Treating mental disorders

Abstract

A method for treating mental disorders such as schizophrenia, depression and bipolar disorder comprises applying peripheral blood from a patient or subject to an apheresis column loaded with a solid support comprising a binding reagent capable of specifically binding to a chemokine receptor, optionally the chemokine receptor CCR9, CCR1, CCR3 and/or CCR5 immobilized directly or indirectly on the support thus removing chemokine receptor, optionally CCR9, CCR1, CCR3 and/or 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 bipolar disorder 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 a binding reagent capable of specifically binding to chemokine receptor CCR9 immobilized directly or indirectly on the support, whereby one or more cells expressing the chemokine receptor CCR9 are removed from the peripheral blood of the subject, and wherein the applied blood is returned to the subject to thereby treat bipolar disorder.

2. The method of claim 1, wherein the binding reagent is an agonist or an antagonist of CCR9.

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

4. The method of claim 1, wherein the chemokine is TECK (CCL25).

5. The method of claim 1, wherein the one or more cells are eosinophils, lymphocytes, basophils, neutrophils, mast cells, or monocytes.

6. The method of claim 1, wherein the subject has increased levels of expression of CCR9 as compared to a subject that does not have bipolar disorder.

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

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1abinding of eotaxin to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

(2) FIG. 1bbinding of CCR3-antibody to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

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

(4) FIG. 3The overall leukapheresis system.

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

(6) FIG. 5aResults of in vitro depletion tests performed on the biotinylated eotaxin coupled matrix showing ability to eliminate CCR3-expressing cells from blood from a healthy donor.

(7) FIG. 5bResults 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.

(8) FIG. 6Sequence and biotinylation, via a spacer group, of mature protein eotaxin derivative containing C-terminal amide.

(9) FIG. 7Sequence and biotinylation, of RANTES derivative.

(10) FIG. 8Example of gating criteria for CCR2 expressing monocytes.

(11) FIG. 9Frequency of CCR9 expressing monocytes in two patients with bipolar disorder (BP) and in 20 healthy controls (HC). Blood was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells.

(12) FIG. 10Binding of the chemokine bTECK (CCL25) to blood monocytes from a patient with BP. Blood was incubated with bTECK and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(13) FIG. 11Depletion of CCR9 expressing monocytes and with Sepharose Streptavidin-matrix conjugated with bTECK. Blood cells from a patient with bipolar disorder were incubated with bTECK-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bTECK-matrix (Before Depletion).

DESCRIPTION OF PREFERRED EMBODIMENTS

(14) Inflammation is an important component of mental disorders such as schizophrenia, depression and bipolar disorder and may involve increased levels of CCR1, CCR3 and/or CCR5 intracellular signalling via CCL11 binding. CCL11 is a ligand for CCR1, CCR3 and/or CCR5, a receptor expressed preferentially on Th2 lymphocytes, mast cells and eosinophils. Higher serum levels of CCL11 in mental disorders such as schizophrenia, depression and bipolar disorder suggest that this disease may be associated with a Th1/Th2 imbalance with a shift toward a Th2 immune response.

(15) In mental disorders, such as Schizophrenia, depression and bipolar disorder, research has focused on finding diagnostic biomarkers to improve classification of disease and treatment of patients. It has been shown in patient plasma samples that high pro-inflammatory cytokine and chemokine expression correlate with depression and fatigue (Plasma Protein Biomarkers for Depression and Schizophrenia by Multi Analyte Profiling of Case-Control Collections: Domenici E et al, PLoS ONE 5(2): e9166, 2010).

(16) Clozapine, the first atypical antipsychotic, is indicated for the treatment of therapyresistant schizophrenia. It needs to be monitored closely because of its well-known potential side-effects, especially agranulocytosis. Agranulocytosis, also known as Agranulosis or Granulopenia, is an acute condition involving a severe and dangerous leukopenia (lowered white blood cell count) in the circulating blood. This indicates that granulocytes; neutrophils, basophils and eosinophils are of major importance for the schizophrenic disease.

(17) It is shown herein that subjects suffering from mental disorders such as bipolar disorder exhibit highly increased frequency of chemokine receptor expressing cells in the peripheral blood, in particular CCR9 expressing monocytes, compared to healthy controls. It is also shown herein that the CCR9 cells can be removed using a suitable binding reagent, in particular CCL25 (in biotinylated form) immobilized on a suitable matrix.

Example 1

(18) Materials and Methods

(19) Isolation of Peripheral Blood Leukocytes.

(20) Heparinized peripheral blood from healthy blood donors 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.

(21) Chemokines.

(22) The leukocytes were incubated for 30 min in the dark at 4 C. with biotinylated and Alexa647 Fluor labeled eotaxin (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.

(23) Flow Cytometry Assay.

(24) 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.

(25) Neutrophils/eosinophils were investigated for their expression of CCR3 (FIG. 1b) and their ability to bind eotaxin (FIG. 1a). CCR3 expression was noted on all neutrophils/eosinophils with the majority of neutrophils/eosinophils expressing high levels, using an anti-CCR3 antibody (FIG. 1b). The eotaxin binding to neutrophils/eosinophils shown in FIG. 1a corresponds to the CCR3.sup.hi expressing population shown in FIG. 1b. Thus, eotaxin binds favourably to CCR3.sup.hi expressing cells.

Example 2Tailored Leukapheresis

(26) Column Design and Properties

INTRODUCTION

(27) 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.

(28) The Column

(29) The column is intended to be used as a leukapheresis treatment for mental disorders such as schizophrenia, depression and bipolar disorder. It will specifically remove CCR9, CCR1, CCR3 and/or CCR5-expressing leukocytes, in particular eosinophils, through the use of a binding reagent, more specifically a biotinylated eotaxin containing resin, exploiting the CCR9, CCR1, CCR3 and/or CCR5-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose BigBeads matrix and biotinylated eotaxin bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(30) The plastic house (FIG. 2)

(31) 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. 4. 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.

(32) Streptavidin Sepharose BigBeads

(33) 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.

(34) Binding Reagent

(35) Coupled to the matrix is the third component of the device, the binding reagent that binds specifically to CCR9, CCR1, CCR3 and/or CCR5. Chemokines such as eotaxin 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 CCR9, CCR1, CCR3 and/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.

(36) The Apheresis System

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

(38) The Circuit

(39) The system is illustrated in FIG. 3. 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.

(40) The 4008 ADS Pump

(41) 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.

(42) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 4. 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.

(43) Legend for FIG. 4: 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

(44) 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.

(45) Leukapheresis Time and Flow Rate

(46) 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.

(47) Storage Conditions

(48) 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.

(49) Transport Conditions

(50) 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.

(51) In-Vitro Depletion of Target Cell PopulationsEotaxin

(52) To investigate the ability to eliminate CCR3-expressing cells, in vitro tests have been performed on the eotaxin coupled matrix. Blood was collected from blood donors and passed through the magnetic column device containing eotaxin coupled MACS beads. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR3-expressing cells.

(53) The results demonstrate significant depletion of the target population CCR3-expressing neutrophils/eosinophils post matrix perfusion. Depletion tests were performed on blood from a healthy donor. The results are shown in FIG. 5a.

(54) In conclusion, the in-vitro results demonstrate a specific reduction of around 25% of the CCR9, CCR1, CCR3 and/or CCR5-expressing cells by the column. Non-CCR9, CCR1, CCR3 and/or CCR5-expressing cells remained unaffected (data not shown).

(55) In-Vitro Depletion of Target Cell PopulationsRANTES

(56) 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.

(57) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 3:

(58) TABLE-US-00006 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-CO2H

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

(60) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 7. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

(61) 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. 5b.

(62) 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 3Eotaxin Derivatives

(63) Eotaxin has been produced with Lys73 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 eotaxin on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of eoxtaxin, including a 23 amino acid leader sequence (signal peptide) is set forth as SEQ ID NO: 1,

(64) TABLE-US-00007 MKVSAALLWLLLIAAAFSPQGLAGPASVPTTCCFNLANRK IPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKW VQDSMKYLDQKSPTPKP

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

(66) TABLE-US-00008 GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQK AVIFKTKLAKDICADPKKKWVQDSMKYLDQKSPTPKP

(67) 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.

(68) Thus, eoxtaxin derivatised at the -amino side chain functionality of Lys73 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker) to avoid diketopiperazine formation during the synthesis. The molecule is shown schematically in FIG. 6.

(69) A biotin eotaxin 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.

(70) Once synthesised, the activity of the various eoxtaxin 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 CCR3 receptor.

Examples 4 to 7

(71) General Protocols for Chemokine Synthesis

(72) Assembly:

(73) 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.

(74) Removal of Dde Protection:

(75) 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.

(76) Labelling Steps:

(77) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(78) 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.

(79) 2. Capping

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

(81) 3. Fmoc Deprotection

(82) 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.

(83) 4. Couple Biotin-OSu

(84) 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.

(85) Cleavage:

(86) 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.

(87) Purification Protocol:

(88) 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].

(89) Folding Protocol:

(90) 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 4biotinMCP-2 (CCL8)

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

(92) 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.

(93) The linear amino acid sequence (SEQ ID NO: 4) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(94) TABLE-US-00009 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2 X = pyroGlu or Gln

(95) 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:

(96) TABLE-US-00010 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2 X1 = pyroGlu or Gln X75 = K(ivDde)

(97) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 5). 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: 6):

(98) TABLE-US-00011 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).

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

(100) Functional Assay Data:

(101) 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 5Biotineotaxin (Ccl11)

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

(103) 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.

(104) The linear amino acid sequence (SEQ ID NO: 7) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(105) TABLE-US-00012 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2

(106) 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).

(107) 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:

(108) TABLE-US-00013 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2

(109) FmocLys(ivDde)-OH was incorporated as residue 73 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 8). 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: 9):

(110) TABLE-US-00014 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2 X is K(PEG-Biotin)

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

(112) Functional Assay Data:

(113) 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 6biotinRANTES (CCL5)

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

(115) 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.

(116) The linear amino acid sequence (SEQ ID NO: 10) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(117) TABLE-US-00015 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(118) 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:

(119) TABLE-US-00016 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN X is K(ivDde)

(120) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 11). 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: 12).

(121) TABLE-US-00017 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH

(122) 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))

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

(124) Functional Assay Data:

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

Example 7Biotinteck (Ccl25)

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

(127) 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.

(128) The linear amino acid sequence (SEQ ID NO: 13) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(129) TABLE-US-00018 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNKVF-OH X1 = pyroGlu or Gln X64 = Norleucine

(130) 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 (SEQ ID NO: 14):

(131) TABLE-US-00019 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-RESIN X1 = pyroGlu or Gln X64 = Norleucine X72 = K(Dde)

(132) FmocLys(ivDde)-OH was incorporated as residue 72 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 (SEQ ID NO: 15).

(133) TABLE-US-00020 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-OH X1 = pyroGlu or Gln X64 = norleucine 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)

(134) 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.

(135) Functional Assay Data:

(136) 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 8Diagnosis and Treatment of Bipolar Disorder (Bp)

(137) Materials and Methods

(138) 1. Flow Cytometric Analysis of Peripheral Blood

(139) 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).

(140) TABLE-US-00021 TABLE 2 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter Streptavidin PE, APC Biolegend CD16 PE Cy7 BD Biosciences CCR9 APC R&D Systems HLADR APC Cy7 Biolegend CD3 V450 BD Biosciences CD19 V500 BD Biosciences
2. Chemokine Binding Test

(141) 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).

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

(143) 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.

(144) Results and Discussion

(145) 1. Flow Cytometric Analysis of Peripheral Blood

(146) White blood cells from two patients with bipolar disorder (BP) were analysed with flow cytometry. Both patients exhibited a highly increased frequency of CCR9 expressing monocytes (FIG. 9).

(147) 2. Chemokine Binding Test

(148) The CCR9 receptor binds to the chemokine TECK (CCL25) which is mainly expressed in the gut but potentially also in the central nervous system (CNS). Migration of immune cells towards TECK mediates inflammation.

(149) The monocytes from a patient with BP bound biotinylated TECK (bTECK) (FIG. 10).

(150) 3. Cell Depletion

(151) The majority of the CCR9 expressing monocytes were depleted with bTECK-conjugated Sepharose Streptavidin Matrix (FIG. 11).

(152) We conclude that the frequency of monocytes that express the chemokine receptor CCR9 is highly increased in bipolar disorder. These monocytes bind the ligand bTECK, and can be removed with Sepharose Streptavidin matrix conjugated with bTECK.

(153) 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.

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