Diagnosing and treating inflammatory diseases

Abstract

A method of diagnosing, monitoring progression of, or monitoring treatment of inflammatory bowel disease comprises determining the levels of CD14.sup.+HLA-DR.sup.hi monocytes or monocytes expressing CCR7 or CCR9 or both CCR7 and CCR9 in a sample obtained from a subject, wherein high levels of CD14.sup.+HLA-DR.sup.hi monocytes or monocytes expressing CCR7 or CCR9 or both CCR7 and CCR9, or increased levels of CD14.sup.+HLA-DR.sup.hi monocytes or monocytes expressing CCR7 or CCR9 or both CCR7 and CCR9 compared to control, indicate the presence or progression of inflammatory bowel disease. Similar methods for diagnosing irritable bowel syndrome are also described. Various companion therapeutic methods and useful binding reagents are also described.

Claims

1. A method for treating inflammatory bowel disease comprising: applying peripheral blood from a patient diagnosed as having inflammatory bowel disease to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to a marker of CD14.sup.+HLA-DR.sup.hi monocytes comprising CD14, wherein the one or more binding reagents are immobilized directly or indirectly on the support thus removing CD14.sup.+HLA-DR.sup.hi monocytes from the peripheral blood of the patient; and returning the peripheral blood from which CD14.sup.+HLA-DR.sup.hi monocytes are removed to the patient.

2. The method of claim 1 wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease.

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

4. The method of claim 1, further comprising selecting a patient for treatment, the patient having an increase in the level of chemokine receptors comprising CD14+ cells, or an increased fraction/cell number of CD14+CCR9 positive cells.

5. The method of claim 1 wherein the apheresis column is loaded with a solid support comprising at least two binding reagents capable of specifically binding to at least two markers of CD14.sup.+HLA-DR.sup.hi monocytes, wherein at least one of said markers comprises CD14.

6. The method of claim 1 wherein the binding reagents are selected from an anti-CD14 antibody or a modified version thereof.

7. A method for treating inflammatory bowel disease comprising: applying peripheral blood from a patient diagnosed as having inflammatory bowel disease to an apheresis column loaded with a solid support comprising one or more binding reagents capable of specifically binding to a marker of CD14.sup.+HLA-DR.sup.hi monocytes is selected from CD14, CCR7, and CCR9 wherein the binding reagent is immobilized directly or indirectly on the support thus removing CD14.sup.+HLA-DR.sup.hi monocytes from the peripheral blood of the patient; and returning the peripheral blood from which CD14.sup.+HLA-DR.sup.hi monocytes are removed to the patient, and wherein the binding reagents is selected from CCL19 or a modified version thereof, and wherein the modified CCL19 chemokine comprises the amino acid sequence of SEQ ID NO: 4, and wherein the amino acid residue at position 78 of SEQ ID NO: 4 is biotinylated, optionally via a spacer group, to permit immobilization of the chemokine on a solid support.

8. The method of claim 7, wherein the amino acid residue at position 78 of SEQ ID NO: 4 is a lysine residue which is biotinylated via a polyethylene glycol (PEG) spacer group, to permit immobilization of the chemokine on a solid support.

9. The method of claim 8, wherein the PEG spacer is 3,6-dioxo aminooctanoic acid.

Description

DESCRIPTION OF THE FIGURES

(1) A. Diagnosing and Treating Inflammatory Bowel Disease and Irritable Bowel Syndrome

(2) FIG. 1. Representative flow cytometry plots showing the gating strategies used throughout the study for the CD14+HLA-DRhi (lower left), CD14loCD16+ (lower middle) and CD14+CD16− (lower right) monocyte populations.

(3) FIG. 2. CD14+HLA-DRhi monocytes are increased and correlate to disease activity in patients with ulcerative colitis and Crohn's disease.

(4) (A) Frequency of CD14+HLA-DRhi monocytes in peripheral blood of IBD patients compared to controls, as determined with flow cytometry. Bars represent mean values ±SEM from controls (n=11) and patients (n=31) with active ulcerative colitis (n=20; UC-DAI 6-12) or Crohn's disease (n=11; HBI 8-16) (p=0.006).

(5) (B) Regression analysis of CD14+HLA-DRhi monocytes and clinical disease activity in patients with ulcerative colitis (p=0.024; r2=0.072). Data represents measurements (n=84) from 28 unique patients at different time points during treatment.

(6) (C) Regression analysis of CD14+HLA-DRhi monocytes and clinical disease activity in patients with Crohn's disease (p=0.016; r2=0.190). Data represents measurements (n=29) from 11 unique patients at different time points during treatment FIG. 3. CD14+HLA-DRhi monocytes are targets for therapy in IBD.

(7) CD14+HLA-DRhi monocyte levels in IBD patients during treatment with (A) GMA apheresis (n=18), (B) corticosteroids (n=16) or (C) anti-TNF-α biological therapy (n=14). Control patient reference levels (n=11) are included in all graphs. (D) IBD patients receiving GMA or corticosteroid therapy, divided into remission (n=12) and non-remission (n=7) patients as well as healthy controls (n=11). Error bars represent group mean values ±SEM.

(8) FIG. 4. CD14+HLA-DRhi monocytes produce high levels of inflammatory mediators.

(9) (A) Representative flow cytometry plots depicting CD14+HLA-DRhi and CD14+HLADR

(10) lo purity after flow cytometry sorting.

(11) (B) PCR analysis of TNF-α in CD14+HLA-DRhi monocytes after LPS activation for 2 hrs

(12) (n=4, p=0.0047).

(13) (C) Functional grouping of target transcripts from PCR array analysis of CD14+HLADR

(14) hi and CD14+HLA-DRlo monocytes from three independent healthy donors after LPS activation for 6 hrs.

(15) (D) The 20 target transcripts that represented the strongest up- and down-regulation in PCR array analyses of CD14+HLA-DRhi monocytes as compared to the CD14+HLADR

(16) lo population after LPS activation for 6 hrs. Fold changes range between 347.3-10.9 and −10.3-232.3, respectively.

(17) FIG. 5. The relative chemokine receptor expression in CD14+HLA-DRhi monocytes.

(18) (A) The CD14+HLA-DRhi subset is distinguished from CD14loCD16+ and CD14+CD16−

(19) monocytes by their expression of CCR7 and CCR9. (B) Chemokine receptors whose

(20) expression levels do not differentiate the CD14+HLA-DRhi population. (C) Control staining showing the difference in expression of CD16 and HLA-DR as well as morphology defined by side-scatter (SSC) and forward-scatter (FSC) appearance between the monocyte populations discussed in this study. Data presented are from one representative IBD patient.

(21) FIG. 6. CCR9-CCL25 functional interaction assay in CD14+HLA-DRhi monocytes

(22) Depletion of CCR9-expressing CD14+HLA-DRhi monocytes from IBD patients using

(23) CCL25-coated microbeads (n=5; p=0.0112).

(24) FIGS. 7a, 7b & 7c—the binding of biotinylated CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor;

(25) FIGS. 8a, 8b & 8c—the binding of biotinylized CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a patient with CD;

(26) FIG. 9—The plastic house and top showing the distribution plate (2) and safety filter units (3 and 4).

(27) FIG. 10—The overall leukapheresis system.

(28) FIG. 11—The pump with air detector and optical detector (4).

(29) FIG. 12—Depletion of CCR9-expressing cell populations in one blood donor. Total cell populations are unaffected after the column passage.

(30) FIG. 13—Depletion of CCR9-expressing cell populations in one IBD patient. Total cell populations are unaffected after the column passage.

(31) FIG. 14—HPLC of purified folded Biotin-TECK(Nleu).

(32) FIG. 15—Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu).

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

(34) FIG. 17—Expression of CCR9 expressing monocytes in seven patients with Crohn's disease (CD) and in 20 healthy controls (HC). Blood from patients with CD and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells.

(35) FIG. 18—Binding of the chemokine bTECK (CCL25) to monocytes from a patient with Crohn's disease. Blood from a patient with CD was incubated with bTECK and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(36) FIG. 19—Depletion of CCR9 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bTECK. Blood cells from a patient with CD 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).

(37) FIG. 20—The CCR9 expressing monocytes show a 80% expression of HLADRhi. The monocytes were characterized as CD14 positive cells.

(38) FIG. 21—Expression of CCR9 expressing monocytes in 2 patients with Irritable bowel syndrome (IBS) and in 20 healthy controls (HC). Blood from patients with IBS and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells.

(39) FIG. 22—Binding of the chemokine bTECK (CCL25) to monocytes from a patient with IBS. Blood from a patient with IBS was incubated with bTECK and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(40) FIG. 23—Depletion of CCR9 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bTECK. Blood cells from a patient with IBS 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).

(41) FIG. 24—Expression of CCR9 expressing monocytes in 4 patients with Ulcerative Colitis (UC), 2 patients with Irritable bowel syndrome (IBS) and 8 patients with Crohns Disease (Crohns). Blood from patients was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells.

(42) FIG. 25a—The CCR9 expressing monocytes show a 25% expression of HLADRhi.

(43) FIG. 25b—The CCR9 expressing monocytes which also have a high expression of HLADR can be depleted with bTECK conjugated sepharose matrix. The monocytes were characterized as CD14 positive cells.

(44) B. Treating Conditions Associated with Metabolic Syndrome

(45) FIGS. 26a, 226b & 26c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(46) FIG. 27a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(47) FIG. 27b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(48) FIG. 28a—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(49) FIG. 28b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.

(50) FIG. 29—Sequence (SED ID NO: 8) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(51) FIG. 30—Sequence (SED ID NO: 8) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(52) FIG. 31—Sequence (SED ID NO: 8) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(53) FIG. 32—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(54) FIG. 33—Sequence (SED ID NO: 13) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(55) FIG. 34—Sequence (SED ID NO: 14) and biotinylation, of RANTES derivative

(56) FIG. 35—Example of gating criteria for CCR2-expressing monocytes.

(57) FIG. 36—Expression of CCR2 expressing monocytes in 9 patients with diabetes mellitus (DM) and in 20 healthy controls (HC). Blood from patients with DM and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells.

(58) FIG. 37—Binding of the chemokine bMCP1 (CCL2) to monocytes from 3 patients with Diabetes Mellitus and in 20 healthy controls (HC). Blood from 3 patient with DM was incubated with bMCP1 and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(59) FIG. 38—Depletion of CCR2 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bMCP1. Blood cells from a patient with DM were incubated with bMCP1—Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bMCP1-matrix (Before Depletion).

(60) FIG. 39—Expression of CCR2 expressing B cells in seven patients with adiposis dolorosa (AD) and in 20 healthy controls (HC). Blood from patients with AD and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The B cells were characterized as CD19 positive cells.

(61) FIG. 40—Binding of the chemokine bMCP1 (CCL2) to B cells from seven patient with Adiposis Dolorosa. Blood from a patient with AD was incubated with bMCP1 and analysed with flow cytometry. The B cells were characterized as CD19 positive cells.

(62) FIG. 41—Depletion of CCR2 expressing B cells with Sepharose Streptavidin-matrix conjugated with bMCP1. Blood cells from a patient with AD were incubated with bMCP1-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bMCP1-matrix (Before Depletion).

(63) FIG. 42—Increased frequency of CCR1 expressing monocytes in 4 patients with Adiposis dolorosa compared to healthy controls.

(64) FIG. 43—Depletion of CCR1 expressing moncoytes with SepharoseStrepavidin-matrix-bRANTES. Blood cells from a healthy control were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion).

(65) FIG. 44—Increased frequency of CCR4 expressing T cells in four patients with type 2 Diabetes.

(66) FIG. 45—Increased frequency of CCR5 expressing T cells in four patients with type 2 Diabetes.

(67) C. Treating Inflammatory Arthritis

(68) FIGS. 46a, 46b & 46c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(69) FIG. 47a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(70) FIG. 47b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(71) FIG. 48a—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(72) FIG. 48b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells (CCR1, 3 or 5) from peripheral blood taken from a healthy donor.

(73) FIG. 49—Sequence (SED ID NO: 24) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(74) FIG. 50—Sequence (SED ID NO: 24) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(75) FIG. 51—Sequence (SED ID NO: 25) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(76) FIG. 52—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(77) FIG. 53—Sequence (SED ID NO: 27) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(78) FIG. 54—Sequence and (SED ID NO: 34) biotinylation, of RANTES derivative

(79) FIG. 55—example of gating criteria for CCR2 expressing monocytes.

(80) FIG. 56a—Expression of CCR1 receptor on monocytes in 2 patients with Rheumatoid arthritis (RE).

(81) FIG. 56b—Expression of CCR2 receptor on monocytes in 2 patients with Rheumatoid arthritis (RE).

(82) FIG. 56c—Expression of CCR5 receptor on T cells in 2 patients with Rheumatoid arthritis (RE). Blood from patients with RE was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells and T cells were characterized as CD3 positive cells.

(83) FIG. 57a—Binding of the chemokine bMCP1 (CCL2) to monocytes from a patient with Rheumatoid arthritis. Blood from a patient with RE was incubated with bMCP1 and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(84) FIG. 57b—Binding of the chemokine bRANTES (CCL5) to T cells from a patient with Rheumatoid arthritis. Blood from a patient with RE was incubated with bRANTES and analysed with flow cytometry. The T cells were characterized as CD3 positive cells.

(85) FIG. 58a—Depletion of CCR2 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bMCP1.

(86) FIG. 58b—Depletion of CCR5 expressing T cells with Sepharose Streptavidin-matrix conjugated with bRANTES. Blood cells from a patient with Rheumatoid arthritis were incubated with b chemokine-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 b-chemokine-matrix (Before Depletion).

(87) D. Treating Cancer

(88) FIG. 59—Results 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.

(89) FIG. 60—Example of gating criteria for CCR2 expressing monocytes

(90) FIG. 61—Percentage 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.

(91) FIG. 62—Expression 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.

(92) FIG. 63—Binding 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.

(93) FIG. 64—Depletion 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).

(94) FIG. 65—Frequency of CCR4 positive Tregs in eight healthy controls (HC), two patients with Pancreatic Cancer (PC) and one patient with Urinay 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.

(95) FIG. 66—CCR4 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.

(96) FIG. 67—Binding 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.

(97) FIG. 68—Depletion 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).

(98) E. Treating Mental Disorders

(99) FIG. 69a—binding of eotaxin to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

(100) FIG. 69b—binding of CCR3-antibody to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

(101) FIG. 70a—Results 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.

(102) FIG. 70b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.

(103) FIG. 71—Sequence (SED ID NO: 78) and biotinylation, via a spacer group, of mature protein eotaxin derivative containing C-terminal amide.

(104) FIG. 72—Sequence (SED ID NO: 86) and biotinylation, of RANTES derivative.

(105) FIG. 73—Example of gating criteria for CCR2 expressing monocytes.

(106) FIG. 74—Frequency 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.

(107) FIG. 75—Binding 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.

(108) FIG. 76—Depletion 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).

(109) F. Treating Conditions Associated with Allergy

(110) FIG. 77a—binding of eotaxin to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

(111) FIG. 77b—binding of CCR3-antibody to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

(112) FIG. 78a—Results 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.

(113) FIG. 78b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.

(114) FIG. 79—Sequence (SED ID NO: 93) and biotinylation, via a spacer group, of mature protein eotaxin derivative containing C-terminal amide.

(115) FIG. 80—Sequence (SED ID NO: 97) and biotinylation, of RANTES derivative

(116) FIGS. 81a, 81b & 81c—the binding of IL-8 by CD4+, CD8+ T-cells and CD16+ neutrophils respectively, obtained from peripheral blood of a healthy donor;

(117) FIG. 82—example of gating criteria for CCR2 expressing monocytes.

(118) FIG. 83a—Frequency of neutrophils that express CXCR1 and CXCR2.

(119) FIG. 83b—Frequency of monocytes that express CCR2.

(120) FIG. 83c—Frequency of eosinophils that express CCR3.

(121) FIG. 84a—Binding of the chemokine bEotaxin (CCL11) to granulocytes from patient with allergy. Blood from a patient with allergy was incubated with bEotaxin and analysed with flow cytometry. Granuocytes were characterized based on size and granularity.

(122) FIG. 84b—Binding of the chemokine bIL8 to neutrophils from a patient with allergy. Blood from a patient with allergy was incubated with bIL8 and analysed with flow cytometry. The neutrophils were characterized as CD16 positive cells.

(123) FIG. 85a—Depletion of CXCR1 and CXCR2 expressing neutrophils with Sepharose Streptavidin-matrix conjugated with bIL8.

(124) FIG. 85b—Depletion of CCR3 expressing granulocytes with Sepharose Streptavidin-matrix conjugated with bEotaxin.

(125) FIG. 85c—Depletion of CCR2 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bMCP1. Blood cells from a patient with allergy were incubated with biotinylated-Chemokine-SepharoseStreptavidin-matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bMCP1-matrix (Before Depletion).

(126) FIG. 86—Increased frequency of CCR3 expressing monocytes in blood from 4 allergic patients compared to 20 healthy controls. Analysed with flow cytometry.

(127) FIG. 87—Depletion of CCR3 expressing monocytes in one allergic patient with Sepharose StreptavidinMatrix-bEotaxin.

(128) FIG. 88—Increased frequency of eosinophils in blood from four allergic patients compared to twenty healthy controls. Analysed with flow cytometry. Eosinophils were characterized as CD16 negative granulocytes.

(129) G. Treating Inflammatory Skin Diseases

(130) FIGS. 89a, 89b & 89c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(131) FIGS. 89d, 89e & 89f—the binding of biotinylized CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor;

(132) FIGS. 89g, 89h & 89i—the binding of biotinylized CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a patient with CD;

(133) FIG. 90a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(134) FIG. 90b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(135) FIG. 91a—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(136) FIG. 91b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood of a healthy donor.

(137) FIG. 91c—Results of in vitro depletion tests performed on the biotinylated MIP-3a coupled matrix showing ability to eliminate CCR6-expressing lymphocytes from blood from three healthy donors.

(138) FIG. 91d—Depletion of CCR9-expressing cell populations in one blood donor. Total cell populations are unaffected after the column passage.

(139) FIG. 91e—Depletion of CCR9-expressing cell populations in one IBD patient. Total cell populations are unaffected after the column passage.

(140) FIG. 92—Sequence (SED ID NO: 113) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(141) FIG. 93—Sequence (SED ID NO: 113) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(142) FIG. 94—Sequence (SED ID NO: 114) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(143) FIG. 95—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(144) FIG. 96—Sequence (SED ID NO: 118) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(145) FIG. 97—Sequence (SED ID NO: 126) and biotinylation, of RANTES derivative

(146) FIG. 98—HPLC of purified folded Biotin-TECK(Nleu).

(147) FIG. 99—Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu).

(148) FIG. 100—example of gating criteria for CCR2 expressing monocytes.

(149) FIG. 101—Frequency of CCR4 expressing T cells in one patient with psoriasis. The expression of chemokine receptors and specific cell markers were analysed with flow cytometry.

(150) FIG. 102—Expression of CCR4 compared to binding of bMDC to blood T cells from a patient with psoriasis. The expression of chemokine receptors, binding of chemokine, and specific cell markers were analysed with flow cytometry.

(151) FIG. 103—Depletion of CCR4 expressing T cells with Sepharose Streptavidin-matrix conjugated with bMDC. Blood cells from a healthy control were incubated with biotinylated MDC-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bMDC-matrix (Before Depletion).

(152) FIG. 104—Expression of CXCR1 and CXCR2 on neutrophils from a patient with psoriasis. The expression of chemokine receptors, binding of chemokine and specific cell markers were analysed with flow cytometry.

(153) FIG. 105—Binding of the chemokine bIL-8 to neutrophils in blood from a psoriasis patient. Blood from a psoriasis patient was incubated with bIL-8 and analysed with flow cytometry. The neutrophils were characterized as CD16 positive cells.

(154) FIG. 106—Depletion of CXCR2 expressing neutrophils with Sepharose Streptavidin-matrix conjugated with bIL-8. Blood cells from a psoriasis patient were incubated with bIL-8 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 bIL-8-matrix (Before Depletion).

(155) H. Treating Multiple Sclerosis

(156) FIGS. 107a, 107b & 107c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(157) FIG. 108a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(158) FIG. 108b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(159) FIG. 109a—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(160) FIG. 109b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood of a healthy donor.

(161) FIG. 109c—Results of in vitro depletion tests performed on the biotinylated MIP-3a coupled matrix showing ability to eliminate CCR6-expressing lymphocytes from blood from three healthy donors.

(162) FIG. 110—Sequence (SED ID NO: 145) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(163) FIG. 111—Sequence (SED ID NO: 145) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(164) FIG. 112—Sequence (SED ID NO: 146) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(165) FIG. 113—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(166) FIG. 114—Sequence (SED ID NO: 150) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(167) FIG. 115—Sequence (SED ID NO: 160) and biotinylation, of RANTES derivative

(168) FIG. 116—example of gating criteria for CCR2 expressing monocytes.

(169) FIG. 117a—Frequency of CCR2 positive T cells. Bars represent mean and SEM of T cells that express CCR2 in 2 patients and 20 healthy controls.

(170) FIG. 117b—Frequency of CCR6 positive T cells. Bars represent mean and SEM of T cells that express CCR6 in 5 patients and 20 healthy controls. The expression of chemokine receptors and specific cell markers were analysed with flow cytometry. The T cells were characterized as CD3 positive.

(171) FIG. 118a—Binding of the chemokine bMCP-1 to T cells. Bar represents mean frequency and SEM of MCP-1 binding T cells in 5 patients with MS.

(172) FIG. 118b—Binding of the chemokine bMIP3a to T cells. Bar represents frequency of MIP3a-binding T cells in 1 patient with MS. Blood was incubated with biotinylated chemokine and analysed with flow cytometry. The T cells were characterized as CD3 positive.

(173) FIG. 119a—Depletion of CCR2 expressing T cells with Sepharose Streptavidin-matrix conjugated with bMCP-1.

(174) FIG. 119b—Depletion of CCR6 expressing T cells with Sepharose Streptavidin-matrix conjugated with bMIP3a. Blood cells from a patient with MS were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion).

(175) I. Treating Cardiovascular Disease

(176) FIGS. 120a, 120b & 120c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(177) FIG. 121a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(178) FIG. 121b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(179) FIG. 122—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(180) FIG. 123—Sequence (SED ID NO: 176) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(181) FIG. 124—Sequence (SED ID NO: 176) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(182) FIG. 125—Sequence (SED ID NO: 177) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(183) FIG. 126—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(184) FIG. 127—Sequence (SED ID NO: 181) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(185) FIG. 128—example of gating criteria for CCR2 expressing monocytes.

(186) FIG. 129—Frequency of CCR1 expressing monocytes in 20 healthy controls and one patient with atherosclerosis (AS). The expression of chemokine receptors and specific cell markers were analysed with flow cytometry.

(187) FIG. 130—Binding of bRANTES to blood monocytes from a patient with atherosclerosis. The binding of chemokine and expression of specific cell markers were analysed with flow cytometry.

(188) FIG. 131—Depletion of CCR1 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bRANTES. Blood cells from an atherosclerosis patient were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with biotinylated-chemokine-matrix (Before Depletion).

(189) FIG. 132—Expression of CCR2 on monocytes from one patient with atherosclerosis. The expression of chemokine receptors, binding of chemokine and specific cell markers were analysed with flow cytometry.

(190) FIG. 133—Binding of the chemokine bMCP-1 to monocytes. Bars represent frequency of bMCP-1 binding monocytes and CCR2 expressing monocytes in blood from a patient with atherosclerosis. Blood was incubated with biotinylated chemokine and analysed with flow cytometry.

(191) FIG. 134—Depletion of CCR2 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bMCP-1. Blood cells from a patient with atherosclerosis were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with b-chemokine-matrix (Before Depletion).

(192) J. Treating Primary Sclerosing Cholangitis

(193) FIGS. 135a, 135b & 135c—the binding of biotinylated CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor;

(194) FIGS. 136a, 136b & 136c—the binding of biotinylized CCL25 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a patient with CD;

(195) FIG. 137—Depletion of CCR9-expressing cell populations in one blood donor. Total cell populations are unaffected after the column passage.

(196) FIG. 138—Depletion of CCR9-expressing cell populations in one IBD patient. Total cell populations are unaffected after the column passage

(197) FIG. 139—Sequence (SEQ ID NO: 195) and biotinylation of RANTES derivative

(198) FIG. 140—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.

(199) FIG. 141—HPLC of purified folded Biotin-TECK (Nleu)

(200) FIG. 142—Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu).

(201) FIG. 143—Example of gating criteria for CCR2 expressing monocytes

(202) FIG. 144a—Expression of CCR9 expressing monocytes in 4 patients with Primary sclerosing cholangitis (PSC) and in 20 healthy controls (HC).

(203) FIG. 144b—Expression of CCR9 in activated (HLADRhi) monocytes in 4 patients with Primary sclerosing cholangitis (PSC) and in 20 healthy controls (HC). Blood from patients with PSC and healthy controls was analysed for the expression of various chemokine receptors by flow cytometry. The monocytes were characterized as CD14 positive cells, and the activated monocytes are characterized as highly expressed HLADR CD14 positive cells.

(204) FIG. 145—Binding of the chemokine bTECK (CCL25) to monocytes from a patient with Primary sclerosing cholangitis. Blood from a patient with PSC was incubated with bTECK and analysed with flow cytometry. The monocytes were characterized as CD14 positive cells.

(205) FIG. 146a—Depletion of CCR9 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bTECK.

(206) FIG. 146b—Depletion of activated CCR9 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bTECK. Blood cells from a patient with PSC were incubated with bTECK-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix with Phosphate Buffer Saline. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bTECK-matrix (Before Depletion).

(207) K. Treating Respiratory Conditions

(208) FIGS. 147a, 147b & 147c—the binding of biotinylized MIP-1α by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor;

(209) FIGS. 148a, 148b & 148c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor;

(210) FIGS. 149a, 149b & 149c—the binding of IL-8 by CD4+, CD8+ T-cells and CD16+ monocytes respectively, obtained from peripheral blood of a healthy donor

(211) FIG. 150a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(212) FIG. 150b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(213) FIG. 151a—binding of eotaxin to neutrophils/eosinophils (dashed line) in peripheral blood. The graph represents a summary of four tests.

(214) FIG. 151b—binding of CCR3-antibody to neutrophils/eosinophils (line) in peripheral blood. The graph represents a summary of four tests.

(215) FIG. 152a—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(216) FIG. 152b—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.

(217) FIG. 152c—Results 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.

(218) FIG. 153—Sequence (SEQ ID NO: 211) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(219) FIG. 154—Sequence (SEQ ID NO: 211) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(220) FIG. 155—Sequence (SEQ ID NO: 212) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(221) FIG. 156—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(222) FIG. 157—Sequence (SEQ ID NO: 216) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(223) FIG. 158—Sequence (SEQ ID NO: 225) and biotinylation, of RANTES derivative.

(224) FIG. 159—Sequence (SEQ ID NO: 218) and biotinylation, via a spacer group, of mature protein eotaxin derivative containing C-terminal amide.

(225) FIG. 160—Example of gating criteria for CCR2 expressing monocytes

(226) FIG. 161—Frequency of CCR1 expressing monocytes in 20 healthy controls and 2 patients with sarcoidosis. The expression of chemokine receptors and specific cell markers were analysed with flow cytometry.

(227) FIG. 162—Expression of CCR1 compared to binding of bRANTES to blood monocytes from a patient with sarcoidosis. The expression of chemokine receptors, binding of chemokine, and specific cell markers were analysed with flow cytometry.

(228) FIG. 163—Depletion of CCR1 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bRANTES. Blood cells from a healthy control were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with biotinylated-chemokine-matrix (Before Depletion).

(229) FIG. 164—Expression of CCR2 on monocytes from two patients with sarcoidosis. The expression of chemokine receptors, binding of chemokine and specific cell markers were analysed with flow cytometry.

(230) FIG. 165—Binding of the chemokine bMCP-1 to monocytes. Bars represent frequency of MCP-1 binding monocytes and CCR2 expressing monocytes in blood from a patient with sarcoidosis. Blood was incubated with biotinylated chemokine and analysed with flow cytometry.

(231) FIG. 166—Depletion of CCR2 expressing monocytes with Sepharose Streptavidin-matrix conjugated with bMCP-1. Blood cells from a healthy control were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion).

(232) FIG. 167a—Frequency of CCR7 expressing T cells. Bars represent frequency of T cells that express CCR7 in 2 patients and 20 healthy controls. The expression of chemokine receptors and specific cell markers were analysed with flow cytometry. The T cells were characterized as CD3 positive.

(233) FIG. 167b—Frequency of central memory T cells in one patient with sarcoidosis. The central memory T cells were characterized as CD3 positive, CD4 positive, CD45RA negative, CCR7 positive cells.

(234) FIG. 168—Depletion of CCR7 expressing T cells with Sepharose Streptavidin-matrix conjugated with bMIP3b. Blood cells from a patient with Sarciodosis were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion).

(235) L. Treating Conditions Associated with Sepsis

(236) FIGS. 169a, 169b & 169c—the binding of biotinylized MCP-1 by CD4+, CD8+ T-cells and CD14+ monocytes respectively, obtained from peripheral blood of a healthy donor.

(237) FIGS. 169d, 169e & 169f—the binding of IL-8 by CD4+, CD8+ T-cells and CD16+ neutrophils respectively, obtained from peripheral blood of a healthy donor

(238) FIG. 170a—binding of MCP-1 to monocytes (dashed line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(239) FIG. 170b—binding of CCR2-antibody to monocytes (line) in peripheral blood taken from IBD patients. The graph represents a summary of four tests.

(240) FIG. 171—Results of in vitro depletion tests performed on the bMCP-1 coupled matrix showing ability to eliminate CCR2-expressing cells from blood from three healthy donors.

(241) FIG. 172—Sequence (SEQ ID NO: 241) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification.

(242) FIG. 173—Sequence (SEQ ID NO: 241) and biotinylation, via a spacer group, of mature protein MCP-1 derivative containing Gln to pyroGlu modification and Met to Norleu substitution.

(243) FIG. 174—Sequence (SEQ ID NO: 242) and biotinylation, via a spacer group, of truncated MCP-1 derivative containing Met to Norleu substitution.

(244) FIG. 175—Alignment of MCP-1 (residues 25-99 of SEQ ID NO: 11) and MCP-5 (residues 24-104 of SEQ ID NO: 10) amino acid sequences.

(245) FIG. 176—Sequence (SEQ ID NO: 246) and biotinylation, via a spacer group, of (C-terminal) truncated MCP-5 derivative containing Ile to Lys modification.

(246) FIG. 177—example of gating criteria for CCR2 expressing monocytes.

(247) FIG. 178—Frequency of neutrophils in peripheral blood of RDS patents and healthy controls (HC). Bars represent mean and SEM of CD16 positive granulocytes in blood from 8 RDS patients and 20 HC. Blood was analysed for the expression of cell specific markers with flow cytometry.

(248) FIG. 179—Expression of CXCR1 and CXCR2 on neutrophils from patients with RDS. Blood was analysed for the expression of chemokine receptors by flow cytometry. Bars represent mean and SEM of CXCR1 positive and CXCR2 positive neutrophils in 6 RDS patients. Blood was analysed for the expression of cell specific markers by flow cytometry and neutrophils were characterized as CD16 expressing granulocytes.

(249) FIG. 180—Binding of biotinylated IL-8 (bIL-8) to neutrophils from a healthy control. Bar represent percentage of neutrophils that bind biotinylated IL-8, analysed with flow cytometry.

(250) FIG. 181—Depletion of neutrophils with a CXCR1-antibody and MACS. Bars represent percentage of CXCR1 positive neutrophils before and after MACS. Peripheral blood from a RDS patient was used for the experiment.

(251) FIG. 182a—Frequency of CCR5 expressing neutrophils in patients with RDS (n=6) and healthy controls (n=20) Bars represent mean and SEM of CCR5 expressing neutrophils, analysed with flow cytometry.

(252) FIG. 182b—Frequency of CCR5 expressing cells in blood from healthy controls and RDS patients and in BALF from RDS patient. Bars represent mean and SEM of CCR5 expressing neutrophils, analysed with flow cytometry.

(253) FIG. 183—Binding of RANTES and CCR5 (T cells and neutrophils) or RANTES and CCR1 (monocytes). Bars represent percentage of positive cells in blood from healthy control.

(254) FIG. 184—Depletion of CCR5 expressing neutrophils with an anti-CCR5 antibody and MACS.

(255) FIG. 185—Sequence (SEQ ID NO: 253) and biotinylation of RANTES derivative

(256) FIG. 186—Results of in vitro depletion tests performed on the biotinylated RANTES coupled matrix showing ability to eliminate chemokine receptor-expressing cells from peripheral blood taken from a healthy donor.

(257) The various embodiments of the invention will now be described in more detail by reference to the following non-limiting embodiments and examples:

DESCRIPTION OF PREFERRED EMBODIMENTS

(258) A. Diagnosing and Treating Inflammatory Bowel Disease and Irritable Bowel Syndrome

Example 1

CCR9-Expressing CD14+HLA-DRhi Blood Monocytes Promote Intestinal Inflammation in IBD

(259) Introduction

(260) Crohn's disease (CD) and ulcerative colitis (UC) (Inflammatory bowel diseases; IBD) are chronic, inflammatory disorders of the gastrointestinal tract resulting from a disrupted balance between the mucosal immune system and commensal flora. To date, the immunological pathophysiology behind IBD remains poorly understood. Traditionally, adaptive immunity was believed to play an important role in the onset of IBD. Studies in patients and animal models have shown that CD is driven by T helper 1-signaling with IL-12 and IFN-γ production, whereas UC is characterized by T helper 2-responses and IL-13. (1) However, the Th1/Th2 paradigm has been questioned over the last decade..sup.(2) Since the discovery of the NOD2/CARD15 susceptibility locus that encodes a pattern recognition receptor mainly expressed on dendritic cells and monocytes, the focus of IBD research has shifted towards innate immunity..sup.(3,4) Currently, innate mechanisms are believed to be responsible for the onset of acute mucosal inflammation in genetically susceptible individuals, whereas the chronic state might be maintained by adaptive elements..sup.(5)

(261) Monocytes are bone marrow-derived leukocytes of the myeloid lineage that migrate to the tissue and differentiate into macrophages or dendritic cells (DCs). Increased turnover rates and elevated levels of circulating monocytes have been demonstrated in IBD..sup.(6, 7) Furthermore, monocytes have the ability to migrate to the inflamed mucosa and mediate inflammation, but the phenotype of these monocytes as well as the mechanisms underlying this relocation remains to be elucidated..sup.(8-10) Currently, two main human monocyte subpopulations have been characterized. The CD14+CD16− cells have been shown to produce the regulatory cytokine IL-10 and are most commonly referred to as classical monocytes. The CD14loCD16+ subset is characterized by production of pro-inflammatory cytokines as well as high surface expression of inflammatory markers such as CD43..sup.(11-13) However, a larger degree of heterogeneity among human monocyte populations with regards to their surface antigen expression has lately been observed..sup.(14) Monocyte HLA-DR expression has been demonstrated to play an important role in conditions characterized by immune responses against bacterial agents..sup.(15, 16) Although the CD14+CD16− subset has been reported to express HLA-DR, the specific contribution of CD14+HLA-DRhi monocytes to intestinal inflammation has not been studied. Since it is well established that induction of colitis in human as well as in animal models requires the presence of bacteria, we set out to study CD14+HLA-DRhi monocytes in patients with chronic intestinal inflammation..sup.(17)

(262) Materials and Methods

(263) Patients

(264) In total, 51 IBD patients (UC=31; CD=20) were included in this study (Table 13). The patients were monitored during treatment with corticosteroids (n=16), the anti-TNF-α antibodies infliximab or adalimumab (Remicade® or Humira®; n=17), or Granulocyte/Monocyte apheresis (GMA; Adacolumn®; n=18). Four to six biopsies from affected rectum and sigmoideum were collected together with blood samples before the start of treatment, followed by weekly sampling for four consecutive weeks. Patients were assessed using the UC-DAI (UC) and Harvey-Bradshaw (HBI; CD) indices. Clinical remission was reviewed at week 11 post-treatment and defined as <3 for UC-DAI and <5 for HBI..sup.(8, 41) Fourteen controls without IBD were included in the study. All patients were enrolled through formal consent and the study was approved by the regional ethics committee.

(265) Leukocyte Isolation and Activation

(266) For flow cytometry studies, peripheral blood mononuclear cells (PBMC) were obtained from heparinised whole blood by incubation in hypotonic buffer (160 mM NH4Cl, mM Tris-HCl, pH=7.4). For PCR and CCL25 depletion experiments, PBMC were obtained from anti-coagulated healthy donor buffy coats by density gradient centrifugation using Ficoll-Paque (GE Healthcare). For PCR experiments, CD14+ monocytes were negatively isolated using Monocyte Isolation Kit II (Miltenyi Biotec). Monocytes were subsequently activated with LPS (lipopolysaccharide; Sigma) (200 ng/mL/106 cells) for 2 hrs (TNF-α PCR) or 6 hrs (PCR array) in RPMI medium (Hyclone) supplemented with 1% L-glutamine and 1% PEST (penicillin-streptomycin).

(267) Flow Cytometry

(268) PBMC were stained for flow cytometry analysis or sorting using combinations of the antibody conjugates described in Table 14. All stainings were carried out according to the instructions of the manufacturer for the respective antibody conjugate. An IgG2a-FITC (BD Biosciences) isotype control was used to define CCR9 positivity. Flow cytometry analyses and sorting experiments were carried out using a FACSAria cytometer and data was analysed using FACSDiva software (BD Biosciences).

(269) PCR Experiments

(270) For TNF-α PCR experiments, RNA isolation was performed using TRIZOL reagent (Invitrogen). For the CCL25 experiment, intestinal biopsies were collected through flexible sigmoidoscopy from UC patients and immediately submerged in RNAlater (Ambion). RNA was subsequently isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. For the TNF-α and CCL25 experiments, 100 ng RNA per sample was included in a reverse transcriptase reaction using iScript cDNA synthesis kit (Bio-Rad). For PCR array analyses, RNA was isolated from sorted CD14+HLA-DRhi and CD14+HLA-DRlo populations using RNeasy Mini Kit (Qiagen). For each of the analyzed populations, equal amounts of RNA from three independent donors were pooled and cDNA was synthesized using RT2 First Strand Kit (SABiosciences) from 150 ng of RNA. Subsequently, cDNA was put into a RT2 qPCR Master Mix (SABiosciences) reaction and loaded onto a Human Inflammatory Response and Autoimmunity 96-well PCR array plate according to the instructions of the manufacturer (SABiosciences). In all PCR experiments, quantitative PCR was performed on an iCyclerIQ Optical System using 2×IQ SYBR Green supermix and iCycler IQ Optical System Software v3.1 (Bio-Rad) for data retrieval. In the TNF-α and CCL25 experiments, expression levels were normalized to RNA polymerase II using either the 2-ΔCt (TNF-α; FIG. 4B) or 2-ΔCt (CCL25) methods. Primers used were TNF-α forward (5′-CTCTCTCCCCTGGAAAGGAC-3′, SEQ ID NO: 262); TNF-α reverse (5′-GCCAGAGGGCTGATTAGAGA-3′, SEQ ID NO: 263); CCL25 forward (5′-AAGGTTTTTGCAAAGCTCCA-3′, SEQ ID NO: 264); CCL25 reverse (5′-TACTGCTGCTGATGGGATTG-3′, SEQ ID NO: 265); RPII forward (5′-GCACCACGTCCAATGACAT-3′, SEQ ID NO: 266); RPII reverse (5′-GTGCGGCTGCTTCCATAA-3′, SEQ ID NO: 267). For PCR array analyses, expression levels were normalized to the arithmetic mean expression of the B2M, HPRT1, RPL13A, GAPDH and ACTB housekeeping genes using the 2-ΔCt method.

(271) CCL25 Depletion Assay

(272) Biotinylated CCL25 (Almac Sciences) was bound to a solid support consisting of a streptavidin-sepharose matrix (GE Healthcare). PBMC from six healthy donors was perfused through the device and CCR9 expression was analysed before and after using flow cytometry.

(273) Statistical Analyses

(274) All group analyses were carried out using two-tailed dependent Student's t-test (FIGS. 2-4 and 6) or two-tailed independent Student's t-test (FIG. 3). Regression analyses were performed using ordinal regression test for non-parametric data (FIG. 2B-C). All calculations were carried out in GraphPad Prism v5 software (GraphPad Software, Inc.). Values of p≦0.05 were regarded as significant and depicted as follows: p≦0.05=*, p<0.01=**, p≦0.001=***. In all figures, error bars represent ±SEM.

(275) Ethical Considerations

(276) The study was approved by the Stockholm Regional Ethical Review Board in Stockholm, Sweden. The ethical approval applies to all central from which patients were recruited (South Hospital, Stockholm, Sweden; Karolinska Hospital, Stockholm, Sweden; Danderyd Hospital, Stockholm, Sweden). All patients were enrolled through formal written consent.

(277) Results

(278) The Frequency of CD14+HLA-DRhi Monocytes Correlates to Clinical Disease Activity in Ulcerative Colitis and Crohn's Disease

(279) To investigate the role of CD14+HLA-DRhi monocytes in the IBD patients, we used flow cytometry to identify the population in peripheral blood (FIG. 1). When analyzing blood from patients and controls we found that active inflammation in the colon correlated to a significantly higher frequency of these monocytes compared with the control group (FIG. 2A, p=0.006).

(280) In order to assess the correlation between monocyte levels and disease activity, we carried out regression analyses of CD14+HLA-DRhi frequency against two commonly used disease activity indices for the assessment of ulcerative colitis and Crohn's disease, respectively. We could observe a significant correlation with disease activity in both UC (UCDAI) (FIG. 2B; p=0.0137, r2=0.072) and CD (Harvey-Bradshaw) (FIG. 2C; p=0.0182, r2=0.1898). Thus, the frequency of circulating pro-inflammatory CD14+HLA-DRhi monocytes correlates with established disease activity indices of IBD.

(281) CD14+HLA-DRhi Monocytes are Potential Therapeutic Targets and Markers of Inflammation in Colitis

(282) Next, we investigated whether the CD14+HLA-DRhi population was affected by conventional IBD therapy. Patients with active intestinal inflammation who received either corticosteroids or anti-TNF-α antibodies (infliximab or adalimumab) were monitored for five consecutive weeks. A patient group treated with Granulocyte/Monocyte apheresis was included for comparison considering the selective removal of monocytes associated with Adacolumn®. (18) When plotting these treatment regimes separately, the patient group receiving GMA therapy accounted for the most prominent decrease (FIG. 3A). The monocyte population was also attenuated already after one week of therapy among corticosteroid patients. The suppression was maintained, reaching levels well below those of healthy control patients at week 4 (FIG. 3B, p<0.05). The decreased population of CD14+HLA-DRhi during treatment was not influenced by the diagnosis UC or CD, extension of the disease in the colon, concomitant azathioprine treatment or gender (data not shown). Interestingly, biological therapy with antibodies against TNF-α did not significantly affect the proportion of CD14+HLA-DRhi monocytes (FIG. 3C). Among these patients, CD14+HLADRhi never reached the reference level observed in the controls.

(283) Lastly, we investigated whether the CD14+HLA-DRhi population was selectively affected in patients achieving long-term remission at week 11 post-treatment. Among patients receiving corticosteroids or GMA apheresis, the monocyte population was significantly decreased in those who later achieved or maintained remission. This was not observed among the non-remission patients (FIG. 3D).

(284) CD14+HLA-DRhi Monocytes Produce High Levels of Inflammatory Mediators

(285) With the purpose of investigating the capacity of CD14+HLA-DRhi monocytes to produce inflammatory mediators, monocytes from healthy blood donors were cultured in the presence of lipopolysaccharide (LPS). The HLA-DRhi population and its CD14+HLA-DRlo counterpart were subsequently sorted with flow cytometry (FIG. 4A). The two cell populations were investigated with regards to the production of the proinflammatory cytokine TNF-α. Interestingly, the CD14+HLA-DRhi population produced 500-fold increased levels of TNF-α transcripts upon LPS-stimulation compared to the CD14+HLA-DRlo cells (FIG. 4B). Furthermore, PCR array analyses were carried out on sorted CD14+HLA-DRhi monocytes from three independent donors after activation with LPS in order to establish the distinctive phenotype of the population. In accordance with our hypothesis several gene transcripts described as being involved in monocyte-mediated immune responses were up-regulated in the CD14+HLA-DRhi monocytes. Increased gene expression was mainly found among chemotactic cytokines and genes involved in the humoral immune response (FIG. 4C). The most prominent fold change difference between the CD14+HLADR hi and the CD14+HLA-DRlo monocytes was observed for the chemotactic cytokine CCL4. The transcript with the most apparent down-regulation among HLA-DRhi monocytes was the HDAC4 gene that encodes a histone deacetylase that functions as a transcriptional repressor..sup.(19) Together, this data shows that CD14+HLA-DR.sup.hi monocytes have strong proinflammatory potential.

(286) CD14+HLA-DRhi Monocytes Express the Gut-Homing Chemokine Receptor CCR9

(287) Next, we studied the surface expression of various chemokine receptors on CD14+HLA-DRhi monocytes in relation to the CD14+CD16− and CD14loCD16+ subsets. Although we could observe significant overlap between many of these markers in terms of their expression in the respective subsets, the CD14+HLA-DRhi population was clearly distinguished by its expression of CCR7 and CCR9 (FIG. 5A). CCR7 is mainly described as a lymph-node homing receptor for dendritic cells and T helper cells, and has previously not been reported to be expressed on circulating monocytes..sup.(20) CCR9 has been shown to be important in lymphocyte homing to the gut through interactions with its ligand CCL25, expressed in the intestinal mucosa..sup.(21, 22) Although CCR9-CCL25 interactions have been well characterized in T helper cells, their role in monocytes is unclear. The general CD14+ monocyte population exhibited significantly higher CCR9 expression compared to CD4+ T lymphocytes, which together with CD8+ T lymphocytes have been described as the main CCR9 expressing cell types (p<0.05)..sup.(23) When comparing CCR9 expression in CD14+HLA-DRhi with CD14loCD16+ and CD14+CD16− monocytes, the HLA-DRhi subset displayed significantly increased levels (FIG. 5A). In contrast, the expression of CCR2, a chemokine receptor responsible for general monocyte migration, was not increased on the HLA-DRhi monocytes indicating that gut-homing phenotype constitutes a specific feature of the monocytes rather than reflecting general immunological activation (FIG. 5B)..sup.(24) We also analyzed colonic mucosal tissue biopsies with real-time PCR and found that the CCR9 ligand, CCL25/TECK, was expressed in the colon. To establish a functional interaction between CCL25 and CCR9, we carried out depletion experiments by perfusing peripheral blood cells through a solid support containing CCL25-coated sepharose beads. The frequency of CCR9-positive CD14+HLA-DRhi monocytes was significantly reduced after encounter with the CCL25-coated sepharose beads, showing that CCR9 on CD14+HLA-DRhi monocytes could bind CCL25 and be removed from the blood (p<0.05; FIG. 6). In conclusion, pro-inflammatory CD14+HLA-DRhi monocytes express high levels of the gut-homing chemokine receptor CCR9 which directs them to the site of mucosal inflammation.

(288) Discussion

(289) In this study, we identify CCR9-expressing CD14+HLA-DRhi blood monocytes as an important player in intestinal inflammation. The expression of HLA-DR on monocytes is vital to the inflammatory response and has been shown to determine the efficacy of antigen presentation to T helper cells. (25, 26) Monocytes with high expression of HLA-DR have also been shown to infiltrate the joints of patients with rheumatoid arthritis, an inflammatory disease successfully treated with TNF-α antibodies..sup.(27) In addition, carrying the class II HLA-DRB1*0103 allele correlates with an increased risk for developing ulcerative colitis..sup.(28) Several studies have suggested that monocytes per se are targeted by conventional IBD therapy..sup.(6, 29, 30) Our results suggest that specific down-regulation of the HLA-DRhi subpopulation may be an important mechanism behind resolution of the inflammation. In this study, patients treated with Granulocyte/Monocyte apheresis were added for comparison since Adacolumn® is the only treatment specifically targeting circulating monocytes. These cells are removed through Fcγ receptor binding to the cellulose acetate beads in the column, leaving circulating T-cells unaffected..sup.(18) Corticosteroid therapy mediates a decrease in the number of circulating CD14+HLA-DRhi monocytes that is comparable to GMA (FIG. 3B). This suppression seems to be important for the induction of remission, since significant decrease of CD14+HLA-DRhi monocytes is only observed in the patients achieving long-term remission (FIG. 3D). Interestingly, patients subjected to biological treatment did not display a decrease of pro-inflammatory monocytes (FIG. 3C). We speculate that by removing TNF-α, one of the main products of these monocytes, autocrine feed-back mechanisms leading to cellular activation might be induced. The observation underscores the difference in mode of action between anti-TNF-α antibodies and corticosteroids and should be further investigated since anti-TNF-α failure may partly depend on the monocytes' production of additional pro-inflammatory chemokines, cytokines and integrin receptors counterbalancing TNF-α suppression.

(290) Classically, leukocyte populations have been defined through their capability to produce inflammatory mediators such as cytokines and chemokines. In order to gain a functional understanding of how CD14+HLA-DRhi monocytes mediate inflammation, we investigated their pro-inflammatory potential at the mRNA level compared to their HLA-DRlo-expressing counterpart. In this context, the HLA-DRhi subset produced markedly elevated levels of gene transcripts associated with activation and pro-inflammatory phenotype. The population displayed a 500-fold increase of TNF-α transcript levels, which establishes the HLA-DRhi subset as one of the most important producers of this cytokine. Other genes were also investigated by PCR array analysis, revealing the highest up-regulation in CCL4, CCL3 and IL-1β, all cytokines previously described to be involved in the recruitment of inflammatory cells to the intestinal mucosa in IBD (FIG. 4D)..sup.(31-35) The most prominent difference was observed in the CCL4/MIP-1 gene, with up-regulated transcript levels of more than 300-fold. The inflammatory role of CCL4 was reported by Bystry and colleagues; activated T helper cells were demonstrated to efficiently migrate towards a CCL4 tissue gradient through CCR5 interaction..sup.(31) Thus, our finding that CCL4 transcripts were produced in high levels by CD14+HLA-DR.sup.hi monocytes supports their inflammatory potential..sup.(36) The transcript with the most apparent down-regulation in HLA-DRhi monocytes was the HDAC4 gene that encodes a histone deacetylase that functions as a transcriptional repressor..sup.(19) Together, these data indicate that CD14+HLA-DRhi is a transcriptionally active subset that readily expresses genes important for mediating mucosal immune responses.

(291) On the surface level, CD14+HLA-DRhi monocytes only partially express CD16, suggesting that the population constitutes a separate subset that is not included in its entirety when defining pro-inflammatory monocytes through their expression of CD14lo CD16+ (FIG. 5C). The population is further defined by its expression of CCR7 and the gut-homing receptor CCR9, which clearly distinguishes HLA-DRhi monocytes from the CD14+CD16− and CD14loCD16+ subsets (FIG. 5).

(292) In the context of intestinal inflammation, interactions between CCR9-expressing T cells and CCL25 (TECK) expressed in the gut epithelium have been implicated as an important mechanism for recruiting circulating lymphocytes to the intestinal mucosa..sup.(21) However, whether this mechanism also applies for the extensive infiltration of blood monocytes to the intestinal mucosa observed during inflammation has never been studied..sup.(37) It was recently shown that CCR9-expressing monocytes are increased in peripheral blood of patients with rheumatoid arthritis. However, the role of these cells in IBD remains unclear. In accordance with the results from this study, we show that the CD14+CD16− and CD14loCD16+ subsets express similar levels of CCR9. (38) Those levels were notably superseded by the CCR9 expression on CD14+HLA-DRhi monocytes (FIG. 5A). Surprisingly, this expression was considerably higher than that observed on T cells, which is considered to be the main CCR9− carrying cell type..sup.(23) In contrast, the expression of CCR2, another chemokine receptor important for monocyte relocation in several disease groups, was not increased on HLA-DRhi monocytes (FIG. 5B)..sup.(24) This suggests that the specific increase in CCR9 expression among CD14+HLA-DRhi may reflect a gut-specific phenotype, rather than a generally activated subset. The ligand for CCR9, CCL25, was found to be expressed in mucosal tissue by QT-PCR analysis, which is supported by other reports identifying CCL25 in the colon..sup.(22, 39) In conclusion, these data suggest that monocytes in general and CD14+HLA-DRhi in particular possess the ability to relocate to the intestinal mucosa through CCR9-CCL25 interactions.

(293) T cells have been shown to acquire their CCR9 expression through retinoic-acid dependent imprinting by mesenterial lymph node dendritic cells..sup.(40) The issue of whether CCR9 expression on monocytes is acquired through similar mechanisms seems controversial, especially since blood monocytes are not known to traffic lymph nodes to the same extent as T cells. Here, we show that CD14+HLA-DRhi monocytes are defined by their high expression of CCR7, a marker mainly described as a lymph-node homing receptor for dendritic cells and T helper cells (FIG. 5A)..sup.(20) Therefore, it is tempting to speculate that CCR7-expressing CD14+HLA-DRhi monocytes traffic the lymph nodes to a higher extent than has previously been known, and that CCR9 imprinting in these monocytes may occur through mechanisms similar to those reported in T cells. Being beyond the scope of this study, the mechanisms behind CCR9 induction in monocytes, as well as the functional role of their CCR7 expression, need to be addressed in the future.

(294) In this study, we have shown that CD14+HLA-DRhi blood monocytes are increased in patients with active intestinal inflammation, and correlate with disease severity as defined by the UC-DAI and HBI disease activity indices. Furthermore, these monocytes produce major amounts of inflammatory mediators and express high levels of the gut-homing receptor CCR9. In summary, these findings indicate that CD14+HLA-DRhi blood monocytes play an important role in IBD and that future studies evaluating these monocytes as specific targets for IBD therapy are highly motivated.

REFERENCES

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Tables

(296) TABLE-US-00114 TABLE 13 Patient demography. Gender Male 32 Female 19 Age mean 37.9 Diagnosis Ulcerative colitis 31 Crohn's disease 20 Extension Extensive 25 Left-sided 19 Proctitis 5 Ileocekal 2 Intervention Corticosteroids.sup.1,2 16 Anti-TNF-α.sup.3,5 17 GMA apheresis.sup.4,5 18 Azathioprine Yes 21 No 30 .sup.1Fifteen patients were introduced to 20-45 mg prednisone followed by tapering of 5 mg weekly. .sup.2One patient received topical corticosteroids for ulcerative proctosigmoiditis. .sup.3Anti-TNF-α treatment was administered either as infusions of 5 mg/kg infliximab week 0, 2 and 6 or subcutaneous injections of 80 mg Adalimumab week 0 followed by 40 mg every other week. .sup.4In the GMA apheresis group, each patient received a total of 5-8 Adacolumn ® leukocytapheresis session 1-2 times weekly. .sup.5Some patients were receiving baseline corticosteroid medication

(297) TABLE-US-00115 TABLE 14 Flow cytometry antibodies used in the study Marker Conjugate Clone Manufacturer CD4 Pacific Blue RPA-T4 BD CD14 APC M5E2 BD CD16 PE-Cy7 3G8 BD HLA-DR PerCP L243 BD CCR1 Alexa Fluor 647 TG4 Biolegend CCR2 PerCP-Cy5.5 TG5 Biolegend CCR3 PE 5E8 Biolegend CCR5 PE HM-CCR5 Biolegend CCR6 PerCP-Cy5.5 11A9 BD CCR7 PerCP-Cy5.5 TG8 Biolegend CCR9 APC 112509 R&D Systems CCR10 PE 314305 R&D Systems CXCR1 APC 8F1 Biolegend CXCR5 PerCP-Cy5.5 TG2 Biolegend CXCR6 PE 56811 R&D Systems

Example 2

Affinity of Blood Cells to CCL25

(298) Materials and Methods

(299) Isolation of peripheral blood leukocytes. Heparinized peripheral blood from healthy blood donors or IBD patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(300) Chemokines. The leukocytes were incubated for 30 min in the dark at 4° C. with the following biotinylated and Alexa647 Fluor® labeled chemokines: CCL25 (in concentrations of 0.1 ng/μL, 0.5 ng/μL and 5 ng/μL). 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.

(301) Flow cytometry assay. The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

(302) In the experiment with biotinylated CCL25 it was found that neither T-cells (CD4+ lymphocytes; CD8+ lymphocytes) nor monocytes (CD14+ monocytes) from the peripheral blood of a healthy donor (FIGS. 7a, 7b and 7c) bound to the biotinylated chemokine. In contrast, about 80% of the CD8+ lymphocytes and about 90% of the CD4+ lymphocytes and the monocytes from a patient with Crohn's disease bound to CCL25 (FIGS. 8a, 8b and 8c).

Example 3

Preparation of a Chemokine Column for Blood Cell Apheresis

(303) To streptavidin cross-linked agarose (ProZyme, San Leandro, Calif., U.S.A.) beads in the range from 75 μm to 300 g suspended (200 ml, ˜50%, v/v) in an aqueous solution of 25 mM sodium phosphate (pH 7.0) and 150 mM NaCl was added a solution of 75 μg biotinylated CCL25 (Almac Sciences) in the same buffer at 22° C. and slowly stirred by hand for 3 min. After standing for another 20 min, the support was filtered off, washed thrice with neutral aqueous sodium phosphate/sodium chloride and filled into a glass column (i.d. 25 mm, length 12 cm).

Example 4

Separation of Monocytes from Peripheral Blood of a Healthy Donor with the Chemokine Column of Example 3

(304) Heparinized peripheral blood from a healthy male donor was analyzed by flow cytometry for CD4+ lymphocytes, CD8+ lymphocytes and CD14 monocytes. 100 ml of the blood was filtered through the column at a rate of about 8 ml per min and washed with FACS buffer. The filtered blood was analyzed for the same cells. It was found that about 95% of the monocytes had been retained by the column whereas more than 90% each of CD4+ and CD8+ lymphocytes had been recovered.

Example 5

Tailored Leukapheresis

(305) Column Design and Properties

(306) Introduction

(307) 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.

(308) The Column

(309) The column is intended to be used as a leukapheresis treatment for inflammatory bowel disease, including Crohn's disease (CD) and ulcerative cholitis (UC). It will specifically remove CD14, HLA-DR, CCR9 and/or CCR7-expressing gut-homing leukocytes through the use of suitable binding reagents contained on a resin, exploiting the CD14, HLA-DR, CCR9 and/or CCR7-binding reagent interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and binding reagent bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(310) The Plastic House (FIG. 9)

(311) 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. 9. 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.

(312) Streptavidin Sepharose™ BigBeads

(313) 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.

(314) bTECK

(315) Coupled to the matrix is the third component of the device, in this example the bTECK. This bTECK peptide is a synthetic, engineered version of the human chemokine TECK, which is truncated and biotinylated, but retains its binding activity to the TECK receptor CCR9. By biotinylating the engineered TECK, 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 4×10-14 M. The calculated ratio of streptavidin:biotin binding sites in the column is 10:1. Therefore, the coupling between the matrix and bTECK will be immediate, minimising the risk of bTECK decoupling from the matrix.

(316) The Apheresis System

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

(318) The Circuit

(319) The system is illustrated in FIG. 10. 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.

(320) The 4008 ADS Pump

(321) 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.

(322) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

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

(324) 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.

(325) Leukapheresis Time and Flow Rate

(326) 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.

(327) Storage Conditions

(328) 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.

(329) Transport Conditions

(330) 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.

Example 6

Non-Clinical Studies

(331) In-Vitro Depletion of Target Cell Populations

(332) To investigate the ability to eliminate CCR9-expressing cells, in vitro tests have been performed on the bTECK coupled matrix. Blood was collected from blood donors and inflammatory bowel disease patients and passed through the column device containing bTECK coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR9-expressing cells.

(333) The results demonstrate significant depletion of the target population CD14− positive CCR9-expressing cells post matrix perfusion; while total CD14-positive cells remain unchanged. Depletion tests were performed on blood from healthy donors and IBD patients confirming similar effects. The results are shown in FIGS. 12 and 13 respectively. In conclusion, the in-vitro results demonstrate a specific reduction of 50-75% of the CCR9-expressing cells by the column. Non-CCR9-expressing cells remained unaffected.

Example 7

TECK-PEG-Biotin Synthesis Summary

(334) Target Molecule:

(335) TECK (Met to Nleu substitution) derivatised at the ε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(336) Modifications:

(337) 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 ε-amino functionality and the biotin.

(338) The linear amino acid sequence (SEQ ID NO: 1) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(339) TABLE-US-00116 HXGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNKVF-OH
X1=pyroGlu or Gln
X64=Norleucine
The engineered TECK sequence was assembled on a solid support, using Fmoc protocols for solid-phase peptide synthesis (SEQ ID NO: 2):

(340) TABLE-US-00117 HXGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-RESIN
X1=pyroGlu or Gln
X64=Norleucine
X72=K(Dde)
FmocLys(Dde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein.
Met to Nle substitution.
N-terminal Gln to pyroglutamic acid substitution.
Removal of Dde Protection:
The Dde protecting group was removed by treatment of all resin (2.5 g) with a solution of 2% hydrazine in DMF (100 ml) over 1 hour period to afford 2.0 g resin.
Labelling Steps:
1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid
Resin (1.5 g) was swollen in DMF (2 ml) and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38 g, 1 mmol), DIC solution (2 ml, 0.5M in DMF) and HOCt solution (2 ml, 0.5M in DMF) was added. The mixture was sonicated for 2 hours and then washed with DMF.
2. Cap
The resin was capped with 0.5M acetic anhydride/DMF solution (20 ml) for 5 minutes and then washed with DMF.
3. Fmoc Deprotection
Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.
4. Couple Biotin-OSu

(341) A solution of Biotin-NHS ester (341 mg, 1 mmol) and DIPEA (348 ul, 2 mmol) 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. Dry resin obtained=1.5 g.

(342) Cleavage:

(343) Dry peptide resin (1.5 g) and the mixture was cleaved with TFA (30 ml) containing a scavenger cocktail consisting of TIS, thioanisole, water, EDT and phenol and the mixture was stirred at room temperature for 6 hours. The solution was filtered into cold ether and the resin rinsed with TFA. The peptide was centrifuged, washed with ether, centrifuged and lyophilised to give 1.0 g crude peptide.

(344) Folding Protocol:

(345) Crude peptide (100 mg) was dissolved into 6M GnHCl (233 ml) and then rapidly diluted to 2M GnHCl concentration by the addition of 50 mM TRIS pH8 (467 ml) containing 0.5 mM GSSG and 5 mM GSH. The mixture was stirred at room temperature for 2.5 days and then analysed by HPLC (Jupiter C18, 250×4.6 mm column, 10-60% B over 30 minutes. HPLC analysis confirmed the formation of desired product as well as mis-folded by-products.

(346) Purification:

(347) The folded protein was purified by reverse phase HPLC using a Jupiter C18, 250×21 mm column, 9 ml/min, 10-60% B over 50 minutes. 11.1 mg of pure folded Nle-TECK-Biotin was afforded.

(348) FIG. 14 shows HPLC of purified folded Biotin-TECK(Nleu). The protein eluted in a single peak at 21.6 mins.

(349) FIG. 15 shows Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu). The expected mass was 8959.4 Da.

(350) Functional Assay Data:

(351) TECK-Biotin-Nle 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 native TECK is 67.87 nM.

(352) The final active chemokine thus has the following sequence (SEQ ID NO: 3):

(353) TABLE-US-00118 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR HRKVCGNPKSREVQRAXKLLDARNXVF-OH
X1=pyroGlu
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).

Example 8

Synthesis of BiotinMIP-3b (CCL19)

(354) Assembly:

(355) 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.

(356) Removal of Dde Protection:

(357) 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.

(358) Labelling Steps:

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

(360) 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.

(361) 2. Capping

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

(363) 3. Fmoc deprotection

(364) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(365) 4. Couple Biotin-OSu

(366) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml 2 mmol) 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.

(367) Cleavage:

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

(369) Purification Protocol:

(370) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(371) Folding Protocol:

(372) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

(373) Target Molecule:

(374) MIP-3b derivatised at the e-amino side chain functionality of Lys(78) with Biotin (TFA salt)

(375) Modifications:

(376) Human MIP-3b 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.

(377) The linear amino acid sequence (SEQ ID NO: 4) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):

(378) TABLE-US-00119 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGR QLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
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)

(379) The engineered MIP-3b 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:

(380) TABLE-US-00120 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGR QLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN
X is FmocLys(ivDde)

(381) FmocLys(ivDde)-OH was incorporated as residue 78 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).

(382) TABLE-US-00121 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGR QLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2

(383) X is K(Biotin)

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

(385) Functional Assay Data:

(386) biotinMip-3b 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-3b is 1.6 nM.

Example 9

Treatment of Crohn's Disease (CD)

(387) Materials and Methods

(388) 1. Flow Cytometric Analysis of Peripheral Blood

(389) 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 (RT) and stained with antibodies (Table 15) 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).

(390) TABLE-US-00122 TABLE 15 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

(391) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 15). 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).

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

(393) 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 μM) 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 15), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(394) Results and Discussion

(395) Crohn's Disease (CD)

(396) 1. Flow Cytometric Analysis of Peripheral Blood

(397) White blood cells from CD patients was analysed with flow cytometry. The patients exhibited increased numbers of circulating CCR9 expressing monocytes, a mean of 13% compared to approximately 7% in healthy blood (FIG. 17).

(398) 2. Chemokine Binding Test

(399) CCR9 binds to the gut homing chemokine TECK (CCL25) and is important for cell migration to the small intestine. In accordance with the CCR9 expression, 14% of the monocytes bind to the biotinylated TECK (bTECK) (FIG. 18).

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

(401) 89% of the CCR9 expressing monocytes were efficiently depleted with bTECK-conjugated Sepharose Streptavidin Matrix. Before depletion there were 7.2% CCR9 expressing monocytes and after depletion 0.8% (FIG. 19).

(402) 80% of the CCR9 expressing monocytes also have a high expression of HLADR suggesting a proinflammatory phenotype (FIG. 20).

(403) We conclude that monocytes in CD (CD14.sup.+HLA-DR.sup.hi monocytes) express CCR9 and bind the ligand bTECK. Furthermore, the majority of the CCR9 expressing monocytes can be removed with Sepharose Streptavidin matrix conjugated with bTECK.

Example 10

Diagnosis and Treatment of IBS

(404) The inventors have surprisingly found that Irritable Bowel Syndrome (IBS) patients, or subject suffering from IBS, display an increased frequency (or level) of chemokine receptor expressing cells, in particular monocytes, more specifically CCR9 expressing monocytes. Thus, IBS patients may display inflammation that is comparable to that shown by patients suffering from IBD. Irritable bowel syndrome (IBS) is a condition characterized by chronic abdominal pain, discomfort, bloating, and alteration of bowel habits. It is currently diagnosed on the basis of symptoms only. Accordingly, identification of a pro-inflammatory component provides new avenues for treatment and diagnosis of this debilitating condition.

(405) Materials and Methods

(406) 1. Flow Cytometric Analysis of Peripheral Blood

(407) 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 (RT) and stained with antibodies (Table 16) 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).

(408) TABLE-US-00123 TABLE 16 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

(409) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 16). 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).

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

(411) 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 μM) 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 16), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(412) Results and Discussion

(413) Irritable Bowel Syndrome (IBS)

(414) White blood cells from IBS patients was analysed with flow cytometry. The patients exhibited increased numbers of circulating CCR9 expressing monocytes compared to healthy blood (FIG. 21).

(415) Irritable bowel syndrome is a condition characterized by chronic abdominal pain, discomfort, bloating, and alteration of bowel habits. It is currently diagnosed on the basis of symptoms only. The symptoms of IBS are very similar to IBD, Crohns disease (CD) and Ulcerative Colitis (UC), which also show an increase in CCR9 expressing monocytes (FIG. 24).

(416) CCR9 binds to the gut homing chemokine TECK (CCL25) and is important for cell migration to the small intestine. The monocytes in IBS patient blood bind to the biotinylated TECK (bTECK) (FIG. 22).

(417) Depletion of CCR9 expressing monocytes in IBS patient using bTECK conjugated sepharose streptavidin matrix is shown in FIG. 23.

(418) About 25% of the CCR9 expressing monocytes also have a high expression of HLADR suggesting a proinflammatory phenotype (FIG. 25a). These cells can be depleted using bTECK conjugated sepharose streptavidin matrix (FIG. 25b).

(419) We conclude that monocytes in IBS express CCR9 and bind the ligand bTECK. Furthermore, the majority of the CCR9 expressing monocytes can be removed with Sepharose Streptavidin matrix conjugated with bTECK.

(420) B. Treating Conditions Associated with Metabolic Syndrome

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

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

(423) Materials and Methods

(424) Isolation of Peripheral Blood Leukocytes.

(425) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(426) Chemokines.

(427) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labeled MCP-1 (in concentrations 10 ng/μL and 50 ng/μL). 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.

(428) Flow Cytometry Assay.

(429) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 11

Binding of Monocytes to MCP-1

(430) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 26a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 26b and 26c).

Example 12

(431) Monocytes were investigated for their expression of CCR2 (FIG. 27b) and their ability to bind MCP-1 (FIG. 27a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 27b). The MCP-1 binding to monocytes shown in FIG. 27a corresponds to the CCR2.sup.hi expressing population shown in FIG. 27b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 13

Tailored Leukapheresis

(432) Column Design and Properties

(433) Introduction

(434) 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.

(435) The Column

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

(437) The Plastic House (FIG. 9)

(438) 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. 9. 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.

(439) Streptavidin Sepharose™ BigBeads

(440) 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.

(441) Binding Reagent

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

(443) The Apheresis System

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

(445) The Circuit

(446) The system is illustrated in FIG. 10. 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.

(447) The 4008 ADS Pump

(448) 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.

(449) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

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

(451) 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.

(452) Leukapheresis Time and Flow Rate

(453) 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.

(454) Storage Conditions

(455) 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.

(456) Transport Conditions

(457) 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.

(458) In-Vitro Depletion of Target Cell Populations

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

(460) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 28a.

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

(462) 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.

(463) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 14:

(464) TABLE-US-00124 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK NRQVCANPEKKWVREYINSLEKS-CO2H

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

(466) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 34. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines. The results demonstrate significant depletion of the target population chemokine receptor-expressing cells post matrix perfusion. Depletion tests were performed on blood from a healthy donor. The results are shown in FIG. 28b.

(467) 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 14

MCP1 Derivatives

(468) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 7,

(469) TABLE-US-00125 MKVSAALLCLLLIAATFIPQGLAQPDAINAPVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIVAKEICADPKQ KWVQDSMDHLDKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 8,

(470) TABLE-US-00126 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSXDHL DKQTQTPKT
X=Met or Norleucine
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.

(471) Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 29.

(472) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 30, see also SEQ ID NO: 8.

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

Example 15

Synthesis of a Ccr2 Antagonist Biotinmcp-1 which Binds to the Receptor without Activation

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

(475) The amino acid sequence of the truncated version is set forth as SEQ ID NO: 9:

(476) TABLE-US-00127 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(477) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the e-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 31. The PEG spacer will be 3,6,-dioxoaminooctanoic acid.

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

Example 16

Demonstrate Removal of Ccr2 Expressing Cells Using an Alternative Chemokine Ligand to mcp-1

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

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

(481) TABLE-US-00128 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG
The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 11

(482) TABLE-US-00129 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(483) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 32. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 12:

(484) TABLE-US-00130 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL
In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 13:

(485) TABLE-US-00131 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL
Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 33.

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

Examples 17 to 19

Synthesis of Additional Chemokines

(487) General Protocols

(488) Assembly:

(489) 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.

(490) Removal of Dde Protection:

(491) 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.

(492) Labelling Steps:

(493) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG) Resin was swollen in DMF and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38 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.
2. Capping
The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.
3. Fmoc Deprotection
Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.
4. Couple Biotin-OSu
A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.
Cleavage:

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

(495) Purification Protocol:

(496) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(497) Folding Protocol:

(498) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 17

BiotinMCP-1 (CCL2)

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

(500) Modifications:

(501) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

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

(503) TABLE-US-00132 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln
The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(504) TABLE-US-00133 SEQ ID NO: 16 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(505) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine.

(506) TABLE-US-00134 SEQ ID NO: 17 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu or Gln
X75 is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

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

(508) Functional Assay Data:

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

Example 18

BiotinRANTES (CCL5)

(510) Target Molecule:

(511) RANTES derivatised at the ε-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(512) Modifications:

(513) 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.

(514) The linear amino acid sequence (SEQ ID NO: 18) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(515) TABLE-US-00135 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(516) 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:

(517) TABLE-US-00136 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN

(518) X is K(ivDde)

(519) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 19). 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: 20).

(520) TABLE-US-00137 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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))

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

(522) Functional Assay Data:

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

Example 19

BiotinMCP-2 (CCL8)

(524) Target Molecule:

(525) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(526) Modifications:

(527) 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 ε-amino functionality and the biotin.

(528) The linear amino acid sequence (SEQ ID NO: 21) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(529) TABLE-US-00138 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(530) 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:

(531) TABLE-US-00139 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(532) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 22). 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: 23):

(533) TABLE-US-00140 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).

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

(535) Functional Assay Data:

(536) 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 20

Diagnosis and Treatment of Diabetes Mellitus (DM)

(537) Materials and Methods

(538) 1. Flow Cytometric Analysis of Peripheral Blood

(539) 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 (RT) and stained with antibodies (Table 17) 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).

(540) TABLE-US-00141 TABLE 17 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter CCR5 PE Biolegend CCR3 PE Biolegend Streptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend CD3 V450 BD Biosciences CD19 V500 BD Biosciences CD16 PE Cy7 BD Biosciences CCR4 PerCP Cy5.5 BD Biosciences
2. Chemokine Binding Test

(541) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 17). 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).

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

(543) 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 μM) 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 17), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(544) Results and Discussion

(545) Diabetes Mellitus (DM)

(546) 1. Flow Cytometric Analysis of Peripheral Blood

(547) White blood cells from DM patients were analysed with flow cytometry. About 80% of the monocytes are shown to express the chemokine receptor CCR2 (FIG. 36) based upon flow cytometry data and binding by an anti-CCR2 antibody. CCR2 and its ligand MCP1 (CCL2) are important for monocyte migration and infiltration into the inflamed tissue. CCR2 is expressed on the majority of the monocytes in both diabetes patients and healthy controls. The expression of the CCR2 receptor on monocytes is not increased in diabetes patients; however, the monocytes in diabetes are potentially different in their pro-inflammatory profile with regards to other mediators. Furthermore, the ligand MCP-1 is secreted by inflamed and damaged tissue, such as the pancreas in diabetes, and will attract CCR2 expressing monocytes. In healthy individuals, this signal is absent and the monocytes will not migrate into the tissue even though they express CCR2. Thus, migration of pro-inflammatory CCR2 expressing monocytes is rather regulated by the level of the ligand MCP-1 than the level of CCR2 expression.

(548) FIG. 44 shows an increased frequency of CCR4 expressing T cells in four patients with type 2 Diabetes compared to healthy controls. FIG. 23 shows an increased frequency of CCR5 expressing T cells in four patients with type 2 Diabetes compared to healthy controls. These cells may be depleted using suitable chemokines such as CCL17 (TARC) and CCL22 (MDC), which bind CCR4 only and CCL5 (RANTES) which binds CCR5.

(549) 2. Chemokine Binding Test

(550) In accordance with the CCR2 expression, all the monocytes binds to biotinylated MCP1 (bMCP1) (FIG. 37).

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

(552) 21% of the CCR2 expressing monocytes were efficiently depleted with bMCP1-conjugated Sepharose Streptavidin Matrix. Before depletion there were 80% CCR2 expressing monocytes and after depletion 63% (FIG. 38).

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

Example 21

Diagnosis and Treatment of Adiposis Dolorosa

(554) Materials and Methods

(555) 1. Flow Cytometric Analysis of Peripheral Blood

(556) 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 (RT) and stained with antibodies (Table 18) 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).

(557) TABLE-US-00142 TABLE 18 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter Streptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend CD16 PE Cy7 BD Biosciences HLADR APC Cy7 Biolegend CD3 V450 BD Biosciences CD19 V500 BD Biosciences
2. Chemokine Binding Test

(558) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 18). 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).

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

(560) 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 μM) 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 18), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(561) Results and Discussion

(562) 1. Flow Cytometric Analysis of Peripheral Blood

(563) White blood cells from AD patients was analysed with flow cytometry. The patients exhibited two fold increased numbers of circulating CCR2 expressing B cells (mean of 1.8% compared to approximately 0.7% in healthy blood) (FIG. 39).

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

(565) 2. Chemokine Binding Test

(566) CCR2 binds to the chemokine MCP1 (CCL2) and is important for cell recruitment to inflammation sites. 65% of the B cells bind to the biotinylated MCP1 (bMCP1) (FIG. 40).

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

(568) 67% of the CCR2 expressing B cells were efficiently depleted with bMCP1-conjugated Sepharose Streptavidin Matrix. Before depletion there were 3% CCR2 expressing B cells and after depletion 1% (FIG. 41).

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

(570) FIG. 43 shows depletion of CCR1 expressing moncoytes with SepharoseStrepavidin-matrix-bRANTES. Blood cells from a healthy control were incubated with biotinylated chemokine-Sepharose Streptavidin-matrix. Unbound cells were retrieved by washing the matrix. The cells (After Depletion) were then analysed with flow cytometry and compared with cells that had not been incubated with bchemokine-matrix (Before Depletion). Thus, monocytes expressing CCR1 are upregulated in AD. These cells can be effectively depleted using a suitable chemokine, in this case biotinylated CCL5 (RANTES).

(571) C. Treating Inflammatory Arthritis

(572) The expression of MCP-1 precedes the development of MA. RA in patients with autoantibodies against citrullinated peptides and the MCP-1 levels remain higher during active disease.

(573) This implies as an important inflammatory mediator of disease and that MCP-1 levels is a marker of disease activity for RA and in patients with juvenile RA. The increased levels of MCP-1 in the inflammatory joint will recruit proinflammatory monocytes, machropages to the joint to further increase the inflammation. Thus, removing MCP-1 recruitable cells by tailored leukapheresis is a promising treatment.

(574) In animal models of RA (adjuvant-induced arthritis model) local and systemic elevated levels of the chemokine RANTES has been observed. This implies that proinflammatory cells recruited by the increased expression of RANTES in the joint is a contributing mechanism to the arthritis disease.

(575) Thus, in some phases of the disease MCP-1 or RANTES may be the dominant chemokine recruiting different sets of proinflammatory cells to the joint indicating different stages of the inflammatory disease. By investigating chemokines, and the expression of chemokine receptors on circulating proinflammatory cells the inflammatory stage and direction can be determined, supporting a personalized choice for removing a particular disease creating cell population from the circulation by tailored chemokine leukapheresis. RA is a systemic autoimmune disease and can affect any area of the body, including the nervous system, the lungs, the vasculature and the heart. The joints become inflamed and infiltration of inflammatory cells lead to cartilage destruction. The infiltration of cells is regulated by chemokine secretion in the synovial (“Molecular aspects of rheumatoid arthritis: chemokines in the joints of patients”: Iwamoto T et al, Minireview FEBS Journal 275 (2008) 4448-4455).

(576) Chemocentryx have ongoing clinical studies on a CCR1 antagonist, CCX354, to target the chemokine-infiltration mechanism in RA. Results indicate problems with dose-finding. (“Pharmacokinetic and pharmacodynamic evaluation of novel CCR1 antagonist CCX354 in healthy human subjects: implications for selection of clinical dose”: Dairaghi D J et al, Nature Volume 89 number 5, May 2011).

(577) It is shown herein that subjects suffering from rheumatoid arthritis contain chemokine receptor expressing cells in the peripheral blood, in particular CCR1 and CCR2 expressing monocytes. It is also shown herein that the CCR2 and CCR1 cells can be removed using a suitable binding reagent, in particular CCL2 to remove CCR2 expressing cells and CCL5 (RANTES) (in biotinylated form) immobilized on a suitable matrix to remove CCR1 expressing cells. Similarly, it is shown herein that CCR5-expressing lymphocytes, in particular T-lymphocytes can be depleted in subjects suffering from rheumatoid arthritis using a suitable binding reagent, in particular CCL5 (RANTES), in biotinylated form, immobilized on a suitable matrix. Leukocytes with pro inflammatory functions are in the circulation and are recruited to the site of inflammation, i.e, the joint, by the expression of local chemokines allowing transmigration of effector leukocytes into the joint. Thus, normal expression levels in the presence of local production of chemokines will promote inflammation. In a healthy subject where there is no production of local chemokines there is no extravasation of leukocytes to the joint. Therefore, subjects may have an increased expression level of chemokine receptor, increased number of chemokine receptor expressing cells or the chemokine receptor expression may be normal but the patient has an increased level of local chemokine production.

(578) Chemokines can be measured using immunological techniques such as ELISA. Such approaches may be multiplexed as needed. The chemokine levels are increased in the joint, attracting a particular chemokine receptor expressing cell. Although the number of cells may not be increased in the circulation, that transmigration into the joint will worsen the joint inflammation. In blood the decrease in the chemokine receptor expressing population targeted by the column will be used for monitoring treatment as discussed herein.

Examples 22 and 23

Materials and Methods

(579) Isolation of Peripheral Blood Leukocytes.

(580) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(581) Chemokines.

(582) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labelled MCP-1 (in concentrations 10 ng/μL and 50 ng/μL). 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.

(583) Flow Cytometry Assay.

(584) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 22

Binding of Monocytes to MCP-1

(585) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 46a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 46b and 46c).

Example 23

(586) Monocytes were investigated for their expression of CCR2 (FIG. 47b) and their ability to bind MCP-1 (FIG. 47a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 47b). The MCP-1 binding to monocytes shown in FIG. 47a corresponds to the CCR2.sup.hi expressing population shown in FIG. 47b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 24

Tailored Leukapheresis

(587) Column Design and Properties

(588) Introduction

(589) 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.

(590) The Column

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

(592) The Plastic House (FIG. 9)

(593) 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. 9. 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.

(594) Streptavidin Sepharose™ BigBeads

(595) 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.

(596) Binding Reagent

(597) Coupled to the matrix is the third component of the device, one or more binding reagents that bind specifically to CCR2, CCR1, CCR3 or CCR5. One or more chemokines selected from the group consisting of: MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 and RANTES may be employed. These peptides may be synthetic, engineered versions of the human chemokine, which are truncated and biotinylated, but retain binding activity to the CCR2, CCR1, CCR3 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 4×10.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.

(598) The Apheresis System

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

(600) The Circuit

(601) The system is illustrated in FIG. 10. 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.

(602) The 4008 ADS Pump

(603) 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.

(604) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. 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.

(605) Legend for FIG. 11:

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

(607) 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.

(608) Leukapheresis Time and Flow Rate

(609) 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.

(610) Storage Conditions

(611) 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.

(612) Transport Conditions

(613) 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.

(614) In-Vitro Depletion of Target Cell Populations

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

(616) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 48a.

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

(618) 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 magnetic column device containing biotinylated RANTES coupled MACS beads. 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. 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. 48b.

(619) 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).

(620) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 34:

(621) TABLE-US-00143 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-CO2H

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

(623) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 54. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

Example 25

MCP1 Derivatives

(624) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 25). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 24,

(625) TABLE-US-00144 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 25,

(626) TABLE-US-00145 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(627) 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.

(628) Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 49.

(629) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 50.

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

Example 26

Synthesis of a Ccr2 Antagonist Biotinmcp-1 which Binds to the Receptor without Activation

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

(632) The amino acid sequence of the truncated version is set forth as SED ID NO: 26:

(633) TABLE-US-00146 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(634) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 51. The PEG spacer will be 3,6,-dioxoaminooctanoic acid.

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

Example 27

Demonstrate Removal of Ccr2 Expressing Cells Using an Alternative Chemokine Ligand to mcp-1

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

(637) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 27

(638) TABLE-US-00147 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(639) The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 28

(640) TABLE-US-00148 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(641) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 52. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 29:

(642) TABLE-US-00149 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL
In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 27:

(643) TABLE-US-00150 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(644) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 53.

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

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

Examples 28 to 31

Chemokine Synthesis

(647) General Protocols

(648) Assembly:

(649) 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.

(650) Removal of Dde Protection:

(651) 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.

(652) Labelling Steps:

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

(654) 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.

(655) 2. Capping

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

(657) 3. Fmoc Deprotection

(658) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(659) 4. Couple Biotin-OSu

(660) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(661) Cleavage:

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

(663) Purification Protocol:

(664) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(665) Folding Protocol:

(666) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 28

BiotinMCP-1 (CCL2)

(667) Target Molecule:

(668) MCP-1 derivatised at the ε-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(669) Modifications:

(670) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(671) The linear amino acid sequence (SEQ ID NO: 31) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(672) TABLE-US-00151 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

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

(674) TABLE-US-00152 SEQ ID NO: 32 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(675) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine.

(676) TABLE-US-00153 SEQ ID NO: 33 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu or Gln
X75 is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

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

(678) Functional Assay Data:

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

Example 29

BiotinRANTES (CCL5)

(680) Target Molecule:

(681) RANTES derivatised at the ε-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(682) Modifications:

(683) 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.

(684) The linear amino acid sequence (SEQ ID NO: 34) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(685) TABLE-US-00154 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(686) 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:

(687) TABLE-US-00155 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(688) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 35). 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: 36).

(689) TABLE-US-00156 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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))

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

(691) Functional Assay Data:

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

Example 30

BiotinMCP-2 (CCL8)

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

(694) Modifications:

(695) 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 ε-amino functionality and the biotin.

(696) The linear amino acid sequence (SEQ ID NO: 37) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(697) TABLE-US-00157 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln
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:

(698) TABLE-US-00158 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln X75=K(ivDde)

(699) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 38). 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: 39):

(700) TABLE-US-00159 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-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).

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

(702) Functional Assay Data:

(703) 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 31

BiotinEotaxin (CCL11)

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

(705) Modifications:

(706) 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 ε-amino functionality and the biotin.

(707) The linear amino acid sequence (SEQ ID NO: 40) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(708) TABLE-US-00160 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLA KDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
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).

(709) 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:

(710) TABLE-US-00161 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(ivDde)

(711) FmocLys(ivDde)-OH was incorporated as residue 73 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 41). 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: 42):

(712) TABLE-US-00162 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(PEG-Biotin)

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

(714) Functional Assay Data:

(715) 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 32

Diagnosis and Treatment of Inflammatory Arthritis

(716) Materials and Methods

(717) 1. Flow Cytometric Analysis of Peripheral Blood

(718) 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 (RT) and stained with antibodies (Table 19) 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).

(719) TABLE-US-00163 TABLE 19 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter CCR5 PE Biolegend Streptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend CD16 PE Cy7 BD Biosciences CCR1 Alexa Fluor Biolegend 647 CD3 V450 BD Biosciences CD19 V500 BD Biosciences
2. Chemokine Binding Test

(720) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 19). 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).

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

(722) 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 μM) 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 19), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(723) Results and Discussion

(724) 1. Flow Cytometric Analysis of Peripheral Blood

(725) White blood cells from patients with RE were analysed for the expression of chemokine receptors with flow cytometry. The majority of the monocytes express the chemokine receptors CCR1 (FIG. 56a) and CCR2 (FIG. 56b) and 40% of the T cells express CCR5 (FIG. 56b), based upon flow cytometry data and binding by anti-CCR1, anti-CCR2 and anti-CCR5 antibodies.
2. Chemokine Binding Test

(726) The ligand for CCR2 is MCP-1 (CCL2) and the ligand for CCR1 and CCR5 is RANTES (CCL5). Both MCP1 and RANTES are expressed in the synovial fluid of patients with rheumatoid arthritis and are associated with migration of inflammatory immune cells into the joint.

(727) Circulating blood cells from RE patient bound to biotinylated MCP1 (bMCP1) and biotinylated RANTES (bRANTES) (FIGS. 57a and 57b).

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

(729) A majority of the CCR2 expressing monocytes were efficiently depleted with bMCP1-conjugated Sepharose Streptavidin Matrix. (FIG. 58a). 64% of the CCR5 expressing T cells were efficiently depleted with bRANTES-conjugated Sepharose Streptavidin Matrix. (FIG. 58b).

(730) We conclude that circulating immune cells in rheumatoid arthritis patients express CCR1, CCR2 and CCR5 and bind the ligands bMCP1 and bRANTES. Furthermore, 94% of the CCR2 and 64% CCR5 expressing cells can be removed with Sepharose Streptavidin matrix conjugated with the corresponding biotinylated chemokine.

(731) D. Treating Cancer

(732) 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.

(733) 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.

(734) 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.

(735) 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.

(736) 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.

(737) 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 33

Tailored Leukapheresis

(738) Column Design and Properties

(739) Introduction

(740) 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.

(741) The Column

(742) 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.

(743) The Plastic House (FIG. 9)

(744) 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.

(745) Streptavidin Sepharose™ BigBeads

(746) 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.

(747) Binding Reagent

(748) 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 4×10.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.

(749) The Apheresis System

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

(751) The Circuit

(752) The system is illustrated in FIG. 10. 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.

(753) The 4008 ADS Pump

(754) 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.

(755) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

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

(757) 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.

(758) Leukapheresis Time and Flow Rate

(759) 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.

(760) Storage Conditions

(761) 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.

(762) Transport Conditions

(763) 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.

(764) In-Vitro Depletion of Target Cell Populations

(765) 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.

(766) 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. 59.

(767) 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 34A

Treatment of Chronic Lymphatic Leukemia (CLL) Patient

(768) Materials and Methods

(769) 1. Flow Cytometric Analysis of Peripheral Blood

(770) 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 (RT) and stained with antibodies (Table 20) 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).

(771) TABLE-US-00164 TABLE 20 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
2. Chemokine Binding Test

(772) 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 (RT) and stained with cell specific antibodies together with biotinylated chemokine (1 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 20). 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).

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

(774) 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 μM) 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 μm nylon filter (BD Falcon™ Cell Strainer). The flow through cells were stained with antibodies (Table 20), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(775) Results and Discussion

(776) 1. Flow Cytometric Analysis of Peripheral Blood

(777) 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. 61). 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.

(778) 2. Chemokine Binding Test

(779) All the B cells were shown to express the chemokine receptor CCR7 (FIG. 62) 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. 63).

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

(781) 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. 64).

(782) 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 34B

Treatment of Cancers Via Removal of CCR4 Expressing Tregs Using Biotinylated-MDC (CCL22)

(783) Materials and Methods

(784) 1. Flow Cytometric Analysis of Peripheral Blood

(785) 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 (RT) and stained with antibodies (Table 21) 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).

(786) TABLE-US-00165 TABLE 21 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
2. Chemokine Binding Test

(787) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 21). 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).

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

(789) 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 μM) 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 21), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(790) Results and Discussion

(791) 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. 65). In both UBC and PC patients, CCR4 was highly upregulated on Tregs compared with the total T cell population (FIG. 66).

(792) The ligand for CCR4 is the chemokine MDC (CCL22). In accordance with the CCR4 expression, the Tregs bound to biotinylated MDC (bMDC) (FIG. 67).

(793) The CCR4 expressing T cells could be depleted with bMDC-conjugated Sepharose Streptavidin Matrix (FIG. 68).

(794) 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 35 to 42

General Protocols

(795) Synthesis of Chemokines

(796) Assembly:

(797) 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.

(798) Removal of Dde Protection:

(799) 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.

(800) Labelling Steps:

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

(802) 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.

(803) 2. Capping

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

(805) 3. Fmoc deprotection

(806) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(807) 4. Couple Biotin-OSu

(808) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(809) Cleavage:

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

(811) Purification Protocol:

(812) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(813) Folding Protocol:

(814) 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, 250×4.6 mm column, 10-60% B over 30 minutes). Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 35

BiotinMCP-2 (CCL8)

(815) Target Molecule:

(816) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(817) Modifications:

(818) 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 ε-amino functionality and the biotin.

(819) The linear amino acid sequence (SEQ ID NO: 43) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(820) TABLE-US-00166 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKE RWVRDSMKHLDQIFQNLXP-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, in particular K(PEG-Biotin)

(821) 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:

(822) TABLE-US-00167 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(823) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 44). 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: 45).

(824) TABLE-US-00168 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(PEG-Biotin)

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

(826) Functional Assay Data:

(827) 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 36

BiotinRANTES (CCL5)

(828) Target Molecule:

(829) RANTES derivatised at the e-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(830) Modifications:

(831) 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.

(832) The linear amino acid sequence (SEQ ID NO: 46) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(833) TABLE-US-00169 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(834) 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:

(835) TABLE-US-00170 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X=K(ivDde)

(836) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 47). 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: 48).

(837) TABLE-US-00171 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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)

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

(839) Functional Assay Data:

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

Example 37

BiotinMIP-3a (CCL20)

(841) Target Molecule:

(842) MIP-3a derivatised at the e-amino side chain functionality of Lys(68) with PEG-Biotin (TFA salt)

(843) Modifications:

(844) 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 ε-amino functionality and the biotin.

(845) The linear amino acid sequence (SEQ ID NO: 49) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 68 (K):

(846) TABLE-US-00172 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVKNM-OH

(847) 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:

(848) TABLE-US-00173 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVXNM-RESIN
X=K(ivDde)

(849) FmocLys(ivDde)-OH was incorporated as residue 68 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 50). 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: 51).

(850) TABLE-US-00174 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVXNM-OH
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)
Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMip-3a: obtained=8396.4 Da; expected 8397.0 Da.
Functional Assay Data:

(851) BiotinMIP-3a 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-3a is 1.0 nM.

Example 38

BiotinSDF-1a (CXCL12)

(852) Target Molecule:

(853) SDF-1a derivatised at the e-amino side chain functionality of Lys(64) with Biotin (TFA salt)

(854) Modifications:

(855) 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.

(856) The linear amino acid sequence (SEQ ID NO: 52) is shown, prior to attachment of the biotin molecule at amino acid 64 (K):

(857) TABLE-US-00175 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNR QVCIDPKLKWIQEYLEKALN-OH

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

(859) TABLE-US-00176 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKXALN-RESIN

(860) X=K(ivDde)

(861) FmocLys(ivDde)-OH was incorporated as residue 64 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 53). 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: 54).

(862) TABLE-US-00177 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEXALN-OH
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)

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

(864) Functional Assay Data:

(865) biotinSDF-1a 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-1a is 12.0 nM.

Example 39

BiotinMDC (CCL22)

(866) Target Molecule:

(867) MDC derivatised at the e-amino side chain functionality of Lys(66) with PEG-Biotin (TFA salt)

(868) Modifications:

(869) 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 ε-amino functionality and the biotin.

(870) The linear amino acid sequence (SEQ ID NO: 55) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 66 (K):

(871) TABLE-US-00178 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTF RDKEICADPRVPWVKMILNKLSQ-NH.sub.2

(872) 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:

(873) TABLE-US-00179 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRD KEICADPRVPWVKMILNXLSQ-RESIN
X=K(ivDde)

(874) FmocLys(ivDde)-OH was incorporated as residue 66 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 56). 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: 57).

(875) TABLE-US-00180 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFR DKEICADPRVPWVKMILNXLSQ-NH.sub.2
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)

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

(877) Functional Assay Data:

(878) 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 40

BiotinTARC (CCL17)

(879) Target Molecule:

(880) TARC derivatised at the e-amino side chain functionality of Lys(72) with PEG-Biotin (TFA salt)

(881) Modifications:

(882) 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 ε-amino functionality and the biotin.

(883) The linear amino acid sequence (SEQ ID NO: 58) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(884) TABLE-US-00181 H-ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQGRA ICSDPNNKRVKNAVKYLQSLERSX-NH.sub.2
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)

(885) 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:

(886) TABLE-US-00182 H-ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQG RAICSDPNNKRVKNAVKYLQSLERSX-RESIN
X=K(ivDde)

(887) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 59). 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: 60).

(888) TABLE-US-00183 H-ARGTNVGRECCLEYFKGAIPLRKLKTWYQTSEDCSRDAIVFVTVQG RAICSDPNNKRVKNAVKYLQSLERSX-NH.sub.2
X=K(PEG-Biotin)

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

(890) Functional Assay Data:

(891) 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 41

BiotinMIP-3b (CCL19)

(892) Target Molecule:

(893) MIP-3b derivatised at the e-amino side chain functionality of Lys(78) with Biotin (TFA salt)

(894) Modifications:

(895) Human MIP-3b 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.

(896) The linear amino acid sequence (SEQ ID NO: 61) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):

(897) TABLE-US-00184 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
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)

(898) The engineered MIP-3b 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:

(899) TABLE-US-00185 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN
X is FmocLys(ivDde)

(900) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 62). 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: 63).

(901) TABLE-US-00186 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
X is K(Biotin)

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

(903) Functional Assay Data:

(904) biotinMip-3b 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-3b is 1.6 nM.

Example 42

BiotinITAC (CXCL11)

(905) Target Molecule:

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

(907) Modifications:

(908) 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.

(909) The linear amino acid sequence (SEQ ID NO: 64) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(910) TABLE-US-00187 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNF-OH

(911) 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:

(912) TABLE-US-00188 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNFX-RESIN
X is PEG-K(ivDde)

(913) 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 ε-amino side chain functionality of the additional Lys (SEQ ID NO: 66). 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: 67).

(914) TABLE-US-00189 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKEN KGQRCLNPKSKQARLIIKKVERKNFX-OH
X is PEG-K(Biotin)

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

(916) Functional Assay Data:

(917) 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 43

BiotinTECK (CCL25)

(918) Target Molecule:

(919) TECK (Met to Nleu substitution) derivatised at the ε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(920) Modifications:

(921) 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 ε-amino functionality and the biotin.

(922) The linear amino acid sequence (SEQ ID NO: 68) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(923) TABLE-US-00190 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR HRKVCGNPKSREVQRAXKLLDARNXVF-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, e.g. K(PEG-Biotin)

(924) 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:

(925) TABLE-US-00191 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-RESIN
X1=pyroGlu or Gln
X64=Norleucine
X72=K(ivDde)

(926) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 69). 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: 70).

(927) TABLE-US-00192 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-OH
X1=pyroGlu or Gln
X64=Norleucine
X72 is K(PEG-Biotin)

(928) 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.

(929) Functional Assay Data:

(930) 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 44

BiotinCTAC (CCL27)

(931) Target Molecule:

(932) CTAC derivatised at the e-amino side chain functionality of Lys(87) with PEG-Biotin (TFA salt)

(933) Modifications:

(934) 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 ε-amino functionality and the biotin.

(935) The linear amino acid sequence (SEQ ID NO: 71) is shown, prior to attachment of the PEG spacer and biotin molecules:

(936) TABLE-US-00193 FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLAQR SICIHPQNPSLSQWFEHQERKLHGTLPKLNFGMLRKXG
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)

(937) 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:

(938) TABLE-US-00194 H-FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLA QRSICIHPQNPSLSQWFEHQERKLHGTLPKLNFGMLRKXG-RESIN
X=K(ivDde)

(939) FmocLys(ivDde)-OH was incorporated as residue 87 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 72). 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: 73).

(940) TABLE-US-00195 H-FLLPPSTACCTQLYRKPLSDKLLRKVIQVELQEADGDCHLQAFVLHLA QRSICIHPQNPSLSQWFEHQERKLHGTLPKLNFGMLRKXG-OH
X=K(PEG-Biotin)

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

(942) Functional Assay Data:

(943) 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 45

BiotinIP-10 (CXCL10)

(944) Target Molecule:

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

(946) Modifications:

(947) 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.

(948) The linear amino acid sequence (SEQ ID NO: 74) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(949) TABLE-US-00196 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSP-OH

(950) 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:

(951) TABLE-US-00197 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSPX-RESIN
X is K(ivDde), optionally attached via a spacer such as PEG, e.g.—PEG-K(ivDde)

(952) 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 ε-amino side chain functionality of the additional Lys (SEQ ID NO: 75). 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: 76):

(953) TABLE-US-00198 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKK GEKRCLNPESKAIKNLLKAVSKERSKRSPX-OH
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)

(954) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIP-110: obtained=9141.0 Da; expected 9141.9 Da.

(955) Functional Assay Data:

(956) 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.

(957) E. Treating Mental Disorders

(958) 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.

(959) 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).

(960) 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.

(961) 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 46

Materials and Methods

(962) Isolation of Peripheral Blood Leukocytes.

(963) 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.

(964) Chemokines.

(965) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labeled eotaxin (in concentrations 10 ng/μL and 50 ng/μL). 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.

(966) Flow Cytometry Assay.

(967) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

(968) Neutrophils/eosinophils were investigated for their expression of CCR3 (FIG. 69b) and their ability to bind eotaxin (FIG. 69a). CCR3 expression was noted on all neutrophils/eosinophils with the majority of neutrophils/eosinophils expressing high levels, using an anti-CCR3 antibody (FIG. 69b). The eotaxin binding to neutrophils/eosinophils shown in FIG. 1a corresponds to the CCR3.sup.hi expressing population shown in FIG. 69b. Thus, eotaxin binds favourably to CCR3.sup.hi expressing cells.

Example 47

Tailored Leukapheresis

(969) Column Design and Properties

(970) Introduction

(971) 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.

(972) The Column

(973) 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.

(974) The Plastic House (FIG. 9)

(975) 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. 9. 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.

(976) Streptavidin Sepharose™ BigBeads

(977) 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.

(978) Binding Reagent

(979) 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 4×10.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.

(980) The Apheresis System

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

(982) The Circuit

(983) The system is illustrated in FIG. 10. 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.

(984) The 4008 ADS Pump

(985) 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.

(986) 1A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

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

(988) 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.

(989) Leukapheresis Time and Flow Rate

(990) 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.

(991) Storage Conditions

(992) 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.

(993) Transport Conditions

(994) 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.

(995) In-Vitro Depletion of Target Cell Populations—Eotaxin

(996) 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.

(997) 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. 70a.

(998) 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).

(999) In-Vitro Depletion of Target Cell Populations—RANTES

(1000) 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.

(1001) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 79:

(1002) TABLE-US-00199 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-CO2H

(1003) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(1004) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 72. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines. The results demonstrate significant depletion of the target population chemokine receptor-expressing cells post matrix perfusion. Depletion tests were performed on blood from a healthy donor. The results are shown in FIG. 70b.

(1005) 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 48

Eotaxin Derivatives

(1006) 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: 77,

(1007) TABLE-US-00200 MKVSAALLWL LLIAAAFSPQ GLAGPASVPT TCCFNLANRK IPLQRL ESYRRITSGKCPQK AVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP

(1008) The amino acid sequence of the mature protein is set forth as SEQ ID NO: 78,

(1009) TABLE-US-00201 GPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLA KDICADPKKKW VQDSMKYLDQ KSPTPKP

(1010) 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.

(1011) Thus, eoxtaxin derivatised at the ε-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. 71.

(1012) 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. 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 49 to 52

General Protocols for Chemokine Synthesis

(1013) Assembly:

(1014) 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.

(1015) Removal of Dde Protection:

(1016) 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.

(1017) Labelling Steps:

(1018) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(1019) 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.

(1020) 2. Capping

(1021) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(1022) 3. Fmoc Deprotection

(1023) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1024) 4. Couple Biotin-OSu

(1025) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(1026) Cleavage:

(1027) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(1028) Purification Protocol:

(1029) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(1030) Folding Protocol:

(1031) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 49

BiotinMCP-2 (CCL8)

(1032) Target Molecule:

(1033) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1034) Modifications:

(1035) 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 ε-amino functionality and the biotin.

(1036) The linear amino acid sequence (SEQ ID NO: 80) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1037) TABLE-US-00202 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(1038) 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:

(1039) TABLE-US-00203 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(1040) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 81). 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: 82):

(1041) TABLE-US-00204 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).

(1042) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(1043) Functional Assay Data:

(1044) 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 50

BiotinEotaxin (CCL11)

(1045) Target Molecule:

(1046) Eotaxin derivatised at the e-amino side chain functionality of Lys(73) with PEG-Biotin (TFA salt)

(1047) Modifications:

(1048) 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 ε-amino functionality and the biotin.

(1049) The linear amino acid sequence (SEQ ID NO: 83) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(1050) TABLE-US-00205 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
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).

(1051) 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:

(1052) TABLE-US-00206 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(ivDde)

(1053) FmocLys(ivDde)-OH was incorporated as residue 73 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 84). 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: 85):

(1054) TABLE-US-00207 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(PEG-Biotin)

(1055) Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) data of purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3 Da.

(1056) Functional Assay Data:

(1057) 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 51

BiotinRANTES (CCL5)

(1058) Target Molecule:

(1059) RANTES derivatised at the e-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(1060) Modifications:

(1061) Human RANTES corresponding to residues 1-68, is initially expressed as 91 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The single methionine (Met67) within the sequence was mutated to lysine, to mitigate against oxidation of this residue during the chain assembly, which was observed during the synthesis of the natural sequence derivative. This Met to Lys substitution provided a lysine at position 67 which was modified through biotinylation on the resin. The linear amino acid sequence (SEQ ID NO: 86) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(1062) TABLE-US-00208 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(1063) 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:

(1064) TABLE-US-00209 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(1065) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 87). 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: 88).

(1066) TABLE-US-00210 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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))

(1067) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(1068) Functional Assay Data:

(1069) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 52

BiotinTECK (CCL25)

(1070) Target Molecule:

(1071) TECK (Met to Nleu substitution) derivatised at the ε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(1072) Modifications:

(1073) 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 ε-amino functionality and the biotin.

(1074) The linear amino acid sequence (SEQ ID NO: 89) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(1075) TABLE-US-00211 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNKVF-OH
X1=pyroGlu or Gln
X64=Norleucine

(1076) 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: 90):

(1077) TABLE-US-00212 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKRH RKVCGNPKSREVQRAXKLLDARNXVF-RESIN
X1=pyroGlu or Gin
X64=Norleucine
X72=K(Dde)

(1078) 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: 91).

(1079) TABLE-US-00213 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)

(1080) 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.

(1081) Functional Assay Data:

(1082) 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 53

Diagnosis and Treatment of Bipolar Disorder (BP)

(1083) Materials and Methods

(1084) 1. Flow Cytometric Analysis of Peripheral Blood

(1085) 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 (RT) and stained with antibodies (Table 22) 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).

(1086) TABLE-US-00214 TABLE 22 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

(1087) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 22). 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).

(1088) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1089) 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 μM) 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 22), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(1090) Results and Discussion

(1091) 1. Flow Cytometric Analysis of Peripheral Blood

(1092) 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. 74).

(1093) 2. Chemokine Binding Test

(1094) 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.

(1095) The monocytes from a patient with BP bound biotinylated TECK (bTECK) (FIG. 75).

(1096) 3. Cell Depletion

(1097) The majority of the CCR9 expressing monocytes were depleted with bTECK-conjugated Sepharose Streptavidin Matrix (FIG. 76).

(1098) 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.

(1099) F. Treating Conditions Associated with Allergy

(1100) A range of allergic conditions include an inflammatory component. Eosinophilia appears to be central to allergy related conditions. For example, Kim et al., (Respiratory Medicine (2010) 104, 1436-1443) show that asthma is characterized by eosinophilic inflammation and Th1 response and that none atopic asthma (NAA) patients showed higher percentage eosinophils and Eotaxin levels than atopic asthma and healthy controls. The inventors have identified removal of CCR-3 expressing cells, up-regulated in allergic inflammation, as a suitable therapeutic target. The inventors show, see FIG. 88 that eosinophils are increased in frequency in (peripheral blood of) allergic subjects compared to healthy subjects.

(1101) It is shown herein (see FIG. 86) that subjects or patients suffering from allergies displayed increased frequency of CCR3 expressing monocytes and that these cells can be depleted using a suitable reagent (FIG. 87) such as eotaxin (CCL11). It is further shown herein that subjects suffering from allergic conditions contain chemokine receptor expressing cells in the peripheral blood. Subjects suffering from allergic conditions contain CXCR1 and CXCR2 expressing neutrophils, CCR2 expressing monocytes and CCR3 expressing eosinophils. The expression of these receptors was not increased in allergic patients; however, the cells expressing the relevant receptors are increased in number in the inflammatory tract of patients with allergic disease. Moreover, the cells are potentially different in their pro-inflammatory profile with regards to other mediators. Therefore it may be beneficial for the subjects/patients to remove these cells from the circulation in order to prevent and reduce the influx of cells to the inflammatory tract. It is also shown herein that a potentially therapeutic proportion of the relevant chemokine receptor expressing cells can be removed using a suitable binding reagent. Thus, CXCR1 and CXCR2 expressing cells may be depleted from peripheral blood using IL-8 as binding reagent, in particular biotinylated IL-8 conjugated to a sepharose straptvidin matrix. CCR2 expressing cells may be depleted from peripheral blood using MCP-1 (CCL2) as binding reagent, in particular biotinylated MCP-1 conjugated to a sepharose straptvidin matrix. CCR3 expressing cells may be depleted from peripheral blood using eotaxin (CCL11) as binding reagent, in particular biotinylated eotaxin conjugated to a sepharose straptvidin matrix.

Example 54

Materials and Methods

(1102) Isolation of Peripheral Blood Leukocytes.

(1103) 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.

(1104) Chemokines.

(1105) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labeled eotaxin (in concentrations 10 ng/μL and 50 ng/μL). 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.

(1106) Flow Cytometry Assay.

(1107) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

(1108) Neutrophils/eosinophils were investigated for their expression of CCR3, (FIG. 77b) and their ability to bind eotaxin (FIG. 77a). CCR3, expression was noted in all neutrophils/eosinophils with the majority of neutrophils/eosinophils expressing high levels, using an anti-CCR3, antibody (FIG. 77b). The eotaxin binding to neutrophils/eosinophils shown in FIG. 77a corresponds to the CCR3.sup.hi expressing population shown in FIG. 77b. Thus, eotaxin binds favourably to CCR3.sup.hi expressing cells.

Example 55

Tailored Leukapheresis

(1109) Column Design and Properties

(1110) Introduction

(1111) 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.

(1112) The Column

(1113) The column is intended to be used as a leukapheresis treatment for an allergic condition. It will specifically remove CCR3, CXCR1, CXCR2 and/or CCR2-expressing leukocytes, in particular eosinophils, through the use of a binding reagent, more specifically a biotinylated eotaxin containing resin, exploiting the CCR3, CXCR1, CXCR2 and/or CCR2-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.

(1114) The Plastic House (FIG. 9)

(1115) 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. 9. 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.

(1116) Streptavidin Sepharose™ BigBeads

(1117) 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.

(1118) Binding Reagent

(1119) Coupled to the matrix is the third component of the device, the binding reagent that binds specifically to CCR3, CXCR1, CXCR2 and/or CCR2. 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 CCR3, CXCR1, CXCR2 and/or CCR2 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 4×10.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.

(1120) The Apheresis System

(1121) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(1122) The Circuit

(1123) The system is illustrated in FIG. 10. 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.

(1124) The 4008 ADS Pump

(1125) 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.

(1126) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

(1127) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(1128) 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.

(1129) Leukapheresis Time and Flow Rate

(1130) 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.

(1131) Storage Conditions

(1132) 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.

(1133) Transport Conditions

(1134) 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.

(1135) In-Vitro Depletion of Target Cell Populations—Eotaxin

(1136) 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 a 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.

(1137) 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. 78a.

(1138) In conclusion, the in-vitro results demonstrate a specific reduction of around 25% of the CCR3-expressing cells by the column. Non-CCR3-expressing cells remained unaffected (data not shown).

(1139) In-Vitro Depletion of Target Cell Populations—RANTES

(1140) To investigate the ability to eliminate CCR1, 3 or 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.

(1141) 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. 78b.

(1142) The in-vitro results demonstrate a specific reduction of the majority of the chemokine receptor 1, 3 or 5-expressing cells by the column.

(1143) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 97:

(1144) TABLE-US-00215 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-CO2H

(1145) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(1146) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 80. The protein was folded and disulphide bonds formed between the first and third cysteine residues in the sequence and between the 2nd and 4th cysteines.

Example 56

Eotaxin Derivatives

(1147) Eotaxin has been produced with position 73, thought to be a lysine residue, 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: 92,

(1148) TABLE-US-00216 MKVSAALLWL LLIAAAFSPQ GLAGPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 93,

(1149) TABLE-US-00217 GPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP

(1150) 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.

(1151) Thus, eoxtaxin derivatised at the ε-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. 79.

(1152) 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.

(1153) 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 functional cell-based assay on human CCR3 receptor.

Example 57

Affinity of Blood Cells to Biotinylated IL-8

(1154) Materials and Methods

(1155) Isolation of Peripheral Blood Leukocytes.

(1156) Heparinized peripheral blood from healthy blood donors or IBD patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(1157) Chemokines.

(1158) The leukocytes were incubated for 30 min in the dark at 4° C. with the following biotinylated and Alexa647 Fluor® labeled chemokines: CCL25 (in concentrations of 0.1 ng/μL, 0.5 ng/μL and 5 ng/μL), MIP-1α or MCP-1 (in concentrations 10 ng/μL and 50 ng/μL). 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.

(1159) Flow Cytometry Assay.

(1160) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

(1161) In FIG. 81 the binding to biotinylated IL-8 (CXCL8) of CD4+ lymphocytes (FIG. 81a), CD8+ lymphocytes (FIG. 81a) and CD16+ neutrophils (FIG. 81c) obtained from healthy donors is shown. After 30 min of incubation all CD16+ neutrophils bound to IL-8. In contrast no binding was observed with CD4+ lymphocytes and CD8+ lymphocytes.

Examples 58 to 63

Chemokine Synthesis

(1162) General Protocols

(1163) Assembly:

(1164) 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.

(1165) Removal of Dde Protection:

(1166) 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.

(1167) Labelling Steps:

(1168) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(1169) 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.

(1170) 2. Capping

(1171) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(1172) 3. Fmoc Deprotection

(1173) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1174) 4. Couple Biotin-OSu

(1175) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(1176) Cleavage:

(1177) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(1178) Purification Protocol:

(1179) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(1180) Folding Protocol:

(1181) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 58

BiotinMCP-1 (CCL2)

(1182) Target Molecule:

(1183) MCP-1 derivatised at the ε-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1184) Modifications:

(1185) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(1186) The linear amino acid sequence (SEQ ID NO: 100) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1187) TABLE-US-00218 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

(1188) The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(1189) TABLE-US-00219 SEQ ID NO: 101 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(1190) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine.

(1191) TABLE-US-00220 SEQ ID NO: 102 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu or Gln
X75 is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

(1192) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(1193) Functional Assay Data:

(1194) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 59

BiotinRANTES (CCL5)

(1195) Target Molecule:

(1196) RANTES derivatised at the ε-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(1197) Modifications:

(1198) 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.

(1199) The linear amino acid sequence (SEQ ID NO: 97) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(1200) TABLE-US-00221 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(1201) 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:

(1202) TABLE-US-00222 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(1203) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 98). 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: 99).

(1204) TABLE-US-00223 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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))

(1205) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(1206) Functional Assay Data:

(1207) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 60

BiotinMCP-2 (CCL8)

(1208) Target Molecule:

(1209) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1210) Modifications:

(1211) 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 ε-amino functionality and the biotin.

(1212) The linear amino acid sequence (SEQ ID NO: 94) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1213) TABLE-US-00224 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(1214) 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:

(1215) TABLE-US-00225 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(1216) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 95). 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: 96):

(1217) TABLE-US-00226 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-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).

(1218) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(1219) Functional Assay Data:

(1220) 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 61

BiotinIL-8 (CXCL8)

(1221) Target Molecule:

(1222) IL-8 derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(1223) Modifications:

(1224) Human IL-8 corresponding to residues 1-77, is initially expressed as 99 amino acids comprising the chemokine fold, and a 22 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. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(1225) The linear amino acid sequence (SEQ ID NO: 103) is shown, prior to attachment of the PEG spacer and biotin molecules:

(1226) TABLE-US-00227 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIV KLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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)

(1227) The engineered IL-8 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:

(1228) TABLE-US-00228 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIV KLSDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(1229) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 104). 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: 105):

(1230) TABLE-US-00229 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIV KLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)

(1231) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8: obtained=9416.9 Da; expected 9417.0 Da.

(1232) Functional Assay Data:

(1233) BiotinIL-8 was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 18.9 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 62

BiotinIL-8 (6-78)

(1234) Target Molecule:

(1235) IL-8 (6-78) derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(1236) Modifications:

(1237) Truncated form of IL-8 corresponding to residues 6-77, the first five N-terminal residues have been removed and an additional lysine was inserted at the C-terminus at position 78, and modified through biotinylation on the resin. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(1238) The linear amino acid sequence (SEQ ID NO: 106) is shown, prior to attachment of the PEG spacer and biotin molecules:

(1239) TABLE-US-00230 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSD GRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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

(1240) The engineered IL-8 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:

(1241) TABLE-US-00231 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDG RELCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(1242) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 107). 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: 108):

(1243) TABLE-US-00232 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDG RELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)

(1244) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8 (6-78): obtained=8880.50 Da; expected 8880.4 Da.

(1245) Functional Assay Data:

(1246) BiotinIL-8 (6-78) was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 6.1 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 63

BiotinEotaxin (CCL11)

(1247) Target Molecule:

(1248) Eotaxin derivatised at the e-amino side chain functionality of Lys(73) with PEG-Biotin (TFA salt)

(1249) Modifications:

(1250) 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 ε-amino functionality and the biotin.

(1251) The linear amino acid sequence (SEQ ID NO: 109) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(1252) TABLE-US-00233 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLA KDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
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).

(1253) 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:

(1254) TABLE-US-00234 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLA KDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(ivDde)

(1255) FmocLys(ivDde)-OH was incorporated as residue 73 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 110). 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: 111):

(1256) TABLE-US-00235 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLA KDICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(PEG-Biotin)

(1257) Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) data of purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3 Da.

(1258) Functional Assay Data:

(1259) 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 64

Treatment of Allergy Using CXCR1, CXCR2, CCR2 and CCR3 and Biotinylated IL8, Eotaxin and MCP1

(1260) Materials and Methods

(1261) 1. Flow Cytometric Analysis of Peripheral Blood

(1262) 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 (RT) and stained with antibodies (Table 23) 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).

(1263) TABLE-US-00236 TABLE 23 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CCR3 PE Biolegend CCR2 PerCPCy5.5 Biolegend CXCR1 APC Biolegend CXCR2 PE Biolegend CD16 PECy7 BD Streptavdin APC BD CD14 FITC Beckman Coulter
2. Chemokine Binding Test

(1264) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 23). 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).

(1265) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1266) 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 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 23), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(1267) Results and Discussion

(1268) 1. Flow Cytometric Analysis of Peripheral Blood

(1269) White blood cells from patients with allergy (three of the patients were allergic to birch and one patient was allergic to cats, dogs and grass) were analysed for cell surface markers with flow cytometry. The neutrophils expressed the chemokine receptors CXCR1 and CXCR2, the monocytes expressed CCR2 and the eosinophils expressed CCR3, based upon flow cytometry data (FIG. 83a-c).

(1270) The expression of these receptors was not increased in allergic patients; however, the cells are increased in the inflammatory tract of patients with allergic disease. Moreover, the cells are potentially different in their pro-inflammatory profile with regards to other mediators. Therefore it could be beneficial for the patients to remove these cells from the circulation in order to prevent and reduce the influx of cells to the inflammatory tract. CCR3 expressing monocyte levels are increased in allergic patients (FIG. 86).

(1271) 2. Chemokine Binding Test

(1272) The ligand for CXCR1 and CXCR2 is IL-8, the ligand for CCR3 is eotaxin, and the ligand for CCR2 is MCP-1. Circulating blood cells from patients with allergy bind to biotinylated IL8 (bIL8) and biotinylated Eotaxin (bEotaxin) (FIGS. 85a and b).

(1273) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1274) About 50 percent of the CXCR1 and CXCR2 expressing neutrophils were efficiently depleted with bIL8-conjugated Sepharose Streptavidin Matrix (FIG. 85a). 44% of the CCR3 expressing granulocytes were efficiently depleted with bEotaxin-conjugated Sepharose Streptavidin Matrix (FIG. 85b). 50 percent of the CCR2 expressing monocytes were efficiently depleted with bMCP-conjugated Sepharose Streptavidin Matrix (FIG. 85c).

(1275) CCR3 expressing monocytes can be depleted using biotinylated eotaxin (FIG. 87. We conclude that the immune cells from allergic patients express the CXCR1, CXCR2, CCR2 and CCR3 receptors. The cells can bind their respective ligand and can be removed with Sepharose Streptavidin matrix conjugated with the corresponding biotinylated chemokine.

(1276) G. Treating Inflammatory Skin Diseases

(1277) Patients with psoriasis and AD have an increased number of circulating CCR2 expressing monocytes or increased proinflammtory properties of CCR2 expressing monocytes compared to healthy controls. When investigating the skin from patients with psoriasis and AD there is a selective infiltration of CCR2 expressing monocytes in affected skin areas. To support this notion keratinocytes produce high amounts of MCP-1 thus recruiting CCR2 expressing cells.

(1278) The expression of RANTES is increased in psoriatic lesions, produced by the keratinocytes. Skin infiltrating T cell infiltrates are an important cell population involved in disease development. Supporting these ideas is that CCR6 deficient mice fail to develop IL-23 induced, IL-22 dependent psoriasis-like inflammation demonstrating the importance recruitment of CCR6 expressing T cells for the development of the disease. Moreover animal models of contact dermatitis can be abolished using RANTES antagonists.

(1279) It is shown herein that subjects suffering from inflammatory skin disorders such as psoriasis exhibit increased frequency of chemokine receptor expressing cells in the peripheral blood, in particular CCR4 expressing cells such as CCR4 expressing T lymphocytes, compared to healthy controls. It is also shown herein that the CCR4 expressing cells can be removed using a suitable binding reagent, in particular MDC (in biotinylated form) immobilized on a suitable matrix. Similarly, it is shown herein that CXCR1 and CXCR2-expressing cells, in particular neutrophils, can be depleted in psoriasis patients using a suitable binding reagent, in particular IL-8, in biotinylated form, immobilized on a suitable matrix.

Examples 65 and 66

Materials and Methods

(1280) Isolation of Peripheral Blood Leukocytes.

(1281) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(1282) Chemokines.

(1283) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labeled chemokine (e.g. MCP-1) (in concentrations 10 ng/μL and 50 ng/μL). 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.

(1284) Flow Cytometry Assay.

(1285) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 65

Binding of Monocytes to MCP-1

(1286) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 89a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 89b and 89c).

Example 66

(1287) Monocytes were investigated for their expression of CCR2 (FIG. 90b) and their ability to bind MCP-1 (FIG. 90a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 90b). The MCP-1 binding to monocytes shown in FIG. 90a corresponds to the CCR2.sup.hi expressing population shown in FIG. 90b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 67

Affinity of Blood Cells to CCL25

(1288) In the experiment with biotinylated CCL25 it was found that neither T-cells (CD4+ lymphocytes; CD8+ lymphocytes) nor monocytes (CD14+ monocytes) from the peripheral blood of a healthy donor (FIGS. 89d, 89e and 89f) bound to the biotinylated chemokine. In contrast, about 80% of the CD8+ lymphocytes and about 90% of the CD4+ lymphocytes and the monocytes from a patient with Crohn's disease bound to CCL25 (FIGS. 89g, 89h and 89i).

Example 68

Tailored Leukapheresis

(1289) Column Design and Properties

(1290) Introduction

(1291) 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.

(1292) The Column

(1293) The column is intended to be used as a leukapheresis treatment for inflammatory skin disease. It will specifically remove CCR2-expressing leukocytes, in particular monocytes, through the use of a binding reagent, more specifically an MCP-1, MCP-2, MCP-3, MCP-4 and/or MCP-5 containing resin, exploiting the CCR2-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and one or more of bMCP-1 bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(1294) The Plastic House (FIG. 9)

(1295) 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. 9. 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.

(1296) Streptavidin Sepharose™ BigBeads

(1297) 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.

(1298) Binding Reagent

(1299) Coupled to the matrix is the third component of the device, one or more binding reagents that bind specifically to CCR4, CXCR1, CXCR2, CCR2, CCR6, CCR3, CCR5, CCR1, CCR9 or ChemR23. One or more chemokines selected from the group consisting of: MCP-1. MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, RANTES, CCL25 and/or Chemerin 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 CCR4, CXCR1, CXCR2, CCR2, CCR6, CCR3, CCR5, CCR1, CCR9 or ChemR23 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 4×10.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.

(1300) The Apheresis System

(1301) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(1302) The Circuit

(1303) The system is illustrated in FIG. 10. 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.

(1304) The 4008 ADS Pump

(1305) 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.

(1306) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 22

(1307) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(1308) 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.

(1309) Leukapheresis Time and Flow Rate

(1310) 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.

(1311) Storage Conditions

(1312) 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.

(1313) Transport Conditions

(1314) 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.

(1315) In-Vitro Depletion of Target Cell Populations—MCP-1

(1316) To investigate the ability to eliminate CCR2-expressing cells, in vitro tests have been performed on the bMCP-1 coupled matrix. Blood was collected from blood donors and passed through the column device containing bMCP-1 coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR2-expressing cells.

(1317) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 91a.

(1318) In conclusion, the in-vitro results demonstrate a specific reduction of up to 80% of the CCR2-expressing cells by the column. Notably, individuals with fewer CCR2 expressing cells initially achieved lower depletion. The remaining levels of monocytes were around 20-30% in each case, irrespective of the starting point. Non-CCR2-expressing cells remained unaffected (data not shown).

(1319) In-Vitro Depletion of Target Cell Populations—RANTES

(1320) 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 a magnetic column device containing biotinylated RANTES coupled to MACs beads. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR1, 3 and 5-expressing cells.

(1321) 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. 91b.

(1322) The in-vitro results demonstrate a specific reduction of around 20% of the chemokine receptor-expressing cells by the column. Non-CCR1, 3 or 5-expressing cells remained unaffected (data not shown).

(1323) In-Vitro Depletion of Target Cell Populations—MIP-3a

(1324) To investigate the ability to eliminate CCR6-expressing cells, in vitro tests have been performed on the biotinylated MIP-3a coupled matrix. Blood was collected from blood donors and passed through the column device containing biotinylated MIP-3a coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR6-expressing cells.

(1325) 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. 91c.

(1326) 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).

(1327) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 119:

(1328) TABLE-US-00237 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK NRQVCANPEKKWVREYINSLEKS-CO2H

(1329) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(1330) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 12. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

(1331) In-Vitro Depletion of Target Cell Populations—TECK

(1332) To investigate the ability to eliminate CCR9-expressing cells, in vitro tests have been performed on the bTECK coupled matrix. Blood was collected from blood donors and IBD patients and passed through the column device containing bTECK coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR9-expressing cells.

(1333) The results demonstrate significant depletion of the target population CD14− positive CCR9-expressing cells post matrix perfusion; while total CD14-positive cells remain unchanged. Depletion tests were performed on blood from healthy donors and IBD patients confirming similar effects. The results are shown in FIGS. 91d and 6e respectively.

(1334) In conclusion, the in-vitro results demonstrate a specific reduction of 50-75% of the CCR9-expressing cells by the column. Non-CCR9-expressing cells remained unaffected.

Example 69

Mcp1 Derivatives

(1335) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 112,

(1336) TABLE-US-00238 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 113,

(1337) TABLE-US-00239 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(1338) 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.

(1339) Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 92.

(1340) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 93.

(1341) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 70

Synthesis of a Ccr2 Antagonist Biotinmcp-1 which Binds to the Receptor without Activation

(1342) Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995, 181, 63) has been shown for an MCP-1 derivative truncated at the N-terminus. In particular, deletion of residues 1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis of CCR2 positive cells. (inhibition of chemotaxis IC50 20 nM)

(1343) The amino acid sequence of the truncated version is set forth as SEQ ID NO: 114:

(1344) TABLE-US-00240 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(1345) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 94. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 71

Demonstrate Removal of Ccr2 Expressing Cells Using an Alternative Chemokine Ligand to Mcp-1

(1346) CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 in addition to MCP-1. MCP-5 only binds CCR2 and should be selective in its removal of CCR2 expressing cells. MCP5 is a mouse chemokine shown to chemotact human CCR2 cells with EC50<3 nM.

(1347) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 115

(1348) TABLE-US-00241 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG
The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 116

(1349) TABLE-US-00242 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(1350) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 95. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 117:

(1351) TABLE-US-00243 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL
In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 118:

(1352) TABLE-US-00244 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(1353) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 96.

(1354) Once synthesised, the activity of the various biotinMCP-5 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 72

TECK-PEG-Biotin Synthesis Summary

(1355) Target Molecule:

(1356) TECK (Met to Nleu substitution) derivatised at the ε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(1357) Modifications:

(1358) 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 ε-amino functionality and the biotin.

(1359) The linear amino acid sequence (SEQ ID NO: 120) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(1360) TABLE-US-00245 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR HRKVCGNPKSREVQRAXKLLDARNXVF-OH
X=pyroGlu
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, e.g. K(PEG-Biotin)

(1361) The engineered TECK sequence was assembled on a solid support, using Fmoc protocols for solid-phase peptide synthesis:

(1362) TABLE-US-00246 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR HRKVCGNPKSREVQRAXKLLDARNXVF-RESIN
X1=pyroGlu
X64=Norleucine
X72=K(ivDde)

(1363) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 121).

(1364) Met to Nle substitution.

(1365) N-terminal Gln to pyroglutamic acid substitution.

(1366) Removal of Dde Protection:

(1367) The Dde protecting group was removed by treatment of all resin (2.5 g) with a solution of 2% hydrazine in DMF (100 ml) over 1 hour period to afford 2.0 g resin.

(1368) Labelling Steps:

(1369) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid

(1370) Resin (1.5 g) was swollen in DMF (2 ml) and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38 g, 1 mmol), DIC solution (2 ml, 0.5M in DMF) and HOCt solution (2 ml, 0.5M in DMF) was added. The mixture was sonicated for 2 hours and then washed with DMF.

(1371) 2. Cap

(1372) The resin was capped with 0.5M acetic anhydride/DMF solution (20 ml) for 5 minutes and then washed with DMF.

(1373) 3. Fmoc Deprotection

(1374) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1375) 4. Couple Biotin-OSu

(1376) A solution of Biotin-NHS ester (341 mg, 1 mmol) and DIPEA (348 ul) 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. Dry resin obtained=1.5 g.

(1377) Cleavage:

(1378) Dry peptide resin (1.5 g) and the mixture was cleaved with TFA (30 ml) containing a scavenger cocktail consisting of TIS, thioanisole, water, EDT and phenol and the mixture was stirred at room temperature for 6 hours. The solution was filtered into cold ether and the resin rinsed with TFA. The peptide was centrifuged, washed with ether, centrifuged and lyophilised to give 1.0 g crude peptide.

(1379) Folding Protocol:

(1380) Crude peptide (100 mg) was dissolved into 6M GnHCl (233 ml) and then rapidly diluted to 2M GnHCl concentration by the addition of 50 mM TRIS pH8 (467 ml) containing 0.5 mM GSSG and 5 mM GSH. The mixture was stirred at room temperature for 2.5 days and then analysed by HPLC (Jupiter C18, 250×4.6 mm column, 10-60% B over 30 minutes. HPLC analysis confirmed the formation of desired product as well as mis-folded by-products.

(1381) Purification:

(1382) The folded protein was purified by reverse phase HPLC using a Jupiter C18, 250×21 mm column, 9 ml/min, 10-60% B over 50 minutes. 11.1 mg of pure folded Nle-TECK-Biotin was afforded.

(1383) FIG. 98 shows HPLC of purified folded Biotin-TECK(Nleu). The protein eluted in a single peak at 21.6 mins.

(1384) FIG. 99 shows Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu). The expected mass was 8959.4 Da.

(1385) Functional Assay Data:

(1386) TECK-Biotin-Nle 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 native TECK is 67.87 nM.

(1387) The desired active chemokine comprises the amino acid sequence of SEQ ID NO: 122:

(1388) TABLE-US-00247 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR HRKVCGNPKSREVQRAXKLLDARNXVF-OH
X1=pyroGlu
X64=Norleucine
X72 is K(PEG-Biotin)

Examples 73 to 79

Chemokine Synthesis

(1389) General Protocols

(1390) Assembly:

(1391) 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.

(1392) Removal of Dde Protection:

(1393) 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.

(1394) Labelling Steps:

(1395) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(1396) 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.

(1397) 2. Capping

(1398) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(1399) 3. Fmoc deprotection

(1400) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1401) 4. Couple Biotin-OSu

(1402) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(1403) Cleavage:

(1404) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(1405) Purification Protocol:

(1406) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(1407) Folding Protocol:

(1408) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 73

BiotinMCP-1 (CCL2)

(1409) Target Molecule:

(1410) MCP-1 derivatised at the ε-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1411) Modifications:

(1412) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(1413) The linear amino acid sequence (SEQ ID NO: 123) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1414) TABLE-US-00248 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

(1415) The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(1416) TABLE-US-00249 SEQ ID NO: 124 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(1417) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine.

(1418) TABLE-US-00250 SEQ ID NO: 125 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu or Gln
X75 is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

(1419) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(1420) Functional Assay Data:

(1421) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 74

BiotinRANTES (CCL5)

(1422) Target Molecule:

(1423) RANTES derivatised at the ε-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(1424) Modifications:

(1425) 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.

(1426) The linear amino acid sequence (SEQ ID NO: 126) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(1427) TABLE-US-00251 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(1428) 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:

(1429) TABLE-US-00252 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(1430) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 127). 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: 128).

(1431) TABLE-US-00253 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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))

(1432) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(1433) Functional Assay Data:

(1434) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 75

BiotinMCP-2 (CCL8)

(1435) Target Molecule:

(1436) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1437) Modifications:

(1438) 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 ε-amino functionality and the biotin.

(1439) The linear amino acid sequence (SEQ ID NO: 129) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1440) TABLE-US-00254 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(1441) 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:

(1442) TABLE-US-00255 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(1443) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 130). 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: 131):

(1444) TABLE-US-00256 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).

(1445) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(1446) Functional Assay Data:

(1447) 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 76

BiotinIL-8 (CXCL8)

(1448) Target Molecule:

(1449) IL-8 derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(1450) Modifications:

(1451) Human IL-8 corresponding to residues 1-77, is initially expressed as 99 amino acids comprising the chemokine fold, and a 22 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. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(1452) The linear amino acid sequence (SEQ ID NO: 132) is shown, prior to attachment of the PEG spacer and biotin molecules:

(1453) TABLE-US-00257 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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)

(1454) The engineered IL-8 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:

(1455) TABLE-US-00258 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(1456) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 133). 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: 134):

(1457) TABLE-US-00259 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)
Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8: obtained=9416.9 Da; expected 9417.0 Da.
Functional Assay Data:

(1458) BiotinIL-8 was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 18.9 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.1 nM.

Example 77

BiotinIL-8 (6-78)

(1459) Target Molecule:

(1460) IL-8 (6-78) derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(1461) Modifications:

(1462) Truncated form of IL-8 corresponding to residues 6-77, the first five N-terminal residues have been removed and an additional lysine was inserted at the C-terminus at position 78, and modified through biotinylation on the resin. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(1463) The linear amino acid sequence (SEQ ID NO: 135) is shown, prior to attachment of the PEG spacer and biotin molecules:

(1464) TABLE-US-00260 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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

(1465) The engineered IL-8 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:

(1466) TABLE-US-00261 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(1467) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 136). 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: 137):

(1468) TABLE-US-00262 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)

(1469) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8 (6-78): obtained=8880.50 Da; expected 8880.4 Da.

(1470) Functional Assay Data:

(1471) BiotinIL-8 (6-78) was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 6.1 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.1 nM.

Example 78

BiotinMIP-3a (CCL20)

(1472) Target Molecule:

(1473) MIP-3a derivatised at the e-amino side chain functionality of Lys(68) with PEG-Biotin (TFA salt)

(1474) Modifications:

(1475) 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 ε-amino functionality and the biotin.

(1476) The linear amino acid sequence (SEQ ID NO: 138) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 68 (K):

(1477) TABLE-US-00263 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVKNM-OH

(1478) 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:

(1479) TABLE-US-00264 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVXNM-RESIN
X=K(ivDde)

(1480) FmocLys(ivDde)-OH was incorporated as residue 68 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 139). 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: 140).

(1481) TABLE-US-00265 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSVC ANPKQTWVKYIVRLLSKKVXNM-OH
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)

(1482) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMip-3a: obtained=8396.4 Da; expected 8397.0 Da.

(1483) Functional Assay Data:

(1484) BiotinMIP-3a 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-3a is 1.0 nM.

Example 79

BiotinMDC (CCL22)

(1485) Target Molecule:

(1486) MDC derivatised at the e-amino side chain functionality of Lys(66) with PEG-Biotin (TFA salt)

(1487) Modifications:

(1488) 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 ε-amino functionality and the biotin.

(1489) The linear amino acid sequence (SEQ ID NO: 141) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 66 (K):

(1490) TABLE-US-00266 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRD KEICADPRVPWVKMILNKLSQ-NH.sub.2

(1491) 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:

(1492) TABLE-US-00267 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFRD KEICADPRVPWVKMILNXLSQ-RESIN
X=K(ivDde)

(1493) FmocLys(ivDde)-OH was incorporated as residue 66 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 142). 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: 143).

(1494) TABLE-US-00268 H-GPYGANMEDSVCCRDYVRYRLPLRVVKHFYWTSDSCPRPGVVLLTFR DKEICADPRVPWVKMILNXLSQ-NH.sub.2
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)

(1495) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMDC: obtained=8456.1 Da; expected 8456.9 Da.

(1496) Functional Assay Data:

(1497) 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 80

Diagnosis and Treatment of Inflammatory Skin Disease

(1498) Materials and Methods

(1499) 1. Flow Cytometric Analysis of Peripheral Blood

(1500) 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 (RT) and stained with antibodies (Table 24) 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).

(1501) TABLE-US-00269 TABLE 24 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CCR4 PerCpCy5.5 BD CXCR1 APC Biolegend CXCR2 PE Biolegend CD16 PECy7 BD CD3 Horizon V450 BD Streptavdin APC BD
2. Chemokine Binding Test

(1502) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 24). 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).

(1503) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1504) 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 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 24), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(1505) Results and Discussion

(1506) 1. Flow Cytometric Analysis of Peripheral Blood—CCR4

(1507) White blood cells from one patient with psoriasis were analysed for the expression of chemokine receptors with flow cytometry. The patient exhibited an increased frequency of CCR4 expressing T cells compared to healthy controls, based upon flow cytometry data and binding by an anti-CCR4 antibody (FIG. 101).

(1508) 2. Chemokine Binding Test—CCR4

(1509) The chemokine receptor CCR4 is necessary for T cell migration to the skin, which leads to inflammation. The ligand for CCR4 is the chemokine MDC (CCL22) which is expressed in inflamed skin lesions.

(1510) The T cells could bind biotinylated MDC (bMDC) to a similar extent as the chemokine receptor expression (FIG. 102).

(1511) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine—CCR4

(1512) The CCR4 expressing T cells could be efficiently depleted with biotinylated MDC conjugated Sepharose Streptavidin Matrix (FIG. 103).

(1513) 1. Flow Cytometric Analysis of Peripheral Blood—CXCR1 and CXCR2

(1514) The chemokine receptors CXCR1 and CXCR2 were expressed on neutrophils (FIG. 104), based upon flow cytometry data and binding of an anti-CXCR1 and anti-CXCR2 antibody. Th17 cells produce IL-17 causing IL-8 release and attract neutrophils to sites of skin inflammation. Therefore eliminating neutrophils is predicted to be beneficial for treatment of inflammatory skin conditions such as psoriasis.

(1515) 2. Chemokine Binding Test—CXCR1 and CXCR2

(1516) The ligand for CXCR1 and CXCR2 is IL-8 that mediate migration of neutrophils in inflammation. In accordance with the receptor expression, biotinylated IL-8 (bIL-8) could bind to blood neutrophils from a psoriasis patient (FIG. 105).

(1517) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine—CXCR1 and CXCR2

(1518) The CXCR2 expressing neutrophils could be efficiently depleted with bIL-8 conjugated Sepharose Streptavidin Matrix (FIG. 106).

(1519) Conclusions

(1520) We conclude that the frequency of CCR4 expressing T cells is enhanced in psoriasis. The CCR4 expressing T cells and CXCR1 and CXCR2 expressing neutrophils could bind their respective chemokines, and could be efficiently depleted with biotinylated chemokine-Sepharose Streptavidin-matrix.

(1521) H. Treating Multiple Sclerosis

(1522) In secondary progressive MS microglia/MØ present at border of plaques produce chemokines MCP-1 and CXCL10 responsible for attracting CCR2 and CXCR3 expressing cells including macrophages and astrocytes.

(1523) It is shown herein that subjects suffering from MS exhibit increased frequency of chemokine receptor expressing cells in the peripheral blood, in particular CCR2 and CCR6 expressing T lymphocytes, compared to healthy controls. It is also shown herein that the CCR2 cells can be removed using a suitable binding reagent, in particular MCP-1 (in biotinylated form) immobilized on a suitable matrix. Similarly, it is shown herein that (the additional) CCR6-expressing cells can be depleted using a suitable binding reagent, in particular CCL20 (MIP-31), in biotinylated form, immobilized on a suitable matrix. The CCL5 levels are significantly elevated in cerebrospinal fluid of MS patients with relapsing disease demonstrating that circulating CCL5 is involved in recruiting CCL5 binding cells to the brain. These findings are supported by the enrichment of T cells in cerebrospinal fluid expressing CCR5 and CCR6 suggesting an active accumulation due to a chemokine gradient of CCL5. Therefore eliminating cells normally attracted to the brain by providing an extracorpeal source of CCL5 attached to a column will be useful for the treatment of MS.

Examples 81 and 82

Materials and Methods

(1524) Isolation of Peripheral Blood Leukocytes.

(1525) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(1526) Chemokines.

(1527) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labeled MCP-1 (in concentrations 10 ng/μL and 50 ng/μL). 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.

(1528) Flow Cytometry Assay.

(1529) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 81

Binding of Monocytes to MCP-1

(1530) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 107a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 107b and 107c).

Example 82

(1531) Monocytes were investigated for their expression of CCR2 (FIG. 108b) and their ability to bind MCP-1 (FIG. 108a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 108b). The MCP-1 binding to monocytes shown in FIG. 108a corresponds to the CCR2hi expressing population shown in FIG. 108b. Thus, MCP-1 binds favourably to CCR2hi expressing cells.

Example 83

Tailored Leukapheresis

(1532) Column Design and Properties

(1533) Introduction

(1534) 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.

(1535) The Column

(1536) The column is intended to be used as a leukapheresis treatment for multiple sclerosis. It will specifically remove CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9-expressing leukocytes, in particular monocytes, through the use of a binding reagent, more specifically an MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES containing resin, exploiting the CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and one or more of biotinylated MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(1537) The Plastic House (FIG. 9)

(1538) 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. 9. 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.

(1539) Streptavidin Sepharose™ BigBeads

(1540) 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.

(1541) Binding Reagent

(1542) Coupled to the matrix is the third component of the device, the one or more binding reagents that bind specifically to CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9. One or more chemokines such as those selected from the group consisting of MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, MIP-3alpha, MIG (CXCL9), IP10 (CXCL10), CXCL11 (I-TAC), CCL25 and RANTES may be employed. These peptides may be synthetic, engineered versions of the human chemokine, which are truncated and biotinylated, but retain binding activity to the CCR2, CCR6, CCR3, CCR5, CCR1, CXCR3 and/or CCR9 receptor. By biotinylating the engineered chemokine, it is able to bind to the streptavidin molecules in the Sepharose™ matrix. The biotin-streptavidin binding is known be one of the strongest biological interactions with a Kd in the order of 4×10.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.

(1543) The Apheresis System

(1544) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(1545) The Circuit

(1546) The system is illustrated in FIG. 10. 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.

(1547) The 4008 ADS Pump

(1548) 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.

(1549) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

(1550) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(1551) 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.

(1552) Leukapheresis Time and Flow Rate

(1553) 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.

(1554) Storage Conditions

(1555) 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.

(1556) Transport Conditions

(1557) 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.

(1558) In-Vitro Depletion of Target Cell Populations—MCP-1

(1559) To investigate the ability to eliminate CCR2-expressing cells, in vitro tests have been performed on the bMCP-1 coupled matrix. Blood was collected from blood donors and passed through the column device containing bMCP-1 coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR2-expressing cells.

(1560) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 109a.

(1561) In conclusion, the in-vitro results demonstrate a specific reduction of up to 80% of the CCR2-expressing cells by the column. Notably, individuals with fewer CCR2 expressing cells initially achieved lower depletion. The remaining levels of monocytes were around 20-30% in each case, irrespective of the starting point. Non-CCR2-expressing cells remained unaffected (data not shown).
In-Vitro Depletion of Target Cell Populations—RANTES

(1562) To investigate the ability to eliminate CCR1, 3 and 5-expressing cells, in vitro tests have been performed on the biotinylated RANTES coupled matrix. Blood was collected from blood donors and passed through the column device containing biotinylated RANTES coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR1, 3 and 5-expressing cells.

(1563) 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. 109b.

(1564) The in-vitro results demonstrate a specific reduction of around 20% of the chemokine receptor-expressing cells by the column. Non-CCR1, 3 or 5-expressing cells remained unaffected (data not shown).

(1565) In-Vitro Depletion of Target Cell Populations—MIP-3Alpha

(1566) To investigate the ability to eliminate CCR6-expressing cells, in vitro tests have been performed on the biotinylated MIP-3a coupled matrix. Blood was collected from blood donors and passed through the column device containing biotinylated MIP-3a coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR6-expressing cells.

(1567) 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. 109c.

(1568) 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).

(1569) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 160:

(1570) TABLE-US-00270 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-CO2H

(1571) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(1572) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 115. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

Example 84

MCP1 Derivatives

(1573) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 144). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 144,

(1574) TABLE-US-00271 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 145,

(1575) TABLE-US-00272 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSXDHL DKQTQTPKT
X=Met or Nleu

(1576) 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.

(1577) Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 110.

(1578) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 111 and in SEQ ID NO: 145.

(1579) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 85

Synthesis of a Ccr2 Antagonist Biotinmcp-1 which Binds to the Receptor without Activation

(1580) Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995, 181, 63) has been shown for an MCP-1 derivative truncated at the N-terminus. In particular, deletion of residues 1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis of CCR2 positive cells (inhibition of chemotaxis IC50 20 nM)

(1581) The amino acid sequence of the truncated version is set forth as SED ID NO: 146:

(1582) TABLE-US-00273 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(1583) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 112. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 86

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

(1584) CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 in addition to MCP-1. MCP-5 only binds CCR2 and should be selective in its removal of CCR2 expressing cells. MCP5 is a mouse chemokine shown to chemotact human CCR2 cells with EC50<3 nM.

(1585) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 147

(1586) TABLE-US-00274 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG
The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 148

(1587) TABLE-US-00275 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(1588) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 113. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 149:

(1589) TABLE-US-00276 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL

(1590) In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 150:

(1591) TABLE-US-00277 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(1592) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 114.

(1593) Once synthesised, the activity of the various biotinMCP-5 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Examples 87 to 94

General Protocols for Chemokine Synthesis

(1594) Assembly:

(1595) 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.

(1596) Removal of Dde Protection:

(1597) 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.

(1598) Labelling Steps:

(1599) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(1600) 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.

(1601) 2. Capping

(1602) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(1603) 3. Fmoc Deprotection

(1604) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1605) 4. Couple Biotin-OSu

(1606) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(1607) Cleavage:

(1608) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(1609) Purification Protocol:

(1610) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(1611) Folding Protocol:

(1612) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 87

BiotinMCP-1 (CCL2)

(1613) Target Molecule:

(1614) MCP-1 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1615) Modifications:

(1616) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(1617) The linear amino acid sequence (SEQ ID NO: 151) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1618) TABLE-US-00278 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu

(1619) The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(1620) TABLE-US-00279 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu
X75=K(ivDde)

(1621) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 152). 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: 153):

(1622) TABLE-US-00280 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu
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).

(1623) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(1624) Functional Assay Data:

(1625) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 88

BiotinMCP-2 (CCL8)

(1626) Target Molecule:

(1627) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1628) Modifications:

(1629) 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 ε-amino functionality and the biotin.

(1630) The linear amino acid sequence (SEQ ID NO: 154) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1631) TABLE-US-00281 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu

(1632) 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:

(1633) TABLE-US-00282 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu
X75=K(ivDde)

(1634) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 155). 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: 156):

(1635) TABLE-US-00283 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu
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).

(1636) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(1637) Functional Assay Data:

(1638) 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 89

BiotinEotaxin (CCL11)

(1639) Target Molecule:

(1640) Eotaxin derivatised at the e-amino side chain functionality of Lys(73) with PEG-Biotin (TFA salt)

(1641) Modifications:

(1642) 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 ε-amino functionality and the biotin.

(1643) The linear amino acid sequence (SEQ ID NO: 157) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(1644) TABLE-US-00284 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
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).

(1645) 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:

(1646) TABLE-US-00285 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(ivDde)

(1647) FmocLys(ivDde)-OH was incorporated as residue 73 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 158). 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: 159):

(1648) TABLE-US-00286 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(PEG-Biotin)

(1649) Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) data of purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3 Da.

(1650) Functional Assay Data:

(1651) 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 90

BiotinRANTES (CCL5)

(1652) Target Molecule:

(1653) RANTES derivatised at the e-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(1654) Modifications:

(1655) 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.

(1656) The linear amino acid sequence (SEQ ID NO: 160) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(1657) TABLE-US-00287 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK NRQVCANPEKKWVREYINSLEKS-OH

(1658) 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:

(1659) TABLE-US-00288 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(1660) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 161). 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: 162).

(1661) TABLE-US-00289 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEXS-OH
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))

(1662) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(1663) Functional Assay Data:

(1664) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 91

BiotinMIP-3a (CCL20)

(1665) Target Molecule:

(1666) MIP-3a derivatised at the e-amino side chain functionality of Lys(68) with PEG-Biotin (TFA salt)

(1667) Modifications:

(1668) 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 ε-amino functionality and the biotin.

(1669) The linear amino acid sequence (SEQ ID NO: 163) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 68 (K):

(1670) TABLE-US-00290 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSV CANPKQTWVKYIVRLLSKKVKNM-OH

(1671) 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:

(1672) TABLE-US-00291 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSV CANPKQTWVKYIVRLLSKKVXNM-RESIN
X=K(ivDde)

(1673) FmocLys(ivDde)-OH was incorporated as residue 68 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 164). 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: 165).

(1674) TABLE-US-00292 H-ASNFDCCLGYTDRILHPKFIVGFTRQLANEGCDINAIIFHTKKKLSV CANPKQTWVKYIVRLLSKKVXNM-OH
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)

(1675) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMip-3a: obtained=8396.4 Da; expected 8397.0 Da.

(1676) Functional Assay Data:

(1677) BiotinMIP-3a 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-3a is 1.0 nM.

Example 92

BiotinTECK (CCL25)

(1678) Target Molecule:

(1679) TECK (Met to Nleu substitution) derivatised at the ε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(1680) Modifications:

(1681) 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 ε-amino functionality and the biotin.

(1682) The linear amino acid sequence (SEQ ID NO: 166) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(1683) TABLE-US-00293 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPKR HRKVCGNPKSREVQRAXKLLDARNKVF-OH
X1=pyroGlu or Gln
X64=Norleucine

(1684) 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:

(1685) TABLE-US-00294 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPK RHRKVCGNPKSREVQRAXKLLDARNXVF-RESIN
X1=pyroGlu or Gln
X64=Norleucine
X72=K(Dde)

(1686) TABLE-US-00295 NPKSREVQRANleKLLDARNK(ivDde)VF-RESIN

(1687) FmocLys(ivDde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 167). 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.

(1688) The final active chemokine thus has the following sequence (SEQ ID NO: 168):

(1689) TABLE-US-00296 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLP KRHRKVCGNPKSREVQRAXKLLDARNXVF-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)

(1690) 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.

(1691) Functional Assay Data:

(1692) 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 93

BiotinITAC (CXCL11)

(1693) Target Molecule:

(1694) ITAC derivatised with Biotin at the e-amino side chain functionality of an additional Lysine inserted at the C-terminus after a PEG spacer (TFA salt)

(1695) Modifications:

(1696) 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.

(1697) The linear amino acid sequence (SEQ ID NO: 169) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(1698) TABLE-US-00297 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLK ENKGQRCLNPKSKQARLIIKKVERKNF-OH

(1699) 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:

(1700) TABLE-US-00298 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLK ENKGQRCLNPKSKQARLIIKKVERKNFX-RESIN
X is PEG-K(ivDde)

(1701) 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 ε-amino side chain functionality of the additional Lys (SEQ ID NO: 170). 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: 171).

(1702) TABLE-US-00299 H-FPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLK ENKGQRCLNPKSKQARLIIKKVERKNFX-OH
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)

(1703) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinITAC: obtained=8866.5 Da; expected 8860.6 Da.

(1704) Functional Assay Data:

(1705) 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 94

BiotinIP-10 (CXCL10)

(1706) Target Molecule:

(1707) IP-10 derivatised with Biotin at the e-amino side chain functionality of an additional Lysine inserted at the C-terminus after a PEG spacer (TFA salt)

(1708) 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.

(1709) The linear amino acid sequence (SEQ ID NO: 172) is shown, prior to attachment of the PEG spacer, additional lysine and biotin molecules:

(1710) TABLE-US-00300 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMK KKGEKRCLNPESKAIKNLLKAVSKERSKRSP-OH

(1711) 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:

(1712) TABLE-US-00301 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKK KGEKRCLNPESKAIKNLLKAVSKERSKRSPX-RESIN
X is K(ivDde), optionally attached via a spacer such as PEG, e.g. -PEG-K(ivDde)

(1713) 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 ε-amino side chain functionality of the additional Lys (SEQ ID NO: 173). 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: 174):

(1714) TABLE-US-00302 H-VPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKK KGEKRCLNPESKAIKNLLKAVSKERSKRSPX-OH
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)

(1715) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIP-110: obtained=9141.0 Da; expected 9141.9 Da.

(1716) Functional Assay Data:

(1717) BiotinIP-10 was tested for agonist activity in an Aequorin assay against hCXCR3, (Euroscreen) and an EC50 value of 8.7 nM was reported. c.f. EC50 for recombinant native IP-10 is 4.4 nM.

Example 95

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

(1718) Materials and Methods

(1719) 1. Flow Cytometric Analysis of Peripheral Blood

(1720) Peripheral blood from patients with Multiple Sclerosis and healthy controls was collected in heparin tubes. The red blood cells were lysed using Fix Buffer (Phosphate Buffer Saline (PBS) citrate with 4% paraformaldehyde) for four minutes at 37° C. and Lysing buffer (PBS with 10 mM Tris and 160 mM NH.sub.4Cl, pH 7.5) for 15 min at 37° C. The cells were washed in PBS with 2% Bovine Growth Serum, incubated with 10% human serum for 15 min at room temperature (RT) and stained with antibodies (Table 25) at 4° C. for 30 min. The cells were analysed with flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences).

(1721) TABLE-US-00303 TABLE 25 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD3 V450 BD Biosciences CCR6 PE BD Biosciences Streptavidin PE, APC Biolegend CCR2 PerCP Cy5.5 Biolegend
2. Chemokine Binding Test

(1722) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 25). 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).

(1723) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1724) 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 μM) 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 25), analysed with flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(1725) Results and Discussion

(1726) 1. Flow Cytometric Analysis of Peripheral Blood

(1727) White blood cells from patients with Multiple Sclerosis (MS) were analysed for the expression of chemokine receptors with flow cytometry. The MS patients exhibited an increased frequency of circulating T cells that expressed the chemokine receptor CCR2, 15% compared to approximately 5% in healthy blood (FIG. 117a), based upon flow cytometry data and binding by an anti-CCR2 antibody. Furthermore, the patients had an increased frequency of T cells that expressed CCR6 (FIG. 117b).

(1728) 2. Chemokine Binding Test

(1729) CCR2 binds to the chemokine MCP-1 that mediate migration and infiltration of inflammatory cells to various tissues. The ligand for CCR6 is MIP3a (CCL20) that can mediate migration of T cells into the CNS. Both these receptors are important in the inflammatory process. In accordance with the CCR2 and CCR6 expression, the T cells bound the biotinylated MCP-1 (bMCP-1) (FIG. 118a) and bMIP3a (FIG. 118b).

(1730) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1731) The CCR2 expressing T cells could be efficiently depleted with bMCP-1-conjugated Sepharose Streptavidin Matrix (FIG. 119a), and the CCR6 expressing T cells could be depleted with bMIP3a-conjugated Sepharose Streptavidin Matrix (FIG. 119b)

(1732) We conclude that the frequency of T cells that express CCR2 and CCR6 is enhanced in MS. These T cells can bind the ligands MCP-1 and MIP3a. Furthermore, the majority of the CCR2 and CCR6 expressing T cells can be removed with Sepharose Streptavidin matrix conjugated with the corresponding biotinylated chemokine.

(1733) I. Treating Cardiovascular Disease

(1734) MCP-1 is secreted from endothelial cells and smooth muscle cells and plays a critical role in the development of cardiovascular diseases. It has been shown (Niu and Kolattakudy. Clinical Science (2009) 117, 95-109) that MCP-1, by its chemotactic activity, causes diapedesis of monocytes from the lumen to the subendothelial space where they become foam cells, initiating fatty streak formation that leads to atherosclerotic plaque formation. Inflammatory macrophages probably play a role in plaque rupture and the resulting ischaemic episode as well as restenosis after angioplasty. There is strong evidence that MCP-1 plays a major role in myocarditis, ischaemia/reperfusion injury in the heart and in transplant rejection. MCP-1 also plays a role in cardiac repair and manifests protective effects under certain conditions. Such protective effects may be due to the induction of protective ER (endoplasmic reticulum) stress chaperones by MCP-1. Under sustained ER stress caused by chronic exposure to MCP-1, the protection would break down resulting in the development of heart failure. MCP-1 is also involved in ischaemic angiogenesis. In mice, blocking of MCP-1 or CCR2 show beneficial effects against cardiovascular disease. In particular, MCP-1 knockout (KO) mice or CCR2 KO mice have reduced atherosclerosis, whereas MCP-1 overexpression increases foam cell formation and atherosclerosis. On this basis, the inventors have selected CCR2 expressing cells as a target for treatment of cardiovascular disease using specific binding interactions with CCR2 expressing cells.

(1735) It is shown herein that subjects suffering from atherosclerosis exhibit increased frequency of chemokine receptor expressing cells in the peripheral blood, in particular CCR1 expressing monocytes, compared to healthy controls. It is also shown herein that the CCR1 cells can be removed using a suitable binding reagent, in particular RANTES (in biotinylated form) immobilized on or in a suitable matrix. Similarly, it is shown herein that CCR2-expressing cells can be depleted in atherosclerosis patients using a suitable binding reagent, in particular MCP-1, in biotinylated form, immobilized on or in a suitable matrix.

Examples 96 and 97

Materials and Methods

(1736) Isolation of Peripheral Blood Leukocytes.

(1737) Heparinized peripheral blood from healthy blood donors 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.

(1738) Chemokines.

(1739) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labeled MCP-1 (in concentrations 10 ng/μL and 50 ng/μL). 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.

(1740) Flow Cytometry Assay.

(1741) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 96

Binding of Monocytes to MCP-1

(1742) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 120a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 120b and 120c).

Example 97

(1743) Monocytes were investigated for their expression of CCR2 (FIG. 121b) and their ability to bind MCP-1 (FIG. 121a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 121b). The MCP-1 binding to monocytes shown in FIG. 121a corresponds to the CCR2.sup.hi expressing population shown in FIG. 121b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 98

Tailored Leukapheresis

(1744) Column Design and Properties

(1745) Introduction

(1746) 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.

(1747) The Column

(1748) The column is intended to be used as a leukapheresis treatment for a cardiovascular disease. It will specifically remove CCR1, CCR2 and/or CCR7-expressing leukocytes, in particular monocytes, through the use of a binding reagent, more specifically an MCP-1, MCP-2, MCP-3, MCP-4 and/or MCP-5 containing resin, or a CCL19 containing resin, exploiting the CCR1, CCR2 and/or CCR7-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and the binding reagent bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(1749) The Plastic House (FIG. 9)

(1750) 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. 9. 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.

(1751) Streptavidin Sepharose™ BigBeads

(1752) 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.

(1753) Binding Reagent

(1754) Coupled to the matrix is the third component of the device, the binding reagent that binds specifically to CCR1, CCR2 and/or CCR7. Chemokines such as MCP-1. MCP-2, MCP-3, MCP-4, MCP-5, CCL19 and/or CCL5 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 CCR1, CCR2 and/or CCR7 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 4×10.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.

(1755) The Apheresis System

(1756) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(1757) The Circuit

(1758) The system is illustrated in FIG. 10. 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.

(1759) The 4008 ADS Pump

(1760) 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.

(1761) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

(1762) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(1763) 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.

(1764) Leukapheresis Time and Flow Rate

(1765) 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.

(1766) Storage Conditions

(1767) 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.

(1768) Transport Conditions

(1769) 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.

(1770) In-Vitro Depletion of Target Cell Populations

(1771) To investigate the ability to eliminate CCR2-expressing cells, in vitro tests have been performed on the bMCP-1 coupled matrix. Blood was collected from blood donors and passed through the column device containing bMCP-1 coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR2-expressing cells.

(1772) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 122.

(1773) In conclusion, the in-vitro results demonstrate a specific reduction of up to 80% of the CCR2-expressing cells by the column. Notably, individuals with fewer CCR2 expressing cells initially achieved lower depletion. The remaining levels of monocytes were around 20-30% in each case, irrespective of the starting point. Non-CCR2-expressing cells remained unaffected (data not shown).

Example 99

MCP1 Derivatives

(1774) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 175,

(1775) TABLE-US-00304 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 176,

(1776) TABLE-US-00305 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(1777) 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.

(1778) Thus, MCP-1 derivatised at the e-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 123.

(1779) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 124.

(1780) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 100

Synthesis of a Ccr2 Antagonist Biotinmcp-1 which Binds to the Receptor without Activation

(1781) Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995, 181, 63) has been shown for an MCP-1 derivative truncated at the N-terminus. In particular, deletion of residues 1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis of CCR2 positive cells. (inhibition of chemotaxis IC50 20 nM) The amino acid sequence of the truncated version is set forth as SED ID NO: 177:

(1782) TABLE-US-00306 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(1783) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 125. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 101

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

(1784) CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 in addition to MCP-1. MCP-5 only binds CCR2 and should be selective in its removal of CCR2 expressing cells. MCP5 is a mouse chemokine shown to chemotact human CCR2 cells with EC50<3 nM.

(1785) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 178

(1786) TABLE-US-00307 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(1787) The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 179

(1788) TABLE-US-00308 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(1789) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 126. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 180:

(1790) TABLE-US-00309 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL

(1791) In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 181:

(1792) TABLE-US-00310 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(1793) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 127. Once synthesised, the activity of the various biotinMCP-5 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in (aequorin) functional cell-based assay on human CCR2 receptor.

Examples 102 to 105

Chemokine Synthesis

(1794) General Protocols

(1795) Assembly:

(1796) 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.

(1797) Removal of Dde Protection:

(1798) 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.

(1799) Labelling Steps:

(1800) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(1801) 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.

(1802) 2. Capping

(1803) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(1804) 3. Fmoc Deprotection

(1805) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1806) 4. Couple Biotin-OSu

(1807) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(1808) Cleavage:

(1809) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(1810) Purification Protocol:

(1811) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(1812) Folding Protocol:

(1813) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 102

BiotinMCP-1 (CCL2)

(1814) Target Molecule:

(1815) MCP-1 derivatised at the ε-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1816) Modifications:

(1817) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(1818) The linear amino acid sequence (SEQ ID NO: 182) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1819) TABLE-US-00311 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV AKEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

(1820) The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(1821) TABLE-US-00312 SEQ ID NO: 183 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV AKEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(1822) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine.

(1823) TABLE-US-00313 SEQ ID NO: 184 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIV AKEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu or Gln
X75 is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

(1824) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(1825) Functional Assay Data:

(1826) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 103

BiotinRANTES (CCL5)

(1827) Target Molecule:

(1828) RANTES derivatised at the ε-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(1829) Modifications:

(1830) 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.

(1831) The linear amino acid sequence (SEQ ID NO: 185) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(1832) TABLE-US-00314 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK RQVCANPEKKWVREYINSLEKS-OH

(1833) 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:

(1834) TABLE-US-00315 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK NRQVCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(1835) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 186). 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: 187).

(1836) TABLE-US-00316 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRK NRQVCANPEKKWVREYINSLEXS-OH
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))

(1837) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(1838) Functional Assay Data:

(1839) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 104

BiotinMCP-2 (CCL8)

(1840) Target Molecule:

(1841) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1842) Modifications:

(1843) 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 ε-amino functionality and the biotin.

(1844) The linear amino acid sequence (SEQ ID NO: 188) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1845) TABLE-US-00317 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(1846) 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:

(1847) TABLE-US-00318 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(1848) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 189). 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: 190):

(1849) TABLE-US-00319 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKR GKEVCADPKERWVRDSMKHLDQIFQNLXP-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).

(1850) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(1851) Functional Assay Data:

(1852) 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 105

BiotinMIP-3b (CCL19)

(1853) Target Molecule:

(1854) MIP-3b derivatised at the e-amino side chain functionality of Lys(78) with Biotin (TFA salt)

(1855) Modifications:

(1856) Human MIP-3b 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.

(1857) The linear amino acid sequence (SEQ ID NO: 191) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):

(1858) TABLE-US-00320 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRG RQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
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)

(1859) The engineered MIP-3b 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:

(1860) TABLE-US-00321 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRG RQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN
X is FmocLys(ivDde)

(1861) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 192). 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: 193).

(1862) TABLE-US-00322 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRG RQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
X is K(Biotin)

(1863) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMIP-3b: obtained=9148.8 Da; expected 9149.7 Da.

(1864) Functional Assay Data:

(1865) biotinMip-3b 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-3b is 1.6 nM.

Example 106

Diagnosis and Treatment of Cardiovascular Disease (P117760US00) Using CCR1, CCR2 and Biotinylated MCP-1, and RANTES

(1866) Materials and Methods

(1867) 1. Flow Cytometric Analysis of Peripheral Blood

(1868) 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 (RT) and stained with antibodies (Table 26) 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).

(1869) TABLE-US-00323 TABLE 26 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CCR1 Alexa flour 647 Biolegend CCR2 PerCPCy5.5 Biolegend Streptavdin APC BD CD14 FITC Beckman Coulter
2. Chemokine Binding Test

(1870) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 26). 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).

(1871) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1872) 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 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 26), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(1873) Results and Discussion

(1874) 1. Flow Cytometric Analysis of Peripheral Blood

(1875) White blood cells from one patient with atherosclerosis were analysed for the expression of chemokine receptors with flow cytometry. The patients exhibited increased frequency of monocytes that expressed the receptor CCR1 based upon flow cytometry data and binding of an anti-CCR1 antibody (FIG. 13).

(1876) In addition to CCR1, the monocytes express the chemokine receptor CCR2 (FIG. 16), based upon flow cytometry data and binding of an anti-CCR2 antibody.

(1877) 2. Chemokine Binding Test

(1878) The ligand for CCR1 is the chemokine RANTES. RANTES is involved in the recruitment of monocytes to atherosclerotic lesions. The monocytes from a patient with atherosclerosis bound biotinylated RANTES (bRANTES) to the same extent as the chemokine receptor expression (FIG. 14).

(1879) The ligand for CCR2 is MCP-1 that is increased in atherosclerotic inflammation. In accordance with the CCR2 expression, biotinylated MCP-1 (bMCP-1) could bind to blood monocytes from an atherosclerosis patient (FIG. 17).

(1880) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(1881) The CCR1 expressing monocytes could be efficiently depleted with bRANTES-conjugated Sepharose Streptavidin Matrix (FIG. 15).

(1882) The CCR2 expressing monocytes could be depleted with bMCP1-conjugated Sepharose Streptavidin Matrix (FIG. 18).

(1883) We conclude that the frequency of CCR1 expressing monocytes is enhanced in atherosclerosis. The CCR2 receptor is expressed on monocytes from atherosclerosis patients to the same extent as in the healthy controls, but the CCR2 expressing cells could differ in their pro-inflammatory profile in the patients compared to healthy controls. The CCR1 and CCR2 expressing monocytes bind the chemokines that corresponded with the chemokine receptor expression, and could be efficiently depleted with the biotinylated chemokine-Sepharose Streptavidin-matrix.

(1884) J. Treating Primary Sclerosing Cholangitis

(1885) In patients with PSC it has been shown that expression of CCL25, CXCL12, CCL21 or CCL5 in hepatic endothelium mediates recruitment of CCR9, CXCR4, CCR7 and/or CCR5+ lymphocytes to the liver. CCR9, CXCR4, CCR7 and/or CCR5+ lymphocytes have been activated in the gut and homing to the liver might contribute to the strong correlation between IBD and PSC. (Eksteen B 2004)

(1886) In several inflammatory liver diseases, including PSC, an upregulation of CXCL12 (SDF-1) was seen in biliary epithelial cells when compared to healthy controls. Increased levels of CXCL12 were also seen in the plasma and the CXCL12 receptor CXCR4 was present on most liver infiltrating lymphocytes. (Terada R 2003) Neolymphoid tissue develops in the liver in patients with PSC, contributing to the chronic inflammation. Expression of the chemokine CCL21 recruits CCR7+ lymphocytes to this lymphoid tissue. (Grant A 2002).

(1887) Polymorphisms in the promoter region of the gene coding for RANTES (CCL5) can lead to increased expression of this chemokine. A significant difference in the presence of the −28 G allele was seen when 124 patients with PSC were compared to 362 healthy controls. (Henckaerts 2006)

(1888) Thus, the invention focuses on the following Chemokine receptor-Chemokine pairs of interest:

(1889) CCR9-CCL25

(1890) CXCR4-CXCL12

(1891) CCR7-CCL21, CCL19

(1892) CCR5-CCL5

(1893) The inventors have also determined that PSC patients include higher levels of CCR9 expressing monocytes in the blood. The disease relevant monocytes may also have high expression of HLA-DR (referred to herein as HLA-DR.sup.hi). The treatments and diagnostic methods herein may accordingly be applicable in particular to monocytes and to HLA-DR.sup.hi monocytes.

(1894) Materials and Methods

(1895) Isolation of Peripheral Blood Leukocytes.

(1896) Heparinized peripheral blood from healthy blood donors or IBD patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(1897) Chemokines.

(1898) The leukocytes were incubated for 30 min in the dark at 4° C. with the following biotinylated and Alexa647 Fluor® labeled chemokines: CCL25 (in concentrations of 0.1 ng/μL, 0.5 ng/μL and 5 ng/μL). 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.

(1899) Flow Cytometry Assay.

(1900) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 107

Affinity of Blood Cells to CCL25

(1901) In the experiment with biotinylated CCL25 it was found that neither T-cells (CD4+ lymphocytes; CD8+ lymphocytes) nor monocytes (CD14+ monocytes) from the peripheral blood of a healthy donor (FIGS. 135a, 135b and 135c) bound to the biotinylated chemokine. In contrast, about 80% of the CD8+ lymphocytes and about 90% of the CD4+ lymphocytes and the monocytes from a patient with Crohn's disease bound to CCL25 (FIGS. 136a, 136b and 136c).

Example 108

Preparation of a Chemokine Column for Blood Cell Apheresis

(1902) To streptavidin cross-linked agarose (ProZyme, San Leandro, Calif., U.S.A.) beads in the range from 75 μm to 300μ suspended (200 ml, ˜50%, v/v) in an aqueous solution of 25 mM sodium phosphate (pH 7.0) and 150 mM NaCl was added a solution of 75 μg biotinylated MIP-1α (Almac Sciences) in the same buffer at 22° C. and slowly stirred by hand for 3 min. After standing for another 20 min, the support was filtered off, washed thrice with neutral aqueous sodium phosphate/sodium chloride and filled into a glass column (i.d. 25 mm, length 12 cm).

Example 109

Separation of Monocytes from Peripheral Blood of a Healthy Donor with the Chemokine Column of Example 108

(1903) Heparinized peripheral blood from a healthy male donor was analyzed by flow cytometry for CD4+ lymphocytes, CD8+ lymphocytes and CD14 monocytes. 100 ml of the blood was filtered through the column at a rate of about 8 ml per min and washed with FACS buffer. The filtered blood was analyzed for the same cells. It was found that about 95% of the monocytes had been retained by the column whereas more than 90% each of CD4+ and CD8+ lymphocytes had been recovered.

Example 110

Tailored Leukapheresis

(1904) Column Design and Properties

(1905) Introduction

(1906) 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.

(1907) The Column

(1908) The column is intended to be used as a leukapheresis treatment for primary sclerosing cholangitis. It will specifically remove CCR9-expressing gut-homing leukocytes through the use of a bTECK containing resin, exploiting the CCR9-TECK interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and bTECK bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(1909) The Plastic House (FIG. 9)

(1910) 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. 9. 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.

(1911) Streptavidin Sepharose™ BigBeads

(1912) 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.

(1913) bTECK

(1914) Coupled to the matrix is the third component of the device, the bTECK. This bTECK peptide is a synthetic, engineered version of the human chemokine TECK, which is truncated and biotinylated, but retains its binding activity to the TECK receptor CCR9. By biotinylating the engineered TECK, 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 4×10.sup.−14 M. The calculated ratio of streptavidin:biotin binding sites in the column is 10:1. Therefore, the coupling between the matrix and bTECK will be immediate, minimising the risk of bTECK decoupling from the matrix.

(1915) The Apheresis System

(1916) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(1917) The Circuit

(1918) The system is illustrated in FIG. 10. 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.

(1919) The 4008 ADS Pump

(1920) 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.

(1921) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

(1922) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(1923) 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.

(1924) Leukapheresis Time and Flow Rate

(1925) 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.

(1926) Storage Conditions

(1927) 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.

(1928) Transport Conditions

(1929) 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.

Example 111

Non-Clinical Studies

(1930) In-Vitro Depletion of Target Cell Populations

(1931) To investigate the ability to eliminate CCR9-expressing cells, in vitro tests have been performed on the bTECK coupled matrix. Blood was collected from blood donors and primary sclerosing cholangitis patients and passed through the column device containing bTECK coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR9-expressing cells. The results demonstrate significant depletion of the target population CD14− positive CCR9-expressing cells post matrix perfusion; while total CD14-positive cells remain unchanged. Depletion tests were performed on blood from healthy donors and IBD patients confirming similar effects. The results are shown in FIGS. 137 and 138 respectively.

(1932) In conclusion, the in-vitro results demonstrate a specific reduction of 50-75% of the CCR9-expressing cells by the column. Non-CCR9-expressing cells remained unaffected. To investigate the ability to eliminate CCR5-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 CCR5-expressing cells.

(1933) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 195:

(1934) TABLE-US-00324 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVT RKNRQVCANPEKKWVREYINSLEKS-CO2H

(1935) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(1936) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 139. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

(1937) 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. 140.

(1938) The in-vitro results demonstrate a specific reduction of around 20% of the chemokine receptor-expressing cells by the column. Non-CCR5-expressing cells remained unaffected (data not shown).

Example 112

TECK-PEG-Biotin Synthesis Summary

(1939) Target Molecule:

(1940) TECK (Met to Nleu substitution) derivatised at the ε-amino side chain functionality of Lys72 with PEG-Biotin (TFA salt)

(1941) Modifications:

(1942) 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 ε-amino functionality and the biotin.

(1943) The linear amino acid sequence (SEQ ID NO: 194) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 72 (K):

(1944) TABLE-US-00325 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPK RHRKVCGNPKSREVQRAXKLLDARNKVF-OH
X1=pyroGlu or Gln
X64=Norleucine

(1945) The engineered TECK sequence was assembled on a solid support, using Fmoc protocols for solid-phase peptide synthesis (SEQ ID NO: 196):

(1946) TABLE-US-00326 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPK RHRKVCGNPKSREVQRAXKLLDARNXVF-RESIN
X1=pyroGlu or Gln
X64=Norleucine
X72=K(Dde)

(1947) FmocLys(Dde)-OH was incorporated as residue 72 to facilitate site-specific labelling at this position of the protein.

(1948) Met to Nle substitution.

(1949) N-terminal Gln to pyroglutamic acid substitution.

(1950) Removal of Dde Protection:

(1951) The Dde protecting group was removed by treatment of all resin (2.5 g) with a solution of 2% hydrazine in DMF (100 ml) over 1 hour period to afford 2.0 g resin.

(1952) Labelling Steps:

(1953) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid

(1954) Resin (1.5 g) was swollen in DMF (2 ml) and then a solution of Fmoc-8-amino-3,6-dioctanoic acid (0.38 g, 1 mmol), DIC solution (2 ml, 0.5M in DMF) and HOCt solution (2 ml, 0.5M in DMF) was added. The mixture was sonicated for 2 hours and then washed with DMF.

(1955) 2. Cap

(1956) The resin was capped with 0.5M acetic anhydride/DMF solution (20 ml) for 5 minutes and then washed with DMF.

(1957) 3. Fmoc Deprotection

(1958) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1959) 4. Couple Biotin-OSu

(1960) A solution of Biotin-NHS ester (341 mg, 1 mmol) and DIPEA (348 ul, 2 mmol) 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. Dry resin obtained=1.5 g.

(1961) Cleavage:

(1962) Dry peptide resin (1.5 g) and the mixture was cleaved with TFA (30 ml) containing a scavenger cocktail consisting of TIS, thioanisole, water, EDT and phenol and the mixture was stirred at room temperature for 6 hours. The solution was filtered into cold ether and the resin rinsed with TFA. The peptide was centrifuged, washed with ether, centrifuged and lyophilised to give 1.0 g crude peptide.

(1963) Folding Protocol:

(1964) Crude peptide (100 mg) was dissolved into 6M GnHCl (233 ml) and then rapidly diluted to 2M GnHCl concentration by the addition of 50 mM TRIS pH8 (467 ml) containing 0.5 mM GSSG and 5 mM GSH. The mixture was stirred at room temperature for 2.5 days and then analysed by HPLC (Jupiter C18, 250×4.6 mm column, 10-60% B over 30 minutes. HPLC analysis confirmed the formation of desired product as well as mis-folded by-products.

(1965) Purification:

(1966) The folded protein was purified by reverse phase HPLC using a Jupiter C18, 250×21 mm column, 9 ml/min, 10-60% B over 50 minutes. 11.1 mg of pure folded Nle-TECK-Biotin was afforded.

(1967) FIG. 141 shows HPLC of purified folded Biotin-TECK(Nleu). The protein eluted in a single peak at 21.6 mins.

(1968) FIG. 142 shows Electrospray ionisation with tandem mass spectrometry (ES/MS) data of purified folded Biotin-TECK(Nleu). The expected mass was 8959.4 Da.

(1969) Functional Assay Data:

(1970) TECK-Biotin-Nle 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 native TECK is 67.87 nM.

(1971) The final active chemokine thus has the following sequence (SEQ ID NO: 197):

(1972) TABLE-US-00327 H-XGVFEDCCLAYHYPIGWAVLRRAWTYRIQEVSGSCNLPAAIFYLPK RHRKVCGNPKSREVQRAXKLLDARNXVF-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)

Examples 112 to 115

Further Embodiments

(1973) General Protocols

(1974) Assembly:

(1975) 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.

(1976) Removal of Dde Protection:

(1977) 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.

(1978) Labelling Steps:

(1979) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(1980) 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.

(1981) 2. Capping

(1982) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(1983) 3. Fmoc Deprotection

(1984) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(1985) 4. Couple Biotin-OSu

(1986) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml, 2 mmol) 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.

(1987) Cleavage:

(1988) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(1989) Purification Protocol:

(1990) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(1991) Folding Protocol:

(1992) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 112

BiotinMCP-2 (CCL8)

(1993) Target Molecule:

(1994) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(1995) Modifications:

(1996) 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 ε-amino functionality and the biotin.

(1997) The linear amino acid sequence (SEQ ID NO: 198) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(1998) TABLE-US-00328 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTK RGKEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(1999) 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:

(2000) TABLE-US-00329 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTK RGKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(2001) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 199). 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: 200):

(2002) TABLE-US-00330 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTK RGKEVCADPKERWVRDSMKHLDQIFQNLXP-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).

(2003) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(2004) Functional Assay Data:

(2005) 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 113

BiotinRANTES (CCL5)

(2006) Target Molecule:

(2007) RANTES derivatised at the e-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(2008) Modifications:

(2009) 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.

(2010) The linear amino acid sequence (SEQ ID NO: 201) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(2011) TABLE-US-00331 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEMS-OH

(2012) 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:

(2013) TABLE-US-00332 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(2014) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 202). 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: 203):

(2015) TABLE-US-00333 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEXS-OH
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))

(2016) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(2017) Functional Assay Data:

(2018) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 114

BiotinSDF-1a (CXCL12)

(2019) Target Molecule:

(2020) SDF-1a derivatised at the e-amino side chain functionality of Lys(64) with Biotin (TFA salt)

(2021) Modifications:

(2022) 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.

(2023) The linear amino acid sequence (SEQ ID NO: 204) is shown, prior to attachment of the biotin molecule at amino acid 64 (K):

(2024) TABLE-US-00334 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEKALN-OH

(2025) The engineered SDF-1a sequence was assembled on a solid support (Wang resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(2026) TABLE-US-00335 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEXALN-RESIN
X=K(ivDde)

(2027) FmocLys(ivDde)-OH was incorporated as residue 64 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 205). 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: 206):

(2028) TABLE-US-00336 H-KPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQ VCIDPKLKWIQEYLEXALN-OH
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)

(2029) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinSDF-1a: obtained=8055.5 Da; expected 8057.5 Da.

(2030) Functional Assay Data:

(2031) biotinSDF-1a 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-1a is 12.0 nM.

Example 115

BiotinMIP-3b (CCL19)

(2032) Target Molecule:

(2033) MIP-3b derivatised at the e-amino side chain functionality of Lys(78) with Biotin (TFA salt)

(2034) Modifications:

(2035) Human MIP-3b 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.

(2036) The linear amino acid sequence (SEQ ID NO: 207) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):

(2037) TABLE-US-00337 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSK-NH.sub.2

(2038) The engineered MIP-3b 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:

(2039) TABLE-US-00338 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN
X is K(ivDde)

(2040) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 208). 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: 209):

(2041) TABLE-US-00339 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQ LCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
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)

(2042) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMIP-3b: obtained=9148.8 Da; expected 9149.7 Da.

(2043) Functional Assay Data:

(2044) biotinMip-3b 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-3b is 1.6 nM.

Example 116

Treatment and Diagnosis of Primary Sclerosing Cholangitis (PSC)

(2045) Materials and Methods

(2046) 1. Flow Cytometric Analysis of Peripheral Blood

(2047) 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 (RT) and stained with antibodies (Table 27) 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).

(2048) TABLE-US-00340 TABLE 27 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

(2049) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 27). 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).

(2050) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(2051) 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 μM) 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 27), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(2052) Results and Discussion

(2053) Primary Sclerosing Cholangitis (PSC)

(2054) 1. Flow Cytometric Analysis of Peripheral Blood

(2055) White blood cells from PSC patients was analysed with flow cytometry. The patients exhibited increased numbers of CCR9 expressing monocytes, a mean of 11% compared to approximately 7% in healthy blood (FIG. 144a). 51% of the CCR9 expressing monocytes in PSC have a high expression of HLADR compared to 35% in healthy blood. (FIG. 144b)

(2056) 2. Chemokine Binding Test

(2057) CCR9 binds to the chemokine TECK (CCL25) which is mainly expressed in the gut but is also reported to be upregulated in PSC liver. 27% of the monocytes bind to the biotinylated TECK (bTECK) (FIG. 145).

(2058) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(2059) 44% of the CCR9 expressing monocytes (FIG. 146a.) and 39% of the CCR9 expressing HLADRhi monocytes (FIG. 146b.) were specifically depleted with bTECK-conjugated Sepharose Streptavidin Matrix.

(2060) We conclude that monocytes in PSC express CCR9 and bind the ligand bTECK. Furthermore, the CCR9 expressing monocytes can be removed with Sepharose Streptavidin matrix conjugated with bTECK.

(2061) K. Treating Respiratory Conditions

(2062) Plasma levels of MCP-1 and MIP-1α were monitored in 26 patients with active pulmonary sarcoidosis over a two year period. During this period, the authors show that levels of these cytokines were closely related to the clinical course of the disease. The authors conclude that plasma MCP-1 and MIP-1a levels are useful indicators of clinical severity of sarcoidosis, and that levels “may reflect subclinical evidence of extrathoracic sarcoidosis and may play a role in initiating monocyte migration into the tissue”. Hashimoto S et al, Clin Exp Immunol, 1998 Serum MCP-1 levels were measured in 47 sarcoidosis patients and 10 healthy controls. Chemokine levels were significantly higher in the patient group, and more specifically, correlated positively with patients in early disease stages. Furthermore, MCP-1 was shown to be specifically expressed by macrophages associated with sarcoid lymph nodes. Iyonaga K et al, Sarcoidosis Vasc Diffuse Lung Dis, 1998 The inflammatory cytokines TNF-a, IL-8, MCP-1, MMP9 and GRO-a were measured in 100 COPD patients and 50 matched healthy smokers. These values were subsequently correlated to the BODE index of COPD disease severity. The largest difference in these biomarkers what observed in serum levels of MCP-1 which were significantly increased in the COPD group. The authors conclude that serum MCP-1 levels may be a clinical candidate for distinguishing between healthy smokers and patients with stable COPD. Liu S F et al, Respirology, 2009 TNF-alpha, IL-8, MMP-9, MCP-1, TIMP-1 and TIMP-2 were measured in 20 COPD patients, 10 asymptomatic smokers and 10 non-smoker healthy controls. The authors found highly reproducible and statistically significant elevations of plasma IL-8 among the COPD patients compared to the other groups. No other correlations were observed. Shaker S B et al, Clin Respir J, 2008.

(2063) It is shown herein that subjects suffering from respiratory conditions such as sarcoidosis exhibit increased frequency of chemokine receptor expressing cells in the peripheral blood. Subjects with sarcoidosis exhibit increased frequency of CCR1 expressing cells such as CCR1 expressing monocytes, compared to healthy controls. It is also shown herein that the CCR1 expressing cells can be removed using a suitable binding reagent, in particular RANTES (in biotinylated form) immobilized on a suitable matrix. Similarly, it is shown herein that the monocytes also express CCR2. The CCR2 expressing monocytes can be depleted in sarcoidosis patients using a suitable binding reagent, in particular MCP-1, in biotinylated form, immobilized on a suitable matrix. It is also shown herein that subjects suffering from respiratory conditions such as sarcoidosis exhibit increased frequency of CCR7 expressing cells such as CCR7 expressing lymphocytes, and also central memory T cells, compared to healthy controls. It is also shown herein that the CCR7 expressing cells can be removed using a suitable binding reagent, in particular MIP3b (in biotinylated form) immobilized on a suitable matrix.

(2064) On this basis the inventors have selected a range of chemokine receptors to use as targets for treatment according to the methods of the invention.

Examples 117 to 125

Materials and Methods

(2065) Isolation of Peripheral Blood Leukocytes.

(2066) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(2067) Chemokines.

(2068) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labelled chemokine (CCL5, CCL2, CXCL8) (in concentrations 10 ng/μL 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.

(2069) Flow Cytometry Assay.

(2070) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 117

Binding of Monocytes to MIP-1α

(2071) In the experiment with biotinylated MIP-1α it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 147c), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 147a and 147b).

Example 118

Binding of Monocytes to MCP-1

(2072) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 148a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 148b and 148c).

Example 119

Affinity of Blood Cells to Biotinylated IL-8

(2073) In FIG. 149 the binding to biotinylated IL-8 (CXCL8) of CD4+ lymphocytes (FIG. 149a), CD8+ lymphocytes (FIG. 149b) and CD16+ neutrophils (FIG. 149c) obtained from healthy donors is shown. After 30 min of incubation all CD16+ neutrophils bound to IL-8. In contrast no binding was observed with CD4+ lymphocytes and CD8+ lymphocytes.

Example 120

(2074) Monocytes were investigated for their expression of CCR2 (FIG. 150b) and their ability to bind MCP-1 (FIG. 150a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 150b). The MCP-1 binding to monocytes shown in FIG. 148a corresponds to the CCR2.sup.hi expressing population shown in FIG. 150b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 121

(2075) Neutrophils/eosinophils were investigated for their expression of CCR3, (FIG. 147b) and their ability to bind eotaxin (FIG. 151a). CCR3, expression was noted in all neutrophils/eosinophils with the majority of neutrophils/eosinophils expressing high levels, using an anti-CCR3, antibody (FIG. 15b). The eotaxin binding to neutrophils/eosinophils shown in FIG. 151a corresponds to the CCR3.sup.hi expressing population shown in FIG. 151b. Thus, eotaxin binds favourably to CCR3.sup.hi expressing cells.

Example 122

Preparation of a Chemokine Column for Blood Cell Apheresis

(2076) To streptavidin cross-linked agarose (ProZyme, San Leandro, Calif., U.S.A.) beads in the range from 75 μm to 300μ suspended (200 ml, ˜50%, v/v) in an aqueous solution of 25 mM sodium phosphate (pH 7.0) and 150 mM NaCl was added a solution of 75 μg biotinylated MIP-1α (Almac Sciences) in the same buffer at 22° C. and slowly stirred by hand for 3 min. After standing for another 20 min, the support was filtered off, washed thrice with neutral aqueous sodium phosphate/sodium chloride and filled into a glass column (i.d. 25 mm, length 12 cm).

Example 123

Separation of Monocytes from Peripheral Blood of a Healthy Donor with the Chemokine Column of Example 122

(2077) Heparinized peripheral blood from a healthy male donor was analyzed by flow cytometry for CD4+ lymphocytes, CD8+ lymphocytes and CD14 monocytes. 100 ml of the blood was filtered through the column at a rate of about 8 ml per min and washed with FACS buffer. The filtered blood was analyzed for the same cells. It was found that about 95% of the monocytes had been retained by the column whereas more than 90% each of CD4+ and CD8+ lymphocytes had been recovered.

Example 124

Preparation of Streptavidin Conjugated Magnetic Beads Complexed with Biotinylated MIP-1α

(2078) An aqueous suspension of streptavidin conjugated magnetic beads (MagCellect Streptavidin Ferrofluid, 1 ml; R&D Systems, Minneapolis, Minn., U.S.A.) was mixed with 30 μg of MIP-1α (Almac Sciences) in 50 ml of 25 mM sodium phosphate (pH 7.0) and 150 mM NaCl and slowly stirred for 1 hour. The particles were washed thrice with 20 ml portions the same solvent and stored in suspension at 4° C.

Example 125

Separation of CD14+ Monocytes from Peripheral Blood of a Healthy Donor with the Streptavidin Magnetic Beads of Example 124

(2079) 100 ml of heparinized blood from the healthy donor of Example 124 was mixed with the streptavidin conjugated magnetic beads complexed with biotinylated MIP-1α and slowly stirred for 40 min. The particles were separated from the blood by a magnetic separator, and the blood analyzed for CD14+ monocytes and CD4+ and CD8+ lymphocytes. While essentially no CD14+ monocytes could be detected, CD4+ and CD8+ lymphocytes were present in roughly the original amounts.

Example 126

Tailored Leukapheresis

(2080) Column Design and Properties

(2081) Introduction

(2082) 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.

(2083) The Column

(2084) The column is intended to be used as a leukapheresis treatment for respiratory conditions, in particular sarcoidosis and Chronic Obstructive Pulmonary Disease (COPD). It will specifically remove CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7-expressing leukocytes, in particular monocytes, through the use of a binding reagent containing resin, exploiting the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and one or more biotinylated chemokine bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(2085) The Plastic House (FIG. 9)

(2086) 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. 9. 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.

(2087) Streptavidin Sepharose™ BigBeads

(2088) 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.

(2089) Binding Reagent

(2090) Coupled to the matrix is the third component of the device, one or more binding reagents that bind specifically to CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7. One or more chemokines may be employed. These peptides may be synthetic, engineered versions of the human chemokine, which are truncated and biotinylated, but retain binding activity to the CCR2, CCR1, CCR3, CCR5, CXCR1, CXCR2 and/or CCR7 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 4×10.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.

(2091) The Apheresis System

(2092) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(2093) The Circuit

(2094) The system is illustrated in FIG. 10. 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.

(2095) The 4008 ADS Pump

(2096) 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.

(2097) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

(2098) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(2099) 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.

(2100) Leukapheresis Time and Flow Rate

(2101) 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.

(2102) Storage Conditions

(2103) 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.

(2104) Transport Conditions

(2105) 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.

(2106) In-Vitro Depletion of Target Cell Populations

(2107) To investigate the ability to eliminate CCR2-expressing cells, in vitro tests have been performed on the bMCP-1 coupled matrix. Blood was collected from blood donors and passed through the column device containing bMCP-1 coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR2-expressing cells.

(2108) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 152a.

(2109) The in-vitro results demonstrate a specific reduction of up to 80% of the CCR2-expressing cells by the column. Notably, individuals with fewer CCR2 expressing cells initially achieved lower depletion. The remaining levels of monocytes were around 20-30% in each case, irrespective of the starting point. Non-CCR2-expressing cells remained unaffected (data not shown).

(2110) 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.

(2111) The RANTES molecule was synthesized by Almac. The amino acid sequence of the biotinylated RANTES molecule is set forth as SEQ ID NO: 225:

(2112) TABLE-US-00341 H2N- SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVC ANPEKKWVREYINSLEKS-CO2H

(2113) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67.

(2114) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 158. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines.

(2115) 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. 152b.

(2116) 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).

(2117) In-Vitro Depletion of Target Cell Populations

(2118) 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 column device (including a magnetic separator) containing eotaxin coupled matrix (MACS beads). Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR3-expressing cells. 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. 152a.

(2119) In conclusion, the in-vitro results demonstrate a specific reduction of around 25% of the CCR3-expressing cells by the column. Non-CCR3-expressing cells remained unaffected (data not shown).

Example 127

MCP1 Derivatives

(2120) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 210,

(2121) TABLE-US-00342 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 211,

(2122) TABLE-US-00343 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(2123) 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.

(2124) Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 153.

(2125) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 154.

(2126) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 128

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

(2127) Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995, 181, 63) has been shown for an MCP-1 derivative truncated at the N-terminus. In particular, deletion of residues 1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis of CCR2 positive cells. (inhibition of chemotaxis IC50 20 nM)

(2128) The amino acid sequence of the truncated version is set forth as SED ID NO: 212:

(2129) TABLE-US-00344 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(2130) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 155. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 129

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

(2131) CCR2 also binds chemokines MCP-2, MCP-3, MCP-4, MCP-5, and HCC-4 in addition to MCP-1. MCP-5 only binds CCR2 and should be selective in its removal of CCR2 expressing cells. MCP5 is a mouse chemokine shown to chemotact human CCR2 cells with EC50<3 nM.

(2132) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 213

(2133) TABLE-US-00345 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(2134) The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 214

(2135) TABLE-US-00346 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(2136) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 156. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 215:

(2137) TABLE-US-00347 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL
In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 216:

(2138) TABLE-US-00348 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFKL

(2139) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 157.

Example 130

Eotaxin Derivatives

(2140) Eotaxin has been produced with residue 73 (thought to be a lysine) as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 9). 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: 217,

(2141) TABLE-US-00349 MKVSAALLWL LLIAAAFSPQ GLAGPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 218,

(2142) TABLE-US-00350 GPASVPT TCCFNLANRK IPLQRLESYR RITSGKCPQK AVIFKTKLAK DICADPKKKW VQDSMKYLDQ KSPTPKP

(2143) 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.

(2144) Thus, eotaxin derivatised at the ε-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. 159.

(2145) 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. 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 functional cell-based assay on human CCR3 receptor.

(2146) Once synthesised, the activity of the various biotinMCP-5 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in functional cell-based assay on human CCR2 receptor.

Examples 131 to 137

Chemokine Synthesis

(2147) General Protocols

(2148) Assembly:

(2149) 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.

(2150) Removal of Dde Protection:

(2151) 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.

(2152) Labelling Steps:

(2153) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(2154) 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.

(2155) 2. Capping

(2156) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(2157) 3. Fmoc Deprotection

(2158) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(2159) 4. Couple Biotin-OSu

(2160) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(2161) Cleavage:

(2162) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(2163) Purification Protocol:

(2164) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(2165) Folding Protocol:

(2166) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 131

BiotinMCP-1 (CCL2)

(2167) Target Molecule:

(2168) MCP-1 derivatised at the ε-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(2169) Modifications:

(2170) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(2171) The linear amino acid sequence (SEQ ID NO: 219) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(2172) TABLE-US-00351 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTI VAKEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

(2173) The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(2174) TABLE-US-00352 SEQ ID NO: 220 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTI VAKEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(2175) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein. Subsequent removal of the ivDde protecting group, followed by coupling of the PEG spacer and Biotin, was carried out as described in the general protocol section. Cleavage, purification and folding protocols were carried out as described to furnish the desired active chemokine.

(2176) TABLE-US-00353 SEQ ID NO: 221 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTI VAKEICADPKQKWVQDSMDHLDKQTQTPXT-NH.sub.2
X1=pyroGlu or Gln
X75 is an amino acid residue that can be biotinylated, such as lysine, ornithine or diaminopropionic acid and optionally is biotinylated, optionally via a spacer molecule such as PEG, optionally K(PEG-Biotin)

(2177) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(2178) Functional Assay Data:

(2179) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 132

BiotinRANTES (CCL5)

(2180) Target Molecule:

(2181) RANTES derivatised at the ε-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(2182) Modifications:

(2183) 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.

(2184) The linear amino acid sequence (SEQ ID NO: 225) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(2185) TABLE-US-00354 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-OH

(2186) 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:

(2187) TABLE-US-00355 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(2188) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 226). 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: 227).

(2189) TABLE-US-00356 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEXS-OH
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))

(2190) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(2191) Functional Assay Data:

(2192) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 133

BiotinMCP-2 (CCL8)

(2193) Target Molecule:

(2194) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(2195) Modifications:

(2196) 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 ε-amino functionality and the biotin.

(2197) The linear amino acid sequence (SEQ ID NO: 222) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(2198) TABLE-US-00357 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTK RGKEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(2199) 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:

(2200) TABLE-US-00358 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTK RGKEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(2201) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 223). 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: 224):

(2202) TABLE-US-00359 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTK RGKEVCADPKERWVRDSMKHLDQIFQNLXP-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).

(2203) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(2204) Functional Assay Data:

(2205) 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 134

BiotinMIP-3b (CCL19)

(2206) Target Molecule:

(2207) MIP-3b derivatised at the e-amino side chain functionality of Lys(78) with Biotin (TFA salt)

(2208) Modifications:

(2209) Human MIP-3b 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.

(2210) The linear amino acid sequence (SEQ ID NO: 228) is shown, prior to attachment of the biotin molecule at amino acid 78 (K):

(2211) TABLE-US-00360 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRG RQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
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)

(2212) The engineered MIP-3b 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:

(2213) TABLE-US-00361 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRG RQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-RESIN
X is FmocLys(ivDde)

(2214) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 229). 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: 230).

(2215) TABLE-US-00362 H-GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRG RQLCAPPDQPWVERIIQRLQRTSAKMKRRSSX-NH.sub.2
X is K(Biotin)

(2216) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMIP-3b: obtained=9148.8 Da; expected 9149.7 Da.

(2217) Functional Assay Data:

(2218) biotinMip-3b 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-3b is 1.6 nM.

Example 135

BiotinIL-8 (CXCL8)

(2219) Target Molecule:

(2220) IL-8 derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(2221) Modifications:

(2222) Human IL-8 corresponding to residues 1-77, is initially expressed as 99 amino acids comprising the chemokine fold, and a 22 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. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(2223) The linear amino acid sequence (SEQ ID NO: 231) is shown, prior to attachment of the PEG spacer and biotin molecules:

(2224) TABLE-US-00363 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIV KLSDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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)

(2225) The engineered IL-8 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:

(2226) TABLE-US-00364 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIV KLSDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(2227) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 232). 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: 233):

(2228) TABLE-US-00365 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)

(2229) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8: obtained=9416.9 Da; expected 9417.0 Da.

(2230) Functional Assay Data:

(2231) BiotinIL-8 was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 18.9 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 136

BiotinIL-8 (6-78)

(2232) Target Molecule:

(2233) IL-8 (6-78) derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(2234) Modifications:

(2235) Truncated form of IL-8 corresponding to residues 6-77, the first five N-terminal residues have been removed and an additional lysine was inserted at the C-terminus at position 78, and modified through biotinylation on the resin. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(2236) The linear amino acid sequence (SEQ ID NO: 234) is shown, prior to attachment of the PEG spacer and biotin molecules:

(2237) TABLE-US-00366 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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

(2238) The engineered IL-8 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:

(2239) TABLE-US-00367 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(2240) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 235). 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: 236):

(2241) TABLE-US-00368 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)

(2242) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8 (6-78): obtained=8880.50 Da; expected 8880.4 Da.

(2243) Functional Assay Data:

(2244) BiotinIL-8 (6-78) was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 6.1 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 137

BiotinEotaxin (CCL11)

(2245) Target Molecule:

(2246) Eotaxin derivatised at the e-amino side chain functionality of Lys(73) with PEG-Biotin (TFA salt)

(2247) Modifications:

(2248) 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 ε-amino functionality and the biotin.

(2249) The linear amino acid sequence (SEQ ID NO: 237) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 73 (K):

(2250) TABLE-US-00369 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
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).

(2251) 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:

(2252) TABLE-US-00370 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(ivDde)

(2253) FmocLys(ivDde)-OH was incorporated as residue 73 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 238). 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: 239):

(2254) TABLE-US-00371 H-GPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKD ICADPKKKWVQDSMKYLDQKSPTPXP-NH.sub.2
X is K(PEG-Biotin)

(2255) Electrospray ionisation with tandem mass spectrometry (ESi-TOF-MS) data of purified folded biotinEotaxin: obtained=8731.3 Da; expected 8731.3 Da.

(2256) Functional Assay Data:

(2257) 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 138

Diagnosis and Treatment of Sarcoidosis

(2258) Materials and Methods

(2259) 1. Flow Cytometric Analysis of Peripheral Blood

(2260) 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 (RT) and stained with antibodies (Table 28) 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).

(2261) TABLE-US-00372 TABLE 29 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CCR1 Alexa flour Biolegend 647 CCR2 PerCPCy5.5 Biolegend CCR7 PerCpCy5.5 Biolegend CD4 V500 BD CD3 Horizon V450 BD Streptavdin APC BD CD14 FITC Beckman Coulter CD45RA PECy7 BD
2. Chemokine Binding Test

(2262) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 29). 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).

(2263) 3. Cell Depletion by Matrix Conjugated with Biotinylated Chemokine

(2264) 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 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 29), analysed by flow cytometry and compared with cells from peripheral blood that had not been incubated with the chemokine-matrix.

(2265) Results and Discussion

(2266) White blood cells from 2 patients with sarcoidosis were analysed for the expression of chemokine receptors with flow cytometry. The patients exhibited increased frequency of monocytes that expressed the receptor CCR1 based upon flow cytometry data and binding of an anti-CCR1 antibody (FIG. 161).

(2267) The ligand for CCR1 is the chemokine RANTES that also binds to CCR5 expressed on T cells. RANTES is expressed in the lungs where it mediates migration of inflammatory cells. The monocytes bind biotinylated RANTES to the same extent as the chemokine receptor expression (FIG. 162).

(2268) The CCR1 expressing monocytes could be efficiently depleted with bRANTES-conjugated Sepharose Streptavidin Matrix (FIG. 163).

(2269) In addition to CCR1, the monocytes express the chemokine receptor CCR2 (FIG. 164), based upon flow cytometry data and binding of an anti-CCR2 antibody.

(2270) The ligand for CCR2 is MCP-1 that mediate migration of monocytes in inflammation. In accordance with the CCR2 expression, biotinylated MCP-1 (bMCP-1) could bind to blood monocytes from a sarcoidosis patient (FIG. 165).

(2271) The CCR2 expressing monocytes could be depleted with bMCP1-conjugated Sepharose Streptavidin Matrix (FIG. 166).

(2272) The sarcoidosis patients exhibit an increased frequency of circulating T cells that express the chemokine receptor CCR7 (FIG. 167a), based upon flow cytometry data and binding by an anti-CCR7 antibody. Furthermore, the frequency of central memory T cells, which are characterized as CCR7 positive, is increased in sarcoidosis. (FIG. 167b). Central memory T cells contribute to inflammation by mounting a fast and strong immune response the second time the inflammation is triggered, and may be responsible for relapsing sarcoidosis.

(2273) The ligand for CCR7 is MIP3b. The CCR7 expressing T cells could be efficiently depleted with bMIP3b-conjugated Sepharose Streptavidin Matrix (FIG. 168)

(2274) We conclude that the frequency of CCR1 expressing monocytes and T cells that express CCR7 is enhanced in Sarcoidosis. The CCR2 receptor is expressed on monocytes from sarcoidosis patients to the same extent as in the healthy controls, but the CCR2 expressing cells could differ in their pro-inflammatory profile in the patients compared to healthy controls. Both monocytes and T cells bind the chemokines that corresponded with the expression of the chemokine receptor, and could be efficiently depleted with the corresponding biotinylated chemokine-Sepharose Streptavidin-matrix.

(2275) L. Treating Conditions Associated with Sepsis

(2276) Sepsis is defined as a systemic inflammation syndrome (SIRS) in response to an infection. If not successfully treated, it may lead to the potentially lethal Multiple Organ Dysfunction Syndrome. Thus, the invention may be aimed at treating SIRS and/or Multiple Organ Dysfunction Syndrome. In short, increased vascular permeability due to released inflammatory cytokines leads to decreased blood pressure. As a consequence, there is insufficient circulation in important organs such as kidneys and lungs, which may lead to organ dysfunction.

(2277) The exact cellular mechanism of the progress of SIRS is still unknown, but it has been shown that neutrophils show impaired migrational capacity into infected tissue in patients suffering from sepsis. The neutrophils act as the first line of defense against invading pathogens. If neutrophils cannot efficiently migrate into infected tissue the infection will persists which contributes to the systemic inflammation response.

(2278) In addition, in a later stage of SIRS, activated neutrophils migrate in to healthy tissue in remote organs (not affected by the infection) causing tissue destruction. The combination of organ dysfunction and tissue destruction may be fatal for the patient. Factors causing increased vascular permeability:

(2279) Proinflammatory molecules such interleukin1-beta, interleukin 6 and tumor necrosing factor-alpha, released by macrophages in the liver and the spleen and granulas released by mast cells.

(2280) Role of MCP-1/CCL2 and the CCR2 receptor in sepsisNeutrophils stimulated by bacterial antigen lipopolysaccharide through Toll-Like Receptors upregulate MCP-1 receptor CCR2. Neutrophils do not normally express CCR2. Interestingly, migration of neutrophils into healthy tissue is mediated through CCR2.

(2281) It has been shown that CCR2 plays an important role in the recruitment of neutrophils in a variety of models of inflammation in addition it has been shown that a CCR2 antagonist reduces the severity of acute lung injury.

(2282) It may be possible to protect the patient from tissue destruction in remote organs by removing activated neutrophils through the MCP-1/CCR2 interaction.

(2283) Chemokine receptor-Chemokine pairs of relevance to the present invention thus include:

(2284) CCR2-MCP-1

(2285) CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8-CXCR2

(2286) CXCL8-CXCR1

(2287) CCR5-CCL5, CCL3, CCL8

(2288) The invention may also be directed to treating RDS, including but not limited to sepsis-associated RDS. The RDS treated according to the invention is typically acute respiratory distress syndrome (ARDS). Three clinical settings account for 75% of ARDS cases: sepsis, severe multiple trauma and aspiration of saliva/gastric contents. Sepsis is the most common cause of ARDS (referred to herein as sepsis-associated RDS). Some cases of ARDS are linked to large volumes of fluid used during resuscitation post trauma. Other causes include shock, near-drowning, multiple transfusions and inhalation of irritants or toxic fumes that damage the alveolar epithelium. It is shown herein that patients suffering from respiratory distress syndrome (RDS) exhibit an increase in circulating neutrophils compared to healthy controls. The neutrophils express characteristic chemokine receptors including CXCR1, CXCR2 and CCR5. This provides a therapeutic approach to treat this condition, by removal of CXCR1, CXCR2 and/or CCR5 expressing cells using a suitable binding reagent. Moreover, it is also shown herein that CCR5 expressing neutrophils are highly increased in bronchoalveolar lavage fluid (BALF) in patients suffering from RDS. Thus, the invention may be applied to treat sepsis and/or RDS, including but not limited to sepsis-associated RDS.

(2289) Materials and Methods

(2290) Isolation of Peripheral Blood Leukocytes.

(2291) Heparinized peripheral blood from healthy blood donors or inflammatory bowel disease (IBD) patients was fixed with 4% paraformaldehyde for 4 minutes, hemolyzed for 15 minutes with a 0.83% ammonium chloride solution and washed twice in FACS buffer to obtain a suspension of blood leukocytes.

(2292) Chemokines.

(2293) The leukocytes were incubated for 30 min in the dark at 4° C. with biotinylated and Alexa647 Fluor® labelled MCP-1 or IL-8 (in concentrations 10 ng/μL and 50 ng/μL). 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.

(2294) Flow Cytometry Assay.

(2295) The flow cytometry assay was performed on a two laser FACS Calibur cytometer (BD Immunocytometry systems, San José, Ca, USA). Ten thousand cells were counted and analysed in each sample. For data analyses, Cell Quest Pro software from Becton Dickinson was used.

Example 139

Binding of Monocytes to MCP-1

(2296) In the experiment with biotinylated MCP-1 it was found that about 90% of the monocytes obtained from peripheral blood of healthy donors had bound to the cytokine after 30 min of incubation (FIG. 169a), whereas CD4+ and CD8+ lymphocytes had not bound (FIGS. 169b and 169c).

Example 140

Affinity of Blood Cells to Biotinylated IL-8

(2297) In FIG. 169 the binding to biotinylated IL-8 (CXCL8) of CD4+ lymphocytes (FIG. 1d), CD8+ lymphocytes (FIG. 169e) and CD16+ neutrophils (FIG. 169f) obtained from healthy donors is shown. After 30 min of incubation all CD16+ neutrophils bound to IL-8. In contrast no binding was observed with CD4+ lymphocytes and CD8+ lymphocytes.

Example 141

(2298) Monocytes were investigated for their expression of CCR2 (FIG. 170b) and their ability to bind MCP-1 (FIG. 170a). CCR2 expression was noted an all monocytes with the majority of monocytes expressing high levels, using an anti-CCR2 antibody (FIG. 170b). The MCP-1 binding to monocytes shown in FIG. 170a corresponds to the CCR2.sup.hi expressing population shown in FIG. 170b. Thus, MCP-1 binds favourably to CCR2.sup.hi expressing cells.

Example 142

Tailored Leukapheresis

(2299) Column Design and Properties

(2300) Introduction

(2301) 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.

(2302) The Column

(2303) The column is intended to be used as a leukapheresis treatment for sepsis. It will specifically remove CCR2, CXCR1, CXCR2 and/or CCR5-expressing leukocytes, in particular monocytes, through the use of a binding reagent, more specifically an MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 and/or CXCL8 containing resin or a CCL5, CCL3 or CCL8 containing resin, exploiting the CCR2, CXCR1, CXCR2 and/or CCR5-chemokine interaction. The column consists of three combined components, the plastic house, the streptavidin (SA) Sepharose™ BigBeads matrix and one or more of biotinylated MCP-1, MCP-2, MCP-3, MCP-4, MCP-5, CXCL8, CCL5, CCL3 or CCL8 bound to the matrix. The treatment is conducted using the same techniques as a standard apheresis procedure.

(2304) The Plastic House (FIG. 9)

(2305) 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. 9. 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.

(2306) Streptavidin Sepharose™ BigBeads

(2307) 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.

(2308) Binding Reagent

(2309) Coupled to the matrix is the third component of the device, one or more binding reagents that bind specifically to CCR2, CXCR1, CXCR2 and/or CCR5. One or more chemokines selected from the group consisting of: MCP-1, MCP-2, MCP-3, MCP-4, MCP-5 and CXCL8 may be employed and/or CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7 and CXCL8 or CCL5, CCL3 or CCL8 (bind CCR5). These peptides may be synthetic, engineered versions of the human chemokine, which are truncated and biotinylated, but retain binding activity to the CCR2, CXCR1, CXCR2 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 4×10.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.

(2310) The Apheresis System

(2311) To conduct the leukapheresis the following components are needed; the column, tubing system, and a 4008 ADS pump (Fresenius Medical Care).

(2312) The Circuit

(2313) The system is illustrated in FIG. 10. 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.

(2314) The 4008 ADS Pump

(2315) 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.

(2316) A schematic diagram of the pump, showing the air detector and optical filter is shown in FIG. 11. If the pump system detects air bubbles and optical fluctuations or if extracorporeal pressure values are out of the set range, then the pump stops immediately and a visual/audible alarm are emitted.

LEGEND FOR FIG. 11

(2317) 1. Monitor 2. Holder for waste bag 3. Modules (left to right—Blood pump, ACD pump, Air detector) 4. Reserve places for further modules 5. Absorber holder 6. Drip detector 7. IV pole
Preparation of the Patient

(2318) 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.

(2319) Leukapheresis Time and Flow Rate

(2320) 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.

(2321) Storage Conditions

(2322) 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.

(2323) Transport Conditions

(2324) 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.

(2325) In-Vitro Depletion of Target Cell Populations

(2326) To investigate the ability to eliminate CCR2-expressing cells, in vitro tests have been performed on the bMCP-1 coupled matrix. Blood was collected from blood donors and passed through the column device containing bMCP-1 coupled matrix. Blood samples were taken before and after column passage and analyzed by flow cytometry (FACS) for the depletion of CCR2-expressing cells.

(2327) The results demonstrate significant depletion of the target population CCR2-expressing monocytes post matrix perfusion. Depletion tests were performed on blood from three healthy donors. The results are shown in FIG. 171.

(2328) The in-vitro results demonstrate a specific reduction of up to 80% of the CCR2-expressing cells by the column. Notably, individuals with fewer CCR2 expressing cells initially achieved lower depletion. The remaining levels of monocytes were around 20-30% in each case, irrespective of the starting point. Non-CCR2-expressing cells remained unaffected (data not shown).

Example 143

MCP1 Derivatives

(2329) MCP-1 has been produced with residue 75 as the site of biotinylation on the chemokine (numbering based upon the mature protein having the amino acid sequence of SEQ ID NO: 2). Biotinylation permits immobilization of MCP-1 on a solid support (via a biotin-avidin interaction). The basic amino acid sequence of MCP-1, including a 23 amino acid leader sequence is set forth as SEQ ID NO: 240,

(2330) TABLE-US-00373 MKVSAALLCL LLIAATFIPQ GLAQPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT
The amino acid sequence of the mature protein is set forth as SEQ ID NO: 241,

(2331) TABLE-US-00374 QPDAINA PVTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSXDHL DKQTQTPKT
X=Met or Nleu

(2332) 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.

(2333) Thus, MCP-1 derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt) will be synthesised. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. The Gln at the N-terminus of the proteins is subject to pyroGlu formation under physiological conditions. Thus the first glutamine (Gln1) of the sequence will be substituted with pyroglutamine. The molecule will be synthesised as a C-terminal amide (via synthesis on an amide linker). The molecule is shown schematically in FIG. 172.

(2334) A biotinMCP-1 Met to Nleu analogue will also be synthesised. The single methionine within the sequence will be altered to Norleucine, to mitigate against oxidation of this residue during the chain assembly and improve stability of the final product. This molecule is shown schematically in FIG. 173 and in SEQ ID NO: 241.

(2335) Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 144

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

(2336) Antagonist Activity (J-H Gong and I. Clark-Lewis, J. Exp. Med., 1995, 181, 63) has been shown for an MCP-1 derivative truncated at the N-terminus. In particular, deletion of residues 1-8, results in binding to CCR2 with Kd 8.3 nM. This protein was unable to cause chemotaxis of CCR2 positive cells. (inhibition of chemotaxis IC50 20 nM)

(2337) The amino acid sequence of the truncated version is set forth as SED ID NO: 242:

(2338) TABLE-US-00375 VTCCYNFTN RKISVQRLAS YRRITSSKCP KEAVIFKTIV AKEICADPKQ KWVQDSMDHL DKQTQTPKT

(2339) A derivative of this truncated version will be synthesised comprising residues 9 to 76 of the mature protein (MCP-1 9-76) with Met64 to Nleu substitution and derivatised at the ε-amino side chain functionality of Lys75 with PEG-Biotin (TFA salt). This molecule is shown schematically in FIG. 174. The PEG spacer will be 3,6,-dioxoaminooctanoic acid. Once synthesised, the activity of the various biotinMCP-1 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Example 145

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

(2340) CCR2 also binds chemokines MCP-2, MCP-3, MCP-4 and MCP-5 in addition to MCP-1. MCP-5 only binds CCR2 and should be selective in its removal of CCR2 expressing cells. MCP5 is a mouse chemokine shown to chemotact human CCR2 cells with EC50<3 nM.

(2341) The full length amino acid sequence, including the signal peptide, is set forth as SEQ ID NO: 243

(2342) TABLE-US-00376 MKISTLLCLL LIATTISPQV LAGPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG
The amino acid sequence of N-terminal processed MCP-5 chemokine is 82 amino acids long and is set forth as SEQ ID NO: 244

(2343) TABLE-US-00377 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFILEP SCLG

(2344) An amino acid sequence alignment suggests that MCP-5 has a C-terminal extension when compared to the amino acid sequence of MCP-1. The results of this alignment are shown in FIG. 175. On this basis a C-terminal truncated version of MCP-5 will be synthesised. This truncated version will comprise MCP-5 residues 1-76, set forth as SEQ ID NO: 245:

(2345) TABLE-US-00378 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFIL
In the truncated version, Ile75 to be substituted with Lys, set forth as SEQ ID NO: 246:

(2346) TABLE-US-00379 GPDAVSTP VTCCYNVVKQ KIHVRKLKSY RRITSSQCPR EAVIFRTILD KEICADPKEK WVKNSINHLD KTSQTFXL
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)

(2347) Following substitution, the substituted version will be biotinylated at position 75, a lysine or other suitable residue such as ornithine or diaminopropanoic acid via A PEG spacer (3,6,-dioxoaminooctanoic acid). The protein will be synthesised on an amide linker to yield a C-terminal amide derivative. This molecule is shown schematically in FIG. 11. Once synthesised, the activity of the various biotinMCP-5 derivatives will be determined in cell-based assays. In particular, agonist and antagonist properties will be determined in aequorin functional cell-based assay on human CCR2 receptor.

Further Chemokine Examples

General Protocols

(2348) Assembly:

(2349) 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.

(2350) Removal of Dde Protection:

(2351) 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.

(2352) Labelling Steps:

(2353) 1. Couple Fmoc-8-Amino-3,6-Dioctanoic Acid (PEG)

(2354) 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.

(2355) 2. Capping

(2356) The resin was capped with acetic anhydride solution (0.5 M in DMF, 10 ml) for 5 minutes and then washed with DMF.

(2357) 3. Fmoc Deprotection

(2358) Fmoc deprotection was carried out by treatment with 20% piperidine in DMF solution (2×50 ml) for 15 minutes each. The resin was washed with DMF.

(2359) 4. Couple Biotin-OSu

(2360) A solution of Biotin-OSu (341 mg, 1 mmol) and DIPEA (348 ml) 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.

(2361) Cleavage:

(2362) Dry resin was treated with TFA (10 ml) containing a scavenger cocktail consisting of TIS (500 ml), thioanisole (500 ml), water (500 ml), DMS (500 ml), EDT (250 ml), NH.sub.4I (500 mg) and phenol (500 mg) 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.

(2363) Purification Protocol:

(2364) The crude peptide was purified by reverse phase HPLC (RP-HPLC) using a Jupiter C18, 250×21 mm column, 9 ml/min, eluting with an optimised gradient [Buffer A: water containing 0.1% TFA, Buffer B: acetonitrile containing 0.1% TFA].

(2365) Folding Protocol:

(2366) 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, 250×4.6 mm column, 10-60% B over 30 minutes. Purification by RP-HPLC using an optimised gradient afforded the desired product.

Example 146

BiotinMCP-1 (CCL2)

(2367) Target Molecule:

(2368) MCP-1 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(2369) Modifications:

(2370) Human MCP-1 corresponding to residues 1-76, is initially expressed as 99 amino acids comprising the chemokine fold, and a 23 amino acid signal peptide which is cleaved off. The Gln at the N-terminus of the protein is subject to pyroGlu formation under physiological conditions. Thus Gln1 of the sequence was substituted with pyroglutamine to prevent mixed species of N-terminal Gln and pyroGlu being generated. This improves the yield of synthesis and ensures a homogeneous chemokine preparation through column manufacture and use. The naturally occurring lysine at position 75 was modified through biotinylation on the resin. A PEG spacer was incorporated between the ε-amino functionality and the biotin.

(2371) The linear amino acid sequence (SEQ ID NO: 247) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(2372) TABLE-US-00380 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPKT-NH.sub.2
X=pyroGlu or Gln

(2373) The engineered MCP-1 sequence was assembled on a solid support (Rink Amide resin), using Fmoc protocols for solid-phase peptide synthesis as described in the general protocols section:

(2374) TABLE-US-00381 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-RESIN
X1=pyroGlu or Gln
X75=K(ivDde)

(2375) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 248). 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: 249):

(2376) TABLE-US-00382 H-XPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVA KEICADPKQKWVQDSMDHLDKQTQTPXT-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).

(2377) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-1: obtained=9032.8 Da; expected 9034.4 Da.

(2378) Functional Assay Data:

(2379) biotinMCP-1 was tested for agonist activity in an Aequorin assay against hCCR2b, (Euroscreen) and an EC50 value of 9.6 nM was reported. c.f. EC50 for recombinant native MCP-1 is 3.1 nM.

Example 147

BiotinMCP-2 (CCL8)

(2380) Target Molecule:

(2381) MCP-2 derivatised at the e-amino side chain functionality of Lys(75) with PEG-Biotin (TFA salt)

(2382) Modifications:

(2383) 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 ε-amino functionality and the biotin.

(2384) The linear amino acid sequence (SEQ ID NO: 250) is shown, prior to attachment of the PEG spacer and biotin molecules at amino acid 75 (K):

(2385) TABLE-US-00383 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLKP-NH.sub.2
X=pyroGlu or Gln

(2386) 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:

(2387) TABLE-US-00384 H-XPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRG KEVCADPKERWVRDSMKHLDQIFQNLXP-NH.sub.2
X1=pyroGlu or Gln
X75=K(ivDde)

(2388) FmocLys(ivDde)-OH was incorporated as residue 75 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 251). 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: 252):

(2389) TABLE-US-00385 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).

(2390) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinMCP-2: obtained=9263.6 Da; expected 9263.8 Da.

(2391) Functional Assay Data:

(2392) 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 148

BiotinRANTES (CCL5)

(2393) Target Molecule:

(2394) RANTES derivatised at the e-amino side chain functionality of Lys(67) with Biotin (TFA salt)

(2395) Modifications:

(2396) 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.

(2397) The linear amino acid sequence (SEQ ID NO: 253) is shown, prior to attachment of the biotin molecule at amino acid 67 (K):

(2398) TABLE-US-00386 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEKS-OH

(2399) 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:

(2400) TABLE-US-00387 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-RESIN
X is K(ivDde)

(2401) FmocLys(ivDde)-OH was incorporated as residue 67 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 254). 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: 255).

(2402) TABLE-US-00388 H-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQ VCANPEKKWVREYINSLEXS-OH
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))

(2403) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinRANTES: obtained=8068.9 Da; expected 8070.2 Da.

(2404) Functional Assay Data:

(2405) BiotinRANTES was tested for agonist activity in an Aequorin assay against hCCR5, (Euroscreen) and an EC50 value of 0.5 nM was reported.

Example 149

BiotinIL-8 (CXCL8)

(2406) Target Molecule:

(2407) IL-8 derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(2408) Modifications:

(2409) Human IL-8 corresponding to residues 1-77, is initially expressed as 99 amino acids comprising the chemokine fold, and a 22 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. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(2410) The linear amino acid sequence (SEQ ID NO: 256) is shown, prior to attachment of the PEG spacer and biotin molecules:

(2411) TABLE-US-00389 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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

(2412) The engineered IL-8 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:

(2413) TABLE-US-00390 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(2414) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 257). 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: 258):

(2415) TABLE-US-00391 H-AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKL SDGRELCLDPKENWVQRVVEKFLKRAENSK(PEG-Biotin)-NH.sub.2

(2416) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8: obtained=9416.9 Da; expected 9417.0 Da.

(2417) Functional Assay Data:

(2418) BiotinIL-8 was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 18.9 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 150

BiotinIL-8 (6-78)

(2419) Target Molecule:

(2420) IL-8 (6-78) derivatised at the e-amino side chain functionality of Lys(78) with PEG-Biotin (TFA salt)

(2421) Modifications:

(2422) Truncated form of IL-8 corresponding to residues 6-77, the first five N-terminal residues have been removed and an additional lysine was inserted at the C-terminus at position 78, and modified through biotinylation on the resin. A PEG spacer was incorporated between the e-amino functionality and the biotin.

(2423) The linear amino acid sequence (SEQ ID NO: 259) is shown, prior to attachment of the PEG spacer and biotin molecules:

(2424) TABLE-US-00392 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
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

(2425) The engineered IL-8 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:

(2426) TABLE-US-00393 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-RESIN
X is K(ivDde)

(2427) FmocLys(ivDde)-OH was incorporated as residue 78 to facilitate site-specific labelling at this position of the protein (SEQ ID NO: 260). 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: 261):

(2428) TABLE-US-00394 H-SAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRE LCLDPKENWVQRVVEKFLKRAENSX-NH.sub.2
X is K(PEG-Biotin)

(2429) Electrospray ionisation with tandem mass spectrometry (ESI-TOF-MS) data of purified folded biotinIL-8 (6-78): obtained=8880.50 Da; expected 8880.4 Da.

(2430) Functional Assay Data:

(2431) BiotinIL-8 (6-78) was tested for agonist activity in an Aequorin assay against hCXCR1, (Euroscreen) and an EC50 value of 6.1 nM was reported. c.f. EC50 for recombinant native IL-8 is 4.2 nM.

Example 151

Diagnosis and Treatment of RDS

(2432) Materials and Methods

(2433) 1. Flow Cytometric Analysis of Peripheral Blood Peripheral blood from patients and healthy controls was collected in heparin tubes.

(2434) 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 (RT) and stained with antibodies (Table 30) at 4° C. for 30 min. The cells were analysed with flow cytometry on a FACS Canto flow cytometer with the FACSDiva software (BD Biosciences).

(2435) TABLE-US-00395 TABLE 30 List of antibodies for flow cytometric analysis. Antibody Fluorophore Supplier CD14 FITC Beckman Coulter CCR5 PE Biolegend CXCR2 PE Biolegend Streptavidin PE, APC Biolegend CD16 PE Cy7 BD Biosciences CXCR1 APC Biolegend CD3 V450 BD Biosciences CD14 V500 BD Biosciences
2. Chemokine Binding Test

(2436) 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 μM) or the corresponding chemokine receptor antibody at 4° C. for 30 min (Table 30). 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).

(2437) 3. Cell Depletion with Antibody and Magnetic Activated Cell Sorting (MACS)

(2438) Cells were prepared from peripheral blood (section 1) and kept in MACS buffer (PBS pH 7.2 supplemented with 2 mM EDTA and 0.5% bovine serum albumin (BSA)). The cells were incubated with an anti-CXCR1-APC antibody for 30 min at 4° C. Next, the cells were incubated with anti-APC microbeads for 15 min, 4° C. (Miltenyi Biotec). The cells were run through MACS® columns (Miltenyi Biotec) to enable binding of the CXCR1 positive cells.

(2439) The cells were then analysed with flow cytometry.

(2440) Results and Discussion

(2441) 1. Flow Cytometric Analysis of Peripheral Blood

(2442) White blood cells from patients with respiratory distress syndrome (RDS) were analysed for cell surface markers with flow cytometry. The patients exhibited an increased frequency of circulating neutrophils, 73% compared to 45% in healthy controls (FIG. 178).

(2443) 2. Chemokine Binding Test

(2444) The neutrophils expressed the chemokine receptors CXCR1 and CXCR2 based upon flow cytometry data and binding by anti-CXCR1 and CXCR2 antibodies (FIG. 179). Both these receptors bind IL-8, a very important pro-inflammatory chemokine that mediates migration of neutrophils to site of infection.

(2445) Biotinylated IL-8 (bIL-8) could bind to the neutrophils to the same extent as the receptor-specific antibody anti-CXCR1 (FIG. 180).

(2446) 3. Cell Depletion with Antibody and Magnetic Activated Cell Sorting (MACS)

(2447) Furthermore, the neutrophils could be efficiently depleted with an anti-CXCR1 antibody and Magnetic Activated Cell Sorting (MACS) (FIG. 181).

(2448) We conclude that the frequency of neutrophils is increased in sepsis patients. The neutrophils express the IL-8 receptors CXCR1 and CXCR2 and can bind the ligand IL-8. Furthermore, the neutrophils can be depleted with an anti-CXCR1-antibody and MACS.

Example 152

In-Vitro Depletion of Target Cell Populations (CCR5)

(2449) To investigate the ability to eliminate CCR5-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 CCR5-expressing cells.

(2450) The RANTES molecule was synthesized by Almac. The amino acid sequence of the RANTES molecule, prior to biotinylation, is set forth as SEQ ID NO: 253:

(2451) TABLE-US-00396 H2N-SPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKN RQVCANPEKKWVREYINSLEKS-CO2H

(2452) This molecule has the naturally occurring methionine at position 67 replaced with lysine to facilitate biotinylation at position 67. The biotinylated version is shown as SEQ ID NO: 16.

(2453) The side-chain of Lys 67 was directly biotinylated to given the protein primary structure shown in FIG. 185. The protein was folded and disulphide bonds formed between the first and third cysteine in the sequence and between the 2nd and 4th cysteines. The results demonstrate significant depletion of the target population chemokine receptor-expressing cells post matrix perfusion. Depletion tests were performed on blood from a healthy donor. The results are shown in FIG. 186.

(2454) The in-vitro results demonstrate a specific reduction of around 20% of the chemokine receptor-expressing cells by the column. Non-CCR5-expressing cells remained unaffected (data not shown).

(2455) 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.