WO2003022885A1 - Chimiokines - Google Patents
Chimiokines Download PDFInfo
- Publication number
- WO2003022885A1 WO2003022885A1 PCT/GB2002/004083 GB0204083W WO03022885A1 WO 2003022885 A1 WO2003022885 A1 WO 2003022885A1 GB 0204083 W GB0204083 W GB 0204083W WO 03022885 A1 WO03022885 A1 WO 03022885A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- chemokine
- chemokines
- apcs
- regulatory
- Prior art date
Links
- 108010012236 Chemokines Proteins 0.000 title claims abstract description 264
- 102000019034 Chemokines Human genes 0.000 title claims abstract description 260
- 238000000034 method Methods 0.000 claims abstract description 89
- 210000003289 regulatory T cell Anatomy 0.000 claims abstract description 82
- 230000028993 immune response Effects 0.000 claims abstract description 55
- 210000000612 antigen-presenting cell Anatomy 0.000 claims abstract description 42
- 239000000203 mixture Substances 0.000 claims abstract description 36
- 241000124008 Mammalia Species 0.000 claims abstract description 30
- 230000007115 recruitment Effects 0.000 claims abstract description 29
- 230000001965 increasing effect Effects 0.000 claims abstract description 23
- 230000005714 functional activity Effects 0.000 claims abstract description 21
- 230000003247 decreasing effect Effects 0.000 claims abstract description 20
- 210000004027 cell Anatomy 0.000 claims description 117
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 89
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 81
- 230000004913 activation Effects 0.000 claims description 44
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 claims description 37
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 claims description 37
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 claims description 37
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 36
- 239000002975 chemoattractant Substances 0.000 claims description 23
- 239000003112 inhibitor Substances 0.000 claims description 19
- 238000011282 treatment Methods 0.000 claims description 19
- 210000004443 dendritic cell Anatomy 0.000 claims description 17
- 210000002540 macrophage Anatomy 0.000 claims description 17
- 201000010099 disease Diseases 0.000 claims description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 11
- 230000003834 intracellular effect Effects 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 238000009826 distribution Methods 0.000 claims description 8
- 230000005784 autoimmunity Effects 0.000 claims description 7
- 239000003814 drug Substances 0.000 claims description 7
- 239000003085 diluting agent Substances 0.000 claims description 6
- 239000003937 drug carrier Substances 0.000 claims description 6
- 230000001404 mediated effect Effects 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- 206010052779 Transplant rejections Diseases 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 4
- 230000030786 positive chemotaxis Effects 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- 238000013518 transcription Methods 0.000 claims description 4
- 230000035897 transcription Effects 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 3
- 238000011321 prophylaxis Methods 0.000 claims description 3
- 230000004481 post-translational protein modification Effects 0.000 claims description 2
- 238000013519 translation Methods 0.000 claims description 2
- 239000003550 marker Substances 0.000 claims 3
- 102100035304 Lymphotactin Human genes 0.000 abstract description 2
- 102000000021 Chemokine CCL1 Human genes 0.000 abstract 1
- 108010055288 Chemokine CCL1 Proteins 0.000 abstract 1
- 101000804764 Homo sapiens Lymphotactin Proteins 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 38
- 230000014509 gene expression Effects 0.000 description 32
- 230000000694 effects Effects 0.000 description 30
- 239000000523 sample Substances 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 22
- 239000000427 antigen Substances 0.000 description 21
- 239000002158 endotoxin Substances 0.000 description 21
- 229920006008 lipopolysaccharide Polymers 0.000 description 21
- 108091007433 antigens Proteins 0.000 description 20
- 102000036639 antigens Human genes 0.000 description 20
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 16
- 208000014197 Fetal anticonvulsant syndrome Diseases 0.000 description 15
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 15
- 102000014914 Carrier Proteins Human genes 0.000 description 14
- 210000000952 spleen Anatomy 0.000 description 14
- 230000003393 splenic effect Effects 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 13
- 108091008324 binding proteins Proteins 0.000 description 12
- 238000000338 in vitro Methods 0.000 description 12
- 238000010232 migration assay Methods 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- 238000003491 array Methods 0.000 description 11
- 230000006870 function Effects 0.000 description 11
- 102000009410 Chemokine receptor Human genes 0.000 description 10
- 108050000299 Chemokine receptor Proteins 0.000 description 10
- 238000004132 cross linking Methods 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 230000000638 stimulation Effects 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- 239000006228 supernatant Substances 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 9
- 108010021083 hen egg lysozyme Proteins 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 230000001105 regulatory effect Effects 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 239000012190 activator Substances 0.000 description 8
- 230000012292 cell migration Effects 0.000 description 8
- 238000002649 immunization Methods 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 235000018102 proteins Nutrition 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 102000027005 chemokine binding proteins Human genes 0.000 description 7
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- 230000005012 migration Effects 0.000 description 7
- 238000013508 migration Methods 0.000 description 7
- 150000007523 nucleic acids Chemical group 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 102000005962 receptors Human genes 0.000 description 7
- 108020003175 receptors Proteins 0.000 description 7
- 230000003844 B-cell-activation Effects 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000006043 T cell recruitment Effects 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 238000001727 in vivo Methods 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 230000009261 transgenic effect Effects 0.000 description 6
- 102100032367 C-C motif chemokine 5 Human genes 0.000 description 5
- 241000699660 Mus musculus Species 0.000 description 5
- 239000011324 bead Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 210000004408 hybridoma Anatomy 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 238000002955 isolation Methods 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 230000000069 prophylactic effect Effects 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 108010040471 CC Chemokines Proteins 0.000 description 4
- 102000001902 CC Chemokines Human genes 0.000 description 4
- 108010021064 CTLA-4 Antigen Proteins 0.000 description 4
- 102000008203 CTLA-4 Antigen Human genes 0.000 description 4
- 229940045513 CTLA4 antagonist Drugs 0.000 description 4
- 101710172503 Chemokine-binding protein Proteins 0.000 description 4
- 102100027286 Fanconi anemia group C protein Human genes 0.000 description 4
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 description 4
- 108060003951 Immunoglobulin Proteins 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 235000001014 amino acid Nutrition 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000012530 fluid Substances 0.000 description 4
- 238000009396 hybridization Methods 0.000 description 4
- 210000000987 immune system Anatomy 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 102000018358 immunoglobulin Human genes 0.000 description 4
- 230000008611 intercellular interaction Effects 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 210000004698 lymphocyte Anatomy 0.000 description 4
- 238000002493 microarray Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000011830 transgenic mouse model Methods 0.000 description 4
- 108091093088 Amplicon Proteins 0.000 description 3
- 108050006947 CXC Chemokine Proteins 0.000 description 3
- 102000019388 CXC chemokine Human genes 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 101000582950 Homo sapiens Platelet factor 4 Proteins 0.000 description 3
- 108010014251 Muramidase Proteins 0.000 description 3
- 102000016943 Muramidase Human genes 0.000 description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 3
- 102100030304 Platelet factor 4 Human genes 0.000 description 3
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 3
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 3
- 108700019146 Transgenes Proteins 0.000 description 3
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 108091008503 chemokine binding proteins Proteins 0.000 description 3
- 238000007405 data analysis Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 239000008121 dextrose Substances 0.000 description 3
- 230000003467 diminishing effect Effects 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 3
- 230000003325 follicular Effects 0.000 description 3
- 210000002443 helper t lymphocyte Anatomy 0.000 description 3
- 230000028996 humoral immune response Effects 0.000 description 3
- 230000008348 humoral response Effects 0.000 description 3
- 230000001976 improved effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 229960000274 lysozyme Drugs 0.000 description 3
- 239000004325 lysozyme Substances 0.000 description 3
- 235000010335 lysozyme Nutrition 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 206010028417 myasthenia gravis Diseases 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 230000002265 prevention Effects 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 102000006306 Antigen Receptors Human genes 0.000 description 2
- 108010083359 Antigen Receptors Proteins 0.000 description 2
- 102000019260 B-Cell Antigen Receptors Human genes 0.000 description 2
- 108010012919 B-Cell Antigen Receptors Proteins 0.000 description 2
- 241000588832 Bordetella pertussis Species 0.000 description 2
- 102100025074 C-C chemokine receptor-like 2 Human genes 0.000 description 2
- 102100023698 C-C motif chemokine 17 Human genes 0.000 description 2
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 2
- 102100036848 C-C motif chemokine 20 Human genes 0.000 description 2
- 102100036845 C-C motif chemokine 22 Human genes 0.000 description 2
- -1 CCLl 9 (MIP3beta) Proteins 0.000 description 2
- 108010061299 CXCR4 Receptors Proteins 0.000 description 2
- 102000012000 CXCR4 Receptors Human genes 0.000 description 2
- 102000000844 Cell Surface Receptors Human genes 0.000 description 2
- 108010001857 Cell Surface Receptors Proteins 0.000 description 2
- 108010082169 Chemokine CCL17 Proteins 0.000 description 2
- 108010078239 Chemokine CX3CL1 Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 102000013818 Fractalkine Human genes 0.000 description 2
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 2
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000716068 Homo sapiens C-C chemokine receptor type 6 Proteins 0.000 description 2
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 description 2
- 108010091358 Hypoxanthine Phosphoribosyltransferase Proteins 0.000 description 2
- 102100029098 Hypoxanthine-guanine phosphoribosyltransferase Human genes 0.000 description 2
- 108090001007 Interleukin-8 Proteins 0.000 description 2
- 102000004890 Interleukin-8 Human genes 0.000 description 2
- 102000003896 Myeloperoxidases Human genes 0.000 description 2
- 108090000235 Myeloperoxidases Proteins 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 239000012979 RPMI medium Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 108091006088 activator proteins Proteins 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 2
- 229940037003 alum Drugs 0.000 description 2
- 230000007503 antigenic stimulation Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- 238000005422 blasting Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000012636 effector Substances 0.000 description 2
- 230000005684 electric field Effects 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000004108 freeze drying Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000012224 gene deletion Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000011005 laboratory method Methods 0.000 description 2
- 210000005210 lymphoid organ Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 230000005305 organ development Effects 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 229920003023 plastic Polymers 0.000 description 2
- YIQPUIGJQJDJOS-UHFFFAOYSA-N plerixafor Chemical compound C=1C=C(CN2CCNCCCNCCNCCC2)C=CC=1CN1CCCNCCNCCCNCC1 YIQPUIGJQJDJOS-UHFFFAOYSA-N 0.000 description 2
- 229960002169 plerixafor Drugs 0.000 description 2
- 229920000729 poly(L-lysine) polymer Polymers 0.000 description 2
- 239000004417 polycarbonate Substances 0.000 description 2
- 229920000515 polycarbonate Polymers 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000007112 pro inflammatory response Effects 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 230000003252 repetitive effect Effects 0.000 description 2
- 210000005212 secondary lymphoid organ Anatomy 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229910052710 silicon Inorganic materials 0.000 description 2
- 239000010703 silicon Substances 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 208000035408 type 1 diabetes mellitus 1 Diseases 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 description 1
- XZKIHKMTEMTJQX-UHFFFAOYSA-N 4-Nitrophenyl Phosphate Chemical compound OP(O)(=O)OC1=CC=C([N+]([O-])=O)C=C1 XZKIHKMTEMTJQX-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101100457315 Arabidopsis thaliana MIP3 gene Proteins 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 230000028728 B cell mediated immunity Effects 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108050005711 C Chemokine Proteins 0.000 description 1
- 102000017483 C chemokine Human genes 0.000 description 1
- 102100037853 C-C chemokine receptor type 4 Human genes 0.000 description 1
- 101710149863 C-C chemokine receptor type 4 Proteins 0.000 description 1
- 102100036850 C-C motif chemokine 23 Human genes 0.000 description 1
- 101710085496 C-X-C motif chemokine 2 Proteins 0.000 description 1
- 101150011672 CCL9 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 101710104316 Cell surface-binding protein Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108010083701 Chemokine CCL22 Proteins 0.000 description 1
- 108010055166 Chemokine CCL5 Proteins 0.000 description 1
- 102100035294 Chemokine XC receptor 1 Human genes 0.000 description 1
- 102000000503 Collagen Type II Human genes 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 description 1
- 208000016192 Demyelinating disease Diseases 0.000 description 1
- 101710094214 Duffy receptor Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100023688 Eotaxin Human genes 0.000 description 1
- 101710139422 Eotaxin Proteins 0.000 description 1
- 208000009386 Experimental Arthritis Diseases 0.000 description 1
- 102000009109 Fc receptors Human genes 0.000 description 1
- 108010087819 Fc receptors Proteins 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- VSRCAOIHMGCIJK-SRVKXCTJSA-N Glu-Leu-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O VSRCAOIHMGCIJK-SRVKXCTJSA-N 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 101000684275 Homo sapiens ADP-ribosylation factor 3 Proteins 0.000 description 1
- 101100005560 Homo sapiens CCL23 gene Proteins 0.000 description 1
- 101000804783 Homo sapiens Chemokine XC receptor 1 Proteins 0.000 description 1
- 101000889276 Homo sapiens Cytotoxic T-lymphocyte protein 4 Proteins 0.000 description 1
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 description 1
- 101001135199 Homo sapiens Partitioning defective 3 homolog Proteins 0.000 description 1
- 101001098557 Homo sapiens Proteinase-activated receptor 3 Proteins 0.000 description 1
- 101001130437 Homo sapiens Ras-related protein Rap-2b Proteins 0.000 description 1
- 101000815628 Homo sapiens Regulatory-associated protein of mTOR Proteins 0.000 description 1
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 1
- 101000652747 Homo sapiens Target of rapamycin complex 2 subunit MAPKAP1 Proteins 0.000 description 1
- 101000648491 Homo sapiens Transportin-1 Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 108090001005 Interleukin-6 Proteins 0.000 description 1
- 102100026236 Interleukin-8 Human genes 0.000 description 1
- 108010018951 Interleukin-8B Receptors Proteins 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- 102000043129 MHC class I family Human genes 0.000 description 1
- 108091054437 MHC class I family Proteins 0.000 description 1
- 102000043131 MHC class II family Human genes 0.000 description 1
- 108091054438 MHC class II family Proteins 0.000 description 1
- 102000047918 Myelin Basic Human genes 0.000 description 1
- 101710107068 Myelin basic protein Proteins 0.000 description 1
- 101100386053 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cys-3 gene Proteins 0.000 description 1
- 101100117488 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) mip-1 gene Proteins 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 101150006573 PAN1 gene Proteins 0.000 description 1
- 102000004861 Phosphoric Diester Hydrolases Human genes 0.000 description 1
- 108090001050 Phosphoric Diester Hydrolases Proteins 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102100037133 Proteinase-activated receptor 3 Human genes 0.000 description 1
- 102100031421 Ras-related protein Rap-2b Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 238000011803 SJL/J (JAX™ mice strain) Methods 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 230000006052 T cell proliferation Effects 0.000 description 1
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 1
- 102000009843 Thyroglobulin Human genes 0.000 description 1
- 108010034949 Thyroglobulin Proteins 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- 102000009618 Transforming Growth Factors Human genes 0.000 description 1
- 102100028748 Transportin-1 Human genes 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 102100038138 WD repeat-containing protein 26 Human genes 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003429 anti-cardiolipin effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 210000000628 antibody-producing cell Anatomy 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000006472 autoimmune response Effects 0.000 description 1
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000002306 biochemical method Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 230000020411 cell activation Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000003588 centroblast Anatomy 0.000 description 1
- 210000002711 centrocyte Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 239000012050 conventional carrier Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 108091092330 cytoplasmic RNA Proteins 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 1
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000011223 gene expression profiling Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 229940094991 herring sperm dna Drugs 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 230000037417 hyperactivation Effects 0.000 description 1
- 230000009610 hypersensitivity Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000002637 immunotoxin Effects 0.000 description 1
- 239000002596 immunotoxin Substances 0.000 description 1
- 229940051026 immunotoxin Drugs 0.000 description 1
- 231100000608 immunotoxin Toxicity 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000005923 long-lasting effect Effects 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 238000010208 microarray analysis Methods 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- 238000004377 microelectronic Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000000865 mononuclear phagocyte system Anatomy 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000002966 oligonucleotide array Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 210000003024 peritoneal macrophage Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000000206 photolithography Methods 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000131 polyvinylidene Polymers 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 210000004986 primary T-cell Anatomy 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 210000001948 pro-b lymphocyte Anatomy 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000003133 propidium iodide exclusion Methods 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002287 radioligand Substances 0.000 description 1
- 108010014186 ras Proteins Proteins 0.000 description 1
- 102000016914 ras Proteins Human genes 0.000 description 1
- 230000008707 rearrangement Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- 229960002175 thyroglobulin Drugs 0.000 description 1
- 206010043778 thyroiditis Diseases 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/521—Chemokines
- C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
- G01N33/5047—Cells of the immune system
- G01N33/505—Cells of the immune system involving T-cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/21—Chemokines, e.g. MIP-1, MIP-2, RANTES, MCP, PF-4
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
Definitions
- the present invention relates to chemokines, compositions and methods effective to modulate the immune response in a mammal, via the recruitment of regulatory T-cells to antigen presenting cells (APCs).
- the present invention relates to modulating an immune response by increasing or decreasing or otherwise altering the functional activity of any one or more of the chemokines CCLl, CCL4, CCL3, CCL2, and XCLl.
- Uses of chemokines, compositions and methods of the present invention are also described.
- Chemokines (chemoattractant cytokines) were initially identified as cytokines profoundly induced by pro-inflammatory stimuli that control the recruitment of leukocytes into inflammatory foci. They are now emerging as important regulators of cellular trafficking in development and homeostasis. In addition, the chemokines also seem to be critical for the coordinated movement of dendritic cells and lymphocytes necessary to generate long lasting antigen specific immunity.
- chemokine superfamily unrecognised 10 years ago, now has over 40 different members, classified into different subfamilies on the basis of conserved structural features. Most chemokines contain four cysteine residues per monomer which are crosslinked Cysl-> Cys3 and Cys2->Cys4. The backbone of the chemokine molecule consists of beta strands while the N and C termini of the protein appear to have a less ordered structure. All chemokines for which the structure has been determined exist as non-covalently-linked dimers in solution.
- the arrangement of the cysteine residues within the chemokine protein sequence forms the basis for the structural classification of chemokines.
- Chemokines in which the Cl and C2 cysteine residues are separated by a single amino acid are the CXC chemokines; examples include IL-8 and PF4.
- CC chemokines the Cl and C2 cysteine residues are adjacent, examples include RANTES, MCP-1 (CCL2) and eotaxin.
- the T cell chemoattractant chemokine lymphotactin (XCLl) is the sole C chemokine with only one cysteine residue in the N terminal half of the protein.
- the only described CXXX3C chemokine is fractalkine, which has three amino acids separating Cl and C2.
- Fractalkine is also unusual in being the only chemokine that exists both as a membrane bound protein with the CXXX3C chemokine domain sitting on top of a mucin stalk and as a cleaved soluble molecule (Bazan et al.1997).
- chemokine superfamily has been made on the basis of amino acid sequence comparisons. For instance, a subgroup of CXC chemokines shares a three amino acid motif ELR (Glu-Leu-Arg) immediately preoceeding the first cysteine residue. Most of these ELR CXC chemokines bind the chemokine receptor CXCR2 and can act as neutrophil chemoattractants. A number of recently described CC chemokines have six rather than the more usual four cysteine residues and some have an extended amino terminus relative to other CC chemokines. The six-cysteine CC chemokine family includes the human chemokines 1-309 (CCLl) and Secondary Lymphoid organ Chemokine (SLC).
- CCLl human chemokines 1-309
- SLC Secondary Lymphoid organ Chemokine
- Chemokines mediate their effects on cells via specific cell surface receptors that contain seven transmembrane spanning regions(7TM receptors). Chemokine receptors are coupled to G proteins that transmit intracellular signals via linked phosphodiesterases. Upon binding their cognate chemokine ligands, chemokine receptors initiate changes in the intracellular concentrations of calcium and cAMP. Chemokine receptors also transmit signals to the small GTP binding proteins of the Ras and Rho families leading to rearrangement of actin bundles, pseudopod formation and cell migration.
- chemokine receptors appear to be restricted to only limited amount of cell types eg. CCR6 is expressed on immature dendritic cells and some activated T cells. Most cellular chemokine receptors bind their cognate chemokine ligands with affinities in the nanomolar range. Most chemokine receptors have multiple chemokine ligands eg. CCR4 binds Macrophage Derived Chemokine (MDC now called CCL22) and Thymus and Activation Regulated Chemokine (TARC).
- MDC Macrophage Derived Chemokine
- TARC Thymus and Activation Regulated Chemokine
- the Duffy antigen is a 7TM receptor expressed on erythrocytes which binds a wide range of chemokines with low affinity (Kd ⁇ 10-6).
- the Duffy receptor is thought to act as a clearance receptor for chemokines.
- Chemokines are involved in the regulation of biological functions as diverse as angiogenesis, inflammation, lymphoid organ development and the orchestration of immune responses. They have been shown to be central to regulating the migration of cells of the immune system through various secondary lymphoid organs. Gene deletion models, in which chemokines or their receptors were targeted, highlight such functions although it is unclear whether the resulting phenotype is a consequence of altered lymphoid organ development or a direct effect on specific cell populations. Chemokines are thought to mediate interaction between T helper cells, effector cells and antigen presenting cells (APC), which are central to the generation of immune responses. The interaction between APC and T cells is a critical determinant of the ensuing immunological response.
- APC antigen presenting cells
- the present inventors realised that if the mechanism by which regulatory T-cell recruitment to APCs could be elucidated, then a method for modulating immune responses, and for the treatment or prevention of diseases may be established.
- the present invention addresses the problem of providing substances, methods and compositions effective to modulate an immune response in a mammal by affecting the recruitment of regulatory T-cells to APCs.
- APCs are defined here as professional antigen presenting cells. However, the regulatory effect can be on the B cell in the function of an antigen presenting cell or in the function as an effector cell of the B cell mediated immunity.
- B cells can act as APC in order to recruit T cell help.
- the present inventors have used gene expression profiling described infra, to study the changes in chemokine expression in response to physiological activation. Using this, and other methods described herein, several chemokines were identified which are involved in the modulation of the immune response via the recruitment of regulatory T-cells to APCs.
- chemokines described herein can be used to modulate the immune response.
- the present invention provides a method for modulating the immune response in a vertebrate comprising the step of increasing or decreasing, or otherwise altering, the functional activity of at least one chemokine secreted by antigen presenting cells (APCs) on their activation, and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APCs.
- APCs antigen presenting cells
- the present invention provides a method for modulating an immune response in a vertebrate comprising the steps of:
- step (a) testing one or more chemokines for their ability to be secreted by APCs on their activation, and also which are capable of acting as chemoattractant/s for the recruitment of regulatory T-cells to APCs.
- step (b) Selecting the one or more chemokines which possess the characteristics defined in step (a),
- step (c) Modulating the functional activity of the one or more chemokines selected according to step (b) in the vertebrate.
- chemokine in the context of the present invention includes any cytokine which is involved in immune or and/or pro-inflammatory responses.
- Suitable antigen presenting cells for use in the method of the present invention include any one or more selected from the group consisting of: B cells, dendritic cells and macrophages.
- the vertebrate is a mammal.
- the chemokines secreted by the activated APCs are any one or more selected from the group consisting of: CCLl, CCL4, CCL2, CCL3 and XCLl.
- the chemokine is CCL4 and/or CCLl.
- the chemokine is CCL4.
- the term 'increasing or decreasing the functional activity of at least one chemokine' in the context of the present invention includes within its scope increasing or decreasing the expression and/or intracellular or extracellular distribution, and/or activity of at least one chemokine as described herein.
- Increasing the expression may occur as a result of increasing mRNA expression, or by increasing gene transcription using methods known to those skilled in the art. Those skilled will appreciate that there are many suitable methods to increase or decrease the expression of a nucleic acid sequence encoding a chemokine as herein described.
- chemokines may be increased or decreased by increasing or decreasing the levels of chemokine mRNA respectively by post transcriptional modulation.
- interfering RNA may be used as a method to decrease chemokine RNA levels.
- Increasing or decreasing the intracellular distribution may occur as a result of the addition of chemokine binding proteins to the intracellular environment.
- the intracellular distribution may be increased, decreased or altered by the addition or removal of signal sequences and/or leader sequences to the chemokine. Techniques used in such procedures will be familiar to those skilled in the art.
- Increasing or decreasing the activity of the chemokines can be brought about by bringing the chemokines into contact with inhibitors of chemokines, or activators of chemokines and/or chemokine binding molecules.
- the term 'contact' in the context of the present invention means does not require a physical contact.
- Suitable inhibitors and activators include but are not limited to inhibitors of chemokine receptors.
- the small molecule AMD-3100 inhibits the CXCR4 receptor.
- co-factors or chemokine binding molecules may affect their activity. Examples include antibodies and fragments thereof (for example FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody). It will be appreciated that this list is by no means exhaustive.
- the functional activity of one or more chemokines is modulated using any one of more of the methods selected from the group consisting of: administering a pharmaceutically effective amount of those one or more chemokine/s to the vertebrate; administering a pharmaceutically effective amount of one or more inhibitor/s of those one or more chemokines to the vertebrate; modulating the transcription of those one or more chemokines in the vertebrate; modulating the translation of those one or more chemokines in the vertebrate; modulating the post-translational modification of those one or more chemokines in the vertebrate and modulating the intracellular or extracellular distribution of those one or more chemokines in the vertebrate.
- the functional activity of one or more chemokines is modulated by administering a pharmaceutically effective amount of one or more inhibitor/s of those one or more chemokines to the vertebrate.
- the one or more chemokine inhibitor/s are selected from the group consisting of: chemical chemokine inhibitors, anti -chemokine antibodies and dominant negative mutants of those one or more chemokines described herein.
- the functional activity of one chemokine may be modulated, or of several chemokines may be modulated.
- these chemokines may act in isolation or synergistically. In addition there may be functional redundancy in the activity of chemokines.
- the present invention provides the use of a chemokine which is secreted by antigen presenting cells (APCs) on their activation and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APCs, in the preparation of a medicament for modulating the immune response in a vertebrate.
- APCs antigen presenting cells
- the chemokines secreted by the activated APCs are any one or more selected from the group consisting of: CCLl, CCL4, CCL2, CCL3, and XCLl.
- the chemokine is CCL4 and or CCLl.
- the chemokine is CCL4.
- the vertebrate is a mammal.
- chemokines according to the present invention may act independently of each other, in an additive or subtractive manner or synergistically.
- there may be functional redundancy that is, one chemokine may act in preference to another chemokine in the recruitment of T cells to APCs.
- APCs include the following: dendritic cells, macrophages and B cells.
- dendritic cells include the following: dendritic cells, macrophages and B cells.
- B cells include the following: dendritic cells, macrophages and B cells.
- the present inventors have found, using methods described herein, that the chemokines of the present invention modulate the immune response via the recruitment of regulatory T-cells.
- the present invention provides a method for selecting one or more isolated regulatory T-cells capable of modulating the immune response in a vertebrate, comprising the step/s of: (a) preparing a population of T-cells; and (b) exposing said cells to activated antigen presenting cells (APCs); and/or at least one chemokine which is secreted by APCs on their activation, and
- step (c) selecting those T-cells which can migrate to those APCs or chemokines defined in step (b).
- the subset of regulatory T-cells referred to and/or selected are CD25+. In an especially preferred embodiment, they are CD25+ and CD4+. In an alternative embodiment, they are CD25+ and CD8+.
- a population of regulatory T-cells, and APCs such as B-cells, macrophages and monocytes can be prepared using methods known to those skilled in the art including FACs sorting, MACsorting (sorting with magnetic beads), immunoprecipation and standard chromatographic techniques. All these methods are familiar to one skilled in the art and suitable methods will be described in detail herein. Generally, the ability of a population of T-cells to migrate will be measured by techniques known to those skilled in the art, including, standard cell migration assays. Those skilled in the art will be aware of other suitable methods.
- the present invention provides the use of one or more isolated regulatory T cell/s which are capable of migrating towards antigen presenting cells (APCs) by chemoattraction mediated by at least one chemokine secreted by APCs on their activation, in the preparation of a medicament for modulating an immune response in a vertebrate.
- APCs antigen presenting cells
- the regulatory T-cells are CD25+ positive. In an especially preferred embodiment, they are CD25+ and CD4+ positive. In an alternative embodiment, they are CD25+ and CD8+ positive.
- the chemokines will be at least one selected from the group consisting of CCLl, CCL4, CCL3, CCL2, and XCLl. More preferably, the chemokines will be CCLl and/or CCL4. In a preferred embodiment of this aspect of the invention, the regulatory T-cells will be capable of migrating towards B-cells.
- chemokines which are functionally active according to the method of the present invention, or the regulatory T-cells may form therapeutically useful compositions.
- the present invention provides a composition comprising at least one chemokine which is secreted by antigen presenting cells (APCs) on their activation, and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APCs, or a chemokine binding molecule and a pharmaceutically acceptable carrier, diluent or exipient.
- APCs antigen presenting cells
- At least one chemokine is selected from the group consisting of: CCLl, CCL4, CCL2, CCL3, and XCLl.
- at least one chemokine is CCL4 or CCLl .
- at least one chemokine is CCL4. It will be appreciated that in any composition comprising many chemokines, they may act synergistically, independently of one another, or there may be functional redundancy in the mixture.
- the composition comprises a chemokine binding molecule.
- the present invention provides a composition comprising one or more regulatory T-cells as herein described and a pharmaceutically acceptable carrier, diluent or exipient.
- the present invention provides the use of at least one chemokine, regulatory T-cells, or a composition according to the present invention in the prophylaxis and/or treatment of disease.
- the term 'disease' includes amy one or more of the following: cancer, auto-immunity, transplant rejection, infections. It will be appreciated that the list is not exhaustive.
- FIG. 1 Chemokine expression profile of primary B cells, dendritic cells and macrophages before and after stimulation
- a-c Radioautographs of arrays probed with labelled cDNA obtained from splenic B cells of MD4 transgenic mice, after the following 24h treatments (a) control, mock incubation (b) 500ng/ml hen egg lysozyme (HEL) (c) 50 ⁇ g/ml LPS.
- HEL hen egg lysozyme
- FIG. 1 marks the position of DNA spots of chemokines relevant to the analysis: 1 : CCL22, 2: XCR1, 3: CCL2, 4: CCL3, 5: CCL4, 6:CCL5.
- Arrow set 2 marks the position of other genes relevant to the analysis: a: CCR6, b:CX 3 CR, c:CRD6, d:PAR3, e:Ig , f: Ig .
- (d) Summaries of DNA array analysis of primary mouse splenic B cells (Ig HEL transgenic/ MD4), blood B cells (wildtype, C57bl/6xBalb/c), bone marrow derived dendritic cells and peritoneal macrophages (wildtype, C57bl/6xBalb/c). In each case, cDNA preparations from control and activated cells are being compared. The stimulation used in the various cases is denoted on the left side for each particular case.
- Ig x-linked was by 24h treatment with lOO ⁇ g/ml anti-mouse IgM F(ab)s. Grey scale indicates degree of expression; from light grey for undetectable to black for saturated, (e) Concentration of CCL3 and CCL4 in the supernatants of control B cells (white bars) and B cells activated by Ig cross-linking (grey bars) at 24h and 48h.
- FIG. 1 Analysis of the migration of CD25 +ve cell from spleens of un-immunised mice
- (a-f) Representative examples of trans-well migration assays using T-cell column enriched primary splenic cells from Balb/c mice and 500ng/ml of the respective chemokine as chemo-attractant. Shown in red is a FACS histogram of the CD25 stained migrated cells. Each histogram contains the data from four pooled migration wells. Unstained input cells are shown for comparison in black, (a) XCLl, (b) CCL2, (c) CCL3, (d) CCL4, (e) CCL5 and (f) CCL22.
- a-c Histograms of the CD4 +ve cells migrated towards specific stimuli stained for CD25 in the following trans-well migration assays (FACS gated for CD4 +VC cells only): (a) input cells, (b) cells migrated towards the supernatant of activated B cells and (c) cells migrated towards CCL4.
- d Summary of three independent migration inhibition experiments using purified T cells (approx. 90% enriched for CD4 +ve cells by depletion) prepared from three independent mice.
- Frozen spleen sections were prepared from nu-/nu- mice (a) without treatment; (b) 14 days after reconstitution with 5xl0 6 splenic T cells; (c) 14 days after reconstitution with 5xl0 6 splenic T cells depleted of CD25 +ve cells; (d,e) 7 days after i.v. anti-CCL4 treatment on day 0, 1 and 2 (d) without prior reconstitution with T cells and (e) after injection of 10 6 purified splenic T cells from age and gender matched Balb/c mice. Further, frozen spleen sections were prepared from Balb/c mice 7 days after (f ) i.v. injection of anti-CCL4 antibody on day 0, 1 and 2; (g) i.p.
- FIG. 5 The effect of regulatory T cells on B cell proliferation and blasting.
- B cells were activated with 50 ⁇ g/ml LPS and incubated with specific T cell populations, which were activated by anti-CD3 cross-linking,
- Anti-CCL4 treated mice develop IgG switched auto-antibodies. Serial dilutions of the blood taken from 7days after the following treatments (three animals in each group): control (open grey squares); i.p. immunisation with OxCSA at day 0 (filled black squares); i.v. anti-CCL4 treatment on days 0, 1 and 2 (open red circles); i.p. immunisation with OxCSA on day 0 followed by i.v.
- Figure 7 shows a time course of the relative expression of chemokines upon B cell activation.
- B cells were activated by cross-linking of surface immunoglobulin with anti-mouse IgM F(ab)2 (15 ⁇ g/ml) to analyse the induction of chemokine expression.
- Supernatants were harvested at 6 hourly intervals and assayed for the presence of the chemokines CCLl, CCL3, and CCL22 by ELISA. (+) denotes activated supernatant ; (-) supernatant of non-activated controls.
- Chemokine A chemokine in the context of the present invention includes any cytokine which plays a role in an immune response and/or pro-inflammatory response. Examples include CCL3 (MIP1 alpha), CCL4 (mip 1 beta), MIP2, CCLl 9 (MIP3beta), CCL20 (MIP3 alpha), CXCL8 (IL8). Structurally, they are small disulphide-linked polypeptides of typically 60-70 amino acids (7-lOkDa). They act at a nanomolar concentration. Those skilled in the art should appreciate that the reference model used herein is the mouse. In humans and other mammals the functional homologues to the chemokines herein described may have different names.
- chemokines with different functions have been given the same name in different mammals. That is, non-functional homologues in different mammals may have same names.
- the term chemokine includes within its scope functional homologues (as described above) of the chemokines herein described.
- Antibody An antibody (for example IgG, IgM, IgA, IgD or IgE) or fragment (such as a FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody) whether derived from any species naturally producing an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria).
- antibody for example IgG, IgM, IgA, IgD or IgE
- fragment such as a FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody
- Antigen Presenting Cell As a first step in mounting an antibody response, foreign antigens are engulfed non-specifically by macrophages or other cells of the reticuloendothelial system. These cells include macrophages and dendritic cells. B cells can also act as antigen presenting cells, either by internalising antigen specifically via their antigen receptor or non-specifically via fluid phase uptake. These cells are collectively known as antigen presenting cells.
- chemokine in the context of the present invention, refers to the function a chemokine normally performs in its native mammalian environment. These roles include mediators of chemoattraction or repulsion, mediators of cell-cell interactions and effects directly on the target cell.
- T-cell recruitment In order to elicit an effective immune response in a mammal T- cells must come into contact with APCs.
- the attraction of T-cells by APCs is known as T-cell recruitment, and it ocurrs on activation of APCs. It is mediated via chemokines of the present invention such as CCL4, CCL3, CCLl and CCL2.
- APCs Activation of Antigen Presenting Cells Occurs rapidly in an immune response. Once activated, APCs express more MHC class I and II, more Fc receptors and more co- stimulatory adhesion molecules. They also produce more cytokines such as IL-1, IL-6, TNFalpha and other mediators. APC activation is essential for the antigen processing and presentation necessary to elicit an immune response. Activation can be brought about by several methods including treatment with LPS (lipopolysaccharide), or cross- linking the surface with antibodies or F(ab)s, for example cross-linking the surface immunoglobulin (Ig) of B cells with anti-Ig F(ab)s.
- LPS lipopolysaccharide
- Ig surface immunoglobulin
- Modulating an immune response includes either enhancing or diminishing a mammals reaction to foreign or to self-antigen within the mammalian body and/or ex vivo.
- the term means at least either enhancing or diminishing a mammals reaction to foreign or to self-antigen within the mammalian body.
- the immune system involves a vast variety of molecules, which are capable of interacting with one another, as well as molecules which themselves are not directly involved in immune responses per se. Thus, certain interactions may be enhanced and others diminished such that apparently a mammals reaction to foreign or self antigen remains unaltered. Changes in immune responses of this sort are also contemplated according to the present invention.
- modulating the immune response according to the present invention includes within its scope the substantial prevention or purposeful induction of autoimmunity.
- a systematic analysis of the changes in the chemokine and related receptor expression before and after activation by physiological stimuli is performed in order to identify those chemokines involved in the recruitment of regulatory T cells to APCs.
- LPS is used for activation.
- cross linking of surface Ig or antigen is used for the activation of B cells.
- the use of DNA array technology is employed for this purpose.
- the chemokines are detected in the supernatant of the cells by chemokine specific ELISA.
- the samples (for example, members of a library, or PCR products or oligonucleotides) are generally fixed or immobilised onto a solid phase, preferably a solid substrate, to limit diffusion and admixing of the samples.
- the samples may be immobilised to a substantially planar solid phase, including membranes and non- porous substrates such as plastic and glass.
- the samples are preferably arranged in such a way that indexing (i.e., reference or access to a particular sample) is facilitated.
- the samples are applied as spots in a grid formation.
- Common assay systems may be adapted for this purpose.
- an array may be immobilised on the surface of a microplate, either with multiple samples in a well or with a single sample in each well, or fibre optic bundles.
- the solid substrate may be a membrane, such as a nitrocellulose or nylon or polyvinylidene membrane (for example, membranes used in blotting experiments).
- Alternative substrates include gold, glass, plastic, or silica based substrates.
- the substrates may be coated with a reagent(s) for example poly-L-lysine or polyacrylamide.
- the samples are immobilised by any suitable method known in the art, for example, by charge interactions, or by chemical coupling to the walls or bottom of the wells, or the surface of the membrane.
- Other means of arranging and immobilising may be used, for example, pipetting, drop-touch, piezoelectric means, ink-jet and bubblejet technology or electrostatic application.
- photolithography may be utilised to arrange and fix the samples on the chip.
- Nomenclature has developed in the art, and is used herein, whereby the samples (for example, members of a library, or PCR products or oligonucleotides) immobilised on the array are termed probes and the sample (for example, libraries, or PCR products, or DNA, or cDNA, or RNA, or oligonucleotides) for hybridisation with the array is termed target.
- the probes may be arranged by being "spotted” onto the solid substrate; this may be done by hand or by making use of robotics to deposit the sample.
- Array formats may be described as, for example, macroarrays, microarrays, high-density oligonucleotide arrays and microelectronic arrays.
- targets and/or probes may be labelled with any readily detectable reporter, for example, a fluorescent, bioluminescent, phosphorescent, or radioactive reporter. Labelling of probes and targets is disclosed in Shalon et al., 1996, Genome Res 6:639-45. Arrays are based on the same steps. An array of the probe sample is constructed. The target sample is labelled. The labelled target sample is hybridised to the arrayed probe samples and the relative signals are measured.
- any readily detectable reporter for example, a fluorescent, bioluminescent, phosphorescent, or radioactive reporter.
- the raw data from a microarray experiment typically are images, which need to be transformed into gene expression matrices (tables where rows represent for example genes, columns represent for example various samples such as tissues or experimental conditions, and numbers in each cell for example characterise the expression level of the particular gene in the particular sample). These matrices have to be analysed further, if any knowledge about the underlying biological processes is to be extracted.
- Methods of data analysis including supervised and unsupervised data analysis as well as bioinformatic approaches) are disclosed in Brazma and Nilo J (2000) FEBS Lett 480:17-24.
- a custom made DNA array may be used.
- a custom made DNA array containing probes for 29 chemokine genes as well as two additional putative chemokine genes was generated using the methods described above. Also included were probes for 61 non-chemokine related genes implicated in regulating cell migration.
- probes for 61 non-chemokine related genes implicated in regulating cell migration.
- Suitable methods for the preparation of a uniform population of APCs are generally known to those skilled in the art and include FACs sorting, MACsorting and standard chromatographic techniques as well as the use of isolated cells from trangenic animals.
- the FACS allows cells (lymphocytes) to be separated and isolated through the use of tagged antibodies or anti-immunoglobulins.
- the sample mixture is funneled through a nozzle which isolates cells and charges them.
- At the base of the FACS there are 2 variably charged deflection plates and cell collectors.
- the individual cells in a resting lymphocyte pool must be isolated. This is accomplished by suspending the cells in a fluid (eg., saline). This suspension of cells is then forced through a fine, high-pressure nozzle or fluidic diluting system which distributes the cells into a single-file line or flow cell (Kidd and Nicholson 229).
- Light is an integral part of the FACS technique. Light striking each cell is scattered.
- FACSs have two types of data collecting hardware: light scatter sensors and photomultiplier tubes (PMTs).
- the light scatter sensors measure the light that is scattered by each cell from two different angles.
- the forward angle light scatter sensor (FALS) gathers light scattered in the forward direction. This type of scattered light gives a clue as to a cell's size.
- Right angle, orthogonal, or side light scatter (SS) sensors gather light scattered at 90° from the original direction of the light source. This light reveals cell granularity, refractiveness, and the presence of intracellular structures that reflect light (Darzynkiewicz, et al. 335).
- Scatter sensors are useful in distinguishing cells based on the cells' different structures.
- Neutrophiles for example, display more SS than lymphocytes (Kidd and Nicholson 229).
- different cell lineages or cells at different stages of development i.e., pre B cells versus plasma B cells
- PMTs detect fluorescent emissions from the fluorescent dyes on antibodies bound to cells or from auto-fluorescence of the cells.
- the cells are passed through an electric field generated by oppositely- charged plates. By changing the direction of the electric field between the plates, selected cells can be directed into precise collection areas.
- the use of APCs isolated from transgenic animals may be employed.
- Ig HEL (MD4) trangenic mice all express the same anti-hen egg lysozyme (HEL) immunoglobulin transgene and can be activated by HEL.
- HEL anti-hen egg lysozyme
- APCs Methods for the activation of APCs are known to those skilled in the art (Antibodies, Harlow and Lane, Cold Spring Harbour, 1998). Purified APCs can be activated by
- Ig (MD4) B cells can be activated by anti-hen egg lysozyme (HEL) immunoglobulin.
- HEL anti-hen egg lysozyme
- purified B cells from the blood of wild type mice can be activated by exposure to anti-mouse Ig F(Ab)s or LPS. It will be appreciated that the choice of ligand used to activate the purified APCs depends upon the APC sample used.
- transwell migration assays can be performed using methods known to one skilled in the art, and detailed in (Antibodies, Harlow and Lane, Cold Spring Harbour, 1998). Those skilled in the art will be aware of other suitable methods.
- a typical protocol is detailed below: Migration assays are carried out in 6.5mm diameter, 5.0 ⁇ m pore size polycarbonate membrane filter trans-well plates (Costar Corning). 0.6 ml RPMI medium containing the chemokine (500ng/ml) and/or antibody (2 ⁇ g/ml) or supernatant of in vitro activated B cells was placed in the lower chamber. 0.1 ml of T lymphocyte suspension (lxlO 7 cells/ml) was added to the upper filters. Following incubation at 37°C 10% CO 2 for 3 hours the cells migrated into the lower chamber were stained with anti-CD4 PE (Pharmingen) and anti-CD25 FITC (Pharmingen) and analysed by FACS and quantified using beads.
- chemokines which are secreted by APCs on their activation, and which are capable of acting as a chemo-attractant for the recruitment of regulatory T-cells are identified.
- the chemokines are any one or more selected from the group consisting of: CCLl, CCL4, CCL3, CCL2, and XCLl.
- the chemokine/s are CCL4 and/or CCLl.
- at least one chemokine which is secreted by APCs on their activation, and which is capable of acting as a chemo-attractant for the recruitment of regulatory T cells is CCL4.
- the chemokines of the present invention comprise a narrow range of those chemokines already known in the art. These chemokines have been found, surprisingly, to modulate immune responses in a mammal, and this effect is brought about by the action of these chemokines in recruiting regulatory T-cells to APCs.
- chemokines as described herein may be effective in regulatory T cell recruitment in isolation, or when acting in conjunction with one or more further chemokines. That is, a given subset of chemokines may show regulatory T-cell recruitment effects whereas each chemokine in isolation may show negligible effects. Furthermore, one skilled in the art will appreciate that two or more chemokines may act synergistically in their effects. Those skilled in the art will appreciate that within a mixture of chemokines, one or several chemokines may influence the recruitment effects as herein described of one or more further chemokines within that mixture, be it by synergistic, additive, subtractive or other modulating effects.
- modulating the immune response involves either enhancing or diminishing a mammals reaction to foreign or to self-antigen within the body.
- the immune system involves a vast variety of molecules, which are capable of interacting with one another as well as molecules which are not involved in immune responses. Thus, certain interactions may be enhanced and others diminshed such that apparently a mammals reaction to foreign or self antigen remains unaltered. Changes in immune responses of this sort are also crossmplated according to the present invention.
- modulating the immune response includes within its scope the substantial prevention or purposeful induction of autoimmunity.
- the present invention provides a method for modulating the immune response in a mammal comprising the step of increasing or decreasing, or otherwise altering the functional activity of at least one chemokine secreted by antigen presenting cells (APCs) on their activation, and which is capable of acting as a chemoattractant for the recruitment of regulatory T-cells to APC.
- APCs antigen presenting cells
- chemokine in the context of the present invention refers to the function the chemokine normally performs in its native mammalian environment. These may include roles such as chemoattractants, mediators of cell-cell interactions and so on.
- chemokine includes within its scope functional homologues (as described above) of the chemokines herein described.
- the term 'increasing or decreasing the functional activity of at least one chemokine' in the context of the present invention includes within its scope increasing or decreasing the expression and/or activity of at least one chemokine as described herein. Otherwise altering' the functional activity may result from adjusting the intracellular and/or extracellular distribution of the chemokine/s, or the nucleic acid encoding them.
- Increasing or decreasing the expression may occur as a result of increasing or decreasing mRNA expression respectively, or by increasing or decreasing gene transcription respectively using methods known to those skilled in the art.
- Increasing or decreasing the expression and/or functional activity of a chemokine may also be brought about via posttranslational modications using methods known to those skilled in the art.
- intracellular distribution or extracellular distribution may occur as a result of the addition of nucleic acid binding molecules, or chemokine binding molecules, to the inta or extracellular environment, using techniques known to one skilled in the art.
- Increasing or decreasing the activity of the chemokines can be brought about by bringing the chemokines into contact with inhibitors of the chemokines, or activators of chemokines and/or chemokine binding molecules.
- contact in the context of the present invention means does not require a physical contact.
- a functional contact, that is where the presence of the inhibitor or activator or chemokine binding protein affects the activity of the chemokine is sufficient.
- Suitable chemokine binding molecules include antibodies as herein defined and chemokine dominant negative mutants.
- Antibodies may be prepared using standard laboratory techniques. Either recombinant proteins or those derived from natural sources can be used to generate antibodies. For example, the protein (or "immunogen") is administered to challenge a mammal such as a monkey, goat, rabbit or mouse. The resulting antibodies can be collected as polyclonal sera, or antibody- producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner to produce a hybridoma), which cells then produce monoclonal antibodies.
- the antigen protein is either used alone or conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised in an animal, as described above.
- Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al. (1992) J. Biol. Chem., 267: 4815).
- the serum is titered against protein antigen by ELISA or alternatively by dot or spot blotting (Boersma and Van Leeuwen (1994) J. Neurosci. Methods, 51: 317).
- the serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al. (1982) Cell, 28: 477.
- monoclonal antibodies may be prepared using any candidate antigen, preferably bound to a carrier, as described by Arnheiter et al. (1981) Nature, 294, 278.
- Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced. Nevertheless, monoclonal antibodies may be described as being “raised against” or “induced by” a protein.
- monoclonal antibodies are tested for function and specificity by any of a number of means. Similar procedures can also be used to test recombinant antibodies produced by phage display or other in vitro selection technologies. Monoclonal antibody-producing hybridomas (or polyclonal sera) can be screened for antibody binding to the immunogen, as well. Particularly preferred immunological tests include enzyme-linked immunoassays (ELISA), immunoblotting and immunoprecipitation (see Voller, (1978) Diagnostic Horizons, 2: 1, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller et al. (1978) J. Clin. Pathol., 31: 507; U.S. Reissue Pat. No.
- ELISA enzyme-linked immunoassays
- Suitable activators or inhibitors of chemokines which may be of use in the method of the present invention include but are not limited to inhibitors of chemokine receptors.
- the small molecule AMD-3100 inhibits the CXCR4 receptor.
- One skilled in the art will be aware of other suitable inhibitors or activators.
- Increasing or decreasing the functional activity of the chemokines of the present invention leads to a modulation of the recruitment of regulatory T-cells to APCs, and therefore a concomitant modulation of the immune response.
- a further aspect of the present invention provides the use of one or more isolated regulatory T cell/s which are capable of migrating towards antigen presenting cell/s (APCs) by chemoattraction mediated by at least one chemokine secreted by APCs on their activation, in the preparation of a medicament for modulating an immune response in a vertebrate.
- APCs antigen presenting cell/s
- the vertebrate is a mammal.
- T cells can be isolated and purified using methods known to those skilled in the art. These include standard chromatographic methods including immunoprecipitation, and FACs sorting.
- a suitable source of T cells is spleens from un-immunised mice.
- One skilled in the art will be aware of other suitable sources of T cells.
- An example of a protocol for the isolation of T-cells is described below:
- T lymphocytes were prepared from Balb/c mice using Lympholyte M followed by a T cell column (Cedarlane Laboratories). CD4 +ve T lymphocytes enriched by negative selection using MACS reagents (Miltenyi biotech) and antibodies to CD 19, CD l ib, CD8a, CD80, CD86 and CD 16/32 (Pharmingen).
- the present invention provides a method for selecting one or more regulatory T cells capable of modulating the immune response in a vertebrate comprising the steps of:- (a) preparing a population of T cells, and
- step (c) Selecting those T cells which can migrate towards those APCs or chemokines defined in step (b).
- a population of T-cells is prepared as herein described.
- the selection step (c) also preferably involves a cell-migration assay, followed by the physical selection of those cells which have migrated towards the chemokines or APCs as described above.
- step (b), (that is exposing the cells to activated APCs) also includes exposing the cells to supematants from in vitro activated APCs.
- at least one chemokine used is selected from the group consisting of CCLl, CCL4, CCL3 and CCL2, and XCLl.
- Suitable APCs for use in the method of this aspect of the present invention include macrophages, dendritic cells and B cells, which can be isolated using standard laboratory techniques as herein described.
- the vertebrate is a mammal.
- Those regulatory T-cells selected according to the method of the present invention are preferably CD25+ positive. Even more preferably they are CD25+ and CD4+ positive. In an alternative embodiment of this aspect of the invention, they are CD25+ and CD8+.
- the present invention provides a composition comprising at least one chemokine, or chemokine binding protein, according to the present invention, and a pharmaceutically acceptable carrier, diluent or exipient.
- the present invention provides a composition comprising at least one or more regulatory T cells as herein described, and a pharmaceutically acceptable carrier, diluent or exipient.
- Chemokines, chemokine binding proteins, T-cells and compositions according to the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
- chemokines chemokine binding proteins
- T-cells T-cells and compositions prepared according to the invention involve the administration of the above to a recipient mammal, such as a human.
- chemokines, binding proteins thereof of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human.
- the chemokines, binding proteins and T-cells may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures using methods known to those skilled in the art.
- the selected chemokines, or binding proteins thereof, or T-cells of the present invention will typically find use in preventing, suppressing or treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune disorders (which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis), and in preventing transplant rejection.
- autoimmune disorders which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis
- depletion of the regulatory T cells or interference with their recruitment may result in an enhanced immune response which may be of particular use in the treatment of infections which otherwise escape a normal immune response.
- the additional administration of regulatory T cells of the present invention may result in a dampened immune response or even complete suppression of an immune response.
- chemokines of the present invention may lead to the generation of an autoimmune response, which may be of use in the destruction of certain cells types. This may be of particular use in the treatment of cancer.
- chemokines or regulatory T cells as herein described may be useful for modulating a immune response in regions of a vertebrate where they are not normally located.
- one or more chemokines used as herein described may be perfused, injected, or the nucleic acid encoding them expressed within a tissue of a vertebrate, using techniques known to those skilled in the art.
- the presence of a chemokine and/or a regulatory T-cell as described herein, in such an ectopic environment may be useful in the modulation of an immune response during for example, transplant rejection and the like.
- prevention involves administration of the protective composition prior to the induction of the disease.
- suppression refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.
- Treatment involves administration of the protective composition after disease symptoms become manifest.
- EAE in mouse and rat serves as a model for MS in human.
- the demyelinating disease is induced by administration of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et al., eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al. (1987) J. Immunol., 138: 179).
- the selected chemokines, or binding proteins thereof, or T-cells of the present invention will be utilised in purified form together with pharmacologically appropriate carriers.
- these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.
- Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
- Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).
- the selected chemokines, or binding proteins thereof, or T-cells of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the chemokines, or binding proteins thereof, or T-cells of the present invention or even combinations of selected chemokines, or binding proteins thereof, according to the present invention.
- immunotherapeutic drugs such as cylcosporine, methotrexate, adriamycin or cisplatinum
- Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in conjunction with the chemokines, or binding proteins thereof, or T-cells of the present invention or even combinations of selected chemokines, or binding proteins thereof, according to the present invention.
- the route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art.
- the selected antibodies, receptors or binding proteins thereof of the invention can be administered to any patient in accordance with standard techniques.
- the administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter.
- the dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.
- the selected chemokines, or binding proteins, of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use.
- Known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of functional activity loss and that use levels may have to be adjusted upward to compensate.
- compositions containing the present selected chemokines, or binding proteins thereof, or T-cells of the present invention or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
- an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a "therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.00005 to 5.0 mg of selected chemokine or chemokine binding-protein per kilogram of body weight, with doses of 0.0005 to 2.0 mg/kg/dose being more commonly used.
- compositions containing the present selected polypeptides or cocktails thereof may also be administered in similar or slightly lower dosages.
- a composition containing one or more selected chemokines, or binding proteins thereof, or T-cells according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal.
- the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.
- Blood from a mammal may be combined extracorporeally with the selected antibodies, cell-surface receptors or binding proteins thereof whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
- Regulatory T cells isolated from a patient may be enriched by migration.
- these cells may be treated such that their responsiveness to one or more chemokines is modulated.
- Such cells may subsequently introduced into the same or a different patient.
- adoptive transfer of ex vivo enriched regulatory T cells from the patient him/herself or a donor is also contemplated herein. The invention is further described, for the purposes of illustration only, in the following examples which are in no way limiting of the invention.
- chemokines changes. Whilst some of these changes can be deduced from studies performed with the affimetrix oligonucleotide expression array (R Glynne et al, Nature 403, 672-6 (2000)), a systematic analysis of changes in the chemokine and related receptor expression during B cell differentiation, after B cell activation and during cell-cell interaction had not until now been performed. For this purpose the present inventors developed a custom made DNA array containing probes for 29 chemokine genes as well as two additional putative chemokine genes. Also included were probes for 61 non-chemokine related genes implicated in regulating cell migration.
- splenic B cells from Ig HEL transgenic mice were stimulated for 24-48h with HEL.
- splenic B cells from wild-type Balb/c mice were stimulated with either anti mouse Ig F(ab)s or LPS.
- purified B cells from the blood of C57/B16 mice were stimulated for 24-48h with either lOO ⁇ g/ml anti mouse Ig F(ab)s or LPS.
- B lymphocytes were prepared from the spleen of Ig HEL transgenic (MD4) mice or blood of C57B16xBalb/c mice using Lympholyte M or Mammal, followed by a B cell column (Cedarlane Laboratories).
- the cells were activated for 24 hours using either 500ng/ml of hen egg lysozyme (Sigma) or 50 ⁇ g of anti-mouse IgM F(ab) 2 (Jackson labs). Macrophages were harvested by peritoneal lavage and further enriched by adhesion to tissue culture plates. Bone-marrow-derived dendritic cells obtained as described [K. Inaba, et al. J. Exp. Med. 76, 1693 (1992)]. Both APC types were activated with 250ng/ml LPS (Sigma). The cDNA preparations obtained from these samples were hybridised to the DNA arrays.
- RNA from 10 cells was reverse transcribed using ( ⁇ 32 P) dATP (Amersham) and a mix of gene specific primers. After removal of unincorporated nucleotides by G50 sephadex column centrifugation, the probe was denatured and then added directly to the 'Expresshyb' solution (ClontechyiOO ⁇ g herring sperm DNA, in which the blot was pre-hybridised for lhr.
- CCL5 is the only chemokine detected in un-stimulated splenic B cells (Fig. la, d), while un-stimulated blood B cells also express CCLl 8 and CXCL4, suggesting a possible functional difference (Fig. Id).
- Stimulation with antigen Fig. lb, d
- cross- linking of surface Ig Fig. Id
- LPS Fig.lc, d
- Antigen activation and Ig cross-linking but not LPS activation leads to a marked induction of CCL3 and CCL4 in both cell populations (Fig.lc, d).
- CCL2 In splenic B cells CCL2, CCL22 and XCLl are also up-regulated. All the other 26 chemokines remain unaffected by both specific and unspecific stimulation.
- ELISA assays for CCL3 and CCL4 using supernatant derived from either non-activated or surface Ig cross- linked B cells. High levels of both chemokines can be detected upon B cell activation (Fig.le).
- chemokines likely to play a recruitment role early in an immune response we then enquired whether other antigen presenting cell types show a similar activation dependent chemokine/chemokine receptor profile.
- CCL2, CCL3 and CCL4 three of the five chemokines up-regulated in activated B cells (CCL2, CCL3 and CCL4) are also induced upon activation of dendritic cells and macrophages (Fig. Id).
- CCL22 which is only slightly induced upon activation in B cells and could not be detected in macrophages, appears to be constitutively present in dendritic cells with a further increase in expression upon activation.
- both macrophages and dendritic cells appear to constitutively express CCL9/10 and the putative chemokine nap212.
- CCLl 7 can only be detected in dendritic cells whilst CCL20 can only be detected in macrophages.
- Migration assays were carried out in 6.5mm diameter, 5.0 ⁇ m pore size polycarbonate membrane filter trans-well plates (Costar Corning). 0.6 ml RPMI medium containing the chemokine (500ng/ml) and/or antibody (2 ⁇ g/ml) or supernatant of in vitro activated B cells was placed in the lower chamber. 0.1 ml of T lymphocyte suspension (1x10 cells/ml) was added to the upper filters.
- MCP-1 has been demonstrated to play a major role in the polarisation of the immune response (L Gu et al, Nature 404, 407-8 (2000)).
- XCLl was found to be an inhibitor of T-cell proliferation (C Cerdan et al, Blood 96 (420-8) (2000)).
- the present inventors were interested in the signals that promote cell-cell interaction at the initiation of an immune response. Therefore a more detailed analysis of cell migration induced by chemokines that show activation dependent expression in B cells and professional APC (Fig.2b-g) was performed. Using T cells purified from spleens of un-immunised mice it was observed that CCL4 induced the migration of a substantial population of CD25 +ve cells (Fig.2e). This was somewhat surprising since the number of CD25 +ve activated T cells in an un-immunised mouse spleen would be expected to be low. Furthermore, it was found that the other chemokines whose expression is activation dependent in B cells also can recruit CD25 +ve cells albeit with markedly reduced efficiency (Fig2b-g). CCL22 and XCLl are comparably poor at attracting T cells in general (Fig.2a,b,g).
- CD4 +ve CD25 +ve could not be stimulated by CD3 cross- linking confirming that they are indeed anergic.
- CD4 +ve CD25 +ve cells migrating to CCL4 are anergic. This coupled with the fact that depletion of CD25 +ve cells from the input population led to a loss of CD4 +ve migrated cells makes it unlikely that CCL4 non-selectively attracts T cells and subsequently activates them, leading to expression of CD25 and CTLA4.
- regulatory T cells can influence a normal B cell immune response by reconstituting nu-/nu- mice with either CD25 depleted T cells or all T cells.
- PALS peri-arteriolar lymphoid sheets
- Germinal centres can be detected by day 6-7 when they are characterised by displacement of small, non-dividing IgD +ve , PNA l0 follicular B cells by the rapidly proliferating IgD "ve , PNA hl blasts.
- the T cell compartment of nu-/nu- mice was reconstituted with either 'total' T cells or with T cells depleted of their CD25 expressing sub-populations.
- the transferred cells were obtained from age, gender and background matched Balb/c mice.
- T lymphocytes from the spleen of Balb/c mice were prepared using Lympholyte M followed by a T cell column (Cedarlane Laboratories).
- CD25+ cells were depleted by FACsorting.
- nu- /nu- mice were injected i.v with 5xl0 6 -10 7 'total' T cells or CD25 depleted T cells. For the temporal depletion of CCL4, mice were injected i.v.
- mice When mice are reconstituted with CD25 depleted T cells found an abundance of germinal centres all of which contained a substantial number of IgGl switched cells were found (Fig.4c). In addition, a dramatic increase in IgGl switched cells was found in extra follicular areas (Fig. 4c). In contrast to this, few germinal centres were seen in mice reconstituted with total T cells and these never contained any IgG switched cells (Fig.4b). However, it is clear that reconstitution with total T cells alone can cause inconsistent but usually low level switching of B cells to IgGl in the spleen (Fig. 4b). This effect is transient with a return to normal levels after 3 weeks.
- Example 7 Since our studies implicated CCL4 as the most effective chemo-attractant for regulatory T cells, which can clearly effect the humoral immune response, the role of CCL4 in vivo was studied.
- the chemokine was temporarily depleted by injection of an anti-serum, which neutralises the biological activity of CCL4 in migration assays using primary T cells (Fig.3f). It was not possible to detect any change in the spleen of any of the analysed nu-/nu- mice after injection of anti-CCL4 serum (Fig. 4d).
- mice were reconstituted with 'total' T cells prior to anti- CCL4 serum injection, a dramatic effect on the kinetics of the humoral response and the architecture of the spleen was observed (Fig. 4e).
- the same effect was observed in Balb/c mice treated with the neutralising anti-serum.
- Fig. 4f By day 7 nearly all follicles developed large germinal centres (Fig. 4f). This is in marked contrast to a normal immunisation (Fig. 4g), in which a minority of follicles contain nascent germinal centres.
- the presence of isotype switched cells in the germinal centres reflects their mature nature (Fig. 4f,h).
- Combinations of cells were incubated for 60-84h in RPMI (10% FCS): 5xl0 5 B cells, 4xl0 5 FACsorted CD25 + or CD25 " CD4+ cells, 2xl0 5 splenic 'input' T cells and cells migrated to CCL4.
- Wells were pre-coated with 2 ⁇ g/ml anti-CD3e (Pharmingen) and LPS was used at 25 ⁇ g/ml. Cells were counted by FACS using beads for normalisation. Two independent sources of regulatory T cells, FACS sorted small CD4 +ve CD25 +ve cells and T cells that had actually migrated to CCL4 in vitro.
- Activation of B cells leads to the production of a chemokine that recruits regulatory T cells, which can directly inhibit B cell activation. Interuption of this pathway by either the removal of the regulatory cells themselves or the chemokine that recruits them leads to the generation of auto-antibodies. Plates were coated with either 50 ⁇ l of 5 ⁇ g/ml linear dsDNA in TE pH7.4 following pre-treatment with 5 ⁇ g/ml Poly-L-Lysine in water, or lOO ⁇ g/ml cardiolipin in absolute ethanol, or 250ng/ml myeloperoxidase in bicarbonate buffer pH9.3.
- IgGl level measurement plates were incubated overnight at 4°C with 8 ⁇ g/ml of IgGl capture antibody (Pharmingen) in sterile 0.1M NaHCO 3 . Plates were blocked with 10% FCS in PBS. 3 fold sera dilution steps were incubated, washed and bound antibody was detected using 2 ⁇ g/ml biotinylated anti-mouse IgG, followed by 0.5 ⁇ g/ml Streptavidin-HRP or Avidin-AP (myeloperoxidase plates). Colorimetric detection was by ABTS or pNPP.
- Example 9 The inventors have shown that upon activation B cells and professional APC up-regulate CCLl and/or CCL2 and/or CCL3 and/or CCL4.
- CCL4 is the most efficient chemo-attractant for regulatory T cells in vitro. It is possible that in vivo synergistic or additive effects amongst these chemokines may come into play. However, the profound effect of CCL4 depletion suggests that this chemokine plays a central role in vivo. Regulatory T cells have been implicated in modulating T cell function and results show regulatory T cell recruitment occurs at the level of APC activation. Furthermore, these cells are not only recruited efficiently by B cells, but directly affect the humoral response.
- regulatory T cells can clearly influence T helper cells our findings suggest an additional more direct effect on B cells. Indeed, our data indicates that regulatory T cells can affect B cell function in vitro in the absence of helper T cells (Fig. 5).
- the functional interaction between regulatory T cells and APC is further supported by a number of gene deletion models involving genes implicated in this network. Whilst having diverse phenotypes resulting from pleiotropic effects, all lead to exaggerated lympho-proliferation and in many cases to auto-immunity. These include CD25, CTLA-4 and TGF ⁇ all of which are markers for regulatory T cells.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Hematology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0121579.7A GB0121579D0 (en) | 2001-09-06 | 2001-09-06 | Chemokines |
GB0121579.7 | 2001-09-06 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2003022885A1 true WO2003022885A1 (fr) | 2003-03-20 |
Family
ID=9921627
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2002/004083 WO2003022885A1 (fr) | 2001-09-06 | 2002-09-06 | Chimiokines |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB0121579D0 (fr) |
WO (1) | WO2003022885A1 (fr) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004050706A2 (fr) * | 2002-12-03 | 2004-06-17 | Medical Research Council | Lymphocytes t regulateurs |
WO2006083289A2 (fr) * | 2004-06-04 | 2006-08-10 | Duke University | Methodes et compositions ameliorant l'immunite par depletion in vivo de l'activite cellulaire immunosuppressive |
WO2007085464A1 (fr) * | 2006-01-26 | 2007-08-02 | Universität Duisburg-Essen | Cellules cd88 t régulatrices et cytotoxiques |
WO2018049120A1 (fr) * | 2016-09-09 | 2018-03-15 | The General Hospital Corporation | Cellules présentatrices d'antigène ex vivo ou cellules t cd positives activées pour le traitement du cancer |
EP3447066A1 (fr) * | 2017-08-21 | 2019-02-27 | Herlev Hospital | Nouveaux composés ccl2 pour utilisation dans le traitement anti-cancéreux |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113893331A (zh) * | 2020-07-06 | 2022-01-07 | 中国农业科学院特产研究所 | 一种诱导母鹿生茸的产品及其制备方法和诱导母鹿生茸的方法和应用 |
-
2001
- 2001-09-06 GB GBGB0121579.7A patent/GB0121579D0/en not_active Ceased
-
2002
- 2002-09-06 WO PCT/GB2002/004083 patent/WO2003022885A1/fr not_active Application Discontinuation
Non-Patent Citations (7)
Title |
---|
ANDRIAN VON U H ET AL: "T-CELL FUNCTION AND MIGRATION TWO SIDES OF THE SAME COIN", NEW ENGLAND JOURNAL OF MEDICINE, THE, MASSACHUSETTS MEDICAL SOCIETY, WALTHAM, MA, US, vol. 343, no. 14, 5 October 2000 (2000-10-05), pages 1020 - 1034, XP001029001, ISSN: 0028-4793 * |
BYSTRY R S ET AL: "B cells and professional APCs recruit regulatory T cells via CCL4.", NATURE IMMUNOLOGY. UNITED STATES DEC 2001, vol. 2, no. 12, December 2001 (2001-12-01), pages 1126 - 1132, XP001120877, ISSN: 1529-2908 * |
IELLEM ANDREA ET AL: "Unique chemotactic response profile and specific expression of chemokine receptors CCR4 and CCR8 by CD4+CD25+ regulatory T cells.", JOURNAL OF EXPERIMENTAL MEDICINE, vol. 194, no. 6, 17 September 2001 (2001-09-17), pages 847 - 853, XP002223042, ISSN: 0022-1007 * |
JONULEIT H ET AL: "Dendritic cells as a tool to induce anergic and regulatory T cells", TRENDS IN IMMUNOLOGY, ELSEVIER, CAMBRIDGE, GB, vol. 22, no. 7, 1 July 2001 (2001-07-01), pages 394 - 400, XP004247295, ISSN: 1471-4906 * |
READ S ET AL: "CD4regulatory T cells", CURRENT OPINION IN IMMUNOLOGY, CURRENT BIOLOGY LTD,, vol. 13, no. 6, 1 December 2001 (2001-12-01), pages 644 - 649, XP004311242, ISSN: 0952-7915 * |
READ S ET AL: "Cytotoxic T lymphocyte-associated antigen 4 plays an essential role in the function of CD25(+)CD4(+) regulatory cells that control intestinal inflammation.", THE JOURNAL OF EXPERIMENTAL MEDICINE. UNITED STATES 17 JUL 2000, vol. 192, no. 2, 17 July 2000 (2000-07-17), pages 295 - 302, XP002223043, ISSN: 0022-1007 * |
TAKAHASHI T ET AL: "Immunologic self-tolerance maintained by CD4+CD25+ regulatory T cells constitutively expressing cytotoxic T lymphocyte-associated antigen 4", JOURNAL OF EXPERIMENTAL MEDICINE, TOKYO, JP, vol. 192, no. 2, 2000, pages 303 - 309, XP002174070, ISSN: 0022-1007 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004050706A2 (fr) * | 2002-12-03 | 2004-06-17 | Medical Research Council | Lymphocytes t regulateurs |
WO2004050706A3 (fr) * | 2002-12-03 | 2004-09-16 | Medical Res Council | Lymphocytes t regulateurs |
WO2006083289A2 (fr) * | 2004-06-04 | 2006-08-10 | Duke University | Methodes et compositions ameliorant l'immunite par depletion in vivo de l'activite cellulaire immunosuppressive |
WO2006083289A3 (fr) * | 2004-06-04 | 2006-12-21 | Univ Duke | Methodes et compositions ameliorant l'immunite par depletion in vivo de l'activite cellulaire immunosuppressive |
WO2007085464A1 (fr) * | 2006-01-26 | 2007-08-02 | Universität Duisburg-Essen | Cellules cd88 t régulatrices et cytotoxiques |
WO2018049120A1 (fr) * | 2016-09-09 | 2018-03-15 | The General Hospital Corporation | Cellules présentatrices d'antigène ex vivo ou cellules t cd positives activées pour le traitement du cancer |
US11364264B2 (en) | 2016-09-09 | 2022-06-21 | The General Hospital Corporation | Ex vivo antigen-presenting cells or activated CD-positive T cells for treatment of cancer |
EP3447066A1 (fr) * | 2017-08-21 | 2019-02-27 | Herlev Hospital | Nouveaux composés ccl2 pour utilisation dans le traitement anti-cancéreux |
WO2019038235A1 (fr) * | 2017-08-21 | 2019-02-28 | Herlev Hospital | Nouveaux peptides ccl2 destinés à être utilisés en thérapie anticancéreuse |
Also Published As
Publication number | Publication date |
---|---|
GB0121579D0 (en) | 2001-10-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8889124B2 (en) | Tolerogenic populations of dendritic cells | |
AU2003287257B2 (en) | Inhibition of IL-17 production | |
US20100203053A1 (en) | Method and composition for treating immune complex associated disorders | |
JP2006514004A5 (fr) | ||
WO2004050706A2 (fr) | Lymphocytes t regulateurs | |
AU2002324937A1 (en) | Method and composition for treating immune complex associated disorders | |
AU2002315115A2 (en) | Methods of treating and preventing colitis involving IL-13 and NK-T cells | |
EP1552462A1 (fr) | Procedes de traitement et de prevention d'une colite dans laquelle sont impliquees l'interleukine 13 et les cellules nk-t | |
WO2003022885A1 (fr) | Chimiokines | |
US20050287142A1 (en) | Remedies or preventives for rheumatoid arthritis | |
WO1999025734A2 (fr) | APPAUVRISSEMENT DE LA CELLULE Th2, COMPOSITIONS ET METHODES EN RAPPORT | |
EP1274450A2 (fr) | Utilisations de cytokine; compositions; et methodes | |
Song et al. | Specialized CC-chemokine secretion by Th1 cells in destructive autoimmune myocarditis | |
WO2007035551A1 (fr) | Methodes d'identification des agonistes et des antagonistes gpr83 capables de moduler la fonction regulatrice des lymphocytes t | |
JP2007505141A (ja) | Cd99アンタゴニストを用いる慢性関節リウマチの処置 | |
WO2007095345A2 (fr) | Procédés et compositions permettant de réguler des maladies neurologiques | |
Smith | Characterisation of T cell surface phenotype and effector function in a surrogate model of rheumatoid arthritis | |
Peters | The role of Th1 and Th2 cytokines in the regulation of collagen induced arthritis | |
ZA200401576B (en) | Method and composition for treating immune complex associated disorders. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BY BZ CA CH CN CO CR CU CZ DE DM DZ EC EE ES FI GB GD GE GH HR HU ID IL IN IS JP KE KG KP KR LC LK LR LS LT LU LV MA MD MG MN MW MX MZ NO NZ OM PH PL PT RU SD SE SG SI SK SL TJ TM TN TR TZ UA UG US UZ VC VN YU ZA ZM |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW MZ SD SL SZ UG ZM ZW AM AZ BY KG KZ RU TJ TM AT BE BG CH CY CZ DK EE ES FI FR GB GR IE IT LU MC PT SE SK TR BF BJ CF CG CI GA GN GQ GW ML MR NE SN TD TG US |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
122 | Ep: pct application non-entry in european phase | ||
NENP | Non-entry into the national phase |
Ref country code: JP |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: JP |