic subjects. It is also shown herein that MCP-1 can be used to reduce CCR2-expressing B cell levels in subjects suffering from AD. It is also shown herein that levels of CCR2 expressing leukocytes in particular B lymphocytes are increased in AD patients. This provides a target for leukapheresis treatment and diagnosis according to the invention.

(28) Materials and Methods

(29) Isolation of Peripheral Blood Leukocytes.

(30) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (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.

(31) Chemokines.

(32) The leukocytes were incubated for 30 min in the dark at 4 C. with biotinylated and Alexa647 Fluor labeled MCP-1 (in concentrations 10 ng/L and 50 ng/L). The cells were then washed with FACS-buffer and analyzed by flow cytometry. All chemokines used in the Examples were provided by Almac Sciences Scotland Ltd, Edinburgh, Scotland.

(33) Flow Cytometry Assay.

(34) 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

Binding of Monocytes to MCP-1

(35) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 1a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 1b and 1c).

Example 2

(36) Monocytes were investigated for their expression of CCR2 (FIG. 2b) and their ability to bind MCP-1 (FIG. 2a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 2b). The MCP-1 binding to monocytes shown in FIG. 2a corresponds to the CCR2.sup.hi expressing population shown in FIG. 2b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 3

Tailored Leukapheresis

(37) Column Design and Properties

(38) Introduction

(39) Apheresis is an established treatment used for depletion of blood components, such as antibodies, low-density lipoproteins (LDL) and blood cells. 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.

(40) The Column

(41) The column is intended to be used as a leukapheresis treatment for a condition associated with metabolic syndrome. It will specifically remove CCR2, CCR1, CCR3, CCR4 or CCR5-expressing leukocytes, in particular monocytes, through the use of a binding reagent, more specifically an MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 and/or RANTES containing resin, exploiting the CCR2, CCR1, CCR3, CCR4 or CCR5-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose BigBeads matrix and one or more of biotinylated MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 and RANTES bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(42) The Plastic House (FIG. 3)

(43) 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 FIG. 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.

(44) Streptavidin Sepharose BigBeads

(45) The second component in the device is the affinity matrix called streptavidin Sepharose BigBeads (Sepharose GE Healthcare, Sweden). Sepharose is a cross linked, beaded-form of agarose, which is a polysaccharide extracted from seaweed. Sepharose 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.

(46) Binding Reagent

(47) Coupled to the matrix is the third component of the device, one or more binding reagents that bind specifically to CCR2, CCR1, CCR3, CCR4 or CCR5. One or more hemokines selected from the group consisting of: MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, RANTES, CCL17 (TARC) and CCL22 (MDC) may be employed. These peptides may be synthetic, engineered versions of the human chemokine, which are truncated and biotinylated, but retain binding activity to the CCR2, CCR1, CCR3, CCR4 or CCR5 receptor. By biotinylating the engineered chemokine, it is able to bind to the streptavidin molecules in the Sepharose matrix. The biotin-streptavidin binding is known be one of the strongest biological interactions with a Kd in the order of 410.sup.14 M. The calculated ratio of streptavidin:biotin binding sites in the column is 10:1. Therefore, the coupling between the matrix and chemokine will be immediate, minimising the risk of chemokine decoupling from the matrix.

(48) The Apheresis System

(49) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(50) The Circuit

(51) The system is illustrated in FIG. 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.

(52) The 4008 ADS Pump

(53) An apheresis pump, from Fresenius Medical Care, 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.

(54) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 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.

LEGEND FOR FIG. 5

(55) 1. Monitor 2. Holder for waste bag 3. Modules (left to rightBlood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(56) 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.

(57) Leukapheresis Time and Flow Rate

(58) The apheresis system should be operated at a flow rate of 30-60 mL/min. A treatment is finalised after 1800 mL of blood has been circulated.

(59) Storage Conditions

(60) 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.

(61) Transport Conditions

(62) 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.

(63) In-Vitro Depletion of Target Cell Populations

(64) To investigate the ability to eliminate CCR2-expressing cells, in vitro tests have been performed on the bMCP-1 coupled matrix. Blood was collected from blood donors and passed through the column device containing bMCP-1 coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR2-expressing cells.

(65) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 6a.

(66) The in-vitro results demonstrate a specific reduction of up to 80% of the CCR2-expressing cells by the column. Notably, individuals with fewer CCR2 expressing cells initially achieved lower depletion. The remaining levels of monocytes were around 20-30% in each case, irrespective of the starting point. Non-CCR2-expressing cells remained unaffected (data not shown).

(67) To investigate the ability to eliminate CCR1, 3 and 5-expressing cells, in vitro tests have been performed on the biotinylated RANTES coupled matrix. Blood was collected from blood donors and passed through the column device containing biotinylated RANTES coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR1, 3 or 5-expressing cells.

(68) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 8:

(69) TABLE-US-00005 H2N- SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC ANPEKKWVREYINSLEKS- CO2H

(70) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(71) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 12. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

(72) 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 FIG. 6b.

(73) The in-vitro results demonstrate a specific reduction of around 20% of the chemokine receptor-expressing cells by the column. Non-CCR1, 3 and 5-expressing cells remained unaffected (data not shown).

Example 4

MCP1 Derivatives

(74) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 1,

(75) MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KVVVQDSMDHL DKQTQTPKT

(76) The amino acid sequence of the mature protein is set forth as SEQ ID NO: 2,

(77) QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSXDHL DKQTQTPKT

(78) X=Met or Norleucine

(79) The inventors have determined that chemokines may display improved binding properties where the chemokine is biotinylated via a spacer group. The spacer may prevent the biotin group from impacting on the binding affinity of the chemokine.

(80) Thus, MCP-1 derivatised at the -amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 7.

(81) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 8, see also SEQ ID NO: 2.

(82) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor. The relevant molecules have been synthesised, see Example 7 below.

Example 5

Synthesis of a CCR2 Antagonist biotinMCP-1 which Binds to the Receptor without Activation

(83) Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995, 181, 63) has been shown for an MCP-1 derivative truncated at the N-terminus. In particular, deletion of residues 1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis of CCR2 positive cells. (inhibition of chemotaxis IC50 20 nM)

(84) The amino acid sequence of the truncated version is set forth as SEQ ID NO: 3:

(85) TABLE-US-00006 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(86) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the -amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 9. The PEG spacer will be 3,6,-dioxoaminooctanoic acid.

(87) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 6

Demonstrate Removal of CCR2 Expressing Cells Using an Alternative Chemokine Ligand to MCP-1

(88) CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 in addition to MCP-1. MCP-5 only binds CCR2 and should be selective in its removal of CCR2 expressing cells. MCP5 is a mouse chemokine shown to chemotact human CCR2 cells with EC50<3 nM.

(89) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 4

(90) TABLE-US-00007 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(91) The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 5

(92) TABLE-US-00008 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(93) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 10. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 6:

(94) TABLE-US-00009 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL

(95) In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 7:

(96) TABLE-US-00010 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(97) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 11.

(98) Once synthesised, the activity of the various biotinMCP-5 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Examples 7 to 9

Synthesis of Additional Chemokines

(99) General Protocols

(100) Assembly:

(101) 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.

(102) Removal of Dde Protection:

(103) The Dde protecting group was removed by treatment of resin with a solution of 2.5% hydrazine in DMF (200 ml) over a 2 hour period. The resin was then washed with DMF.

(104) Labelling Steps:

(105) 1. Couple Fmoc-8-amino-3,6-dioctanoic acid (PEG)

(106) Resin was swollen in DMF and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38 g, 1 mmol), DIC solution (2 ml, 0.5 M in DMF) and OxymaPure solution (2 ml, 0.5 M in DMF) was added. The mixture was sonicated for 3 hours and then washed with DMF.

(107) 2. Capping

(108) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(109) 3. Fmoc Deprotection

(110) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (250 ml) for 15 minutes each. The resin was washed with DMF.

(111) 4. Couple Biotin-OSu

(112) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 l) in DMF (10 ml) was added to the resin and the mixture was sonicated for 3 hours. The resin was washed thoroughly with DMF and DCM then dried in vacuo.

(113) Cleavage:

(114) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 thioanisole (500 l), water (500 l), DMS (500 l), EDT (250 l), NH.sub.4I (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.

(115) Purification Protocol:

(116) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 25021 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(117) Folding Protocol:

(118) Pure peptide (10 mg) was dissolved into 6M GnHCl (16 ml) and then rapidly diluted to 2M GnHCl concentration by the addition of 50 mM TRIS pH 8.5 (84 ml) containing 0.3 mM GSSG and 3 mM GSH. The mixture was stirred at room temperature for 24 hours and then analysed by RP-HPLC (Jupiter C18, 2504.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 7

biotinMCP-1 (CCL2)

(119) Target Molecule: MCP-1 Derivatised at the -Amino Side Chain Functionality of Lys(75) with PEG-Biotin (TFA Salt)

(120) Modifications: Human MCP-1 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 Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln 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.

(121) The linear amino acid sequence (SEQ ID NO:9) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(122) TABLE-US-00011 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

(123) The engineered MCP-1 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:

(124) TABLE-US-00012 SEQ ID NO: 10 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(125) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. 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.

(126) TABLE-US-00013 SEQ ID NO: 11 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2

(127) X1=pyroGlu or Gln

(128) X75 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, optionally K(PEG-Biotin)

(129) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(130) Functional Assay Data:

(131) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 8

biotinRANTES (CCL5)

(132) Target Molecule: RANTES Derivatised at the -Amino Side Chain Functionality of Lys(67) with Biotin (TFA Salt)

(133) Modifications: 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.

(134) The linear amino acid sequence (SEQ ID NO: 12) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(135) TABLE-US-00014 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(136) 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:

(137) TABLE-US-00015 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(138) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 13). 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: 14).

(139) TABLE-US-00016 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH

(140) 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))

(141) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(142) Functional Assay Data:

(143) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 9

biotinMCP-2 (CCL8)

(144) Target Molecule: MCP-2 Derivatised at the -Amino Side Chain Functionality of Lys(75) with PEG-Biotin (TFA Salt)

(145) Modifications: 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 Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln 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.

(146) The linear amino acid sequence (SEQ ID NO: 15) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(147) TABLE-US-00017 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(148) 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:

(149) TABLE-US-00018 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(150) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 16). 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: 17):

(151) TABLE-US-00019 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=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).

(152) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(153) Functional Assay Data:

(154) 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 50.9 nM. c.f. EC50 for recombinant native MCP-2 is 23.5 nM (partial agonist).

Example 10

Diagnosis and Treatment of Diabetes Mellitus (DM)

(155) Materials and Methods

(156) 1. Flow Cytometric Analysis of Peripheral Blood

(157) 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 10 mM Tris and 160 mM NH.sub.4Cl, pH 7.5) for 15 min at 37 C. The cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum for 15 min at room temperature (RD and stained with antibodies (Table 2) at 4 C. for 30 min. The cells were analysed by flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences).

(158) TABLE-US-00020 TABLE 2 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter CCR5 PE Biolegend CCR3 PE Biolegend Streptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend CD3 V450 BD Biosciences CD19 V500 BD Biosciences CD16 PE Cy7 BD Biosciences CCR4 PerCP Cy5.5 BD Biosciences
2. Chemokine Binding Test

(159) 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 10 mM Tris and 160 mM NH4Cl, pH 7.5) for 15 min at 37 C. The cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum 15 min at room temperature (RD and stained with cell specific antibodies together with biotinylated chemokine (1 M) or the corresponding chemokine receptor antibody at 4 C. for 30 min (Table 2). The 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).

(160) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(161) Cells were prepared from peripheral blood (section 1). 1 mL Sepharose BigBeads matrix conjugated with 0.4 mg/mL Streptavidin (GE Healthcare) was washed in 50 mL PBS and added to a 5 mL polystyrene tube (BD Falcon). Biotinylated chemokine (1 M) 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 40 um nylon filter (BD Falcon 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.

(162) Results and Discussion

(163) Diabetes Mellitus (DM)

(164) 1. Flow Cytometric Analysis of Peripheral Blood

(165) White blood cells from DM patients were analysed with flow cytometry. About 80% of the monocytes are shown to express the chemokine receptor CCR2 (FIG. 14) based upon flow cytometry data and binding by an anti-CCR2 antibody. CCR2 and its ligand MCP1 (CCL2) are important for monocyte migration and infiltration into the inflamed tissue. CCR2 is expressed on the majority of the monocytes in both diabetes patients and healthy controls. The expression of the CCR2 receptor on monocytes is not increased in diabetes patients; however, the monocytes in diabetes are potentially different in their pro-inflammatory profile with regards to other mediators. Furthermore, the ligand MCP-1 is secreted by inflamed and damaged tissue, such as the pancreas in diabetes, and will attract CCR2 expressing monocytes. In healthy individuals, this signal is absent and the monocytes will not migrate into the tissue even though they express CCR2. Thus, migration of pro-inflammatory CCR2 expressing monocytes is rather regulated by the level of the ligand MCP-1 than the level of CCR2 expression.

(166) FIG. 22 shows an increased frequency of CCR4 expressing T cells in four patients with type 2 Diabetes compared to healthy controls. FIG. 23 shows an increased frequency of CCR5 expressing T cells in four patients with type 2 Diabetes compared to healthy controls. These cells may be depleted using suitable chemokines such as CCL17 (TARC) and CCL22 (MDC), which bind CCR4 only and CCL5 (RANTES) which binds CCR5.

(167) 2. Chemokine Binding Test

(168) In accordance with the CCR2 expression, all the monocytes binds to biotinylated MCP1 (bMCP1) (FIG. 15).

(169) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(170) 21% of the CCR2 expressing monocytes were efficiently depleted with bMCP1-conjugated Sepharose Streptavidin Matrix. Before depletion there were 80% CCR2 expressing monocytes and after depletion 63% (FIG. 16).

(171) We conclude that monocytes in DM express CCR2 and bind the ligand bMCP1. Furthermore, 20% of the CCR2 expressing monocytes can be removed with Sepharose Streptavidin matrix conjugated with bMCP1.

Example 11

Diagnosis and Treatment of Adiposis Dolorosa

(172) Materials and Methods

(173) 1. Flow Cytometric Analysis of Peripheral Blood

(174) 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 10 mM Tris and 160 mM NH.sub.4Cl, pH 7.5) for 15 min at 37 C. The cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum for 15 min at room temperature (RD and stained with antibodies (Table 3) at 4 C. for 30 min. The cells were analysed by flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences).

(175) TABLE-US-00021 TABLE 3 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter Streptavidin PE, ARC Biolegend CCR2 PerCP Cy5.5 Biolegend CD16 PE Cy7 BD Biosciences HLADR APC Cy7 Biolegend CD3 V450 BD Biosciences CD19 V500 BD Biosciences
2. Chemokine Binding Test

(176) 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 10 mM Tris and 160 mM NH4Cl, pH 7.5) for 15 min at 37 C. The cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum 15 min at room temperature (RD and stained with cell specific antibodies together with biotinylated chemokine (1 M) or the corresponding chemokine receptor antibody at 4 C. for 30 min (Table 3). The 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).

(177) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(178) Cells were prepared from peripheral blood (section 1). 1 mL Sepharose BigBeads matrix conjugated with 0.4 mg/mL Streptavidin (GE Healthcare) was washed in 50 mL PBS and added to a 5 mL polystyrene tube (BD Falcon). Biotinylated chemokine (1 M) 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 40 um nylon filter (BD Falcon Cell Strainer). The flow through cells were stained with antibodies (Table 3), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(179) Results and Discussion

(180) 1. Flow Cytometric Analysis of Peripheral Blood

(181) White blood cells from AD patients was analysed with flow cytometry. The patients exhibited two fold increased numbers of circulating CCR2 expressing B cells (mean of 1.8% compared to approximately 0.7% in healthy blood) (FIG. 17).

(182) FIG. 20 shows an increased frequency of CCR1 expressing monocytes in 4 patients with Addipois dolorosa compared to healthy controls.

(183) 2. Chemokine Binding Test

(184) CCR2 binds to the chemokine MCP1 (CCL2) and is important for cell recruitment to inflammation sites. 65% of the B cells bind to the biotinylated MCP1 (bMCP1) (FIG. 18).

(185) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(186) 67% of the CCR2 expressing B cells were efficiently depleted with bMCP1-conjugated Sepharose Streptavidin Matrix. Before depletion there were 3% CCR2 expressing B cells and after depletion 1% (FIG. 19).

(187) We conclude that B cells in AD express CCR2 and bind the ligand bMCP1. Furthermore, 67% of the CCR2 expressing B cells can be removed with Sepharose Streptavidin matrix conjugated with bMCP1.

(188) FIG. 21 shows depletion of CCR1 expressing moncoytes with SepharoseStrepavidin-matrix-bRANTES. Blood cells from a healthy control were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion). Thus, monocytes expressing CCR1 are upregulated in AD. These cells can be effectively depleted using a suitable chemokine, in this case biotinylated CCL5 (RANTES).

(189) The various embodiments of the present invention are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the various embodiments of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Moreover, all embodiments described herein are considered to be broadly applicable and combinable with any and all other consistent embodiments, as appropriate.

(190) Various publications are cited herein, the disclosures of which are incorporated by reference in their entireties.