Treating multiple sclerosis

Abstract

A method for treating multiple sclerosis 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, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 immobilized directly or indirectly on the support thus removing one or more chemokine receptor, optionally CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 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 multiple sclerosis in a subject in need thereof 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 the chemokine receptor CCR2 are removed from the peripheral blood of the subject, wherein blood applied to the apheresis column is circulated back into the subject's systemic circulation, and whereby the multiple sclerosis is treated.

2. The method of claim 1, wherein the multiple sclerosis is selected from active and stable relapsing-remitting multiple sclerosis, primary progressive relapsing multiple sclerosis, secondary progressive relapsing multiple sclerosis, and progressive relapsing multiple sclerosis.

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 selected from monocytes, lymphocytes, neutrophils, macrophages, eosinophils, and basophils.

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 multiple sclerosis.

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 of a healthy donor.

(9) FIG. 6cResults of in vitro depletion tests performed on the biotinylated MIP-3a coupled matrix showing ability to eliminate CCR6-expressing lymphocytes from blood from three healthy donors.

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

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

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

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

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

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

(16) FIG. 13example of gating criteria for CCR2 expressing monocytes

(17) FIG. 14aFrequency of CCR2 positive T cells. Bars represent mean and SEM of T cells that express CCR2 in 2 patients and 20 healthy controls.

(18) FIG. 14bFrequency of CCR6 positive T cells. Bars represent mean and SEM of T cells that express CCR6 in 5 patients and 20 healthy controls. The expression of chemokine receptors and specific cell markers were analysed with flow cytometry. The T cells were characterized as CD3 positive.

(19) FIG. 15aBinding of the chemokine bMCP-1 to T cells. Bar represents mean frequency and SEM of MCP-1 binding T cells in 5 patients with MS.

(20) FIG. 15bBinding of the chemokine bMIP3a to T cells. Bar represents frequency of MIP3a-binding T cells in 1 patient with MS. Blood was incubated with biotinylated chemokine and analysed with flow cytometry. The T cells were characterized as CD3 positive.

(21) FIG. 16aDepletion of CCR2 expressing T cells with Sepharose Streptavidin-matrix conjugated with bMCP-1.

(22) FIG. 16bDepletion of CCR6 expressing T cells with Sepharose Streptavidin-matrix conjugated with bMIP3a. Blood cells from a patient with MS 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).

DESCRIPTION OF PREFERRED EMBODIMENTS

(23) In secondary progressive MS microglia/M present at border of plaques produce chemokines MCP-1 and CXCL10 responsible for attracting CCR2 and CXCR3 expressing cells including macrophages and astrocytes.

(24) It is shown herein that subjects suffering from MS exhibit increased frequency of chemokine receptor expressing cells in the peripheral blood, in particular CCR2 and CCR6 expressing T lymphocytes, compared to healthy controls. It is also shown herein that the CCR2 cells can be removed using a suitable binding reagent, in particular MCP-1 (in biotinylated form) immobilized on a suitable matrix. Similarly, it is shown herein that (the additional) CCR6-expressing cells can be depleted using a suitable binding reagent, in particular CCL20 (MIP-31), in biotinylated form, immobilized on a suitable matrix.

(25) The CCL5 levels are significantly elevated in cerebrospinal fluid of MS patients with relapsing disease demonstrating that circulating CCL5 is involved in recruiting CCL5 binding cells to the brain. These findings are supported by the enrichment of T cells in cerebrospinal fluid expressing CCR5 and CCR6 suggesting an active accumalation due to a chemokine gradient of CCL5. Therefore eliminating cells normally attracted to the brain by providing an extracorpeal source of CCL5 attached to a column will be useful for the treatment of MS.

Examples 1 and 2

(26) Materials and Methods

(27) Isolation of Peripheral Blood Leukocytes.

(28) 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.

(29) Chemokines.

(30) 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.

(31) Flow Cytometry Assay.

(32) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San Jos, 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 1Binding of Monocytes to MCP-1

(33) 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

(34) 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 CCR2hi expressing population shown in FIG. 2b. Thus, MCP-1 binds favourably to CCR2hi expressing cells.

Example 3Tailored Leukapheresis

(35) Column Design and Properties

(36) Introduction

(37) 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.

(38) The Column

(39) The column is intended to be used as a leukapheresis treatment for multiple sclerosis. It will specifically remove CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9-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, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES containing resin, exploiting the CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9-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, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(40) The Plastic House (FIG. 3)

(41) 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.

(42) Streptavidin Sepharose BigBeads

(43) 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.

(44) Binding Reagent

(45) Coupled to the matrix is the third component of the device, the one or more binding reagents that bind specifically to CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9. One or more chemokines such as those selected from the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES 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, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 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.

(46) The Apheresis System

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

(48) The Circuit

(49) 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.

(50) The 4008 ADS Pump

(51) 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.

(52) 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

(53) 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

(54) 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.

(55) Leukapheresis Time and Flow Rate

(56) 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.

(57) Storage Conditions

(58) 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.

(59) Transport Conditions

(60) 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.

(61) In-Vitro Depletion of Target Cell PopulationsMCP-1

(62) 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.

(63) 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.

(64) In conclusion, 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).

(65) In-Vitro Depletion of Target Cell PopulationsRANTES

(66) 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 and 5-expressing cells.

(67) 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.

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

(69) In-Vitro Depletion of Target Cell PopulationsMIP-3alpha

(70) To investigate the ability to eliminate CCR6-expressing cells, in vitro tests have been performed on the biotinylated MIP-3a coupled matrix. Blood was collected from blood donors and passed through the column device containing biotinylated MIP-3a coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR6-expressing cells.

(71) The results demonstrate significant depletion of the target population CCR6-expressing lymphocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 6c.

(72) The in-vitro results demonstrate a specific reduction of up to around 15% of the CCR6-expressing cells by the column. Non-CCR6-expressing cells remained unaffected (data not shown).

(73) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 17:

(74) TABLE-US-00011 H2N- SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC ANPEKKWVREYINSLEKS-CO2H

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

(76) 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.

Example 4MCP1 Derivatives

(77) 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,

(78) TABLE-US-00012 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

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

(80) TABLE-US-00013 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSXDHL DKQTQTPKT

(81) X=Met or Nleu

(82) 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.

(83) 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.

(84) 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 and in SEQ ID NO: 2.

(85) 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 5Synthesis of a CCR2 Antagonist biotinMCP-1 which Binds to the Receptor without Activation

(86) 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)

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

(88) TABLE-US-00014 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(89) 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.

(90) 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 6Demonstrate Removal of CCR2 Expressing Cells Using an Alternative Chemokine Ligand to MCP-1

(91) 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.

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

(93) TABLE-US-00015 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(94) 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

(95) TABLE-US-00016 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(96) 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:

(97) TABLE-US-00017 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL

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

(99) TABLE-US-00018 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(100) 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.

(101) 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 14

(102) General Protocols for Chemokine Synthesis

(103) Assembly:

(104) Chemical synthesis of chemokines was performed using standard Fmoc solid phase peptides synthesis (SPPS) techniques on an ABI 433 peptide synthesiser. DIC (0.5M in DMF) and OxymaPure (0.5M in DMF) were used for activation, acetic anhydride (0.5M 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.

(105) Removal of Dde Protection:

(106) 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.

(107) Labelling Steps:

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

(109) 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, 0.5M in DMF) and OxymaPure solution (2 ml, 0.5M in DMF) was added. The mixture was sonicated for 3 hours and then washed with DMF.

(110) 2. Capping

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

(112) 3. Fmoc Deprotection

(113) 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.

(114) 4. Couple Biotin-OSu

(115) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (3481 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.

(116) Cleavage:

(117) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 l), 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.

(118) Purification Protocol:

(119) 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].

(120) Folding Protocol:

(121) 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 7biotinMCP-1 (CCL2)

(122) Target Molecule: MCP-1 derivatised at the -amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(123) 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.

(124) The linear amino acid sequence (SEQ ID NO: 8) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(125) TABLE-US-00019 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV AKEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2

(126) X=pyroGlu

(127) 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:

(128) TABLE-US-00020 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV AKEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN

(129) X1=pyroGlu

(130) X75=K(ivDde)

(131) FmocLys(ivDde)-OH was incorporated as residue 75 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 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: 10):

(132) TABLE-US-00021 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV AKEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2

(133) X1=pyroGlu

(134) 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).

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

(136) Functional Assay Data:

(137) 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 8biotinMCP-2 (CCL8)

(138) Target Molecule: MCP-2 derivatised at the -amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(139) 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.

(140) The linear amino acid sequence (SEQ ID NO: 11) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(141) TABLE-US-00022 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2

(142) X=pyroGlu

(143) 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:

(144) TABLE-US-00023 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2

(145) X1=pyroGlu

(146) X75=K(ivDde)

(147) FmocLys(ivDde)-OH was incorporated as residue 75 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 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: 13):

(148) TABLE-US-00024 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2

(149) X1=pyroGlu

(150) 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).

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

(152) Functional Assay Data:

(153) 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 9biotinEotaxin (CCL11)

(154) Target Molecule: Eotaxin derivatised at the -amino side chain functionality of Lys(73) with PEG-Biotin (TFA salt)

(155) Modifications: Human eotaxin corresponding to residues 1-74, is initially expressed as 97 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The naturally occurring lysine at position 73 was modified through biotinylation on the resin. A PEG spacer was incorporated between the -amino functionality and the biotin.

(156) The linear amino acid sequence (SEQ ID NO: 14) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(157) TABLE-US-00025 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKL AKDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2

(158) 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).

(159) The engineered eotaxin 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:

(160) TABLE-US-00026 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLA KDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2

(161) X is K(ivDde)

(162) FmocLys(ivDde)-OH was incorporated as residue 73 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 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: 16):

(163) TABLE-US-00027 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLA KDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2

(164) X is K(PEG-Biotin)

(165) Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) data of purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3 Da.

(166) Functional Assay Data:

(167) biotinEotaxin was tested for activity in an Aequorin assay against hCCR3, (Euroscreen) and was shown to be an antagonist with an EC50 value of 211.8 nM. c.f. EC50 for recombinant native eotaxin is 10.7 nM (agonist).

Example 10biotinRANTES (CCL5)

(168) Target Molecule: RANTES derivatised at the -amino side chain functionality of Lys(67) with Biotin (TFA salt)

(169) 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.

(170) The linear amino acid sequence (SEQ ID NO: 17) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(171) TABLE-US-00028 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNR QVCANPEKKWVREYINSLEKS-OH

(172) 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:

(173) TABLE-US-00029 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN

(174) X is K(ivDde)

(175) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 18). 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: 19).

(176) TABLE-US-00030 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH

(177) 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))

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

(179) Functional Assay Data:

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

Example 11biotinMIP-3 (CCL20)

(181) Target Molecule: MIP-3 derivatised at the -amino side chain functionality of Lys(68) with PEG-Biotin (TFA salt)

(182) Modifications: Human MIP-3 corresponding to residues 1-70, is initially expressed as 96 amino acids comprising the chemokine fold, and a 26 amino acid signal peptide which is cleaved off. The naturally occurring lysine at position 68 was modified through biotinylation on the resin. A PEG spacer was incorporated between the -amino functionality and the biotin.

(183) The linear amino acid sequence (SEQ ID NO: 20) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 68 (K):

(184) TABLE-US-00031 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVKNM-OH

(185) The engineered MIP-3 sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(186) TABLE-US-00032 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVXNM-RESIN

(187) X=K(ivDde)

(188) FmocLys(ivDde)-OH was incorporated as residue 68 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 21). 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: 22).

(189) TABLE-US-00033 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVXNM-OH

(190) 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, in particular K(PEG-Biotin)

(191) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMip-3: obtained=8396.4 Da; expected 8397.0 Da.

(192) Functional Assay Data:

(193) BiotinMIP-3 was tested for agonist activity in an Aequorin assay against hCCR6, (Euroscreen) and an EC50 value of 1.6 nM was reported. c.f. EC50 for recombinant native MIP-3 is 1.0 nM.

Example 12biotinTECK (CCL25)

(194) Target Molecule: TECK (Met to Nleu substitution) derivatised at the -amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(195) Modifications: 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 Gln 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 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 72 was modified through biotinylation on the resin. A PEG spacer was incorporated between the -amino functionality and the biotin.

(196) The linear amino acid sequence (SEQ ID NO: 23) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(197) TABLE-US-00034 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNKVF-OH

(198) X1=pyroGlu or Gln

(199) X64=Norleucine

(200) The engineered TECK sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(201) TABLE-US-00035 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-RESIN

(202) X1=pyroGlu or Gln

(203) X64=Norleucine

(204) X72=K(Dde)

(205) TABLE-US-00036 NPKSREVQRANIeKLLDARNK(ivDde)VF-RESIN

(206) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 24). 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.

(207) The final active chemokine thus has the following sequence (SEQ ID NO: 25):

(208) TABLE-US-00037 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-OH

(209) X1=pyroGlu or Gln

(210) X64=norleucine

(211) 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)

(212) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinTECK(Met to Nleu substitution): obtained=8958.5 Da; expected 8959.6 Da.

(213) Functional Assay Data:

(214) biotinTECK(Met to Nleu substitution) was tested for agonist activity in an Aequorin assay against hCCR9, (Euroscreen) and an EC50 value of 63.6 nM was reported. c.f. EC50 for recombinant native TECK is 67.9 nM.

Example 13biotinITAC (CXCL11)

(215) Target Molecule: ITAC derivatised with Biotin at the -amino side chain functionality of an additional Lysine inserted at the C-terminus after a PEG spacer (TFA salt)

(216) Modifications: Human ITAC corresponding to residues 1-73, is initially expressed as 94 amino acids comprising the chemokine fold, and a 21 amino acid signal peptide which is cleaved off. A PEG spacer and an additional lysine were inserted at the C-terminus, and modified through biotinylation on the resin. The PEG spacer was incorporated at the C-terminus between the protein and the additional lysine.

(217) The linear amino acid sequence (SEQ ID NO: 26) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(218) TABLE-US-00038 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNF-OH

(219) The engineered ITAC sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(220) TABLE-US-00039 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNFX-RESIN

(221) X is PEG-K(ivDde)

(222) Fmoc-12-amino-4,7,10-trioxadodecanoic acid followed by FmocLys(ivDde)-OH were incorporated at the C-terminus to facilitate site-specific labelling with biotin at the -amino side chain functionality of the additional Lys (SEQ ID NO: 27). 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: 28).

(223) TABLE-US-00040 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNFX-OH

(224) 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) and may be attached via a spacer molecule, e.g. PEG-K(Biotin)

(225) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinITAC: obtained=8866.5 Da; expected 8860.6 Da.

(226) Functional Assay Data:

(227) biotinITAC was tested for agonist activity in an Aequorin assay against hCXCR3, (Euroscreen) and an EC50 value of 15.7 nM was reported. c.f. EC50 for recombinant native ITAC is 0.7 nM.

Example 14biotinIP-10 (CXCL10)

(228) Target Molecule: IP-10 derivatised with Biotin at the -amino side chain functionality of an additional Lysine inserted at the C-terminus after a PEG spacer (TFA salt)

(229) Modifications: Human IP-10 corresponding to residues 1-77, is initially expressed as 98 amino acids comprising the chemokine fold, and a 21 amino acid signal peptide which is cleaved off. A PEG spacer and an additional lysine were inserted at the C-terminus, and modified through biotinylation on the resin. The PEG spacer was incorporated at the C-terminus between the protein and the additional lysine.

(230) The linear amino acid sequence (SEQ ID NO: 29) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(231) TABLE-US-00041 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSP-OH

(232) The engineered IP-10 sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(233) TABLE-US-00042 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSPX-RESIN

(234) X is K(ivDde), optionally attached via a spacer such as PEG, e.g. -PEG-K(ivDde)

(235) Fmoc-8-amino-3,6-dioctanoic acid followed by FmocLys(ivDde)-OH were incorporated at the C-terminus to facilitate site-specific labelling with biotin at the -amino side chain functionality of the additional Lys (SEQ ID NO: 30). 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. The final active chemokine thus has the following sequence (SEQ ID NO: 31):

(236) TABLE-US-00043 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSPX-OH

(237) 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) and may be attached via a spacer molecule, e.g. PEG-K(Biotin)

(238) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIP-10: obtained=9141.0 Da; expected 9141.9 Da.

(239) Functional Assay Data:

(240) BiotinIP-10 was tested for agonist activity in an Aequorin assay against hCXCR3, (Euroscreen) and an EC50 value of 8.7 nM was reported. c.f. EC50 for recombinant native IP-10 is 4.4 nM.

Example 15MS Diagnosis and Treatment Based Upon CCR2 and CCR6 Expressing T-Cells

(241) Materials and Methods

(242) 1. Flow Cytometric Analysis of Peripheral Blood

(243) Peripheral blood from patients with Multiple Sclerosis 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 (RT) and stained with antibodies (Table 2) at 4 C. for 30 min. The cells were analysed with flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences).

(244) TABLE-US-00044 TABLE 2 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD3 V450 BD Biosciences CCR6 PE BD Biosciences Streptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend
2. Chemokine Binding Test

(245) 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 (RT) 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).

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

(247) 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 with flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(248) Results and Discussion

(249) 1. Flow Cytometric Analysis of Peripheral Blood

(250) White blood cells from patients with Multiple Sclerosis (MS) were analysed for the expression of chemokine receptors with flow cytometry. The MS patients exhibited an increased frequency of circulating T cells that expressed the chemokine receptor CCR2, 15% compared to approximately 5% in healthy blood (FIG. 14a), based upon flow cytometry data and binding by an anti-CCR2 antibody. Furthermore, the patients had an increased frequency of T cells that expressed CCR6 (FIG. 14b).

(251) 2. Chemokine Binding Test

(252) CCR2 binds to the chemokine MCP-1 that mediate migration and infiltration of inflammatory cells to various tissues. The ligand for CCR6 is MIP3a (CCL20) that can mediate migration of T cells into the CNS. Both these receptors are important in the inflammatory process. In accordance with the CCR2 and CCR6 expression, the T cells bound the biotinylated MCP-1 (bMCP-1) (FIG. 15a) and bMIP3a (FIG. 15b).

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

(254) The CCR2 expressing T cells could be efficiently depleted with bMCP-1-conjugated Sepharose Streptavidin Matrix (FIG. 16a), and the CCR6 expressing T cells could be depleted with bMIP3a-conjugated Sepharose Streptavidin Matrix (FIG. 16b)

(255) We conclude that the frequency of T cells that express CCR2 and CCR6 is enhanced in MS. These T cells can bind the ligands MCP-1 and MIP3a. Furthermore, the majority of the CCR2 and CCR6 expressing T cells can be removed with Sepharose Streptavidin matrix conjugated with the corresponding biotinylated chemokine.

(256) The various embodiments of the present invention is 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.

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