Treating cancer

Abstract

A method for treating cancer comprising 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 CCR7, CCR5, CCR6, CCR8, CXCR4, CXCR7, CCR4, CCR9, CCR10, CXCR3 or CXCR5 or to a Treg receptor immobilized directly or indirectly on the support thus removing one or more chemokine receptor, optionally CCR7, CCR5, CCR6, CCR8, CXCR4, CXCR7, CCR4, CCR9, CCR10, CXCR3 or CXCR5 or Treg receptor 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 cancer in a subject in need thereof, which comprises: 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 chemokine receptor CCR7 immobilized directly or indirectly on the support, whereby one or more cells expressing chemokine receptor CCR7 are removed from the peripheral blood of the subject; and returning the applied peripheral blood back to the subject, whereby the cancer is treated, and wherein the cancer is a leukemia.

2. The method of claim 1, wherein the levels of circulating tumour cells are reduced in the subject.

3. The method of claim 1, wherein the incidence of tumour metastasis is reduced in the subject.

4. The method of claim 1, wherein the leukemia is selected from chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and leukemic phase of lymphoma.

5. The method of claim 1, wherein the binding reagent is an agonist or an antagonist of CCR7.

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

7. The method of claim 6, wherein the chemokine is selected from CCL19 and CCL21.

8. The method of claim 7, wherein the chemokine is CCL19 (MIP-3b) or CCL21 (SLC) and the chemokine receptor is CCR7.

9. The method of claim 1, wherein the one or more cells are B cells, regulatory T cells, tumour cells, central memory T lymphocytes, or regulatory T lymphocytes.

10. The method of claim 1, wherein the subject has increased levels of expression of CCR7 as compared to a subject that does not have cancer.

11. The method of claim 1, wherein 20-90% of the subject's blood is applied to the column in a single treatment.

12. The method of claim 1, wherein treating cancer comprises: (a) reducing levels of circulating tumour cells in the subject, (b) reducing the incidence of tumour metastasis in the subject, (c) removing regulatory T lymphocytes from the peripheral blood of the subject, (d) debulking in Acute Myeloid Leukemia (AML), or Acute Lymphoblastic Leukemia (ALL) in the subject before harvest for autologous bone marrow/stem cell transplantation, or (e) any combination of (a)-(d).

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1The plastic house and top showing the distribution plate (2) and safety filter units (3 and 4).

(2) FIG. 2The overall leukapheresis system.

(3) FIG. 3The pump with air detector and optical detector (4).

(4) FIG. 4Results of in vitro depletion tests performed on the biotinylated MIP-3alpha coupled matrix showing ability to eliminate CCR6-expressing lymphocytes from blood from three healthy donors.

(5) FIG. 5Example of gating criteria for CCR2 expressing monocytes

(6) FIG. 6Percentage of different leukocyte populations in a patient with Chronic Lymphatic Leukemia and in a healthy control (HC). Blood from a patient with CLL and a healthy control were analysed for the expression of cell specific markers with flow cytometry. The B cells were characterized as CD19 positive, the T cells as CD3 positive, granulocytes as CD16 positive and monocytes as CD14 positive.

(7) FIG. 7Expression of CCR7 on B cells from a patient with Chronic Lymphatic Leukemia. Blood from a patient with CCL was analysed for the expression of various chemokine receptors by flow cytometry. The B cells were characterized as CD19+ lymphocytes.

(8) FIG. 8Binding of the biotinylated chemokine MIP3b (CCL19) to B cells from a patient with Chronic Lymphatic Leukemia. Blood from a patient with CLL was incubated with bMIP3b and analysed by flow cytometry. The B cells were characterized as CD19+ lymphocytes.

(9) FIG. 9Depletion of B cells with Sepharose Streptavidin matrix conjugated with bMIP3b. Blood cells from a patient with CLL were incubated with bMIP3b-SepharoseStreptavidin matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed by flow cytometry and compared with cells that had not been incubated with the MIP3b-matrix (Before Depletion).

(10) FIG. 10Frequency of CCR4 positive Tregs in eight healthy controls (HC), two patients with Pancreatic Cancer (PC) and one patient with Urinary Bladder Cancer (UBC). The expression of chemokine receptors and specific cell markers was analyzed with flow cytometry. The Tregs were defined as CD4 positive, CD25 positive (hi), CD127 negative cells.

(11) FIG. 11CCR4 expression on Tregs compared to T cells in UBC (left) and PC (right). The expression of chemokine receptors and specific cell markers was analysed with flow cytometry in two patients with PC and one patient with UBC.

(12) FIG. 12Binding of the chemokine bMDC to Tregs. Blood cells from a patient with PC were incubated with biotinylated chemokine or an anti-CCR4 antibody and analyzed with flow cytometry.

(13) FIG. 13Depletion of CCR4 expressing T cells with Sepharose Streptavidin-matrix conjugated with biotinylated MDC (bMDC). Blood cells from a patient with UBC were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The unbound 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

(14) The mechanism behind seeding of metastatic cells in to particular organs is by the expression of chemokines in the organ recruiting chemokine receptor expressing tumor cells. For example metastatic colon cancer cells express CCR6 and they home to liver because CCL20 is expressed in the liver allowing entrance of CCR6 expressing cancer cells.

(15) In non Hodgkin lymphomas (T-NHL) the expression of CCR7 indicates a higher lymphatic dissemination which was demonstrated to migrate towards CCL21, the preferred chemokine for entrance of cells in to lymphoid organs.

(16) Lung cancer metastases seem to be dependent on the expression of CXCR4 on tumour cells and the local expression of the corresponding chemokine CXCL12 (SDF-1) for the successful metastasis. In line with these findings development of small molecules for inhibition of CXCR4 mediated metastasis is under development.

(17) Regulatory T cells (Tregs) are upregulated in many cancers as an immune escape mechanism induced by the tumor. In order to avoid immune recognition and elimination tumors activate and recruit Tregs. The production of chemokines by the tumor results in recruitment of circulating Tregs and therefore protection against immune recognition and elimination. Tregs may be recruited to the tumour by CCL17 and CCL22 binding to CCR4 expressed on the Treg. In addition CCR8 mediated recruitment by CCL1 may occur. Since many treatment regimes with chemotherapy involve activation of the immune system, eliminating Tregs in the circulation by leukapheresis may thus favour immune recognition and elimination of tumor cells, thereby enhancing cancer therapy.

(18) In chronic leukemic disorders the number of circulating tumor cells inhibit normal hematopoeis and therefore cause symptoms. The increased production of chemokines such as CCL3 correlates with poor prognosis (Blood. 2011 Feb. 3; 117(5):1662-9. Epub 2010 Nov. 29). Thus elimination of chemokine receptor expressing leukemic cells will favorably affect the disease in terms of symptoms and disease progression.

(19) It is shown herein that cancer patients, in particular subjects suffering from UBC and PC, exhibit an increased frequency of CCR4 expressing circulating Tregs and that this response is specific to Tregs (and does not apply to other T lymphocytes). CCR4 expressing cells may be thus be targeted in order to treat cancer. Treatment may rely upon suitable binding reagents such as CCL22 (MDC) and derivatives thereof, as described herein in further detail. It is also shown herein that subject suffering from leukemias, such as CLL, have a highly increased number of circulating B cells. The B cells express characteristic chemokine receptors, such as CCR7. It is also shown herein that CCR7 expressing B cells may be efficiently depleted using MIP3b as a specific binding reagent in a leukapheresis method.

Example 1Tailored Leukapheresis

(20) Column Design and Properties

(21) Introduction

(22) 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.

(23) The Column

(24) The column is intended to be used as a leukapheresis treatment for cancer. It will specifically remove CCR5, CCR6, CCR7, CCR8, CXCR4, CXCR7, CCR4, CCR9, CCR10, CXCR3 and/or CXCR5-expressing cells, such as tumour cells and leukocytes, such as regulatory T lymphocytes, through the use of a binding reagent, more specifically an MIP-3alpha (CCL20), CCL19, CCL21, CCL1, CXCL11, CXCL12, CCL25 (TECK), CCL27 (CTACK), CCL28 (MEC), CXCL9 (MIG), CXCL10 (IP10), CXCL13 (BCA-1), CCL17 (TARC) and CCL22 (MDC) containing resin, exploiting the CCR5, CCR6, CCR7, CCR8, CXCR4, CXCR7, CCR4, CCR9, CCR10, CXCR3 and/or CXCR5-chemokine interaction. Treg receptor expressing cells, such as CLA receptor, CCR4 or CCR8 expressing cells may be specifically removed through the use of an appropriate binding reagent, more specifically an SELE, CCL17, CCL22 and/or CCL1 containing resin. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose BigBeads matrix and one or more of biotinylated MIP-3alpha (CCL20), CCL19, CCL21, CCL1, CXCL11, CXCL12, CCL25 (TECK), CCL27 (CTACK), CCL28 (MEC), CXCL9 (MIG), CXCL10 (IP10), CXCL13 (BCA-1), CCL17 (TARC) and CCL22 (MDC) and/or SELE, CCL17, CCL22 and/or CCL1 bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(25) The Plastic House (FIG. 1)

(26) 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. 1. 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.

(27) Streptavidin Sepharose BigBeads

(28) 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.

(29) Binding Reagent

(30) Coupled to the matrix is the third component of the device, one or more binding reagents that bind specifically to CCR5, CCR6, CCR7, CCR8, CXCR4, CXCR7, CCR4, CCR9, CCR10, CXCR3 and/or CXCR5 and/or to CLA receptor, CCR4 and/or CCR8 where Tregs are specifically targeted. One or more chemokines selected from the group consisting of: MIP-3alpha (CCL20), CCL19, CCL21, CCL1, CXCL11, CXCL12, CCL25 (TECK), CCL27 (CTACK), CCL28 (MEC), CXCL9 (MIG), CXCL10 (IP10), CXCL13 (BCA-1), CCL17 (TARC) and CCL22 (MDC) may be employed. Alternatively, SELE, CCL17, CCL22 and/or CCL1 may be employed to target Tregs. These peptides may be synthetic, engineered versions of the human chemokine, which are truncated and biotinylated, but retain binding activity to the CCR5, CCR6, CCR7, CCR8, CXCR4, CXCR7, CCR4, CCR9, CCR10, CXCR3 and/or CXCR5 receptor (or CLA receptor, CCR4 and/or CCR8 where Tregs are targeted). 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.

(31) The Apheresis System

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

(33) The Circuit

(34) The system is illustrated in FIG. 2. 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.

(35) The 4008 ADS Pump

(36) 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.

(37) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 3. 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.

(38) Legend for FIG. 3: 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

(39) Preparation of the Patient

(40) 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.

(41) Leukapheresis Time and Flow Rate

(42) 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.

(43) Storage Conditions

(44) 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.

(45) Transport Conditions

(46) 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.

(47) In-Vitro Depletion of Target Cell Populations

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

(49) 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. 4.

(50) 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).

Example 2ATreatment of Chronic Lymphatic Leukemia (CLL) Patient

(51) Materials and Methods

(52) 1. Flow Cytometric Analysis of Peripheral Blood

(53) 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 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).

(54) TABLE-US-00023 TABLE 2 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter Streptavidin PE, APC Biolegend CCR7 PerCP Cy5.5 Biolegend CD16 PE Cy7 BD Biosciences CD3 V450 BD Biosciences CD19 V500 BD Biosciences

(55) 2. Chemokine Binding Test

(56) 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 for 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).

(57) 3. Cell Depletion By Matrix Conjugated with Biotinylated Chemokine

(58) 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 m 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.

(59) Results and Discussion

(60) 1. Flow Cytometric Analysis of Peripheral Blood

(61) White blood cells from a CLL patient were analysed by flow cytometry. The patient had a highly increased number of circulating B cells; 92% compared to approximately 2% in healthy blood (FIG. 6). This finding is common in CLL where malignant B cells undergo extensive proliferation, accumulate in the bone marrow and blood and crowd out healthy blood cells.

(62) 2. Chemokine Binding Test

(63) All the B cells were shown to express the chemokine receptor CCR7 (FIG. 7) based upon flow cytometry data and binding by an anti-CCR7 antibody. CCR7 is important for lymph node homing which is mediated by binding to chemokines such as MIP3b (CCL19) and SLC (CCL21) that are expressed in lymphoid tissue. In accordance with the CCR7 expression, all the B cells bound to a biotinylated MIP3b (bMIP3b) (FIG. 8).

(64) 3. Cell Depletion By Matrix Conjugated with Biotinylated Chemokine

(65) The B cells were efficiently depleted using bMIP3b-conjugated Sepharose Streptavidin Matrix. Before depletion the B cells constituted 89.1% of the cells and after depletion 26.4% (FIG. 9).

(66) We conclude that B cells in CLL express CCR7 and bind the ligand bMIP3b. Furthermore the majority (62.7%) of the CCR7 expressing B cells can be removed using a Sepharose Streptavidin matrix conjugated with bMIP3b.

Example 2BTreatment of Cancers Via Removal of CCR4 Expressing Tregs Using Biotinylated-MDC (CCL22)

(67) Materials and Methods

(68) 1. Flow Cytometric Analysis of Peripheral Blood

(69) 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).

(70) TABLE-US-00024 TABLE 3 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CCR4 PerCP Cy5.5 BD Biosciences CD127 PE Cy7 Biolegend CD4 V500 Biolegend CD25 APCCy7 Biolegend CD3 Pacific blue BD Biosciences Streptavdin PerCpCy5.5 BD Biosciences CD25 FITC BD Biosciences

(71) 2. Chemokine Binding Test

(72) 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).

(73) 3. Cell Depletion By Matrix Conjugated with Biotinylated Chemokine

(74) 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.

(75) Results and Discussion

(76) T regulatory cells (Tregs) are a subpopulation of T cells that suppress the immune system in order to maintain immune homeostasis. In cancer, the Tregs can inhibit an effective immune response against the tumor and thus removal of the Tregs could lead to a better immune activation against the cancer cells. White blood cells from patients with Urinary Bladder Cancer (UBC) and Pancreas Cancer (PC) were analyzed for the expression of chemokine receptors with flow cytometry. The cancer patients exhibited an increased frequency of circulating T regulatory cells (Tregs) that expressed the chemokine receptor CCR4, based upon flow cytometry data and binding by an anti-CCR4 antibody (FIG. 10).

(77) In both UBC and PC patients, CCR4 was highly upregulated on Tregs compared with the total T cell population (FIG. 11).

(78) The ligand for CCR4 is the chemokine MDC (CCL22). In accordance with the CCR4 expression, the Tregs bound to biotinylated MDC (bMDC) (FIG. 12).

(79) The CCR4 expressing T cells could be depleted with bMDC-conjugated Sepharose Streptavidin Matrix (FIG. 13).

(80) We conclude that the frequency of Tregs that express CCR4 is enhanced in PC and UBC. These cells can bind the ligand bMDC. The Tregs express significantly more CCR4 than conventional T cells and can thus be specifically deleted with Sepharose Streptavidin matrix conjugated with bMDC.

Examples 3 to 10: General ProtocolsSynthesis of Chemokines

(81) Assembly:

(82) 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.

(83) Removal of Dde Protection:

(84) 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.

(85) Labelling Steps:

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

(87) 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.

(88) 2. Capping

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

(90) 3. Fmoc deprotection

(91) 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.

(92) 4. Couple Biotin-OSu

(93) 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.

(94) Cleavage:

(95) 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.

(96) Purification Protocol:

(97) 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].

(98) Folding Protocol:

(99) 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 3BiotinMCP-2 (CCL8)

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

(101) 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.

(102) The linear amino acid sequence (SEQ ID NO: 1) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(103) TABLE-US-00025 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2

(104) X1=pyroGlu or Gln

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

(106) 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:

(107) TABLE-US-00026 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2

(108) X1=pyroGlu or Gln

(109) X75=K(ivDde)

(110) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 2). 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: 3).

(111) TABLE-US-00027 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2

(112) X1=pyroGlu or Gln

(113) X75=K(PEG-Biotin)

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

(115) Functional Assay Data:

(116) 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 4BiotinRANTES (CCL5)

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

(118) 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.

(119) The linear amino acid sequence (SEQ ID NO: 4) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

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

(121) 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:

(122) TABLE-US-00029 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNR QVCANPEKKWVREYINSLEXS-RESIN

(123) X=K(ivDde)

(124) FmocLys(ivDde)-OH was incorporated as residue 67 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 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).

(125) TABLE-US-00030 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNR QVCANPEKKWVREYINSLEXS-OH

(126) X is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated (e.g. K-biotin), optionally via a spacer molecule such as PEG, in particular K(PEG-Biotin)

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

(128) Functional Assay Data:

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

Example 5BiotinMIP-3 (CCL20)

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

(131) Modifications: Human MIP-3a 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.

(132) The linear amino acid sequence (SEQ ID NO: 7) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 68 (K):

(133) TABLE-US-00031 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSV CANPKQTWVKYIVRLLSKKVKNM-OH

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

(135) TABLE-US-00032 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSV CANPKQTWVKYIVRLLSKKVXNM-RESIN

(136) X=K(ivDde)

(137) FmocLys(ivDde)-OH was incorporated as residue 68 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).

(138) TABLE-US-00033 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSV CANPKQTWVKYIVRLLSKKVXNM-OH

(139) 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)

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

(141) Functional Assay Data:

(142) 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 6BiotinSDF-1 (CXCL12)

(143) Target Molecule: SDF-1 derivatised at the s-amino side chain functionality of Lys(64) with Biotin (TFA salt)

(144) Modifications: Truncated form of human SDF-1a corresponding to residues 1-67 of the mature protein, which encompasses the sequence corresponding to the chemokine fold. The full length mature protein is 72 amino acids (the signal peptide is 21 amino acids in a 93 amino acid immature protein). The naturally occurring lysine at position 64 was modified through biotinylation on the resin.

(145) The linear amino acid sequence (SEQ ID NO: 10) is shown, prior to attachment of the biotin molecule at amino acid 64 (K):

(146) TABLE-US-00034 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALN-OH

(147) The engineered SDF-1 sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(148) TABLE-US-00035 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKXALN-RESIN

(149) X=K(ivDde)

(150) FmocLys(ivDde)-OH was incorporated as residue 64 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).

(151) TABLE-US-00036 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEXALN-OH

(152) 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, especially K(Biotin)

(153) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinSDF-1: obtained =8055.5 Da; expected 8057.5 Da.

(154) Functional Assay Data:

(155) biotinSDF-1 was tested for agonist activity in an Aequorin assay against hCXCR4, (Euroscreen) and an EC50 value of 17.3 nM was reported. c.f. EC50 for recombinant native SDF-1 is 12.0 nM.

Example 7BiotinMDC (CCL22)

(156) Target Molecule: MDC derivatised at the -amino side chain functionality of Lys(66) with PEG-Biotin (TFA salt)

(157) Modifications: Human MDC corresponding to residues 1-69, is initially expressed as 93 amino acids comprising the chemokine fold, and a 24 amino acid signal peptide which is cleaved off. The naturally occurring lysine at position 66 was modified through biotinylation on the resin. A PEG spacer was incorporated between the -amino functionality and the biotin.

(158) The linear amino acid sequence (SEQ ID NO: 13) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 66 (K):

(159) TABLE-US-00037 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFR DKEICADPRVPWVKMILNKLSQ-NH.sub.2

(160) The engineered MDC 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:

(161) TABLE-US-00038 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFR DKEICADPRVPWVKMILNXLSQ-RESIN

(162) X=K(ivDde)

(163) FmocLys(ivDde)-OH was incorporated as residue 66 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 14). 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).

(164) TABLE-US-00039 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFR DKEICADPRVPWVKMILNXLSQ-NH.sub.2

(165) 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, especially K(PEG-Biotin)

(166) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMDC: obtained =8456.1 Da; expected 8456.9 Da.

(167) Functional Assay Data:

(168) BiotinMDC was tested for agonist activity in an Aequorin assay against hCCR4, (Euroscreen) and an EC50 value of 4.5 nM was reported. c.f. EC50 for recombinant native MDC is 3.6 nM.

Example 8BiotinTARC (CCL17)

(169) Target Molecule: TARC derivatised at the s-amino side chain functionality of Lys(72) with PEG-Biotin (TFA salt)

(170) Modifications: Human TARC corresponding to residues 1-71, is initially expressed as 94 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. An additional lysine was inserted at the C-terminus, at position 72, and modified through biotinylation on the resin. A PEG spacer was incorporated between the -amino functionality and the biotin.

(171) The linear amino acid sequence (SEQ ID NO: 16) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(172) TABLE-US-00040 H-ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGR AICSDPNNKRVKNAVKYLQSLERSX-NH.sub.2

(173) X is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated (e.g. K-biotin), optionally via a spacer molecule such as PEG, in particular K(PEG-Biotin)

(174) The engineered TARC 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:

(175) TABLE-US-00041 H-ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQG RAICSDPNNKRVKNAVKYLQSLERSX-RESIN

(176) X=K(ivDde)

(177) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 17). 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: 18).

(178) TABLE-US-00042 H-ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQG RAICSDPNNKRVKNAVKYLQSLERSX-NH.sub.2

(179) X=K(PEG-Biotin)

(180) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinTARC: obtained=8577.2 Da; expected 8577.8 Da.

(181) Functional Assay Data:

(182) BiotinTARC was tested for agonist activity in an Aequorin assay against hCCR4, (Euroscreen) and an EC50 value of 3.1 nM was reported. c.f. EC50 for recombinant native TARC is 2.6 nM.

Example 9BiotinMIP-3 (CCL19)

(183) Target Molecule: MIP-3 derivatised at the -amino side chain functionality of Lys(78) with Biotin (TFA salt)

(184) Modifications: Human MIP-3 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. An additional lysine was inserted at the C-terminus, at position 78, and modified through biotinylation on the resin.

(185) The linear amino acid sequence (SEQ ID NO: 19) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):

(186) TABLE-US-00043 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2

(187) X is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated (e.g. K-biotin), optionally via a spacer molecule such as PEG, in particular K(PEG-Biotin)

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

(189) TABLE-US-00044 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN

(190) X is FmocLys(ivDde)

(191) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 20). 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: 21).

(192) TABLE-US-00045 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2

(193) X is K(Biotin)

(194) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMIP-3: obtained=9148.8 Da; expected 9149.7 Da.

(195) Functional Assay Data:

(196) biotinMip-3 was tested for agonist activity in an Aequorin assay against hCCR7, (Euroscreen) and an EC50 value of 11.0 nM was reported. c.f. EC50 for recombinant native MIP-3 is 1.6 nM.

Example 10BiotinITAC (CXCL11)

(197) 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)

(198) 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.

(199) The linear amino acid sequence (SEQ ID NO: 22) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(200) TABLE-US-00046 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNF-OH

(201) 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:

(202) TABLE-US-00047 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNFX-RESIN

(203) X is PEG-K(ivDde)

(204) 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 c-amino side chain functionality of the additional Lys (SEQ ID NO: 24). 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: 25).

(205) TABLE-US-00048 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNFX-OH

(206) X is PEG-K(Biotin)

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

(208) Functional Assay Data:

(209) 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 11BiotinTECK (CCL25)

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

(211) 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.

(212) The linear amino acid sequence (SEQ ID NO: 26) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(213) TABLE-US-00049 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-OH

(214) X1=pyroGlu or Gln

(215) X64=Norleucine

(216) 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, e.g. K(PEG-Biotin)

(217) 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:

(218) TABLE-US-00050 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-RESIN

(219) X1=pyroGlu or Gln

(220) X64=Norleucine

(221) X72=K(ivDde)

(222) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 27). 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: 28).

(223) TABLE-US-00051 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-OH

(224) X1=pyroGlu or Gln

(225) X64=Norleucine

(226) X72 is K(PEG-Biotin)

(227) 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.

(228) Functional Assay Data:

(229) 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 12BiotinCTAC (CCL27)

(230) Target Molecule: CTAC derivatised at the -amino side chain functionality of Lys(87) with PEG-Biotin (TFA salt)

(231) Modifications: Human CTAC corresponding to residues 1-88, is initially expressed as 112 amino acids comprising the chemokine fold, and a 24 amino acid signal peptide which is cleaved off. The Met(87) within the sequence was mutated to lysine to provide a lysine at position 87 which was modified through biotinylation on the resin. A PEG spacer was incorporated between the -amino functionality and the biotin.

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

(233) TABLE-US-00052 FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQR SICIHPQNPSLSQWFEHQERKLHGTLPKLNFGMLRKXG

(234) 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)

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

(236) TABLE-US-00053 H-FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLA QRSICIHPQNPSLSQWFEHQERKLHGTLPKLNFGMLRKXG-RESIN

(237) X=K(ivDde)

(238) FmocLys(ivDde)-OH was incorporated as residue 87 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 30). 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: 31).

(239) TABLE-US-00054 H-FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLA QRSICIHPQNPSLSQWFEHQERKLHGTLPKLNFGMLRKXG-OH

(240) X=K(PEG-Biotin)

(241) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinCTAC: obtained=10513.4 Da; expected 10514.2 Da.

(242) Functional Assay Data:

(243) BiotinCTAC was tested for agonist activity in an Aequorin assay against hCCR10, (Euroscreen) and an EC50 value of 49.4 nM was reported. c.f. EC50 for recombinant native CTAC is 33.5 nM.

Example 13BiotinIP-10 (CXCL10)

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

(245) 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.

(246) The linear amino acid sequence (SEQ ID NO: 32) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(247) TABLE-US-00055 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSP-OH

(248) 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:

(249) TABLE-US-00056 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSPX-RESIN

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

(251) 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: 33). 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: 34):

(252) TABLE-US-00057 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSPX-OH

(253) 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)

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

(255) Functional Assay Data:

(256) 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.

(257) 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.

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