US20160228570A1 - Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies - Google Patents
Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies Download PDFInfo
- Publication number
- US20160228570A1 US20160228570A1 US14/987,308 US201614987308A US2016228570A1 US 20160228570 A1 US20160228570 A1 US 20160228570A1 US 201614987308 A US201614987308 A US 201614987308A US 2016228570 A1 US2016228570 A1 US 2016228570A1
- Authority
- US
- United States
- Prior art keywords
- seq
- composition
- cii
- antibody
- fragment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 208000036487 Arthropathies Diseases 0.000 title claims abstract description 11
- 208000012659 Joint disease Diseases 0.000 title claims abstract description 11
- 238000003745 diagnosis Methods 0.000 title claims abstract description 7
- 238000002560 therapeutic procedure Methods 0.000 title description 8
- 239000000203 mixture Substances 0.000 claims abstract description 51
- 239000012634 fragment Substances 0.000 claims abstract description 34
- 229920001436 collagen Polymers 0.000 claims abstract description 18
- 102000008186 Collagen Human genes 0.000 claims abstract description 16
- 108010035532 Collagen Proteins 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 9
- 238000011282 treatment Methods 0.000 claims abstract description 9
- 238000003776 cleavage reaction Methods 0.000 claims description 20
- 102000037865 fusion proteins Human genes 0.000 claims description 20
- 108020001507 fusion proteins Proteins 0.000 claims description 20
- 230000007017 scission Effects 0.000 claims description 20
- 102100026720 Interferon beta Human genes 0.000 claims description 14
- 108090000467 Interferon-beta Proteins 0.000 claims description 14
- 102000004127 Cytokines Human genes 0.000 claims description 10
- 108090000695 Cytokines Proteins 0.000 claims description 10
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 claims description 8
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 claims description 8
- 230000006337 proteolytic cleavage Effects 0.000 claims description 7
- 102000014150 Interferons Human genes 0.000 claims description 6
- 108010050904 Interferons Proteins 0.000 claims description 6
- 102000002274 Matrix Metalloproteinases Human genes 0.000 claims description 6
- 108010000684 Matrix Metalloproteinases Proteins 0.000 claims description 6
- 102100030416 Stromelysin-1 Human genes 0.000 claims description 6
- 229940079322 interferon Drugs 0.000 claims description 6
- 102100026802 72 kDa type IV collagenase Human genes 0.000 claims description 5
- 101000627872 Homo sapiens 72 kDa type IV collagenase Proteins 0.000 claims description 5
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 claims description 5
- 101000990912 Homo sapiens Matrilysin Proteins 0.000 claims description 5
- 101000990902 Homo sapiens Matrix metalloproteinase-9 Proteins 0.000 claims description 5
- 101000990908 Homo sapiens Neutrophil collagenase Proteins 0.000 claims description 5
- 101000990915 Homo sapiens Stromelysin-1 Proteins 0.000 claims description 5
- 101000577874 Homo sapiens Stromelysin-2 Proteins 0.000 claims description 5
- 102100030417 Matrilysin Human genes 0.000 claims description 5
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 claims description 5
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 claims description 5
- 102100030411 Neutrophil collagenase Human genes 0.000 claims description 5
- 102000035195 Peptidases Human genes 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 5
- 239000004365 Protease Substances 0.000 claims description 5
- 102100028848 Stromelysin-2 Human genes 0.000 claims description 5
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 claims description 5
- 229920002674 hyaluronan Polymers 0.000 claims description 5
- 239000003053 toxin Substances 0.000 claims description 5
- 102000003298 tumor necrosis factor receptor Human genes 0.000 claims description 5
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims description 4
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 claims description 4
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 claims description 4
- 102000014962 Monocyte Chemoattractant Proteins Human genes 0.000 claims description 4
- 108010064136 Monocyte Chemoattractant Proteins Proteins 0.000 claims description 4
- 229940112869 bone morphogenetic protein Drugs 0.000 claims description 4
- 231100000765 toxin Toxicity 0.000 claims description 4
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 claims description 3
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 3
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 2
- 102000019034 Chemokines Human genes 0.000 claims description 2
- 108010012236 Chemokines Proteins 0.000 claims description 2
- 229920002567 Chondroitin Polymers 0.000 claims description 2
- 102000004190 Enzymes Human genes 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 claims description 2
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 2
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 2
- 102000012479 Serine Proteases Human genes 0.000 claims description 2
- 108010022999 Serine Proteases Proteins 0.000 claims description 2
- 229920002385 Sodium hyaluronate Polymers 0.000 claims description 2
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 claims description 2
- 229960001338 colchicine Drugs 0.000 claims description 2
- 108010057085 cytokine receptors Proteins 0.000 claims description 2
- 102000003675 cytokine receptors Human genes 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 230000007760 free radical scavenging Effects 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 229960003160 hyaluronic acid Drugs 0.000 claims description 2
- 229960004171 hydroxychloroquine Drugs 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- 230000003071 parasitic effect Effects 0.000 claims description 2
- 244000052769 pathogen Species 0.000 claims description 2
- 229940010747 sodium hyaluronate Drugs 0.000 claims description 2
- 150000003431 steroids Chemical class 0.000 claims description 2
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 claims 2
- 229960001388 interferon-beta Drugs 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 11
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 1450
- 206010039073 rheumatoid arthritis Diseases 0.000 description 43
- 210000000845 cartilage Anatomy 0.000 description 36
- 108090000765 processed proteins & peptides Proteins 0.000 description 34
- 238000010186 staining Methods 0.000 description 22
- 230000027455 binding Effects 0.000 description 19
- 210000004027 cell Anatomy 0.000 description 17
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 14
- 239000002953 phosphate buffered saline Substances 0.000 description 14
- 210000002683 foot Anatomy 0.000 description 13
- 102000004196 processed proteins & peptides Human genes 0.000 description 13
- 241000699670 Mus sp. Species 0.000 description 12
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 12
- 206010003246 arthritis Diseases 0.000 description 11
- 238000010367 cloning Methods 0.000 description 11
- 210000001503 joint Anatomy 0.000 description 11
- 229920001184 polypeptide Polymers 0.000 description 11
- 201000008482 osteoarthritis Diseases 0.000 description 10
- 238000001262 western blot Methods 0.000 description 10
- 150000001413 amino acids Chemical group 0.000 description 9
- 230000002441 reversible effect Effects 0.000 description 9
- 239000000427 antigen Substances 0.000 description 8
- 230000006378 damage Effects 0.000 description 8
- 230000002757 inflammatory effect Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 8
- 238000002965 ELISA Methods 0.000 description 7
- 206010061218 Inflammation Diseases 0.000 description 7
- 235000001014 amino acid Nutrition 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 230000004054 inflammatory process Effects 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 230000008685 targeting Effects 0.000 description 7
- 239000013598 vector Substances 0.000 description 7
- 230000004481 post-translational protein modification Effects 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 5
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 210000002744 extracellular matrix Anatomy 0.000 description 5
- 230000001965 increasing effect Effects 0.000 description 5
- 230000002285 radioactive effect Effects 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 230000009870 specific binding Effects 0.000 description 5
- -1 superoxide radicals Chemical class 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 4
- 230000000903 blocking effect Effects 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 230000021615 conjugation Effects 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 210000004969 inflammatory cell Anatomy 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 208000014674 injury Diseases 0.000 description 4
- 210000003127 knee Anatomy 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000007800 oxidant agent Substances 0.000 description 4
- 239000001301 oxygen Substances 0.000 description 4
- 229910052760 oxygen Inorganic materials 0.000 description 4
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 4
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 208000009386 Experimental Arthritis Diseases 0.000 description 3
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 101001054328 Mus musculus Interferon beta Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 239000002671 adjuvant Substances 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 230000004941 influx Effects 0.000 description 3
- 229940100601 interleukin-6 Drugs 0.000 description 3
- 230000004807 localization Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 150000007523 nucleic acids Chemical group 0.000 description 3
- 230000001590 oxidative effect Effects 0.000 description 3
- CMFNMSMUKZHDEY-UHFFFAOYSA-N peroxynitrous acid Chemical compound OON=O CMFNMSMUKZHDEY-UHFFFAOYSA-N 0.000 description 3
- 238000002823 phage display Methods 0.000 description 3
- 239000008194 pharmaceutical composition Substances 0.000 description 3
- 150000003254 radicals Chemical class 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000008409 synovial inflammation Effects 0.000 description 3
- 201000004595 synovitis Diseases 0.000 description 3
- 241000701447 unidentified baculovirus Species 0.000 description 3
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 2
- GIANIJCPTPUNBA-QMMMGPOBSA-N (2s)-3-(4-hydroxyphenyl)-2-nitramidopropanoic acid Chemical compound [O-][N+](=O)N[C@H](C(=O)O)CC1=CC=C(O)C=C1 GIANIJCPTPUNBA-QMMMGPOBSA-N 0.000 description 2
- HMLGSIZOMSVISS-ONJSNURVSA-N (7r)-7-[[(2z)-2-(2-amino-1,3-thiazol-4-yl)-2-(2,2-dimethylpropanoyloxymethoxyimino)acetyl]amino]-3-ethenyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid Chemical compound N([C@@H]1C(N2C(=C(C=C)CSC21)C(O)=O)=O)C(=O)\C(=N/OCOC(=O)C(C)(C)C)C1=CSC(N)=N1 HMLGSIZOMSVISS-ONJSNURVSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 2
- 238000011735 C3H mouse Methods 0.000 description 2
- 102000000503 Collagen Type II Human genes 0.000 description 2
- 108010041390 Collagen Type II Proteins 0.000 description 2
- 108010071942 Colony-Stimulating Factors Proteins 0.000 description 2
- 241000134884 Ericales Species 0.000 description 2
- 108010008165 Etanercept Proteins 0.000 description 2
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000913074 Homo sapiens High affinity immunoglobulin gamma Fc receptor I Proteins 0.000 description 2
- 101000926206 Homo sapiens Putative glutathione hydrolase 3 proenzyme Proteins 0.000 description 2
- 102000051628 Interleukin-1 receptor antagonist Human genes 0.000 description 2
- 108700021006 Interleukin-1 receptor antagonist Proteins 0.000 description 2
- 102100026018 Interleukin-1 receptor antagonist protein Human genes 0.000 description 2
- 101710144554 Interleukin-1 receptor antagonist protein Proteins 0.000 description 2
- 102000003810 Interleukin-18 Human genes 0.000 description 2
- 108090000171 Interleukin-18 Proteins 0.000 description 2
- 239000000232 Lipid Bilayer Substances 0.000 description 2
- 102000009571 Macrophage Inflammatory Proteins Human genes 0.000 description 2
- 108010009474 Macrophage Inflammatory Proteins Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 208000007534 Patellar Dislocation Diseases 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 102100034060 Putative glutathione hydrolase 3 proenzyme Human genes 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108010039491 Ricin Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 108700012920 TNF Proteins 0.000 description 2
- 102000018594 Tumour necrosis factor Human genes 0.000 description 2
- 108050007852 Tumour necrosis factor Proteins 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 229960002964 adalimumab Drugs 0.000 description 2
- 229960004238 anakinra Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- RZEKVGVHFLEQIL-UHFFFAOYSA-N celecoxib Chemical compound C1=CC(C)=CC=C1C1=CC(C(F)(F)F)=NN1C1=CC=C(S(N)(=O)=O)C=C1 RZEKVGVHFLEQIL-UHFFFAOYSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 210000001612 chondrocyte Anatomy 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- IDLFZVILOHSSID-OVLDLUHVSA-N corticotropin Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)NC(=O)[C@@H](N)CO)C1=CC=C(O)C=C1 IDLFZVILOHSSID-OVLDLUHVSA-N 0.000 description 2
- 229960000258 corticotropin Drugs 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000006731 degradation reaction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229960000403 etanercept Drugs 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 230000036252 glycation Effects 0.000 description 2
- 210000000548 hind-foot Anatomy 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 229960000598 infliximab Drugs 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000037231 joint health Effects 0.000 description 2
- 238000013150 knee replacement Methods 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 238000004091 panning Methods 0.000 description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 230000007026 protein scission Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- RZJQGNCSTQAWON-UHFFFAOYSA-N rofecoxib Chemical compound C1=CC(S(=O)(=O)C)=CC=C1C1=C(C=2C=CC=CC=2)C(=O)OC1 RZJQGNCSTQAWON-UHFFFAOYSA-N 0.000 description 2
- 238000002603 single-photon emission computed tomography Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000008961 swelling Effects 0.000 description 2
- 238000007910 systemic administration Methods 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- LNPDTQAFDNKSHK-UHFFFAOYSA-N valdecoxib Chemical compound CC=1ON=C(C=2C=CC=CC=2)C=1C1=CC=C(S(N)(=O)=O)C=C1 LNPDTQAFDNKSHK-UHFFFAOYSA-N 0.000 description 2
- CBOJBBMQJBVCMW-BTVCFUMJSA-N (2r,3r,4s,5r)-2-amino-3,4,5,6-tetrahydroxyhexanal;hydrochloride Chemical compound Cl.O=C[C@H](N)[C@@H](O)[C@H](O)[C@H](O)CO CBOJBBMQJBVCMW-BTVCFUMJSA-N 0.000 description 1
- FJQZXCPWAGYPSD-UHFFFAOYSA-N 1,3,4,6-tetrachloro-3a,6a-diphenylimidazo[4,5-d]imidazole-2,5-dione Chemical compound ClN1C(=O)N(Cl)C2(C=3C=CC=CC=3)N(Cl)C(=O)N(Cl)C12C1=CC=CC=C1 FJQZXCPWAGYPSD-UHFFFAOYSA-N 0.000 description 1
- UGBLISDIHDMHJX-UHFFFAOYSA-N 1-(4-fluorophenyl)-4-[4-(2-methoxyphenyl)piperazin-1-yl]butan-1-one;hydrochloride Chemical compound [Cl-].COC1=CC=CC=C1N1CC[NH+](CCCC(=O)C=2C=CC(F)=CC=2)CC1 UGBLISDIHDMHJX-UHFFFAOYSA-N 0.000 description 1
- VKUYLANQOAKALN-UHFFFAOYSA-N 2-[benzyl-(4-methoxyphenyl)sulfonylamino]-n-hydroxy-4-methylpentanamide Chemical compound C1=CC(OC)=CC=C1S(=O)(=O)N(C(CC(C)C)C(=O)NO)CC1=CC=CC=C1 VKUYLANQOAKALN-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 108090001008 Avidin Proteins 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108700012434 CCL3 Proteins 0.000 description 1
- 108010029697 CD40 Ligand Proteins 0.000 description 1
- 101150013553 CD40 gene Proteins 0.000 description 1
- 102100032937 CD40 ligand Human genes 0.000 description 1
- 101100275473 Caenorhabditis elegans ctc-3 gene Proteins 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 102000000013 Chemokine CCL3 Human genes 0.000 description 1
- 102000001187 Collagen Type III Human genes 0.000 description 1
- 108010069502 Collagen Type III Proteins 0.000 description 1
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 1
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 1
- 102100021866 Hepatocyte growth factor Human genes 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000945318 Homo sapiens Calponin-1 Proteins 0.000 description 1
- 101000960954 Homo sapiens Interleukin-18 Proteins 0.000 description 1
- 101000599048 Homo sapiens Interleukin-6 receptor subunit alpha Proteins 0.000 description 1
- 101000652736 Homo sapiens Transgelin Proteins 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- HEFNNWSXXWATRW-UHFFFAOYSA-N Ibuprofen Chemical compound CC(C)CC1=CC=C(C(C)C(O)=O)C=C1 HEFNNWSXXWATRW-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102100037850 Interferon gamma Human genes 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 108010074328 Interferon-gamma Proteins 0.000 description 1
- 102000003814 Interleukin-10 Human genes 0.000 description 1
- 108090000174 Interleukin-10 Proteins 0.000 description 1
- 108090000177 Interleukin-11 Proteins 0.000 description 1
- 102000003815 Interleukin-11 Human genes 0.000 description 1
- 108010065805 Interleukin-12 Proteins 0.000 description 1
- 102000013462 Interleukin-12 Human genes 0.000 description 1
- 102000003816 Interleukin-13 Human genes 0.000 description 1
- 108090000176 Interleukin-13 Proteins 0.000 description 1
- 108090000172 Interleukin-15 Proteins 0.000 description 1
- 102000003812 Interleukin-15 Human genes 0.000 description 1
- 101800003050 Interleukin-16 Proteins 0.000 description 1
- 102000049772 Interleukin-16 Human genes 0.000 description 1
- 108050003558 Interleukin-17 Proteins 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 102100039898 Interleukin-18 Human genes 0.000 description 1
- 102100039879 Interleukin-19 Human genes 0.000 description 1
- 108050009288 Interleukin-19 Proteins 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 102100030703 Interleukin-22 Human genes 0.000 description 1
- 108010002386 Interleukin-3 Proteins 0.000 description 1
- 102000000646 Interleukin-3 Human genes 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 102100039897 Interleukin-5 Human genes 0.000 description 1
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 1
- 108010002586 Interleukin-7 Proteins 0.000 description 1
- 102100021592 Interleukin-7 Human genes 0.000 description 1
- 102000004890 Interleukin-8 Human genes 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108010002335 Interleukin-9 Proteins 0.000 description 1
- 102000000585 Interleukin-9 Human genes 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 241001262617 Japonica Species 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 1
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 101710091439 Major capsid protein 1 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010008364 Melanocortins Proteins 0.000 description 1
- 102000005741 Metalloproteases Human genes 0.000 description 1
- 108010006035 Metalloproteases Proteins 0.000 description 1
- CMWTZPSULFXXJA-UHFFFAOYSA-N Naproxen Natural products C1=C(C(C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-UHFFFAOYSA-N 0.000 description 1
- 241000737052 Naso hexacanthus Species 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 108010082093 Placenta Growth Factor Proteins 0.000 description 1
- 102100035194 Placenta growth factor Human genes 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 102100024952 Protein CBFA2T1 Human genes 0.000 description 1
- 108700033844 Pseudomonas aeruginosa toxA Proteins 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 101710108790 Stromelysin-1 Proteins 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 230000005867 T cell response Effects 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 101000709029 Toxoplasma gondii Rhomboid-like protease 5 Proteins 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102100030742 Transforming growth factor beta-1 proprotein Human genes 0.000 description 1
- 102100031013 Transgelin Human genes 0.000 description 1
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000002917 arthritic effect Effects 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 229940110331 bextra Drugs 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 239000000090 biomarker Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000008355 cartilage degradation Effects 0.000 description 1
- 229940047495 celebrex Drugs 0.000 description 1
- 229960000590 celecoxib Drugs 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 229940111134 coxibs Drugs 0.000 description 1
- 230000009260 cross reactivity Effects 0.000 description 1
- 239000003255 cyclooxygenase 2 inhibitor Substances 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 210000000750 endocrine system Anatomy 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- BCQZXOMGPXTTIC-UHFFFAOYSA-N halothane Chemical compound FC(F)(F)C(Cl)Br BCQZXOMGPXTTIC-UHFFFAOYSA-N 0.000 description 1
- 229960003132 halothane Drugs 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940018991 hyalgan Drugs 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- XMBWDFGMSWQBCA-UHFFFAOYSA-N hydrogen iodide Chemical compound I XMBWDFGMSWQBCA-UHFFFAOYSA-N 0.000 description 1
- TUJKJAMUKRIRHC-UHFFFAOYSA-N hydroxyl Chemical compound [OH] TUJKJAMUKRIRHC-UHFFFAOYSA-N 0.000 description 1
- 229960001680 ibuprofen Drugs 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000002134 immunopathologic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000006749 inflammatory damage Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 108090000681 interleukin 20 Proteins 0.000 description 1
- 102000004114 interleukin 20 Human genes 0.000 description 1
- 108010074108 interleukin-21 Proteins 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 150000002540 isothiocyanates Chemical class 0.000 description 1
- 210000000281 joint capsule Anatomy 0.000 description 1
- 208000018937 joint inflammation Diseases 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 230000003859 lipid peroxidation Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002865 melanocortin Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- OHDXDNUPVVYWOV-UHFFFAOYSA-N n-methyl-1-(2-naphthalen-1-ylsulfanylphenyl)methanamine Chemical compound CNCC1=CC=CC=C1SC1=CC=CC2=CC=CC=C12 OHDXDNUPVVYWOV-UHFFFAOYSA-N 0.000 description 1
- 229960002009 naproxen Drugs 0.000 description 1
- CMWTZPSULFXXJA-VIFPVBQESA-N naproxen Chemical compound C1=C([C@H](C)C(O)=O)C=CC2=CC(OC)=CC=C21 CMWTZPSULFXXJA-VIFPVBQESA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- GQPLMRYTRLFLPF-UHFFFAOYSA-N nitrous oxide Inorganic materials [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- KHIWWQKSHDUIBK-UHFFFAOYSA-N periodic acid Chemical compound OI(=O)(=O)=O KHIWWQKSHDUIBK-UHFFFAOYSA-N 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 229940072689 plaquenil Drugs 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000000700 radioactive tracer Substances 0.000 description 1
- 239000003642 reactive oxygen metabolite Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 229960000371 rofecoxib Drugs 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical class O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- JJAHTWIKCUJRDK-UHFFFAOYSA-N succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate Chemical compound C1CC(CN2C(C=CC2=O)=O)CCC1C(=O)ON1C(=O)CCC1=O JJAHTWIKCUJRDK-UHFFFAOYSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 210000001258 synovial membrane Anatomy 0.000 description 1
- 229940036220 synvisc Drugs 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 230000007838 tissue remodeling Effects 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001228 trophic effect Effects 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 229960002004 valdecoxib Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 229940087652 vioxx Drugs 0.000 description 1
- BPICBUSOMSTKRF-UHFFFAOYSA-N xylazine Chemical compound CC1=CC=CC(C)=C1NC1=NCCCS1 BPICBUSOMSTKRF-UHFFFAOYSA-N 0.000 description 1
- 229960001600 xylazine Drugs 0.000 description 1
Images
Classifications
-
- A61K47/48538—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6843—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a material from animals or humans
-
- A61K47/48415—
-
- A61K47/48423—
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6813—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin the drug being a peptidic cytokine, e.g. an interleukin or interferon
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
- A61K47/6817—Toxins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
- A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
- A61K51/04—Organic compounds
- A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
- A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
- A61K51/1093—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies
- A61K51/1096—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody conjugates with carriers being antibodies radioimmunotoxins, i.e. conjugates being structurally as defined in A61K51/1093, and including a radioactive nucleus for use in radiotherapeutic applications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/55—Fab or Fab'
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
Definitions
- the present invention relates to a novel antibody and its use in the diagnosis and therapy of inflammatory diseases of the joints such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- RA rheumatoid arthritis
- OA osteoarthritis
- RA rheumatoid arthritis
- OA osteoarthritis
- RA activated leukocytes
- OA activated chondrocytes
- Pro-inflammatory cytokine blockade such as anti-TNFa and IL-1Ra is therefore currently used to treat arthritic conditions, mainly RA.
- RA is a classic inflammatory form of arthritis, which is a chronic autoimmune disease with extensive synovial inflammation. Influx of activated leukocytes infiltrating the inflamed synovial membrane results in up-regulation of inflammatory cytokines such as TNFa, interleukin-1 (IL-1) and interleukin-6 (IL-6) leading to increase in the levels of matrix metalloproteases (MMP).
- IL-1 interleukin-1
- IL-6 interleukin-6
- ROS reactive oxidant species
- SO 2 superoxide radicals
- H 2 O 2 hydrogen peroxide
- OH hydroxyl radicals
- HOCl hypochlorous acid
- NO nitric oxide
- ONOO peroxynitrite
- sequential oxidative reactions generate reactive oxidants such as advanced glycation end-products (AGE).
- AGE advanced glycation end-products
- synovial inflammation in OA is not as extensive as in RA and inflammatory cells are not significant in numbers, low grade synovitis is nearly a constant feature in OA.
- Abnormal mechanical force appears to stimulate chondrocytes to produce the same inflammatory mediators and ROS as the infiltrated leukocytes present in inflamed RA joints leading to post translational modifications of CII.
- the presence of strong staining of nitrotyrosine and low antioxidative capacity in the degenerative region of OA cartilage compared with the intact region from the same sample suggests a possible correlation between oxidative damage and cartilage degradation.
- RA rheumatoid arthritis
- OA osteoarthritis
- CII post-translationally modified Collagen II
- composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) in which the antibody or fragment thereof is conjugated to a pharmaceutically active moiety.
- CII oxidised Collagen II
- the present invention therefore provides a novel approach to the targeting of drugs to self-epitopes on Collagen II that are a normal component of the tissue but which become immunogenic after post-translational modification by free radicals as part of a disease process affecting Collagen II.
- the antibody may be a polyclonal antibody or a monoclonal antibody. It may be a human or humanized or chimeric antibody with sequences, residues or domains derived from more than one animal species. Fragments of antibodies include Fc, Fab, scFv, single domain (dAb) antibody, diabody, minibody, and scFv-Fc fragments.
- the antibody comprises CDR sequences in the Variable Heavy (VH) Chains and Variable Light (VL) chains as shown in Table 1.
- VH Variable Heavy
- VL Variable Light
- CDRH2 and CDRH3 are in the VH chain and CDRL2 and CDRL3 are in the VL chain.
- the antibody may be an scFv selected from the group consisting of the following:
- the scFv may comprise a sequence as shown in Table 2.
- the scFv is 1-11E.
- Oxidised Collagen II is post-translationally modified Collagen II (CII) that has been oxidised by non enzymatic glycation or by reactive oxidant species (ROS) which may include superoxide radical (O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (OH), hypochlorous acid (HOCl), nitric oxide (NO) and peroxynitrite (ONOO).
- ROS reactive oxidant species
- the antigen may therefore be HOCl-Collagen II or Ribose-Collagen II.
- the antibody or fragment thereof is conjugated to the pharmaceutically active moiety which may be a peptide or peptide-based molecule by any suitable means.
- the pharmaceutically active moiety is a peptide or peptide-based molecule the conjugation may be by means of a peptide bond, including the insertion of one or more amino acid residues.
- conjugation of a peptide or a peptide-based molecule may be achieved by any generally convenient chemical means or biological means (see for example, Wu & Senter Nature Biotechnology , volume 23 (9) pages 1137-1146 (2005); “ Chemistry of Protein Conjugation and Crosslinking ” by S. S. Wong, CRC Press Inc. (1991)).
- Chemical conjugation typically uses a bifunctional chemical reagent, for example glutaraldehyde can link molecules to the N-terminus of a peptide, carbodiimide can link molecules to the C-terminus of a peptide, succinimide esters (e.g. MBS, SMCC) can bind free amino groups and cysteine residues, benzidine links to tyrosine residues, periodate attaches to carbohydrate groups and isothiocyanate can also link molecules to antibodies.
- a bifunctional chemical reagent for example glutaraldehyde can link molecules to the N-terminus of a peptide, carbodiimide can link molecules to the C-terminus of a peptide, succinimide esters (e.g. MBS, SMCC) can bind free amino groups and cysteine residues, benzidine links to tyrosine residues, periodate attaches to carbohydrate groups and isothiocyanate can also link molecules to antibodies.
- a fusion protein may be synthesised using standard recombinant molecular biology techniques (see for example, Sambrook et al “ Molecular Cloning: A Laboratory Manual ”, 3 rd edition, CSHL Press, (2001); Trachsel et al Arthritis Research & Therapy , volume 9 (1) R9 (2007); Nagai Arthritis & Rheumatism , volume 54 (10) pages 3126-3134 (2006)). Methods for producing fusion proteins are described in the Examples herein.
- the insertion of additional amino acid residues between the antibody or fragment thereof and the pharmaceutically active moiety may represent a site for cleavage by a protease.
- the proteolytic cleavage site may comprise any protease specific cleavage site.
- the proteolytic cleavage site may include, but is not limited to, a matrix metalloproteinase (MMP) cleavage site, a serine protease cleavage site, a site cleavable by a parasitic protease derived from a pathogenic organism (Zhang et al., J. Mol. Biol.
- MMP matrix metalloproteinase
- the MMP cleavage site may comprise any amino acid sequence which is cleavable by a MMP.
- the amino acid sequence of the MMP cleavage site may be encoded by a nucleic acid sequence coding for an MMP sequence as shown in FIGS. 5A, 5B, 5C , and 5 D or a sequence of nucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto.
- the nucleic acid sequence encoding the MMP cleavage site comprises the minimum number of residues required for recognition and cleavage by MMP.
- a MMP cleavage site may comprise a number of amino acid residues recognisable by MMP. Moreover, the amino acids of the MMP site may be linked by one or more peptide bonds which are cleavable, proteolytically, by MMP.
- MMPs which may cleave the MMP site include, but are not limited to, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 (Yu and Stamenkovic, Genes and Dev. 14, 163-176 (2000); Nagase and Fields, Biopolymers, 40, 399-416 (1996); Massova et al., J. Mol. Model. 3, 17-30 (1997); reviewed in Vu and Werb, Genes and Dev. 14, 2123-2133 (2000)).
- the sequences of the protein cleavage sites of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 and MMP10 are shown in FIGS. 5A, 5B, 5C, and 5D
- the proteolytic cleavage site of the present invention is cleaved at sites of inflammation and tissue remodeling. More preferably, the proteolytic cleavage site of the present invention is a MMP cleavage site e.g. any one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 as shown in FIGS. 5A, 5B, 5C, and 5D .
- MMP cleavage site e.g. any one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 as shown in FIGS. 5A, 5B, 5C, and 5D .
- the pharmaceutically active moiety may comprise one or more molecules which may be the same or different, one or more radioisotopes which may be the same or different, or one or more non-radioactive elements which may be the same or different.
- the pharmaceutically active moiety may comprise a polypeptide or non-polypeptide molecule.
- references to a polypeptide include a peptide and vice versa unless the context specifies otherwise.
- the polypeptide may be an antibody or a fragment thereof, such as an anti-TNFalpha monoclonal antibody (for example infliximab or adalimumab), a soluble p75 TNF receptor molecule (for example etanercept) or a IL-1 receptor antagonist (for example anakinra).
- an anti-TNFalpha monoclonal antibody for example infliximab or adalimumab
- a soluble p75 TNF receptor molecule for example etanercept
- IL-1 receptor antagonist for example anakinra
- the composition will therefore comprise a bispecific antibody which may be a diabody (scFv with a linker which is too short to allow pairing between VH and VL and therefore the domains are forced to pair with the complementary domain of another scFv to create two antigen binding site), a minibody (composed of two scFv moieties linked via a constant heavy chain region (CH3)), a scFv-Fc molecule, or an intact antibody molecule containing the two separate binding regions.
- a bispecific antibody which may be a diabody (scFv with a linker which is too short to allow pairing between VH and VL and therefore the domains are forced to pair with the complementary domain of another scFv to create two antigen binding site), a minibody (composed of two scFv moieties linked via a constant heavy chain region (CH3)), a scFv-Fc molecule, or an intact antibody molecule containing the two separate binding regions.
- CH3 constant heavy chain
- a bispecific antibody may comprise a first binding region specific for modified Collagen II (CII) and a second binding region specific for anti-TNFa.
- CCII modified Collagen II
- the polypeptide is a TNF receptor (TNFR) antibody fusion protein, typically a TNFR2-Fc fusion protein.
- TNFR TNF receptor
- a bispecific antibody of the invention may also further comprise another pharmaceutically active moiety.
- a composition of the invention may comprise a first binding region specific for modified Collagen II, a second binding region specific for CD64, and a toxin, such as Ricin A.
- the polypeptide may be a growth factor (e.g. TGF ⁇ , epidermal growth factor (EGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), colony stimulating factor (CSF) granulocyte/macrophage colony stimulating factor (GM-CSF), hepatocyte growth factor, insulin-like growth factor, placenta growth factor); differentiation factor, cytokine molecule, for example an interleukin, (e.g.
- IFN- ⁇ , IFN- ⁇ and IFN- ⁇ tumour necrosis factor
- IGIF IFN- ⁇ inducing factor
- BMP bone morphogenetic protein
- chemokine for example a MIP (Macrophage Inflammatory Protein) e.g.
- MIP1 ⁇ and MIP1 ⁇ a MCP (Monocyte Chemotactic Protein) e.g. MCP1, 2 or 3; RANTES (regulated upon activation normal T-cell expressed and secreted)); a trophic factor; a cytokine inhibitor; a cytokine receptor (for example, CD20, CD40, CD40L, CD64); a free-radical scavenging enzyme (e.g. superoxide dismutase or catalase), or a toxin (for example Ricin A toxin, or Pseudomonas exotoxin A), or an active fragment or portion thereof.
- the polypeptide is an interferon, typically IFN- ⁇ .
- antibody-fusion proteins include but are not limited to, antibody-TNFalpha, antibody-GM-CSF, and antibody-IL2 fusion proteins
- the pharmaceutically active polypeptide may be derived from the species to be treated e.g. human origin for the treatment of humans.
- composition may also comprise further peptide sequences which can target the composition inside a cell.
- intracellular targeting sequences include, but are not limited to, the TAT sequence YGRKKRQRRR (SEQ ID NO: 126) (see for example, Cohen-Saidon et al Blood , volume 102 (7), pages 2506-2512 (2003)).
- peptide mimetics includes, but is not limited to, agents having a desired peptide backbone conformation embedded into a non-peptide skeleton which holds the peptide in a particular conformation. Peptide mimetics, which do not have some of the drawbacks of peptides, are of interest in those cases where peptides are not suitable in medicine.
- Peptide mimetics may comprise a peptide backbone which is of the L or D conformation.
- Examples of peptide mimetics include melanocortin, adrenocorticotrophin hormone (ACTH) and other peptide mimetic agents which play a role in the central nervous system, endocrine system, in signal transduction and in infection and immunity.
- ACTH adrenocorticotrophin hormone
- the pharmaceutically active agent may comprise a chemical compound such as a chemotherapeutic agent or other synthetic drug.
- the pharmaceutically active agent may comprise a peptide nucleic acid (PNA) sequence e.g. a poly-lysine sequence which binds to nucleic acids and permeabilises lipid bilayers (Wyman et al., Biological Chemistry, 379, 1045-1052 (1998)) or a KALA peptide which facilitates transfer through lipid bilayers (Wyman et al., Biochemistry, 36, 3008-3017 (1997)).
- PNA peptide nucleic acid
- the non-polypeptide may be a glycosaminoglycan molecule, such as glucosamine (suitably, glucosamine HCl) or chondroitin.
- the non-polypeptide molecule may be a non-steroidal anti-inflammatory drug (NSAID) such as a non-selective NSAID or a selective NSAID.
- NSAID non-steroidal anti-inflammatory drug
- non-selective NSAIDs include aspirin, ibuprofen, and naproxen.
- selective NSAIDs also called COX-2 inhibitors
- celecoxib Celebrex®
- rofecoxib Vioxx®
- valdecoxib Bosecoxib
- steroids such as cortisol
- polymeric molecules such as sodium hyaluronate or hyaluronic acid (for example hyaluronan (Hyalgan®) and hylan-GF-20 (Synvisc®)
- drug substances such as colchicine or hydroxychloroquine (Plaquenil®).
- Non-polypeptides may be conjugated to the antibody or fragment thereof using a linker that may be a labile bond in order to permit release of the pharmaceutically active substance.
- a linker that may be a labile bond in order to permit release of the pharmaceutically active substance.
- a hydrazone bond may be used where the drug is released under acidic conditions, or a disulfide bond which is reduced to release the drug, or also a peptide bond which is cleaved enzymatically by a protease as described above.
- the composition may comprise a radioactive element or a non-radioactive element.
- the radioisotope may be an alpha particle-emitting radionuclide such as 213 Bi or 211 At, a beta particle-emitting radionuclide such as 131 I, 90 Y, 177 Lu or 67 Cu, a gamma radiation-emitting radionuclide such as 99m Tc, 123 I or 111 In, or a positron-emitting radionuclide such as 18 F, 64 Cu, 68 Ga, 86 Y or 124 I. Radioisotopes may be used in order to render the composition detectably labelled for diagnostic uses of the composition.
- the non-radioactive element may be Au, Fe, Cu, Pt or Ag.
- a composition of the first aspect for use in medicine includes a composition of the first aspect for use in the treatment of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- This aspect of the invention therefore extends to a method of treatment of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA), comprising the step of administering to a subject a composition of the first aspect of the invention.
- the present invention therefore also includes the use of a composition of the first aspect of the invention in the manufacture of a medicament for the treatment of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- a composition of the first aspect of the invention may therefore be formulated as a pharmaceutical composition.
- a pharmaceutical composition may comprise a diluent, excipient, adjuvant and/or physiologically acceptable buffer.
- the pharmaceutical composition may be administered in any effective, convenient manner effective for treating a disease as described above including, for instance, administration by oral, topical, intravenous, intramuscular, intra-articular, intranasal, or intradermal routes among others.
- the composition of the invention may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic.
- the injection may suitably be made into the joint affected by the disease.
- the daily dosage of the composition of the invention will be from 0.01 mg/kg body weight, typically around 1 mg/kg.
- the physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependant on factors including the age, weight, sex and response of the individual.
- the above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention
- a method for the diagnosis of an arthropathy comprising the steps of administering a detectably labelled composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) to a subject and subsequently detecting the composition.
- This aspect of the invention therefore extends to a detectably labelled composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) for use in the diagnosis of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- Such embodiments also extend to the use of such compositions in the manufacture of an agent for the diagnosis of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- the detectable label may be a radioactive or a fluorescent label.
- the radioisotope may be an alpha particle-emitting radionuclide such as 213 Bi or 211 At, a beta particle-emitting radionuclide such as 131 I 90 Y, 177 Lu or 67 Cu, a gamma radiation-emitting radionuclide such as 99m Tc, 123 I or 111 In, or a positron-emitting radionuclide such as 18 F, 64 Cu, 68 Ga, 86 Y or 124 I. Radioisotopes may be used in order to render the composition detectably labelled for diagnostic uses of the composition.
- fluorescent dyes such as Alexa Fluor 488 or the Cy3 monofunctional N-hydroxysuccinimide (NHS) ester could also be used.
- composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) and a detectable label.
- CII oxidised Collagen II
- RA is the most common chronic inflammatory autoimmune disease, with disability occurring usually within 10 years. Over activation of the inflammatory pathway leads to synovitis, joint damage and destruction. Key players in the joint inflammation are inflammatory cytokines such as TNFa and IL-1.
- TNFa inflammatory cytokines
- IL-1 receptor antagonist IL-1 receptor antagonist
- CII is the best candidate to target therapy to the joint one needs to find a way to target the drugs solely to the inflamed joints.
- the present studies show the development of a targeting antibody that will specifically recognise collagen type II that has been modified by ROS present in inflamed joint which then allows targeting to inflammatory damage joint independently of the aetiology.
- FIG. 1 a panel of human scFvs was developed ( FIG. 1 ) that bind only to CII which was modified in vitro by HOCl or glycation, known reactive oxidants in RA. Most importantly this scFv binds only to damaged cartilage but not to normal cartilage ( FIG. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H ) and inversely correlates with the staining of safranin-O for the integrity of cartilage-specific proteoglycan.
- a composition of the invention comprises mouse interferon-beta (IFN- ⁇ ), the scFv 1-11E and a MMP cleavage site.
- IFN- ⁇ mouse interferon-beta
- MCS multiple cloning sites
- mIFNb mouse interferon b
- Mouse interferon-beta is typically amplified using primers having the sequences shown in SEQ ID NO: 129 (forward) and SEQ ID NO: 130 (reverse).
- 1-11E is typically amplified using primers having the sequences shown in SEQ ID NO: 131 (forward) and SEQ ID NO: 132 (reverse), wherein 1-11E is amplified with NotI/ApaI ends to include a histidine (His) tag.
- 1-11E is typically then cloned into FastBac1.AH mIFN-b/MMP/SP/His and cut with Not/Apa to liberate SP/His.
- the mIFN-beta/His construct is typically cloned by amplifying mIFN-b with HindIII/ApaI using primers having the sequence shown in SEQ ID NO: 129 (forward) and SEQ ID NO: 133 (reverse).
- constructs are then typically transformed into DH10Bac cells (Invitrogen).
- a composition of the invention comprises TNF receptor 2-Fc (TNFR2Fc), an scFv (either 1-11E or C7 as a negative control) and a MMP cleavage site.
- TNF receptor 2-Fc TNF receptor 2-Fc
- scFv either 1-11E or C7 as a negative control
- MCS multiple cloning sites
- Mouse TNFR2-Fc is typically amplified using primers having the sequences shown in SEQ ID NO: 141 (forward) and SEQ ID NO: 142 (reverse).
- 1-11E is typically amplified using primers having the sequences shown in SEQ ID NO: 131 (forward) and SEQ ID NO: 132 (reverse), wherein 1-11E is amplified with NotI/ApaI ends to include a histidine (His) tag.
- infected High 5 cells are grown for 3 days at 27° C.
- the supernatant is typically then collected and run on an SDS-PAGE gel.
- Recombinant proteins can be detected by Western blot, for example using anti-tetra-His antibody (Qiagen) and anti-mouse HRP (Sigma).
- FIG. 1 shows representative ELISA of unique scFvs
- FIG. 2 shows Western Blotting with scFv 1-11E probe
- FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H show specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with OA in photographs (A) to (H).
- FIG. 4 shows the localisation of scFv 1-11E in inflamed paw.
- FIGS. 5A, 5B, 5C, and 5D show the sequences of the protein cleavage sites of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 and MMP10.
- FIG. 6 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with OA.
- FIG. 7 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with RA.
- FIGS. 8A, 8B, 8C, 8D, and 8E show: (A) and (B) histological staining of the right paw; (C) and (D) 1-11E staining of cartilage in the paw; (E) staining with a non-relevant scFv; all in a mouse RA model.
- FIG. 9 shows staining of a joint surface injury in a mouse OA model.
- FIGS. 10A and 10B show the construction of (A) an IFN- ⁇ /1-11E fusion protein; and (B) a TNFR2Fc/scFv fusion protein.
- FIG. 11 shows the protocol used for expression of the IFN- ⁇ /1-11E fusion protein and the TNFR2Fc/scFv fusion protein.
- FIG. 12 is a Western blot of the IFN- ⁇ /1-11E fusion protein.
- FIGS. 13A, 13B, and 13C is a Western blots of (A) and (B) TNFR2Fc/1-11E fusion proteins; (C) a TNFR2Fc/C7 fusion protein.
- CII was prepared from bovine cartilage as in Miller (Miller, Biochemistry 11(26): 4903-4909, 1972) and subsequently exposed to reactive oxygen generating systems as previously described (Nissim A, 2005). Briefly, CII was modified with ( ⁇ OH), HOCl (Hawkins CL, 2001; Hawkins CL, 2002), (ONOO + ), or 2M ribose by ON incubation at 37° C. Bovine serum albumin (BSA, Sigma) was also modified as above and was used as control antigen.
- BSA Bovine serum albumin
- Phage display antibody technology (Winter G et al, Annu. Rev. Immunol. 12: 433-455, 1994) was used to raise a single chain fragment variable (scFv) that binds only to CII that has been post-translationally modified by free radicals.
- scFv single chain fragment variable
- HOCl modified CII was used as a target for panning as binding to HOCl modified CII was strongest in RA sera (Nissim A, 2005). Glycated CII was used in parallel.
- immunotubes (Nunc-Immuno Tubes, Maxi-Sorp, Nunc, Denmark) were coated with 10 ⁇ g/ml CII in phosphate-buffered saline (PBS). After blocking with 2% argue in PBS (MPBS) coated tubes were exposed for 2 hours to 10 13 transforming units (tu) of the phage library in 2% MPBS. Unbound phage were then transferred to a second immunotube previously coated with HOCl or ribose-modified CII for a further 2 hours incubation at room temperature. Modified CII-bound phage were then used to infect E. coli TG-1 and rescued by helper phage as described (Harrison J. L, 1996). The panning process was repeated three times and E. coli TG-1 was infected with the final phage eluted after the third round and individual ampicillin-resistant colonies (phage clones) were selected for further analysis.
- PBS phosphate-buffered saline
- the reaction was monitored in an ELISA reader at 450 nm with a reference wavelength of 650 nm using GENios plate reader (TECAN, Theale Court, Reading UK) and Magellan software (TECAN, Theale Court, Reading UK)
- the reactive phage clones obtained from E. coli TG-1 bacteria were used to infect E. coli HB2151 non-suppressor bacterial strain to obtain soluble scFv. After overnight induction with 1 mM IPTG at 30° C., the antibody fragments, derived from the V H 3 family, were harvested from the supernatant and periplasmic space as described (Harrison J. L, 1996) and purified on a protein A affinity column (GE Healthcare Ltd, Little Chalfont, Buckinghamshire).
- Binding of purified scFv to modified CII was first analyzed by ELISA as above except that mouse anti-myc tag antibody (Santa Cruz Biotechnology, INC, Wembley, UK) followed by anti-mouse-HRP conjugate (Sigma, Dorset, UK) were used to probe bound scFv.
- Clone 1-11E binds to modified CII (glycated, HOCl and to some extent to peroxynitrated CII), clone 6-11D binds to both native and modified CII and clone 12E that does not bind to any form of CII. None of these scFv bound to native or free radical modified BSA, or to collagen type III (data not shown).
- 1-11E binds several CII fragments between 50 and 150 kDa as well as to a band >250 kDa which resulted from CII cross linking due to the ROS reactivity ( FIG. 2 lane 2-5). 1-11E also binds to native CII corresponding to a band below 150KDa ( FIG. 2 lane 1). Binding to native CII in Western blotting was also seen in sera from RA patients that did not bind to native CII in ELISA but only to ROS modified CII in ELISA (for example sera 33 (Nissim A, 2005)).
- osteochondral sample was obtained from the femoral condyle of a patient (female, 63 years old) undergoing prosthetic knee replacement for OA.
- One sample of normal human cartilage was obtained post-mortem from a preserved area of a knee with unicompartimental OA undergoing joint replacement (female, 54 years old).
- cartilage was fixed overnight at 4° C. in 4% paraformaldehyde, decalcified for 15 days in 0.5M EDTA at 4° C., washed in PBS, and embedded in paraffin according to standard protocols. Safranin O staining was performed according to standard protocols (Rosenberg, 1971). All samples were obtained in accordance with institutional policies and regulations.
- Immunostaining was performed using the selected scFv (10 ⁇ g/ml and 1 ⁇ g/ml) as well as control commercial mouse anti-CII antibodies (diluted 1:100 and 1:1000 dilution; Chemicon International, Chandlers Ford, Hampshire, UK) and polyclonal anti-CII antibodies (diluted 1:100, 1:1000) from collagen induced arthritis (CIA) mice.
- ScFv or control antibodies were added to the slide in blocking buffer (0.5% BSA in PBS plus 0.05% sodium azide) and left overnight at 4° C.
- the cartilage extracellular matrix is a complex structure where several molecules interact to form a structural and functional unit. There is therefore the chance that the tertiary and quaternary structure of collagens in the intact tissue may alter the specificity of binding of the phage antibodies that had been selected in vitro.
- the capacity of anti-ROS-modified CII scFv to bind to CII within the matrix complex structure and to present immunoreactivity with damaged OA cartilage as opposed to normal cartilage was tested. 1-11E stained the extracellular matrix of cartilage tissue that displayed marked features of OA ( FIG. 3A, 3B ) with mostly pericellular staining ( FIG.
- FIG. 3B shows severe damage of the extracellular matrix determined with reduced staining with safranin O
- FIG. 3C shows no staining by scFv 1-11E was detected when using histologically normal cartilage from normal cartilage
- FIG. 3F polyclonal antibodies from CIA mice bound the OA cartilage in both the damaged and non-damaged regions strongly ( FIG. 3F ) and weakly stained with safranin O ( FIG. 3C ).
- a commercial anti-CII mAb did not stain the damaged cartilage areas stained by 1-11E on an adjacent section ( FIG. 3G ) but intensely stained a histologically normal cartilage ( FIG. 3H ), suggesting that the epitope recognised by the commercial antibody is lost in the OA section.
- FIG. 6 also shows staining of OA cartilage.
- synovial inflammation in OA is not as extensive as in RA and inflammatory cells are not significant in numbers, low grade synovitis is nearly a constant feature in OA.
- Abnormal mechanical force appears to stimulate chrondocytes to produce some of the same inflammatory mediators and ROS as the infiltrating leukocytes present in inflamed RA joints, leading to post-translational modifications of CII in OA.
- FIG. 6 confirms the results shown in FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H and shows that staining of OA cartilage section is only pericellular, around the chrondocytes.
- a further sample was obtained from a patient (female, 47 years old) undergoing total right knee replacement for RA. Fixing and staining protocols were as described above.
- FIG. 7 shows staining of the RA cartilage.
- RA reactive oxygen species
- ROS reactive oxygen species
- mice Male C3H mice (age 17-19 weeks) were used. 100 mg of dessicated non-viable T.B. strain H37RA (Difco 231141) was added to 30 ml of incomplete Freunds adjuvant (IFA, Difco 263910) to form complete Freunds adjuvant (CFA). An equal volume of CFA was added to a 2 mg/ml solution (in PBS) of methylated BSA (mBSA) (Sigma A1009). The mixture was then emulsified on ice using an Ultra-Turrax T25 homogeniser at 13500-20500 rpm until a fluffy milky consistency was obtained. Mice were anaesthetised with Hypnorm, and 100 ⁇ l of 1 mg/ml (i.e.
- mice were anaesthetised with nitrous/oxygen and halothane, and inflammation was induced by injecting 50 ⁇ l of 1 mg/ml (i.e. 50 ⁇ g) mBSA in PBS into the animals' left hind paw. As a control, 50 ⁇ l PBS was injected into the right hind paw. Inflammation was measured using callipers to measure the paw thickness. Swelling was seen only in the right paws from 24 hours, and persisted until 1 week later. 2 weeks later, the swelling had totally subsided.
- mice Four and 22 hours later the mice were anaesthetized by ip injection of Ketamine/Xylazine.
- the mice were imaged on a NanoSPECT/CT scanner (Bioscan Inc, Washington, USA) using a four-detector/36 ⁇ 1.4 mm pinhole configuration. 30-50,000 counts were acquired for the SPECT study over 20-50 minutes.
- FIG. 4 shows a representative image acquired 22 hours after injection of the radiotracer. Increased uptake of radioactivity is clearly seen in the rear left (inflamed) paw relative to the right (normal paw).
- mice were sensitized with mBSA (100 ⁇ g) in CFA intradermally at the base of the tail, and challenged either intra-articularly (both knees) or intra-plantarly (right, saline left) with 500 ⁇ g mBSA in saline 14 days later.
- FIGS. 8A, 8B, 8C, 8D, and 8E Staining of cartilage is shown in FIGS. 8A, 8B, 8C, 8D, and 8E .
- FIGS. 8A and 8B 12 hours post challenge with mBSA, the right paw was grossly inflamed in the subplantar region (seen by haematoxylin and eosin (H&E) staining), as shown in FIGS. 8A and 8B .
- the cartilage in the mBSA paws is uniformly and strongly stained by 1-11E, as shown in FIGS. 8C and 8D .
- the left paw injected by saline had no subplantar inflammation. Some cartilage within the left paw joints was stained heterogeneously, perhaps associated with spontaneous osteoarthritis.
- mice Seven week old C57BL/6 male mice were utilized for these experiments (Dell'Accio F et al, Arthritis Res Ther. 2006; 8(5):R139). The mice were anesthetized and subjected to medial para-patellar arthrotomy. The patellar groove was exposed by lateral patellar dislocation. A longitudinal full thickness injury was made in the patellar groove using a custom made device in which the length of a 26G needle was limited by a glass bead (injured knee). The patellar dislocation was then reduced and the joint capsule and the skin sutured in separate layers. The animals were killed after 4 weeks and the knees dissected for histological and histochemical analysis.
- Staining methods are as set out in Example 6 above, except that rabbit anti-myc followed by anti-rabbit-HRP were used to avoid cross-reactivity with mouse antibody in the tissue.
- pFastBac1 pFastBac1.
- AH was created from pFastBac1 (Invitrogen) by cutting out BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI.
- Mouse interferon b (mIFNb) was cloned into the HindIII-EcoRI sites, followed by a MMP cleavage site and 1-11E which were cloned into the NotI and ApaI sites as shown in FIG. 10A .
- the MMP cleavage site can be cleaved by MPP-1 and MMP-3.
- Mouse interferon-beta was amplified with the following primers:
- mIFNBHindFOR (SEQ ID NO: 129) 5′ gct aag ctt atg aac aac agg tgg atT 3′ HindIII Start * reverse: mIFNBEcoRIREV (SEQ ID NO: 130) 5′ CGC GAA TTC GTT TTG GAA GTT TCT GGT 3′
- NotI1-11Efor (SEQ ID NO: 131) 5′cag GC GGC CGC a ATG GCC GAG GTG CAG CTG 3′ NotI * Start reverse: 1-11EApaRev (SEQ ID NO: 132) 5′CTTGGGCCCTCAATGGTGGTGGTGATGGTGTCTAGACCGTTTGATTT CCACCTT 3′
- the mIFN-beta/His construct was cloned by amplifying mIFN-b with HindIII/ApaI with the following primers:
- IFN-beta/MMP/1-11E/His (50.4 kDa) (SEQ ID NO: 134) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNEFGGGGSPLGLWAGGGSA 200 AAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 250 WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 300 AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS 350 VGDRVTITCRASQSISSYLN
- the IFN-beta portion is from amino acids 1 to 182 as follows:
- the MMP linker portion is from amino acids 183 to 202 as follows:
- the 1-11E portion is from amino acids 203 to 446 as follows:
- the His tag is from amino acids 448 to 502 as follows:
- the IFN-beta portion is from amino acids 1 to 184 as follows:
- the His tag is from amino acids 185 to 190 as follows:
- the protocol for expression of the constructs is shown in FIG. 11 .
- the constructs were transformed into competent DH10Bac cells (Invitrogen) to generate bacmid vectors.
- Recombinant bacmid vectors were confirmed by blue-white screening and PCR according to Invitrogen instructions.
- Bacmid DNA was transfected into Sf9 insect cells using cellfectin according to Invitrogen instructions.
- P1 Baculovirus (P1) was harvested from the supernatant of transfected cells, and used to infect fresh Sf9 cells to amplify the virus stocks. P3 virus was used to infect High 5 insect cells for 72 hours, and the supernatant was collected and run on an SDS-PAGE gel. Recombinant proteins were detected by Western blot using anti-tetra-His antibody (Qiagen) and anti-mouse HRP (Sigma).
- test expression of the fusion constructs is shown in FIG. 12 .
- pFastBac1 pFastBac1.
- AH was created from pFastBac1 (Invitrogen) by cutting out BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI.
- TNFR2Fc was cloned into the HindIII-EcoRI sites, followed by a MMP cleavage site and scFv (1-11E or C7) which were cloned into the NotI and ApaI sites as shown in FIG. 10B .
- Mouse TNFR2-Fc was amplified with the following primers:
- forward primer (SEQ ID NO: 141) 5′ GCT aag ctt ATG GCG CCC GCC GCC CTC 3′ reverse primer: (SEQ ID NO: 142) 5′ CTTGAATTCTTTACCCAGAGACCGGGA 3′ 1-11E was amplified with the same primers as above for the INFb.
- TNFR2Fc/MMP/1-11E The sequence of TNFR2Fc/MMP/1-11E is as follows:
- TNFR2Fc portion is as follows:
- the MMP linker portion is as follows:
- the 1-11E portion is as follows:
- the His tag is as follows:
- HEL Hen Egg Lysosyme
- TNFR2Fc portion is as follows:
- the MMP linker portion is as follows:
- the C7 portion is as follows:
- the His tag is as follows:
- the protocol for expression of the constructs is shown in FIG. 11 and is as described above for the IFN-beta/1-11E fusion protein.
- Infected Hi-5 cells were grown for 3 days at 27° C. After 3 days, different 100, 50, 25 and 12 microliter aliquots of cell supernatant were taken for Western blot analysis. Fusion protein was probed with anti-His tag antibodies. As shown in FIGS. 13A, 13B, and 13C the apparent molecular weight of the TNFR2Fc fusion proteins is slightly above 75 kDa which reflects the predicted 50 kDa TNFR2Fc plus 30 kDa scFv.
Landscapes
- Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Optics & Photonics (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Rheumatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention provides a composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) in which the antibody or fragment thereof is conjugated to a pharmaceutically active moiety. The invention also provides a composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) and a detectable label. The invention further provides the use of such compositions in medicine, in particular for the treatment of an arthropathy, and in methods of diagnosis.
Description
- The present invention relates to a novel antibody and its use in the diagnosis and therapy of inflammatory diseases of the joints such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- The final outcome of most rheumatic conditions, the leading cause of disabilities in the western world, is breakdown of articular cartilage. This breakdown is the final outcome of inflammatory events in both rheumatoid arthritis (RA) and osteoarthritis (OA) mediated by either influx of activated leukocytes (RA) or activated chondrocytes (OA). Pro-inflammatory cytokine blockade such as anti-TNFa and IL-1Ra is therefore currently used to treat arthritic conditions, mainly RA. These treatments however, are not consistently effective and the number of patients that fail anti-TNF therapy is increasing. Especially for anti-TNFa treatment there is a risk of serious infections and malignancies. These systemic side effects could be minimised by the development of technologies to target therapeutic agents specifically to the inflamed tissues, but has so far been impeded by the lack of proper target epitope(s) that would be present uniquely in the diseased joint and not in the healthy joint.
- RA is a classic inflammatory form of arthritis, which is a chronic autoimmune disease with extensive synovial inflammation. Influx of activated leukocytes infiltrating the inflamed synovial membrane results in up-regulation of inflammatory cytokines such as TNFa, interleukin-1 (IL-1) and interleukin-6 (IL-6) leading to increase in the levels of matrix metalloproteases (MMP). Moreover, infiltrated inflammatory cells consume increased amounts of oxygen, resulting in the generation of reactive oxidant species (ROS) including superoxide radicals (O2), hydrogen peroxide (H2O2), hydroxyl radicals (OH), hypochlorous acid (HOCl), nitric oxide (NO) and peroxynitrite (ONOO). In addition, sequential oxidative reactions generate reactive oxidants such as advanced glycation end-products (AGE). The combined activities of MMP and ROS may be the cause of the excessive degradation of the extracellular matrix leading to cartilage destruction.
- The immuno-pathological events following the ROS reactivity with cartilage specific collagen type II (CII) protein have been studied recently. A substantial increase in binding of RA sera to CII after chemical post-translational modification in vitro by ROS has been demonstrated in comparison to binding to native non-modified CII, which is significantly greater than in non-RA sera. Post-translational modification in the acute and chronic inflammation by ROS has also been postulated by the presence of other ROS damaged proteins and auto-antibodies against other auto-antigens that are post-translationally modified by ROS. Generation of neoantigenic epitopes on modified CII has been reported in Nissim et al Arthritis & Rheumatism, volume 52 (12) pages 3829-3838 (2005)). Antibodies against IgG-AGE and a T cell response against IgG modified by HOCl and peroxynitrite have also been observed.
- Although synovial inflammation in OA is not as extensive as in RA and inflammatory cells are not significant in numbers, low grade synovitis is nearly a constant feature in OA. Abnormal mechanical force appears to stimulate chondrocytes to produce the same inflammatory mediators and ROS as the infiltrated leukocytes present in inflamed RA joints leading to post translational modifications of CII. There is a report of elevated levels of nitrated CII peptide in sera of patients with OA. The presence of strong staining of nitrotyrosine and low antioxidative capacity in the degenerative region of OA cartilage compared with the intact region from the same sample suggests a possible correlation between oxidative damage and cartilage degradation. As in RA, indirect involvement of oxidative stress has also been evidenced in OA by the fact that: (i) OA is strongly linked with age and in aged cartilage there is accumulation of AGE; and (ii) there is accumulation of lipid peroxidation product and nitrotyrosine.
- There is a need for improved means for diagnosing inflammatory diseases of the joints and for improved therapies for arthropathies such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- It has been found that an antibody raised against post-translationally modified Collagen II (CII) can specifically target the antibody to the sites of inflammation in the joints. This degree of specificity is important since native CII may be present in both inflamed and healthy joints also.
- According to a first aspect of the invention, there is provided a composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) in which the antibody or fragment thereof is conjugated to a pharmaceutically active moiety.
- The present invention therefore provides a novel approach to the targeting of drugs to self-epitopes on Collagen II that are a normal component of the tissue but which become immunogenic after post-translational modification by free radicals as part of a disease process affecting Collagen II.
- The antibody may be a polyclonal antibody or a monoclonal antibody. It may be a human or humanized or chimeric antibody with sequences, residues or domains derived from more than one animal species. Fragments of antibodies include Fc, Fab, scFv, single domain (dAb) antibody, diabody, minibody, and scFv-Fc fragments.
- In one embodiment of the invention, the antibody comprises CDR sequences in the Variable Heavy (VH) Chains and Variable Light (VL) chains as shown in Table 1. CDRH2 and CDRH3 are in the VH chain and CDRL2 and CDRL3 are in the VL chain.
-
TABLE 1 CDRH2 CDRH3 CDRL2 CDRL3 DISSTGSYTAYADSVKG GAGSFDY AASALQS QQSSSTPTT (SEQ ID NO: 1) (SEQ ID NO: 39) (SEQ ID NO: 61) (SEQ ID NO: 86) AISAAGTATAYADSVKG GYDTFDY AASSLQS QQNYGYPNT (SEQ ID NO: 2) (SEQ ID NO: 40) (SEQ ID NO: 62) (SEQ ID NO: 87) SISNSGSYTDYADSVKG GYGSFDY AASTLQS QQANSSPDT (SEQ ID NO: 3) (SEQ ID NO: 41) (SEQ ID NO: 63) (SEQ ID NO: 88) SINNYGSNTAYADSVKG GYSSFDY AASYLQS QQTSSSPDT (SEQ ID NO: 4) (SEQ ID NO: 42) (SEQ ID NO: 64) (SEQ ID NO: 89) SINNYGSNTAYADSVKG GYSSFDY AASYLQS QQTSSSPDT (SEQ ID NO: 4) (SEQ ID NO: 42) (SEQ ID NO: 64) (SEQ ID NO: 89) SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID NO: 43) (SEQ ID NO: 65) (SEQ ID NO: 90) SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID NO: 43) (SEQ ID NO: 65) (SEQ ID NO: 90) SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID NO: 43) (SEQ ID NO: 65) (SEQ ID NO: 90) SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID NO: 43) (SEQ ID NO: 65) (SEQ ID NO: 90) SISYTGNSTDYASVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID NO: 43) (SEQ ID NO: 65) (SEQ ID NO: 90) NIATDGTTTYYADSVKG NSTYFDY SASTLQS QQAATSPTT (SEQ ID NO: 6) (SEQ ID NO: 44) (SEQ ID NO: 66) (SEQ ID NO: 91) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) SISYTGDSTYYADSVKG NYSAFDY YASYLQS QQADSTPTT (SEQ ID NO: 8) (SEQ ID NO: 46) (SEQ ID NO: 65) (SEQ ID NO: 90) SINDSGTTTYYADSVKG NYSAFDY AASDLQS QQSDSAPTT (SEQ ID NO: 9) (SEQ ID NO: 46) (SEQ ID NO: 67) (SEQ ID NO: 93) SIDSAGASTYYADSVKG NYSAFDY NASSLQS QQSDTYPST (SEQ ID NO: 10) (SEQ ID NO: 46) (SEQ ID NO: 68) (SEQ ID NO: 94) SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQSYASPTT (SEQ ID NO: 8) (SEQ ID NO: 46) (SEQ ID NO: 69) (SEQ ID NO: 95) SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQSYASPTT (SEQ ID NO: 8) (SEQ ID NO: 46) (SEQ ID NO: 69) (SEQ ID NO: 95) SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQTGSYPTT (SEQ ID NO: 8) (SEQ ID NO: 46) (SEQ ID NO: 69) (SEQ ID NO: 96) SINATGYGTYYADSVKG NYSDFDY SASALQS QQGDSYPTT (SEQ ID NO: 11) (SEQ ID NO: 47) (SEQ ID NO: 70) (SEQ ID NO: 97) SINSNGTDTYYADSVKG NYSDFDY TASALQS QQGYGAPTT (SEQ ID NO: 12) (SEQ ID NO: 47) (SEQ ID NO: 71) (SEQ ID NO: 98) SISATGSSTYYADSVKG NYSDFDY SASDLQS QQSSYTPTT (SEQ ID NO: 13) (SEQ ID NO: 47) (SEQ ID NO: 72) (SEQ ID NO: 99) SISATGSSTYYADSVKG NYSDFDY SASDLQS QQSSYTPTT (SEQ ID NO: 13) (SEQ ID NO: 47) (SEQ ID NO: 72) (SEQ ID NO: 99) SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 100) SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 100) SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 100) SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 100) SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID NO: 48) (SEQ ID NO: 74) (SEQ ID NO: 102) SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID NO: 48) (SEQ ID NO: 74) (SEQ ID NO: 102) SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID NO: 48) (SEQ ID NO: 74) (SEQ ID NO: 102) SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID NO: 48) (SEQ ID NO: 74) (SEQ ID NO: 102) SIDTTGTTTYFADSVKG NYSSFDY SASYLQS QQGYSAPTT (SEQ ID NO: 17) (SEQ ID NO: 48) (SEQ ID NO: 75) (SEQ ID NO: 103) TISYSGNNTYYADSVKG NYSSFDY TASSLQS QQGYTSPTT (SEQ ID NO: 18) (SEQ ID NO: 48) (SEQ ID NO: 76) (SEQ ID NO: 104) SIDAGGNGTYYADSVKG NYSSFDY TASNLQS QQNNYYPTT (SEQ ID NO: 19) (SEQ ID NO: 48) (SEQ ID NO: 77) (SEQ ID NO: 105) SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 106) SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 106) SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 106) SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 106) SIDSAGNATYYADSVKG NYSSFDY AASTLQS TSNYPTTQQ (SEQ ID NO: 20) (SEQ ID NO: 48) (SEQ ID NO: 78) (SEQ ID NO: 107) SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID NO: 49) (SEQ ID NO: 79) (SEQ ID NO: 108) SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID NO: 49) (SEQ ID NO: 79) (SEQ ID NO: 108) SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID NO: 49) (SEQ ID NO: 79) (SEQ ID NO: 108) SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID NO: 49) (SEQ ID NO: 79) (SEQ ID NO: 108) SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID NO: 49) (SEQ ID NO: 79) (SEQ ID NO: 108) SIATTGDNTYYADSVKG NYSYFDY TASTLQS QQAAGNPTT (SEQ ID NO: 22) (SEQ ID NO: 50) (SEQ ID NO: 80) (SEQ ID NO: 109) AINAYGGSTYYADSVKG NYSYFDY AASSLQS QQGSDYPTT (SEQ ID NO: 23) (SEQ ID NO: 50) (SEQ ID NO: 62) (SEQ ID NO: 110) AINAYGGSTYYADSVKG NYSYFDY AASSLQS QQGSDYPTT (SEQ ID NO: 23) (SEQ ID NO: 50) (SEQ ID NO: 62) (SEQ ID NO: 110) SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID NO: 50) (SEQ ID NO: 76) (SEQ ID NO: 111) SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID NO: 50) (SEQ ID NO: 76) (SEQ ID NO: 111) TIDTAGSYTDYADSVKG NYSYFDY GASTLQS QQSTASPST (SEQ ID NO: 25) (SEQ ID NO: 50) (SEQ ID NO: 81) (SEQ ID NO: 112) SISNNGSSTYYADSVKG NYSYFDY AASNLQS QQTSSYPTT (SEQ ID NO: 26) (SEQ ID NO: 50) (SEQ ID NO: 82) (SEQ ID NO: 113) SIAYGGAGTDYADSVKG NYTAFDY AASYLQS QQGAGSPST (SEQ ID NO: 27) (SEQ ID NO: 51) (SEQ ID NO: 64) (SEQ ID NO: 114) AIANTGSATNYADSVKG NYTAFDY DASTLQS QQRNTSPTT (SEQ ID NO: 28) (SEQ ID NO: 51) (SEQ ID NO: 83) (SEQ ID NO: 115) SISTAGTYTDYADSVKG NYTDFDY SASYLQS QQSNTSPAT (SEQ ID NO: 29) (SEQ ID NO: 52) (SEQ ID NO: 75) (SEQ ID NO: 116) SISTAGTYTDYADSVKG NYTDFDY SASYLQS QQSNTSPAT (SEQ ID NO: 29) (SEQ ID NO: 52) (SEQ ID NO: 75) (SEQ ID NO: 116) SINDTGYTTYYADSVKG NYTYFDY TASTLQS QQAYTAPTT (SEQ ID NO: 30) (SEQ ID NO: 53) (SEQ ID NO: 80) (SEQ ID NO: 117) SIASSGTTTYYADSVKG SYADFDY AASNLQS QQADTYPTT (SEQ ID NO: 31) (SEQ ID NO: 54) (SEQ ID NO: 82) (SEQ ID NO: 118) TITSTGAATAYADSVKG SYATFDY AASYLQS QQAANSPDT (SEQ ID NO: 32) (SEQ ID NO: 55) (SEQ ID NO: 64) (SEQ ID NO: 119) AIDGTGYGTAYADSVKG SYDTFDY GASSLQS QQTSDYPNT (SEQ ID NO: 33) (SEQ ID NO: 56) (SEQ ID NO: 84) (SEQ ID NO: 120) SIANAGTATYYADSVKG SYSNFDY SASTLQS QQASTSPTT (SEQ ID NO: 34) (SEQ ID NO: 57) (SEQ ID NO: 66) (SEQ ID NO: 121) SIDSAGDSTYYADSVKG SYSYFDY TASYLQS QQASDYPTT (SEQ ID NO: 35) (SEQ ID NO: 58) (SEQ ID NO: 85) (SEQ ID NO: 122) SISSSGDTTYYADSVKG SYSYFDY TASTLQS QQSSSNPTT (SEQ ID NO: 36) (SEQ ID NO: 58) (SEQ ID NO: 80) (SEQ ID NO: 123) SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID NO: 59) (SEQ ID NO: 75) (SEQ ID NO: 124) SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID NO: 59) (SEQ ID NO: 75) (SEQ ID NO: 124) SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID NO: 59) (SEQ ID NO: 75) (SEQ ID NO: 124) SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID NO: 59) (SEQ ID NO: 75) (SEQ ID NO: 124) SIDASGANTAYADSVKG TYGTFDY SASYLQS QQSATTPDT (SEQ ID NO: 38) (SEQ ID NO: 60) (SEQ ID NO: 75) (SEQ ID NO: 125) - In one embodiment of the invention, the antibody may be an scFv selected from the group consisting of the following:
- 3-11A, 6-6E, 1-7G, 3-7B, 6-9D, 1-1C, 1-8D, 1-3G, 4-12C, 6-3E, 6-9A, 1-12A, 4-6A, 4-8A, 4-9F, 4-4H, 3-3A, 3-6F, 6-10H, 12E, 3-5G, 3-4D, 3-5D, 6-4E, 3-6B, 3-6G, 4-11F, 6-7H, 1-11E, 1-2F, 1-6H, 3-8D, 1-4D, 4-2F, 3-3B, 3-5C, 6-9C, 4G, 3-12F, 3-4G, 6-11F, 6-11H, 3-2C, 5B, 6-10G, 1-4H, 4-5A, 4-1B, 4-12D, 6-4B, 1-2E, 1-7F, 1-10F, 1-9G, 4-1C, 6-7G, 3-7H, 6-1F, 6-3B, 4H, 3-9A, 6-10D, 3-5H, 3-2F, 1-6G, 3-11H, 6-9F, 3-9D, 4-3H, 3-3E, 3-10C, 3-11E, 6-8C, 6-11D, 4-5H, 6-5F, 6-7F, 1-10D
- These scFvs are listed in Table 3 in the Examples below and comprise the CDRH2, CDRH3, CDRL2 and CDRL3 sequences shown in Table 1.
- In one embodiment of the invention, the scFv may comprise a sequence as shown in Table 2.
-
TABLE 2 Clone I.D. VH-CDR2 VH-CDR3 VL-CDR2 VL-CDR3 1-2E SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID NO: 49) (SEQ ID NO: 79) (SEQ ID NO: 108) 1-11E SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID NO: 48) (SEQ ID NO: 73) (SEQ ID NO: 100) 1-4D SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) 1-12A SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID NO: 45) (SEQ ID NO: 65) (SEQ ID NO: 92) 12E SINDSGTTTYYADSVKG NYSAFDY AASDLQS QQSDSAPTT (SEQ ID NO: 9) (SEQ ID NO: 46) (SEQ ID NO: 67) (SEQ ID NO: 93) 3-9A TIDTAGSYTDYADSVKG NYSYFDY GASTLQS QQSTASPST (SEQ ID NO: 25) (SEQ ID NO: 50) (SEQ ID NO: 81) (SEQ ID NO: 112) 3-5C SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID NO: 48) (SEQ ID NO: 70) (SEQ ID NO: 101) 3-11E SIDSAGDSTYYADSVKG SYSYFDY TASYLQS QQASDYPTT (SEQ ID NO: 35) (SEQ ID NO: 58) (SEQ ID NO: 85) (SEQ ID NO: 122) 3-2F AIANTGSATNYADSVKG NYTAFDY DASTLQS QQRNTSPTT (SEQ ID NO: 28) (SEQ ID NO: 51) (SEQ ID NO: 83) (SEQ ID NO: 115) 3-6G SINSNGTDTYYADSVKG NYSDFDY TASALQS QQGYGAPTT (SEQ ID NO: 12) (SEQ ID NO: 47) (SEQ ID NO: 71) (SEQ ID NO: 98) 3-9D SIASSGTTTYYADSVKG SYADFDY AASNLQS QQADTYPTT (SEQ ID NO: 31) (SEQ ID NO: 54) (SEQ ID NO: 82) (SEQ ID NO: 118) 6-3B SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID NO: 50) (SEQ ID NO: 76) (SEQ ID NO: 111) 6-11D SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID NO: 59) (SEQ ID NO: 75) (SEQ ID NO: 124) 6-9F SINDTGYTTYYADSVKG NYTYFDY TASTLQS QQAYTAPTT (SEQ ID NO: 30) (SEQ ID NO: 53) (SEQ ID NO: 80) (SEQ ID NO: 117) 6-7G SIATTGDNTYYADSVKG NYSYFDY TASTLQS QQAAGNPTT (SEQ ID NO: 22) (SEQ ID NO: 50) (SEQ ID NO: 80) (SEQ ID NO: 109) - Typically, the scFv is 1-11E.
- Oxidised Collagen II (CII) is post-translationally modified Collagen II (CII) that has been oxidised by non enzymatic glycation or by reactive oxidant species (ROS) which may include superoxide radical (O2), hydrogen peroxide (H2O2), hydroxyl radical (OH), hypochlorous acid (HOCl), nitric oxide (NO) and peroxynitrite (ONOO).
- The antigen may therefore be HOCl-Collagen II or Ribose-Collagen II.
- The antibody or fragment thereof is conjugated to the pharmaceutically active moiety which may be a peptide or peptide-based molecule by any suitable means. Where the pharmaceutically active moiety is a peptide or peptide-based molecule the conjugation may be by means of a peptide bond, including the insertion of one or more amino acid residues.
- The conjugation of a peptide or a peptide-based molecule may be achieved by any generally convenient chemical means or biological means (see for example, Wu & Senter Nature Biotechnology, volume 23 (9) pages 1137-1146 (2005); “Chemistry of Protein Conjugation and Crosslinking” by S. S. Wong, CRC Press Inc. (1991)).
- Chemical conjugation typically uses a bifunctional chemical reagent, for example glutaraldehyde can link molecules to the N-terminus of a peptide, carbodiimide can link molecules to the C-terminus of a peptide, succinimide esters (e.g. MBS, SMCC) can bind free amino groups and cysteine residues, benzidine links to tyrosine residues, periodate attaches to carbohydrate groups and isothiocyanate can also link molecules to antibodies.
- Alternatively, a fusion protein may be synthesised using standard recombinant molecular biology techniques (see for example, Sambrook et al “Molecular Cloning: A Laboratory Manual”, 3rd edition, CSHL Press, (2001); Trachsel et al Arthritis Research & Therapy, volume 9 (1) R9 (2007); Nagai Arthritis & Rheumatism, volume 54 (10) pages 3126-3134 (2006)). Methods for producing fusion proteins are described in the Examples herein.
- In certain embodiments of the invention, the insertion of additional amino acid residues between the antibody or fragment thereof and the pharmaceutically active moiety may represent a site for cleavage by a protease. The proteolytic cleavage site may comprise any protease specific cleavage site. The proteolytic cleavage site may include, but is not limited to, a matrix metalloproteinase (MMP) cleavage site, a serine protease cleavage site, a site cleavable by a parasitic protease derived from a pathogenic organism (Zhang et al., J. Mol. Biol. 289, 1239-1251 (1999); Voth et al., Molecular and Biochemical Parasitology, 93, 31-41 (1998); Yoshioka et al., Folia Pharmacologica Japonica, 110, 347-355 (1997); Tort et al., Advances in Parasitology, 43, 161-266 (1999); McKerrow, International Journal for Parasitology, 29, 833-837 (1999); Young et al., International Journal for Parasitology, 29, 861-867 (1999); Coombs and Mottram, Parasitology, 114, 61-80 (1997)) or a site cleavable by the proteins of the complement cascade (Carroll, Annu. Rev. Immunol. 16, 545-568 (1998); Williams et al., Ann. Allergy, 60, 293-300 (1988)).
- The MMP cleavage site may comprise any amino acid sequence which is cleavable by a MMP. The amino acid sequence of the MMP cleavage site may be encoded by a nucleic acid sequence coding for an MMP sequence as shown in
FIGS. 5A, 5B, 5C , and 5D or a sequence of nucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identity, using the default parameters of the BLAST computer program provided by HGMP, thereto. Preferably, the nucleic acid sequence encoding the MMP cleavage site comprises the minimum number of residues required for recognition and cleavage by MMP. - A MMP cleavage site may comprise a number of amino acid residues recognisable by MMP. Moreover, the amino acids of the MMP site may be linked by one or more peptide bonds which are cleavable, proteolytically, by MMP. MMPs which may cleave the MMP site include, but are not limited to, MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 (Yu and Stamenkovic, Genes and Dev. 14, 163-176 (2000); Nagase and Fields, Biopolymers, 40, 399-416 (1996); Massova et al., J. Mol. Model. 3, 17-30 (1997); reviewed in Vu and Werb, Genes and Dev. 14, 2123-2133 (2000)). The sequences of the protein cleavage sites of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 and MMP10 are shown in
FIGS. 5A, 5B, 5C, and 5D . - Preferably, the proteolytic cleavage site of the present invention is cleaved at sites of inflammation and tissue remodeling. More preferably, the proteolytic cleavage site of the present invention is a MMP cleavage site e.g. any one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 as shown in
FIGS. 5A, 5B, 5C, and 5D . - The pharmaceutically active moiety may comprise one or more molecules which may be the same or different, one or more radioisotopes which may be the same or different, or one or more non-radioactive elements which may be the same or different.
- In some embodiments of the invention, the pharmaceutically active moiety may comprise a polypeptide or non-polypeptide molecule. References to a polypeptide include a peptide and vice versa unless the context specifies otherwise.
- The polypeptide may be an antibody or a fragment thereof, such as an anti-TNFalpha monoclonal antibody (for example infliximab or adalimumab), a soluble p75 TNF receptor molecule (for example etanercept) or a IL-1 receptor antagonist (for example anakinra). In such embodiments of the invention, the composition will therefore comprise a bispecific antibody which may be a diabody (scFv with a linker which is too short to allow pairing between VH and VL and therefore the domains are forced to pair with the complementary domain of another scFv to create two antigen binding site), a minibody (composed of two scFv moieties linked via a constant heavy chain region (CH3)), a scFv-Fc molecule, or an intact antibody molecule containing the two separate binding regions.
- For example, a bispecific antibody may comprise a first binding region specific for modified Collagen II (CII) and a second binding region specific for anti-TNFa.
- In one embodiment, the polypeptide is a TNF receptor (TNFR) antibody fusion protein, typically a TNFR2-Fc fusion protein.
- A bispecific antibody of the invention may also further comprise another pharmaceutically active moiety. For example, a composition of the invention may comprise a first binding region specific for modified Collagen II, a second binding region specific for CD64, and a toxin, such as Ricin A.
- Alternatively, the polypeptide may be a growth factor (e.g. TGFβ, epidermal growth factor (EGF), platelet derived growth factor (PDGF), nerve growth factor (NGF), colony stimulating factor (CSF) granulocyte/macrophage colony stimulating factor (GM-CSF), hepatocyte growth factor, insulin-like growth factor, placenta growth factor); differentiation factor, cytokine molecule, for example an interleukin, (e.g. IL1 IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20 or IL-21, either a or (3), an interferon (e.g. IFN-α, IFN-β and IFN-γ), tumour necrosis factor (TNF), IFN-γ inducing factor (IGIF), a bone morphogenetic protein (BMP); a chemokine (for example a MIP (Macrophage Inflammatory Protein) e.g. MIP1α and MIP1β; a MCP (Monocyte Chemotactic Protein) e.g. MCP1, 2 or 3; RANTES (regulated upon activation normal T-cell expressed and secreted)); a trophic factor; a cytokine inhibitor; a cytokine receptor (for example, CD20, CD40, CD40L, CD64); a free-radical scavenging enzyme (e.g. superoxide dismutase or catalase), or a toxin (for example Ricin A toxin, or Pseudomonas exotoxin A), or an active fragment or portion thereof. Typically, the polypeptide is an interferon, typically IFN-β.
- See for example, Trachsel et al Arthritis Research & Therapy, volume 9 (1) R9 (2007) reporting antibody-IL10 fusion protein; Nagai Arthritis & Rheumatism, volume 54 (10) pages 3126-3134 (2006) reporting antibody-toxin fusion protein.
- Other examples of antibody-fusion proteins, include but are not limited to, antibody-TNFalpha, antibody-GM-CSF, and antibody-IL2 fusion proteins
- The pharmaceutically active polypeptide may be derived from the species to be treated e.g. human origin for the treatment of humans.
- The composition may also comprise further peptide sequences which can target the composition inside a cell. Such intracellular targeting sequences include, but are not limited to, the TAT sequence YGRKKRQRRR (SEQ ID NO: 126) (see for example, Cohen-Saidon et al Blood, volume 102 (7), pages 2506-2512 (2003)).
- As used herein “peptide mimetics” includes, but is not limited to, agents having a desired peptide backbone conformation embedded into a non-peptide skeleton which holds the peptide in a particular conformation. Peptide mimetics, which do not have some of the drawbacks of peptides, are of interest in those cases where peptides are not suitable in medicine.
- Peptide mimetics may comprise a peptide backbone which is of the L or D conformation. Examples of peptide mimetics include melanocortin, adrenocorticotrophin hormone (ACTH) and other peptide mimetic agents which play a role in the central nervous system, endocrine system, in signal transduction and in infection and immunity.
- The pharmaceutically active agent may comprise a chemical compound such as a chemotherapeutic agent or other synthetic drug. Alternatively, the pharmaceutically active agent may comprise a peptide nucleic acid (PNA) sequence e.g. a poly-lysine sequence which binds to nucleic acids and permeabilises lipid bilayers (Wyman et al., Biological Chemistry, 379, 1045-1052 (1998)) or a KALA peptide which facilitates transfer through lipid bilayers (Wyman et al., Biochemistry, 36, 3008-3017 (1997)).
- The non-polypeptide may be a glycosaminoglycan molecule, such as glucosamine (suitably, glucosamine HCl) or chondroitin. Alternatively, the non-polypeptide molecule may be a non-steroidal anti-inflammatory drug (NSAID) such as a non-selective NSAID or a selective NSAID. Examples of non-selective NSAIDs include aspirin, ibuprofen, and naproxen. Examples of selective NSAIDs (also called COX-2 inhibitors) include celecoxib (Celebrex®), rofecoxib (Vioxx®) and valdecoxib (Bextra®)). Other substances may include steroids, such as cortisol, or polymeric molecules such as sodium hyaluronate or hyaluronic acid (for example hyaluronan (Hyalgan®) and hylan-GF-20 (Synvisc®)), or drug substances such as colchicine or hydroxychloroquine (Plaquenil®).
- Non-polypeptides may be conjugated to the antibody or fragment thereof using a linker that may be a labile bond in order to permit release of the pharmaceutically active substance. For example, a hydrazone bond may be used where the drug is released under acidic conditions, or a disulfide bond which is reduced to release the drug, or also a peptide bond which is cleaved enzymatically by a protease as described above.
- In some embodiments, the composition may comprise a radioactive element or a non-radioactive element. The radioisotope may be an alpha particle-emitting radionuclide such as 213Bi or 211At, a beta particle-emitting radionuclide such as 131I, 90Y, 177Lu or 67Cu, a gamma radiation-emitting radionuclide such as 99mTc, 123I or 111In, or a positron-emitting radionuclide such as 18F, 64Cu, 68Ga, 86Y or 124I. Radioisotopes may be used in order to render the composition detectably labelled for diagnostic uses of the composition.
- Alternatively, the non-radioactive element may be Au, Fe, Cu, Pt or Ag.
- Combinations of the various elements and substances described above may also be included as desired.
- According to a second aspect of the invention, there is provided a composition of the first aspect for use in medicine. This aspect of the invention includes a composition of the first aspect for use in the treatment of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA). This aspect of the invention therefore extends to a method of treatment of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA), comprising the step of administering to a subject a composition of the first aspect of the invention. The present invention therefore also includes the use of a composition of the first aspect of the invention in the manufacture of a medicament for the treatment of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- A composition of the first aspect of the invention may therefore be formulated as a pharmaceutical composition. Suitably, a pharmaceutical composition may comprise a diluent, excipient, adjuvant and/or physiologically acceptable buffer.
- The pharmaceutical composition may be administered in any effective, convenient manner effective for treating a disease as described above including, for instance, administration by oral, topical, intravenous, intramuscular, intra-articular, intranasal, or intradermal routes among others. In therapy or as a prophylactic, the composition of the invention may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. The injection may suitably be made into the joint affected by the disease.
- For administration to mammals, and particularly humans, it is expected that the daily dosage of the composition of the invention will be from 0.01 mg/kg body weight, typically around 1 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual which will be dependant on factors including the age, weight, sex and response of the individual. The above dosages are exemplary of the average case. There can, of course, be instances where higher or lower dosages are merited, and such are within the scope of this invention
- According to a third aspect of the present invention, there is provided a method for the diagnosis of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA), comprising the steps of administering a detectably labelled composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) to a subject and subsequently detecting the composition. This aspect of the invention therefore extends to a detectably labelled composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) for use in the diagnosis of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA). Such embodiments also extend to the use of such compositions in the manufacture of an agent for the diagnosis of an arthropathy, such as rheumatoid arthritis (RA) and osteoarthritis (OA).
- The detectable label may be a radioactive or a fluorescent label. In some embodiments the radioisotope may be an alpha particle-emitting radionuclide such as 213Bi or 211At, a beta particle-emitting radionuclide such as 131I 90Y, 177Lu or 67Cu, a gamma radiation-emitting radionuclide such as 99mTc, 123I or 111In, or a positron-emitting radionuclide such as 18F, 64Cu, 68Ga, 86Y or 124I. Radioisotopes may be used in order to render the composition detectably labelled for diagnostic uses of the composition.
- For diagnostic purposes, fluorescent dyes such as Alexa Fluor 488 or the Cy3 monofunctional N-hydroxysuccinimide (NHS) ester could also be used.
- According to a fourth aspect of the invention, there is provided a composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) and a detectable label.
- RA is the most common chronic inflammatory autoimmune disease, with disability occurring usually within 10 years. Over activation of the inflammatory pathway leads to synovitis, joint damage and destruction. Key players in the joint inflammation are inflammatory cytokines such as TNFa and IL-1. The efficacy of anti-TNFa monoclonal antibodies (Infliximab and Adalimumab), soluble p75 TNF receptors (Etanercept) and IL-1 receptor antagonist (Anakinra) in the treatment of RA patients unresponsive to traditional therapy is now well established but unfortunately might be associated with an increase in serious infection and malignancies. It is therefore becoming very important to develop targeted delivery of anti-proinflammatory drugs to the inflamed joint rather than systemic administration because cytokines exert their function as auto or paracrine factors with high concentrations only in close vicinity of the producing cell. Systemic administration of sufficient blocking agents that can block the local high physiological concentration will likely cause severe side effects.
- Although CII is the best candidate to target therapy to the joint one needs to find a way to target the drugs solely to the inflamed joints. The present studies show the development of a targeting antibody that will specifically recognise collagen type II that has been modified by ROS present in inflamed joint which then allows targeting to inflammatory damage joint independently of the aetiology.
- By employing the phage display human antibody library, a panel of human scFvs was developed (
FIG. 1 ) that bind only to CII which was modified in vitro by HOCl or glycation, known reactive oxidants in RA. Most importantly this scFv binds only to damaged cartilage but not to normal cartilage (FIG. 3A, 3B, 3C, 3D, 3E, 3F, 3G, 3H ) and inversely correlates with the staining of safranin-O for the integrity of cartilage-specific proteoglycan. Most importantly, when inflammation was induced in only one paw in CH3 mice with antigen induced arthritis model 1-11E diabody localized only to the inflamed paw without any background to any of the other healthy paws (FIG. 4 ). This strongly supports the specificity of 1-11E for damaged CII in vivo in inflammation setting and therefore have potential for targeting anti-TNFa, other pro-inflammatory cytokine blockade or cartilage regenerating factors to inflamed joints. This approach is significantly different from targeting citrullinated peptides that appear as a good biomarker for disease in RA but could not be used as a targeting molecule as its tissue expression is not joint specific. - Preferred features of the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis.
- In one embodiment, a composition of the invention comprises mouse interferon-beta (IFN-β), the scFv 1-11E and a MMP cleavage site. Such a composition can be produced by creating pFastBac1.AH by cutting out a BamHI/HindIII fragment containing multiple cloning sites (MCS) from pFastBac1 (Invitrogen) and replacing this fragment with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI, cloning mouse interferon b (mIFNb) into the HindIII-EcoRI sites and cloning MMP and 1-11E into the NotI and ApaI site as shown in
FIG. 10A . - Mouse interferon-beta is typically amplified using primers having the sequences shown in SEQ ID NO: 129 (forward) and SEQ ID NO: 130 (reverse). 1-11E is typically amplified using primers having the sequences shown in SEQ ID NO: 131 (forward) and SEQ ID NO: 132 (reverse), wherein 1-11E is amplified with NotI/ApaI ends to include a histidine (His) tag.
- 1-11E is typically then cloned into FastBac1.AH mIFN-b/MMP/SP/His and cut with Not/Apa to liberate SP/His. The mIFN-beta/His construct is typically cloned by amplifying mIFN-b with HindIII/ApaI using primers having the sequence shown in SEQ ID NO: 129 (forward) and SEQ ID NO: 133 (reverse).
- The constructs are then typically transformed into DH10Bac cells (Invitrogen).
- In another embodiment, a composition of the invention comprises TNF receptor 2-Fc (TNFR2Fc), an scFv (either 1-11E or C7 as a negative control) and a MMP cleavage site. Such a composition can be produced by creating pFastBac1.AH from pFastBac1 (Invitrogen) by cutting out a BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing this fragment with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI, cloning TNFR2Fc into the HindIII-EcoRI sites and cloning a MMP cleavage site and scFv (1-11E or C7) into the NotI and ApaI sites as shown in
FIG. 10B . - Mouse TNFR2-Fc is typically amplified using primers having the sequences shown in SEQ ID NO: 141 (forward) and SEQ ID NO: 142 (reverse). 1-11E is typically amplified using primers having the sequences shown in SEQ ID NO: 131 (forward) and SEQ ID NO: 132 (reverse), wherein 1-11E is amplified with NotI/ApaI ends to include a histidine (His) tag.
- Expression of the constructs is typically carried out using a protocol set out in
FIG. 11 . Such a protocol typically involves the following steps: -
- 1. Transforming the constructs into competent DH10Bac cells (Invitrogen) to generate bacmid vectors.
- 2. Confirming recombinant bacmid vectors by blue-white screening and PCR, typically according to Invitrogen instructions.
- 3. Transfecting bacmid DNA into Sf9 insect cells using cellfectin, typically according to Invitrogen instructions.
- 4. Harvesting baculovirus (P1) from the supernatant of transfected cells.
- 5. Using the harvested baculovirus to infect fresh Sf9 cells to amplify the virus stocks.
- 6. Using P3 virus to infect High 5 insect cells, typically for 72 hours.
- In one embodiment, infected High 5 cells are grown for 3 days at 27° C.
- The supernatant is typically then collected and run on an SDS-PAGE gel. Recombinant proteins can be detected by Western blot, for example using anti-tetra-His antibody (Qiagen) and anti-mouse HRP (Sigma).
- The invention will now be described by way of reference to the following Examples which are present for the purposes of illustration only and are not to be construed as being limiting on the present invention. Reference is also made in the Examples to the following drawings in which:
-
FIG. 1 shows representative ELISA of unique scFvs -
FIG. 2 shows Western Blotting with scFv 1-11E probe -
FIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H show specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with OA in photographs (A) to (H). -
FIG. 4 shows the localisation of scFv 1-11E in inflamed paw. -
FIGS. 5A, 5B, 5C, and 5D show the sequences of the protein cleavage sites of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 and MMP10. -
FIG. 6 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with OA. -
FIG. 7 shows specific binding to damaged human cartilage tissue by anti-ROS-modified CII scFv in patients with RA. -
FIGS. 8A, 8B, 8C, 8D, and 8E show: (A) and (B) histological staining of the right paw; (C) and (D) 1-11E staining of cartilage in the paw; (E) staining with a non-relevant scFv; all in a mouse RA model. -
FIG. 9 shows staining of a joint surface injury in a mouse OA model. -
FIGS. 10A and 10B show the construction of (A) an IFN-β/1-11E fusion protein; and (B) a TNFR2Fc/scFv fusion protein. -
FIG. 11 shows the protocol used for expression of the IFN-β/1-11E fusion protein and the TNFR2Fc/scFv fusion protein. -
FIG. 12 is a Western blot of the IFN-β/1-11E fusion protein. -
FIGS. 13A, 13B, and 13C is a Western blots of (A) and (B) TNFR2Fc/1-11E fusion proteins; (C) a TNFR2Fc/C7 fusion protein. - CII was prepared from bovine cartilage as in Miller (Miller, Biochemistry 11(26): 4903-4909, 1972) and subsequently exposed to reactive oxygen generating systems as previously described (Nissim A, 2005). Briefly, CII was modified with (−OH), HOCl (Hawkins CL, 2001; Hawkins CL, 2002), (ONOO+), or 2M ribose by ON incubation at 37° C. Bovine serum albumin (BSA, Sigma) was also modified as above and was used as control antigen.
- Phage display antibody technology (Winter G et al, Annu. Rev. Immunol. 12: 433-455, 1994) was used to raise a single chain fragment variable (scFv) that binds only to CII that has been post-translationally modified by free radicals.
- A human semi-synthetic scFv library constructed from a single human framework for VH (DP-47 and JH4) and VL (DPK9 and JK1) was employed, in which diversity was incorporated in CDR3 and CDR2 (de Wildt R. M et al, Nat. Biotechnol. 18(9): 989-994, 2000). To select for phage binding to modified CII and not to native non-modified CII, subtractive selection was performed using native non-modified CII for subtraction. HOCl modified CII was used as a target for panning as binding to HOCl modified CII was strongest in RA sera (Nissim A, 2005). Glycated CII was used in parallel. Briefly, immunotubes (Nunc-Immuno Tubes, Maxi-Sorp, Nunc, Denmark) were coated with 10 μg/ml CII in phosphate-buffered saline (PBS). After blocking with 2% marvel in PBS (MPBS) coated tubes were exposed for 2 hours to 1013 transforming units (tu) of the phage library in 2% MPBS. Unbound phage were then transferred to a second immunotube previously coated with HOCl or ribose-modified CII for a further 2 hours incubation at room temperature. Modified CII-bound phage were then used to infect E. coli TG-1 and rescued by helper phage as described (Harrison J. L, 1996). The panning process was repeated three times and E. coli TG-1 was infected with the final phage eluted after the third round and individual ampicillin-resistant colonies (phage clones) were selected for further analysis.
- Screening for positive anti-modified CII phage clones was first performed by enzyme-linked immunosorbent assay (ELISA), as previously described (Harrison J. L, 1996). Microtiter plate (Nunc, Paisley, UK) wells were coated with 10 μg/ml native or modified CII and incubated with 100 μl phage suspension for 90 minutes. In addition, native and modified BSA were used as negative control. After removal of the supernatants, the amount of bound phage was determined using peroxidase-labeled anti-M13 antibodies (GE Healthcare: Ltd, Little Chalfont, Buckinghamshire) and developed by using 100
mM - The entire scFv DNA fragment of each modified CII bound phage clone was sequenced using the primers LMB-3 (5′-C AGGAAACAGCTATGAC) (SEQ ID NO: 127) and Fd-Seq (5′-GAATTTTCTGTATGAGG) (SEQ ID NO: 128). Sequences were analyzed using Chromas (Technelysium Pty Ltd) and VBASE (http://vbase.mrc-cpe.cam.ac.uk), to identify unique scFv sequences as shown in Table 3.
-
TABLE 3 Clone Antigen CDRH2 CDRH3 CDRL2 CDRL3 3-11A HOCl-CII DISSTGSYTAYADSVKG GAGSFDY AASALQS QQSSSTPTT (SEQ ID NO: 1) (SEQ ID (SEQ ID (SEQ ID NO: NO: 39) NO: 61) 86) 6-6E HOCl-CII AISAAGTATAYADSVKG GYDTFDY AASSLQS QQNYGYPNT (SEQ ID NO: 2) (SEQ ID (SEQ ID (SEQ ID NO: NO: 40) NO: 62) 87) 1-7G Ribose-CII SISNSGSYTDYADSVKG GYGSFDY AASTLQS QQANSSPDT (SEQ ID NO: 3) (SEQ ID (SEQ ID (SEQ ID NO: NO: 41) NO: 63) 88) 3-7B HOCl-CII SINNYGSNTAYADSVKG GYSSFDY AASYLQS QQTSSSPDT (SEQ ID NO: 4) (SEQ ID (SEQ ID (SEQ ID NO: NO: 42) NO: 64) 89) 6-9D HOCl-CII SINNYGSNTAYADSVKG GYSSFDY AASYLQS QQTSSSPDT (SEQ ID NO: 4) (SEQ ID (SEQ ID (SEQ ID NO: NO: 42) NO: 64) 89) 1-1C Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) T-8D Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 1-3G Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 4-12C Ribose-CII SISYTGNSTDYADSVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 6-3E HOCl-CII SISYTGNSTDYASVKG GYTAFDY YASYLQS QQADSTPTT (SEQ ID NO: 5) (SEQ ID (SEQ ID (SEQ ID NO: NO: 43) NO: 65) 90) 6-9A HOCl-CII NIATDGTTTYYADSVKG NSTYFDY SASTLQS QQAATSPTT (SEQ ID NO: 6) (SEQ ID (SEQ ID (SEQ ID NO: NO: 44) NO: 66) 91) I-12A Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-6A Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-8A Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-9F Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 4-4H Ribose-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 3-3A HOCl-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 3-6F HOCl-CII SISNSGTNTDYADSVKG NYASFDY YASYLQS QQGSASPST (SEQ ID NO: 7) (SEQ ID (SEQ ID (SEQ ID NO: NO: 45) NO: 65) 92) 6-10H HOCl-CII SISYTGDSTYYADSVKG NYSAFDY YASYLQS QQADSTPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 65) 90) 12E Unknown SINDSGTTTYYADSVKG NYSAFDY AASDLQS QQSDSAPTT (SEQ ID NO: 9) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 67) 93) 3-5G HOCl-CII SIDSAGASTYYADSVKG NYSAFDY NASSLQS QQSDTYPST (SEQ ID NO: 10) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 68) 94) 3-4D HOCl-CII SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQSYASPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 69) 95) 3-5D HOCl-CII SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQSYASPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 69) 95) 6-4E HOCl-CII SISYTGDSTYYADSVKG NYSAFDY TASNLQS QQTGSYPTT (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID NO: NO: 46) NO: 69) 96) 3-6B HOCl-CII SINATGYGTYYADSVKG NYSDFDY SASALQS QQGDSYPTT (SEQ ID NO: 11) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 70) 97) 3-6G HOCl-CII SINSNGTDTYYADSVKG NYSDFDY TASALQS QQGYGAPTT (SEQ ID NO: 12) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 71) 98) 4-11F Ribose-CII SISATGSSTYYADSVKG NYSDFDY SASDLQS QQSSYTPTT (SEQ ID NO: 13) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 72) 99) 6-7H HOCl-CII SISATGSSTYYADSVKG NYSDFDY SASDLQS QQSSYTPTT (SEQ ID NO: 13) (SEQ ID (SEQ ID (SEQ ID NO: NO: 47) NO: 72) 99) 1-11E Ribose-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 1-2F Ribose-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 1-6H Ribose-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 3-8D HOCl-CII SIDDSGATTYYADSVKG NYSSFDY YASSLQS QQAANYPTT (SEQ ID NO: 14) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 100) 1-4D Ribose-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 4-2F Ribose-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 3-3B HOCl-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 3-5C HOCl-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 6-9C HOCl-CII SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 4G Unknown SIASTGDSTYYADSVKG NYSSFDY SASALQS QQASNYPTT (SEQ ID NO: 15) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 70) 101) 3-12F HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 3-4G HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 6-11F HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 6-11H HOCl-CII SISTNGSSTYYADSVKG NYSSFDY DASGLQS QQGDTSPTT (SEQ ID NO: 16) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 74) 102) 3-2C HOCl-CII SIDTTGTTTYFADSVKG NYSSFDY SASYLQS QQGYSAPTT (SEQ ID NO: 17) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 75) 103) 5B Unknown TISYSGNNTYYADSVKG NYSSFDY TASSLQS QQGYTSPTT (SEQ ID NO: 18) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 76) 104) 6-10G HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY TASNLQS QQNNYYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 77) 105) 1-4H Ribose-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 4-5A HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 4-1B HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 4-12D HOCl-CII SIDAGGNGTYYADSVKG NYSSFDY YASSLQS QQSDAYPTT (SEQ ID NO: 19) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 73) 106) 6-4B HOCl-CII SIDSAGNATYYADSVKG NYSSFDY AASTLQS TSNYPTTQQ (SEQ ID NO: 20) (SEQ ID (SEQ ID (SEQ ID NO: NO: 48) NO: 78) 107) 1-2E Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 1-7F Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 1-10F Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 1-9G Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 4-1C Ribose-CII SITDSGDTTYYADSVKG NYSTFDY SASSLQS QQSNATPTT (SEQ ID NO: 21) (SEQ ID (SEQ ID (SEQ ID NO: NO: 49) NO: 79) 108) 6-7G HOCl-CII SIATTGDNTYYADSVKG NYSYFDY TASTLQS QQAAGNPTT (SEQ ID NO: 22) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 80) 109) 3-7H HOCl-CII AINAYGGSTYYADSVKG NYSYFDY AASSLQS QQGSDYPTT (SEQ ID NO: 23) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 62) 110) 6-1F HOCl-CII AINAYGGSTYYADSVKG NYSYFDY AASSLQS QQGSDYPTT (SEQ ID NO: 23) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 62) 110) 6-3B HOCl-CII SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 76) 111) 4H Unknown SIATTGTSTTYADSVKG NYSYFDY TASSLQS QQGSTAPTT (SEQ ID NO: 24) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 76) 111) 3-9A HOCl-CII TIDTAGSYTDYADSVKG NYSYFDY GASTLQS QQSTASPST (SEQ ID NO: 25) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 81) 112) 6-10D HOCl-CII SISNNGSSTYYADSVKG NYSYFDY AASNLQS QQTSSYPTT (SEQ ID NO: 26) (SEQ ID (SEQ ID (SEQ ID NO: NO: 50) NO: 82) 113) 3-5H HOCl-CII SIAYGGAGTDYADSVKG NYTAFDY AASYLQS QQGAGSPST (SEQ ID NO: 27) (SEQ ID (SEQ ID (SEQ ID NO: NO: 51) NO: 64) 114) 3-2F HOCl-CII AIANTGSATNYADSVKG NYTAFDY DASTLQS QQRNTSPTT (SEQ ID NO: 28) (SEQ ID (SEQ ID (SEQ ID NO: NO: 51) NO: 83) 115) 1-6G Ribose-CII SISTAGTYTDYADSVKG NYTDFDY SASYLQS QQSNTSPAT (SEQ ID NO: 29) (SEQ ID (SEQ ID (SEQ ID NO: NO: 52) NO: 75) 116) 3-11H HOCl-CII SISTAGTYTDYADSVKG NYTDFDY SASYLQS QQSNTSPAT (SEQ ID NO: 29) (SEQ ID (SEQ ID (SEQ ID NO: NO: 52) NO: 75) 116) 6-9F HOCl-CII SINDTGYTTYYADSVKG NYTYFDY TASTLQS QQAYTAPTT (SEQ ID NO: 30) (SEQ ID (SEQ ID (SEQ ID NO: NO: 53) NO: 80) 117) 3-9D HOCl-CII SIASSGTTTYYADSVKG SYADFDY AASNLQS QQADTYPTT (SEQ ID NO: 31) (SEQ ID (SEQ ID (SEQ ID NO: NO: 54) NO: 82) 118) 4-3H Ribose-CII TITSTGAATAYADSVKG SYATFDY AASYLQS QQAANSPDT (SEQ ID NO: 32) (SEQ ID (SEQ ID (SEQ ID NO: NO: 55) NO: 64) 119) 3-3E HOCl-CII AIDGTGYGTAYADSVKG SYDTFDY GASSLQS QQTSDYPNT (SEQ ID NO: 33) (SEQ ID (SEQ ID (SEQ ID NO: NO: 56) NO: 84) 120) 3-10C HOCl-CII SIANAGTATYYADSVKG SYSNFDY SASTLQS QQASTSPTT (SEQ ID NO: 34) (SEQ ID (SEQ ID (SEQ ID NO: NO: 57) NO: 66) 121) 3-11E HOCl-CII SIDSAGDSTYYADSVKG SYSYFDY TASYLQS QQASDYPTT (SEQ ID NO: 35) (SEQ ID (SEQ ID (SEQ ID NO: NO: 58) NO: 85) 122) 6-8C HOCl-CII SISSSGDTTYYADSVKG SYSYFDY TASTLQS QQSSSNPTT (SEQ ID NO: 36) (SEQ ID (SEQ ID (SEQ ID NO: NO: 58) NO: 80) 123) 6-11D HOCl-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 4-5H Ribose-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 6-5F HOCl-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 6-7F HOCl-CII SIDTGGSYTDYADSVKG SYTTFDY SASYLQS QQGSNSPTT (SEQ ID NO: 37) (SEQ ID (SEQ ID (SEQ ID NO: NO: 59) NO: 75) 124) 1-10D Ribose-CII SIDASGANTAYADSVKG TYGTFDY SASYLQS QQSATTPDT (SEQ ID NO: 38) (SEQ ID (SEQ ID (SEQ ID NO: NO: 60) NO: 75) 125) - The reactive phage clones obtained from E. coli TG-1 bacteria were used to infect E. coli HB2151 non-suppressor bacterial strain to obtain soluble scFv. After overnight induction with 1 mM IPTG at 30° C., the antibody fragments, derived from the
V H3 family, were harvested from the supernatant and periplasmic space as described (Harrison J. L, 1996) and purified on a protein A affinity column (GE Healthcare Ltd, Little Chalfont, Buckinghamshire). Binding of purified scFv to modified CII was first analyzed by ELISA as above except that mouse anti-myc tag antibody (Santa Cruz Biotechnology, INC, Wembley, UK) followed by anti-mouse-HRP conjugate (Sigma, Dorset, UK) were used to probe bound scFv. - Anti-Modified CII scFv Raised by Phage Display Human Antibody Library
- After three rounds of subtractive selection 82 phage clones specific to either glycated CII or HOCl modified CII were selected out of which 42 clones had unique sequences. 15 representative clones with different binding patterns but with good expression were then studied for further analysis (
FIG. 1 and Table 1). As shown inFIG. 1 , out of these 15 clones there were 9 clones with stronger binding to modified CII, 3 clones bound to all forms of CII and and 3 clones had no binding reactivity to any form of CII. Three scFvs that have different binding characteristics were then further studied: Clone 1-11E binds to modified CII (glycated, HOCl and to some extent to peroxynitrated CII), clone 6-11D binds to both native and modified CII and clone 12E that does not bind to any form of CII. None of these scFv bound to native or free radical modified BSA, or to collagen type III (data not shown). - Western blot using scFv as probe and modified or native CII as target antigens was done as described (Nissim A, 2005). Briefly, modified and native CII (2 μg of each) were run on a 7.5% denaturing SDS gel and electroblotted into a nitrocellulose membrane. After blocking with 2% MPBS, membranes were incubated with 10 μg/ml purified scFv in 2% MPBS for 2 hr at room temperature, followed by incubation with mouse anti-myc tag (Santa Cruz Biotechnology, INC, Wembley, UK) and then with anti-mouse-HRP (Sigma, Dorset). Membranes were washed three times with 0.1% Tween PBS (5 min each) and three times with PBS (5 min each) before development with ECL (GE Healthcare Ltd, Little Chalfont, Buckinghamshire).
- Comparative Analysis of Human RA Serum and scFv Binding to CII by Western Blotting
- 1-11E binds several CII fragments between 50 and 150 kDa as well as to a band >250 kDa which resulted from CII cross linking due to the ROS reactivity (
FIG. 2 lane 2-5). 1-11E also binds to native CII corresponding to a band below 150KDa (FIG. 2 lane 1). Binding to native CII in Western blotting was also seen in sera from RA patients that did not bind to native CII in ELISA but only to ROS modified CII in ELISA (for example sera 33 (Nissim A, 2005)). - One osteochondral sample was obtained from the femoral condyle of a patient (female, 63 years old) undergoing prosthetic knee replacement for OA. One sample of normal human cartilage was obtained post-mortem from a preserved area of a knee with unicompartimental OA undergoing joint replacement (female, 54 years old). In both cases, cartilage was fixed overnight at 4° C. in 4% paraformaldehyde, decalcified for 15 days in 0.5M EDTA at 4° C., washed in PBS, and embedded in paraffin according to standard protocols. Safranin O staining was performed according to standard protocols (Rosenberg, 1971). All samples were obtained in accordance with institutional policies and regulations.
- For immunostaining, 5 mm thick sections were cut, deparaffinized and hydrated according to standard protocols. After endogenous peroxidase quenching in 0.5% hydrogen peroxide for 15 min antigen retrieval was done by 45 min incubation of slides with 3 mg/ml pepsin (Zymed, Chandlers Ford, Hampshire, UK) at 37° C. followed by two washes with PBS. Endogenous avidin activity was blocked using a commercially available kit (Vector Laboratories, Orton Southgate, Peterborough, UK) according to the manufacturer's instructions. This was followed by 30 min blocking with 0.5% BSA. Immunostaining was performed using the selected scFv (10 μg/ml and 1 μg/ml) as well as control commercial mouse anti-CII antibodies (diluted 1:100 and 1:1000 dilution; Chemicon International, Chandlers Ford, Hampshire, UK) and polyclonal anti-CII antibodies (diluted 1:100, 1:1000) from collagen induced arthritis (CIA) mice. ScFv or control antibodies were added to the slide in blocking buffer (0.5% BSA in PBS plus 0.05% sodium azide) and left overnight at 4° C. When scFv were used for probing, next day slides were washed with PBS for 2 minutes and incubated for 30 minutes with anti-myc tag mouse antibodies to bind to the myc tag incorporated at the carboxy terminal end of the scFv (diluted 1:200, Santa Cruz Biotechnology Inc, Wembley, UK). After two washes as above anti-mouse biotinylated antibodies were added (Vector kit PK-6102) followed by two washes with PBS and development with DAB substrate (DAKO, Ely, Cambridgeshire, UK) and nuclear counterstaining with Mayer's haematoxylin. Slides were finally dehydrated and mounted with DPX mount (BDH, London, UK)
- Specific Binding to Damaged Human Cartilage Tissue by Anti-ROS-Modified CII scFv
- The cartilage extracellular matrix is a complex structure where several molecules interact to form a structural and functional unit. There is therefore the chance that the tertiary and quaternary structure of collagens in the intact tissue may alter the specificity of binding of the phage antibodies that had been selected in vitro. To determine binding specificity in the intact tissue, the capacity of anti-ROS-modified CII scFv to bind to CII within the matrix complex structure and to present immunoreactivity with damaged OA cartilage as opposed to normal cartilage was tested. 1-11E stained the extracellular matrix of cartilage tissue that displayed marked features of OA (
FIG. 3A, 3B ) with mostly pericellular staining (FIG. 3B ), with severe damage of the extracellular matrix determined with reduced staining with safranin O (FIG. 3C ). No staining by scFv 1-11E was detected when using histologically normal cartilage from normal cartilage (FIG. 3D, 3E ). By contrast, polyclonal antibodies from CIA mice bound the OA cartilage in both the damaged and non-damaged regions strongly (FIG. 3F ) and weakly stained with safranin O (FIG. 3C ). A commercial anti-CII mAb did not stain the damaged cartilage areas stained by 1-11E on an adjacent section (FIG. 3G ) but intensely stained a histologically normal cartilage (FIG. 3H ), suggesting that the epitope recognised by the commercial antibody is lost in the OA section. -
FIG. 6 also shows staining of OA cartilage. Although synovial inflammation in OA is not as extensive as in RA and inflammatory cells are not significant in numbers, low grade synovitis is nearly a constant feature in OA. Abnormal mechanical force appears to stimulate chrondocytes to produce some of the same inflammatory mediators and ROS as the infiltrating leukocytes present in inflamed RA joints, leading to post-translational modifications of CII in OA.FIG. 6 confirms the results shown inFIGS. 3A, 3B, 3C, 3D, 3E, 3F, 3G, and 3H and shows that staining of OA cartilage section is only pericellular, around the chrondocytes. - A further sample was obtained from a patient (female, 47 years old) undergoing total right knee replacement for RA. Fixing and staining protocols were as described above.
-
FIG. 7 shows staining of the RA cartilage. In RA, infiltrating inflammatory cells consume increased amounts of oxygen, resulting in the generation of reactive oxygen species (ROS), which may cause excessive degradation of the extracellular matrix leading to cartilage destruction and chemical post-translational modification of CII by ROS.FIG. 7 shows uniteral staining of RA cartilage across the section. This is due to the high influx of immune cells that produce high levels of ROS. - Out of the unique scFv assessed for specific binding to modified CII as well as best expression in bacteria, the most promising scFv, 1-11E of 25 kDa, was engineered to a larger fragment of 55 KDa. The linker between the VH and VL was shortened by digesting the phagemid vector with XhoI and SalI and relegation. This results in bivalent diabody, a superior molecule with an increased half life (Hudson, 2005) built from two scFv. Expression and screening of diabody binders was done as above. Molecular weight profile of the resulted expressed diabody was analyzed by gel filtration.
- Male C3H mice (age 17-19 weeks) were used. 100 mg of dessicated non-viable T.B. strain H37RA (Difco 231141) was added to 30 ml of incomplete Freunds adjuvant (IFA, Difco 263910) to form complete Freunds adjuvant (CFA). An equal volume of CFA was added to a 2 mg/ml solution (in PBS) of methylated BSA (mBSA) (Sigma A1009). The mixture was then emulsified on ice using an Ultra-Turrax T25 homogeniser at 13500-20500 rpm until a fluffy milky consistency was obtained. Mice were anaesthetised with Hypnorm, and 100 μl of 1 mg/ml (i.e. 100 μg) mBSA in CFA was injected over 2-3 separate sites intradermally. 1 week later, the immunisation was repeated as previously, except that no bacteria were added (i.e. IFA/mBSA). Two weeks after the 2nd immunisation, mice were anaesthetised with nitrous/oxygen and halothane, and inflammation was induced by injecting 50 μl of 1 mg/ml (i.e. 50 μg) mBSA in PBS into the animals' left hind paw. As a control, 50 μl PBS was injected into the right hind paw. Inflammation was measured using callipers to measure the paw thickness. Swelling was seen only in the right paws from 24 hours, and persisted until 1 week later. 2 weeks later, the swelling had totally subsided.
- 50 μg of 1-11E diabody was radiolabelled with 20 MBq of sodium [I-125] iodide (GE Healthcare, Amersham, UK) using the iodogen method (Perbio Science, Cramlingham, UK) and diluted in PBS to a final volume of 240 μl. Radiochemical purity was determined by thin-layer chromatography on silica gel (ITLC, Pall Corporation, Portsmouth, UK) using 85% methanol as mobile phase. A volume of 100 μl of the labeled diabody was injected intravenously via the tail vein into two arthritis-bearing C3H mice 24 hours after injection of the mBSA. Four and 22 hours later the mice were anaesthetized by ip injection of Ketamine/Xylazine. The mice were imaged on a NanoSPECT/CT scanner (Bioscan Inc, Washington, USA) using a four-detector/36×1.4 mm pinhole configuration. 30-50,000 counts were acquired for the SPECT study over 20-50 minutes.
- Imaging of 1-11E Localisation into the Inflamed Paw
- SPECT and CT images from the NanoSPECT/CT camera were fused and displayed using PMOD software.
FIG. 4 shows a representative image acquired 22 hours after injection of the radiotracer. Increased uptake of radioactivity is clearly seen in the rear left (inflamed) paw relative to the right (normal paw). - Staining of cartilage was observed in the mouse mBSA model described in Example 8 above, except that C57BL mice were used.
- Mice were sensitized with mBSA (100 μg) in CFA intradermally at the base of the tail, and challenged either intra-articularly (both knees) or intra-plantarly (right, saline left) with 500 μg mBSA in saline 14 days later.
- Staining of cartilage is shown in
FIGS. 8A, 8B, 8C, 8D, and 8E . - Paw:
- 12 hours post challenge with mBSA, the right paw was grossly inflamed in the subplantar region (seen by haematoxylin and eosin (H&E) staining), as shown in
FIGS. 8A and 8B . The cartilage in the mBSA paws is uniformly and strongly stained by 1-11E, as shown inFIGS. 8C and 8D . The left paw injected by saline had no subplantar inflammation. Some cartilage within the left paw joints was stained heterogeneously, perhaps associated with spontaneous osteoarthritis. - Staining was observed in mice with joint surface injury.
- Seven week old C57BL/6 male mice were utilized for these experiments (Dell'Accio F et al, Arthritis Res Ther. 2006; 8(5):R139). The mice were anesthetized and subjected to medial para-patellar arthrotomy. The patellar groove was exposed by lateral patellar dislocation. A longitudinal full thickness injury was made in the patellar groove using a custom made device in which the length of a 26G needle was limited by a glass bead (injured knee). The patellar dislocation was then reduced and the joint capsule and the skin sutured in separate layers. The animals were killed after 4 weeks and the knees dissected for histological and histochemical analysis.
- Staining methods are as set out in Example 6 above, except that rabbit anti-myc followed by anti-rabbit-HRP were used to avoid cross-reactivity with mouse antibody in the tissue.
- As shown in
FIG. 9 , there is strong staining at the site of the injury. - Cloning of IFN-beta/1-11E
- pFastBac1.AH was created from pFastBac1 (Invitrogen) by cutting out BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI.
- Mouse interferon b (mIFNb) was cloned into the HindIII-EcoRI sites, followed by a MMP cleavage site and 1-11E which were cloned into the NotI and ApaI sites as shown in
FIG. 10A . The MMP cleavage site can be cleaved by MPP-1 and MMP-3. - Mouse interferon-beta was amplified with the following primers:
-
forward: mIFNBHindFOR (SEQ ID NO: 129) 5′ gct aag ctt atg aac aac agg tgg atT 3′ HindIII Start * reverse: mIFNBEcoRIREV (SEQ ID NO: 130) 5′ CGC GAA TTC GTT TTG GAA GTT TCT GGT 3′ - 1-11E was amplified with the following primers:
-
forward: NotI1-11Efor: (SEQ ID NO: 131) 5′cag GC GGC CGC a ATG GCC GAG GTG CAG CTG 3′NotI * Start reverse: 1-11EApaRev (SEQ ID NO: 132) 5′ CTTGGGCCCTCAATGGTGGTGGTGATGGTGTCTAGACCGTTTGATTT CCACCTT 3′ - 1-11E was amplified with NotI/ApaI ends to include a histidine (His) tag and then cloned into FastBac1.AH mIFN-b/MMP/SP/His and cut with Not/Apa to liberate SP/His.
- The mIFN-beta/His construct was cloned by amplifying mIFN-b with HindIII/ApaI with the following primers:
-
forward (this primer is the same as the primer used for cloning IFN-b/MMP/1-11E/His): (SEQ ID NO: 129) mIFNBHindFOR 5′ gct aag ctt atg aac aac agg tgg atT 3′ HindIII Start * reverse primer: (SEQ ID NO: 133) mIFN-bApaRev 5′ CTTGGGCCCTCAATGGTGGTGGTGATGGTGTCTAGAGTTTTGGAAGTTTCT GGT 3′ - These constructs were transformed into DH10Bac cells from Invitrogen and the sequence was confirmed as follows:
-
IFN-beta/MMP/1-11E/His (50.4 kDa) (SEQ ID NO: 134) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNEFGGGGSPLGLWAGGGSA 200 AAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 250 WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 300 AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS 350 VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSG 400 SGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKVEIKRDIHHHH 450 HH* - Within this sequence, the IFN-beta portion is from
amino acids 1 to 182 as follows: -
(SEQ ID NO: 135) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQN - The MMP linker portion is from amino acids 183 to 202 as follows:
- The 1-11E portion is from amino acids 203 to 446 as follows:
-
(SEQ ID NO: 137) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE 250 WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC 300 AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS 350 VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSG 400 SGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKVEIKRDI - The His tag is from amino acids 448 to 502 as follows:
-
(SEQ ID NO: 138) HHHH 450 HH* IFN-beta/His (23.2 kDa) (SEQ ID NO: 139) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNDIHHHHHH* - Within this sequence, the IFN-beta portion is from
amino acids 1 to 184 as follows: -
(SEQ ID NO: 140) MNNRWILHAAFLLCFSTTALSINYKQLQLQERTNIRKCQELLEQLNGKIN 50 LTYRADFKIPMEMTEKMQKSYTAFAIQEMLQNVFLVFRNNFSSTGWNETI 100 VVRLLDELHQQTVFLKTVLEEKQEERLTWEMSSTALHLKSYYWRVQRYLK 150 LMKYNSYAWMVVRAEIFRNFLIIRRLTRNFQNDI - The His tag is from
amino acids 185 to 190 as follows: - The protocol for expression of the constructs is shown in
FIG. 11 . - Briefly, the constructs were transformed into competent DH10Bac cells (Invitrogen) to generate bacmid vectors. Recombinant bacmid vectors were confirmed by blue-white screening and PCR according to Invitrogen instructions. Bacmid DNA was transfected into Sf9 insect cells using cellfectin according to Invitrogen instructions.
- Baculovirus (P1) was harvested from the supernatant of transfected cells, and used to infect fresh Sf9 cells to amplify the virus stocks. P3 virus was used to infect High 5 insect cells for 72 hours, and the supernatant was collected and run on an SDS-PAGE gel. Recombinant proteins were detected by Western blot using anti-tetra-His antibody (Qiagen) and anti-mouse HRP (Sigma).
- The test expression of the fusion constructs is shown in
FIG. 12 . - Fusion Proteins: 1-11E/C7 with TNFR2-Fc
- pFastBac1.AH was created from pFastBac1 (Invitrogen) by cutting out BamHI/HindIII fragment containing multiple cloning sites (MCS), and replacing with a linker to give another MCS of BamHI-KpnI-HindIII-ApaI.
- TNFR2Fc was cloned into the HindIII-EcoRI sites, followed by a MMP cleavage site and scFv (1-11E or C7) which were cloned into the NotI and ApaI sites as shown in
FIG. 10B . - Mouse TNFR2-Fc was amplified with the following primers:
-
forward primer: (SEQ ID NO: 141) 5′ GCT aag ctt ATG GCG CCC GCC GCC CTC 3′reverse primer: (SEQ ID NO: 142) 5′ CTTGAATTCTTTACCCAGAGACCGGGA 3′1-11E was amplified with the same primers as above for the INFb. - The sequence of TNFR2Fc/MMP/1-11E is as follows:
-
(SEQ ID NO: 143) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK---EFGGGGSPLGL WAGGGSAAA---MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW VRQAPGKGLEWVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSL RAEDTAVYYCAKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQM TQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKV EIKRDIHHHHHH - Of this sequence, the TNFR2Fc portion is as follows:
-
(SEQ ID NO: 144) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK 50 AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS 100 CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG 150 VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA 200 VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC 250 VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 300 DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK 350 EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR 400 VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK* - The MMP linker portion is as follows:
- The 1-11E portion is as follows:
-
(SEQ ID NO: 146) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSSIDDSGATTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AKNYSSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSTDIQMTQSPSSLSAS VGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYYASSLQSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQAANYPTTFGQGTKVEIKRDI - The His tag is as follows:
- As a negative control a non specific scFv was developed that binds to Hen Egg Lysosyme (HEL). Clone C7 was the best expressed and was taken forward for TNFR2Fc fusion as done for 1-11E.
-
-
(SEQ ID NO: 147) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGKEFGGGGSPLGLWAG GGSAAAMAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG KGLEWVSTISYAGASTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCAKTSTSFDYWGQGTLVTVSTDIQMTQSPSSLSASVGDRVTITCRAS QSISSYLNWYQQKPGKAPKLLIYNASYLQSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQAYAGPYTFGQGTKVEIKRDIHHHHHH* - Of this sequence, the TNFR2Fc portion is as follows:
-
(SEQ ID NO: 148) MAPAALWVALVFELQLWATGHTVPAQVVLTPYKPEPGYECQISQEYYDRK 50 AQMCCAKCPPGQYVKHFCNKTSDTVCADCEASMYTQVWNQFRTCLSCSSS 100 CTTDQVEIRACTKQQNRVCACEAGRYCALKTHSGSCRQCMRLSKCGPGFG 150 VASSRAPNGNVLCKACAPGTFSDTTSSTDVCRPHRICSILAIPGNASTDA 200 VCAPESDGSPPLKECPPCAAPDLLGGPSVFIFPPKIKDVLMISLSPMVTC 250 VVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQ 300 DWMSGKEFKCKVNNRALPSPIEKTISKPRGPVRAPQVYVLPPPAEEMTKK 350 EFSLTCMITGFLPAEIAVDWTSNGRTEQNYKNTATVLDSDGSYFMYSKLR 400 VQKSTWERGSLFACSVVHEGLHNHLTTKTISRSLGK* - The MMP linker portion is as follows:
- The C7 portion is as follows:
-
(SEQ ID NO: 150) MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWV STISYAGASTAYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK TSTSFDYWGQGTLVTVSTDIQMTQSPSSLSASVGDRVTITCRASQSISSY LNWYQQKPGKAPKLLIYNASYLQSGVPSRFSGSGSGTDFTLTISSLQPED FATYYCQQAYAGPYTFGQGTKVEIKRDI - The His tag is as follows:
- The protocol for expression of the constructs is shown in
FIG. 11 and is as described above for the IFN-beta/1-11E fusion protein. - Infected Hi-5 cells were grown for 3 days at 27° C. After 3 days, different 100, 50, 25 and 12 microliter aliquots of cell supernatant were taken for Western blot analysis. Fusion protein was probed with anti-His tag antibodies. As shown in
FIGS. 13A, 13B, and 13C the apparent molecular weight of the TNFR2Fc fusion proteins is slightly above 75 kDa which reflects the predicted 50 kDa TNFR2Fc plus 30 kDa scFv.
Claims (25)
1. A composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) in which the antibody or fragment thereof is conjugated to a pharmaceutically active moiety, wherein the antibody or fragment thereof comprises CDR sequences in the Variable Heavy (VH) chains and Variable Light (VL) chains as shown below, wherein the CDRH1 and CDRL1 sequences are the same as those of scFv 1-11E;
2. A composition as claimed in claim 1 , in which the antibody is a polyclonal antibody or a monoclonal antibody.
3. A composition as claimed in claim 1 , in which the antibody fragment is a Fc, Fab, scFv, single domain (dAb) antibody, diabody, minibody, or scFv-Fc fragment.
4. (canceled)
5. A composition as claimed in claim 1 , in which the antibody is an scFv selected from the group consisting of:
1-12A, 4-6A, 4-8A, 4-9F, 4-4H, 3-3A, 3-6F, 3-9D, 3-11E, 6-11D, 4-5H, 6-5F, and 6-7F.
6. (canceled)
7. A composition as claimed in claim 1 , in which the composition comprises a proteolytic cleavage site between the antibody or fragment thereof and the pharmaceutically active moiety.
8. A composition as claimed in claim 7 , in which the proteolytic cleavage site is a matrix metalloproteinase (MMP) cleavage site, a serine protease cleavage site, or a site cleavable by a parasitic protease derived from a pathogenic organism.
9. A composition as claimed in claim 8 , in which the proteolytic cleavage site is a MMP cleavage site.
10. A composition as claimed in claim 9 , in which the MMP cleavage site is one or more of MMP1, MMP2, MMP3, MMP7, MMP8, MMP9 or MMP10 as shown in FIG. 5 .
11. A composition as claimed in claim 1 , in which the pharmaceutically active moiety is an antibody or a fragment thereof, a growth factor, a differentiation factor, a cytokine molecule, an interferon, a bone morphogenetic protein (BMP);
a chemokine, a MCP (Monocyte Chemotactic Protein), a cytokine inhibitor; a cytokine receptor, a free-radical scavenging enzyme or a toxin, or an active fragment or portion thereof.
12. A composition as claimed in claim 11 , in which the pharmaceutically active moiety is an interferon.
13. A composition as claimed in claim 12 , in which the pharmaceutically active moiety is interferon beta (IFN-β).
14. (canceled)
15. A composition as claimed in claim 11 , in which the pharmaceutically active moiety is a TNF receptor (TNFR) antibody fusion protein.
16. A composition as claimed in claim 15 , in which the pharmaceutically active moiety is TNFR2-Fc.
17. (canceled)
18. A composition as claimed in claim 1 , in which the pharmaceutically active moiety is a glycosaminoglycan molecule, chondroitin, a non-steroidal anti-inflammatory drug (NSAID), a steroid, sodium hyaluronate or hyaluronic acid, colchicine or hydroxychloroquine.
19. A composition comprising an antibody or fragment thereof against oxidised Collagen II (CII) and a detectable label, wherein the antibody or fragment thereof comprises CDR sequences in the Variable Heavy (VH) chains and Variable Light (VL) chains as shown below, wherein the CDRH1 and CDRL1 sequences are the same as those of scFv 1-11E;
20. A composition as claimed in claim 19 , in which the detectable label is a radionuclide or a dye.
21. A composition as claimed in claim 20 , in which the detectable label is a dye.
22-23. (canceled)
24. A method of treatment of an arthropathy, comprising the step of administering to a subject a composition as claimed in claim 1 .
25. (canceled)
26. A method for the diagnosis of an arthropathy comprising the steps of administering a composition of claim 1 and subsequently detecting the composition.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14/987,308 US20160228570A1 (en) | 2007-05-03 | 2016-01-04 | Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies |
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0708585.5A GB0708585D0 (en) | 2007-05-03 | 2007-05-03 | Novel antibody and use in diagnosis and therapy of arthropathies |
GB0708585.5 | 2007-05-03 | ||
PCT/GB2008/001526 WO2008135734A1 (en) | 2007-05-03 | 2008-05-02 | Novel antibody and use in diagnosis and therapy of arthropathies |
US59867110A | 2010-02-11 | 2010-02-11 | |
US14/987,308 US20160228570A1 (en) | 2007-05-03 | 2016-01-04 | Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies |
Related Parent Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2008/001526 Continuation WO2008135734A1 (en) | 2007-05-03 | 2008-05-02 | Novel antibody and use in diagnosis and therapy of arthropathies |
US12/598,671 Continuation US9844602B2 (en) | 2007-05-03 | 2008-05-02 | Antibody and use in diagnosis and therapy of arthropathies |
Publications (1)
Publication Number | Publication Date |
---|---|
US20160228570A1 true US20160228570A1 (en) | 2016-08-11 |
Family
ID=38198689
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/598,671 Expired - Fee Related US9844602B2 (en) | 2007-05-03 | 2008-05-02 | Antibody and use in diagnosis and therapy of arthropathies |
US14/987,308 Abandoned US20160228570A1 (en) | 2007-05-03 | 2016-01-04 | Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies |
US15/844,974 Abandoned US20180221505A1 (en) | 2007-05-03 | 2017-12-18 | Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US12/598,671 Expired - Fee Related US9844602B2 (en) | 2007-05-03 | 2008-05-02 | Antibody and use in diagnosis and therapy of arthropathies |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/844,974 Abandoned US20180221505A1 (en) | 2007-05-03 | 2017-12-18 | Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies |
Country Status (5)
Country | Link |
---|---|
US (3) | US9844602B2 (en) |
EP (1) | EP2155256B1 (en) |
ES (1) | ES2663381T3 (en) |
GB (1) | GB0708585D0 (en) |
WO (1) | WO2008135734A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10053515B2 (en) | 2016-01-22 | 2018-08-21 | Merck Sharp & Dohme Corp. | Anti-coagulation factor XI antibodies |
US10676536B2 (en) | 2016-06-14 | 2020-06-09 | Merck Sharp & Dohme Corp. | Anti-coagulation factor XI antibodies |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2694488A1 (en) * | 2007-07-31 | 2009-02-05 | Medimmune, Llc | Multispecific epitope binding proteins and uses thereof |
FR2932182A1 (en) * | 2008-06-10 | 2009-12-11 | Commissariat Energie Atomique | CYCLOPEPTIDES USEFUL AS INHIBITORS OF MMP PROTEASE ENZYMES |
GB0908613D0 (en) | 2009-05-20 | 2009-06-24 | Immunocore Ltd | T Cell Reseptors |
WO2012047583A2 (en) * | 2010-09-27 | 2012-04-12 | Janssen Biotech, Inc. | Antibodies binding human collagen ii |
US8906649B2 (en) | 2010-09-27 | 2014-12-09 | Janssen Biotech, Inc. | Antibodies binding human collagen II |
US20140294813A1 (en) * | 2013-03-15 | 2014-10-02 | Abbvie, Inc. | TNF Binding Proteins |
GB201413357D0 (en) | 2014-07-28 | 2014-09-10 | Philogen Spa | Antibodies for treatment and diagnosis |
BR112018074748A2 (en) | 2016-06-02 | 2019-03-06 | Immunocore Limited | bispecific therapeutic composition of t-cell redirection, and gp100 positive cancer treatment method in a patient |
WO2017211731A1 (en) | 2016-06-06 | 2017-12-14 | F. Hoffmann-La Roche Ag | Fusion proteins for ophthalmology with increased eye retention |
JP2020508319A (en) * | 2017-02-24 | 2020-03-19 | フィロジェン ソチエタ ペル アツィオーニ | Immune complexes with optimized linkers and orientation |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5541295A (en) | 1993-02-12 | 1996-07-30 | The Board Of Governors For Higher Education State Of Rhode Island And Providence Plantations | Detection of type II collagen and its peptides |
US6642007B1 (en) * | 1998-11-02 | 2003-11-04 | Pfizer Inc. | Assays for measurement of type II collagen fragments in urine |
FI20050814A0 (en) | 2005-08-11 | 2005-08-11 | Procollagen Oy | Procedure for observing autoantibodies formed in rheumatoid arthritis |
ES2483724T3 (en) | 2009-03-30 | 2014-08-07 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Biomarkers, methods and kits for the diagnosis of rheumatoid arthritis |
JP2014516155A (en) | 2011-05-13 | 2014-07-07 | アンスティチュ ナショナル ドゥ ラ サンテ エ ドゥ ラ ルシェルシュ メディカル | Methods for diagnosis of early rheumatoid arthritis |
-
2007
- 2007-05-03 GB GBGB0708585.5A patent/GB0708585D0/en not_active Ceased
-
2008
- 2008-05-02 EP EP08737154.8A patent/EP2155256B1/en not_active Not-in-force
- 2008-05-02 ES ES08737154.8T patent/ES2663381T3/en active Active
- 2008-05-02 WO PCT/GB2008/001526 patent/WO2008135734A1/en active Application Filing
- 2008-05-02 US US12/598,671 patent/US9844602B2/en not_active Expired - Fee Related
-
2016
- 2016-01-04 US US14/987,308 patent/US20160228570A1/en not_active Abandoned
-
2017
- 2017-12-18 US US15/844,974 patent/US20180221505A1/en not_active Abandoned
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10053515B2 (en) | 2016-01-22 | 2018-08-21 | Merck Sharp & Dohme Corp. | Anti-coagulation factor XI antibodies |
US10584179B2 (en) | 2016-01-22 | 2020-03-10 | Merck Sharp & Dohme Corp. | Anti-coagulation factor XI antibodies |
US10676536B2 (en) | 2016-06-14 | 2020-06-09 | Merck Sharp & Dohme Corp. | Anti-coagulation factor XI antibodies |
US11479615B2 (en) | 2016-06-14 | 2022-10-25 | Merck Sharp & Dohme Llc | Anti-coagulation factor XI antibodies |
US11485794B2 (en) | 2016-06-14 | 2022-11-01 | Merck Sharp & Dohme Llc | Anti-coagulation factor XI antibodies |
US11512142B2 (en) | 2016-06-14 | 2022-11-29 | Merck Sharp & Dohme Llc | Anti-coagulation factor XI antibodies |
US11661460B2 (en) | 2016-06-14 | 2023-05-30 | Merck Sharp & Dohme Llc | Anti-coagulation factor XI antibodies |
Also Published As
Publication number | Publication date |
---|---|
WO2008135734A1 (en) | 2008-11-13 |
US20110129415A1 (en) | 2011-06-02 |
GB0708585D0 (en) | 2007-06-13 |
US20180221505A1 (en) | 2018-08-09 |
ES2663381T3 (en) | 2018-04-12 |
US9844602B2 (en) | 2017-12-19 |
EP2155256A1 (en) | 2010-02-24 |
EP2155256B1 (en) | 2017-11-29 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20180221505A1 (en) | Novel Antibody And Use In Diagnosis And Therapy Of Arthropathies | |
AU2018214147B2 (en) | Modified Antibody Compositions, Methods of Making and Using Thereof | |
TWI614030B (en) | Molecular constructs with targeting and effector moieties | |
US20170056519A1 (en) | Fusion proteins for treating pathological blood clots | |
ES2614260T3 (en) | Use of antibodies against the interleukin-4 receptor and compositions thereof | |
TWI617319B (en) | Fusion proteins for treating pathological blood clots | |
KR101413480B1 (en) | Sparc binding peptides and uses thereof | |
JP2020073470A (en) | Method for treating crohn's disease by using anti-il23 antibody | |
JP6240195B2 (en) | Anti-CD26 antibody and use thereof | |
JP2007511593A (en) | Method for treating multiple sclerosis by inhibiting IL-17 activity | |
ES2588484T3 (en) | Anti-ADAMTS-5 antibody, derivatives and uses thereof | |
US20210371533A1 (en) | Methods of treating inflammatory diseases by blocking galectin-3 | |
KR20170004011A (en) | Novel specific-binding polypeptides and uses thereof | |
WO2021218947A1 (en) | Anti-novel coronavirus monoclonal antibody and application thereof | |
CA2927525C (en) | Human antibody against aggrecanase-type adamts species for therapeutics of aggrecanase-related diseases | |
CN110267680B (en) | Preventive and/or therapeutic agent for infectious disease or inflammatory disease | |
WO2013103783A1 (en) | Murine il-13 antibodies | |
US20240199762A1 (en) | Anti-fibroblast activation protein antibodies | |
US20220275077A1 (en) | Il-38-specific antibodies | |
JP2022543844A (en) | Compositions and uses of antagonists of humoral immunosuppressive factors for the treatment of humoral immunosuppressive diseases | |
AU2016211890B2 (en) | Methods and Compositions for Regulating Iron Homeostasis by Modulation of BMP-6 | |
CN116970078A (en) | anti-TrkA antibody and application thereof in pets | |
TW202417478A (en) | Anti-pcrv and psl bispecifics for treatment of bronchiectasis | |
EP3344659A1 (en) | Molecular constructs for preventing the formation of blood clot and/or treating thrombosis |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: QUEEN MARY & WESTFIELD COLLEGE, UNITED KINGDOM Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:NISSIM, AHUVA;CHERNAJOVSKY, YUTI;FAURHOLM, BJARNE;AND OTHERS;SIGNING DATES FROM 20091211 TO 20110207;REEL/FRAME:037462/0742 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |