CA2882022A1 - Monoclonal antibodies against hmgb1 - Google Patents
Monoclonal antibodies against hmgb1 Download PDFInfo
- Publication number
- CA2882022A1 CA2882022A1 CA2882022A CA2882022A CA2882022A1 CA 2882022 A1 CA2882022 A1 CA 2882022A1 CA 2882022 A CA2882022 A CA 2882022A CA 2882022 A CA2882022 A CA 2882022A CA 2882022 A1 CA2882022 A1 CA 2882022A1
- Authority
- CA
- Canada
- Prior art keywords
- antibody
- antigen
- binding fragment
- mab
- hmgb1
- 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
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 354
- 230000027455 binding Effects 0.000 claims abstract description 315
- 239000012634 fragment Substances 0.000 claims abstract description 242
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 241
- 239000000427 antigen Substances 0.000 claims abstract description 237
- 108091007433 antigens Proteins 0.000 claims abstract description 237
- 102000036639 antigens Human genes 0.000 claims abstract description 237
- 229920001184 polypeptide Polymers 0.000 claims abstract description 195
- 238000000034 method Methods 0.000 claims abstract description 91
- 102000004127 Cytokines Human genes 0.000 claims abstract description 43
- 108090000695 Cytokines Proteins 0.000 claims abstract description 43
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims abstract description 37
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 35
- 230000002757 inflammatory effect Effects 0.000 claims abstract description 15
- 230000004913 activation Effects 0.000 claims abstract description 13
- 241000282414 Homo sapiens Species 0.000 claims description 112
- 125000000539 amino acid group Chemical group 0.000 claims description 89
- 210000004027 cell Anatomy 0.000 claims description 79
- 210000004408 hybridoma Anatomy 0.000 claims description 72
- 241001529936 Murinae Species 0.000 claims description 54
- 150000007523 nucleic acids Chemical class 0.000 claims description 44
- 102000039446 nucleic acids Human genes 0.000 claims description 42
- 108020004707 nucleic acids Proteins 0.000 claims description 42
- 238000002965 ELISA Methods 0.000 claims description 37
- 102000055207 HMGB1 Human genes 0.000 claims description 36
- 108700010013 HMGB1 Proteins 0.000 claims description 34
- 101150021904 HMGB1 gene Proteins 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 28
- 206010040047 Sepsis Diseases 0.000 claims description 21
- 238000012360 testing method Methods 0.000 claims description 15
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 10
- 206010003246 arthritis Diseases 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 206010025135 lupus erythematosus Diseases 0.000 claims description 5
- 208000006673 asthma Diseases 0.000 claims description 4
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 208000028867 ischemia Diseases 0.000 claims description 4
- 206010034674 peritonitis Diseases 0.000 claims description 4
- 206010001052 Acute respiratory distress syndrome Diseases 0.000 claims description 3
- 208000009137 Behcet syndrome Diseases 0.000 claims description 3
- 206010006895 Cachexia Diseases 0.000 claims description 3
- 208000009329 Graft vs Host Disease Diseases 0.000 claims description 3
- 206010033645 Pancreatitis Diseases 0.000 claims description 3
- 201000004681 Psoriasis Diseases 0.000 claims description 3
- 208000013616 Respiratory Distress Syndrome Diseases 0.000 claims description 3
- 208000011341 adult acute respiratory distress syndrome Diseases 0.000 claims description 3
- 201000000028 adult respiratory distress syndrome Diseases 0.000 claims description 3
- 208000024908 graft versus host disease Diseases 0.000 claims description 3
- 201000006417 multiple sclerosis Diseases 0.000 claims description 3
- 208000006545 Chronic Obstructive Pulmonary Disease Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 101100339431 Arabidopsis thaliana HMGB2 gene Proteins 0.000 claims 29
- 238000003018 immunoassay Methods 0.000 claims 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 claims 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 claims 1
- 102000017727 Immunoglobulin Variable Region Human genes 0.000 claims 1
- 108010067060 Immunoglobulin Variable Region Proteins 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 45
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 37
- 102000003390 tumor necrosis factor Human genes 0.000 description 35
- 101001025337 Homo sapiens High mobility group protein B1 Proteins 0.000 description 34
- 150000001413 amino acids Chemical class 0.000 description 33
- 238000002474 experimental method Methods 0.000 description 33
- 102000053637 human HMGB1 Human genes 0.000 description 33
- 241000699666 Mus <mouse, genus> Species 0.000 description 28
- 239000002773 nucleotide Substances 0.000 description 27
- 125000003729 nucleotide group Chemical group 0.000 description 27
- 239000000523 sample Substances 0.000 description 25
- 108091023037 Aptamer Proteins 0.000 description 24
- 241000699670 Mus sp. Species 0.000 description 24
- 102100022128 High mobility group protein B2 Human genes 0.000 description 22
- 101001045791 Homo sapiens High mobility group protein B2 Proteins 0.000 description 22
- 241000700159 Rattus Species 0.000 description 19
- 108090000623 proteins and genes Proteins 0.000 description 19
- 230000000770 proinflammatory effect Effects 0.000 description 18
- 230000004071 biological effect Effects 0.000 description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 15
- 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
- 230000004083 survival effect Effects 0.000 description 14
- 238000003556 assay Methods 0.000 description 13
- 239000003814 drug Substances 0.000 description 13
- 230000005764 inhibitory process Effects 0.000 description 13
- 201000010099 disease Diseases 0.000 description 12
- 230000006870 function Effects 0.000 description 11
- 210000001519 tissue Anatomy 0.000 description 11
- 238000011282 treatment Methods 0.000 description 11
- 238000001262 western blot Methods 0.000 description 11
- 230000001404 mediated effect Effects 0.000 description 10
- 206010061218 Inflammation Diseases 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 230000004968 inflammatory condition Effects 0.000 description 9
- 230000004054 inflammatory process Effects 0.000 description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 108060003951 Immunoglobulin Proteins 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 8
- 102000018358 immunoglobulin Human genes 0.000 description 8
- 238000004519 manufacturing process Methods 0.000 description 8
- 102000040945 Transcription factor Human genes 0.000 description 7
- 108091023040 Transcription factor Proteins 0.000 description 7
- 239000002158 endotoxin Substances 0.000 description 7
- -1 paxlitaxol Chemical compound 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 102000000849 HMGB Proteins Human genes 0.000 description 6
- 108010001860 HMGB Proteins Proteins 0.000 description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 210000000628 antibody-producing cell Anatomy 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000002163 immunogen Effects 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 230000000670 limiting effect Effects 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 208000024891 symptom Diseases 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- 101000739159 Homo sapiens Mammaglobin-A Proteins 0.000 description 5
- 102100037273 Mammaglobin-A Human genes 0.000 description 5
- 101100046526 Mus musculus Tnf gene Proteins 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 231100000673 dose–response relationship Toxicity 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 229920000136 polysorbate Polymers 0.000 description 5
- 230000002829 reductive effect Effects 0.000 description 5
- 230000001225 therapeutic effect Effects 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 101100178203 Arabidopsis thaliana HMGB3 gene Proteins 0.000 description 4
- 108090001008 Avidin Proteins 0.000 description 4
- 241000699802 Cricetulus griseus Species 0.000 description 4
- 208000011231 Crohn disease Diseases 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 230000004568 DNA-binding Effects 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 101150091750 HMG1 gene Proteins 0.000 description 4
- 101100178199 Homo sapiens HMGB2 gene Proteins 0.000 description 4
- 206010035226 Plasma cell myeloma Diseases 0.000 description 4
- 101100177261 Rattus norvegicus Hbp1 gene Proteins 0.000 description 4
- 101100339430 Rattus norvegicus Hmgb1 gene Proteins 0.000 description 4
- 239000005557 antagonist Substances 0.000 description 4
- 239000002260 anti-inflammatory agent Substances 0.000 description 4
- 229940121363 anti-inflammatory agent Drugs 0.000 description 4
- 230000000692 anti-sense effect Effects 0.000 description 4
- 229940098773 bovine serum albumin Drugs 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000003111 delayed effect Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 229920006008 lipopolysaccharide Polymers 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 201000000050 myeloid neoplasm Diseases 0.000 description 4
- 210000001672 ovary Anatomy 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 206010039073 rheumatoid arthritis Diseases 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 239000000829 suppository Substances 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 239000003053 toxin Substances 0.000 description 4
- 231100000765 toxin Toxicity 0.000 description 4
- 108700012359 toxins Proteins 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- HVAUUPRFYPCOCA-AREMUKBSSA-N 2-O-acetyl-1-O-hexadecyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCOC[C@@H](OC(C)=O)COP([O-])(=O)OCC[N+](C)(C)C HVAUUPRFYPCOCA-AREMUKBSSA-N 0.000 description 3
- 238000011725 BALB/c mouse Methods 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine Chemical compound NCCCC[C@@H](N)C(O)=O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 3
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 108010003541 Platelet Activating Factor Proteins 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 102100022978 Sex-determining region Y protein Human genes 0.000 description 3
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000002378 acidificating effect Effects 0.000 description 3
- 230000001919 adrenal effect Effects 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 238000004166 bioassay Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 231100000135 cytotoxicity Toxicity 0.000 description 3
- 230000003013 cytotoxicity Effects 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 208000027866 inflammatory disease Diseases 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 210000003292 kidney cell Anatomy 0.000 description 3
- 231100000225 lethality Toxicity 0.000 description 3
- 238000004949 mass spectrometry Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000002674 ointment Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 229920001223 polyethylene glycol Polymers 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 235000011960 Brassica ruvo Nutrition 0.000 description 2
- 208000037487 Endotoxemia Diseases 0.000 description 2
- 206010014824 Endotoxic shock Diseases 0.000 description 2
- 101710168537 High mobility group protein B1 Proteins 0.000 description 2
- 102100022130 High mobility group protein B3 Human genes 0.000 description 2
- 101001005953 Homo sapiens Putative high mobility group protein B1-like 1 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 108010002352 Interleukin-1 Proteins 0.000 description 2
- 108010048043 Macrophage Migration-Inhibitory Factors Proteins 0.000 description 2
- 102100037791 Macrophage migration inhibitory factor Human genes 0.000 description 2
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 239000004792 Prolene Substances 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100025288 Putative high mobility group protein B1-like 1 Human genes 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 101000997749 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Intrastrand cross-link recognition protein Proteins 0.000 description 2
- 206010040070 Septic Shock Diseases 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 101710188553 Sex-determining region Y protein Proteins 0.000 description 2
- 208000007107 Stomach Ulcer Diseases 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- 102100040247 Tumor necrosis factor Human genes 0.000 description 2
- 208000024780 Urticaria Diseases 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 239000007801 affinity label Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 230000000890 antigenic effect Effects 0.000 description 2
- 230000006907 apoptotic process Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 210000004534 cecum Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000006071 cream Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 235000003599 food sweetener Nutrition 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 208000008384 ileus Diseases 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 210000004698 lymphocyte Anatomy 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 208000031225 myocardial ischemia Diseases 0.000 description 2
- 230000010807 negative regulation of binding Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 230000030786 positive chemotaxis Effects 0.000 description 2
- 210000004986 primary T-cell Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 208000037803 restenosis Diseases 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 239000003765 sweetening agent Substances 0.000 description 2
- 208000011580 syndromic disease Diseases 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000009261 transgenic effect Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- WMZHJZFCFREAFU-UHFFFAOYSA-N 1,3-dimethyl-7-[4-(2-nitroimidazol-1-yl)butyl]purine-2,6-dione Chemical compound C1=2C(=O)N(C)C(=O)N(C)C=2N=CN1CCCCN1C=CN=C1[N+]([O-])=O WMZHJZFCFREAFU-UHFFFAOYSA-N 0.000 description 1
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 description 1
- 206010000228 Abortion infected Diseases 0.000 description 1
- ORILYTVJVMAKLC-UHFFFAOYSA-N Adamantane Natural products C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 1
- 208000035285 Allergic Seasonal Rhinitis Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 208000004881 Amebiasis Diseases 0.000 description 1
- 206010001980 Amoebiasis Diseases 0.000 description 1
- 206010002199 Anaphylactic shock Diseases 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 206010003011 Appendicitis Diseases 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 208000031729 Bacteremia Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000027496 Behcet disease Diseases 0.000 description 1
- 206010006448 Bronchiolitis Diseases 0.000 description 1
- 101100257359 Caenorhabditis elegans sox-2 gene Proteins 0.000 description 1
- 101100257372 Caenorhabditis elegans sox-3 gene Proteins 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010007134 Candida infections Diseases 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 206010007558 Cardiac failure chronic Diseases 0.000 description 1
- 206010007559 Cardiac failure congestive Diseases 0.000 description 1
- 102000053642 Catalytic RNA Human genes 0.000 description 1
- 108090000994 Catalytic RNA Proteins 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 206010008088 Cerebral artery embolism Diseases 0.000 description 1
- 102100040428 Chitobiosyldiphosphodolichol beta-mannosyltransferase Human genes 0.000 description 1
- 102100023508 Chloride intracellular channel protein 4 Human genes 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 208000015943 Coeliac disease Diseases 0.000 description 1
- 206010009895 Colitis ischaemic Diseases 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 208000028399 Critical Illness Diseases 0.000 description 1
- 201000003883 Cystic fibrosis Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 208000001490 Dengue Diseases 0.000 description 1
- 206010012310 Dengue fever Diseases 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 101000697358 Dictyostelium discoideum FACT complex subunit SSRP1 Proteins 0.000 description 1
- 206010013554 Diverticulum Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 101710190174 E3 ubiquitin-protein ligase MYLIP Proteins 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
- 238000008157 ELISA kit Methods 0.000 description 1
- 208000009366 Echinococcosis Diseases 0.000 description 1
- 206010014561 Emphysema Diseases 0.000 description 1
- YCAGGFXSFQFVQL-UHFFFAOYSA-N Endothion Chemical compound COC1=COC(CSP(=O)(OC)OC)=CC1=O YCAGGFXSFQFVQL-UHFFFAOYSA-N 0.000 description 1
- 241000792859 Enema Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 208000000289 Esophageal Achalasia Diseases 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- 101710100450 FACT complex subunit SSRP1 Proteins 0.000 description 1
- 206010016228 Fasciitis Diseases 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000009123 Fibrin Human genes 0.000 description 1
- 108010073385 Fibrin Proteins 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical group CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 201000006353 Filariasis Diseases 0.000 description 1
- 238000000729 Fisher's exact test Methods 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108700004714 Gelonium multiflorum GEL Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 208000024869 Goodpasture syndrome Diseases 0.000 description 1
- 201000005569 Gout Diseases 0.000 description 1
- 101710103216 HMG1/2-like protein Proteins 0.000 description 1
- 102000049983 HMGA1a Human genes 0.000 description 1
- 108700039142 HMGA1a Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 208000032456 Hemorrhagic Shock Diseases 0.000 description 1
- 241000711549 Hepacivirus C Species 0.000 description 1
- 241000700721 Hepatitis B virus Species 0.000 description 1
- 102100037907 High mobility group protein B1 Human genes 0.000 description 1
- 101710168572 High mobility group protein B3 Proteins 0.000 description 1
- 208000017604 Hodgkin disease Diseases 0.000 description 1
- 208000010747 Hodgkins lymphoma Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000891557 Homo sapiens Chitobiosyldiphosphodolichol beta-mannosyltransferase Proteins 0.000 description 1
- 101000906636 Homo sapiens Chloride intracellular channel protein 4 Proteins 0.000 description 1
- 101001006375 Homo sapiens High mobility group nucleosome-binding domain-containing protein 4 Proteins 0.000 description 1
- 101001045794 Homo sapiens High mobility group protein B3 Proteins 0.000 description 1
- 101000866805 Homo sapiens Non-histone chromosomal protein HMG-17 Proteins 0.000 description 1
- 206010020565 Hyperaemia Diseases 0.000 description 1
- 206010020741 Hyperpyrexia Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 208000010159 IgA glomerulonephritis Diseases 0.000 description 1
- 206010021263 IgA nephropathy Diseases 0.000 description 1
- 208000024781 Immune Complex disease Diseases 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 108090000171 Interleukin-18 Proteins 0.000 description 1
- 108090001007 Interleukin-8 Proteins 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 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
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 206010069698 Langerhans' cell histiocytosis Diseases 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- 101150090732 MTT1 gene Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100339426 Mus musculus Hmgb1 gene Proteins 0.000 description 1
- 101100310657 Mus musculus Sox1 gene Proteins 0.000 description 1
- 101100149887 Mus musculus Sox10 gene Proteins 0.000 description 1
- 101100366231 Mus musculus Sox12 gene Proteins 0.000 description 1
- 101100366242 Mus musculus Sox14 gene Proteins 0.000 description 1
- 101100257363 Mus musculus Sox2 gene Proteins 0.000 description 1
- 101100257376 Mus musculus Sox3 gene Proteins 0.000 description 1
- 101100043067 Mus musculus Sox8 gene Proteins 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 206010029240 Neuritis Diseases 0.000 description 1
- 102100031346 Non-histone chromosomal protein HMG-17 Human genes 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 206010030136 Oesophageal achalasia Diseases 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 241001327682 Oncorhynchus mykiss irideus Species 0.000 description 1
- 239000012570 Opti-MEM I medium Substances 0.000 description 1
- 208000010191 Osteitis Deformans Diseases 0.000 description 1
- 206010031252 Osteomyelitis Diseases 0.000 description 1
- 101710105970 PMS1 protein homolog 1 Proteins 0.000 description 1
- 102100037482 PMS1 protein homolog 1 Human genes 0.000 description 1
- 208000027868 Paget disease Diseases 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 208000031481 Pathologic Constriction Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000057297 Pepsin A Human genes 0.000 description 1
- 108090000284 Pepsin A Proteins 0.000 description 1
- 208000008469 Peptic Ulcer Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010043958 Peptoids Proteins 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 206010035742 Pneumonitis Diseases 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 108010040201 Polymyxins Proteins 0.000 description 1
- 229920001214 Polysorbate 60 Polymers 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 206010037423 Pulmonary oedema Diseases 0.000 description 1
- 102000017143 RNA Polymerase I Human genes 0.000 description 1
- 108010013845 RNA Polymerase I Proteins 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 206010063837 Reperfusion injury Diseases 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 101100444397 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ECM32 gene Proteins 0.000 description 1
- 101100187051 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NHP10 gene Proteins 0.000 description 1
- 208000002359 Septic Abortion Diseases 0.000 description 1
- 206010049771 Shock haemorrhagic Diseases 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical group O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 206010057268 Spinal cord paralysis Diseases 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 206010042496 Sunburn Diseases 0.000 description 1
- 101001081186 Tetrahymena pyriformis High mobility group protein Proteins 0.000 description 1
- WKDDRNSBRWANNC-UHFFFAOYSA-N Thienamycin Natural products C1C(SCCN)=C(C(O)=O)N2C(=O)C(C(O)C)C21 WKDDRNSBRWANNC-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 101710187743 Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 1
- 102100033732 Tumor necrosis factor receptor superfamily member 1A Human genes 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 102100038458 Ubinuclein-1 Human genes 0.000 description 1
- 101710094188 Ubinuclein-1 Proteins 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 206010046851 Uveitis Diseases 0.000 description 1
- 206010046914 Vaginal infection Diseases 0.000 description 1
- 201000008100 Vaginitis Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 208000027207 Whipple disease Diseases 0.000 description 1
- 238000012452 Xenomouse strains Methods 0.000 description 1
- 241000269370 Xenopus <genus> Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 201000000621 achalasia Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 229960002964 adalimumab Drugs 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 230000007815 allergy Effects 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002788 anti-peptide Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 206010003230 arteritis Diseases 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 229960005070 ascorbic acid Drugs 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 206010006451 bronchitis Diseases 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 201000003984 candidiasis Diseases 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 210000000748 cardiovascular system Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 206010008118 cerebral infarction Diseases 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 229960003115 certolizumab pegol Drugs 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 208000003167 cholangitis Diseases 0.000 description 1
- 201000001352 cholecystitis Diseases 0.000 description 1
- DHSUYTOATWAVLW-WFVMDLQDSA-N cilastatin Chemical compound CC1(C)C[C@@H]1C(=O)N\C(=C/CCCCSC[C@H](N)C(O)=O)C(O)=O DHSUYTOATWAVLW-WFVMDLQDSA-N 0.000 description 1
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 description 1
- 229960004316 cisplatin Drugs 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 230000009918 complex formation Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 208000025729 dengue disease Diseases 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 201000001981 dermatomyositis Diseases 0.000 description 1
- 239000008121 dextrose Substances 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 208000007784 diverticulitis Diseases 0.000 description 1
- 238000003182 dose-response assay Methods 0.000 description 1
- 239000006196 drop Substances 0.000 description 1
- 208000000718 duodenal ulcer Diseases 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 1
- 206010014599 encephalitis Diseases 0.000 description 1
- 206010014665 endocarditis Diseases 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 239000007920 enema Substances 0.000 description 1
- 229940079360 enema for constipation Drugs 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 208000003401 eosinophilic granuloma Diseases 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 208000001606 epiglottitis Diseases 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- 231100000283 hepatitis Toxicity 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 208000024326 hypersensitivity reaction type III disease Diseases 0.000 description 1
- 210000003767 ileocecal valve Anatomy 0.000 description 1
- 229960002182 imipenem Drugs 0.000 description 1
- ZSKVGTPCRGIANV-ZXFLCMHBSA-N imipenem Chemical compound C1C(SCC\N=C\N)=C(C(O)=O)N2C(=O)[C@H]([C@H](O)C)[C@H]21 ZSKVGTPCRGIANV-ZXFLCMHBSA-N 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 239000003547 immunosorbent Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000003701 inert diluent Substances 0.000 description 1
- 230000003960 inflammatory cascade Effects 0.000 description 1
- 229960000598 infliximab Drugs 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 201000010849 intracranial embolism Diseases 0.000 description 1
- 230000008863 intramolecular interaction Effects 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 201000008222 ischemic colitis Diseases 0.000 description 1
- 210000001503 joint Anatomy 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 229950007278 lenercept Drugs 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 235000009973 maize Nutrition 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 208000027202 mammary Paget disease Diseases 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229960001047 methyl salicylate Drugs 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- ZAHQPTJLOCWVPG-UHFFFAOYSA-N mitoxantrone dihydrochloride Chemical compound Cl.Cl.O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO ZAHQPTJLOCWVPG-UHFFFAOYSA-N 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000002864 mononuclear phagocyte Anatomy 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 210000003928 nasal cavity Anatomy 0.000 description 1
- 239000007923 nasal drop Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 230000001175 peptic effect Effects 0.000 description 1
- 239000000816 peptidomimetic Substances 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 208000028169 periodontal disease Diseases 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 210000004303 peritoneum Anatomy 0.000 description 1
- 238000002823 phage display Methods 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 210000000280 pituicyte Anatomy 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 208000008423 pleurisy Diseases 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 201000006292 polyarteritis nodosa Diseases 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 229940114930 potassium stearate Drugs 0.000 description 1
- ANBFRLKBEIFNQU-UHFFFAOYSA-M potassium;octadecanoate Chemical compound [K+].CCCCCCCCCCCCCCCCCC([O-])=O ANBFRLKBEIFNQU-UHFFFAOYSA-M 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 208000037920 primary disease Diseases 0.000 description 1
- 229940027836 primaxin Drugs 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 201000007094 prostatitis Diseases 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 235000020122 reconstituted milk Nutrition 0.000 description 1
- 210000000664 rectum Anatomy 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 229940116176 remicade Drugs 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 206010039083 rhinitis Diseases 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 210000003296 saliva Anatomy 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 201000000306 sarcoidosis Diseases 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 208000013223 septicemia Diseases 0.000 description 1
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 201000009890 sinusitis Diseases 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 101150055666 sox6 gene Proteins 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000036262 stenosis Effects 0.000 description 1
- 208000037804 stenosis Diseases 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 201000004595 synovitis Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 201000005060 thrombophlebitis Diseases 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 208000025883 type III hypersensitivity disease Diseases 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 208000000143 urethritis Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 210000002229 urogenital system Anatomy 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 235000012431 wafers Nutrition 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 230000003442 weekly effect Effects 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4718—Cytokine-induced proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/06—Antiabortive agents; Labour repressants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/04—Antipruritics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/06—Antipsoriatics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/12—Keratolytics, e.g. wart or anti-corn preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
-
- 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
-
- 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
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
-
- 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
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
- A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
- A61P31/22—Antivirals for DNA viruses for herpes viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
- A61P33/06—Antimalarials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/08—Antiallergic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P5/00—Drugs for disorders of the endocrine system
- A61P5/14—Drugs for disorders of the endocrine system of the thyroid hormones, e.g. T3, T4
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/08—Vasodilators for multiple indications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- 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
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
-
- 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
- C07K2317/70—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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/81—Packaged device or kit
Abstract
In various embodiments, the present invention is drawn to antibodies or antigen-binding fragments thereof that bind to a vertebrate high mobility group box (HMGB) polypeptide, methods of detecting and/or identifying an agent that binds to an HMGB polypeptide, methods of treating a condition in a subject characterized by activation of an inflammatory cytokine cascade and methods of detecting an HMGB polypeptide in a sample.
Description
MONOCLONAL ANTIBODIES AGAINST IIMGB I
This application is a divisional application of Canadian patent application number 2,538,763, filed September 10, 2004.
BACKGROUND OF THE INVENTION
Inflammation is often induced by proinflammatory cytokines, such as tumor necrosis factor (TNF), interlenkin (IL)-1a, IL-1 p, IL-6, macrophage migration inhibitory factor (MIF), and other compounds. These proinflarnmatory cytokines are produced by several different cell types, most importantly immune cells (for example, monocytes, macrophages and neutrophils), but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, and neurons. These proinflammatory cytokines contribute to various disorders during the early stages of an inflammatory cytokine cascade.
The early proinflammatory cytokines (e.g., TNF, LL-1, etc.) mediate inflammation,and induce the late release of high mobility group box 1 (HIVIGB1;
also known as HMG-1 and I-EMG1), a protein that accumulates in serum and mediates delayed lethality and further induction of early proinfiammatory cytokines.
HIVIGEri was first identified as the founding member of a family of DNA-binding 20- proteins, termed high mobility group box (HMGB) proteins, which are critical for DNA structure and stability. It was identified as a ubiquitously expressed nuclear protein that binds double-stranded DNA without sequence specificity. The molecule has three domains: two DNA binding motifs termed HMGB A and HMGB
B boxes, and an acidic carboxyl terminus. The two IIMGB boxes are highly conserved SO amino acid, L-shaped domains. HIVIG boxes are also expressed in other transcription factors including the RNA polymerase I transcription factor human upstream-binding factor and lymphoid-specific factor.
Recent evidence has implicated HMG1 as a cytokine mediator of delayed lethality in endotoxemia (Andersson, U., et at., I Exp. Med. 192(4):565-570
This application is a divisional application of Canadian patent application number 2,538,763, filed September 10, 2004.
BACKGROUND OF THE INVENTION
Inflammation is often induced by proinflammatory cytokines, such as tumor necrosis factor (TNF), interlenkin (IL)-1a, IL-1 p, IL-6, macrophage migration inhibitory factor (MIF), and other compounds. These proinflarnmatory cytokines are produced by several different cell types, most importantly immune cells (for example, monocytes, macrophages and neutrophils), but also non-immune cells such as fibroblasts, osteoblasts, smooth muscle cells, epithelial cells, and neurons. These proinflammatory cytokines contribute to various disorders during the early stages of an inflammatory cytokine cascade.
The early proinflammatory cytokines (e.g., TNF, LL-1, etc.) mediate inflammation,and induce the late release of high mobility group box 1 (HIVIGB1;
also known as HMG-1 and I-EMG1), a protein that accumulates in serum and mediates delayed lethality and further induction of early proinfiammatory cytokines.
HIVIGEri was first identified as the founding member of a family of DNA-binding 20- proteins, termed high mobility group box (HMGB) proteins, which are critical for DNA structure and stability. It was identified as a ubiquitously expressed nuclear protein that binds double-stranded DNA without sequence specificity. The molecule has three domains: two DNA binding motifs termed HMGB A and HMGB
B boxes, and an acidic carboxyl terminus. The two IIMGB boxes are highly conserved SO amino acid, L-shaped domains. HIVIG boxes are also expressed in other transcription factors including the RNA polymerase I transcription factor human upstream-binding factor and lymphoid-specific factor.
Recent evidence has implicated HMG1 as a cytokine mediator of delayed lethality in endotoxemia (Andersson, U., et at., I Exp. Med. 192(4):565-570
-2-(2000)). That work demonstrated that bacterial endotoxin (lipopolysaccharide (LPS)) activates monocytes/macrophages to release FEVIGI as a late response to activation, resulting in elevated serum HMG1 levels that are toxic. Antibodies against 1-IMG1 prevent lethality of endotoxin even when antibody administration is delayed until after the early cytokine response. Like other proinflammatory cytokines, ITIVIG1 is a potent activator of monocytes. Intratracheal application of HMG1 causes acute lung injury, and anti-HMG1 antibodies protect against endotoxin-induced lung edema (Abraham. E., et aL, J. InanunoL 165:2950-2954 (2000)). Serum 1{MG1 levels are elevated in critically ill patients with sepsis or hemorrhagic shock, and levels are significantly higher in non-survivors as compared to survivors.
IIMG1 has also been implicated as a ligand for RAGE, a multi-ligand receptor of the immunoglobulin superfamily. RAGE is expressed on endothelial cells, smooth muscle cells, monocytes, and nerves, and ligand interaction transduces signals through MAP kinase, P21 ras, and NF-k-B. The delayed kinetics of I-appearance during endotoxemia makes it a potentially good therapeutic target, but little is known about the molecular basis of IIMG1 signaling and toxicity.
Therefore, given the importance of HMGB proteins in mediating inflammation, it would be useful to identify antibodies that bind HMGB for diagnostic and therapeutic purposes.
SUMMARY OF THE INVENTION
In various embodiments, the present invention is drawn to antibodies or antigen-binding fragments thereof that bind to a vertebrate high mobility group box (HMGB) polypeptide, methods of detecting and/or identifying an agent that binds to an HMGB polypeptide, methods of treating a condition in a subject characterized by activation of an inflammatory cytokine cascade and methods of detecting an HMGB
polypeptide in a sample.
In one embodiment, the invention is an antibody or antigen-binding fragment thereof that specifically binds to a vertebrate HMGB A box but does not specifically bind to non-A box epitopes of I-IdVIGB, wherein the antibody or antigen-binding
IIMG1 has also been implicated as a ligand for RAGE, a multi-ligand receptor of the immunoglobulin superfamily. RAGE is expressed on endothelial cells, smooth muscle cells, monocytes, and nerves, and ligand interaction transduces signals through MAP kinase, P21 ras, and NF-k-B. The delayed kinetics of I-appearance during endotoxemia makes it a potentially good therapeutic target, but little is known about the molecular basis of IIMG1 signaling and toxicity.
Therefore, given the importance of HMGB proteins in mediating inflammation, it would be useful to identify antibodies that bind HMGB for diagnostic and therapeutic purposes.
SUMMARY OF THE INVENTION
In various embodiments, the present invention is drawn to antibodies or antigen-binding fragments thereof that bind to a vertebrate high mobility group box (HMGB) polypeptide, methods of detecting and/or identifying an agent that binds to an HMGB polypeptide, methods of treating a condition in a subject characterized by activation of an inflammatory cytokine cascade and methods of detecting an HMGB
polypeptide in a sample.
In one embodiment, the invention is an antibody or antigen-binding fragment thereof that specifically binds to a vertebrate HMGB A box but does not specifically bind to non-A box epitopes of I-IdVIGB, wherein the antibody or antigen-binding
-3-fragment inhibits release of a proinflammatory cytokine from a vertebrate cell treated with an HMGB protein.
In certain embodiments, the invention is an antibody produced by murine hybridoma 6E6 HMGB1 mAb, murine hybridoma 6119 131\4GB1 mAb, murine hybridoma 2G7 HMGB1 mAb, murine hybridoma 2E11 HMGB1 mAb, or murine hybridoma 10D4 HMGB1 mAb. In other embodiments, the invention is an antibody or antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to a vertebrate BIVIGB poIypeptide can be inhibited by 6E6 HMGB1 mAb, 6119 HIVIGB1 mAb, 2G7 HMGB1 mAb, 2E11 FIMGB I mAb and/or 1011)4 IIMGB1 mAb. In still other embodiments, the invention is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has the epitopic specificity of 6E6 IIMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 ITIVIGB1 mAb and/or 10D4 IIMGB1 inAb.
In certain embodiments, the invention is an antibody or antigen-binding fragment that binds to a peptide consisting of amino acid residues 46 to 63 of SEQ
ID NO:1, amino acid residues 61 to 78 of SEQ ID NO:1 and/or amino acid residues 151 to 168 of SEQ ID NO:1. In one embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 46 to 63 of SEQ 3D
NO:1, can be inhibited by 2G7 HMGB1 mAb. In another embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 61 to 78 of SEQ ID NO:1, can be inhibited by 6E6 HMGB1 mAb and/or 6H9 HMGB1 mAb. In yet another embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 151 to 168 of SEQ ID NO:1, can be inhibited by 2E11 11MGBI mAb.
In certain embodiments, the invention is an antibody or antigen-binding fragment that comprises the light chain CDRs (CDR1, CDR2 and CDR3) and the heavy chain CDRs (CDR1, CDR2 and CDR3) of an antibody selected from the
In certain embodiments, the invention is an antibody produced by murine hybridoma 6E6 HMGB1 mAb, murine hybridoma 6119 131\4GB1 mAb, murine hybridoma 2G7 HMGB1 mAb, murine hybridoma 2E11 HMGB1 mAb, or murine hybridoma 10D4 HMGB1 mAb. In other embodiments, the invention is an antibody or antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to a vertebrate BIVIGB poIypeptide can be inhibited by 6E6 HMGB1 mAb, 6119 HIVIGB1 mAb, 2G7 HMGB1 mAb, 2E11 FIMGB I mAb and/or 1011)4 IIMGB1 mAb. In still other embodiments, the invention is an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment has the epitopic specificity of 6E6 IIMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 2E11 ITIVIGB1 mAb and/or 10D4 IIMGB1 inAb.
In certain embodiments, the invention is an antibody or antigen-binding fragment that binds to a peptide consisting of amino acid residues 46 to 63 of SEQ
ID NO:1, amino acid residues 61 to 78 of SEQ ID NO:1 and/or amino acid residues 151 to 168 of SEQ ID NO:1. In one embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 46 to 63 of SEQ 3D
NO:1, can be inhibited by 2G7 HMGB1 mAb. In another embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 61 to 78 of SEQ ID NO:1, can be inhibited by 6E6 HMGB1 mAb and/or 6H9 HMGB1 mAb. In yet another embodiment, the invention is an antibody or antigen-binding fragment, wherein the binding of the antibody or antigen-binding fragment to a peptide consisting of amino acid residues 151 to 168 of SEQ ID NO:1, can be inhibited by 2E11 11MGBI mAb.
In certain embodiments, the invention is an antibody or antigen-binding fragment that comprises the light chain CDRs (CDR1, CDR2 and CDR3) and the heavy chain CDRs (CDR1, CDR2 and CDR3) of an antibody selected from the
-4-group consisting of 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 BIMGB1 mAb, 10D4 HMGB1 mAb and 2G7 HMGB1 mAb.
In other embodiments, the invention is murine hybridoma 6E6 HMGB1 mAb, murine hybridoma 6H9 HMGB1 mAb, murine hybridoma 2G7 HMGB1 mAb, murine hybridoma 2E11 HIVIGB1 naAb or murine hybridoma 10D4 HMGB1 mAb.
In another embodiment, the invention is an isolated cell that produces an antibody or antigen-binding fragment that specifically binds to a vertebrate HMGB
A box but does not 'specifically bind to non-A box epitopes of HMGB. In other embodiments, the invention is an isolated cell that produces 6E6 EIMGB1 mAb, HMGB1 mAb, 2G7 IIMGB1 mAb, 2E11 HiVIGB1 mAb or 10D4 HIVIGB1 mAb. In still other embodiments, the invention is an isolated cell that produces an antibody or antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to a vertebrate HIVIGB polypeptide can be inhibited by 6E6 HMGB1 mAb, 6H9 HMGB1 ruAb, 207 HMGB1 mAb, 2E11 HMGB1 inAb and/or 10D4 HMGB1 mAb. In still other embodiments, the invention is an isolated cell that produces an antibody or antigen-binding fragment that has the epitopic specificity of 6E6 FIMGB1 mAb, 6H9 HMGB1 mAb, 207 HMGB1 mAb, 2E11 HMGB1 mAb and/or 10D4 IIMGB1 mAb.
In other embodiments, the invention is a composition that comprises an antibody or antigen-binding fragment of the invention and a pharmaceutically-acceptable excipient.
In another embodiment, the invention is a method of detecting and/or identifying an agent that binds to a vertebrate HMGB polypeptide comprising combining an antibody or antigen-binding fragment of the invention, a test agent and a composition comprising a vertebrate HMGB polypeptide. In the method, the formation of a complex between the antibody or antigen-binding fragment and the polypeptide is detected or measured and a decrease in complex formation, as compared to a suitable control, indicates that the test agent binds to the ITMGB
polypeptide.
In another embodiment, the invention is a method of treating a condition in a subject characterized by activation of an inflammatory cytolcine cascade comprising
In other embodiments, the invention is murine hybridoma 6E6 HMGB1 mAb, murine hybridoma 6H9 HMGB1 mAb, murine hybridoma 2G7 HMGB1 mAb, murine hybridoma 2E11 HIVIGB1 naAb or murine hybridoma 10D4 HMGB1 mAb.
In another embodiment, the invention is an isolated cell that produces an antibody or antigen-binding fragment that specifically binds to a vertebrate HMGB
A box but does not 'specifically bind to non-A box epitopes of HMGB. In other embodiments, the invention is an isolated cell that produces 6E6 EIMGB1 mAb, HMGB1 mAb, 2G7 IIMGB1 mAb, 2E11 HiVIGB1 mAb or 10D4 HIVIGB1 mAb. In still other embodiments, the invention is an isolated cell that produces an antibody or antigen-binding fragment thereof, wherein the binding of the antibody or antigen-binding fragment to a vertebrate HIVIGB polypeptide can be inhibited by 6E6 HMGB1 mAb, 6H9 HMGB1 ruAb, 207 HMGB1 mAb, 2E11 HMGB1 inAb and/or 10D4 HMGB1 mAb. In still other embodiments, the invention is an isolated cell that produces an antibody or antigen-binding fragment that has the epitopic specificity of 6E6 FIMGB1 mAb, 6H9 HMGB1 mAb, 207 HMGB1 mAb, 2E11 HMGB1 mAb and/or 10D4 IIMGB1 mAb.
In other embodiments, the invention is a composition that comprises an antibody or antigen-binding fragment of the invention and a pharmaceutically-acceptable excipient.
In another embodiment, the invention is a method of detecting and/or identifying an agent that binds to a vertebrate HMGB polypeptide comprising combining an antibody or antigen-binding fragment of the invention, a test agent and a composition comprising a vertebrate HMGB polypeptide. In the method, the formation of a complex between the antibody or antigen-binding fragment and the polypeptide is detected or measured and a decrease in complex formation, as compared to a suitable control, indicates that the test agent binds to the ITMGB
polypeptide.
In another embodiment, the invention is a method of treating a condition in a subject characterized by activation of an inflammatory cytolcine cascade comprising
-5-administering to the subject an antibody or antigen-binding fragment of the invention.
In certain embodiments, the condition is sepsis, arthritis or lupus.
In another embodiment, the invention is a method of detecting a vertebrate IIIVIGB polypeptide in a sample. In the method, a sample is contacted with an antibody or antigen-binding fragment of the invention, under conditions suitable for binding of the antibody or fragment to HIVIGB polypeptide present in the sample. If antibody-BMGB complexes or antigen-binding fragment-BMGB complexes are =
detected, their presence is indicative of HMGB polypeptide in the sample.
In another embodiment, the invention is a test kit for use in detecting the presence of a vertebrate HNIGB polypeptide or a portion thereof in a sample.
The test kit comprises an antibody or antigen-binding fragment of the invention and one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or antigen-binding fragment and the BMGB polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the amino acid sequence of a human (Homo sapiens) BIVIGB1 polypeptide (SEQ ID NO:1). The underlined amino acid residues delineate the A
box, B box and acidic tail domains of the HIVIGB1 polypeptide.
FIG. 2A is the amino acid sequence of a polypeptide comprising an A box of human (Homo sapiens) BMGB1 (SEQ ID NO:2). The underlined amino acid residues delineate the A box of the IIMGB1 polypeptide, which is the same for human, rat and mouse.
FIG. 2B is the amino acid sequence of a B box of a human (Homo sapiens) HMGB1 polypeptide (SEQ JD NO:3). The underlined amino acid residues delineate the B box of the HMG131 polypeptide, which is the same for human, rat and mouse.
FIG. 3A is the nucleotide sequence encoding the recombinant CBP-Rat EIMGB1 peptide (SEQ ID NO:4) that was used as an immunogen to generate monoclonal antibodies.
FIG. 3B is the encoded amino acid sequence of the recombinant CBP-Rat IIMGB1 peptide (SEQ ID NO:5) that was used as an immunogen to generate
In certain embodiments, the condition is sepsis, arthritis or lupus.
In another embodiment, the invention is a method of detecting a vertebrate IIIVIGB polypeptide in a sample. In the method, a sample is contacted with an antibody or antigen-binding fragment of the invention, under conditions suitable for binding of the antibody or fragment to HIVIGB polypeptide present in the sample. If antibody-BMGB complexes or antigen-binding fragment-BMGB complexes are =
detected, their presence is indicative of HMGB polypeptide in the sample.
In another embodiment, the invention is a test kit for use in detecting the presence of a vertebrate HNIGB polypeptide or a portion thereof in a sample.
The test kit comprises an antibody or antigen-binding fragment of the invention and one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or antigen-binding fragment and the BMGB polypeptide.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is the amino acid sequence of a human (Homo sapiens) BIVIGB1 polypeptide (SEQ ID NO:1). The underlined amino acid residues delineate the A
box, B box and acidic tail domains of the HIVIGB1 polypeptide.
FIG. 2A is the amino acid sequence of a polypeptide comprising an A box of human (Homo sapiens) BMGB1 (SEQ ID NO:2). The underlined amino acid residues delineate the A box of the IIMGB1 polypeptide, which is the same for human, rat and mouse.
FIG. 2B is the amino acid sequence of a B box of a human (Homo sapiens) HMGB1 polypeptide (SEQ JD NO:3). The underlined amino acid residues delineate the B box of the HMG131 polypeptide, which is the same for human, rat and mouse.
FIG. 3A is the nucleotide sequence encoding the recombinant CBP-Rat EIMGB1 peptide (SEQ ID NO:4) that was used as an immunogen to generate monoclonal antibodies.
FIG. 3B is the encoded amino acid sequence of the recombinant CBP-Rat IIMGB1 peptide (SEQ ID NO:5) that was used as an immunogen to generate
-6-monoclonal antibodies. The CBP affinity tag, which was removed by thrombin cleavage, is indicated in lower case letters and the normal translation initiation amino acid (i.e., M) of HMGB1 is underlined.
FIG. 4A is the nucleotide sequence encoding the VH domain of 6E6 IIIVIGB1 inAb (SEQ NO:6).
FIG. 413 is the encoded amino acid sequence of the VH domain of 6E6 IIMGB1 mAb (SEQ ID NO:7); CDRs are underlined.
FIG. 4C is the nucleotide sequence encoding the VK domain of 6E6 HMGI31 mAb (SEQ ID NO:8).
FIG. 4D is the encoded amino acid sequence of the VK domain of 6E6 HMGB1 mAb (SEQ ID NO:9); CDRs are underlined.
FIG. 5A is the nucleotide sequence encoding the V11 domain of 2E11 IIMGB1 mAb (SEQ ID NO:10).
FIG. 5B is the encoded amino acid sequence of the Vil domain of 2E11 HMGB1 mAb (SEQ JD NO:11); CDRs are underlined.
FIG. 5C is the nucleotide sequence encoding the VK domain of 2E11 IIMGB1 mAb (SEQ ID NO:12).
FIG. 5D is the encoded amino acid sequence of the VK domain of 2E11 IIMGB1 mAb (SEQ ID NO:13); CDRs are underlined.
FIG. 6A is the nucleotide sequence encoding the VH domain of 10D4 1114GB1 mAb (SEQ ID NO:14).
FIG. 6B is the encoded amino acid sequence of the VH domain of 10D4 HIVIGB1 mAb (SEQ ID NO:15); CDRs are underlined.
FIG. 6C is the nucleotide sequence encoding the VK domain of 10D4 IIMGB1 mAb (SEQ ID NO:16).
FIG. 6D is the encoded amino acid sequence of the VK domain of 10D4 HMGB1 mAb (SEQ ID NO:17); CDRs are underlined.
= FIG. 7 is a table summarizing characteristics of various anti-IIMGB1 monoclonal antibodies. Clone names, the imrnunogen used to generate the monoclonal antibody (either rat HMGB1-CBP (SEQ ID NO:5 (see FIG. 3B) or the B
FIG. 4A is the nucleotide sequence encoding the VH domain of 6E6 IIIVIGB1 inAb (SEQ NO:6).
FIG. 413 is the encoded amino acid sequence of the VH domain of 6E6 IIMGB1 mAb (SEQ ID NO:7); CDRs are underlined.
FIG. 4C is the nucleotide sequence encoding the VK domain of 6E6 HMGI31 mAb (SEQ ID NO:8).
FIG. 4D is the encoded amino acid sequence of the VK domain of 6E6 HMGB1 mAb (SEQ ID NO:9); CDRs are underlined.
FIG. 5A is the nucleotide sequence encoding the V11 domain of 2E11 IIMGB1 mAb (SEQ ID NO:10).
FIG. 5B is the encoded amino acid sequence of the Vil domain of 2E11 HMGB1 mAb (SEQ JD NO:11); CDRs are underlined.
FIG. 5C is the nucleotide sequence encoding the VK domain of 2E11 IIMGB1 mAb (SEQ ID NO:12).
FIG. 5D is the encoded amino acid sequence of the VK domain of 2E11 IIMGB1 mAb (SEQ ID NO:13); CDRs are underlined.
FIG. 6A is the nucleotide sequence encoding the VH domain of 10D4 1114GB1 mAb (SEQ ID NO:14).
FIG. 6B is the encoded amino acid sequence of the VH domain of 10D4 HIVIGB1 mAb (SEQ ID NO:15); CDRs are underlined.
FIG. 6C is the nucleotide sequence encoding the VK domain of 10D4 IIMGB1 mAb (SEQ ID NO:16).
FIG. 6D is the encoded amino acid sequence of the VK domain of 10D4 HMGB1 mAb (SEQ ID NO:17); CDRs are underlined.
= FIG. 7 is a table summarizing characteristics of various anti-IIMGB1 monoclonal antibodies. Clone names, the imrnunogen used to generate the monoclonal antibody (either rat HMGB1-CBP (SEQ ID NO:5 (see FIG. 3B) or the B
-7 -box of a human FIMGB1 polypeptide (SEQ ID NO:3 (see FIG. 2B)), the isotype, binding domains for the antibodies and results of in vivo CLP assays are indicated.
FIG. 8 is a histogram depicting inhibition of TNF release by particular anti-FIMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.1 ig,/m1 of recombinant CBP-Rat HMGB1 peptide (SEQ ID
NO:5). Where indicated, 20 u.g/m1 of 6E6 HMGB1 mAb (6E6), 10D4 HMGB1 mAb (10D4), 2E11 HMGB1 mAb (2E11), 9G2 HMGB1 rnAb (9G2) or mouse IgG
control antibody (mIgG) were added. All samples were done in duplicate and error bars are indicated.
FIG. 9 is a histogram depicting inhibition of TNF release by various anti-IIMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.01 ig/m1 or 0.1 pg/m1 of recombinant CBP-Rat HMGB1 peptide (SEQ ID NO:5). Where indicated, 20 pg/m1 of 3G8 HMGB1 mAb (3G8), 1A9 IIMGB1 mAb (1A9), 9G2 HMGB1 mAb (9G2), 6E6 HMGB1 mAb (6E6), 2E11 IfMGB1 mAb (2E11), 10D4 HMGB1 mAb (10D4), 6H9 HMGB1 mAb (6H9) or mouse IgG control antibody (IgG) were added.
FIG. 10 is a graph of the effect of various anti-BMGB1 monoclonal antibodies (6E6 HMGB1 mAb (mAB (6E6)); 2E11 BMGB1 mAb (mAB (2E11));
9G2 HMGB1 mAb (mAB (9G2))) and a control IgG antibody (Ctrl IgG) on survival of mice over time (days) after cecal ligation and puncture (CLP).
FIG. 11 depicts a series of individual Western blots of samples containing either CHO FIMGB1 or CHO HMGB2 and possibly recombinant IIMGB1-His6 (labeled as CHO HMGB2, rec-HMGB1-His6), which were probed with either an anti-His Tag antibody (Anti-His Tag), an anti-HMGB2 antibody (Anti-HMGB2), an anti-HMGB1/2 monoclonal antibody (Anti-IIMGB1/2 mAb) or particular anti-HMGB1 monoclonal antibodies (i.e., 2E11 HMGB1 mAb (CT3-2E11), 1G3 HMGB1 mAb (CT3-1G3), 6H9 HMGB1 mAb (CT3-6H9), 2G7 HMGB1 mAb (CT3-2G7), 2G5 HIvIGB 1 mAb (CT3-2G5) and 6E6 ITMGB 1 mAb (CT3-6E6)).
FIG. 12 is an amino acid sequence alignment of HMGB1 polypeptide sequences from rat (SEQ ID NO:18; labeled "rat # P07155" or "rat" (GenBank Accession No. P07155)), mouse (Mus musculus) (SEQ ID NO:18; labeled "mouse
FIG. 8 is a histogram depicting inhibition of TNF release by particular anti-FIMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.1 ig,/m1 of recombinant CBP-Rat HMGB1 peptide (SEQ ID
NO:5). Where indicated, 20 u.g/m1 of 6E6 HMGB1 mAb (6E6), 10D4 HMGB1 mAb (10D4), 2E11 HMGB1 mAb (2E11), 9G2 HMGB1 rnAb (9G2) or mouse IgG
control antibody (mIgG) were added. All samples were done in duplicate and error bars are indicated.
FIG. 9 is a histogram depicting inhibition of TNF release by various anti-IIMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.01 ig/m1 or 0.1 pg/m1 of recombinant CBP-Rat HMGB1 peptide (SEQ ID NO:5). Where indicated, 20 pg/m1 of 3G8 HMGB1 mAb (3G8), 1A9 IIMGB1 mAb (1A9), 9G2 HMGB1 mAb (9G2), 6E6 HMGB1 mAb (6E6), 2E11 IfMGB1 mAb (2E11), 10D4 HMGB1 mAb (10D4), 6H9 HMGB1 mAb (6H9) or mouse IgG control antibody (IgG) were added.
FIG. 10 is a graph of the effect of various anti-BMGB1 monoclonal antibodies (6E6 HMGB1 mAb (mAB (6E6)); 2E11 BMGB1 mAb (mAB (2E11));
9G2 HMGB1 mAb (mAB (9G2))) and a control IgG antibody (Ctrl IgG) on survival of mice over time (days) after cecal ligation and puncture (CLP).
FIG. 11 depicts a series of individual Western blots of samples containing either CHO FIMGB1 or CHO HMGB2 and possibly recombinant IIMGB1-His6 (labeled as CHO HMGB2, rec-HMGB1-His6), which were probed with either an anti-His Tag antibody (Anti-His Tag), an anti-HMGB2 antibody (Anti-HMGB2), an anti-HMGB1/2 monoclonal antibody (Anti-IIMGB1/2 mAb) or particular anti-HMGB1 monoclonal antibodies (i.e., 2E11 HMGB1 mAb (CT3-2E11), 1G3 HMGB1 mAb (CT3-1G3), 6H9 HMGB1 mAb (CT3-6H9), 2G7 HMGB1 mAb (CT3-2G7), 2G5 HIvIGB 1 mAb (CT3-2G5) and 6E6 ITMGB 1 mAb (CT3-6E6)).
FIG. 12 is an amino acid sequence alignment of HMGB1 polypeptide sequences from rat (SEQ ID NO:18; labeled "rat # P07155" or "rat" (GenBank Accession No. P07155)), mouse (Mus musculus) (SEQ ID NO:18; labeled "mouse
-8-#AAA20508" or "mouse" (Gen-Rank Accession No. AAA20508)) and human (Homo sapiens) (SEQ ID NO:1; labeled "human #AAA64970" or "human" (GenBank Accession No. AAA64970)). The A box and B box domains are underlined and labeled as indicated.
FIG. 13A is a table depicting individual peptides corresponding to particular regions of human 11MGB1, their respective amino acid sequences, molecular weights, calculated masses required to produce a 1 rnM stock solution and available amounts.
FIG. 13B is a histogram depicting the results of HMGB1 peptide binding experiments. Biotinylated peptides corresponding to particular 18 amino acid regions of human HMGB1 and a longer peptide corresponding to amino acid residues 9-85 of human BIVIGB1 (listed in FIG. 13A) were prepared and analyzed for binding to particular anti-HMGB1 monoclonal antibodies (i.e., 2E11. HMGB1 mAb (2E11), 6E6 HMGB1 mAb (6E6), 6119 11MGB1 mAb (6119) and 2G7 BMGB1 mAb (2G7)) by ELISA.
FIG. 14 is a graph depicting the results of anti-HMGB1 monoclonal antibody 1ELISAs. In the ELISAs, particular antilIMGB1 monoclonal antibodies (2E11 IILVIGB1 mAb (2E11), 2G5 B1LVIGB1 inAb (2G5), 2G7 IIMGB1 mAb (2G7) and 6E6 IIMGB1 mAb (6E6)) were used as capture antibodies and a poiyclonal HIVIGB1 antibody was used as the detector antibody.
FIG. 15 is a graph depicting the results of anti-BMGB1 monoclonal antibody ELISAs. In the ELISAs, particular anti-HMGB1 monoclonal antibodies (2E11 HMGB1 mAb (2E11), 2G5 HMGB1 mAb (2G5), 2G71-114GB1 mAb (2G7) and 6E6 mAb (6E6)) were used as capture antibodies and 6E6 HMGB1 mAb was used as the detector antibody.
FIG. 16 is a graph depicting a dose response curve for anti-HMGB1 monoclonal antibody 6E6 HIvIGB1 mAb (6E6; at doses of 11.t.g/mouse, 10 pg/mouse or 100 fig/mouse as labeled) or a control IgG antibody (Control IgG) on survival of mice over time (days) after cecal ligation and puncture (CLP).
FIG. 17 is a sequence alignment of INGB1 polypeptide sequences of an HMGB1 polypeptide expressed in CHO cells (CHOHNIGB1; SEQ ID NO:36); rat
FIG. 13A is a table depicting individual peptides corresponding to particular regions of human 11MGB1, their respective amino acid sequences, molecular weights, calculated masses required to produce a 1 rnM stock solution and available amounts.
FIG. 13B is a histogram depicting the results of HMGB1 peptide binding experiments. Biotinylated peptides corresponding to particular 18 amino acid regions of human HMGB1 and a longer peptide corresponding to amino acid residues 9-85 of human BIVIGB1 (listed in FIG. 13A) were prepared and analyzed for binding to particular anti-HMGB1 monoclonal antibodies (i.e., 2E11. HMGB1 mAb (2E11), 6E6 HMGB1 mAb (6E6), 6119 11MGB1 mAb (6119) and 2G7 BMGB1 mAb (2G7)) by ELISA.
FIG. 14 is a graph depicting the results of anti-HMGB1 monoclonal antibody 1ELISAs. In the ELISAs, particular antilIMGB1 monoclonal antibodies (2E11 IILVIGB1 mAb (2E11), 2G5 B1LVIGB1 inAb (2G5), 2G7 IIMGB1 mAb (2G7) and 6E6 IIMGB1 mAb (6E6)) were used as capture antibodies and a poiyclonal HIVIGB1 antibody was used as the detector antibody.
FIG. 15 is a graph depicting the results of anti-BMGB1 monoclonal antibody ELISAs. In the ELISAs, particular anti-HMGB1 monoclonal antibodies (2E11 HMGB1 mAb (2E11), 2G5 HMGB1 mAb (2G5), 2G71-114GB1 mAb (2G7) and 6E6 mAb (6E6)) were used as capture antibodies and 6E6 HMGB1 mAb was used as the detector antibody.
FIG. 16 is a graph depicting a dose response curve for anti-HMGB1 monoclonal antibody 6E6 HIvIGB1 mAb (6E6; at doses of 11.t.g/mouse, 10 pg/mouse or 100 fig/mouse as labeled) or a control IgG antibody (Control IgG) on survival of mice over time (days) after cecal ligation and puncture (CLP).
FIG. 17 is a sequence alignment of INGB1 polypeptide sequences of an HMGB1 polypeptide expressed in CHO cells (CHOHNIGB1; SEQ ID NO:36); rat
-9-(ratHIVIGB1; SEQ ID NO:18), mouse (musHMGB1; SEQ lD NO:18), human (huHMGB1; SEQ JD NO:74), pig (susHIVIGB1; SEQ ID NO:37) and cow (bosHMGB1; SEQ ID NO:38) FIG. 18A is a nucleotide sequence of a human recombinant H.MGB1 polypeptide containing a 5' 6 HIS tag (rec-IIMGB1-His6; SEQ ID NO:39). Cloning sequences are indicated in lower case.
FIG. 18B is the encoded amino acid sequence of the human recombinant HIVIGB1 polypeptide containing a 5' 6 HIS tag (rec-LIMGBI-His6; SEQ NO:40).
FIG. 19A is the nucleotide sequence encoding the V11 domain of 207 HMGB1 mAb (SEQ ID NO:41).
FIG.19B is the encoded amino acid sequence of the VH domain of 2G7 BMGB1 mAb (SEQ ID NO:42); CDRs are underlined.
FIG. 19C is the nucleotide sequence encoding the Vic domain of 2G7 HMGB1 mAb (SEQ ID NO:43).
FIG. 19D is the encoded amino acid sequence of the VK domain of 2G7 HIVIGB1 mAb (SEQ ID NO:44); CDRs are underlined.
FIG. 20 is a histogram depicting the results of HMGB1 peptide binding experiments. Biotinylated peptides corresponding to either amino acid residues 63 or 61-78 of HMGB1 were prepared and analyzed for binding to 2G7 HMGB1 mAb (2G7) by ELISA.
FIG. 21 is a histogram depicting the results of HMGB1 peptide binding experiments. Biotinylated peptides corresponding to either amino acid residues 63 or 151-168 of HMGB1 were prepared and analyzed for binding to 2E11 BMGB1 mAb (2E11) by ELISA.
FIG. 22 is a histogram depicting the results of HMGD1 and ITMGB2 peptide binding experiments. Peptides corresponding to either amino acid residues 46-63 of human IIMGB1 (labeled "huHMGB1-46-63-B"), amino acid residues 46-63 of human HMGB2 (labeled "huI-EVIGB2-46-63-B"), amino acid residues 53-70 of human BIVIGB1 (labeled "huHMGB1-53-70"), or amino acid residues 61-78 of human HMGB1 (labeled "huHIVIGB1-61-78-B") were prepared and analyzed for binding to 207 1-11VIGB1 mAb (2G7) or avidin by ELISA.
FIG. 23 is a table depicting the results of EIMGB1 and HMGB2 peptide binding experiments. Listed in the table are various peptides, their respective amino acid sequences and whether the peptides bind 2G7 HMGB I mAb. The listed peptides include: a peptide corresponding to amino acid residues 40-57 of human HMGD1 (labeled "Human HMGB1-40-57"), a peptide corresponding to amino acid residues 46-63 of human HIVIGB I (labeled "Human 111VIGB1-46-63-B), a peptide corresponding to amino acid residues 53-70 of human HMGB I (labeled "Human H1\4GBI-53-70"), a peptide corresponding to amino acid residues 46-63 of human HivIGB2 (labeled "Human HIVIGB2-46-63-B"), and a peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 46-63 of human HMGB1 (labeled "Human HMGB1-46-63-scr").
FIG. 24 is a table depicting the results of FIMGB1 peptide binding experiments. Listed in the table are various peptides, their respective amino acid sequences and whether the peptides bind 6E6 HMGB1 mAb. The listed peptides include: a peptide corresponding to amino acid residues 53-70 of human HMGB I
(labeled "Human HMGB1-53-70"), a peptide corresponding to amino acid residues 61-78 of human BMGB I (labeled "Human HMGB1-61-78-B"), a peptide corresponding to amino acid residues 67-84 of human HIVIGB I (labeled "Human HMGB1-67-84"), and a peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 61-78 of human IIIVIGB1 (labeled "Human HIV1GB I -61-78_scr").
FIG. 25 is a table depicting the results of1-11MGB1 peptide binding experiments. Listed in the table are various peptides, their respective amino acid sequences and whether the peptides bind 2E11 HirvIGB1 mAb. The listed peptides include: a peptide corresponding to amino acid residues 143-160 of human HVGB1 (labeled "Human HMGB1-143-160"), a peptide corresponding to amino acid residues 151-168 of human EIMGB I (labeled "Human HIVIGB1-151-168-B"), a peptide corresponding to amino acid residues 157-174 of human HMGB I (labeled "Human HMGB1-157-174"), and a peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 151-168 of human HMGB1 (labeled "Human HMGB I -151-168_scr").
FIG. 26 is a histogram summarizing the results of peptide binding experiments and depicting the mapped epitopes of HMGB1 that are recognized by 2G7 IIMGB1 mAb (2G7), 6E6 HMGB1 mAb (6E6), 2G5 HMGB1 mAb (2G5), 6H9 HMGB1 mAb (6H9) and 2E11 HMGB1 mAb (2E11).
FIG. 27A is a mass spectrum of intact, non-reduced 6E6 HMGB1 mAb.
FIG. 27B is a mass spectrtun of 6E6 HMGB1 mAb, which was reduced by treatment with DTT.
FIG. 27C is amass spectrum of the light chains of 6E6 EIMGB1 mAb, which were reduced by treatment with DTT.
FIG. 27D is a mass spectrum of the heavy chains of 6E6 HMGB1 mAb, which were reduced by treatment with DTT.
FIG. 28 is a graph of the effect of administration of various doses (either 0.004 mg/kg, 0.04 mg/kg or 0.4 mg/kg) of 2G7 HMGB1 mAb or a control IgG
antibody (0.4 mg/kg) on survival of mice over time (days) after cecal ligation and puncture (CLP).
FIG. 29 is a table summarizing CLP survival percentages in mice administered various doses (either 4 mg/kg, 0.4 mg/kg, 0.04 mg/kg or 0.004 mg/kg) of 6E6 HMGB1 mAb (6E6), 2G7 HMGB1 mAb (2G7), or control IgG.
FIG. 30 is the amino acid sequence of a human (Homo sapiens) IIMGB2 polypeptide (SEQ rD NO:54; GenBank Accession No. M83665).
FIG 31A is the amino acid sequence of a human (Homo sapiens) FLIVIGB1 polypeptide (SEQ ID NO:74).
FIG 31B is an A box of a human (Homo sapiens) HMGB1 polypeptide (SEQ
ID NO:75).
FIG 31C is a B box of a human (Homo sapiens) HMGB1 polypeptide (SEQ
ID NO:76).
FIG. 32 is a histogram depicting inhibition of TNF release by various anti-HMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.1 ptg/m1 of recombinant CBP-Rat BIMGB1 peptide (SEQ
NO:5). Where indicated, various IIMGB1 monoclonal antibodies (cultured supernatants) were added to give a final concentration of 13%. The following antibodies were tested: 1A9 HMGB1 mAb (1A9); 2E11 HMGB1 mAb (2E11); 2G5 HMGB1 mAb (2G5); 2G7 HMGB1 mAb (2G7); 3G8 HMGB1 mAb (3G8); 4H11 IEVIGB1 mAb (4H11); 3-5A6 HMGB1 mAb (5A6); 6E6 HMGB1 mAb (6E6); 9G2 HMGB1 mAb (9G2); 4C9 HMGB1 mAb (4C9); and 6119 HMGB1 niAb (6119). The initial dark bar depicts TNF release in the absence of any antibodies.
FIG. 33 is a histogram depicting inhibition of TNF release by various anti-HMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.1 jig/m1 of recombinant CBP-Rat HMGB1 peptide (SEQ ID
NO:5). Where indicated, various HMGB1 monoclonal antibodies (cultured supernatants) were added to give a final concentration of 13%. The following antibodies were tested: 7113 1114GB1 mAb ,(7113); 9113 FIIVIGB1 mAb (9113);
IIMGB I mAb (10D4); 1C3 HMGB1 mAb (1C3); 3E10 HMGB1 mAb (3E10);
4A1 0 IIMGBI mAb (4A1 0); 5C12 HIVIGBI mAb (5C12); and 7G8 BMGB1 mAb (7G8). The initial dark bar depicts TNF release in the absence of any antibodies.
DETAILED DESCRIPTION OF THE INVENTION
In various embodiments, the present invention is drawn to antibodies or antigen-binding fragments thereof that bind to a vertebrate high mobility group box (1IMGB) polypeptide, methods of detecting and/or identifying an agent that binds to an HMGB polyp eptide, methods of treating a condition in a subject characterized by activation of an inflammatory cytoldne cascade and methods of detecting an HMGB
polypeptide in a sample.
Antibodies and Antibody Producing Cells In one embodiment, the present invention encompasses antibodies or antigen-binding fragments thereof that bind to IIMGB polypeptides. The antibodies of the invention can be polyclonal or monoclonal, and the term "antibody" is intended to encompass both polyclonal and monoclonal antibodies. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
In one embodiment, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment thereof. The term "monoclonal antibody"
or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of im_munoreacting with a particular epitope of a polypeptide of the invention. A
monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
The term "antibody" as used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies. Functional fragments include antigen-binding fragments of antibodies that bind to an I-114GB polypeptide (e.g., a mammalian FINIGB
polypeptide (e.g. a mammalian HMGB1 polypeptide)). For example, antibody fragments capable of binding to an HMGB polypeptide or a portion thereof, include, but are not limited to Fv, Fab, Fab' and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.
Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody".
The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., ., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 Bl; Boss etal., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M.S. etal., WO 86/01533; Neuberger, M.S. etal., European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen etal., European Patent No. 0 451 216 Bl; and Padlan, E.A. et aL, EP 0 519 596 Al. See also, Newman, R. etal., Biorechnolog,y, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Patent No. 4,946,778 and Bird, R.E. etal., Science. 242: 423-426 (1988)) regarding single chain antibodies.
Humanized antibodies can be produced using synthetic or recombinant DNA
technology using standard methods or other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., etal., NucL Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101: 297-(1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. 5,514,548; Hoogenboom et al., WO 93/06213).
The antibody can be a humanized antibody comprising one or more iinmunoglobulin chains (e.g., an antibody comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes)). In one embodiment, the antibody or antigen-binding fragment thereof comprises the light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3) of a particular immunoglobulin. In another embodiment, the antibody or antigen-binding fragment further comprises a human framework region.
The antibodies described herein can also be conjugated to an agent. In one embodiment, the agent is a label, for example, a radioisotope, an epitope label (tag), an affinity label (e.g., biotin, avidin), a spin label, an enzyme, a fluorescent group or a chemiluminescent group. Labeled antibodies or antigen-binding fragments of the present invention can be used, e.g., in the diagnostic and/or prognostic methods described herein. In another embodiment, the antibody is conjugated to a drug, toxin or anti-inflammatory agent. Conjugation of a drug, toxin or anti-inflammatory agent to the anti-HMGB antibodies and antigen-binding fragments of the invention allows for targeting of these agents to sites of ITIVIGB expression and/or activity.
Drugs and toxins that can be conjugated to the antibodies of the present invention include, for example, chemotherapeutic agents (e.g., mitomycin C, paxlitaxol, methotrexate, fluorouracil, cisplatin, cyclohexamide), toxins (e.g., ricin, gelonin) and other agents described herein (e.g., the agents described for combination therapy). Anti-inflammatory agents that can be conjugated include, e.g., those described herein.
Antibodies that are specific for an HMGB polypeptide (e.g., a mammalian EdvIGB polypeptide) can be raised against an appropriate immunogen, such as an isolated and/or recombinant HMGB polypeptide or a portion thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express an HMGB
polypeptide, such as 0113 pituicytes, macrophage cells (e.g., RAW 246.7 cells, human macrophage cells), peripheral blood mononuclear cells (PBMCs (e.g., human PBMCs)), primary T cells (e.g., human primary T cells), adrenal cells (e.g., rat adrenal PC-12 cells, human adrenal cells), and kidney cells (e.g., rat primary kidney cells, human primary kidney cells). In addition, cells expressing a recombinant HMGB polypeptide (e.g., a mammalian HMGB polypeptide), such as transfected cells, can be used as an immunogen or in a screen for an antibody that binds thereto (See e.g., Chuntharapai et at., Ininzunol., 152: 1783-1789 (1994);
Chuntharapai et at., U.S. Patent No. 5,440,021).
Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et at., Nature, 256: 495-497 (1975) and Dlr. J.
bizmunoL 6: 511-519 (1976); Milstein et al., Nature 266: 550-552 (1977);
Koprowski et al., U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, as exemplified herein, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as 5P2/0, P3X63Ag8.653 or a heteromyeloma) with antibody-producing cells. Antibody-producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Other suitable methods of producing or isolating antibodies of the requisite specificity (e.g., human antibodies or antigen-binding fragments) can be used, including, for example, methods that select recombinant antibody from a library (e.g., a phage display library). Transgenic animals capable of producing a repertoire of human antibodies (e.g., Xenomouse (Abgenix, Fremont, CA)) can be produced using suitable methods (see e.g., .Takobovits et al., Proc. Natl. Acad. Sci.
USA, 90:
2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258 (1993)). Additional methods that are suitable for production of transgenic animals capable of producing a repertoire of human antibodies have been described (e.g., Lonberg et al., U.S.
Patent No. 5,545,806; Surani et al., U.S. Patent No. 5,545,807; Lonberg et al., W097/13852).
In one embodiment, the antibody or antigen-binding fragment thereof has specificity for an BNIGB polypeptide (e.g., a mammalian HIMGB polypeptide). In a particular embodiment, the antibody or antigen-binding fragment thereof has specificity for an 1-Th4GB1 polypeptide (e.g., a human EMGB1 polypeptide such as depicted in SEQ ID NO:1 and/or SEQ ID NO:74). In another embodiment, the ,antibody or antigen-binding fragment thereof is an IgG or an antigen-binding fragment of an IgG. In another embodiment, the antibody or antigen-binding fragment thereof is an IgG1 or an antigen-binding fragment of an IgGl. In still other embodiments, the antibody or antigen-binding fragment thereof is an IgG2a, IgG2b, IgG3 antibody, or an antigen-binding fragment of any of the foregoing.
In another embodiment, the antibody or antigen-binding fragment can bind to an HIVIGB polypeptide and inhibit (reduce or prevent) one or more functions of the 1-11VIGB polypeptide. Such HMGB functions include, e.g., increasing inflammation (see, e.g., PCT Publication No. WO 02/092004), increasing release of a proinflammatory cytokine from a cell (see, e.g., PCT Publication No. WO
02/092004), binding to RAGE, binding to TLR2, chemoattraction (see, e.g., Degryse et aL, I Cell Biol. I52(6):1197-1206 (2001), and activation of antigen presenting cells (see, e.g., WO 03/026691).
=
In one embodiment, the antibody is a human antibody or an antigen-binding fragment thereof. In another embodiment, the antibody is a humanized antibody or an antigen-binding fragment thereof. In yet another embodiment, the antibody or antigen-binding fragment can inhibit binding of a polypeptide (e.g., RAGE, TLR2) to an HMGB polypeptide and/or inhibit one or more functions mediated by binding of the HMGB polypeptide and the other polypeptide.
In certain embodiments, the antibodies or antigen-binding fragments thereof specifically bind to HiVIGB epitopes or antigenic determinants (e.g., HMGB
epitopes, HMGB A box epitopes, HMGB B box epitopes). As described herein, an antibody or antigen-binding fragment thereof can be screened without undue experimentation for the ability to inhibit release of a proinflammatory cytokine using standard methods. Anti-IIMGB A-box antibodies and anti-BNIGB B box antibodies that can inhibit the production of a proinflanunatory cytokine and/or the release of a proinflammatory cytokine from a cell, and/or inhibit a condition characterized by activation of an inflammatory cytokine cascade, are within the scope of the present =
invention. In one embodiment, the antibody or antigen-binding fragment of the invention can inhibit the production of TNF, IL-1 1. and/or IL-6. In another embodiment, the antibody or antigen-binding fragnent of the invention can inhibit =
the production of TNF (e.g., TNF-a).
As described herein, monoclonal antibodies designated "6E6 HMGB1 mAb", "2E11 HMGB1 mAb", "6H9 FIMGB1 mAb", "10D4 HMGB1 mAb" and "2G7 HMGB1 mAb", all of which bind to HMGB1 have been produced. In addition, other monoclonal antibodies designated "9G2 11MGB1 mAb", "1A9 HMGB1 mAb", "3G8 HMGB1 mAb", "2G5 HMGB1 mAb", "41111 HMGB1 mAb", "7H3 HMGB1 mAb", "3-5A6 HMGB1 mAb", "9G1 HMGB1 mAb", "4C9 HMGB1 mAb", "9113 HMGB1 mAb", "1C3 HMGB1 mAb", "5C12 HMGB1 mAb", "3E10 HMGB1 mAb", "7G8 IEVIGB1 mAb" and "4A10 HMGB1 mAb" have been produced. All but 9G2 HMGB1 mAb and 1A9 HMGB1 mAb have been shown to bind HMGB1. 9G2 HMGB1 mAb and 1A9 HMGB1 mAb appear to bind to the CBP region of the immunogen (which is not cleaved in a small percentage of the immunbgen).
6E6 1{MGB1 mAb, also referred to as 6E6-7-1-1 or 6E6, can be produced by murine hybridoma 6E6 HMGB1 mAb, which was deposited on September 3,2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No.
PTA-5433. The invention relates to murine hybridoma 6E6 HMGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
2E11 HMGB1 mAb, also referred to as 2E11-1-1-2 or 2E11, can be produced by murine hybridoma 2E11 HMGB1 mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No. PTA-5431. The invention relates to murine hybridoma 2E11 HMGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
6H9 11MGB1 mAb, also referred to as 6H9-1-1-2 or 6119, can be produced by murine hybridoma 6119 11MGB1 mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No.
PTA-5434. The invention relates to murine hybridoma 6H9 1114GB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
10D4 IHVIGB I mAb, also referred to as 10D4-1-1-1-2 or 10D4, can be produced by murine hybridoma 10D4 H1MGB1 mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 146 Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No. PTA-5435. The invention relates to murine hybridoma 10D4 BIVIGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
2G7 HMGB1 mAb, also referred to as 3-2G7-1-1-1 or 2G7, can be produced by murine hybridoma 207 IIMGB I mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No.
PTA-5432. The invention relates to murine hybridoma 207 FIMGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
For cultivation of the above identified murine hybridomas (e.g., 6E6 HMGB I mAb, 6H9 HMGB1 mAb, 207 HMGB I mAb, 10D4 HMGB1 mAb, 2G7 HIV1GB I mAb), DMEM, 10% FCS, 1% 1L-6, 1% L-glutamine and 1% Pen-Strep should be added.
902 1-314GB1 mAb, also referred to as 902-7-1-1-1 or 902, can be produced by murine hybridoma 9G2 HMGB1 mAb. The invention relates to murine hybridoma 9G2 HIVIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
1A9 HMGB1 mAb, also referred to as 1A9-1-2-1-4 or 1A9, can be produced by murine hybridoma 1A9 HMGB1 mAb. The invention relates to murine hybridoma 1A9 IIMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
3G8 IIMGB1 mAb, also referred to as 3G8-7-2-1-5 or 3G8, can be produced by murine hybridoma 3G8 HMGB1 mAb. The invention relates to murine hybridoma 3G8 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
2G5 HMGB1 mAb, also referred to as 3-2G5-4-1-2 or 2G5, can be produced by murine hybridoma 2G5 HMGB1 mAb_ The invention relates to murine hybridoma 2G5 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
4H11 HMGB1 mAb, also referred to as 41111. can be produced by murine hybridoma 41111 HMGB mAb. The invention relates to murine hybridoma 4H1 1 HIV1GB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
1 5 7H3 HMGB1 mAb, also referred to as 7H3, can be produced by murine hybridoma 7113 HMGB1 mAb. The invention relates to murine hybridoma 7113 111V1GB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
3-5A6 HMGB1 mAb, also referred to as 3-5A6 or 5A6, can be produced by murine hybridoma 3-5A6 1114GB1 mAb. The invention relates to murine hybridoma 3-5A6 11MGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
9G1 HMGB1 mAb, also referred to as 9G1, can be produced by murine hybridoma 9G1 HMGB1 mAb. The invention relates to murine hybridoma 9G1 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
4C9 HMGB1 mAb, also referred to as 4C9, can be produced by murine hybridoma 4C9 HMGB1 mAb. The invention relates to murine hybridoma 4C9 HIVIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
9H3 IIMGB1 mAb, also referred to as 9H3, can be produced by murine hybridoma 9H3 HMGB1 mAb. The invention relates to murine hybridoma 9113 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
1C3 HMGB1 mAb, also referred to as 1C3-1-1-1-1 or 1C3, can be produced by murine hybridoma 1C3 1-MIGB1 mAb. The invention relates to murine hybridoma 1C3 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
5C12 HMGB1 mAb, also referred to as 5C12-1-1-1-1 or 5C12, can be produced by murine hybridoma 5C12 HMGB1 mAb. The invention relates to murine hybridoma 5C12 HNIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
3E10 HMGB1 mAb, also referred to as 3E10-5-4-1-1 or 3E10, can be produced by murine hybridoma 3E10 IIMGB1 mAb. The invention relates to murine hybridoma 3E10 ILMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
7G8 IIMGB 1 mAb, also referred to as 7G8, can be produced by murine hybridoma 7G8 HMGB1 mAb. The invention relates to marine hybridoma 7G8 111VIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
4A10 HMGB1 mAb, also referred to as 4A10-1-3-1-1 or 4A10, can be produced by murine hybridoma 4A10 HiVIGB I mAb. The invention relates to murine hybridoma 4A10 ITIVIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of 6E6 1-11VIGB1 mAb, 6119 IIMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB I mAb, 2E11 HMGB1 mAb and an antigen-binding fragment of any of the foregoing.
In another embodiment, the antibody or antigen-binding fragment has the same or similar epitopic specificity of an antibody or antigen-binding fragment selected from the group consisting of 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 1-IIVIGB1 mAb, 2E11 HMGB1 mAb and/or an antigen-binding fragment of any of the foregoing. Antibodies or antigen-binding fragments with an epitopic specificity that is the same as, or similar to, that of 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 niAb, 10D4 MVIGB1 mAb and/or 2E11 HilVIGB1 mAb can be identified by a variety of suitable methods. For example, an antibody with the same or similar epitopic specificity as, e.g., 6E6 HIVIGB1 mAb, can be identified based upon the ability to compete with 6E6 IIMGB1 mAb for binding to a FM/1GB
polypeptide (e.g., a mammalian HMGB polypeptide (e.g., a mammalian HMGB1 polypeptide)). In another example, the binding of, e.g., 6E6 IIMGB1 mAb, and the binding of an antibody with the same or similar epitopic specificity for a H_MGB
polypeptide can be inhibited by a single peptide (e.g., a natural peptide, a synthetic peptide). In various embodiments, the peptide can comprise, e.g., 9 to about amino acids, 9 to about 40 amino acids, 9 to about 30 amino acids, 9 to about amino acids or 9 to about 20 amino acids.
As exemplified herein, 18 amino acid peptides corresponding to particular regions of the human HMGB1 polypeptide were shown to bind to various ITIVIGB1 monoclonal antibodies. The studies described herein mapped epitopes within HMGB1 that bind to particular HMGB1 antibodies.
For example, 2E11 HMGB1 raAb was shown to bind a peptide corresponding to amino acids 151-168 of human ITMGB1 (amino acid residues 151-168 of SEQ ID NO:1; i.e., LKEKYEKDIAAYRAKGKP (SEQ ID NO:30)).
Additional studies suggest that 2E11 HMGB1 mAb recognizes an epitope that is present in within amino acid residues 156-161, 155-161, 155-162, 156-162 and/or 156-163, of EIMGB1 (see Example 14).
6E6 HIvIGB1 mAb and 6H9 EIMGB1 mAb were shown to bind to a peptide corresponding to amino acids 61-78 of human HMGB1 (amino acid residues 61-78 of SEQ ID NO:1; i.e., EDMAKADKARYEREIVIKTY (SEQ ID NO:24)).
Additional studies demonstrated that 6E6 HMGB1 mAb recognizes an epitope that is present within amino acid residues 67-78 of HIVIGB1 (see Example 13).
2G7 ELMGB1 mAb was shown to bind a peptide corresponding to amino acids 46-63 of human HMGB1 (amino acid residues 46-63 of SEQ ID NO:1; i.e., SERIVKTMSAKEKGKFEDM (SEQ ID NO:23)) (see Example 10). Further studies demonstrated that 2G7 ITMGB1 mAb recognizes an epitope that is present within amino acid residues 53-63 of HMGB I (see Example 12). In addition, 2G7 EIMGB1 mAb does not bind to amino acid residues 46-63 of HMGB2 (SEQ ID NO:48), notwithstanding only a single amino acid difference between the BlVIGB1 46-63 peptide and the IIMGB2 46-63 peptide (see Example 12). Thus, in one embodiment, the antibodies or antigen-binding fragments of the invention bind to EIMGB1 but not to EIMGB2. In other embodiments, the antibodies or antigen-binding fragments of the invention bind to both HIvIGB1 and HIVIGB2.
These 18 amino acid peptides, or other peptides corresponding to particular regions of HMGB1, could be used in epitopic studies to determine if an antibody or antigen-binding fragment inhibited binding of the peptide to an antibody known to bind that peptide (e.g., 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 niAb, 10D4 BIMGB 1 mAb, 2E11 FEVIGB1 mAb, others antibodies described herein).
Thus, for example, an antibody or antigen-binding fragment to be tested for its epitopic specificity could be assayed with, e.g., 2E11 HMGB1 mAb and a peptide corresponding to amino acids 151-168 of human HMGB1 (which 2E11 HIvIGB1 mAb is known to bind).
In another example, an antibody with the same or similar epitopic specificity as an antibody of the invention (e.g., 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HIVIGB1 mAb, 10D4 HIVGB1 mAb and/or 2E11 HMGB1 mAb) can be identified using a chimeric HMGB polypeptide (see e.g., Banks, G.C., et at., J Biol.
Chem.
274(23):16536-16544 (1999)).
In one embodiment, the antibody or antigen-binding fragment can compete with 6E6 HMGB I mAb, 6H9 IIMGB1 mAb, 2G7 HIVIGB1 mAb, 10D4 BilvIGB1 mAb, 2E11 HMGB1 mAb and/or an antigen-binding fragment of any of the foregoing, for binding to an BIVIGB polypeptide (e.g., a mammalian FINIGB
polypeptide (e.g., a mammalian HMGB1 polypeptide)). Such inhibition of binding can be the result of competition for the same or similar epitope or steric interference (e.g., where antibodies bind overlapping epitopes or adjacent epitopes).
Inhibition by 6E6 IIMGB1 inAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 -?4-mAb, 2E11 HMGB1 mAb, and/or an antigen-binding fragment of any of the foregoing, can also be due to a change in the conformation of the HMGB
polypeptide that is induced upon antibody binding to the FEVIGB polypeptide.
In another embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of 3G8 HMGB1 mAb, 2G5 FIMGB1 mAb, 4H11 HMGB1 mAb, 7113 HIMGB1 mAb, 3-5A6 IIMGB1 mAb, 9G1 HMGB1 mAb, 4C9 HMGB1 mAb, 9H3 HMGB1 mAb, 1C3 EMGB1 mAb, 5C12 H_MGB1 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb, 4A10 HMGB1 mAb, and an antigen-binding fragment of any of the foregoing.
In one embodiment, the antibody or antigen-binding fragment has the epitopic specificity of an antibody or antigen-binding fragment selected from the group consisting of 3G8 IEVIGB1 mAb, 2G5 HMGB1 mAb, 4E111 IIMGB1 mAb, 7113 HMGB1 mAb, 3-5A6 HMGB1 mAb, 9G1 11MGB1 mAb, 4C9 HMGB1 mAb, 9113 BMGB1 mAb, 1C3 HMGB1 mAb, 5C12 HMGB1 mAb, 3E10 1114GB1 mAb, 7G8 HMGB1 mAb, 4A10HMGB1 mAb, and an antigen-binding fragment of any of the foregoing. As described above, antibodies or antigen-binding fragments with an epitopic specificity that is the same as, or similar to, one or more of these antibodies or antigen-binding fragments can be identified by a variety of suitable methods (e.g., ' using methods described herein and/or blown in the art).
In another embodiment, the antibody or antigen-binding fiagment can compete with 3G8 HMGB1 mAb, 2G5 HIVIGB1 mAb, 41111 HMGB1 mAb, 7113 HMGB1 mAb, 3-5A6 HIVIGB1 rnAb, 9G1 HMGB1 mAb, 4C9 HMGB1 mAb, 9113 ITIVIGB1 mAb, 1C3 1-1:MGB1 mAb, 5C12 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb, 4A10 HMGB1 mAb, and/or an antigen-binding fragment of any of the foregoing, for binding to an HMGB polypeptide (e.g., a mammalian HMGB
polypeptide). As described above, inhibition of binding can be the result of competition for the same or similar epitope or steric interference (e.g., where antibodies bind overlapping epitopes or adjacent epitopes). Inhibition can also be due to a change in the confaimation of the HMGB polypeptide that is induced upon binding of the antibody or antigen-binding fragment to the HMGB polypeptide.
In one embodiment, the antibody or antigen-binding fragment thereof comprises the six CDRs (light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3)) of an antibody selected from the group consisting of 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HIIVIGB 1 mAb and 2E11 FIMGB1 mAb. In another embodiment, the antibody is a humanized antibody that comprises the light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3) of an antibody selected from the group consisting of 6E6 HMGB1 mAb, 6H9 HIVIGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb and 2E11 HMGB1 mAb. In other embodiments, the antibody or antigen-binding fragment thereof comprises the six CDRs (light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3)) of any other antibody described herein.
In another embodiment, the antibody or antigen-binding fragment thereof comprises from one to six of the light chain and heavy chain CDRs of an antibody of the invention (e.g., 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 BIMGB1 mAb, 10D4 HMGB1 mAb, 2E11 HMGB1 mAb). For example, the antibody or antigen-binding fragment can comprise one, two, three, four, five or six, of the light chain and heavy chain CDRs. In another embodiment, the antibody or antigen-binding fragment thereof comprises at least one light chain CDR or heavy chain CDR
from one antibody of the invention and at least one light chain CDR or heavy chain CDR
from a different antibody of the invention (e.g., 6E6 HMGB1 mAb, 6119 IIMGB1 mAb, 2G7 HMGB1 mAb, 10D4 FIMGB1 mAb, 2E11 HMGB1 mAb). For example, an antibody or antigen-binding fragment could comprise one or more CDRs from 6E6 11MGB1 mAb and one or more CDRs from 6119 IIMGB1 mAb. Antibodies and antigen-binding fragments combining other combinations of CDRs from different antibodies of the invention are also encompassed.
In another embodiment, the antibody or antigen-binding fragment thereof comprises the six CDRs (light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3)) of an antibody selected from the group consisting of 3G8 HMGB1 mAb, 2G5 IIMGB1 mAb, 41111 HIVIGB1 mAb, 7113 EIMGB1 mAb, 3-5A6 ITh4GB1 mAb, 9G1 HMGB1 mAb, 4C9 111V1GB1 mAb, 9113 mAb, 1C3 HMGB1 mAb, 5C12 HMGB1 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb and 4A10 HMGB1 mAb. In another embodiment, the antibody or antigen-binding fragment thereof comprises from one to six of the light chain and heavy chain CDRs of one of these antibodies.
In certain embodiments, the antibody or antigen-binding fragment comprises one or more CDRs that are at least 80% identical, at least 90% identical, or at least 95% identical, to a CDR of an antibody of the invention (e.g., 6E6 IldvIGB1 mAb, 6H91-EvIGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb, 2E11 HMGB1 mAb).
In other embodiments, the antibody or antigen-binding fragment comprises one or more CDRs that are at least 80% similar, at least 90% similar, or at least 95%
similar, to a CDR of an antibody of the invention. Methods for determining sequence identity and similarity of two polypeptides are described herein and/or are well known in the art.
The invention also relates to a bispecific antibody, or functional fragment thereof (e.g., F(ab')2), which binds to an HMGB polypeptide and at least one other antigen (e.g., tumor antigen, viral antigen). In a particular embodiment, the bispecific antibody, or functional fragment thereof, has the same or similar epitopic specificity as 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb and/or 2E11 HMGB1 raAb, and at least one other antibody.
Bispecific antibodies can be secreted by triomas and hybrid hybridomas.
Generally, =
triomas are fainted by fusion of a hybridoma and a lymphocyte (e.g., antibody-secreting B cell) and hybrid hybridomas are formed by fusion of two hybridomas.
Each of the fused cells (i.e., hybridomas, lymphocytes) produces a monospecific antibody. However, triomas and hybrid hybridomas can produce an antibody containing antigen-binding sites that recognize different antigens. The supernatants of triomas and hybrid hybridomas can be assayed for bispecific antibody using a suitable assay (e.g., ELISA), and bispecific antibodies can be purified using conventional methods. (see, e.g., U.S. Patent No. 5,959,084 (Ring et al.), U.S. Patent No. 5,141,736 (Iwasa etal.), U.S. Patent Nos. 4,444,878, 5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Patent No. 5,496,549 (Yamazaki et al.)).
In one embodiment, the invention relates to an isolated cell that produces an antibody or an antigen-binding fragment of the invention. In a particular embodiment, the isolated antibody-producing cell of the invention is an immortalized cell, such as a hybridoma, heterohybridoma, lymphoblastoid cell or a recombinant cell. The antibody-producing cells of the present invention have uses other than for the production of antibodies. For example, the cell of the present invention can be fused with other cells (such as suitably drug-marked human myeloma, mouse myeloma, human-mouse heteromyelonia or human lymphoblastoid cells) to produce, for example, additional hybridomas, and thus provide for the transfer of the genes encoding the antibody. In addition, the cell can be used as a source of nucleic acids encoding the anti-HMGB immnnoglobulin chains, which can be isolated and expressed (e.g., upon transfer to other cells using any suitable technique (see e.g., Cabilly et al., U.S.
Patent No. 4,816,567, Winter, U.S. Patent No. 5,225,539)). For instance, clones comprising a sequence encoding a rearranged anti-HWIGB light and/or heavy chain can be isolated (e.g., by PCR). In addition, cDNA libraries can be prepared from mRNA isolated from an appropriate cell line, and cDNA clones encoding an anti-=
HMGB immunoglobulin chain(s) can be isolated. Thus, nucleic acids encoding the heavy and/or light chains of the antibodies, or portions thereof, can be obtained and used for the production of the specific immunoglobulin, immunoglobulin chain, or variants thereof (e.g., humanized immunoglobulins) in a variety of host cells or in an in vitro translation system. For example, the nucleic acids, including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or immunoglobulin chain, can be placed into suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host cell genome), to produce a recombinant antibody-producing cell. Thus, in certain embodiments, the invention is a nucleic acid that encodes an antibody or antigen-binding fragment of the invention. In other embodiments, the invention is a vector that comprises a nucleic acid encoding an =
=
antibody or antigen-binding fragment of the invention.
HMGB Polypeptides, HMGB A boxes and HMGB B boxes As described, in one embodiment the invention is an antibody or antigen-binding fragment thereof that binds to an HMGB polypeptide.
As used herein, an "HMGB polypeptide" is a polypeptide that has at least 60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, and most preferably at least 95% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:18 and SEQ JD NO:74 (as determined, for example, using the BLAST program and parameters described herein). In one embodiment, the HMGB polypeptide increases inflammation and/or increases release of a proinflammatory cytokine from a cell. In another embodiment, the HMGB
polypeptide has one of the above biological activities. Typically, the HMGB
polypeptide has both of the above biological activities.
The term "polypeptide" refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. In one embodiment, the HMGB polypeptide is a mammalian HIVIGB polypeptide, for example, a mammalian HMGB polypeptide (e.g., a human HMGB1 polypeptide). In another embodiment, the HMGB
polypeptide contains a B box DNA binding domain and/or an A box DNA binding domain and/or an acidic carboxyl terminus as described herein.
Other examples of HMGB polypeptides are described in GenBank Accession Numbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP 002119, CAA31110, S02826, U00431, X67668, NP 005333 NM 016957, and _ J04179.
Additional examples of IIMGB polypeptides include, but are not limited to mammalian HMG1 ((liMGB1) as described, for example, in GenBank Accession Number U51677), HMG2 ((HMGB2) as described, for example, in GenBank Accession Number M83665), HMG-2A ((HVIGB3, HMG-4) as described, for example, in GenBank Accession Numbers NM 005342 and NP 005333), HIVIG14 _99-(as described, for example, in GenBank Accession Number P05114), HMG17 (as = described, for example, in GenBank Accession Number X13546), HMGI (as described, for example, in GenBank Accession Number L17131), and 11MGY (as described, for example, in GenBank Accession Number M23618); nonmammalian HMG Ti (as described, for example, in GenBank Accession Number X02666) and 1114G T2 (as described, for example, in GenBank Accession Number L32859) (rainbow trout); HMG-X (as described, for example, in GenBank Accession Number D30765) (Xenopus); HMG D (as described, for example, in GenBank Accession Number X71138) and HMG Z (as described, for example, in GenBank Accession Number X71139) (Drosophila); NHP10 protein (HMG protein homolog NITP 1) (as described, for example, in GenBank Accession Number 'Z48008) (yeast);
non-histone chromosomal protein (as described, for example, in GenBank Accession Number 000479) (yeast); HMG 1/2 like protein (as described, for example, in GenBank Accession Number Z11540) (wheat, maize, soybean); upstream binding factor (UBF-1) (as described, for example, in GenBank Accession Number X53390); PMS1 protein homolog 1 (as described, for example, in GenBank Accession Number 1J13695); single-strand recognition protein (SSRP, structure-specific recognition protein) (as described, for example, in GenBank Accession Number M86737); the HMG homolog 1.1)P-1 (as described, for example, in GenBank Accession Number M74017); mammalian sex-determining region Y
protein (SRY, testis-determining factor) (as described, for example, in GenBank Accession Number X53772); fungal proteins: mat-1 (as described, for example, in GenBank Accession Number AB009451), ste 11 (as described, for example, in GenBank Accession Number X53431) and Mc 1; SOX 14 (as described, for example, in GenBank Accession Number AF107043) (as well as SOX 1 (as described, for example, in GenBank Accession Number Y13436), SOX 2 (as described, for example, in GenBank Accession Number Z31560), SOX 3 (as described, for example, in GenBank Accession Number X71135), SOX 6 (as described, for example, in GenBank Accession Number AF309034), SOX 8 (as described, for example, in GenBank Accession Number AF226675), SOX 10 (as described, for example, in GenBank Accession Number AJ001183), SOX 12 (as described, for example, in GenBank Accession Number X73039) and SOX 21 (as described, for example, in GenBank Accession Number AF107044)); lymphoid specific factor (LEF-1) (as described, for example, in GenBank Accession Number X58636); T-cell specific transcription factor (TCF-1) (as described, for example, in GenBank Accession Number X59869); MTT1 (as described, for example, in GenBank Accession Number M62810); and SP100-1-EMG nuclear autoantigen (as described, for example, in GenBank Accession Number U36501).
Other examples of HMGB proteins are polypeptides encoded by HMGB
nucleic acid sequences having GenBank Accession Numbers NG 000897 (HMG1L10) (and in particular by nucleotides 658-1305 of NG j00897); AF076674 (HMG1L1) (and in particular by nucleotides 1-633 of AF076674; AF076676 (111\4G1L4) (and in particular by nucleotides 1-564 of AF076676); AC010149 (HLV1G sequence from BAC clone RP11-395A23) (and in particular by nucleotides 75503-76117 of AC010149); AF165168 (I-IMG1L9) (and in particular by nucleotides 729-968 of AF165168); )34_063129 (L0C122441) (and in particular by nucleotides 319-558 of X.1\1 063129); )34_066789 (L0C139603) (and in particular by nucleotides 1-258 of XM j66789); and AF165167 (RIVIG1L8) (and in particular by nucleotides 456-666 of AF165167).
The antibodies and antigen-binding fragments of the invention bind to an HMGB polypeptide (e.g., one or more of the HMGB polypeptides listed above). In one embodiment, the antibody or antigen-binding fragment thereof binds to a vertebrate FILVIGB polypeptide. In another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian HIV1GB polypeptide (e.g., rat HMGB, mouse I-11\4GB, human HMGB). In still another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian BIVIGB1 polypeptide (e.g., rat HMGB1, mouse HMGB1, human IIMGB1). In a particular embodiment, the antibody or antigen-binding fragment thereof binds to a human HMGB1 polypeptide (e.g., the human 1-11VIGBI polypeptide depicted as SEQ ID NO: 1 or SEQ
ID NO:74).
The compositions and methods of the present invention also feature antibodies to the high mobility group B (HMGB) A box. In one embodiment, the antibody or antigen-binding fragment thereof binds to an HMGB A box but does not specifically bind to non-A box epitopes of EIMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a vertebrate HMGB A box but does not specifically bind to non-A box epitopes of HMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian (e.g., human, rat, mouse) HMGB A box but does not specifically bind to non-A box epitopes of HMGB. In still another embodiment, the antibody or antigen-binding fragment thereof binds to the A box of a IIMGB1 polypeptide (e.g., a mammalian ITMGBI polypeptide (e.g., human HMGB1, rat HMGB 1, mouse HNIGB1)) but does not specifically bind to non-A box epitopes of BMGB1.
As used herein, an "HMGB A box", also referred to herein as an "A box" or "HMG A box", is a protein or polypeptide that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%, sequence identity to an HMGB A box (e.g., an HMGB A
box described herein). In one embodiment, the HMGB A box has one or more of the following biological activities: inhibiting inflammation mediated by HMGB
and/or inhibiting release, of a proinflammatory cytokine from a cell (see, e.g., PCT
Publication No. WO 02/092004.
" In one embodiment, the HMGB A box polypeptide has one of the above biological activities. Typically, the HMGB A box polypeptide has both of the above biological activities. In one embodiment, the A box has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, sequence identity to the A box depicted in FIG. 2A (residues 9-85 of SEQ ID NO:2) or FIG. 31B (SEQ ID NO:75). hi another embodiment, the A box comprises or consists of the amino acid sequence in the corresponding region of an HMGB protein in a mammal. An IIMGB A box is also a recombinantly-produced polypeptide having the same amino acid sequence as the A box sequences described herein. ,The BMGB A box is preferably a vertebrate HMGB A box, for example, a mammalian FEVIGB A box, more preferably, a mammalian HMGB1 A box, for example, a human IIMGBI A box, and most preferably, the HMGB1 A box comprising or consisting of the sequence of the A
box depicted in FIG. 2A (residues 9-85 of SEQ ID NO:2) or FIG. 31B (SEQ ID
NO:75).
An BMGB A box often has no more than about 85 amino acids and no fewer than about 4 amino acids. In other embodiments, an HMGB A box can comprise from 10-85 amino acids, 20-85 amino acids, 30-85 amino acids or 40-85 amino acids. Examples of polypeptides having A box sequences within them include, but are not limited to the HMGB polypeptides described above. The A box sequences in such HMGB polypeptides can be determined and isolated using methods described herein, for example, by sequence comparisons to A boxes described herein and testing for biological activity using methods described herein and/or other methods known in the art.
Additional examples of HMGB A box polyp eptide sequences include the following sequences: PDASVNFSEF SKKCSERWKT MSAKEKGKFE
DMAKADKARY EREMKTYIPP KGET (human ITIVIGB1; SEQ ID NO:55);
DSSVNFAEF SKKCSERWKT MSAKEKSKFE DM_AKSDKARY
DREMKNYVPP KGDK (human HMGB2; SEQ ID NO:56); PEVPVNFAEF
SKKCSERWKT VSGKEKSKFD EMA_KADKVRY DREMKDYGPA KGGK
(human BMGB3; SEQ ID NO:57); PDASVNFSEF SKKCSERWKT
MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (BMG1L10; SEQ ID
NO:58); SDASVNFSEF SNKCSERWK MSAKEKGKFE DMA_KADKTHY
ERQMKTY1PP KGET (B1VIG1L1; SEQ ID NO:59); PDASVNFSEF
SKKCSERWKA MSAKDKGKFE DMAKVDKADY EREMKTYIPP KGET
(FIMG1L4; SEQ ID NO:60); PDASVKFSEF LKKCSETWKT IFAKEKGKFE
DMAKADKAHY EREMKTYIPP KGEK (111,4G sequence from BAC clone RP11-395A23; SEQ ID NO:61); PDASINFSEF SQKCPETWKT TIAKEKGKFE
DMAKADKAHY EREMKTYIPP KGET (BMG1L9; SEQ ID NO:62);
PDASVNSSEF SKKCSERWKTMPTKQGKFE DMAKADRAH (HMG1L8; SEQ
= ED NO:63); PDASVNFSEF SKKCLVRGKT MSAKEKGQFE AMARADKARY
EREMKTY1P PKGET (L0C122441; SEQ ID NO:64); LDASVSFSEF
SNKCSERWKT MS VKEKGKFE DMAKADKACY EREMKIYPYL KGRQ
(L0C139603; SEQ ID NO:65); and GKGDPKKPRG KMSSYAFFVQ
TCREEBKKKH PDASVNFSEF SKKCSERWKT MSAKEKGKFE
DMAKADKARY EREMKTYIPP KGET (human HMGB1 A box; SEQ ID NO:66).
The compositions and methods of the present invention also feature antibodies to the high mobility group B (HMGB) B box. In one embodiment, the antibody or antigen-binding fragment thereof binds to an BIVIGB B box but does not specifically bind to non-B box epitopes of HMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a vertebrate HMGB B box but does not specifically bind to non-B box epitopes of HMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian (e.g., human, rat, mouse) HMGB B box but does not specifically bind to non-B box epitopes of HIVIGB. In still another embodiment, the antibody or antigen-binding fragment thereof binds to the B box of a HI1MGB1 polypeptide (e.g., a mammalian HMGB I polypeptide (e.g., human ILMGB1, rat EIMGB1, mouse FIIVIGB1)) but does not specifically bind to non-B box epitopes ofIEVIGB1.
As used herein, an "HIVIGB B box", also referred to herein as a "B box" or "an HMG B box", is a polypeptide that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, sequence identity to an HMGB1 polypeptide (e.g., an BlVIGB B
box described herein). In one embodiment, the HIVIGB1 box has one or more of the following biological activities: increasing inflammation and/or increasing release of a proinflammatory cytokine from a cell (see, e.g., PCT Publication No. WO
02/092004). In one embodiment, the HMGB B box polypeptide has one of the above biological activities. Typically, the HMGB B box polypeptide has both of the above biological activities. In one embodiment, the HMGB B box has at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%, sequence identity to the B box depicted in FIG. 2B (SEQ ID NO:3) or FIG. 31C (SEQ ID NO:76). In another embodiment, the B box comprises or consists of the amino acid sequence in the corresponding region of an HMGB protein in a mammal. An BIVIGB B box is also a recombinantly-produced polypeptide having the same amino acid sequence as the B box sequences described herein. The BA/1GB B box is preferably a vertebrate HMGB B box, for example, a mammalian HMGB B box, more preferably, a mammalian BNIGB1 B
box, for example, a human HMGB1 B box, and most preferably, the HMGB 1 B box comprising or consisting of the sequence of the B box depicted in FIG. 2B (SEQ
ID
NO:3) or FIG. 31C (SEQ ID NO:76).
An IIMGB B box often has no more than about 85 amino acids and no fewer than about 4 amino acids. Examples of polypeptides having B box sequences within them include, but are not limited to, the HVGB polypeptides described above.
The B box sequences in such polypeptides can be determined and isolated using methods described herein, for example, by sequence comparisons to B boxes described herein and testing for biological activity.
Examples of additional HMGB B box polypeptide sequences include the following sequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP
GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY
(human HMGB1; SEQ ID NO:67); KKDPNAPKRP PSAFFLFCSE IIRPKIKSEHP
GLSIGDTAKK LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY (human HMGB2; SEQ ED NO:68); FKDPNAPKRL PSAFFLFCSE YRPKIKGEBT
GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY
(HIVIG1L10; SEQ ID NO:69); FKDPNAPKRP PSAFFLFCSE YELPICT_KGEHP
GLSIGDVAKK LGEMWNNTAA DDKQPGEKKA AKLKEKYEKD IAAY
(HMG1L1; SEQ ID NO:70); FKDSNAPKRP PSAFLLFCSE YCPKIKGEHP
GLPISDVAKK LVEMWNNTFA DDKQLCEKICA AKLKEKYKKD TATY
(BMG1L4; SEQ ID NO:71); FKDPNAPKRP PSAFFLFCSE YRPK1KGEHP
GLSIGDVVKK LAGMWNNTAA ADKQFYEKKA AKLK_EKYKKD IAAY
(HMG sequence from BAC clone RP11-359A23; SEQ ID NO:72); and DDKQPYEKKA AKLKEKYEKD IAAYRAKGKP DAAKKGVVKA EK (human HMGB1 box; SEQ NO:73).
As described herein, an HMGB polypeptide, an HMGB A box, and an IEVIGB B box, either naturally occurring or non-naturally occurring, encompass polypeptides that have sequence identity to the I-114GB polypeptides, HMGB A
boxes, and/or 1-11VIGB B boxes, described herein. As used herein, two polypeptides (or a region of the polypeptides) are substantially homologous or identical when the amino acid sequences are at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95%
or more, homologous or identical. The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acids or nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity= # of identical positions/total # of positions x 100). In certain embodiments, the length of the HMGB polypeptide, HMGB A box polypeptide, or HMGB B box polypeptide, aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, or 100%, of the length of the reference sequence, for example, the sequences described herein corresponding to an HMGB polypeptide (e.g., SEQ ID NO:1; SEQ ID NO:18, SEQ
ID NO:74), an HMGB A box polypeptide (e.g., residues 9-85 of SEQ ID NO:2, SEQ
ID NO:75) or an 111/1GB B box polypeptide (e.g., SEQ ID NO:3, SEQ ID NO:76).
The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al. (Proc.
Natl.
Acad. Sci. USA, 90:5873-5877 (1993)). Such an algorithm is incorporated into:the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al.
(Nucleic Acids Res., 29:2994-3005 (2001)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site for the National Center for Biotechnology Information) can be used. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62;
and Gap Costs have an Existence of 11 and an Extension of 1.
Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, California) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (Comput.
Appl. Biosci., 10: 3-5, 1994); and FASTA described in Pearson and Lipman (Proc.
Natl. Acad. Sci USA, 85: 2444-2448, 1988).
In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4, and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (Acceltys, San Diego, California), using a gap weight of 50 and a length weight of 3.
Inhibiting Release of Proinflannnatory Cytokines and Methods of Treatment In one embodiment, the present invention is a method of inhibiting release of a proinflammatory cytokine from a mammalian cell. In one embodiment, the method comprises treating the cell with an antibody or antigen-binding fragment of the present invention. Suitable antibodies or antigen-binding fragments are those described herein and include, e.g., 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb, 2E11 HMGB1 mAb, an antibody having the epitopic specificity of 6E6 ITMGB1 mAb, 6119 HMGB1 mAb, 2G7 mAb, 10D4 HMGB1 mAb and/or 2E11 HIVIGB1 mAb, an antibody that can compete with 6E6 HMGB1 mAb, 6H9 EIMGB1 mAb, 2G7 1IMGB1 mAb, 10D4 FIMGB I mAb and/or 2E11 IIMGB1 mAb for binding to a vertebrate high mobility group box (I-LMGB) polypeptide, and an antigen-binding fragment of any of the foregoing.
As used herein, a "cytokine" is a soluble protein or peptide that is naturally produced by mammalian cells and that regulates immune responses and mediates cell-cell interactions. Cytokines can, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. A
proinflammatory cytokine is a cytokine that is capable of causing one or more of the following physiological reactions associated with inflammation or inflammatory conditions:
vasodilation, hyperemia, increased peuiteability of vessels with associated edema, accumulation of granulocytes and mononuclear phagocytes, and deposition of fibrin.
= In some cases, the proinflammatory cytokine can also cause apoptosis. For example, in chronic heart failure, it has been shown that TNF stimulates cardiomyocyte apoptosis (Pulkki, Ann. Med. 29:339-343 (1997); and Tsutsui, et al., Immunol.
Rev.
174:192-209 (2000)). Nonlimiting examples of proinflammatory cytokines are tumor necrosis factor (TNF), interleukin (IL)-1 a, IL-113, EL-6, IL-8, IL-18, interferon y, HMG-1, platelet-activating factor (PAF), and macrophage migration inhibitory factor (1\41F).
In another embodiment, the invention is a method of treating a condition in a subject, wherein the condition is characterized by activation of an inflammatory cytokine cascade comprising administering to the subject an antibody or antigen-binding fragment of the present invention. Suitable antibodies or antigen-binding fragments are those described herein and include, e.g., 6E6 HMGB1 mAb, 6119 FIMGB1 mAb, 2G7 FEVIGB1 mAb, 10D4 IIMGB1 mAb, 2E11 1{MGB1 mAb, an antibody having the epitopic specificity of 6E6 HMGB1 mAb, 6119 HMG131 mAb, 2G7 HMGB1 mAb, 10D4 IIMGB1 mAb and/or 2E11 HMGB1 mAb, an antibody that can compete with 6E6 IIMGB1 mAb, 6H9 IIMGB1 mAb, 2G7 IIMGB1 mAb, 10D4 HMGB1 mAb and/or 2E11 EIMGB1 mAb for binding to a vertebrate high mobility group box (IIMGB) polypeptide, and an antigen-binding fragment of any of the foregoing.
In one embodiment, the method of treatment comprises administering to a subject an effective amount of an antibody or antigen-binding fragment of the invention. As used herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to prevent or decrease an inflammatory response, and/or to ameliorate and/or decrease the longevity of symptoms associated with an inflammatory response. The amount of the composition of the invention that will be effective in the treatment, prevention or management of a particular condition can be determined, for example, by administering the composition to an animal model such as, e.g., the animal models disclosed herein and/or known to those skilled in the art.
In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.
Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors that are known to one of ordinary skill in the art. Such factors include, e.g., the condition or conditions to be treated, the severity of the subject's symptoms, the choice of antibody or antigen-binding fragment to be administered, the subject's age, the subject's body mass, the subject's immune status, the response of the individual subject, and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.
The precise dose to be employed in the fonnulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances.
Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For example, as exemplified herein, using an in vivo cecal ligation and puncture (CLP) assay, a dose response assay for anti-HMGB1 monoclonal antibody 6E6 IIMGB1 inAb (at doses of 1 Jig/mouse, 10 jig/mouse or 100 jig/mouse) was conducted (NG. 16).
For antibodies, the dosage administered to a subject (e.g., a human patient) is typically 0.1 mg/kg to 100 mg/kg of the subject's body weight. Preferably, the dosage administered to a subject is between 0.1 mg/kg and 20 mg/kg of the subject's body weight, more preferably 1 mg/kg to 10 mg/kg of the subject's body weight.
In certain embodiments of the invention, the dosage is at least lmg/kg, or at least 5 mg/kg, or at least 10 mg/kg, or at least 50 mg/kg, or at least 100 mg/kg, or at least 150 mg/kg, of the subject's body weight. Generally, human and humanized antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. For example, an effective amount of an antibody can range from about 0.01 mg/kg to about 5 or
FIG. 18B is the encoded amino acid sequence of the human recombinant HIVIGB1 polypeptide containing a 5' 6 HIS tag (rec-LIMGBI-His6; SEQ NO:40).
FIG. 19A is the nucleotide sequence encoding the V11 domain of 207 HMGB1 mAb (SEQ ID NO:41).
FIG.19B is the encoded amino acid sequence of the VH domain of 2G7 BMGB1 mAb (SEQ ID NO:42); CDRs are underlined.
FIG. 19C is the nucleotide sequence encoding the Vic domain of 2G7 HMGB1 mAb (SEQ ID NO:43).
FIG. 19D is the encoded amino acid sequence of the VK domain of 2G7 HIVIGB1 mAb (SEQ ID NO:44); CDRs are underlined.
FIG. 20 is a histogram depicting the results of HMGB1 peptide binding experiments. Biotinylated peptides corresponding to either amino acid residues 63 or 61-78 of HMGB1 were prepared and analyzed for binding to 2G7 HMGB1 mAb (2G7) by ELISA.
FIG. 21 is a histogram depicting the results of HMGB1 peptide binding experiments. Biotinylated peptides corresponding to either amino acid residues 63 or 151-168 of HMGB1 were prepared and analyzed for binding to 2E11 BMGB1 mAb (2E11) by ELISA.
FIG. 22 is a histogram depicting the results of HMGD1 and ITMGB2 peptide binding experiments. Peptides corresponding to either amino acid residues 46-63 of human IIMGB1 (labeled "huHMGB1-46-63-B"), amino acid residues 46-63 of human HMGB2 (labeled "huI-EVIGB2-46-63-B"), amino acid residues 53-70 of human BIVIGB1 (labeled "huHMGB1-53-70"), or amino acid residues 61-78 of human HMGB1 (labeled "huHIVIGB1-61-78-B") were prepared and analyzed for binding to 207 1-11VIGB1 mAb (2G7) or avidin by ELISA.
FIG. 23 is a table depicting the results of EIMGB1 and HMGB2 peptide binding experiments. Listed in the table are various peptides, their respective amino acid sequences and whether the peptides bind 2G7 HMGB I mAb. The listed peptides include: a peptide corresponding to amino acid residues 40-57 of human HMGD1 (labeled "Human HMGB1-40-57"), a peptide corresponding to amino acid residues 46-63 of human HIVIGB I (labeled "Human 111VIGB1-46-63-B), a peptide corresponding to amino acid residues 53-70 of human HMGB I (labeled "Human H1\4GBI-53-70"), a peptide corresponding to amino acid residues 46-63 of human HivIGB2 (labeled "Human HIVIGB2-46-63-B"), and a peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 46-63 of human HMGB1 (labeled "Human HMGB1-46-63-scr").
FIG. 24 is a table depicting the results of FIMGB1 peptide binding experiments. Listed in the table are various peptides, their respective amino acid sequences and whether the peptides bind 6E6 HMGB1 mAb. The listed peptides include: a peptide corresponding to amino acid residues 53-70 of human HMGB I
(labeled "Human HMGB1-53-70"), a peptide corresponding to amino acid residues 61-78 of human BMGB I (labeled "Human HMGB1-61-78-B"), a peptide corresponding to amino acid residues 67-84 of human HIVIGB I (labeled "Human HMGB1-67-84"), and a peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 61-78 of human IIIVIGB1 (labeled "Human HIV1GB I -61-78_scr").
FIG. 25 is a table depicting the results of1-11MGB1 peptide binding experiments. Listed in the table are various peptides, their respective amino acid sequences and whether the peptides bind 2E11 HirvIGB1 mAb. The listed peptides include: a peptide corresponding to amino acid residues 143-160 of human HVGB1 (labeled "Human HMGB1-143-160"), a peptide corresponding to amino acid residues 151-168 of human EIMGB I (labeled "Human HIVIGB1-151-168-B"), a peptide corresponding to amino acid residues 157-174 of human HMGB I (labeled "Human HMGB1-157-174"), and a peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 151-168 of human HMGB1 (labeled "Human HMGB I -151-168_scr").
FIG. 26 is a histogram summarizing the results of peptide binding experiments and depicting the mapped epitopes of HMGB1 that are recognized by 2G7 IIMGB1 mAb (2G7), 6E6 HMGB1 mAb (6E6), 2G5 HMGB1 mAb (2G5), 6H9 HMGB1 mAb (6H9) and 2E11 HMGB1 mAb (2E11).
FIG. 27A is a mass spectrum of intact, non-reduced 6E6 HMGB1 mAb.
FIG. 27B is a mass spectrtun of 6E6 HMGB1 mAb, which was reduced by treatment with DTT.
FIG. 27C is amass spectrum of the light chains of 6E6 EIMGB1 mAb, which were reduced by treatment with DTT.
FIG. 27D is a mass spectrum of the heavy chains of 6E6 HMGB1 mAb, which were reduced by treatment with DTT.
FIG. 28 is a graph of the effect of administration of various doses (either 0.004 mg/kg, 0.04 mg/kg or 0.4 mg/kg) of 2G7 HMGB1 mAb or a control IgG
antibody (0.4 mg/kg) on survival of mice over time (days) after cecal ligation and puncture (CLP).
FIG. 29 is a table summarizing CLP survival percentages in mice administered various doses (either 4 mg/kg, 0.4 mg/kg, 0.04 mg/kg or 0.004 mg/kg) of 6E6 HMGB1 mAb (6E6), 2G7 HMGB1 mAb (2G7), or control IgG.
FIG. 30 is the amino acid sequence of a human (Homo sapiens) IIMGB2 polypeptide (SEQ rD NO:54; GenBank Accession No. M83665).
FIG 31A is the amino acid sequence of a human (Homo sapiens) FLIVIGB1 polypeptide (SEQ ID NO:74).
FIG 31B is an A box of a human (Homo sapiens) HMGB1 polypeptide (SEQ
ID NO:75).
FIG 31C is a B box of a human (Homo sapiens) HMGB1 polypeptide (SEQ
ID NO:76).
FIG. 32 is a histogram depicting inhibition of TNF release by various anti-HMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.1 ptg/m1 of recombinant CBP-Rat BIMGB1 peptide (SEQ
NO:5). Where indicated, various IIMGB1 monoclonal antibodies (cultured supernatants) were added to give a final concentration of 13%. The following antibodies were tested: 1A9 HMGB1 mAb (1A9); 2E11 HMGB1 mAb (2E11); 2G5 HMGB1 mAb (2G5); 2G7 HMGB1 mAb (2G7); 3G8 HMGB1 mAb (3G8); 4H11 IEVIGB1 mAb (4H11); 3-5A6 HMGB1 mAb (5A6); 6E6 HMGB1 mAb (6E6); 9G2 HMGB1 mAb (9G2); 4C9 HMGB1 mAb (4C9); and 6119 HMGB1 niAb (6119). The initial dark bar depicts TNF release in the absence of any antibodies.
FIG. 33 is a histogram depicting inhibition of TNF release by various anti-HMGB1 monoclonal antibodies. Mouse TNF was induced by stimulating RAW
264.7 cells with 0.1 jig/m1 of recombinant CBP-Rat HMGB1 peptide (SEQ ID
NO:5). Where indicated, various HMGB1 monoclonal antibodies (cultured supernatants) were added to give a final concentration of 13%. The following antibodies were tested: 7113 1114GB1 mAb ,(7113); 9113 FIIVIGB1 mAb (9113);
IIMGB I mAb (10D4); 1C3 HMGB1 mAb (1C3); 3E10 HMGB1 mAb (3E10);
4A1 0 IIMGBI mAb (4A1 0); 5C12 HIVIGBI mAb (5C12); and 7G8 BMGB1 mAb (7G8). The initial dark bar depicts TNF release in the absence of any antibodies.
DETAILED DESCRIPTION OF THE INVENTION
In various embodiments, the present invention is drawn to antibodies or antigen-binding fragments thereof that bind to a vertebrate high mobility group box (1IMGB) polypeptide, methods of detecting and/or identifying an agent that binds to an HMGB polyp eptide, methods of treating a condition in a subject characterized by activation of an inflammatory cytoldne cascade and methods of detecting an HMGB
polypeptide in a sample.
Antibodies and Antibody Producing Cells In one embodiment, the present invention encompasses antibodies or antigen-binding fragments thereof that bind to IIMGB polypeptides. The antibodies of the invention can be polyclonal or monoclonal, and the term "antibody" is intended to encompass both polyclonal and monoclonal antibodies. The terms polyclonal and monoclonal refer to the degree of homogeneity of an antibody preparation, and are not intended to be limited to particular methods of production.
In one embodiment, the antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment thereof. The term "monoclonal antibody"
or "monoclonal antibody composition", as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of im_munoreacting with a particular epitope of a polypeptide of the invention. A
monoclonal antibody composition thus typically displays a single binding affinity for a particular polypeptide of the invention with which it immunoreacts.
The term "antibody" as used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, primatized, veneered or single chain antibodies. Functional fragments include antigen-binding fragments of antibodies that bind to an I-114GB polypeptide (e.g., a mammalian FINIGB
polypeptide (e.g. a mammalian HMGB1 polypeptide)). For example, antibody fragments capable of binding to an HMGB polypeptide or a portion thereof, include, but are not limited to Fv, Fab, Fab' and F(ab')2 fragments. Such fragments can be produced by enzymatic cleavage or by recombinant techniques. For example, papain or pepsin cleavage can generate Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments. Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric gene encoding a F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and hinge region of the heavy chain.
Single chain antibodies, and chimeric, humanized or primatized (CDR-grafted), or veneered antibodies, as well as chimeric, CDR-grafted or veneered single chain antibodies, comprising portions derived from different species, and the like are also encompassed by the present invention and the term "antibody".
The various portions of these antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, nucleic acids encoding a chimeric or humanized chain can be expressed to produce a contiguous protein. See, e.g., ., Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 Bl; Boss etal., U.S. Patent No. 4,816,397; Boss et al., European Patent No. 0,120,694 Bl; Neuberger, M.S. etal., WO 86/01533; Neuberger, M.S. etal., European Patent No. 0,194,276 Bl; Winter, U.S. Patent No. 5,225,539; Winter, European Patent No. 0,239,400 Bl; Queen etal., European Patent No. 0 451 216 Bl; and Padlan, E.A. et aL, EP 0 519 596 Al. See also, Newman, R. etal., Biorechnolog,y, 10: 1455-1460 (1992), regarding primatized antibody, and Ladner et al., U.S. Patent No. 4,946,778 and Bird, R.E. etal., Science. 242: 423-426 (1988)) regarding single chain antibodies.
Humanized antibodies can be produced using synthetic or recombinant DNA
technology using standard methods or other suitable techniques. Nucleic acid (e.g., cDNA) sequences coding for humanized variable regions can also be constructed using PCR mutagenesis methods to alter DNA sequences encoding a human or humanized chain, such as a DNA template from a previously humanized variable region (see e.g., Kamman, M., etal., NucL Acids Res., 17: 5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993); Daugherty, B.L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991); and Lewis, A.P. and J.S. Crowe, Gene, 101: 297-(1991)). Using these or other suitable methods, variants can also be readily produced. In one embodiment, cloned variable regions can be mutated, and sequences encoding variants with the desired specificity can be selected (e.g., from a phage library; see e.g., Krebber et al., U.S. 5,514,548; Hoogenboom et al., WO 93/06213).
The antibody can be a humanized antibody comprising one or more iinmunoglobulin chains (e.g., an antibody comprising a CDR of nonhuman origin (e.g., one or more CDRs derived from an antibody of nonhuman origin) and a framework region derived from a light and/or heavy chain of human origin (e.g., CDR-grafted antibodies with or without framework changes)). In one embodiment, the antibody or antigen-binding fragment thereof comprises the light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3) of a particular immunoglobulin. In another embodiment, the antibody or antigen-binding fragment further comprises a human framework region.
The antibodies described herein can also be conjugated to an agent. In one embodiment, the agent is a label, for example, a radioisotope, an epitope label (tag), an affinity label (e.g., biotin, avidin), a spin label, an enzyme, a fluorescent group or a chemiluminescent group. Labeled antibodies or antigen-binding fragments of the present invention can be used, e.g., in the diagnostic and/or prognostic methods described herein. In another embodiment, the antibody is conjugated to a drug, toxin or anti-inflammatory agent. Conjugation of a drug, toxin or anti-inflammatory agent to the anti-HMGB antibodies and antigen-binding fragments of the invention allows for targeting of these agents to sites of ITIVIGB expression and/or activity.
Drugs and toxins that can be conjugated to the antibodies of the present invention include, for example, chemotherapeutic agents (e.g., mitomycin C, paxlitaxol, methotrexate, fluorouracil, cisplatin, cyclohexamide), toxins (e.g., ricin, gelonin) and other agents described herein (e.g., the agents described for combination therapy). Anti-inflammatory agents that can be conjugated include, e.g., those described herein.
Antibodies that are specific for an HMGB polypeptide (e.g., a mammalian EdvIGB polypeptide) can be raised against an appropriate immunogen, such as an isolated and/or recombinant HMGB polypeptide or a portion thereof (including synthetic molecules, such as synthetic peptides). Antibodies can also be raised by immunizing a suitable host (e.g., mouse) with cells that express an HMGB
polypeptide, such as 0113 pituicytes, macrophage cells (e.g., RAW 246.7 cells, human macrophage cells), peripheral blood mononuclear cells (PBMCs (e.g., human PBMCs)), primary T cells (e.g., human primary T cells), adrenal cells (e.g., rat adrenal PC-12 cells, human adrenal cells), and kidney cells (e.g., rat primary kidney cells, human primary kidney cells). In addition, cells expressing a recombinant HMGB polypeptide (e.g., a mammalian HMGB polypeptide), such as transfected cells, can be used as an immunogen or in a screen for an antibody that binds thereto (See e.g., Chuntharapai et at., Ininzunol., 152: 1783-1789 (1994);
Chuntharapai et at., U.S. Patent No. 5,440,021).
Preparation of immunizing antigen, and polyclonal and monoclonal antibody production can be performed using any suitable technique. A variety of methods have been described (see e.g., Kohler et at., Nature, 256: 495-497 (1975) and Dlr. J.
bizmunoL 6: 511-519 (1976); Milstein et al., Nature 266: 550-552 (1977);
Koprowski et al., U.S. Patent No. 4,172,124; Harlow, E. and D. Lane, 1988, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: Cold Spring Harbor, NY); Current Protocols In Molecular Biology, Vol. 2 (Supplement 27, Summer '94), Ausubel, F.M. et al., Eds., (John Wiley & Sons: New York, NY), Chapter 11, (1991)). Generally, as exemplified herein, a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as 5P2/0, P3X63Ag8.653 or a heteromyeloma) with antibody-producing cells. Antibody-producing cells can be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. The fused cells (hybridomas) can be isolated using selective culture conditions, and cloned by limiting dilution. Cells that produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Other suitable methods of producing or isolating antibodies of the requisite specificity (e.g., human antibodies or antigen-binding fragments) can be used, including, for example, methods that select recombinant antibody from a library (e.g., a phage display library). Transgenic animals capable of producing a repertoire of human antibodies (e.g., Xenomouse (Abgenix, Fremont, CA)) can be produced using suitable methods (see e.g., .Takobovits et al., Proc. Natl. Acad. Sci.
USA, 90:
2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258 (1993)). Additional methods that are suitable for production of transgenic animals capable of producing a repertoire of human antibodies have been described (e.g., Lonberg et al., U.S.
Patent No. 5,545,806; Surani et al., U.S. Patent No. 5,545,807; Lonberg et al., W097/13852).
In one embodiment, the antibody or antigen-binding fragment thereof has specificity for an BNIGB polypeptide (e.g., a mammalian HIMGB polypeptide). In a particular embodiment, the antibody or antigen-binding fragment thereof has specificity for an 1-Th4GB1 polypeptide (e.g., a human EMGB1 polypeptide such as depicted in SEQ ID NO:1 and/or SEQ ID NO:74). In another embodiment, the ,antibody or antigen-binding fragment thereof is an IgG or an antigen-binding fragment of an IgG. In another embodiment, the antibody or antigen-binding fragment thereof is an IgG1 or an antigen-binding fragment of an IgGl. In still other embodiments, the antibody or antigen-binding fragment thereof is an IgG2a, IgG2b, IgG3 antibody, or an antigen-binding fragment of any of the foregoing.
In another embodiment, the antibody or antigen-binding fragment can bind to an HIVIGB polypeptide and inhibit (reduce or prevent) one or more functions of the 1-11VIGB polypeptide. Such HMGB functions include, e.g., increasing inflammation (see, e.g., PCT Publication No. WO 02/092004), increasing release of a proinflammatory cytokine from a cell (see, e.g., PCT Publication No. WO
02/092004), binding to RAGE, binding to TLR2, chemoattraction (see, e.g., Degryse et aL, I Cell Biol. I52(6):1197-1206 (2001), and activation of antigen presenting cells (see, e.g., WO 03/026691).
=
In one embodiment, the antibody is a human antibody or an antigen-binding fragment thereof. In another embodiment, the antibody is a humanized antibody or an antigen-binding fragment thereof. In yet another embodiment, the antibody or antigen-binding fragment can inhibit binding of a polypeptide (e.g., RAGE, TLR2) to an HMGB polypeptide and/or inhibit one or more functions mediated by binding of the HMGB polypeptide and the other polypeptide.
In certain embodiments, the antibodies or antigen-binding fragments thereof specifically bind to HiVIGB epitopes or antigenic determinants (e.g., HMGB
epitopes, HMGB A box epitopes, HMGB B box epitopes). As described herein, an antibody or antigen-binding fragment thereof can be screened without undue experimentation for the ability to inhibit release of a proinflammatory cytokine using standard methods. Anti-IIMGB A-box antibodies and anti-BNIGB B box antibodies that can inhibit the production of a proinflanunatory cytokine and/or the release of a proinflammatory cytokine from a cell, and/or inhibit a condition characterized by activation of an inflammatory cytokine cascade, are within the scope of the present =
invention. In one embodiment, the antibody or antigen-binding fragment of the invention can inhibit the production of TNF, IL-1 1. and/or IL-6. In another embodiment, the antibody or antigen-binding fragnent of the invention can inhibit =
the production of TNF (e.g., TNF-a).
As described herein, monoclonal antibodies designated "6E6 HMGB1 mAb", "2E11 HMGB1 mAb", "6H9 FIMGB1 mAb", "10D4 HMGB1 mAb" and "2G7 HMGB1 mAb", all of which bind to HMGB1 have been produced. In addition, other monoclonal antibodies designated "9G2 11MGB1 mAb", "1A9 HMGB1 mAb", "3G8 HMGB1 mAb", "2G5 HMGB1 mAb", "41111 HMGB1 mAb", "7H3 HMGB1 mAb", "3-5A6 HMGB1 mAb", "9G1 HMGB1 mAb", "4C9 HMGB1 mAb", "9113 HMGB1 mAb", "1C3 HMGB1 mAb", "5C12 HMGB1 mAb", "3E10 HMGB1 mAb", "7G8 IEVIGB1 mAb" and "4A10 HMGB1 mAb" have been produced. All but 9G2 HMGB1 mAb and 1A9 HMGB1 mAb have been shown to bind HMGB1. 9G2 HMGB1 mAb and 1A9 HMGB1 mAb appear to bind to the CBP region of the immunogen (which is not cleaved in a small percentage of the immunbgen).
6E6 1{MGB1 mAb, also referred to as 6E6-7-1-1 or 6E6, can be produced by murine hybridoma 6E6 HMGB1 mAb, which was deposited on September 3,2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No.
PTA-5433. The invention relates to murine hybridoma 6E6 HMGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
2E11 HMGB1 mAb, also referred to as 2E11-1-1-2 or 2E11, can be produced by murine hybridoma 2E11 HMGB1 mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No. PTA-5431. The invention relates to murine hybridoma 2E11 HMGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
6H9 11MGB1 mAb, also referred to as 6H9-1-1-2 or 6119, can be produced by murine hybridoma 6119 11MGB1 mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No.
PTA-5434. The invention relates to murine hybridoma 6H9 1114GB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
10D4 IHVIGB I mAb, also referred to as 10D4-1-1-1-2 or 10D4, can be produced by murine hybridoma 10D4 H1MGB1 mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 146 Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No. PTA-5435. The invention relates to murine hybridoma 10D4 BIVIGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
2G7 HMGB1 mAb, also referred to as 3-2G7-1-1-1 or 2G7, can be produced by murine hybridoma 207 IIMGB I mAb, which was deposited on September 3, 2003, on behalf of Critical Therapeutics, Inc., 675 Massachusetts Avenue, 14th Floor, Cambridge, MA 02139, U.S.A., at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110, U.S.A., under Accession No.
PTA-5432. The invention relates to murine hybridoma 207 FIMGB1 mAb, to the antibody it produces and to nucleic acids encoding the antibody.
For cultivation of the above identified murine hybridomas (e.g., 6E6 HMGB I mAb, 6H9 HMGB1 mAb, 207 HMGB I mAb, 10D4 HMGB1 mAb, 2G7 HIV1GB I mAb), DMEM, 10% FCS, 1% 1L-6, 1% L-glutamine and 1% Pen-Strep should be added.
902 1-314GB1 mAb, also referred to as 902-7-1-1-1 or 902, can be produced by murine hybridoma 9G2 HMGB1 mAb. The invention relates to murine hybridoma 9G2 HIVIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
1A9 HMGB1 mAb, also referred to as 1A9-1-2-1-4 or 1A9, can be produced by murine hybridoma 1A9 HMGB1 mAb. The invention relates to murine hybridoma 1A9 IIMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
3G8 IIMGB1 mAb, also referred to as 3G8-7-2-1-5 or 3G8, can be produced by murine hybridoma 3G8 HMGB1 mAb. The invention relates to murine hybridoma 3G8 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
2G5 HMGB1 mAb, also referred to as 3-2G5-4-1-2 or 2G5, can be produced by murine hybridoma 2G5 HMGB1 mAb_ The invention relates to murine hybridoma 2G5 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
4H11 HMGB1 mAb, also referred to as 41111. can be produced by murine hybridoma 41111 HMGB mAb. The invention relates to murine hybridoma 4H1 1 HIV1GB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
1 5 7H3 HMGB1 mAb, also referred to as 7H3, can be produced by murine hybridoma 7113 HMGB1 mAb. The invention relates to murine hybridoma 7113 111V1GB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
3-5A6 HMGB1 mAb, also referred to as 3-5A6 or 5A6, can be produced by murine hybridoma 3-5A6 1114GB1 mAb. The invention relates to murine hybridoma 3-5A6 11MGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
9G1 HMGB1 mAb, also referred to as 9G1, can be produced by murine hybridoma 9G1 HMGB1 mAb. The invention relates to murine hybridoma 9G1 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
4C9 HMGB1 mAb, also referred to as 4C9, can be produced by murine hybridoma 4C9 HMGB1 mAb. The invention relates to murine hybridoma 4C9 HIVIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
9H3 IIMGB1 mAb, also referred to as 9H3, can be produced by murine hybridoma 9H3 HMGB1 mAb. The invention relates to murine hybridoma 9113 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
1C3 HMGB1 mAb, also referred to as 1C3-1-1-1-1 or 1C3, can be produced by murine hybridoma 1C3 1-MIGB1 mAb. The invention relates to murine hybridoma 1C3 HMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
5C12 HMGB1 mAb, also referred to as 5C12-1-1-1-1 or 5C12, can be produced by murine hybridoma 5C12 HMGB1 mAb. The invention relates to murine hybridoma 5C12 HNIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
3E10 HMGB1 mAb, also referred to as 3E10-5-4-1-1 or 3E10, can be produced by murine hybridoma 3E10 IIMGB1 mAb. The invention relates to murine hybridoma 3E10 ILMGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
7G8 IIMGB 1 mAb, also referred to as 7G8, can be produced by murine hybridoma 7G8 HMGB1 mAb. The invention relates to marine hybridoma 7G8 111VIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
4A10 HMGB1 mAb, also referred to as 4A10-1-3-1-1 or 4A10, can be produced by murine hybridoma 4A10 HiVIGB I mAb. The invention relates to murine hybridoma 4A10 ITIVIGB1 mAb, to the antibody it produces, and to nucleic acids encoding the antibody.
In one embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of 6E6 1-11VIGB1 mAb, 6119 IIMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB I mAb, 2E11 HMGB1 mAb and an antigen-binding fragment of any of the foregoing.
In another embodiment, the antibody or antigen-binding fragment has the same or similar epitopic specificity of an antibody or antigen-binding fragment selected from the group consisting of 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 1-IIVIGB1 mAb, 2E11 HMGB1 mAb and/or an antigen-binding fragment of any of the foregoing. Antibodies or antigen-binding fragments with an epitopic specificity that is the same as, or similar to, that of 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 niAb, 10D4 MVIGB1 mAb and/or 2E11 HilVIGB1 mAb can be identified by a variety of suitable methods. For example, an antibody with the same or similar epitopic specificity as, e.g., 6E6 HIVIGB1 mAb, can be identified based upon the ability to compete with 6E6 IIMGB1 mAb for binding to a FM/1GB
polypeptide (e.g., a mammalian HMGB polypeptide (e.g., a mammalian HMGB1 polypeptide)). In another example, the binding of, e.g., 6E6 IIMGB1 mAb, and the binding of an antibody with the same or similar epitopic specificity for a H_MGB
polypeptide can be inhibited by a single peptide (e.g., a natural peptide, a synthetic peptide). In various embodiments, the peptide can comprise, e.g., 9 to about amino acids, 9 to about 40 amino acids, 9 to about 30 amino acids, 9 to about amino acids or 9 to about 20 amino acids.
As exemplified herein, 18 amino acid peptides corresponding to particular regions of the human HMGB1 polypeptide were shown to bind to various ITIVIGB1 monoclonal antibodies. The studies described herein mapped epitopes within HMGB1 that bind to particular HMGB1 antibodies.
For example, 2E11 HMGB1 raAb was shown to bind a peptide corresponding to amino acids 151-168 of human ITMGB1 (amino acid residues 151-168 of SEQ ID NO:1; i.e., LKEKYEKDIAAYRAKGKP (SEQ ID NO:30)).
Additional studies suggest that 2E11 HMGB1 mAb recognizes an epitope that is present in within amino acid residues 156-161, 155-161, 155-162, 156-162 and/or 156-163, of EIMGB1 (see Example 14).
6E6 HIvIGB1 mAb and 6H9 EIMGB1 mAb were shown to bind to a peptide corresponding to amino acids 61-78 of human HMGB1 (amino acid residues 61-78 of SEQ ID NO:1; i.e., EDMAKADKARYEREIVIKTY (SEQ ID NO:24)).
Additional studies demonstrated that 6E6 HMGB1 mAb recognizes an epitope that is present within amino acid residues 67-78 of HIVIGB1 (see Example 13).
2G7 ELMGB1 mAb was shown to bind a peptide corresponding to amino acids 46-63 of human HMGB1 (amino acid residues 46-63 of SEQ ID NO:1; i.e., SERIVKTMSAKEKGKFEDM (SEQ ID NO:23)) (see Example 10). Further studies demonstrated that 2G7 ITMGB1 mAb recognizes an epitope that is present within amino acid residues 53-63 of HMGB I (see Example 12). In addition, 2G7 EIMGB1 mAb does not bind to amino acid residues 46-63 of HMGB2 (SEQ ID NO:48), notwithstanding only a single amino acid difference between the BlVIGB1 46-63 peptide and the IIMGB2 46-63 peptide (see Example 12). Thus, in one embodiment, the antibodies or antigen-binding fragments of the invention bind to EIMGB1 but not to EIMGB2. In other embodiments, the antibodies or antigen-binding fragments of the invention bind to both HIvIGB1 and HIVIGB2.
These 18 amino acid peptides, or other peptides corresponding to particular regions of HMGB1, could be used in epitopic studies to determine if an antibody or antigen-binding fragment inhibited binding of the peptide to an antibody known to bind that peptide (e.g., 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 niAb, 10D4 BIMGB 1 mAb, 2E11 FEVIGB1 mAb, others antibodies described herein).
Thus, for example, an antibody or antigen-binding fragment to be tested for its epitopic specificity could be assayed with, e.g., 2E11 HMGB1 mAb and a peptide corresponding to amino acids 151-168 of human HMGB1 (which 2E11 HIvIGB1 mAb is known to bind).
In another example, an antibody with the same or similar epitopic specificity as an antibody of the invention (e.g., 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HIVIGB1 mAb, 10D4 HIVGB1 mAb and/or 2E11 HMGB1 mAb) can be identified using a chimeric HMGB polypeptide (see e.g., Banks, G.C., et at., J Biol.
Chem.
274(23):16536-16544 (1999)).
In one embodiment, the antibody or antigen-binding fragment can compete with 6E6 HMGB I mAb, 6H9 IIMGB1 mAb, 2G7 HIVIGB1 mAb, 10D4 BilvIGB1 mAb, 2E11 HMGB1 mAb and/or an antigen-binding fragment of any of the foregoing, for binding to an BIVIGB polypeptide (e.g., a mammalian FINIGB
polypeptide (e.g., a mammalian HMGB1 polypeptide)). Such inhibition of binding can be the result of competition for the same or similar epitope or steric interference (e.g., where antibodies bind overlapping epitopes or adjacent epitopes).
Inhibition by 6E6 IIMGB1 inAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 -?4-mAb, 2E11 HMGB1 mAb, and/or an antigen-binding fragment of any of the foregoing, can also be due to a change in the conformation of the HMGB
polypeptide that is induced upon antibody binding to the FEVIGB polypeptide.
In another embodiment, the antibody or antigen-binding fragment thereof is selected from the group consisting of 3G8 HMGB1 mAb, 2G5 FIMGB1 mAb, 4H11 HMGB1 mAb, 7113 HIMGB1 mAb, 3-5A6 IIMGB1 mAb, 9G1 HMGB1 mAb, 4C9 HMGB1 mAb, 9H3 HMGB1 mAb, 1C3 EMGB1 mAb, 5C12 H_MGB1 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb, 4A10 HMGB1 mAb, and an antigen-binding fragment of any of the foregoing.
In one embodiment, the antibody or antigen-binding fragment has the epitopic specificity of an antibody or antigen-binding fragment selected from the group consisting of 3G8 IEVIGB1 mAb, 2G5 HMGB1 mAb, 4E111 IIMGB1 mAb, 7113 HMGB1 mAb, 3-5A6 HMGB1 mAb, 9G1 11MGB1 mAb, 4C9 HMGB1 mAb, 9113 BMGB1 mAb, 1C3 HMGB1 mAb, 5C12 HMGB1 mAb, 3E10 1114GB1 mAb, 7G8 HMGB1 mAb, 4A10HMGB1 mAb, and an antigen-binding fragment of any of the foregoing. As described above, antibodies or antigen-binding fragments with an epitopic specificity that is the same as, or similar to, one or more of these antibodies or antigen-binding fragments can be identified by a variety of suitable methods (e.g., ' using methods described herein and/or blown in the art).
In another embodiment, the antibody or antigen-binding fiagment can compete with 3G8 HMGB1 mAb, 2G5 HIVIGB1 mAb, 41111 HMGB1 mAb, 7113 HMGB1 mAb, 3-5A6 HIVIGB1 rnAb, 9G1 HMGB1 mAb, 4C9 HMGB1 mAb, 9113 ITIVIGB1 mAb, 1C3 1-1:MGB1 mAb, 5C12 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb, 4A10 HMGB1 mAb, and/or an antigen-binding fragment of any of the foregoing, for binding to an HMGB polypeptide (e.g., a mammalian HMGB
polypeptide). As described above, inhibition of binding can be the result of competition for the same or similar epitope or steric interference (e.g., where antibodies bind overlapping epitopes or adjacent epitopes). Inhibition can also be due to a change in the confaimation of the HMGB polypeptide that is induced upon binding of the antibody or antigen-binding fragment to the HMGB polypeptide.
In one embodiment, the antibody or antigen-binding fragment thereof comprises the six CDRs (light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3)) of an antibody selected from the group consisting of 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HIIVIGB 1 mAb and 2E11 FIMGB1 mAb. In another embodiment, the antibody is a humanized antibody that comprises the light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3) of an antibody selected from the group consisting of 6E6 HMGB1 mAb, 6H9 HIVIGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb and 2E11 HMGB1 mAb. In other embodiments, the antibody or antigen-binding fragment thereof comprises the six CDRs (light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3)) of any other antibody described herein.
In another embodiment, the antibody or antigen-binding fragment thereof comprises from one to six of the light chain and heavy chain CDRs of an antibody of the invention (e.g., 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 BIMGB1 mAb, 10D4 HMGB1 mAb, 2E11 HMGB1 mAb). For example, the antibody or antigen-binding fragment can comprise one, two, three, four, five or six, of the light chain and heavy chain CDRs. In another embodiment, the antibody or antigen-binding fragment thereof comprises at least one light chain CDR or heavy chain CDR
from one antibody of the invention and at least one light chain CDR or heavy chain CDR
from a different antibody of the invention (e.g., 6E6 HMGB1 mAb, 6119 IIMGB1 mAb, 2G7 HMGB1 mAb, 10D4 FIMGB1 mAb, 2E11 HMGB1 mAb). For example, an antibody or antigen-binding fragment could comprise one or more CDRs from 6E6 11MGB1 mAb and one or more CDRs from 6119 IIMGB1 mAb. Antibodies and antigen-binding fragments combining other combinations of CDRs from different antibodies of the invention are also encompassed.
In another embodiment, the antibody or antigen-binding fragment thereof comprises the six CDRs (light chain CDRs (CDR1, CDR2 and CDR3) and heavy chain CDRs (CDR1, CDR2 and CDR3)) of an antibody selected from the group consisting of 3G8 HMGB1 mAb, 2G5 IIMGB1 mAb, 41111 HIVIGB1 mAb, 7113 EIMGB1 mAb, 3-5A6 ITh4GB1 mAb, 9G1 HMGB1 mAb, 4C9 111V1GB1 mAb, 9113 mAb, 1C3 HMGB1 mAb, 5C12 HMGB1 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb and 4A10 HMGB1 mAb. In another embodiment, the antibody or antigen-binding fragment thereof comprises from one to six of the light chain and heavy chain CDRs of one of these antibodies.
In certain embodiments, the antibody or antigen-binding fragment comprises one or more CDRs that are at least 80% identical, at least 90% identical, or at least 95% identical, to a CDR of an antibody of the invention (e.g., 6E6 IldvIGB1 mAb, 6H91-EvIGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb, 2E11 HMGB1 mAb).
In other embodiments, the antibody or antigen-binding fragment comprises one or more CDRs that are at least 80% similar, at least 90% similar, or at least 95%
similar, to a CDR of an antibody of the invention. Methods for determining sequence identity and similarity of two polypeptides are described herein and/or are well known in the art.
The invention also relates to a bispecific antibody, or functional fragment thereof (e.g., F(ab')2), which binds to an HMGB polypeptide and at least one other antigen (e.g., tumor antigen, viral antigen). In a particular embodiment, the bispecific antibody, or functional fragment thereof, has the same or similar epitopic specificity as 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb and/or 2E11 HMGB1 raAb, and at least one other antibody.
Bispecific antibodies can be secreted by triomas and hybrid hybridomas.
Generally, =
triomas are fainted by fusion of a hybridoma and a lymphocyte (e.g., antibody-secreting B cell) and hybrid hybridomas are formed by fusion of two hybridomas.
Each of the fused cells (i.e., hybridomas, lymphocytes) produces a monospecific antibody. However, triomas and hybrid hybridomas can produce an antibody containing antigen-binding sites that recognize different antigens. The supernatants of triomas and hybrid hybridomas can be assayed for bispecific antibody using a suitable assay (e.g., ELISA), and bispecific antibodies can be purified using conventional methods. (see, e.g., U.S. Patent No. 5,959,084 (Ring et al.), U.S. Patent No. 5,141,736 (Iwasa etal.), U.S. Patent Nos. 4,444,878, 5,292,668, 5,523,210 (all to Paulus et al.) and U.S. Patent No. 5,496,549 (Yamazaki et al.)).
In one embodiment, the invention relates to an isolated cell that produces an antibody or an antigen-binding fragment of the invention. In a particular embodiment, the isolated antibody-producing cell of the invention is an immortalized cell, such as a hybridoma, heterohybridoma, lymphoblastoid cell or a recombinant cell. The antibody-producing cells of the present invention have uses other than for the production of antibodies. For example, the cell of the present invention can be fused with other cells (such as suitably drug-marked human myeloma, mouse myeloma, human-mouse heteromyelonia or human lymphoblastoid cells) to produce, for example, additional hybridomas, and thus provide for the transfer of the genes encoding the antibody. In addition, the cell can be used as a source of nucleic acids encoding the anti-HMGB immnnoglobulin chains, which can be isolated and expressed (e.g., upon transfer to other cells using any suitable technique (see e.g., Cabilly et al., U.S.
Patent No. 4,816,567, Winter, U.S. Patent No. 5,225,539)). For instance, clones comprising a sequence encoding a rearranged anti-HWIGB light and/or heavy chain can be isolated (e.g., by PCR). In addition, cDNA libraries can be prepared from mRNA isolated from an appropriate cell line, and cDNA clones encoding an anti-=
HMGB immunoglobulin chain(s) can be isolated. Thus, nucleic acids encoding the heavy and/or light chains of the antibodies, or portions thereof, can be obtained and used for the production of the specific immunoglobulin, immunoglobulin chain, or variants thereof (e.g., humanized immunoglobulins) in a variety of host cells or in an in vitro translation system. For example, the nucleic acids, including cDNAs, or derivatives thereof encoding variants such as a humanized immunoglobulin or immunoglobulin chain, can be placed into suitable prokaryotic or eukaryotic vectors (e.g., expression vectors) and introduced into a suitable host cell by an appropriate method (e.g., transformation, transfection, electroporation, infection), such that the nucleic acid is operably linked to one or more expression control elements (e.g., in the vector or integrated into the host cell genome), to produce a recombinant antibody-producing cell. Thus, in certain embodiments, the invention is a nucleic acid that encodes an antibody or antigen-binding fragment of the invention. In other embodiments, the invention is a vector that comprises a nucleic acid encoding an =
=
antibody or antigen-binding fragment of the invention.
HMGB Polypeptides, HMGB A boxes and HMGB B boxes As described, in one embodiment the invention is an antibody or antigen-binding fragment thereof that binds to an HMGB polypeptide.
As used herein, an "HMGB polypeptide" is a polypeptide that has at least 60%, more preferably, at least 70%, 75%, 80%, 85%, or 90%, and most preferably at least 95% sequence identity, to a sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:18 and SEQ JD NO:74 (as determined, for example, using the BLAST program and parameters described herein). In one embodiment, the HMGB polypeptide increases inflammation and/or increases release of a proinflammatory cytokine from a cell. In another embodiment, the HMGB
polypeptide has one of the above biological activities. Typically, the HMGB
polypeptide has both of the above biological activities.
The term "polypeptide" refers to a polymer of amino acids, and not to a specific length; thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. In one embodiment, the HMGB polypeptide is a mammalian HIVIGB polypeptide, for example, a mammalian HMGB polypeptide (e.g., a human HMGB1 polypeptide). In another embodiment, the HMGB
polypeptide contains a B box DNA binding domain and/or an A box DNA binding domain and/or an acidic carboxyl terminus as described herein.
Other examples of HMGB polypeptides are described in GenBank Accession Numbers AAA64970, AAB08987, P07155, AAA20508, S29857, P09429, NP 002119, CAA31110, S02826, U00431, X67668, NP 005333 NM 016957, and _ J04179.
Additional examples of IIMGB polypeptides include, but are not limited to mammalian HMG1 ((liMGB1) as described, for example, in GenBank Accession Number U51677), HMG2 ((HMGB2) as described, for example, in GenBank Accession Number M83665), HMG-2A ((HVIGB3, HMG-4) as described, for example, in GenBank Accession Numbers NM 005342 and NP 005333), HIVIG14 _99-(as described, for example, in GenBank Accession Number P05114), HMG17 (as = described, for example, in GenBank Accession Number X13546), HMGI (as described, for example, in GenBank Accession Number L17131), and 11MGY (as described, for example, in GenBank Accession Number M23618); nonmammalian HMG Ti (as described, for example, in GenBank Accession Number X02666) and 1114G T2 (as described, for example, in GenBank Accession Number L32859) (rainbow trout); HMG-X (as described, for example, in GenBank Accession Number D30765) (Xenopus); HMG D (as described, for example, in GenBank Accession Number X71138) and HMG Z (as described, for example, in GenBank Accession Number X71139) (Drosophila); NHP10 protein (HMG protein homolog NITP 1) (as described, for example, in GenBank Accession Number 'Z48008) (yeast);
non-histone chromosomal protein (as described, for example, in GenBank Accession Number 000479) (yeast); HMG 1/2 like protein (as described, for example, in GenBank Accession Number Z11540) (wheat, maize, soybean); upstream binding factor (UBF-1) (as described, for example, in GenBank Accession Number X53390); PMS1 protein homolog 1 (as described, for example, in GenBank Accession Number 1J13695); single-strand recognition protein (SSRP, structure-specific recognition protein) (as described, for example, in GenBank Accession Number M86737); the HMG homolog 1.1)P-1 (as described, for example, in GenBank Accession Number M74017); mammalian sex-determining region Y
protein (SRY, testis-determining factor) (as described, for example, in GenBank Accession Number X53772); fungal proteins: mat-1 (as described, for example, in GenBank Accession Number AB009451), ste 11 (as described, for example, in GenBank Accession Number X53431) and Mc 1; SOX 14 (as described, for example, in GenBank Accession Number AF107043) (as well as SOX 1 (as described, for example, in GenBank Accession Number Y13436), SOX 2 (as described, for example, in GenBank Accession Number Z31560), SOX 3 (as described, for example, in GenBank Accession Number X71135), SOX 6 (as described, for example, in GenBank Accession Number AF309034), SOX 8 (as described, for example, in GenBank Accession Number AF226675), SOX 10 (as described, for example, in GenBank Accession Number AJ001183), SOX 12 (as described, for example, in GenBank Accession Number X73039) and SOX 21 (as described, for example, in GenBank Accession Number AF107044)); lymphoid specific factor (LEF-1) (as described, for example, in GenBank Accession Number X58636); T-cell specific transcription factor (TCF-1) (as described, for example, in GenBank Accession Number X59869); MTT1 (as described, for example, in GenBank Accession Number M62810); and SP100-1-EMG nuclear autoantigen (as described, for example, in GenBank Accession Number U36501).
Other examples of HMGB proteins are polypeptides encoded by HMGB
nucleic acid sequences having GenBank Accession Numbers NG 000897 (HMG1L10) (and in particular by nucleotides 658-1305 of NG j00897); AF076674 (HMG1L1) (and in particular by nucleotides 1-633 of AF076674; AF076676 (111\4G1L4) (and in particular by nucleotides 1-564 of AF076676); AC010149 (HLV1G sequence from BAC clone RP11-395A23) (and in particular by nucleotides 75503-76117 of AC010149); AF165168 (I-IMG1L9) (and in particular by nucleotides 729-968 of AF165168); )34_063129 (L0C122441) (and in particular by nucleotides 319-558 of X.1\1 063129); )34_066789 (L0C139603) (and in particular by nucleotides 1-258 of XM j66789); and AF165167 (RIVIG1L8) (and in particular by nucleotides 456-666 of AF165167).
The antibodies and antigen-binding fragments of the invention bind to an HMGB polypeptide (e.g., one or more of the HMGB polypeptides listed above). In one embodiment, the antibody or antigen-binding fragment thereof binds to a vertebrate FILVIGB polypeptide. In another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian HIV1GB polypeptide (e.g., rat HMGB, mouse I-11\4GB, human HMGB). In still another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian BIVIGB1 polypeptide (e.g., rat HMGB1, mouse HMGB1, human IIMGB1). In a particular embodiment, the antibody or antigen-binding fragment thereof binds to a human HMGB1 polypeptide (e.g., the human 1-11VIGBI polypeptide depicted as SEQ ID NO: 1 or SEQ
ID NO:74).
The compositions and methods of the present invention also feature antibodies to the high mobility group B (HMGB) A box. In one embodiment, the antibody or antigen-binding fragment thereof binds to an HMGB A box but does not specifically bind to non-A box epitopes of EIMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a vertebrate HMGB A box but does not specifically bind to non-A box epitopes of HMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian (e.g., human, rat, mouse) HMGB A box but does not specifically bind to non-A box epitopes of HMGB. In still another embodiment, the antibody or antigen-binding fragment thereof binds to the A box of a IIMGB1 polypeptide (e.g., a mammalian ITMGBI polypeptide (e.g., human HMGB1, rat HMGB 1, mouse HNIGB1)) but does not specifically bind to non-A box epitopes of BMGB1.
As used herein, an "HMGB A box", also referred to herein as an "A box" or "HMG A box", is a protein or polypeptide that has at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%, sequence identity to an HMGB A box (e.g., an HMGB A
box described herein). In one embodiment, the HMGB A box has one or more of the following biological activities: inhibiting inflammation mediated by HMGB
and/or inhibiting release, of a proinflammatory cytokine from a cell (see, e.g., PCT
Publication No. WO 02/092004.
" In one embodiment, the HMGB A box polypeptide has one of the above biological activities. Typically, the HMGB A box polypeptide has both of the above biological activities. In one embodiment, the A box has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, sequence identity to the A box depicted in FIG. 2A (residues 9-85 of SEQ ID NO:2) or FIG. 31B (SEQ ID NO:75). hi another embodiment, the A box comprises or consists of the amino acid sequence in the corresponding region of an HMGB protein in a mammal. An IIMGB A box is also a recombinantly-produced polypeptide having the same amino acid sequence as the A box sequences described herein. ,The BMGB A box is preferably a vertebrate HMGB A box, for example, a mammalian FEVIGB A box, more preferably, a mammalian HMGB1 A box, for example, a human IIMGBI A box, and most preferably, the HMGB1 A box comprising or consisting of the sequence of the A
box depicted in FIG. 2A (residues 9-85 of SEQ ID NO:2) or FIG. 31B (SEQ ID
NO:75).
An BMGB A box often has no more than about 85 amino acids and no fewer than about 4 amino acids. In other embodiments, an HMGB A box can comprise from 10-85 amino acids, 20-85 amino acids, 30-85 amino acids or 40-85 amino acids. Examples of polypeptides having A box sequences within them include, but are not limited to the HMGB polypeptides described above. The A box sequences in such HMGB polypeptides can be determined and isolated using methods described herein, for example, by sequence comparisons to A boxes described herein and testing for biological activity using methods described herein and/or other methods known in the art.
Additional examples of HMGB A box polyp eptide sequences include the following sequences: PDASVNFSEF SKKCSERWKT MSAKEKGKFE
DMAKADKARY EREMKTYIPP KGET (human ITIVIGB1; SEQ ID NO:55);
DSSVNFAEF SKKCSERWKT MSAKEKSKFE DM_AKSDKARY
DREMKNYVPP KGDK (human HMGB2; SEQ ID NO:56); PEVPVNFAEF
SKKCSERWKT VSGKEKSKFD EMA_KADKVRY DREMKDYGPA KGGK
(human BMGB3; SEQ ID NO:57); PDASVNFSEF SKKCSERWKT
MSAKEKGKFE DMAKADKARY EREMKTYIPP KGET (BMG1L10; SEQ ID
NO:58); SDASVNFSEF SNKCSERWK MSAKEKGKFE DMA_KADKTHY
ERQMKTY1PP KGET (B1VIG1L1; SEQ ID NO:59); PDASVNFSEF
SKKCSERWKA MSAKDKGKFE DMAKVDKADY EREMKTYIPP KGET
(FIMG1L4; SEQ ID NO:60); PDASVKFSEF LKKCSETWKT IFAKEKGKFE
DMAKADKAHY EREMKTYIPP KGEK (111,4G sequence from BAC clone RP11-395A23; SEQ ID NO:61); PDASINFSEF SQKCPETWKT TIAKEKGKFE
DMAKADKAHY EREMKTYIPP KGET (BMG1L9; SEQ ID NO:62);
PDASVNSSEF SKKCSERWKTMPTKQGKFE DMAKADRAH (HMG1L8; SEQ
= ED NO:63); PDASVNFSEF SKKCLVRGKT MSAKEKGQFE AMARADKARY
EREMKTY1P PKGET (L0C122441; SEQ ID NO:64); LDASVSFSEF
SNKCSERWKT MS VKEKGKFE DMAKADKACY EREMKIYPYL KGRQ
(L0C139603; SEQ ID NO:65); and GKGDPKKPRG KMSSYAFFVQ
TCREEBKKKH PDASVNFSEF SKKCSERWKT MSAKEKGKFE
DMAKADKARY EREMKTYIPP KGET (human HMGB1 A box; SEQ ID NO:66).
The compositions and methods of the present invention also feature antibodies to the high mobility group B (HMGB) B box. In one embodiment, the antibody or antigen-binding fragment thereof binds to an BIVIGB B box but does not specifically bind to non-B box epitopes of HMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a vertebrate HMGB B box but does not specifically bind to non-B box epitopes of HMGB. In another embodiment, the antibody or antigen-binding fragment thereof binds to a mammalian (e.g., human, rat, mouse) HMGB B box but does not specifically bind to non-B box epitopes of HIVIGB. In still another embodiment, the antibody or antigen-binding fragment thereof binds to the B box of a HI1MGB1 polypeptide (e.g., a mammalian HMGB I polypeptide (e.g., human ILMGB1, rat EIMGB1, mouse FIIVIGB1)) but does not specifically bind to non-B box epitopes ofIEVIGB1.
As used herein, an "HIVIGB B box", also referred to herein as a "B box" or "an HMG B box", is a polypeptide that has at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, sequence identity to an HMGB1 polypeptide (e.g., an BlVIGB B
box described herein). In one embodiment, the HIVIGB1 box has one or more of the following biological activities: increasing inflammation and/or increasing release of a proinflammatory cytokine from a cell (see, e.g., PCT Publication No. WO
02/092004). In one embodiment, the HMGB B box polypeptide has one of the above biological activities. Typically, the HMGB B box polypeptide has both of the above biological activities. In one embodiment, the HMGB B box has at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%, sequence identity to the B box depicted in FIG. 2B (SEQ ID NO:3) or FIG. 31C (SEQ ID NO:76). In another embodiment, the B box comprises or consists of the amino acid sequence in the corresponding region of an HMGB protein in a mammal. An BIVIGB B box is also a recombinantly-produced polypeptide having the same amino acid sequence as the B box sequences described herein. The BA/1GB B box is preferably a vertebrate HMGB B box, for example, a mammalian HMGB B box, more preferably, a mammalian BNIGB1 B
box, for example, a human HMGB1 B box, and most preferably, the HMGB 1 B box comprising or consisting of the sequence of the B box depicted in FIG. 2B (SEQ
ID
NO:3) or FIG. 31C (SEQ ID NO:76).
An IIMGB B box often has no more than about 85 amino acids and no fewer than about 4 amino acids. Examples of polypeptides having B box sequences within them include, but are not limited to, the HVGB polypeptides described above.
The B box sequences in such polypeptides can be determined and isolated using methods described herein, for example, by sequence comparisons to B boxes described herein and testing for biological activity.
Examples of additional HMGB B box polypeptide sequences include the following sequences: FKDPNAPKRP PSAFFLFCSE YRPKIKGEHP
GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY
(human HMGB1; SEQ ID NO:67); KKDPNAPKRP PSAFFLFCSE IIRPKIKSEHP
GLSIGDTAKK LGEMWSEQSA KDKQPYEQKA AKLKEKYEKD IAAY (human HMGB2; SEQ ED NO:68); FKDPNAPKRL PSAFFLFCSE YRPKIKGEBT
GLSIGDVAKK LGEMWNNTAA DDKQPYEKKA AKLKEKYEKD IAAY
(HIVIG1L10; SEQ ID NO:69); FKDPNAPKRP PSAFFLFCSE YELPICT_KGEHP
GLSIGDVAKK LGEMWNNTAA DDKQPGEKKA AKLKEKYEKD IAAY
(HMG1L1; SEQ ID NO:70); FKDSNAPKRP PSAFLLFCSE YCPKIKGEHP
GLPISDVAKK LVEMWNNTFA DDKQLCEKICA AKLKEKYKKD TATY
(BMG1L4; SEQ ID NO:71); FKDPNAPKRP PSAFFLFCSE YRPK1KGEHP
GLSIGDVVKK LAGMWNNTAA ADKQFYEKKA AKLK_EKYKKD IAAY
(HMG sequence from BAC clone RP11-359A23; SEQ ID NO:72); and DDKQPYEKKA AKLKEKYEKD IAAYRAKGKP DAAKKGVVKA EK (human HMGB1 box; SEQ NO:73).
As described herein, an HMGB polypeptide, an HMGB A box, and an IEVIGB B box, either naturally occurring or non-naturally occurring, encompass polypeptides that have sequence identity to the I-114GB polypeptides, HMGB A
boxes, and/or 1-11VIGB B boxes, described herein. As used herein, two polypeptides (or a region of the polypeptides) are substantially homologous or identical when the amino acid sequences are at least about 60%, 70%, 75%, 80%, 85%, 90%, or 95%
or more, homologous or identical. The percent identity of two amino acid sequences (or two nucleic acid sequences) can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first sequence). The amino acids or nucleotides at corresponding positions are then compared, and the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity= # of identical positions/total # of positions x 100). In certain embodiments, the length of the HMGB polypeptide, HMGB A box polypeptide, or HMGB B box polypeptide, aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, or 100%, of the length of the reference sequence, for example, the sequences described herein corresponding to an HMGB polypeptide (e.g., SEQ ID NO:1; SEQ ID NO:18, SEQ
ID NO:74), an HMGB A box polypeptide (e.g., residues 9-85 of SEQ ID NO:2, SEQ
ID NO:75) or an 111/1GB B box polypeptide (e.g., SEQ ID NO:3, SEQ ID NO:76).
The actual comparison of the two sequences can be accomplished by well-known methods, for example, using a mathematical algorithm. A preferred, non-limiting example of such a mathematical algorithm is described in Karlin et al. (Proc.
Natl.
Acad. Sci. USA, 90:5873-5877 (1993)). Such an algorithm is incorporated into:the BLASTN and BLASTX programs (version 2.2) as described in Schaffer et al.
(Nucleic Acids Res., 29:2994-3005 (2001)). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTN; available at the Internet site for the National Center for Biotechnology Information) can be used. In one embodiment, the database searched is a non-redundant (NR) database, and parameters for sequence comparison can be set at: no filters; Expect value of 10; Word Size of 3; the Matrix is BLOSUM62;
and Gap Costs have an Existence of 11 and an Extension of 1.
Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, California) sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Additional algorithms for sequence analysis are known in the art and include ADVANCE and ADAM as described in Torellis and Robotti (Comput.
Appl. Biosci., 10: 3-5, 1994); and FASTA described in Pearson and Lipman (Proc.
Natl. Acad. Sci USA, 85: 2444-2448, 1988).
In another embodiment, the percent identity between two amino acid sequences can be accomplished using the GAP program in the GCG software package (Accelrys, San Diego, California) using either a Blossom 63 matrix or a PAM250 matrix, and a gap weight of 12, 10, 8, 6, or 4, and a length weight of 2, 3, or 4. In yet another embodiment, the percent identity between two nucleic acid sequences can be accomplished using the GAP program in the GCG software package (Acceltys, San Diego, California), using a gap weight of 50 and a length weight of 3.
Inhibiting Release of Proinflannnatory Cytokines and Methods of Treatment In one embodiment, the present invention is a method of inhibiting release of a proinflammatory cytokine from a mammalian cell. In one embodiment, the method comprises treating the cell with an antibody or antigen-binding fragment of the present invention. Suitable antibodies or antigen-binding fragments are those described herein and include, e.g., 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb, 10D4 HMGB1 mAb, 2E11 HMGB1 mAb, an antibody having the epitopic specificity of 6E6 ITMGB1 mAb, 6119 HMGB1 mAb, 2G7 mAb, 10D4 HMGB1 mAb and/or 2E11 HIVIGB1 mAb, an antibody that can compete with 6E6 HMGB1 mAb, 6H9 EIMGB1 mAb, 2G7 1IMGB1 mAb, 10D4 FIMGB I mAb and/or 2E11 IIMGB1 mAb for binding to a vertebrate high mobility group box (I-LMGB) polypeptide, and an antigen-binding fragment of any of the foregoing.
As used herein, a "cytokine" is a soluble protein or peptide that is naturally produced by mammalian cells and that regulates immune responses and mediates cell-cell interactions. Cytokines can, either under normal or pathological conditions, modulate the functional activities of individual cells and tissues. A
proinflammatory cytokine is a cytokine that is capable of causing one or more of the following physiological reactions associated with inflammation or inflammatory conditions:
vasodilation, hyperemia, increased peuiteability of vessels with associated edema, accumulation of granulocytes and mononuclear phagocytes, and deposition of fibrin.
= In some cases, the proinflammatory cytokine can also cause apoptosis. For example, in chronic heart failure, it has been shown that TNF stimulates cardiomyocyte apoptosis (Pulkki, Ann. Med. 29:339-343 (1997); and Tsutsui, et al., Immunol.
Rev.
174:192-209 (2000)). Nonlimiting examples of proinflammatory cytokines are tumor necrosis factor (TNF), interleukin (IL)-1 a, IL-113, EL-6, IL-8, IL-18, interferon y, HMG-1, platelet-activating factor (PAF), and macrophage migration inhibitory factor (1\41F).
In another embodiment, the invention is a method of treating a condition in a subject, wherein the condition is characterized by activation of an inflammatory cytokine cascade comprising administering to the subject an antibody or antigen-binding fragment of the present invention. Suitable antibodies or antigen-binding fragments are those described herein and include, e.g., 6E6 HMGB1 mAb, 6119 FIMGB1 mAb, 2G7 FEVIGB1 mAb, 10D4 IIMGB1 mAb, 2E11 1{MGB1 mAb, an antibody having the epitopic specificity of 6E6 HMGB1 mAb, 6119 HMG131 mAb, 2G7 HMGB1 mAb, 10D4 IIMGB1 mAb and/or 2E11 HMGB1 mAb, an antibody that can compete with 6E6 IIMGB1 mAb, 6H9 IIMGB1 mAb, 2G7 IIMGB1 mAb, 10D4 HMGB1 mAb and/or 2E11 EIMGB1 mAb for binding to a vertebrate high mobility group box (IIMGB) polypeptide, and an antigen-binding fragment of any of the foregoing.
In one embodiment, the method of treatment comprises administering to a subject an effective amount of an antibody or antigen-binding fragment of the invention. As used herein, an "effective amount" or "therapeutically effective amount" is an amount sufficient to prevent or decrease an inflammatory response, and/or to ameliorate and/or decrease the longevity of symptoms associated with an inflammatory response. The amount of the composition of the invention that will be effective in the treatment, prevention or management of a particular condition can be determined, for example, by administering the composition to an animal model such as, e.g., the animal models disclosed herein and/or known to those skilled in the art.
In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges.
Selection of the preferred effective dose can be determined (e.g., via clinical trials) by a skilled artisan based upon the consideration of several factors that are known to one of ordinary skill in the art. Such factors include, e.g., the condition or conditions to be treated, the severity of the subject's symptoms, the choice of antibody or antigen-binding fragment to be administered, the subject's age, the subject's body mass, the subject's immune status, the response of the individual subject, and other factors known by the skilled artisan to reflect the accuracy of administered pharmaceutical compositions.
The precise dose to be employed in the fonnulation will also depend on the route of administration, and the seriousness of the condition, and should be decided according to the judgment of the practitioner and each subject's circumstances.
Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems. For example, as exemplified herein, using an in vivo cecal ligation and puncture (CLP) assay, a dose response assay for anti-HMGB1 monoclonal antibody 6E6 IIMGB1 inAb (at doses of 1 Jig/mouse, 10 jig/mouse or 100 jig/mouse) was conducted (NG. 16).
For antibodies, the dosage administered to a subject (e.g., a human patient) is typically 0.1 mg/kg to 100 mg/kg of the subject's body weight. Preferably, the dosage administered to a subject is between 0.1 mg/kg and 20 mg/kg of the subject's body weight, more preferably 1 mg/kg to 10 mg/kg of the subject's body weight.
In certain embodiments of the invention, the dosage is at least lmg/kg, or at least 5 mg/kg, or at least 10 mg/kg, or at least 50 mg/kg, or at least 100 mg/kg, or at least 150 mg/kg, of the subject's body weight. Generally, human and humanized antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. For example, an effective amount of an antibody can range from about 0.01 mg/kg to about 5 or
10 mg/kg administered daily, weekly, biweekly or monthly.
Methods for deteimining whether an antibody or antigen-binding fragment inhibits an inflammatory condition are known to one skilled in the art. For example, inhibition of the release of a proinflanunatory cytokine from a cell can be measured according to methods known to one skilled in the art. For example, as described and exemplified herein, TNF release from a cell can be measured using a standard marine fibroblast L929 (ATCC, American Type Culture Collection, Rocicville, Maryland) cytotoxicity bioassay (Bianchi et al., Journal of Experimental Medicine /83:927-936 (1996)) with the minimum detectable concentration of 30 pg/ml. The L929 cytotoxicity bioassay is carried out as follows. RAW 264.7 cells are cultured in RPMI 1640 medium (Life Technologies, Grand Island, New York) supplemented with 10% fetal bovine serum (Gemini, Catabasas, California), and penicillin and streptomycin (Life Technologies). Polymyxin (Sigma, St. Louis, Missouri) is added at 100 units/ml to suppress the activity of any contaminating LPS. Cells are incubated with the antibodies described herein in Opti-MEM I medium for 8 hours, and conditioned supernatants (containing TNF that has been released from the cells) are collected. TNF that is released from the cells is measured by a standard murine fibroblast L929 (ATCC) cytotoxicity bioassay (Bianchi et al., supra) with the minimum detectable concentration of 30 pg/ml. Recombinant mouse TNF can be obtained from R & D Systems Inc. (Minneapolis, Minnesota) and used as a control in these experiments. Methods for measuring release of other cytoldnes from cells are also known in the art.
An inflammatory condition that is suitable for the methods of treatment described herein can be one in which the inflammatory cytokine cascade is activated.
In one embodiment- the inflammatory cytokine cascade causes a systemic reaction, such as with endotwdc shock. In another embodiment, the inflammatory condition is mediated by a localized inflammatory cytokine cascade, as in rheumatoid arthritis.
Nonlimiting examples of inflammatory conditions that can be usefully treated using the antibodies and antigen-binding fragments of the present invention include, e.g., diseases involving the gastrointestinal tract and associated tissues (such as ileus, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis);
diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C
virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoal and multicellular parasites (such as malaria, filariasis, amebiasis, and hydatid cysts); deiniatological diseases and conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals);
diseases involving the cardiovascular system and associated tissues (such as stenosis, restenosis, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, myocardial ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Retier's syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.
In one embodiment, the condition is selected from the group consisting of sepsis, allogaft rejection, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, restenosis, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia, organic. ischemia, reperfusion ischemia, Behcet's disease, graft versus host disease, Crohn's disease, ulcerative colitis, ileus, multiple sclerosis, and cachexia.
In another embodiment, the condition is selected from the group consisting of sepsis, arthritis (e.g., rheumatoid arthritis), asthma, lupus, psoriasis, inflammatory bowel disease and Crohn's disease.
Preferably the antibodies and antigen-binding fragments are administered to a patient in need thereof in an amount sufficient to inhibit release of proinflammatory cytolcine from a cell and/or to treat an inflammatory condition. In one embodiment, release of the proinflammatory cytoldne is inhibited by at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, or 95%, as assessed using methods described herein or other methods known in the art.
The terms "therapy", "therapeutic" and "treatment", as used herein, refer to ameliorating symptoms associated with a disease or condition, for example, an inflammatory disease or an inflammatory condition, including preventing or delaying the onset of the disease symptoms, and/or lessening the severity or frequency of symptoms of the disease or condition. The terms "subject" and "individual" are defined herein to include animals such as mammals, including but , not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species.
In one embodiment, the animal is a human.
In one embodiment, an excipient can be included with the antibodies and antigen-binding fragments of the invention. The excipient can be selected based on the expected route of administration of the antibodies or antigen-binding fragments in therapeutic applications. The route of administration of the composition depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder such as endotoxic shock, and oral administration may be preferred to treat a gastrointestinal disorder such as a gastric ulcer.
As described above, the dosage of the antibody or antigen-binding fragment to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Depending on the condition, the antibody or antigen-binding fragment can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabucally and transdermally to the patient.
Accordingly, antibodies or antigen-binding fragments designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent and/or edible carrier. The antibodies or antigen-binding fragments may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the antibodies or antigen-binding fragments of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, and the like.
Tablets, pills, capsules, troches, and the like, may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents.
Some examples of binders include microcrystalline cellulose, gum tragacanth, and gelatin. Examples of excipients include starch and lactose. Some examples of disintegrating agents include alginic acid, corn starch, and the like.
Examples of lubricants include magnesium stearate and potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin, and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring, and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
The antibodies and antigen-binding fragments of the present invention can be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the antibodies and antigen-binding fragments of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents, such as water for injection, saline solution, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline (referred to herein as PBS), Hank's solution, Ringer's-= lactate, fixed oils, polyethylene glycols, glycerine, propylene glycol, and other synthetic solvents. Parenteral foimulations may also include antibacterial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), and chelating agents (e.g., EDTA). Buffers, such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride and dextrose, may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic.
Rectal administration includes administering the antibodies and antigen-binding fragments into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120 C, dissolving the antibody or antigen-binding fragment in the glycerin, mixing the heated glycerin, after which purified water may be added, and pouring the hot mixture into a suppository mold.
Transdermal administration includes percutaneous absorption of the antibody or antigen-binding fragment through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, and the like.
The antibodies and antigen-binding fragments of the present invention can be administered nasally to .a subject. As used herein, nasally administering or nasal administration, includes administering the antibodies or antigen-binding fragments to the mucous membranes of the nasal passage or nasal cavity of the subject_ Pharmaceutical compositions for nasal administration of an antibody or antigen-binding fragment include therapeutically effective amounts of the antibody or antigen-binding fragment. Well-known methods for nasal administration include, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream, or powder. Administration of the antibody or antigen-binding fragment may also take place using a nasal tampon or nasal sponge.
As described above, a variety of routes of administration are possible including, for example, oral, dietary, topical, transdernial, rectal, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intraderrnal injection), and inhalation (e.g., intrabronchial, intranasal, oral inhalation, intranasal drops).
Administration can be local or systemic as indicated. The preferred mode of administration can vary depending upon the antibody or antigen-binding fragment to be administered and the particular condition (e.g., disease) being treated, however, oral or parenteral administration is generally preferred.
If desired, the antibodies or antigen-binding fragments described herein can be administered with one or more additional agents (e.g., agents used to treat an inflammatory condition). The antibodies or antigen-binding fragments thereof and additional agent(s) can be present in a single composition or administered as separate compositions. If administered as separate compositions, the antibodies or antigen-binding fragments thereof and additional agent(s) can be co-administered or administered separately.
In one embodiment, the antibodies or antigen-binding fragments of the invention are administered with an anti-inflammatory agent. Such agents are known to one of skill in the art. In one embodiment, the agent is an antagonist of an early sepsis mediator. As used herein, an early sepsis mediator is a proirtflammatory cytokine that is released from cells soon (i.e., within 30-60 min.) after induction of an inflammatory cytolcine cascade (e.g., exposure to LPS). Nonlimiting examples of these cytokines are EL-la, IL-113, IL-6, PAF, and MIR Also included as early sepsis mediators are receptors for these cytokines (for example, tumor necrosis factor receptor type 1) and enzymes required for production of these cytokines, for example, interleukin-113 converting enzyme). Antagonists of any early sepsis mediator, now known or later discovered, can be useful for these embodiments by further inhibiting an inflammatory eytokine cascade.
Nonlimiting examples of antagonists of early sepsis mediators are antisense compounds that bind to the mR_NA of the early sepsis mediator, preventing its expression (see, e.g., Ojwang et al., Biochenzistry 36:6033-6045 (1997);
Parnpfer et al., Biol. Reprod. 52:1316-1326 (1995); U.S. Patent No. 6,228,642; Yahata et al., Antisense Nucleic Acid Drug Dev. 6:55-61 (1996); and Taylor et aL, Antisense Nucleic Acid Drug Dev. 8:199-205 (1998)), ribozymes that specifically cleave the mRNA of the early sepsis mediator (see, e.g., Leavitt et al., Antisense Nucleic Acid Drug Dev. /0:409-414 (2000); Kisich at al., J. ImmunoL 163(4):2008-2016 (1999);
and Hendrix et at., Biochem. J. 314 (Pt. 2):655-661 (1996)), and antibodies that bind to the early sepsis mediator and inhibit their action (see, e.g., Kam and Targan, Expert Opin. Pharmacother. 1:615-622 (2000); Nagahira et at., J. Immunol.
Methods 222:83-92 (1999); Lavine et aL, J. Cereb. Blood Flow Metab. /8:52-58 (1998); and Holmes et at.. Hybridoma 19:363-367 (2000)). An antagonist of an early sepsis mediator, now known or later discovered, is envisioned as within the scope of the invention. The skilled artisan can determine the amount of early sepsis mediator to use for inhibiting any particular inflammatory cytokine cascade without undue experimentation with routine dose-response studies.
Other agents that can be administered with the antibodies and antigen-binding fragments of the invention include, e.g., VitaxinT" and other antibodies targeting avf33 integrin (see, e.g., U.S. Patent No. 5,753,230, PCT
Publication Nos.
WO 00/78815 and WO 02/070007 and anti-IL-9 antibodies (see, e.g., PCT
Publication No. WO 97/08321).
In one embodiment, the antibodies and antigen-binding fragments of the invention are administered with inhibitors of TNF biological activity e.g., inhibitors of TNF-a, biological activity). Such inhibitors of TNF activity include, e.g., peptides, proteins, synthesized molecules, for example, synthetic organic molecules, naturally-occurring molecule, for example, naturally occurring organic molecules, nucleic acid molecules, and components thereof. Preferred examples of agents that inhibit TNF biological activity include infliximab (Remicade; Centocor, Inc., Malvern, Pennsylvania), etanercept (Immunex; Seattle, Washington), adalimumab (D2E7; Abbot Laboratories, Abbot Park Illinois), CDP870 (Pharrnacia Corporation;
Bridgewater, New Jersey) CDP571 (Celltech Group plc, United Kingdom), Lenercept (Roche, Switzerland), and Thalidomide.
In certain embodiments, the present invention is directed to a composition comprising the antibody or antigen-binding fragments described herein, in a pharmaceutically-acceptable excipient. As described above, the excipient included *Trademark with the antibody or antigen-binding fragment in these compositions is selected based on the expected route of administration of the composition. Suitable pharmaceutically-acceptable excipients include those described above and known to those of skill in the art.
In one embodiment, the invention is directed to aptamers of EIMGB (e.g., aptamers of HIVIGB1). As is known in the art, aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., an IIMGB protein, an HMGB box (e.g., an HMGB A box, an BlvIGB B
box), an HMGB polypeptide and/or an HMGB epitope as described herein). A
particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. The chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the universe of all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins).
Aptamers are chemically stable and can be boiled or frozen without loss of activity.
Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications.
For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.
Selection of apatrners that can bind HIVIGB or a fragment thereof (e.g., HMGB1 or a fragment thereof) can be achieved through methods known in the art.
For example, aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C., and Gold, L., Science 249:505-510 (1990)). In the SELEX method, a large library of nucleic acid molecules (e.g., 1015 different molecules) is produced and/or screened with the target molecule (e.g., an HMGB protein, an HMGB box (e.g., an FIMGB A box, an HMGB
B box), an HIVIGB polypeptide and/or an FIMGB epitope as described herein).
The target molecule is allowed to incubate with the library of nucleotide sequences for a period of time. Several methods, known in the art, can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture, which can be discarded. The aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule. The enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this iterative selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule.
Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure, thereby producing aptamers truncated to their core binding domain. See Jayasena,-S.D. Clin. Chem.- 45:1628-1650 (1999) for review of , aptamer technology.
In particular embodiments, the aptamers of the invention have the binding specificity and/or functional activity described herein for the antibodies of the invention. Thus, for example, in certain embodiments, the present invention is drawn to aptamers that have the same or similar binding specificity as described herein for the antibodies of the invention (e.g., binding specificity for a vertebrate HMGB polypeptide, fragment of a vertebrate ITIVIGB polypeptide (e.g., FINIGB A
box, I-IMGB B box), epitopic region of a vertebrate 1111VIGB polypeptide (e.g., epitopic region of HIvIGB1 that is bound by one or more of the antibodies of the invention)). In particular embodiments, the aptamers of the invention can bind to an HMGB polypeptide or fragment thereof and inhibit one or more functions of the HIV1GB polypeptide. As described herein, function of BMGB polypeptides include, e.g., increasing inflammation, increasing release of a proinflammatory cytokine from a cell, binding to RAGE, binding to TLR2, chemoattraction In a particular embodiment, the aptamer binds HMGB1 (e.g., human HMGB1 (e.g., as depicted in SEQ ID NO:1 or SEQ ID NO:74)) or a fragment thereof (e.g., A box (e.g., residues 9-85 of SEQ ID NO:2, SEQ ID NO:75), B box (e.g., SEQ ID NO:3, SEQ ED
NO:76), HMGB1 antibody binding epitope as described herein) and inhibits one or more functions of the EMGB polypeptide (e.g., inhibits release of a proinflam.matory cytokine from a vertebrate cell treated with HIVIGB).
Methods of Diagnosis and/or Prognosis In another embodiment, the invention further provides diagnostic and/or prognostic methods for detecting a vertebrate high mobility group box (1-11VIGB) polypeptide in a sample. In one embodiment of the method, a sample is contacted with an antibody or antigen-binding fragment of the present invention, under conditions suitable for binding of the antibody or fragment to an EIMGB
polypeptide present in the sample. The method further comprises detecting antibody-HMGB
complexes or antigen-binding fragment-HMGB complexes, wherein detection of antibody-HMGB complexes or antigen-binding fragment-HMGB complexes is indicative of the presence of an HIVIGB polypeptide in the sample. Suitable antibodies or antigen-binding fragments for use in these methods include those described herein, e.g., 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 EIMGB1 mAb, 10D4 HMGB1 mAb, 2G7 HMGB1 mAb, an antigen-binding fragment of any of the foregoing.
In another embodiment, the antibody or antigen-binding fragment comprises a detectable label. Labels suitable for use in detection of a complex between an 1-11/1GB polypeptide (e.g., a mammalian HIMGB polypeptide) and an antibody or antigen-binding fragment include, for example, a radioisotope, an epitope label (tag), an affinity label (e.g., biotin, avidin), a spin label, an enzyme, a fluorescent group or a cherniluminescent group.
As described, the antibodies and antigen-binding fragments described herein can be used to detect or measure expression of an IIMGB polypeptide. For example, antibodies of the present invention can be used to detect or measure an H1VIGB
polypeptide in a biological sample (e.g., cells, tissues or body fluids from an individual such as blood, serum, leukocytes (e.g., activated T lymphocytes), bronchoalveolar lavage fluid, saliva, bowel fluid, synovial fluid, biopsy specimens).
In one embodiment, the sample is blood or serum. For example, a sample (e.g., tissue and/or fluid) can be obtained from an individual and a suitable assay can be used to assess the presence or amount of an HMGB polypeptide. Suitable assays include immunological and immunochemical methods such as flow cytometry (e.g., FACS analysis) and immunosorbent assays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), chemiluminescence assays, immunoblot (e.g., western blot), immunocytochemistry and immunohistology. Generally, a sample and an antibody or antigen-binding fragment of the present invention are combined under conditions suitable for the formation of a complex between an HMGB polypeptide and the antibody or antigen-binding fragment thereof, and the formation of said complex is assessed (directly or indirectly). In one embodiment, diagnosis and/or prognosis is done using ELISA
and/or western blot analysis.
As in known in the art, the presence of an increased level of an HMGB
polypeptide (e.g., HI4GB1) in a sample (e.g., a tissue sample) obtained from an individual can be a diagnostic and/or prognostic indicator for monitoring the severity and predicting the likely clinical course of sepsis for a subject exhibiting symptoms associated with conditions characterized by activation of the inflammatory cascade (see U.S. Patent No. 6,303,321).
Thus, in one embodiment, the antibodies and antigen-binding fragments of the invention can be used in diagnostic and prognostic methods for monitoring the severity and/or predicting the likely clinical course of an inflammatory condition associated with HMGB expression (e.g., the conditions described herein). In certain embodiments, the diagnostic and/or prognostic methods comprise measuring the concentration of .1--Th4GB in a sample, preferably a serum sample, and comparing that concentration to a standard for HMGB
representative of a notinal concentration range of HMGB in a like sample, whereby higher levels of HMGB are indicative of poor prognosis or the likelihood of toxic reactions. The diagnostic method may also be applied to other tissue or fluid compartments such as cerebrospinal fluid or urine.
In another embodiment, the invention is a test kit for use in detecting the presence of a vertebrate high mobility group box (FIMGB) polypeptide or portion thereof in a sample. Such test kits can comprise, e.g., an antibody or antigen-binding fragment of the invention and one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or antigen-binding fragment and an FILVIGB polypeptide or portion thereof. The antibody and antigen-binding fragments of the present invention can be provided in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes. The antibodies or antigen-binding fragments thereof, which can be labeled or unlabeled, can be included in the kits with adjunct ingredients (e.g., buffers, such as Tris (Tris(hydroxymethyl)aminomethane), phosphate and carbonate, stabilizers, exciPients, biocides and/or inert proteins, e.g., bovine serum albumin). For example, the antibodies or antigen-binding fragments can be provided as a lyophilized mixture with the adjunct ingredients, or the adjunct ingredients can be separately provided for combination by the user. Generally these adjunct materials will be present in less than about 5% by weight based on the amount of active antibody, and usually will be present in a total amount of at least about 0.001% by weight based on antibody concentration. Where a second antibody or antigen-binding fragment capable of binding to the anti-HNIGB antibody or antigen-binding fragment is employed, such antibody or fragment can be provided in the kit, for instance in a separate vial or container. The second antibody or antigen-binding fragment, if present, is typically labeled, and can be formulated in an analogous manner with the antibody formulations described above. The antibodies, antigen-binding fragments arid/or ancillary reagent of the kit can be packaged separately or together within suitable containment means (e.g., bottle, box, envelope, tube). When the kit comprises a plurality of individually packaged components, the individual packages can be contained within a single larger containment means (e.g., bottle, box, envelope, tube).
Methods of Screening In another embodiment, the invention is a method of detecting or identifying an agent that binds to an HIV1GB polypeptide (e.g., a mammalian HMGB
polypeptide (e.g., an EIMGB1 polypeptide)). In one embodiment, the method of detecting or identifying an agent that binds to an HMGB polypeptide is a competitive binding assay in which the ability of a test agent to inhibit the binding of an antibody or antigen-binding fragment of the invention is assessed. For example, the antibody or antigen-binding fragment can be labeled with a suitable label as described herein, and the amount of labeled antibody or antigen-binding fragment required to saturate the BMGB polypeptide present in the assay can be determined.
For example, a saturating amount of labeled antibody or antigen-binding fragment and various amounts of a test agent can be contacted with an ELIVIGB
polypeptide under conditions suitable for binding, and complex famiation determined. In this type of assay, a decrease in the amount of complex formed between the labeled antibody or antigen-binding fragment and HMGB polypeptide indicates that the test agent binds to the HIVIGB polypeptide. In another embodiment, the IIMGB
polypeptide can be labeled. Suitable labels for labeling antibodies, antigen-binding fragments and/or IIMGB polypeptides include those described above.
A variety of agents, such as Proteins (e.g., antibodies), peptides, peptidomimetics, small organic molecules, nucleic acids and the like, can be tested for binding to an HIVIGB polypeptide (e.g., a mammalian HIVIGB polypeptide (e.g., an HMGB1 polypeptide)). According to the method of the present invention, agents can be individually screened or one or more agents can be tested simultaneously.
Where a mixture of compounds is tested, the compounds selected by the processes described can be separated (as appropriate) and identified using suitable methods (e.g., sequencing, chromatography). The presence of one or more compounds (e.g., a ligand, inhibitor, promoter) in a test sample can also be determined according to these methods.
Agent that bind to an HMGB polypeptide and that are useful in the therapeutic methods described herein can be identified, for example, by screening libraries or collections of molecules, such as, the Chemical Repository of the -5?-National Cancer Institute, in assays described herein or using other suitable methods.
Libraries, such as combinatorial libraries, of compounds (e.g., organic compounds, recombinant or synthetic peptides, "peptoids", nucleic acids) produced by combinatorial chemical synthesis or other methods can be tested (see e.g., Zuckerman, RN. et al., J. Med. Chem., 37: 2678-2685 (1994) and references cited therein; see also, Ohlmeyer, M.H.J. et al., PMC. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S.H. et al., PrOC. Natl. Acad. Sc!. USA 90:6909-6913 (1993), relating to tagged compounds; Rutter, W.J. et al.U.S. Patent No.
5,010,175;
Huebner, V.D. et al., U.S. Patent No. 5,182,366; and Geysen, H.M., U.S. Patent No.
4,833,092). Where compounds selected from a library carry unique tags, identification of individual compounds by chromatographic methods is possible.
The present invention will now be illustrated by the following Examples, which is not intended to be limiting in any way.
Example 1: Materials and Methods Generation of monoclonal antibodies to HiVIGB1 BALB/c mice were intraperitoneally immunized with 20 lig of recombinant CBP-Rat HMGB1 (CBP linked to amino acids 1-215 of rat H1VIGB1; nucleotide sequence of CBP-Rat HMGB1 is depicted as SEQ ID NO:4 and the amino acid sequence is depicted as SEQ ID NO:5 (see FIGS. 3A and 3B)) mixed with Freund's adjuvant at two-week intervals for 6 weeks. A final boost of 10 ug of the CBP-Rat 1-11VIGB1 in PBS was given intravenously after 8 weeks. Four days after the final boost, spleens from the mice were isolated and used for fusion. Fusion was carried out using standard hybridoma technique. The spleen was gently pushed through a cell strainer to obtain a single cell suspension. After extensive washing, spleen cells were mixed with SP2/0 myeloma cells. Polyethylene glycol (PEG) was added slowly, followed by media over a period of five minutes. The cells were washed and resuspended in DMEM containing 20% FCS and HAT, transferred to 96 well plates = and incubated at 37 C with 10% CO2 for 10-14 days.
In other experiments, a human HIvIGB1 B box polypeptide (SEQ ID NO:3;
FIG. 2B) was used as an immuogen. Five female BALB/c mice were intraperitoneally immunized with 10 lig/injection of HMGB1 B box mixed with Freund's adjuvant at three-week intervals. A bleed was obtained from the mice week after each boost. Three weeks after the third boost, a final intravenous injection (10 ug/mouse) of the rat HIvIGB1 B box was given. 72 hours after the final boost, hybridoma fusions were carried out as described above. Hybridomas were cultured in DMEM with 20% FBS, HAT, CondiMed and 1% pen/strep. Positive clones were identified by taking optical readings and identifying those with readings five times that of background.
Antibodies to the CBP-Rat HMGB1 and human HMGB1 B box were screened by limiting dilution and ELISA. ELISA plates were coated with recombinant HMGB1 at 3 pg/ml overnight and blocked with phosphate buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA). Supernatants from the hybridornas were added to the ELISA plates and incubated at room temperature for 30 minutes. The plates were then washed and anti-mouse Ig conjugated with horseradish peroxidase was added. After 30 minutes of incubation at room temperature, the plates were washed and developed. Cells from positive cells were transferred to 24-well plates and cloned by limiting dilution.
HMGB.1 stimulated TNF release The mouse macrophage cell line RAW 264.7 (available from the American Type Culture Collection (ATCC), Manassas, VA) was incubated with various concentrations of111VIGB1 for 4 hours at 37 C in serum-free Opti-IVIEM
(Invitrogen, Carlsbad, CA). The supernatants were harvested and TNF level was measured using an ELISA kit (R&D Systems, Minneapolis, MN). The assay was also performed using heparinized whole blood. In this case, HMGB1 was diluted in Opti-MEM,*
added to 100 p..1 of whole blood to give a final volume of 200 pi, and placed in a U-*Trade-mark bottom 96-well plate. The plates were then incubated for 4 hours at 37 C and plasma was harvested for ELISA analysis. =
To screen for blocking mAbs to HIVIGB1, purified mAbs were diluted in Opti-MEM and mixed with rat HA4GB1 at room temperature. After five minutes, the mixture was transferred into tissue culture wells containing RAW 264.7 cells.
The plates were then incubated for 4 hours at 37 C and supernatants were harvested for ELISA analysis.
SDS-Polyactylanzicle Gel Electrophoresis, Western Blot Analysis and Selectivity of HMGB1 Monoclonal Antibodies For detection of HMGB1 with the HMGB1 mAbs, samples were mixed with 4X NuPAGE LDS Sample Buffer, 10X NuPAGE Sample Reducing Agent (Invitrogen, Carlsbad, CA). The samples were heated in boiling water for 5 minutes, immediately chilled on ice and loaded on an SDS-polyacrylamide gel. Western blot analysis was performed using standard techniques.
For the experiments determining selectivity of the HIVIGB1 monoclonal antibodies (i.e., selectivity for EIMGB1 and/or BMGB2), western blot analysis was performed on samples containing non-recombinant (i.e., natural) HMGB1 from Chinese Hamster Ovary (CHO) cells (FIG. 11; labeled as CHO HMGB1; SEQ ID
N0:36) or samples containing non-recombinant (i.e., natural) HMGB2 from Chinese Hamster Ovary (CHO) cells and some detectable recombinant human HIVIGB1-His6 (FIG. 11; labeled as CHO HMGB2, rec-HMGB1-His6). FIGS. 18A and 18B depict the nucleotide and encoded amino acid sequences of the human recombinant IIMGB1 polypeptide containing a 5' 6 HIS tag (rec-FEMGB1-His6; SEQ ID NOs:39 and 40).
For the samples containing CHO HMGB1, samples contained ¨2.5-5 ng/ 1 of non-recombinant (i.e., natural) FIMGB1 from Chinese Hamster Ovary (CHO) cells. 20 I of the sample (i.e., ¨50-100 ng of HMGB I) was loaded on a gel and subjected to SDS-PAGE. To isolate CHO HMGB1 polypeptide, CHO cells were lysed and subsequently cleared by centrifugation. Anion exchange chromatography and heparin-affinity chromatography were then performed and fractions containing peak HMGB1 imrnunoreactivity but no detectable HMGB2 immunoreactivity were = pooled and used as the source of CRC) B1IvIGB1.
For the samples containing non-recombinant (i.e., natural) HMGB2 from Chinese Hamster Ovary (CHO) cells and some detectable recombinant FIMGB1-His6 (FIG. 11; labeled as CHO HMGB2, rec-IIMGB1-His6), CHO cells transfected with a recombinant HMGB1-His6-expressing plasmid were utilized. To isolate CHO
HMGB2 polypeptide, CHO cells were lysed and subsequently cleared by centrifugation. Anion exchange chromatography and heparin-affinity chromatography were then performed and fractions containing peak FIMGB2 immunoreactivity, but no detectable natural HMGB1 immunoreactivity, were pooled and used as the source of CHO 1-EVIGB2. In some cases, the pooled CHO HIVIGB2 fractions contained detectable amounts of recombinant HMGB-1-His6 polypeptide, however, this recombinant FEVIGB-1-His6 polypeptide was easily distinguished from HMGB2 based on its decreased mobility (and apparent higher molecular weight) when subjected to SDS-PAGE. Using the gel systems that generated the Western blots depicted in FIG. 11, non-recombinant CHO HMGB2 has an apparent molecular weight of-27,000, non-recombinant CHO HMGB1 has an apparent molecular weight of-29,000 and recombinant FIMGB-1-His6 has an apparent molecular weight of ¨31,000. For the CHO HMGB2, rec-TIMGB1-His6 samples, 20 ill of the sample (i.e., ¨10-20 ng of HMGB2) was loaded and subjected to SDS-PAGE.
For the western blots depicted in FIG. 11, either an anti-His Tag antibody (Santa Cruz, CA; 2 !_tojm1), an anti-HMGB2 antibody (Pharmingen, San Diego, CA;
2 jag/m1), an anti-H_MGB1 /2 mAb (MBL International, Watertown, MA; 2 or particular anti-HMGB1 monoclonal antibodies (e.g., 2E11 HMGB1 mAb (CT3-2E11), 1G3 EIMGB1 mAb (CT3-1G3), 6H9 HMGB1 mAb (CT3-6H9), 2G7 HMGB1 mAb (CT3-2G7), 2G5 HIVIGB1 mAb (CT3-2G5) and 6E6 HIVIGB 1 mAb (CT3-6E6); 2 vginil for each) were used.
Sequencing of Monoclonal Antibodies Total RNA was isolated from hybridoma cells using RNeasy MiniKit (Qiagen, Valencia, CA) as described in the kit protocol. The first strand of cDNA
synthesis was performed using ProtoScript First Strand cDNA Synthesis kit (New England Biolabs, Catalog # E6500S) as designed in the kit protocol. 5 pl of cDNA
was added to a PCR reaction (as described in the protocol for Mouse Ig-Primer Set, Catalog # 69831-3, Novagen, Madison, WI) containing 25 pmoles of the appropriate 5' primers (as described in the Novagen Ig-primer set protocol as MuIgGVH5'-A, MuIgGV115'-B, MulgGVH5'-C, MuIgGVH5'-D, MuIgGV15'-E, MuIgGVH5'-F for heavy chain and MuIgGV5'-A, MuIgGVE5'-B, MuIgGVL5'-C, MulgGVL5'-D, MuIgGVL5'-E, MuIgGVL5'-F and MuIgGVL5'-G for light chain) and 3' primers (as described in the Novagen Ig-primer set protocol as MuIgGVH3'-2 for heavy chain and MuIgGVL3'-2 for light chain). The PCR reaction conditions were 1 minute at 94 C, 1 minute at 50 C and 2 minutes at 72 C for 35 cycles, followed by an =
extension at 72 C for 6 minutes. PCR products were cloned into vector TOPO
(Invitrogen, San Diego, CA). DNA sequence analysis was performed by Genaissance Pharmaceuticals (New Haven, CT).
HMGB1 Peptide Binding Experiments Biotinylated peptides bound to React-Bind Streptavidin-Coated Plates (Pierce, Rockford, EL, Catalog # 15501) and non-biotinylated peptides bound to . poly-D-lysine coated ELISA plates were used in anti-peptide ELISAs.
Biotinylated peptides corresponding to particular 18 amino acid regions of human HMGB I, as well as a longer peptide corresponding to amino acid residues 9-85 of human HMGB1, were prepared and analyzed for binding to particular anti-HMGB I
monoclonal antibodies by ELISA. These peptides, and their respective sequences, are depicted in FIG. 13A. Poly-D-lysine coated plates were prepared by adding 100 ill/well of 0.1 mg/mI solution of poly-D-lysine in water. Plates were then incubated at room temperature for 5 minutes and were rinsed with water to remove the solution.
*Trade-mark Briefly, using the molecular weight of the respective peptide, 1 m_M peptide solutions were prepared in pyrogen-free water and diluted in IX phosphate buffered saline. Plate wells were washed three times with 200 ul of PBS and 100 gl of the =
various peptide solutions were added to designated wells. The plates were then covered and incubated for 60 minutes at room temperature. The wells were then washed three times with PBS, 0.05% polyoxyethylenesorbitan (referred to herein as Tween 2OTM) using a volume greater than 100 1. 200 ul of blocking buffer (5%
nonfat dry milk in PBS, 0.05% Tween 2OTM) was added to each of the wells. The plates were covered and incubated for 60 minutes at room temperature. The wells were then washed three times with PBS, 0.05% Tween 2OTM using a volume greater than 100 fa 100 j.d of the primary antibody (e.g., 2E11 HMGB1 mAb, 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb; 2 jig/m1 in blocking buffer) was added to the designated wells and the plates were covered and incubated for 30 minutes at room temperature. The wells were then washed three times with PBS, 0.05% Tween 20Tm using a volume greater than 100 pl.
100 p.1 of the goat anti-mouse horseradish peroxidase (IIRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, Catalog No. 115-035-071; used at a 1:2000 dilution) was added to each of the wells.
The plates were covered, incubated for 30 minutes at room temperature and subsequently washed three times with times with PBS, 0.05% Tween 2OTM using a volume greater than 100 p.1. The plates were developed by adding 50 ul of 1X
(Sigma, St. Louis, MO) to each well, incubating for 10 minutes at room temperature and reading the absorbance at 655 run using Microplate/Manager software and a BioRad Model 680 Plate Reader. Average background signal was subtracted from each of the sample signals.
Cecal Ligation and Puncture Cecal ligation and puncture (CLP) was performed as described previously (Fink and Heard, Surg. Res. 49:186-196 (1990); Wichmann et al., Crit. Care Med.
26:2078-2086 (1998); and Remick et al., Shock 4:89-95 (1995)). Briefly, BALB/c mice were anesthetized with 75 mg/kg ketamine (Fort Dodge, Fort Dodge, Iowa) and 20 mg/kg of xylazine (Bohringer Ingelheim, St. Joseph, MO) intramuscularly. A
midline incision was perfoimed, and the cecum was isolated. A 6-0 prolene suture ligature was placed at a level 5.0 mm from the cecal tip away from the ileocecal valve.
The ligated cecal stump was then punctured once with a 22-gauge needle, without direct extrusion of stool. The cecum was then placed back into its nomial intra-abdominal position. The abdomen was then closed with a running suture of 0 prolene in two layers, peritoneum and fascia separately to prevent leakage of fluid.
All animals were resuscitated with a normal saline solution administered sub-cutaneously at 20 ml/kg of body weight. Each mouse received a subcutaneous injection of imipenem (0.5 mg/mouse) (Primaxin, Merck & Co., Inc., West Point, PA) 30 minutes after the surgery. Animals were then allowed to recuperate.
Mortality was recorded for up to 1 week after the procedure; survivors were followed for 2 weeks to ensure no late mortalities had occurred.
Starting the day after the CLP procedure, 100 pg of particular anti-HIVIGBI
monoclonal antibodies (i.e., 6E6 HMGB1 mAb (mAB (6E6)); 2E11 HMGB1 triAb (mAB (2E11)); 902 II1vIGB1 mAb (mAB (9G2)) and a control IgG antibody were intraperitoneally administered to the mice once or twice a day for a total of treatments. For the data presented in FIG. 16, various doses (11..temouse, 10 pg/mouse or 100 tg/mouse) of 6E6 FIMGB1 mAb or a control IgG antibody were intraperitoneally administered.
Two ELISA methods were perfonned using various HIVIGB1 monoclonal antibodies.
HMGB1 ELISA with Monoclonal (Capture) + Polyclonal (Detectoi) Antibody Pairs In the first method, ELISA plates were coated with a number of purified anti-HMGB1 mAbs (e.g., 2E11 IIMGB1 mAb, 2G5 If_MGB1 mAb, 207 RMGB1 mAb, 6E6 HMGB1 mAb), and incubated overnight at 4 C. The plates were then blocked *Trade-mark with PBS, 1%BSA for one hour at 37 C. After washing, recombinant rat HMGB1 was added at the indicated concentrations, and the plates were incubated at room temperature for 1 hour. The plates were then washed and incubated with rabbit polyclonal antibodies against HMGB1 at 2 jig/m1 (see U.S. Patent Nos.
6,303,321, 6,448,223 and 6,468,533). After 1 hour at room temperature, the plates were washed and incubated for 30 minutes with goat anti-rabbit Ig-I-1PR (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted at 1:1000 in PBS. After washing, the plates were developed with TMB (Invitrogen, San Diego, CA) and absorbance at 655 nm was measured using a plate reader.
HMGB1 ELISA with Monoclonal Antibody Pairs (Detection with 6E6 HMGB1 tnAb) ELISA plates were coated and blocked, and recombinant rat HMGB1 was subsequently added as described above. After washing away the unbound HMGB1 polypeptide, biotinylated 6E6 HMGB1 mAb was added at 2 pg/m1 and incubated for 1 hour at room temperature. Streptavidin-HRP was used to detect bound 6E6 HtVIGB1 inAb and the plates were developed with TMB as described above.
Example 2: Identification and Characterization of Anti-FIMGB1 Monoclonal Antibodies A number of novel anti-FIMGB1 monoclonal antibodies have been isolated and purified from immunizations with either recombinant full-length rat HMGB1(SEQ ID NO:4; FIGS. 3A and 3B) or with a B-box polypeptide of human liMGB1 (SEQ ID NO:3; FIG. 2B). A table summarizing characteristics (clone name, immunogen, isotype, purified antibody binding domain, and results of in vivo CLP assays) of these antibodies is depicted in FIG. 7.
Example 3: Determination of Selectivity of Anti-HMGB1 Monoclonal Antibodies Experiments (e.g., ELISA, Western blot analysis) to determine the selectivity of the HMGB1 monoclonal antibodies revealed that particular anti-HMGB1 monoclonal antibodies are able to bind to the A box portion of HMGB1, the B
box portion of FLMGB1 and/or the whole HMGB1 protein. For example, as depicted in FIG.7, anti-HMGB1 monoclonal antibodies were identified that can bind to the A
box of HMGB1 (e.g., 6E6 HMGB1 mAb, 6H9 HIVIGB1 mAb, 10D4 ILMGB1 mAb, 6H9 HMGB1 mAb, 2G7 ELMGB1 mAb, 2G5 HMGB1 mAb, 41111 HMGB1 mAb, 7113 HMGB1 mAb, 9113 HMGB1 inAb). Other monoclonal antibodies were identified that bind to the B box of HMGB1 (e.g., 2E11 HMGB1 mAb, 3G8 HMGB1 mAb, 3-5A6 IIMGB1 mAb, 9G1 HMGB1 mAb, 4C9 IIMGB1 mAb, 1C3 HMGB1 mAb, 5C12 HMGB1 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb, 4A10 HMGB1 mAb).
Example 4: Determination of Nucleotide and Amino Acid Sequences of Anti-HIVGB1 Monoclonal Antibodies For particular ILMGB1 monoclonal antibodies (6E6 ILMGB1 mAb, 2E11 HMGB1 mAb, 10D4 HLVIGB1 mAb, 2G7 HMGB1 mAb), nucleotide and encoded . amino acid sequences of VH domains and VI; domains, including CDRs, were also obtained (FIGS. 4A-4D, 5A-5D, 6A-6D and 19A-19D).
Example 5: Inhibition of TNF Release by Anti-BNIGB1 Monoclonal Antibodies The ability of particular HMGB1 monoclonal antibodies to inhibit TNF
release was assessed. The results of this study are shown in FIGS. 8 and 9, which are histograms depicting TNF released by RAW 264.7 cells administered only IEVIGB1, ILMGB1 plus particular LIMGB1 monoclonal antibodies, or a control IgG
antibody. FIG. 8 depicts the results of inhibition of 1-114GB1-mediated TNF
release for 6E6 EE14GB1 mAb, 10D4 HMGB1 InAb, 2E11 BEIVIGB1 mAb, 9G2 HIVIGB1 mAb, and a control IgG antibody. FIG. 9 depicts the results of inhibition of HMGB1-mediated TNF release for 3G8 H1\4GB1 mAb, 1A9 HMGB1 mAb, 9G2 HMGB1 mAb, 6E6 ITIVIGB1 mAb, 2E11 HMGB1 mAb, 10D4 HMGB1 mAb, 6H9 HIVIGB1 mAb, or a control IgG antibody. As depicted in FIGS. 8 and 9, particular HMGB1 monoclonal antibodies (e.g., 6E6 HIVIGB1 inAb, 10D4 FEVIGB1 t-nAb) inhibited TNF release, indicating that such antibodies could be used to modulate one or more HIVIGB functions (e.g., as described herein). For example, these blocking antibodies could be used to neutralize the biological activity of FIMGB1 (e.g., HMGB1-mediated activation of the cytokine cascade).
Example 6: Treatment of Septic Mice with Anti-H1VGB1 Monoclonal Antibodies Increases Survival of Mice Mice were subjected to cecal ligation and puncture (CLP), a well characterized model of sepsis caused by perforating a surgically-created cecal diverticulum, that leads to polymicrobial peritonitis and sepsis (Fink and Heard, supra; Wichmann et aL, supra; and Remick et al., supra), and administered - particular anti-H1VIGB1 monoclonal antibodies (6E6 IEVIGB1 mAb, 2E11 FIAIGB1 mAb or 9G2 HMGB1) or a control IgG antibody (100 j_tg of antibody administered twice per day). Survival was monitored for 7 days. The results of this study are shown in FIG. 10, which is a graph of the survival of septic mice treated with either a control antibody or particular anti-IIMGB1 monoclonal antibodies. The results show that anti-HIVIGB1 monoclonal antibodies administered to the mice starting hours after the onset of cecal perforation rescued animals from death, as compared to administration of an IgG control antibody. For 6E6 HMGB1 mAb, the rescue was significant at day 7 (p<0.03 versus control, Fisher's exact test).
A dose response curve for survival of septic mice treated with 6E6 IIMGB1 mAb was also conducted. As depicted in FIG. 16, doses of 1, 10 and 100 ug of HMGB1 mAb were administered to mice. The results demonstrate that a dose of 10 jig of 6E6 IEVIGB1 resulted in the greatest rescue of the septic mice.
Example 7: Selectivity of Anti-HMGB1 Monoclonal Antibodies As described above, western blot analysis was performed using particular anti-IlIvIGB1 monoclonal antibodies. FIG. 11 depicts individual western blots of samples containing either CHO HIvIGB1 or CHO IEVIGB2 and possibly recombinant HMGB1-His6 (labeled as CHO H1VIGB2, rec-HMGB1-His6), which were probed with either an anti-His Tag antibody, an anti-HMGB2 antibody, an anti-HMGB1/2 monoclonal antibody, or particular anti-IEVIGB I monoclonal antibodies (e.g., HMGB1 mAb, 1G3 HIMGB1 mAb, 6H9 HIMGB1 mAb, 2G7 HA/1GB' mAb, 2G5 HMGB1 mAb and 6E6 H1VIGB1 mAb). The results of these experiments reveal that 2G7 HMGB1 mAb binds HMGB1 but does not detectably bind HMGB2, while 2E11 HMGB1 mAb, 1G3 HMGB1 mAb and 6H9 ELMGB1 mAb detect HMGB2 in addition to ELMGB1.
Example 8: HMGB1 Peptide Binding Experiments Biotinylated peptides corresponding to particular 18 amino acid regions of human HMGB I and a longer peptide corresponding to amino acid residues 9-85 of human HMGB1 were prepared and analyzed for binding to particular anti-HMGB1 monoclonal antibodies by ELISA. These peptides and their respective sequences are depicted in FIG. 13A.
The results of these peptide binding experiments are depicted in FIG_ 13B.
As depicted in FIG. 13B, 2E11 HMGB1 mAb bound to a peptide corresponding to amino acid residues 151-168 of human HMGB1 (i.e.', amino acid residues 151-168 SEQ ID NO:1). 6E6 IIMGB1 mAb and 6E19 bound to a peptide corresponding to amino acid residues 61-78 of human HMGB 1 (i.e., amino acid residues 61-78 SEQ
ED NO:1). 2G7 HIMGB1 mAb bound to a peptide corresponding to amino acid residues 46-63 of human HMGB1 (i.e., amino acid residues 46-63 of SEQ ID
NO:1).
In addition, 2G7 HMGB1 mAb and 6E6 IIMGB1 mAb also bound the longer peptide corresponding to amino acid residUes 9-85 of human HMGB1. These experiments demonstrate that particular anti-HMGB1 monoclonal antibodies recognize different epitopes within the HIVIGB1 polypeptide. For example, 6E6 HMGB1 mAb, which was shown to inhibit HMGB 1-mediated TNF release binds to an epitope contained within amino acids 61-78 of HIMGB 1. The discovery of a blocking epitope within this particular region of HIMGB1 could be used to screen for additional blocking agents (e.g., agents that inhibit an HMGB1 function (e.g., HMGB 1 -m e di at e d activation of the cytolcine cascade)).
Example 9: BIVIGB1 ELISA
As depicted in FIGS. 14 and 15, two different ELISA methods were used to examine theproperties of particular anti-HIVIGB1 monoclonal antibodies. In one method, HMGB1 monoclonal antibodies 2E11 IIMGB1 mAb, 2G5 HMGB1 mAb, 2G7 BMGB1 mAb, and 6E6 HMGB1 mAb, were used as capture antibodies and a polyclonal IIMGB1 antibody was used as the detector antibody. In the other ELISA
method, HMGB1 monoclonal antibodies 2E11 FIMGB1 rnAb, 2G5 HMGB1 mAb, 2G7 IIMGB1 mAb, and 6E6 IIMGB1 mAb, were used as capture antibodies and 6E6 FIIVIGB1 mAb was used as the detector antibody. The results from both of the ELISA methods demonstrate that the monoclonal HMGB1 antibodies can detect IIMGB1 and would be suitable for the diagnostic and/or prognostic methods described herein.
Example 10: Binding of 2G7 IIMGB1 mAb to HMGB1 is Inhibited By a Peptide Corresponding to Amino Acid Residues 46-63 of HMGB1 HIVIGB1 peptide binding experiments using 2G7 HMGB1 mAb were conducted. As described, biotinylated synthetic peptides corresponding to either amino acid residues 46-63 of human EINIGB1 or amino acid residues 61-78 of human HMGB1 were prepared and analyzed for binding to 2G7 HMGB1 inAb (2G7) by ELISA. Briefly, 2 m/m1 of 2G7 EIVIGB1 mAb was added to plate wells containing either the HMGB1 46-63 peptide or the HMGB1 61-78 peptide at each of the indicated concentrations (0, 0.33, 1,3, 9, 27, 81 and 243 iuM peptide) for one hour at 25 C to prepare antibody-peptide samples. ELISA plates were coated with 101.tg/m1 of recombinant rat ILMGB1, and incubated overnight at 4 C. The plates were then blocked with reconstituted milk for one hour at 37 C. Antibody-peptide samples were then added at the concentrations listed above and incubated for one hour at room temperature. The plates were then washed and incubated with Streptavidin-HRP. After washing, the plates were developed with TMB
(Invitrogen, San Diego, CA) and absorbance at 655 am was measured using a plate reader.
FIG. 20 depicts the results as percent of maximum signal (y-axis). As FIG.
20 demonstrates, the HMGB1 46-63 peptide inhibited the binding of 2G7 FEMGB1 mAb to bound LIMGB1 at all concentrations (depicted as a decrease in % of maximum signal). In contrast, the HMGB1 61-78 peptide did not inhibit the binding of 2G7 HMGB1 mAb to HMGB1. These experiments further confirm that 2G7 HMGB1 mAb binds to an epitope that is present in amino acids 46-63 of HMGB1.
Example 11: Binding of 2E11 HMGB1 mAb to HMGB1 is Inhibited By Higher Concentrations of a Peptide Corresponding to Amino Acid Residues 151-168 of 1114GB1 peptide binding experiments using 2E11 HMGB1 mAb and biotinylated synthetic peptides corresponding to either amino acid residues 46-63 of human HMGB1 (SEQ ID NO:23) or amino acid residues 151-168 of human FIMGB1 (SEQ ID NO:30) were conducted as described herein The results of these experiments are depicted in FIG. 21 (as percent of maximum signal (y-axis)).
As FIG. 21 demonstrates, the EINIGB1 151-168 peptide significantly inhibited the binding of 2E11 FIMGB1 mAb to bound HMGB1 at concentrations of 9 pM or greater. The HMGB1 151-168 peptide did not significantly inhibit the binding of 2E11 FIMGB1 mAb to PIMGB1 at concentrations of3 uM or below (FIG. 21). In addition, the IBMGB1 46-63 peptide did not inhibit the binding of 2E11 HMGB1 mAb to IT4GB1. These experiments confirm that 2E11 IIMGB1 mAb binds to an epitope that is present in amino acids 151-168 of HMGB1.
Example 12: 2G7 HMGB1 mAb Recognizes an Epitope That is Present in Amino Acids 53-63 of HMGB1 Various synthetic peptides were prepared. These synthetic peptides included a biotinylated peptide corresponding to amino acid residues 46-63 of human IEVIGB1 (SEQ ID NO:23; designated "huH1'dGB1-46-63-B" or "Human HMGB1-46-63-B"), a biotinylated peptide corresponding to amino acid residues 46-63 of human HMGB2 (SEQ ID NO:48; designated "huHlvIGB2-46-63-B" or "Human H1MGB2-46-63-B"), a non-biotinylated peptide corresponding to amino acid residues 53-70 of human HIMGB1 (SEQ NO:47; designated "huHMGB1-53-70" or "Human HMGB1-53-70"), a biotinylated peptide corresponding to amino acid residues 61-78 of human HMGB1 (SEQ ID NO:24; designated "huHMGB1 -61-78-B"), a non-biotinylated peptide corresponding to amino acid residues 40-57 of human HMGB1 (SEQ ID NO:46; designated "Human HMGB1-40-57") and a non-biotinylated peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 46-63 of human HMGB1 (SEQ ID NO:45; designated "Human FIMGB1-46-63-scr"). By ELISA, as described herein, the binding of 2G7 FIMGB1 mAb to these overlapping peptides was analyzed to more specifically ascertain the epitope within HMGB1 that binds to 2G7 HMGB1 mAb. These peptides and their respective sequences are depicted in FIG. 23.
The results of the peptide binding experiments are depicted in FIGS. 22 and 23. As shown in FIGS. 22 and 23, 2G7 HMGB I mAb bound to the HMGB1 46-63 peptide (i.e., amino acid residues 46-63 of SEQ ID NO:1 or SEQ ID NO:23) but did not bind to the corresponding amino acid region of human FIMGB2 (i.e., the HMGB2 46-63 peptide; amino acid residues 46-63 of SEQ ID NO:54 or SEQ ID
NO:48). In addition, 2G7 FilMGB1 mAb bound to the HIVIGB1 53-70 peptide but did not bind the HMGB1 40-57 peptide. 2G7 HMGB1 mAb also did not bind to the peptide consisting of a scrambled sequence of amino acid residues 46-63 of HMGB1. In addition to showing the binding of 2G7 HMGB1 mAb to the various synthetic peptides, FIG. 22 also depicts the binding of avidin to these synthetic peptides.
In Example 8, it was shown that 2G7 LIMGB1 mAb binds to an epitope contained within amino acid residues 46-63 of HNIGB1. Given that 2G7 ITIVIGB1 mAb binds the FEMGB1 46-63 peptide and the HIVIGB I 53-70 peptide, these experiments demonstrate that 2G7 HMGB1 mAb recognizes an epitope present in the amino acid region consisting of amino acid residues 53-63 of HMGB1. These experiments further demonstrate that 2G7 I1MGB1 mAb does not bind to the HMGB2 46-63 peptide, notwithstanding only a single amino acid difference between the HIVIGB1 46-63 peptide and the HMGB2 46-63 peptide. As such, the glycine residue at position 58 of HMGB1 (Gly-58), which is a corresponding serine residue in HMGB2, is an important amino acid residue in the HMGB I epitope recognized by 2G7 HMGB1 mAb (FIG. 23).
Example 13: 6E6 HMGB1 mAb Recognizes an Epitope That is Present in Amino Acids 67-78 of HMGB1 Various synthetic peptides were prepared. These synthetic peptides included a non-biotinylated peptide corresponding to amino acid residues 53-70 of human HMGB1 (SEQ ID NO:47; designated "Human IIMGB1-53-70-B"; described above), a non-biotinylated peptide corresponding to amino acid residues 67-84 of human (SEQ ID NO:50; designated "Human HMGB1-67-84"), a biotinylated peptide corresponding to amino acid residues 61-78 of human HMGB1 (SEQ ID
NO:24 designated "Human IIMGB1-61-78-B") and a non-biotinylated peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 61-78 of human IIMGB1 (SEQ
ID NO:49; designated "Human HMGB1-61-78_scr"). By ELISA, as described herein, the binding of 6E6 HMGB1 mAb to these overlapping peptides was analyzed to more specifically ascertain the epitope within IIMGB1 that binds to 6E6 mAb. These peptides, their respective sequences and which of the peptides were bound by 6E6 HMGB1 mAb are depicted in FIG. 24. =
The results of these peptide binding experiments are depicted in FIG. 24. As shown in FIG. 24, 6E6 HMGB1 mAb bound to the ITIVIGB1 61-78 peptide (SEQ ID
NO:24). Further, 6E6 HMGB1 mAb bound to the HMGB1 67-84 peptide (SEQ ID
NO:50) but did not bind to the FIMGB1 53-70 peptide (SEQ ID NO:47) (FIG. 24).
6E6 HMGB I mAb also did not bind to the peptide consisting of a scrambled sequence of amino acid residues 61-78 of HMGB1 (SEQ ID NO:49) (FIG. 24). In Example 8, it was shown that 6E6 HMGE1 mAb binds to an epitope contained within amino acid residues 61-78 of HMGB1. Given that 6E6 HMGB1 mAb binds to the HMGB1 61-78 peptide and the HMGB1 67-84 peptide, these experiments demonstrate that 6E6 IIIVIGB1 mAb recognizes an epitope present in the amino acid region consisting of amino acid residues 67-78 of 1-EVIGB1.
Example 14: HMGB1 Peptide Binding Experiments with 2E11 HMGB1 mAb Various synthetic peptides were prepared. These synthetic peptides included a biotinylated peptide corresponding to amino acid residues 151-168 of human HMGB1 (SEQ ID NO:30; designated "Human BMGB1-151-168-B"), a non-biotinylated peptide corresponding to amino acid residues 143-160 of human HMGB1 (SEQ ID NO:52; designated "Human I-EVIGB1-143-160"), a non-biotinylated peptide corresponding to amino acid residues 157-174 of human HMGB1 (SEQ ID NO:53; designated "Human HMGB1-157-174"), and a non-biotinylated peptide corresponding to a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 151-of human HMGB1 (SEQ ID NO:51; designated "Human HMGB1-151-168_scr").
By ELISA, as described herein, the binding of 2E11 HMGB1 mAb to these overlapping peptides was analyzed to more specifically ascertain the epitope within HIVIGB1 that binds to 2E11 HMGB1 mAb. These peptides, their respective sequences and which of the peptides were bound by 2E11 HMGB1 mAb are depicted in FIG. 25.
As depicted in FIG. 25, 2E11 HMGB1 mAb bound to the 1-1MGB1 151-168 peptide (SEQ ID NO:30), but did not bind to either the HMGB1 143-160 peptide (SEQ ID NO:52) or the HMGB1 157-174 peptide (SEQ ID NO:53). As is known in the art, there are two types of epitopes or antigenic determinants: linear, sequential or continuous epitopes and non-linear, conformational or discontinuous epitopes.
The dimensions of a typical antibody epitope are often given a's 6 amino acid residues in size, but can be of variable size. These experiments suggest that the epitope recognized by 2E11 HMGB1 mAb is likely to comprise amino acid residues 156-161 of HMGB1. However, 2E11 ITMGB1 mAb may also recognize an epitope that includes flanking amino acids to this region, e.g., amino acid residues 155-161, 155-162, 156-162 and/or 156-163 of HMGB1.
A summary of the peptide binding results depicting the mapped epitopes of HMGB1 that are recognized by various HMGB1 mAbs (e.g., 2G7 HMGB1 mAb, 6E6 HMGB1 mAb, 2G5 HMGB1 mAb, 6H9 HMGB1 mAb, 2E11 HMGB1 mAb) is shown in FIG. 26.
Example 15: Mass Spectrometry of 6E6 HMGB1 mAb The mass of 6E6 HMGB1 inAb was determined by mass spectrometry. 6E6 HMGB1 mAb was sent to Novatia, LLC (Princeton, NJ) for LC/MS analysis.
Briefly, the antibody, either intact or after being treated with DTT (to separate heavy and light chains), was subjected to analysis using a PLRP-s 4000A reverse phase HPLC column (HPLC/ESI-MS system) and mass spectroscopy (Finnigan TSQ7000 mass spectrometer). Mass accuracy for proteins is generally 0.01%, and this accuracy was achieved in measuring the mass of the 6E6 light chain (e.g., 2 Da/23,9I7 Da x 100% 0.008%).
The results of this analysis are shown in FIGS. 27A-27D, which depicts a mass spectrum plot. The total mass of 6E6 HMGB1 mAb was 146.5 kDa (FIG.
27A; depicting mass spectrum for intact 6E6 HMGB1 mAb). The masses of the light and heavy chains of 6E6 IIMGB1 mAb were determined to be 23.9 kDa (FIGS.
27B and 27C) and 49.4 kDa (FIGS. 27B and 27D), respectively. The predicted masses for the light and heavy chains, as calculated using amino acid molecular weights, are 23.9 kDa and 47.9 kDa, respectively.
Example 16: 2G7 EIMGB1 mAb Increases Survival in Septic Mice After Administration of a Single Dose Mice were subjected to cecal ligation and puncture (CLP) as described above. In one experiment, twenty-four hours after surgery, mice were intraperitoneally administered either 0.004 mg/kg, 0.04 mg/kg or 0.4 mg/kg of ILMGB1 mAb (2G7) once per day. In a second experiment, twenty-four hours after .tirgery, mice were administered either 0.04 mg/kg of 2G7 HMGB1 mAb or 0.4 mg/kg of control IgG (IgG control) once per day. Survival was monitored for 14 days for both experiments. The results of the two experiments are combined and presented in FIG. 28. Administration of 0.4 mg/kg of 2G7 HMGB1 mAb resulted in approximately 85% survival at 14 days after CLP, as compared to approximately only 40% survival of mice administered with IgG control at 14 days after CLP
(FIG.
28). Further, as depicted in FIG. 28, administration of 0.04 mg/kg and 0.004 mg/kg of 2G7 HMGB1 mAb resulted in approximately 60% and 50% survival at 14 days after CLP, respectively.
FIG. 29 is a table comparing CLP survival percentages in mice administered various doses (either 4 mg/kg, 0.4 mg/kg, 0.04 mg/kg or 0.004 mg/kg) of 6E6 HIVIGB1 mAb, 2G7 HIVIGB1 mAb or control IgG. As depicted in FIG. 29, the mice were administered the antibodies 4 times a day intraperitoneally. The results demonstrate that administration of a dose of 0.4 mg/kg of either 6E6 HMGB1 mAb or 2G7 HMGB1 mAb resulted in greater than 80% of the septic mice surviving to days post-CLP, as compared to only approximately 40% of the septic mice surviving to 14 days post-CLP when administered control IgG.
Example 17: Inhibition of TNF Release by Anti-HMGB1 Monoclonal Antibodies As in Example 5, the ability of particular FLMGB1 monoclonal antibodies to inhibit TNF release was assessed. The results of this study are shown in FIGS.
and 33, which are histograms depicting TNF released by RAW 264.7 cells administered only H:MGB1 (dark bar), or FEVIGB1 plus particular I-EVIGBI
monoclonal antibodies. FIG. 32 depicts the results of inhibition ofILMGB1-mediated TNF release for 1A9 IIMGB1 mAb (1A9); 2E11 HMGB1 mAb (2E11);
2G5 ilaviGB 1 mAb (2G5); 2G7 HMGB1 mAb (2G7); 3G8 HMGB1 mAb (3G8);
4H11 HMGB1 mAb (4H11); 5A6 HMGB1 mAb (5A6); 6E6 IIMGB1 mAb (6E6);
9G2 FEVIGB1 mAb (9G2); 4C9 HMGB1 mAb (4C9); and 6119 HMGB1 mAb (6119).
FIG. 33 depicts the results of inhibition of HMGB1-mediated TNF release for HMGB1 mAb (7113); 9113 HMGB I mAb (9113); 10D4 FEVIGB1 mAb (10D4); 1C3 IIMGB1 mAb (1C3); 3E10 HMGB1 mAb (3E10); 4A10 HMGB1 mAb (4A10);
5C12 HMGB1 mAb (5C12); and 7G8 ILMGB1 mAb (7G8).
As depicted in FIGS. 32 and 33, and further to the results described in Example 5, particular HMGB1 monoclonal antibodies (e.g., 2E1 1 HMGB1 mAb, 41111 HMGB1 mAb, 6E6 FLMGB1 mAb, 6119 ILMGB1 mAb and 10D4 lEvIGB1 mAb) inhibited TNF release, indicating that such antibodies could be used to modulate one or more FEVIGB functions (e.g., as described herein). For example, these blocking antibodies could be used to neutralize the biological activity of HMGB1 (e.g., I-EVIGB1-mediated activation of the cytokine cascade).
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in foini and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Methods for deteimining whether an antibody or antigen-binding fragment inhibits an inflammatory condition are known to one skilled in the art. For example, inhibition of the release of a proinflanunatory cytokine from a cell can be measured according to methods known to one skilled in the art. For example, as described and exemplified herein, TNF release from a cell can be measured using a standard marine fibroblast L929 (ATCC, American Type Culture Collection, Rocicville, Maryland) cytotoxicity bioassay (Bianchi et al., Journal of Experimental Medicine /83:927-936 (1996)) with the minimum detectable concentration of 30 pg/ml. The L929 cytotoxicity bioassay is carried out as follows. RAW 264.7 cells are cultured in RPMI 1640 medium (Life Technologies, Grand Island, New York) supplemented with 10% fetal bovine serum (Gemini, Catabasas, California), and penicillin and streptomycin (Life Technologies). Polymyxin (Sigma, St. Louis, Missouri) is added at 100 units/ml to suppress the activity of any contaminating LPS. Cells are incubated with the antibodies described herein in Opti-MEM I medium for 8 hours, and conditioned supernatants (containing TNF that has been released from the cells) are collected. TNF that is released from the cells is measured by a standard murine fibroblast L929 (ATCC) cytotoxicity bioassay (Bianchi et al., supra) with the minimum detectable concentration of 30 pg/ml. Recombinant mouse TNF can be obtained from R & D Systems Inc. (Minneapolis, Minnesota) and used as a control in these experiments. Methods for measuring release of other cytoldnes from cells are also known in the art.
An inflammatory condition that is suitable for the methods of treatment described herein can be one in which the inflammatory cytokine cascade is activated.
In one embodiment- the inflammatory cytokine cascade causes a systemic reaction, such as with endotwdc shock. In another embodiment, the inflammatory condition is mediated by a localized inflammatory cytokine cascade, as in rheumatoid arthritis.
Nonlimiting examples of inflammatory conditions that can be usefully treated using the antibodies and antigen-binding fragments of the present invention include, e.g., diseases involving the gastrointestinal tract and associated tissues (such as ileus, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, allergy, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis);
diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alvealitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C
virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoal and multicellular parasites (such as malaria, filariasis, amebiasis, and hydatid cysts); deiniatological diseases and conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals);
diseases involving the cardiovascular system and associated tissues (such as stenosis, restenosis, vasulitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, myocardial ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thryoiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Retier's syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.
In one embodiment, the condition is selected from the group consisting of sepsis, allogaft rejection, arthritis (e.g., rheumatoid arthritis), asthma, atherosclerosis, restenosis, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, myocardial ischemia, organic. ischemia, reperfusion ischemia, Behcet's disease, graft versus host disease, Crohn's disease, ulcerative colitis, ileus, multiple sclerosis, and cachexia.
In another embodiment, the condition is selected from the group consisting of sepsis, arthritis (e.g., rheumatoid arthritis), asthma, lupus, psoriasis, inflammatory bowel disease and Crohn's disease.
Preferably the antibodies and antigen-binding fragments are administered to a patient in need thereof in an amount sufficient to inhibit release of proinflammatory cytolcine from a cell and/or to treat an inflammatory condition. In one embodiment, release of the proinflammatory cytoldne is inhibited by at least 10%, 20%, 25%, 50%, 75%, 80%, 90%, or 95%, as assessed using methods described herein or other methods known in the art.
The terms "therapy", "therapeutic" and "treatment", as used herein, refer to ameliorating symptoms associated with a disease or condition, for example, an inflammatory disease or an inflammatory condition, including preventing or delaying the onset of the disease symptoms, and/or lessening the severity or frequency of symptoms of the disease or condition. The terms "subject" and "individual" are defined herein to include animals such as mammals, including but , not limited to, primates, cows, sheep, goats, horses, dogs, cats, rabbits, guinea pigs, rats, mice or other bovine, ovine, equine, canine, feline, rodent, or murine species.
In one embodiment, the animal is a human.
In one embodiment, an excipient can be included with the antibodies and antigen-binding fragments of the invention. The excipient can be selected based on the expected route of administration of the antibodies or antigen-binding fragments in therapeutic applications. The route of administration of the composition depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder such as endotoxic shock, and oral administration may be preferred to treat a gastrointestinal disorder such as a gastric ulcer.
As described above, the dosage of the antibody or antigen-binding fragment to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Depending on the condition, the antibody or antigen-binding fragment can be administered orally, parenterally, intranasally, vaginally, rectally, lingually, sublingually, bucally, intrabucally and transdermally to the patient.
Accordingly, antibodies or antigen-binding fragments designed for oral, lingual, sublingual, buccal and intrabuccal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent and/or edible carrier. The antibodies or antigen-binding fragments may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the antibodies or antigen-binding fragments of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, and the like.
Tablets, pills, capsules, troches, and the like, may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and flavoring agents.
Some examples of binders include microcrystalline cellulose, gum tragacanth, and gelatin. Examples of excipients include starch and lactose. Some examples of disintegrating agents include alginic acid, corn starch, and the like.
Examples of lubricants include magnesium stearate and potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweetening agents include sucrose, saccharin, and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring, and the like. Materials used in preparing these various compositions should be pharmaceutically pure and non-toxic in the amounts used.
The antibodies and antigen-binding fragments of the present invention can be administered parenterally such as, for example, by intravenous, intramuscular, intrathecal or subcutaneous injection. Parenteral administration can be accomplished by incorporating the antibodies and antigen-binding fragments of the present invention into a solution or suspension. Such solutions or suspensions may also include sterile diluents, such as water for injection, saline solution, bacteriostatic saline (saline containing about 0.9% mg/ml benzyl alcohol), phosphate-buffered saline (referred to herein as PBS), Hank's solution, Ringer's-= lactate, fixed oils, polyethylene glycols, glycerine, propylene glycol, and other synthetic solvents. Parenteral foimulations may also include antibacterial agents (e.g., benzyl alcohol, methyl parabens), antioxidants (e.g., ascorbic acid, sodium bisulfite), and chelating agents (e.g., EDTA). Buffers, such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride and dextrose, may also be added. The parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic.
Rectal administration includes administering the antibodies and antigen-binding fragments into the rectum or large intestine. This can be accomplished using suppositories or enemas. Suppository formulations can be made by methods known in the art. For example, suppository formulations can be prepared by heating glycerin to about 120 C, dissolving the antibody or antigen-binding fragment in the glycerin, mixing the heated glycerin, after which purified water may be added, and pouring the hot mixture into a suppository mold.
Transdermal administration includes percutaneous absorption of the antibody or antigen-binding fragment through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, and the like.
The antibodies and antigen-binding fragments of the present invention can be administered nasally to .a subject. As used herein, nasally administering or nasal administration, includes administering the antibodies or antigen-binding fragments to the mucous membranes of the nasal passage or nasal cavity of the subject_ Pharmaceutical compositions for nasal administration of an antibody or antigen-binding fragment include therapeutically effective amounts of the antibody or antigen-binding fragment. Well-known methods for nasal administration include, for example, as a nasal spray, nasal drop, suspension, gel, ointment, cream, or powder. Administration of the antibody or antigen-binding fragment may also take place using a nasal tampon or nasal sponge.
As described above, a variety of routes of administration are possible including, for example, oral, dietary, topical, transdernial, rectal, parenteral (e.g., intravenous, intraarterial, intramuscular, subcutaneous, intraderrnal injection), and inhalation (e.g., intrabronchial, intranasal, oral inhalation, intranasal drops).
Administration can be local or systemic as indicated. The preferred mode of administration can vary depending upon the antibody or antigen-binding fragment to be administered and the particular condition (e.g., disease) being treated, however, oral or parenteral administration is generally preferred.
If desired, the antibodies or antigen-binding fragments described herein can be administered with one or more additional agents (e.g., agents used to treat an inflammatory condition). The antibodies or antigen-binding fragments thereof and additional agent(s) can be present in a single composition or administered as separate compositions. If administered as separate compositions, the antibodies or antigen-binding fragments thereof and additional agent(s) can be co-administered or administered separately.
In one embodiment, the antibodies or antigen-binding fragments of the invention are administered with an anti-inflammatory agent. Such agents are known to one of skill in the art. In one embodiment, the agent is an antagonist of an early sepsis mediator. As used herein, an early sepsis mediator is a proirtflammatory cytokine that is released from cells soon (i.e., within 30-60 min.) after induction of an inflammatory cytolcine cascade (e.g., exposure to LPS). Nonlimiting examples of these cytokines are EL-la, IL-113, IL-6, PAF, and MIR Also included as early sepsis mediators are receptors for these cytokines (for example, tumor necrosis factor receptor type 1) and enzymes required for production of these cytokines, for example, interleukin-113 converting enzyme). Antagonists of any early sepsis mediator, now known or later discovered, can be useful for these embodiments by further inhibiting an inflammatory eytokine cascade.
Nonlimiting examples of antagonists of early sepsis mediators are antisense compounds that bind to the mR_NA of the early sepsis mediator, preventing its expression (see, e.g., Ojwang et al., Biochenzistry 36:6033-6045 (1997);
Parnpfer et al., Biol. Reprod. 52:1316-1326 (1995); U.S. Patent No. 6,228,642; Yahata et al., Antisense Nucleic Acid Drug Dev. 6:55-61 (1996); and Taylor et aL, Antisense Nucleic Acid Drug Dev. 8:199-205 (1998)), ribozymes that specifically cleave the mRNA of the early sepsis mediator (see, e.g., Leavitt et al., Antisense Nucleic Acid Drug Dev. /0:409-414 (2000); Kisich at al., J. ImmunoL 163(4):2008-2016 (1999);
and Hendrix et at., Biochem. J. 314 (Pt. 2):655-661 (1996)), and antibodies that bind to the early sepsis mediator and inhibit their action (see, e.g., Kam and Targan, Expert Opin. Pharmacother. 1:615-622 (2000); Nagahira et at., J. Immunol.
Methods 222:83-92 (1999); Lavine et aL, J. Cereb. Blood Flow Metab. /8:52-58 (1998); and Holmes et at.. Hybridoma 19:363-367 (2000)). An antagonist of an early sepsis mediator, now known or later discovered, is envisioned as within the scope of the invention. The skilled artisan can determine the amount of early sepsis mediator to use for inhibiting any particular inflammatory cytokine cascade without undue experimentation with routine dose-response studies.
Other agents that can be administered with the antibodies and antigen-binding fragments of the invention include, e.g., VitaxinT" and other antibodies targeting avf33 integrin (see, e.g., U.S. Patent No. 5,753,230, PCT
Publication Nos.
WO 00/78815 and WO 02/070007 and anti-IL-9 antibodies (see, e.g., PCT
Publication No. WO 97/08321).
In one embodiment, the antibodies and antigen-binding fragments of the invention are administered with inhibitors of TNF biological activity e.g., inhibitors of TNF-a, biological activity). Such inhibitors of TNF activity include, e.g., peptides, proteins, synthesized molecules, for example, synthetic organic molecules, naturally-occurring molecule, for example, naturally occurring organic molecules, nucleic acid molecules, and components thereof. Preferred examples of agents that inhibit TNF biological activity include infliximab (Remicade; Centocor, Inc., Malvern, Pennsylvania), etanercept (Immunex; Seattle, Washington), adalimumab (D2E7; Abbot Laboratories, Abbot Park Illinois), CDP870 (Pharrnacia Corporation;
Bridgewater, New Jersey) CDP571 (Celltech Group plc, United Kingdom), Lenercept (Roche, Switzerland), and Thalidomide.
In certain embodiments, the present invention is directed to a composition comprising the antibody or antigen-binding fragments described herein, in a pharmaceutically-acceptable excipient. As described above, the excipient included *Trademark with the antibody or antigen-binding fragment in these compositions is selected based on the expected route of administration of the composition. Suitable pharmaceutically-acceptable excipients include those described above and known to those of skill in the art.
In one embodiment, the invention is directed to aptamers of EIMGB (e.g., aptamers of HIVIGB1). As is known in the art, aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., an IIMGB protein, an HMGB box (e.g., an HMGB A box, an BlvIGB B
box), an HMGB polypeptide and/or an HMGB epitope as described herein). A
particular aptamer may be described by a linear nucleotide sequence and is typically about 15-60 nucleotides in length. The chain of nucleotides in an aptamer form intramolecular interactions that fold the molecule into a complex three-dimensional shape, and this three-dimensional shape allows the aptamer to bind tightly to the surface of its target molecule. Given the extraordinary diversity of molecular shapes that exist within the universe of all possible nucleotide sequences, aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules. In addition to high specificity, aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins).
Aptamers are chemically stable and can be boiled or frozen without loss of activity.
Because they are synthetic molecules, they are amenable to a variety of modifications, which can optimize their function for particular applications.
For example, aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood for use in in vivo applications. In addition, aptamers can be modified to alter their biodistribution or plasma residence time.
Selection of apatrners that can bind HIVIGB or a fragment thereof (e.g., HMGB1 or a fragment thereof) can be achieved through methods known in the art.
For example, aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C., and Gold, L., Science 249:505-510 (1990)). In the SELEX method, a large library of nucleic acid molecules (e.g., 1015 different molecules) is produced and/or screened with the target molecule (e.g., an HMGB protein, an HMGB box (e.g., an FIMGB A box, an HMGB
B box), an HIVIGB polypeptide and/or an FIMGB epitope as described herein).
The target molecule is allowed to incubate with the library of nucleotide sequences for a period of time. Several methods, known in the art, can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture, which can be discarded. The aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule. The enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this iterative selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule.
Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared. Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure, thereby producing aptamers truncated to their core binding domain. See Jayasena,-S.D. Clin. Chem.- 45:1628-1650 (1999) for review of , aptamer technology.
In particular embodiments, the aptamers of the invention have the binding specificity and/or functional activity described herein for the antibodies of the invention. Thus, for example, in certain embodiments, the present invention is drawn to aptamers that have the same or similar binding specificity as described herein for the antibodies of the invention (e.g., binding specificity for a vertebrate HMGB polypeptide, fragment of a vertebrate ITIVIGB polypeptide (e.g., FINIGB A
box, I-IMGB B box), epitopic region of a vertebrate 1111VIGB polypeptide (e.g., epitopic region of HIvIGB1 that is bound by one or more of the antibodies of the invention)). In particular embodiments, the aptamers of the invention can bind to an HMGB polypeptide or fragment thereof and inhibit one or more functions of the HIV1GB polypeptide. As described herein, function of BMGB polypeptides include, e.g., increasing inflammation, increasing release of a proinflammatory cytokine from a cell, binding to RAGE, binding to TLR2, chemoattraction In a particular embodiment, the aptamer binds HMGB1 (e.g., human HMGB1 (e.g., as depicted in SEQ ID NO:1 or SEQ ID NO:74)) or a fragment thereof (e.g., A box (e.g., residues 9-85 of SEQ ID NO:2, SEQ ID NO:75), B box (e.g., SEQ ID NO:3, SEQ ED
NO:76), HMGB1 antibody binding epitope as described herein) and inhibits one or more functions of the EMGB polypeptide (e.g., inhibits release of a proinflam.matory cytokine from a vertebrate cell treated with HIVIGB).
Methods of Diagnosis and/or Prognosis In another embodiment, the invention further provides diagnostic and/or prognostic methods for detecting a vertebrate high mobility group box (1-11VIGB) polypeptide in a sample. In one embodiment of the method, a sample is contacted with an antibody or antigen-binding fragment of the present invention, under conditions suitable for binding of the antibody or fragment to an EIMGB
polypeptide present in the sample. The method further comprises detecting antibody-HMGB
complexes or antigen-binding fragment-HMGB complexes, wherein detection of antibody-HMGB complexes or antigen-binding fragment-HMGB complexes is indicative of the presence of an HIVIGB polypeptide in the sample. Suitable antibodies or antigen-binding fragments for use in these methods include those described herein, e.g., 6E6 HMGB1 mAb, 6H9 HMGB1 mAb, 2G7 EIMGB1 mAb, 10D4 HMGB1 mAb, 2G7 HMGB1 mAb, an antigen-binding fragment of any of the foregoing.
In another embodiment, the antibody or antigen-binding fragment comprises a detectable label. Labels suitable for use in detection of a complex between an 1-11/1GB polypeptide (e.g., a mammalian HIMGB polypeptide) and an antibody or antigen-binding fragment include, for example, a radioisotope, an epitope label (tag), an affinity label (e.g., biotin, avidin), a spin label, an enzyme, a fluorescent group or a cherniluminescent group.
As described, the antibodies and antigen-binding fragments described herein can be used to detect or measure expression of an IIMGB polypeptide. For example, antibodies of the present invention can be used to detect or measure an H1VIGB
polypeptide in a biological sample (e.g., cells, tissues or body fluids from an individual such as blood, serum, leukocytes (e.g., activated T lymphocytes), bronchoalveolar lavage fluid, saliva, bowel fluid, synovial fluid, biopsy specimens).
In one embodiment, the sample is blood or serum. For example, a sample (e.g., tissue and/or fluid) can be obtained from an individual and a suitable assay can be used to assess the presence or amount of an HMGB polypeptide. Suitable assays include immunological and immunochemical methods such as flow cytometry (e.g., FACS analysis) and immunosorbent assays, including enzyme-linked immunosorbent assays (ELISA), radioimmunoassay (RIA), chemiluminescence assays, immunoblot (e.g., western blot), immunocytochemistry and immunohistology. Generally, a sample and an antibody or antigen-binding fragment of the present invention are combined under conditions suitable for the formation of a complex between an HMGB polypeptide and the antibody or antigen-binding fragment thereof, and the formation of said complex is assessed (directly or indirectly). In one embodiment, diagnosis and/or prognosis is done using ELISA
and/or western blot analysis.
As in known in the art, the presence of an increased level of an HMGB
polypeptide (e.g., HI4GB1) in a sample (e.g., a tissue sample) obtained from an individual can be a diagnostic and/or prognostic indicator for monitoring the severity and predicting the likely clinical course of sepsis for a subject exhibiting symptoms associated with conditions characterized by activation of the inflammatory cascade (see U.S. Patent No. 6,303,321).
Thus, in one embodiment, the antibodies and antigen-binding fragments of the invention can be used in diagnostic and prognostic methods for monitoring the severity and/or predicting the likely clinical course of an inflammatory condition associated with HMGB expression (e.g., the conditions described herein). In certain embodiments, the diagnostic and/or prognostic methods comprise measuring the concentration of .1--Th4GB in a sample, preferably a serum sample, and comparing that concentration to a standard for HMGB
representative of a notinal concentration range of HMGB in a like sample, whereby higher levels of HMGB are indicative of poor prognosis or the likelihood of toxic reactions. The diagnostic method may also be applied to other tissue or fluid compartments such as cerebrospinal fluid or urine.
In another embodiment, the invention is a test kit for use in detecting the presence of a vertebrate high mobility group box (FIMGB) polypeptide or portion thereof in a sample. Such test kits can comprise, e.g., an antibody or antigen-binding fragment of the invention and one or more ancillary reagents suitable for detecting the presence of a complex between the antibody or antigen-binding fragment and an FILVIGB polypeptide or portion thereof. The antibody and antigen-binding fragments of the present invention can be provided in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes. The antibodies or antigen-binding fragments thereof, which can be labeled or unlabeled, can be included in the kits with adjunct ingredients (e.g., buffers, such as Tris (Tris(hydroxymethyl)aminomethane), phosphate and carbonate, stabilizers, exciPients, biocides and/or inert proteins, e.g., bovine serum albumin). For example, the antibodies or antigen-binding fragments can be provided as a lyophilized mixture with the adjunct ingredients, or the adjunct ingredients can be separately provided for combination by the user. Generally these adjunct materials will be present in less than about 5% by weight based on the amount of active antibody, and usually will be present in a total amount of at least about 0.001% by weight based on antibody concentration. Where a second antibody or antigen-binding fragment capable of binding to the anti-HNIGB antibody or antigen-binding fragment is employed, such antibody or fragment can be provided in the kit, for instance in a separate vial or container. The second antibody or antigen-binding fragment, if present, is typically labeled, and can be formulated in an analogous manner with the antibody formulations described above. The antibodies, antigen-binding fragments arid/or ancillary reagent of the kit can be packaged separately or together within suitable containment means (e.g., bottle, box, envelope, tube). When the kit comprises a plurality of individually packaged components, the individual packages can be contained within a single larger containment means (e.g., bottle, box, envelope, tube).
Methods of Screening In another embodiment, the invention is a method of detecting or identifying an agent that binds to an HIV1GB polypeptide (e.g., a mammalian HMGB
polypeptide (e.g., an EIMGB1 polypeptide)). In one embodiment, the method of detecting or identifying an agent that binds to an HMGB polypeptide is a competitive binding assay in which the ability of a test agent to inhibit the binding of an antibody or antigen-binding fragment of the invention is assessed. For example, the antibody or antigen-binding fragment can be labeled with a suitable label as described herein, and the amount of labeled antibody or antigen-binding fragment required to saturate the BMGB polypeptide present in the assay can be determined.
For example, a saturating amount of labeled antibody or antigen-binding fragment and various amounts of a test agent can be contacted with an ELIVIGB
polypeptide under conditions suitable for binding, and complex famiation determined. In this type of assay, a decrease in the amount of complex formed between the labeled antibody or antigen-binding fragment and HMGB polypeptide indicates that the test agent binds to the HIVIGB polypeptide. In another embodiment, the IIMGB
polypeptide can be labeled. Suitable labels for labeling antibodies, antigen-binding fragments and/or IIMGB polypeptides include those described above.
A variety of agents, such as Proteins (e.g., antibodies), peptides, peptidomimetics, small organic molecules, nucleic acids and the like, can be tested for binding to an HIVIGB polypeptide (e.g., a mammalian HIVIGB polypeptide (e.g., an HMGB1 polypeptide)). According to the method of the present invention, agents can be individually screened or one or more agents can be tested simultaneously.
Where a mixture of compounds is tested, the compounds selected by the processes described can be separated (as appropriate) and identified using suitable methods (e.g., sequencing, chromatography). The presence of one or more compounds (e.g., a ligand, inhibitor, promoter) in a test sample can also be determined according to these methods.
Agent that bind to an HMGB polypeptide and that are useful in the therapeutic methods described herein can be identified, for example, by screening libraries or collections of molecules, such as, the Chemical Repository of the -5?-National Cancer Institute, in assays described herein or using other suitable methods.
Libraries, such as combinatorial libraries, of compounds (e.g., organic compounds, recombinant or synthetic peptides, "peptoids", nucleic acids) produced by combinatorial chemical synthesis or other methods can be tested (see e.g., Zuckerman, RN. et al., J. Med. Chem., 37: 2678-2685 (1994) and references cited therein; see also, Ohlmeyer, M.H.J. et al., PMC. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S.H. et al., PrOC. Natl. Acad. Sc!. USA 90:6909-6913 (1993), relating to tagged compounds; Rutter, W.J. et al.U.S. Patent No.
5,010,175;
Huebner, V.D. et al., U.S. Patent No. 5,182,366; and Geysen, H.M., U.S. Patent No.
4,833,092). Where compounds selected from a library carry unique tags, identification of individual compounds by chromatographic methods is possible.
The present invention will now be illustrated by the following Examples, which is not intended to be limiting in any way.
Example 1: Materials and Methods Generation of monoclonal antibodies to HiVIGB1 BALB/c mice were intraperitoneally immunized with 20 lig of recombinant CBP-Rat HMGB1 (CBP linked to amino acids 1-215 of rat H1VIGB1; nucleotide sequence of CBP-Rat HMGB1 is depicted as SEQ ID NO:4 and the amino acid sequence is depicted as SEQ ID NO:5 (see FIGS. 3A and 3B)) mixed with Freund's adjuvant at two-week intervals for 6 weeks. A final boost of 10 ug of the CBP-Rat 1-11VIGB1 in PBS was given intravenously after 8 weeks. Four days after the final boost, spleens from the mice were isolated and used for fusion. Fusion was carried out using standard hybridoma technique. The spleen was gently pushed through a cell strainer to obtain a single cell suspension. After extensive washing, spleen cells were mixed with SP2/0 myeloma cells. Polyethylene glycol (PEG) was added slowly, followed by media over a period of five minutes. The cells were washed and resuspended in DMEM containing 20% FCS and HAT, transferred to 96 well plates = and incubated at 37 C with 10% CO2 for 10-14 days.
In other experiments, a human HIvIGB1 B box polypeptide (SEQ ID NO:3;
FIG. 2B) was used as an immuogen. Five female BALB/c mice were intraperitoneally immunized with 10 lig/injection of HMGB1 B box mixed with Freund's adjuvant at three-week intervals. A bleed was obtained from the mice week after each boost. Three weeks after the third boost, a final intravenous injection (10 ug/mouse) of the rat HIvIGB1 B box was given. 72 hours after the final boost, hybridoma fusions were carried out as described above. Hybridomas were cultured in DMEM with 20% FBS, HAT, CondiMed and 1% pen/strep. Positive clones were identified by taking optical readings and identifying those with readings five times that of background.
Antibodies to the CBP-Rat HMGB1 and human HMGB1 B box were screened by limiting dilution and ELISA. ELISA plates were coated with recombinant HMGB1 at 3 pg/ml overnight and blocked with phosphate buffered saline (PBS) supplemented with 1% bovine serum albumin (BSA). Supernatants from the hybridornas were added to the ELISA plates and incubated at room temperature for 30 minutes. The plates were then washed and anti-mouse Ig conjugated with horseradish peroxidase was added. After 30 minutes of incubation at room temperature, the plates were washed and developed. Cells from positive cells were transferred to 24-well plates and cloned by limiting dilution.
HMGB.1 stimulated TNF release The mouse macrophage cell line RAW 264.7 (available from the American Type Culture Collection (ATCC), Manassas, VA) was incubated with various concentrations of111VIGB1 for 4 hours at 37 C in serum-free Opti-IVIEM
(Invitrogen, Carlsbad, CA). The supernatants were harvested and TNF level was measured using an ELISA kit (R&D Systems, Minneapolis, MN). The assay was also performed using heparinized whole blood. In this case, HMGB1 was diluted in Opti-MEM,*
added to 100 p..1 of whole blood to give a final volume of 200 pi, and placed in a U-*Trade-mark bottom 96-well plate. The plates were then incubated for 4 hours at 37 C and plasma was harvested for ELISA analysis. =
To screen for blocking mAbs to HIVIGB1, purified mAbs were diluted in Opti-MEM and mixed with rat HA4GB1 at room temperature. After five minutes, the mixture was transferred into tissue culture wells containing RAW 264.7 cells.
The plates were then incubated for 4 hours at 37 C and supernatants were harvested for ELISA analysis.
SDS-Polyactylanzicle Gel Electrophoresis, Western Blot Analysis and Selectivity of HMGB1 Monoclonal Antibodies For detection of HMGB1 with the HMGB1 mAbs, samples were mixed with 4X NuPAGE LDS Sample Buffer, 10X NuPAGE Sample Reducing Agent (Invitrogen, Carlsbad, CA). The samples were heated in boiling water for 5 minutes, immediately chilled on ice and loaded on an SDS-polyacrylamide gel. Western blot analysis was performed using standard techniques.
For the experiments determining selectivity of the HIVIGB1 monoclonal antibodies (i.e., selectivity for EIMGB1 and/or BMGB2), western blot analysis was performed on samples containing non-recombinant (i.e., natural) HMGB1 from Chinese Hamster Ovary (CHO) cells (FIG. 11; labeled as CHO HMGB1; SEQ ID
N0:36) or samples containing non-recombinant (i.e., natural) HMGB2 from Chinese Hamster Ovary (CHO) cells and some detectable recombinant human HIVIGB1-His6 (FIG. 11; labeled as CHO HMGB2, rec-HMGB1-His6). FIGS. 18A and 18B depict the nucleotide and encoded amino acid sequences of the human recombinant IIMGB1 polypeptide containing a 5' 6 HIS tag (rec-FEMGB1-His6; SEQ ID NOs:39 and 40).
For the samples containing CHO HMGB1, samples contained ¨2.5-5 ng/ 1 of non-recombinant (i.e., natural) FIMGB1 from Chinese Hamster Ovary (CHO) cells. 20 I of the sample (i.e., ¨50-100 ng of HMGB I) was loaded on a gel and subjected to SDS-PAGE. To isolate CHO HMGB1 polypeptide, CHO cells were lysed and subsequently cleared by centrifugation. Anion exchange chromatography and heparin-affinity chromatography were then performed and fractions containing peak HMGB1 imrnunoreactivity but no detectable HMGB2 immunoreactivity were = pooled and used as the source of CRC) B1IvIGB1.
For the samples containing non-recombinant (i.e., natural) HMGB2 from Chinese Hamster Ovary (CHO) cells and some detectable recombinant FIMGB1-His6 (FIG. 11; labeled as CHO HMGB2, rec-IIMGB1-His6), CHO cells transfected with a recombinant HMGB1-His6-expressing plasmid were utilized. To isolate CHO
HMGB2 polypeptide, CHO cells were lysed and subsequently cleared by centrifugation. Anion exchange chromatography and heparin-affinity chromatography were then performed and fractions containing peak FIMGB2 immunoreactivity, but no detectable natural HMGB1 immunoreactivity, were pooled and used as the source of CHO 1-EVIGB2. In some cases, the pooled CHO HIVIGB2 fractions contained detectable amounts of recombinant HMGB-1-His6 polypeptide, however, this recombinant FEVIGB-1-His6 polypeptide was easily distinguished from HMGB2 based on its decreased mobility (and apparent higher molecular weight) when subjected to SDS-PAGE. Using the gel systems that generated the Western blots depicted in FIG. 11, non-recombinant CHO HMGB2 has an apparent molecular weight of-27,000, non-recombinant CHO HMGB1 has an apparent molecular weight of-29,000 and recombinant FIMGB-1-His6 has an apparent molecular weight of ¨31,000. For the CHO HMGB2, rec-TIMGB1-His6 samples, 20 ill of the sample (i.e., ¨10-20 ng of HMGB2) was loaded and subjected to SDS-PAGE.
For the western blots depicted in FIG. 11, either an anti-His Tag antibody (Santa Cruz, CA; 2 !_tojm1), an anti-HMGB2 antibody (Pharmingen, San Diego, CA;
2 jag/m1), an anti-H_MGB1 /2 mAb (MBL International, Watertown, MA; 2 or particular anti-HMGB1 monoclonal antibodies (e.g., 2E11 HMGB1 mAb (CT3-2E11), 1G3 EIMGB1 mAb (CT3-1G3), 6H9 HMGB1 mAb (CT3-6H9), 2G7 HMGB1 mAb (CT3-2G7), 2G5 HIVIGB1 mAb (CT3-2G5) and 6E6 HIVIGB 1 mAb (CT3-6E6); 2 vginil for each) were used.
Sequencing of Monoclonal Antibodies Total RNA was isolated from hybridoma cells using RNeasy MiniKit (Qiagen, Valencia, CA) as described in the kit protocol. The first strand of cDNA
synthesis was performed using ProtoScript First Strand cDNA Synthesis kit (New England Biolabs, Catalog # E6500S) as designed in the kit protocol. 5 pl of cDNA
was added to a PCR reaction (as described in the protocol for Mouse Ig-Primer Set, Catalog # 69831-3, Novagen, Madison, WI) containing 25 pmoles of the appropriate 5' primers (as described in the Novagen Ig-primer set protocol as MuIgGVH5'-A, MuIgGV115'-B, MulgGVH5'-C, MuIgGVH5'-D, MuIgGV15'-E, MuIgGVH5'-F for heavy chain and MuIgGV5'-A, MuIgGVE5'-B, MuIgGVL5'-C, MulgGVL5'-D, MuIgGVL5'-E, MuIgGVL5'-F and MuIgGVL5'-G for light chain) and 3' primers (as described in the Novagen Ig-primer set protocol as MuIgGVH3'-2 for heavy chain and MuIgGVL3'-2 for light chain). The PCR reaction conditions were 1 minute at 94 C, 1 minute at 50 C and 2 minutes at 72 C for 35 cycles, followed by an =
extension at 72 C for 6 minutes. PCR products were cloned into vector TOPO
(Invitrogen, San Diego, CA). DNA sequence analysis was performed by Genaissance Pharmaceuticals (New Haven, CT).
HMGB1 Peptide Binding Experiments Biotinylated peptides bound to React-Bind Streptavidin-Coated Plates (Pierce, Rockford, EL, Catalog # 15501) and non-biotinylated peptides bound to . poly-D-lysine coated ELISA plates were used in anti-peptide ELISAs.
Biotinylated peptides corresponding to particular 18 amino acid regions of human HMGB I, as well as a longer peptide corresponding to amino acid residues 9-85 of human HMGB1, were prepared and analyzed for binding to particular anti-HMGB I
monoclonal antibodies by ELISA. These peptides, and their respective sequences, are depicted in FIG. 13A. Poly-D-lysine coated plates were prepared by adding 100 ill/well of 0.1 mg/mI solution of poly-D-lysine in water. Plates were then incubated at room temperature for 5 minutes and were rinsed with water to remove the solution.
*Trade-mark Briefly, using the molecular weight of the respective peptide, 1 m_M peptide solutions were prepared in pyrogen-free water and diluted in IX phosphate buffered saline. Plate wells were washed three times with 200 ul of PBS and 100 gl of the =
various peptide solutions were added to designated wells. The plates were then covered and incubated for 60 minutes at room temperature. The wells were then washed three times with PBS, 0.05% polyoxyethylenesorbitan (referred to herein as Tween 2OTM) using a volume greater than 100 1. 200 ul of blocking buffer (5%
nonfat dry milk in PBS, 0.05% Tween 2OTM) was added to each of the wells. The plates were covered and incubated for 60 minutes at room temperature. The wells were then washed three times with PBS, 0.05% Tween 2OTM using a volume greater than 100 fa 100 j.d of the primary antibody (e.g., 2E11 HMGB1 mAb, 6E6 HMGB1 mAb, 6119 HMGB1 mAb, 2G7 HMGB1 mAb; 2 jig/m1 in blocking buffer) was added to the designated wells and the plates were covered and incubated for 30 minutes at room temperature. The wells were then washed three times with PBS, 0.05% Tween 20Tm using a volume greater than 100 pl.
100 p.1 of the goat anti-mouse horseradish peroxidase (IIRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, Catalog No. 115-035-071; used at a 1:2000 dilution) was added to each of the wells.
The plates were covered, incubated for 30 minutes at room temperature and subsequently washed three times with times with PBS, 0.05% Tween 2OTM using a volume greater than 100 p.1. The plates were developed by adding 50 ul of 1X
(Sigma, St. Louis, MO) to each well, incubating for 10 minutes at room temperature and reading the absorbance at 655 run using Microplate/Manager software and a BioRad Model 680 Plate Reader. Average background signal was subtracted from each of the sample signals.
Cecal Ligation and Puncture Cecal ligation and puncture (CLP) was performed as described previously (Fink and Heard, Surg. Res. 49:186-196 (1990); Wichmann et al., Crit. Care Med.
26:2078-2086 (1998); and Remick et al., Shock 4:89-95 (1995)). Briefly, BALB/c mice were anesthetized with 75 mg/kg ketamine (Fort Dodge, Fort Dodge, Iowa) and 20 mg/kg of xylazine (Bohringer Ingelheim, St. Joseph, MO) intramuscularly. A
midline incision was perfoimed, and the cecum was isolated. A 6-0 prolene suture ligature was placed at a level 5.0 mm from the cecal tip away from the ileocecal valve.
The ligated cecal stump was then punctured once with a 22-gauge needle, without direct extrusion of stool. The cecum was then placed back into its nomial intra-abdominal position. The abdomen was then closed with a running suture of 0 prolene in two layers, peritoneum and fascia separately to prevent leakage of fluid.
All animals were resuscitated with a normal saline solution administered sub-cutaneously at 20 ml/kg of body weight. Each mouse received a subcutaneous injection of imipenem (0.5 mg/mouse) (Primaxin, Merck & Co., Inc., West Point, PA) 30 minutes after the surgery. Animals were then allowed to recuperate.
Mortality was recorded for up to 1 week after the procedure; survivors were followed for 2 weeks to ensure no late mortalities had occurred.
Starting the day after the CLP procedure, 100 pg of particular anti-HIVIGBI
monoclonal antibodies (i.e., 6E6 HMGB1 mAb (mAB (6E6)); 2E11 HMGB1 triAb (mAB (2E11)); 902 II1vIGB1 mAb (mAB (9G2)) and a control IgG antibody were intraperitoneally administered to the mice once or twice a day for a total of treatments. For the data presented in FIG. 16, various doses (11..temouse, 10 pg/mouse or 100 tg/mouse) of 6E6 FIMGB1 mAb or a control IgG antibody were intraperitoneally administered.
Two ELISA methods were perfonned using various HIVIGB1 monoclonal antibodies.
HMGB1 ELISA with Monoclonal (Capture) + Polyclonal (Detectoi) Antibody Pairs In the first method, ELISA plates were coated with a number of purified anti-HMGB1 mAbs (e.g., 2E11 IIMGB1 mAb, 2G5 If_MGB1 mAb, 207 RMGB1 mAb, 6E6 HMGB1 mAb), and incubated overnight at 4 C. The plates were then blocked *Trade-mark with PBS, 1%BSA for one hour at 37 C. After washing, recombinant rat HMGB1 was added at the indicated concentrations, and the plates were incubated at room temperature for 1 hour. The plates were then washed and incubated with rabbit polyclonal antibodies against HMGB1 at 2 jig/m1 (see U.S. Patent Nos.
6,303,321, 6,448,223 and 6,468,533). After 1 hour at room temperature, the plates were washed and incubated for 30 minutes with goat anti-rabbit Ig-I-1PR (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted at 1:1000 in PBS. After washing, the plates were developed with TMB (Invitrogen, San Diego, CA) and absorbance at 655 nm was measured using a plate reader.
HMGB1 ELISA with Monoclonal Antibody Pairs (Detection with 6E6 HMGB1 tnAb) ELISA plates were coated and blocked, and recombinant rat HMGB1 was subsequently added as described above. After washing away the unbound HMGB1 polypeptide, biotinylated 6E6 HMGB1 mAb was added at 2 pg/m1 and incubated for 1 hour at room temperature. Streptavidin-HRP was used to detect bound 6E6 HtVIGB1 inAb and the plates were developed with TMB as described above.
Example 2: Identification and Characterization of Anti-FIMGB1 Monoclonal Antibodies A number of novel anti-FIMGB1 monoclonal antibodies have been isolated and purified from immunizations with either recombinant full-length rat HMGB1(SEQ ID NO:4; FIGS. 3A and 3B) or with a B-box polypeptide of human liMGB1 (SEQ ID NO:3; FIG. 2B). A table summarizing characteristics (clone name, immunogen, isotype, purified antibody binding domain, and results of in vivo CLP assays) of these antibodies is depicted in FIG. 7.
Example 3: Determination of Selectivity of Anti-HMGB1 Monoclonal Antibodies Experiments (e.g., ELISA, Western blot analysis) to determine the selectivity of the HMGB1 monoclonal antibodies revealed that particular anti-HMGB1 monoclonal antibodies are able to bind to the A box portion of HMGB1, the B
box portion of FLMGB1 and/or the whole HMGB1 protein. For example, as depicted in FIG.7, anti-HMGB1 monoclonal antibodies were identified that can bind to the A
box of HMGB1 (e.g., 6E6 HMGB1 mAb, 6H9 HIVIGB1 mAb, 10D4 ILMGB1 mAb, 6H9 HMGB1 mAb, 2G7 ELMGB1 mAb, 2G5 HMGB1 mAb, 41111 HMGB1 mAb, 7113 HMGB1 mAb, 9113 HMGB1 inAb). Other monoclonal antibodies were identified that bind to the B box of HMGB1 (e.g., 2E11 HMGB1 mAb, 3G8 HMGB1 mAb, 3-5A6 IIMGB1 mAb, 9G1 HMGB1 mAb, 4C9 IIMGB1 mAb, 1C3 HMGB1 mAb, 5C12 HMGB1 mAb, 3E10 HMGB1 mAb, 7G8 HMGB1 mAb, 4A10 HMGB1 mAb).
Example 4: Determination of Nucleotide and Amino Acid Sequences of Anti-HIVGB1 Monoclonal Antibodies For particular ILMGB1 monoclonal antibodies (6E6 ILMGB1 mAb, 2E11 HMGB1 mAb, 10D4 HLVIGB1 mAb, 2G7 HMGB1 mAb), nucleotide and encoded . amino acid sequences of VH domains and VI; domains, including CDRs, were also obtained (FIGS. 4A-4D, 5A-5D, 6A-6D and 19A-19D).
Example 5: Inhibition of TNF Release by Anti-BNIGB1 Monoclonal Antibodies The ability of particular HMGB1 monoclonal antibodies to inhibit TNF
release was assessed. The results of this study are shown in FIGS. 8 and 9, which are histograms depicting TNF released by RAW 264.7 cells administered only IEVIGB1, ILMGB1 plus particular LIMGB1 monoclonal antibodies, or a control IgG
antibody. FIG. 8 depicts the results of inhibition of 1-114GB1-mediated TNF
release for 6E6 EE14GB1 mAb, 10D4 HMGB1 InAb, 2E11 BEIVIGB1 mAb, 9G2 HIVIGB1 mAb, and a control IgG antibody. FIG. 9 depicts the results of inhibition of HMGB1-mediated TNF release for 3G8 H1\4GB1 mAb, 1A9 HMGB1 mAb, 9G2 HMGB1 mAb, 6E6 ITIVIGB1 mAb, 2E11 HMGB1 mAb, 10D4 HMGB1 mAb, 6H9 HIVIGB1 mAb, or a control IgG antibody. As depicted in FIGS. 8 and 9, particular HMGB1 monoclonal antibodies (e.g., 6E6 HIVIGB1 inAb, 10D4 FEVIGB1 t-nAb) inhibited TNF release, indicating that such antibodies could be used to modulate one or more HIVIGB functions (e.g., as described herein). For example, these blocking antibodies could be used to neutralize the biological activity of FIMGB1 (e.g., HMGB1-mediated activation of the cytokine cascade).
Example 6: Treatment of Septic Mice with Anti-H1VGB1 Monoclonal Antibodies Increases Survival of Mice Mice were subjected to cecal ligation and puncture (CLP), a well characterized model of sepsis caused by perforating a surgically-created cecal diverticulum, that leads to polymicrobial peritonitis and sepsis (Fink and Heard, supra; Wichmann et aL, supra; and Remick et al., supra), and administered - particular anti-H1VIGB1 monoclonal antibodies (6E6 IEVIGB1 mAb, 2E11 FIAIGB1 mAb or 9G2 HMGB1) or a control IgG antibody (100 j_tg of antibody administered twice per day). Survival was monitored for 7 days. The results of this study are shown in FIG. 10, which is a graph of the survival of septic mice treated with either a control antibody or particular anti-IIMGB1 monoclonal antibodies. The results show that anti-HIVIGB1 monoclonal antibodies administered to the mice starting hours after the onset of cecal perforation rescued animals from death, as compared to administration of an IgG control antibody. For 6E6 HMGB1 mAb, the rescue was significant at day 7 (p<0.03 versus control, Fisher's exact test).
A dose response curve for survival of septic mice treated with 6E6 IIMGB1 mAb was also conducted. As depicted in FIG. 16, doses of 1, 10 and 100 ug of HMGB1 mAb were administered to mice. The results demonstrate that a dose of 10 jig of 6E6 IEVIGB1 resulted in the greatest rescue of the septic mice.
Example 7: Selectivity of Anti-HMGB1 Monoclonal Antibodies As described above, western blot analysis was performed using particular anti-IlIvIGB1 monoclonal antibodies. FIG. 11 depicts individual western blots of samples containing either CHO HIvIGB1 or CHO IEVIGB2 and possibly recombinant HMGB1-His6 (labeled as CHO H1VIGB2, rec-HMGB1-His6), which were probed with either an anti-His Tag antibody, an anti-HMGB2 antibody, an anti-HMGB1/2 monoclonal antibody, or particular anti-IEVIGB I monoclonal antibodies (e.g., HMGB1 mAb, 1G3 HIMGB1 mAb, 6H9 HIMGB1 mAb, 2G7 HA/1GB' mAb, 2G5 HMGB1 mAb and 6E6 H1VIGB1 mAb). The results of these experiments reveal that 2G7 HMGB1 mAb binds HMGB1 but does not detectably bind HMGB2, while 2E11 HMGB1 mAb, 1G3 HMGB1 mAb and 6H9 ELMGB1 mAb detect HMGB2 in addition to ELMGB1.
Example 8: HMGB1 Peptide Binding Experiments Biotinylated peptides corresponding to particular 18 amino acid regions of human HMGB I and a longer peptide corresponding to amino acid residues 9-85 of human HMGB1 were prepared and analyzed for binding to particular anti-HMGB1 monoclonal antibodies by ELISA. These peptides and their respective sequences are depicted in FIG. 13A.
The results of these peptide binding experiments are depicted in FIG_ 13B.
As depicted in FIG. 13B, 2E11 HMGB1 mAb bound to a peptide corresponding to amino acid residues 151-168 of human HMGB1 (i.e.', amino acid residues 151-168 SEQ ID NO:1). 6E6 IIMGB1 mAb and 6E19 bound to a peptide corresponding to amino acid residues 61-78 of human HMGB 1 (i.e., amino acid residues 61-78 SEQ
ED NO:1). 2G7 HIMGB1 mAb bound to a peptide corresponding to amino acid residues 46-63 of human HMGB1 (i.e., amino acid residues 46-63 of SEQ ID
NO:1).
In addition, 2G7 HMGB1 mAb and 6E6 IIMGB1 mAb also bound the longer peptide corresponding to amino acid residUes 9-85 of human HMGB1. These experiments demonstrate that particular anti-HMGB1 monoclonal antibodies recognize different epitopes within the HIVIGB1 polypeptide. For example, 6E6 HMGB1 mAb, which was shown to inhibit HMGB 1-mediated TNF release binds to an epitope contained within amino acids 61-78 of HIMGB 1. The discovery of a blocking epitope within this particular region of HIMGB1 could be used to screen for additional blocking agents (e.g., agents that inhibit an HMGB1 function (e.g., HMGB 1 -m e di at e d activation of the cytolcine cascade)).
Example 9: BIVIGB1 ELISA
As depicted in FIGS. 14 and 15, two different ELISA methods were used to examine theproperties of particular anti-HIVIGB1 monoclonal antibodies. In one method, HMGB1 monoclonal antibodies 2E11 IIMGB1 mAb, 2G5 HMGB1 mAb, 2G7 BMGB1 mAb, and 6E6 HMGB1 mAb, were used as capture antibodies and a polyclonal IIMGB1 antibody was used as the detector antibody. In the other ELISA
method, HMGB1 monoclonal antibodies 2E11 FIMGB1 rnAb, 2G5 HMGB1 mAb, 2G7 IIMGB1 mAb, and 6E6 IIMGB1 mAb, were used as capture antibodies and 6E6 FIIVIGB1 mAb was used as the detector antibody. The results from both of the ELISA methods demonstrate that the monoclonal HMGB1 antibodies can detect IIMGB1 and would be suitable for the diagnostic and/or prognostic methods described herein.
Example 10: Binding of 2G7 IIMGB1 mAb to HMGB1 is Inhibited By a Peptide Corresponding to Amino Acid Residues 46-63 of HMGB1 HIVIGB1 peptide binding experiments using 2G7 HMGB1 mAb were conducted. As described, biotinylated synthetic peptides corresponding to either amino acid residues 46-63 of human EINIGB1 or amino acid residues 61-78 of human HMGB1 were prepared and analyzed for binding to 2G7 HMGB1 inAb (2G7) by ELISA. Briefly, 2 m/m1 of 2G7 EIVIGB1 mAb was added to plate wells containing either the HMGB1 46-63 peptide or the HMGB1 61-78 peptide at each of the indicated concentrations (0, 0.33, 1,3, 9, 27, 81 and 243 iuM peptide) for one hour at 25 C to prepare antibody-peptide samples. ELISA plates were coated with 101.tg/m1 of recombinant rat ILMGB1, and incubated overnight at 4 C. The plates were then blocked with reconstituted milk for one hour at 37 C. Antibody-peptide samples were then added at the concentrations listed above and incubated for one hour at room temperature. The plates were then washed and incubated with Streptavidin-HRP. After washing, the plates were developed with TMB
(Invitrogen, San Diego, CA) and absorbance at 655 am was measured using a plate reader.
FIG. 20 depicts the results as percent of maximum signal (y-axis). As FIG.
20 demonstrates, the HMGB1 46-63 peptide inhibited the binding of 2G7 FEMGB1 mAb to bound LIMGB1 at all concentrations (depicted as a decrease in % of maximum signal). In contrast, the HMGB1 61-78 peptide did not inhibit the binding of 2G7 HMGB1 mAb to HMGB1. These experiments further confirm that 2G7 HMGB1 mAb binds to an epitope that is present in amino acids 46-63 of HMGB1.
Example 11: Binding of 2E11 HMGB1 mAb to HMGB1 is Inhibited By Higher Concentrations of a Peptide Corresponding to Amino Acid Residues 151-168 of 1114GB1 peptide binding experiments using 2E11 HMGB1 mAb and biotinylated synthetic peptides corresponding to either amino acid residues 46-63 of human HMGB1 (SEQ ID NO:23) or amino acid residues 151-168 of human FIMGB1 (SEQ ID NO:30) were conducted as described herein The results of these experiments are depicted in FIG. 21 (as percent of maximum signal (y-axis)).
As FIG. 21 demonstrates, the EINIGB1 151-168 peptide significantly inhibited the binding of 2E11 FIMGB1 mAb to bound HMGB1 at concentrations of 9 pM or greater. The HMGB1 151-168 peptide did not significantly inhibit the binding of 2E11 FIMGB1 mAb to PIMGB1 at concentrations of3 uM or below (FIG. 21). In addition, the IBMGB1 46-63 peptide did not inhibit the binding of 2E11 HMGB1 mAb to IT4GB1. These experiments confirm that 2E11 IIMGB1 mAb binds to an epitope that is present in amino acids 151-168 of HMGB1.
Example 12: 2G7 HMGB1 mAb Recognizes an Epitope That is Present in Amino Acids 53-63 of HMGB1 Various synthetic peptides were prepared. These synthetic peptides included a biotinylated peptide corresponding to amino acid residues 46-63 of human IEVIGB1 (SEQ ID NO:23; designated "huH1'dGB1-46-63-B" or "Human HMGB1-46-63-B"), a biotinylated peptide corresponding to amino acid residues 46-63 of human HMGB2 (SEQ ID NO:48; designated "huHlvIGB2-46-63-B" or "Human H1MGB2-46-63-B"), a non-biotinylated peptide corresponding to amino acid residues 53-70 of human HIMGB1 (SEQ NO:47; designated "huHMGB1-53-70" or "Human HMGB1-53-70"), a biotinylated peptide corresponding to amino acid residues 61-78 of human HMGB1 (SEQ ID NO:24; designated "huHMGB1 -61-78-B"), a non-biotinylated peptide corresponding to amino acid residues 40-57 of human HMGB1 (SEQ ID NO:46; designated "Human HMGB1-40-57") and a non-biotinylated peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 46-63 of human HMGB1 (SEQ ID NO:45; designated "Human FIMGB1-46-63-scr"). By ELISA, as described herein, the binding of 2G7 FIMGB1 mAb to these overlapping peptides was analyzed to more specifically ascertain the epitope within HMGB1 that binds to 2G7 HMGB1 mAb. These peptides and their respective sequences are depicted in FIG. 23.
The results of the peptide binding experiments are depicted in FIGS. 22 and 23. As shown in FIGS. 22 and 23, 2G7 HMGB I mAb bound to the HMGB1 46-63 peptide (i.e., amino acid residues 46-63 of SEQ ID NO:1 or SEQ ID NO:23) but did not bind to the corresponding amino acid region of human FIMGB2 (i.e., the HMGB2 46-63 peptide; amino acid residues 46-63 of SEQ ID NO:54 or SEQ ID
NO:48). In addition, 2G7 FilMGB1 mAb bound to the HIVIGB1 53-70 peptide but did not bind the HMGB1 40-57 peptide. 2G7 HMGB1 mAb also did not bind to the peptide consisting of a scrambled sequence of amino acid residues 46-63 of HMGB1. In addition to showing the binding of 2G7 HMGB1 mAb to the various synthetic peptides, FIG. 22 also depicts the binding of avidin to these synthetic peptides.
In Example 8, it was shown that 2G7 LIMGB1 mAb binds to an epitope contained within amino acid residues 46-63 of HNIGB1. Given that 2G7 ITIVIGB1 mAb binds the FEMGB1 46-63 peptide and the HIVIGB I 53-70 peptide, these experiments demonstrate that 2G7 HMGB1 mAb recognizes an epitope present in the amino acid region consisting of amino acid residues 53-63 of HMGB1. These experiments further demonstrate that 2G7 I1MGB1 mAb does not bind to the HMGB2 46-63 peptide, notwithstanding only a single amino acid difference between the HIVIGB1 46-63 peptide and the HMGB2 46-63 peptide. As such, the glycine residue at position 58 of HMGB1 (Gly-58), which is a corresponding serine residue in HMGB2, is an important amino acid residue in the HMGB I epitope recognized by 2G7 HMGB1 mAb (FIG. 23).
Example 13: 6E6 HMGB1 mAb Recognizes an Epitope That is Present in Amino Acids 67-78 of HMGB1 Various synthetic peptides were prepared. These synthetic peptides included a non-biotinylated peptide corresponding to amino acid residues 53-70 of human HMGB1 (SEQ ID NO:47; designated "Human IIMGB1-53-70-B"; described above), a non-biotinylated peptide corresponding to amino acid residues 67-84 of human (SEQ ID NO:50; designated "Human HMGB1-67-84"), a biotinylated peptide corresponding to amino acid residues 61-78 of human HMGB1 (SEQ ID
NO:24 designated "Human IIMGB1-61-78-B") and a non-biotinylated peptide consisting of a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 61-78 of human IIMGB1 (SEQ
ID NO:49; designated "Human HMGB1-61-78_scr"). By ELISA, as described herein, the binding of 6E6 HMGB1 mAb to these overlapping peptides was analyzed to more specifically ascertain the epitope within IIMGB1 that binds to 6E6 mAb. These peptides, their respective sequences and which of the peptides were bound by 6E6 HMGB1 mAb are depicted in FIG. 24. =
The results of these peptide binding experiments are depicted in FIG. 24. As shown in FIG. 24, 6E6 HMGB1 mAb bound to the ITIVIGB1 61-78 peptide (SEQ ID
NO:24). Further, 6E6 HMGB1 mAb bound to the HMGB1 67-84 peptide (SEQ ID
NO:50) but did not bind to the FIMGB1 53-70 peptide (SEQ ID NO:47) (FIG. 24).
6E6 HMGB I mAb also did not bind to the peptide consisting of a scrambled sequence of amino acid residues 61-78 of HMGB1 (SEQ ID NO:49) (FIG. 24). In Example 8, it was shown that 6E6 HMGE1 mAb binds to an epitope contained within amino acid residues 61-78 of HMGB1. Given that 6E6 HMGB1 mAb binds to the HMGB1 61-78 peptide and the HMGB1 67-84 peptide, these experiments demonstrate that 6E6 IIIVIGB1 mAb recognizes an epitope present in the amino acid region consisting of amino acid residues 67-78 of 1-EVIGB1.
Example 14: HMGB1 Peptide Binding Experiments with 2E11 HMGB1 mAb Various synthetic peptides were prepared. These synthetic peptides included a biotinylated peptide corresponding to amino acid residues 151-168 of human HMGB1 (SEQ ID NO:30; designated "Human BMGB1-151-168-B"), a non-biotinylated peptide corresponding to amino acid residues 143-160 of human HMGB1 (SEQ ID NO:52; designated "Human I-EVIGB1-143-160"), a non-biotinylated peptide corresponding to amino acid residues 157-174 of human HMGB1 (SEQ ID NO:53; designated "Human HMGB1-157-174"), and a non-biotinylated peptide corresponding to a scrambled amino acid sequence, wherein the amino acid residues that were scrambled were those of amino acid residues 151-of human HMGB1 (SEQ ID NO:51; designated "Human HMGB1-151-168_scr").
By ELISA, as described herein, the binding of 2E11 HMGB1 mAb to these overlapping peptides was analyzed to more specifically ascertain the epitope within HIVIGB1 that binds to 2E11 HMGB1 mAb. These peptides, their respective sequences and which of the peptides were bound by 2E11 HMGB1 mAb are depicted in FIG. 25.
As depicted in FIG. 25, 2E11 HMGB1 mAb bound to the 1-1MGB1 151-168 peptide (SEQ ID NO:30), but did not bind to either the HMGB1 143-160 peptide (SEQ ID NO:52) or the HMGB1 157-174 peptide (SEQ ID NO:53). As is known in the art, there are two types of epitopes or antigenic determinants: linear, sequential or continuous epitopes and non-linear, conformational or discontinuous epitopes.
The dimensions of a typical antibody epitope are often given a's 6 amino acid residues in size, but can be of variable size. These experiments suggest that the epitope recognized by 2E11 HMGB1 mAb is likely to comprise amino acid residues 156-161 of HMGB1. However, 2E11 ITMGB1 mAb may also recognize an epitope that includes flanking amino acids to this region, e.g., amino acid residues 155-161, 155-162, 156-162 and/or 156-163 of HMGB1.
A summary of the peptide binding results depicting the mapped epitopes of HMGB1 that are recognized by various HMGB1 mAbs (e.g., 2G7 HMGB1 mAb, 6E6 HMGB1 mAb, 2G5 HMGB1 mAb, 6H9 HMGB1 mAb, 2E11 HMGB1 mAb) is shown in FIG. 26.
Example 15: Mass Spectrometry of 6E6 HMGB1 mAb The mass of 6E6 HMGB1 inAb was determined by mass spectrometry. 6E6 HMGB1 mAb was sent to Novatia, LLC (Princeton, NJ) for LC/MS analysis.
Briefly, the antibody, either intact or after being treated with DTT (to separate heavy and light chains), was subjected to analysis using a PLRP-s 4000A reverse phase HPLC column (HPLC/ESI-MS system) and mass spectroscopy (Finnigan TSQ7000 mass spectrometer). Mass accuracy for proteins is generally 0.01%, and this accuracy was achieved in measuring the mass of the 6E6 light chain (e.g., 2 Da/23,9I7 Da x 100% 0.008%).
The results of this analysis are shown in FIGS. 27A-27D, which depicts a mass spectrum plot. The total mass of 6E6 HMGB1 mAb was 146.5 kDa (FIG.
27A; depicting mass spectrum for intact 6E6 HMGB1 mAb). The masses of the light and heavy chains of 6E6 IIMGB1 mAb were determined to be 23.9 kDa (FIGS.
27B and 27C) and 49.4 kDa (FIGS. 27B and 27D), respectively. The predicted masses for the light and heavy chains, as calculated using amino acid molecular weights, are 23.9 kDa and 47.9 kDa, respectively.
Example 16: 2G7 EIMGB1 mAb Increases Survival in Septic Mice After Administration of a Single Dose Mice were subjected to cecal ligation and puncture (CLP) as described above. In one experiment, twenty-four hours after surgery, mice were intraperitoneally administered either 0.004 mg/kg, 0.04 mg/kg or 0.4 mg/kg of ILMGB1 mAb (2G7) once per day. In a second experiment, twenty-four hours after .tirgery, mice were administered either 0.04 mg/kg of 2G7 HMGB1 mAb or 0.4 mg/kg of control IgG (IgG control) once per day. Survival was monitored for 14 days for both experiments. The results of the two experiments are combined and presented in FIG. 28. Administration of 0.4 mg/kg of 2G7 HMGB1 mAb resulted in approximately 85% survival at 14 days after CLP, as compared to approximately only 40% survival of mice administered with IgG control at 14 days after CLP
(FIG.
28). Further, as depicted in FIG. 28, administration of 0.04 mg/kg and 0.004 mg/kg of 2G7 HMGB1 mAb resulted in approximately 60% and 50% survival at 14 days after CLP, respectively.
FIG. 29 is a table comparing CLP survival percentages in mice administered various doses (either 4 mg/kg, 0.4 mg/kg, 0.04 mg/kg or 0.004 mg/kg) of 6E6 HIVIGB1 mAb, 2G7 HIVIGB1 mAb or control IgG. As depicted in FIG. 29, the mice were administered the antibodies 4 times a day intraperitoneally. The results demonstrate that administration of a dose of 0.4 mg/kg of either 6E6 HMGB1 mAb or 2G7 HMGB1 mAb resulted in greater than 80% of the septic mice surviving to days post-CLP, as compared to only approximately 40% of the septic mice surviving to 14 days post-CLP when administered control IgG.
Example 17: Inhibition of TNF Release by Anti-HMGB1 Monoclonal Antibodies As in Example 5, the ability of particular FLMGB1 monoclonal antibodies to inhibit TNF release was assessed. The results of this study are shown in FIGS.
and 33, which are histograms depicting TNF released by RAW 264.7 cells administered only H:MGB1 (dark bar), or FEVIGB1 plus particular I-EVIGBI
monoclonal antibodies. FIG. 32 depicts the results of inhibition ofILMGB1-mediated TNF release for 1A9 IIMGB1 mAb (1A9); 2E11 HMGB1 mAb (2E11);
2G5 ilaviGB 1 mAb (2G5); 2G7 HMGB1 mAb (2G7); 3G8 HMGB1 mAb (3G8);
4H11 HMGB1 mAb (4H11); 5A6 HMGB1 mAb (5A6); 6E6 IIMGB1 mAb (6E6);
9G2 FEVIGB1 mAb (9G2); 4C9 HMGB1 mAb (4C9); and 6119 HMGB1 mAb (6119).
FIG. 33 depicts the results of inhibition of HMGB1-mediated TNF release for HMGB1 mAb (7113); 9113 HMGB I mAb (9113); 10D4 FEVIGB1 mAb (10D4); 1C3 IIMGB1 mAb (1C3); 3E10 HMGB1 mAb (3E10); 4A10 HMGB1 mAb (4A10);
5C12 HMGB1 mAb (5C12); and 7G8 ILMGB1 mAb (7G8).
As depicted in FIGS. 32 and 33, and further to the results described in Example 5, particular HMGB1 monoclonal antibodies (e.g., 2E1 1 HMGB1 mAb, 41111 HMGB1 mAb, 6E6 FLMGB1 mAb, 6119 ILMGB1 mAb and 10D4 lEvIGB1 mAb) inhibited TNF release, indicating that such antibodies could be used to modulate one or more FEVIGB functions (e.g., as described herein). For example, these blocking antibodies could be used to neutralize the biological activity of HMGB1 (e.g., I-EVIGB1-mediated activation of the cytokine cascade).
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in foini and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Claims (33)
1. An antibody produced by murine hybridoma 2E11 HMGB1 mAb, deposited as ATCC
Accession Number PTA-5431, or an antigen-binding fragment thereof.
Accession Number PTA-5431, or an antigen-binding fragment thereof.
2. An antibody or antigen-binding fragment thereof, wherein the binding of said antibody or said antigen-binding fragment to a vertebrate high mobility group box (HMGB) polypeptide can be inhibited by 2E11 HMGB1 mAb.
3. An antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment has the epitopic specificity of 2E11 HMGB1 mAb.
4. A murine hybridoma 2E11 HMGB1 mAb, deposited as ATCC Accession Number PTA-5431.
5. An isolated cell that produces 2E11 HMGB1 mAb.
6. The isolated cell of claim 5 wherein said isolated cell is an immortalized B cell, a hybridoma, or a recombinant cell comprising one or more exogenous nucleic acid molecules that encode said antibody or antigen-binding fragment thereof.
7. An isolated cell that produces an antibody or antigen-binding fragment thereof, wherein the binding of said antibody or said antigen-binding fragment to a vertebrate high mobility group box (HMGB) polypeptide can be inhibited by 2E11 HMGB1 mAb.
8. The isolated cell of claim 7, wherein said isolated cell is an immortalized B cell, a hybridoma, or a recombinant cell comprising one or more exogenous nucleic acid molecules that encode said antibody or antigen-binding fragment thereof.
9. An isolated cell that produces an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment has the epitopic specificity of 2E11 HMGB1 mAb.
10. The isolated cell of claim 9, wherein said isolated cell is an immortalized B cell, a hybridoma, or a recombinant cell comprising one or more exogenous nucleic acid molecules that encode said antibody or antigen-binding fragment thereof.
11. An antibody or antigen-binding fragment that binds to a peptide consisting of amino acid residues 151 to 168 of SEQ ID NO:l.
12. The antibody or antigen-binding fragment of claim 11 wherein said antibody or antigen-binding fragment is a monoclonal antibody or an antigen-binding fragment thereof.
13. An antibody or antigen-binding fragment that binds to a peptide consisting of amino acid residues 156 to 161 of SEQ ID NO:1.
14. An antibody or antigen-binding fragment that binds to:
a peptide consisting of amino acid residues 155 to 161 of SEQ ID NO:1;
a peptide consisting of amino acid residues 155 to 162 of SEQ ID NO:1;
a peptide consisting of amino acid residues 156 to 162 of SEQ ID NO:1; or a peptide consisting of amino acid residues 156 to 163 of SEQ ID NO:1.
a peptide consisting of amino acid residues 155 to 161 of SEQ ID NO:1;
a peptide consisting of amino acid residues 155 to 162 of SEQ ID NO:1;
a peptide consisting of amino acid residues 156 to 162 of SEQ ID NO:1; or a peptide consisting of amino acid residues 156 to 163 of SEQ ID NO:1.
15. An antibody or antigen-binding fragment, wherein the binding of said antibody or said antigen-binding fragment to a peptide consisting of amino acid residues 151 to 168 of SEQ ID NO:1 can be inhibited by 2E11 HMGB1 mAb.
16. An antibody or antigen-binding fragment thereof wherein said antibody or fragment comprises the light chain CDRs (CDR1, CDR2 and CDR3) and the heavy chain CDRs (CDR1, CDR2 and CDR3) of 2E11 HMGB1 mAb.
17. The antibody or antigen-binding fragment of claim 16, wherein said antibody or antigen-binding fragment further comprises a human framework region.
18. The antibody or antigen-binding fragment of claim 1, wherein said antibody or antigen-binding fragment is an antigen-binding fragment, and said antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or a Fv fragment.
19. A composition comprising an antibody or antigen-binding fragment thereof and a pharmaceutically-acceptable excipient, wherein said antibody or antigen-binding fragment is 2E11 HMGB1 mAb, or an antigen-binding fragment of any of the foregoing.
20. An antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
a) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
a) 2E11 HMGB1 mAb;
a) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
21. The antibody or antigen-binding fragment of claim 20, wherein said antibody or antigen-binding fragment is a human antibody, a humanized antibody, a chimeric antibody, or an antigen-binding fragment of any of the foregoing.
22. A method of detecting and/or identifying an agent that binds to a vertebrate high mobility group box (HMGB) polypeptide comprising combining:
i) an antibody or antigen-binding fragment that binds HMGB;
ii) a test agent; and iii) a composition comprising a vertebrate HMGB polypeptide;
and detecting or measuring the formation of a complex between said antibody or antigen-binding fragment and said HMGB polypeptide, wherein a decrease in the formation of said complex relative to a suitable control indicates that said test agent binds to said HMGB polypeptide, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
i) an antibody or antigen-binding fragment that binds HMGB;
ii) a test agent; and iii) a composition comprising a vertebrate HMGB polypeptide;
and detecting or measuring the formation of a complex between said antibody or antigen-binding fragment and said HMGB polypeptide, wherein a decrease in the formation of said complex relative to a suitable control indicates that said test agent binds to said HMGB polypeptide, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
23. A use of an antibody or antigen-binding fragment thereof for treating a condition characterized by activation of an inflammatory cytokine cascade, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
24. The use of claim 23, wherein said condition is sepsis, allograft rejection, arthritis, asthma, lupus, adult respiratory distress syndrome, chronic obstructive pulmonary disease, psoriasis, pancreatitis, peritonitis, burns, ischemia, Behcet's disease, graft versus host disease, inflammatory bowel disease, multiple sclerosis, or cachexia.
25. A use of an antibody or antigen-binding fragment thereof for treating sepsis, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
26. A use of an antibody or antigen-binding fragment thereof for treating arthritis, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
27. A use of an antibody or antigen-binding fragment thereof for treating lupus, wherein said antibody or antigen-binding fragment is:
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
a) 2E11 HMGB1 mAb;
b) an antibody having the epitopic specificity of 2E11 HMGB1 mAb;
c) an antibody that can compete with 2E11 HMGB1 mAb for binding to a vertebrate high mobility group box (HMGB) polypeptide; or d) an antigen-binding fragment of a), b), or c).
28. A method of detecting a vertebrate high mobility group box (HMGB) polypeptide in a sample comprising:
a) contacting a sample with an antibody or antigen-binding fragment of claim 17, under conditions suitable for binding of said antibody or fragment to said HMGB polypeptide present in said sample; and b) detecting antibody-HMGB complexes or antigen-binding fragment-HMGB
complexes, wherein detection of said antibody-HMGB complexes or antigen-binding fragment-HMGB complexes is indicative of the presence of HMGB
polypeptide in said sample.
a) contacting a sample with an antibody or antigen-binding fragment of claim 17, under conditions suitable for binding of said antibody or fragment to said HMGB polypeptide present in said sample; and b) detecting antibody-HMGB complexes or antigen-binding fragment-HMGB
complexes, wherein detection of said antibody-HMGB complexes or antigen-binding fragment-HMGB complexes is indicative of the presence of HMGB
polypeptide in said sample.
29. The method of claim 28, wherein said antibody or antigen-binding fragment is 2E11 HMGB1 mAb, or an antigen-binding fragment of any of the foregoing.
30. The method of caim 28, wherein said antibody or antigen-binding fragment comprises a detectable label.
31. The method of claim 28, wherein said detecting of antibody-HMGB
complexes or antigen-binding fragment-HMGB complexes is by immunoassay.
complexes or antigen-binding fragment-HMGB complexes is by immunoassay.
32. The method of claim 31, wherein said immunoassay is an ELISA.
33. A test kit for use in detecting the presence of a vertebrate high mobility group box (HMGB) polypeptide or portion thereof in a sample comprising:
a) an antibody or antigen-binding fragment of claim 17; and b) one or more ancillary reagents suitable for detecting the presence of a complex between said antibody or antigen-binding fragment and said HMGB
polypeptide or portion thereof.
a) an antibody or antigen-binding fragment of claim 17; and b) one or more ancillary reagents suitable for detecting the presence of a complex between said antibody or antigen-binding fragment and said HMGB
polypeptide or portion thereof.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US50256803P | 2003-09-11 | 2003-09-11 | |
US60/502,568 | 2003-09-11 | ||
CA2538763A CA2538763C (en) | 2003-09-11 | 2004-09-10 | Monoclonal antibodies against hmgb1 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2538763A Division CA2538763C (en) | 2003-09-11 | 2004-09-10 | Monoclonal antibodies against hmgb1 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2882022A1 true CA2882022A1 (en) | 2005-03-24 |
Family
ID=34312406
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2538763A Active CA2538763C (en) | 2003-09-11 | 2004-09-10 | Monoclonal antibodies against hmgb1 |
CA2882022A Abandoned CA2882022A1 (en) | 2003-09-11 | 2004-09-10 | Monoclonal antibodies against hmgb1 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2538763A Active CA2538763C (en) | 2003-09-11 | 2004-09-10 | Monoclonal antibodies against hmgb1 |
Country Status (7)
Country | Link |
---|---|
US (4) | US7288250B2 (en) |
EP (1) | EP1668035A2 (en) |
JP (2) | JP4792392B2 (en) |
CN (1) | CN1878793A (en) |
AU (1) | AU2004272607B2 (en) |
CA (2) | CA2538763C (en) |
WO (1) | WO2005026209A2 (en) |
Families Citing this family (73)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6303321B1 (en) | 1999-02-11 | 2001-10-16 | North Shore-Long Island Jewish Research Institute | Methods for diagnosing sepsis |
US7304034B2 (en) | 2001-05-15 | 2007-12-04 | The Feinstein Institute For Medical Research | Use of HMGB fragments as anti-inflammatory agents |
US7696169B2 (en) | 2003-06-06 | 2010-04-13 | The Feinstein Institute For Medical Research | Inhibitors of the interaction between HMGB polypeptides and toll-like receptor 2 as anti-inflammatory agents |
US7288250B2 (en) | 2003-09-11 | 2007-10-30 | Critical Therapeutics, Inc. | Monoclonal antibodies against HMGB1 |
JP4824547B2 (en) * | 2004-02-20 | 2011-11-30 | インテレクト ニュウロサイエンシス,インク. | Monoclonal antibodies and uses thereof |
WO2006083301A2 (en) * | 2004-06-17 | 2006-08-10 | Medimmune, Inc. | Immunogenic compositions comprising hmgb1 polypeptides |
US7470521B2 (en) | 2004-07-20 | 2008-12-30 | Critical Therapeutics, Inc. | RAGE protein derivatives |
EP1812065A4 (en) | 2004-10-22 | 2009-09-02 | Medimmune Inc | High affinity antibodies against hmgb1 and methods of use thereof |
US8129130B2 (en) | 2004-10-22 | 2012-03-06 | The Feinstein Institute For Medical Research | High affinity antibodies against HMGB1 and methods of use thereof |
JP2008523083A (en) * | 2004-12-08 | 2008-07-03 | イムノメディクス, インコーポレイテッド | Methods and compositions for immunotherapy and detection of inflammatory and immune dysregulated diseases, infectious diseases, pathological angiogenesis and cancer |
US20100040607A1 (en) * | 2005-05-13 | 2010-02-18 | Tracey Kevin J | Combination Therapy with Inhibitors of HMGB and Caspase for the Treatment of Inflammatory Diseases |
US8354106B2 (en) | 2005-06-16 | 2013-01-15 | The Feinstein Institute For Medical Research | Antibodies against HMGB1 and fragments thereof |
US20100040608A1 (en) * | 2005-07-18 | 2010-02-18 | Marie Wahren-Herlenius | Use of HMGB1 antagonists for the treatment of inflammatory skin conditions |
US8044179B2 (en) | 2005-09-13 | 2011-10-25 | National Research Council Of Canada | Methods and compositions for modulating tumor cell activity |
WO2007031100A1 (en) * | 2005-09-14 | 2007-03-22 | Ostini, Marco | Active immunotherapy of life-threatening systemic inflammation |
US20100172909A1 (en) * | 2005-10-24 | 2010-07-08 | Masahiro Nishibori | Cerebral edema suppressant |
JP3876325B1 (en) * | 2005-10-24 | 2007-01-31 | 国立大学法人 岡山大学 | Cerebral infarction inhibitor |
AU2006330807A1 (en) * | 2005-11-28 | 2007-07-05 | Medimmune, Llc | Antagonists of HMBG1 and/or rage and methods of use thereof |
JP3882090B1 (en) * | 2006-05-19 | 2007-02-14 | 国立大学法人 岡山大学 | Cerebral vasospasm inhibitor |
CN1850863B (en) * | 2006-05-23 | 2012-07-04 | 中国科学院生物物理研究所 | Antibody to self-antigen and/or intergeneric high conservative antigen, and its preparing method |
JP4982739B2 (en) * | 2006-06-01 | 2012-07-25 | 国立大学法人 東京医科歯科大学 | Preventive and therapeutic agent for polyglutamine disease |
EP2105447A4 (en) * | 2006-12-20 | 2010-05-05 | Shino Test Corp | Avian-derived antibody capable of binding specifically to human hmgb1, immunological determination method for human hmgb1, and immunological determination reagent for human hmgb1 |
JP5285437B2 (en) * | 2007-02-15 | 2013-09-11 | 学校法人福岡大学 | Organ transplant rejection inhibitor containing anti-HMGB-1 antibody |
JP5225109B2 (en) | 2007-02-15 | 2013-07-03 | 国立大学法人 熊本大学 | A therapeutic agent comprising as an active ingredient an antibody that specifically binds to human HMGB-1 |
JP5241518B2 (en) | 2007-02-15 | 2013-07-17 | 国立大学法人九州大学 | Interstitial lung disease therapeutic agent comprising anti-HMGB-1 antibody |
BRPI0911513A2 (en) | 2008-04-30 | 2016-07-12 | Genomix Co Ltd | Methods and Agents for High-Efficiency In vivo Functional Bone Marrow Stem Cell Collection |
ITMI20080799A1 (en) * | 2008-04-30 | 2009-11-01 | Areta Internat S R L | MONOCLONAL ANTIBODIES AMTI HMGA1 PROCEDURE FOR THEIR PREPARATION AND THEIR USE FOR THE QUANTITATIVE DETERMINATION OF HMGA1 |
WO2010029164A1 (en) | 2008-09-11 | 2010-03-18 | Institut Pasteur | Monitoring and inhibiting human immunodeficiency virus infection by modulating hmgb1 dependent triggering of hiv-1 replication and persistence |
CN101798347B (en) * | 2009-03-03 | 2012-05-30 | 中国科学院生物物理研究所 | Monoclonal antibody for resisting high mobility group protein B1 |
CN101891817B (en) * | 2009-03-03 | 2012-11-07 | 中国科学院生物物理研究所 | Monoclonal antibody capable of preventing high mobility group box-1 (HMGB-1) and application thereof |
KR101754433B1 (en) * | 2009-05-08 | 2017-07-05 | 백시넥스 인코포레이티드 | Anti-cd100 antibodies and methods for using the same |
WO2011007876A1 (en) * | 2009-07-16 | 2011-01-20 | Necソフト株式会社 | Nucleic acid molecule capable of binding to hmgb1, and use thereof |
JP2011095014A (en) * | 2009-10-27 | 2011-05-12 | Canon Inc | Immunological measuring method and immunological measuring kit |
WO2011052668A1 (en) | 2009-10-28 | 2011-05-05 | 株式会社ジェノミックス | Tissue-regeneration promoter using recruitment of bone marrow mesenchymal stem cells and/or pluripotent stem cells in blood |
AU2010324506B2 (en) | 2009-11-24 | 2015-02-26 | Alethia Biotherapeutics Inc. | Anti-clusterin antibodies and antigen binding fragments and their use to reduce tumor volume |
KR20120123299A (en) | 2009-12-04 | 2012-11-08 | 제넨테크, 인크. | Multispecific antibodies, antibody analogs, compositions, and methods |
PT2365332E (en) | 2010-03-10 | 2013-08-02 | Pasteur Institut | Hmgb1 and anti-hmgb1 antibodies in hiv infected patients especially with neurological disorders |
WO2012015979A2 (en) | 2010-07-27 | 2012-02-02 | The Regents Of The University Of California | Hmgb1-derived peptides enhance immune response to antigens |
CN102375064A (en) * | 2010-08-26 | 2012-03-14 | 杭州华得森生物技术有限公司 | In-vitro diagnostic kit for detecting HMGA2 (High Mobility Group A) content with enzyme-linked immuno sorbent assay |
JP6154135B2 (en) * | 2010-12-03 | 2017-06-28 | 国立大学法人 岡山大学 | Traumatic neuropathy treatment |
US9045534B2 (en) * | 2011-04-05 | 2015-06-02 | Dia.Pro Diagnostic Bioprobes S.R.L. | HMGB1 specific monoclonal antibodies |
JP6069191B2 (en) * | 2011-04-26 | 2017-02-01 | 株式会社ジェノミックス | Peptides and their use to induce tissue regeneration |
US9561274B2 (en) * | 2011-06-07 | 2017-02-07 | University Of Hawaii | Treatment and prevention of cancer with HMGB1 antagonists |
US9244074B2 (en) | 2011-06-07 | 2016-01-26 | University Of Hawaii | Biomarker of asbestos exposure and mesothelioma |
EP2756002A4 (en) * | 2011-09-16 | 2015-04-08 | Univ Texas | Compositions and methods related to dna damage repair |
DK2766093T3 (en) | 2011-10-11 | 2018-06-06 | Vaccinex Inc | USE OF SEMAPHORIN-4D BINDING MOLECULES FOR MODULATING THE PERMEABILITY OF THE BLOOD-BRAIN BARRIER |
GB2504139B (en) * | 2012-07-20 | 2014-12-31 | Argen X Bv | Antibodies to highly conserved targets produced by the immunisation of Camelidae species |
WO2013091903A1 (en) * | 2011-12-22 | 2013-06-27 | Novo Nordisk A/S | Anti-crac channel antibodies |
AU2013224591A1 (en) | 2012-02-22 | 2014-07-24 | Alethia Biotherapeutics Inc. | Co-use of a clusterin inhibitor with an EGFR inhibitor to treat cancer |
US10213421B2 (en) * | 2012-04-04 | 2019-02-26 | Alkahest, Inc. | Pharmaceutical formulations comprising CCR3 antagonists |
CN102924577B (en) * | 2012-11-28 | 2014-03-26 | 扬州大学 | Chicken high mobility group protein B1 (chHMGB1) antigen polypeptide and anti-chHMGB1 monoclonal antibody |
JP6247646B2 (en) * | 2013-01-28 | 2017-12-13 | 株式会社イーベック | Humanized anti-HMGB1 antibody or antigen-binding fragment thereof |
US9840555B2 (en) | 2013-11-04 | 2017-12-12 | Li-Te Chin | Method for producing human monoclonal antibodies that binds to at least one part of HMGB1 |
ES2811274T3 (en) | 2014-04-18 | 2021-03-11 | Univ Leland Stanford Junior | Humanized and Chimeric Monoclonal Antibodies to CD99 |
US11191808B2 (en) * | 2014-07-04 | 2021-12-07 | Industry-University Cooperation Foundation Hanyang University | Pharmaceutical composition for suppressing cell transplant rejection |
CN108137702B (en) | 2015-08-28 | 2023-01-06 | 艾利妥 | anti-SIGLEC-7 antibodies and methods of use thereof |
KR20190015712A (en) | 2016-05-03 | 2019-02-14 | 더 보드 오브 트러스티스 오브 더 유니버시티 오브 아칸소 | Yeast vaccine vectors comprising immunostimulatory and antigenic polypeptides, and methods of using the same |
US20190284259A1 (en) * | 2016-07-22 | 2019-09-19 | Georgia State University Research Foundation | Monoclonal Antibodies to B Virus And Their Use For Identification Of B Virus Specific Reactive Peptides |
US10730937B2 (en) | 2016-08-09 | 2020-08-04 | National University Corporation Tokyo Medical And Dental University | Antibodies against HMB1, and composition comprising same for treating or preventing alzheimer's disease |
US11065286B2 (en) | 2016-09-21 | 2021-07-20 | Stephen H. Thorne | High mobility group box I mutant |
WO2018079393A1 (en) * | 2016-10-26 | 2018-05-03 | 扶桑薬品工業株式会社 | Disulfide-type hmgb1-specific antibody, method for measuring disulfide-type hmgb1 and kit for said measurement, and measurement method capable of quantitating all of hmgb1 molecules including reduced hmgb1, disulfide-type hmgb1 and thrombin-cleavable hmgb1 and kit for said measurement |
CN111542335A (en) | 2017-12-01 | 2020-08-14 | 斯特姆里姆有限公司 | Therapeutic agent for inflammatory bowel disease |
CN109957015A (en) * | 2019-04-01 | 2019-07-02 | 中国人民解放军海军军医大学国家肝癌科学中心 | A kind of preparation method and applications of HMGB1 blocking antibody |
CN113286618A (en) * | 2019-12-18 | 2021-08-20 | 国立大学法人冈山大学 | Antibody drug conjugates and uses for drug delivery of antibodies |
CA3173213A1 (en) * | 2020-03-31 | 2021-10-07 | Roland B. WALTER | Anti-cd33 antibodies and uses thereof |
WO2021222944A1 (en) * | 2020-04-30 | 2021-11-04 | Board Of Regents, The University Of Texas System | Anti-cd79b antibodies and chimeric antigen receptors and methods of use thereof |
CN115956089A (en) | 2020-05-04 | 2023-04-11 | 加利福尼亚大学董事会 | Inhibitory anti-ENPP 1 antibodies |
US20230295277A1 (en) * | 2020-07-13 | 2023-09-21 | University Of Southern California | Universal car-nk cell targeting various epitopes of hiv-1 gp160 |
CN113151186B (en) * | 2021-02-04 | 2022-02-18 | 上海交通大学 | Monoclonal antibody of anti-human CD271 and application |
TW202305125A (en) | 2021-04-30 | 2023-02-01 | 美商凱立凡爾免疫治療股份有限公司 | Oncolytic viruses for modified mhc expression |
CN113203857B (en) * | 2021-05-06 | 2021-12-31 | 上海奕检医学检验实验室有限公司 | Tumor diagnosis kit |
WO2023137291A1 (en) * | 2022-01-11 | 2023-07-20 | Board Of Regents, The University Of Texas System | Anti-cd94 antibody and chimeric antigen receptor and methods of use thereof |
KR20230139195A (en) * | 2022-03-25 | 2023-10-05 | 부산대학교 산학협력단 | Novel HMGB1-Derived Peptide and Uses Thereof |
Family Cites Families (88)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6468A (en) * | 1849-05-22 | ruteyen | ||
US4678772A (en) | 1983-02-28 | 1987-07-07 | Yissum Research Development Company Of The Hebrew University Of Jerusalem | Compositions containing glycyrrhizin |
JPS6032714A (en) | 1983-08-01 | 1985-02-19 | Teijin Ltd | Stabilized powdery pharmaceutical composition for application to nasal mucous membrane |
US5585344A (en) | 1984-03-19 | 1996-12-17 | The Rockefeller University | Liver-derived receptors for advanced glycosylation endproducts and uses thereof |
JPS62166897A (en) | 1986-01-20 | 1987-07-23 | Toyo Soda Mfg Co Ltd | Monoclonal antibody against intranuclear nonhistone protein |
JPS63135351A (en) | 1986-11-28 | 1988-06-07 | Sanwa Kagaku Kenkyusho Co Ltd | Glycyrrhetic acid derivative, production thereof and antiulcer agent containing said compound as active component |
GB8823869D0 (en) | 1988-10-12 | 1988-11-16 | Medical Res Council | Production of antibodies |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5605690A (en) | 1989-09-05 | 1997-02-25 | Immunex Corporation | Methods of lowering active TNF-α levels in mammals using tumor necrosis factor receptor |
US5859205A (en) | 1989-12-21 | 1999-01-12 | Celltech Limited | Humanised antibodies |
US5545806A (en) | 1990-08-29 | 1996-08-13 | Genpharm International, Inc. | Ransgenic non-human animals for producing heterologous antibodies |
JP2998287B2 (en) | 1991-03-13 | 2000-01-11 | 千寿製薬株式会社 | Glycyrrhetinic acid derivatives |
US5656272A (en) | 1991-03-18 | 1997-08-12 | New York University Medical Center | Methods of treating TNF-α-mediated Crohn's disease using chimeric anti-TNF antibodies |
IT1253431B (en) | 1991-12-02 | 1995-08-08 | Valetudo S R L | PHARMACEUTICAL PREPARATIONS FOR TOPICAL USE FOR THE TREATMENT OF PSORIASIS AND ATOPIC DERMATITIS |
ATE463573T1 (en) | 1991-12-02 | 2010-04-15 | Medimmune Ltd | PRODUCTION OF AUTOANTIBODIES ON PHAGE SURFACES BASED ON ANTIBODIES SEGMENT LIBRARIES |
IT1254321B (en) | 1992-04-10 | 1995-09-14 | Kemiprogress S R L | PHARMACEUTICAL COMPOSITION FOR THE TREATMENT AND PREVENTION OF CUTANEOUS INFLAMMATIONS AND ORAL MUCOSA. |
GB9217316D0 (en) * | 1992-08-14 | 1992-09-30 | Ludwig Inst Cancer Res | Schwann cell mitogenic factor,its preparation and use |
EP0727487A1 (en) | 1995-02-17 | 1996-08-21 | K.U. Leuven Research & Development | Multiple-tumor aberrant growth genes |
JP3472048B2 (en) | 1995-10-09 | 2003-12-02 | 鐘淵化学工業株式会社 | Diagnostics for autoimmune diseases |
DE19548122A1 (en) | 1995-12-21 | 1997-06-26 | Joern Prof Dr Bullerdiek | Nucleic acid sequences of high mobility group protein genes and uses thereof |
US6323329B1 (en) | 1995-12-21 | 2001-11-27 | Jorn Bullerdiek | Nucleic acid sequences of genes encoding high mobility group proteins |
US5864018A (en) | 1996-04-16 | 1999-01-26 | Schering Aktiengesellschaft | Antibodies to advanced glycosylation end-product receptor polypeptides and uses therefor |
US6720472B2 (en) | 1996-07-12 | 2004-04-13 | University Of Medicine And Dentistry Of New Jersey | HMGI proteins in cancer and obesity |
US6171779B1 (en) | 1996-07-12 | 2001-01-09 | University Of Medicine & Dentistry Of New Jersey | HMGI proteins in cancer |
DE69737870T2 (en) | 1996-07-17 | 2008-02-07 | Kaneka Corp. | MEDICAMENTS FOR THE DIAGNOSIS OF AUTOIMMUNE DISEASES |
US7258857B2 (en) | 1996-11-22 | 2007-08-21 | The Trustees Of Columbia University In The City Of New York | Rage-related methods for treating inflammation |
WO2000020621A1 (en) | 1998-10-06 | 2000-04-13 | The Trustees Of Columbia University In The City Of New York | Extracellular novel rage binding protein (en-rage) and uses thereof |
SK146599A3 (en) | 1997-05-07 | 2000-07-11 | Schering Corp | Essentially pure or recombinant protein dtlr2-10, a fused protein, a bond substance, a nucleic acid, an expression vector, a host cell and process for their production |
US20030032090A1 (en) | 1997-05-07 | 2003-02-13 | Schering Corporation, A New Jersey Corporation | Human receptor proteins; related reagents and methods |
IT1291366B1 (en) | 1997-05-14 | 1999-01-07 | Angelini Ricerche Spa | ANTIVIRAL PHARMACEUTICAL COMPOSITION INCLUDING GLYCYRHIZIC ACID AND AT LEAST ONE PROTEIN WITH ANTIVIRAL ACTIVITY |
EP1650219B1 (en) | 1997-10-17 | 2007-12-19 | Genentech, Inc. | Human toll homologues |
US20030027260A1 (en) | 1997-10-17 | 2003-02-06 | Genentech, Inc. | Human Toll homologues |
ES2137125B1 (en) | 1997-11-18 | 2000-08-16 | Vinyals S A Lab Dr | THE USE OF THE ZINC SALT OF THE GLICIRRETIC ACID IN PREPARATIONS AGAINST ACNE AND THE COMPOSITIONS CONTAINING SUCH SALT. |
US6783961B1 (en) | 1999-02-26 | 2004-08-31 | Genset S.A. | Expressed sequence tags and encoded human proteins |
IT1299583B1 (en) | 1998-05-19 | 2000-03-16 | Vander Way Limited | USE OF HMG-I PROTEIN FOR THE PREPARATION OF MEDICATIONS WITH CYTOTOXIC ACTIVITY |
US6303321B1 (en) | 1999-02-11 | 2001-10-16 | North Shore-Long Island Jewish Research Institute | Methods for diagnosing sepsis |
US7151082B2 (en) | 1999-02-11 | 2006-12-19 | The Feinstein Institute For Medical Research | Antagonists of HMG1 for treating inflammatory conditions |
US6177077B1 (en) | 1999-02-24 | 2001-01-23 | Edward L. Tobinick | TNT inhibitors for the treatment of neurological disorders |
AU3395900A (en) | 1999-03-12 | 2000-10-04 | Human Genome Sciences, Inc. | Human lung cancer associated gene sequences and polypeptides |
TWI221082B (en) | 1999-04-14 | 2004-09-21 | Sumitomo Chemical Co | Pesticidal compositions |
EP1187932A2 (en) | 1999-06-04 | 2002-03-20 | Millennium Pharmaceuticals, Inc. | Mammalian toll homologues and uses thereof |
US6794132B2 (en) * | 1999-10-02 | 2004-09-21 | Biosite, Inc. | Human antibodies |
US7572466B1 (en) * | 1999-10-13 | 2009-08-11 | Health Research, Inc. | Oral immunology using plant product containing a non-enteric pathogen antigen |
GB9927332D0 (en) | 1999-11-18 | 2000-01-12 | Leiv Eiriksson Nyfotek As | Novel antibody and uses thereof |
US6677321B1 (en) | 1999-12-09 | 2004-01-13 | Bruce Levin | Methods and compositions for treatment of inflammatory disease |
GB0001704D0 (en) | 2000-01-25 | 2000-03-15 | Glaxo Group Ltd | Protein |
AU2001244711A1 (en) | 2000-03-31 | 2001-10-08 | Mochida Pharmaceutical Co., Ltd. | Tlr/cd14 binding inhibitor |
US6436703B1 (en) | 2000-03-31 | 2002-08-20 | Hyseq, Inc. | Nucleic acids and polypeptides |
EP1283849A2 (en) | 2000-05-25 | 2003-02-19 | Schering Corporation | Human receptor proteins; related reagents and methods |
GB0015325D0 (en) | 2000-06-22 | 2000-08-16 | Danionics As | Electrochemical cells |
JP2004536786A (en) | 2001-03-02 | 2004-12-09 | メディミューン,インコーポレイテッド | Method for preventing or treating inflammatory disease or autoimmune disease by administering an integrin αvβ3 antagonist in combination with another prophylactic or therapeutic agent |
CA2440037C (en) | 2001-03-05 | 2010-02-16 | Transtech Pharma, Inc. | Benzimidazole derivatives for modulating the rage receptor |
CA2440042C (en) | 2001-03-05 | 2011-09-27 | Transtech Pharma, Inc. | Carboxamide derivatives as therapeutic agents |
JP2004528307A (en) | 2001-03-15 | 2004-09-16 | ユニバーシティ オブ ピッツバーグ オブ ザ コモンウェルス システム オブ ハイヤー エデュケーション | Use of pyruvate and / or derivatives thereof for the treatment of inflammatory conditions mediated by cytokines |
ITMI20010562A1 (en) | 2001-03-16 | 2002-09-16 | Marco E Bianchi | HMG1 PROTEIN INHIBITORS OR ANTAGONISTS FOR THE TREATMENT OF VASCULAR DISORDERS |
EP1401281A4 (en) | 2001-05-09 | 2004-12-29 | Univ Yale | Toll/interleukin-1 receptor adaptor protein (tirap) |
MXPA03009995A (en) | 2001-05-09 | 2004-06-30 | Univ Michigan | Use of compositions for treating rosacea. |
US7220723B2 (en) | 2001-05-15 | 2007-05-22 | The Feinstein Institute For Medical Research | Inhibitors of the interaction between HMGB polypeptides and toll-like receptor 2 as anti-inflammatory agents |
US7304034B2 (en) | 2001-05-15 | 2007-12-04 | The Feinstein Institute For Medical Research | Use of HMGB fragments as anti-inflammatory agents |
CA2447576C (en) | 2001-05-15 | 2014-04-08 | North Shore-Long Island Jewish Research Institute | Use of hmg fragments as anti-inflammatory agents |
JP4823465B2 (en) | 2001-07-13 | 2011-11-24 | 株式会社シノテスト | Antibody specifically binding to human HMG-1 and method and reagent for immunoassay of human HMG-1 using this antibody |
US20030032674A1 (en) | 2001-08-13 | 2003-02-13 | Hwang Daniel H. | Use of unsaturated fatty acids to treat severe inflammatory diseases |
EP1435987A4 (en) | 2001-09-07 | 2005-11-23 | Univ Boston | Method and composition for treating immune complex associated disorders |
JP2003088388A (en) | 2001-09-14 | 2003-03-25 | Herikkusu Kenkyusho:Kk | NEW FULL-LENGTH cDNA |
EP1293566A1 (en) * | 2001-09-17 | 2003-03-19 | Societe Des Produits Nestle S.A. | A soluble toll-like receptor |
US20050118688A1 (en) | 2001-12-28 | 2005-06-02 | Hudson Freeze | Novel ligand involved in the transmigration of leukocytes across the endothelium and uses therefor |
CA2476594C (en) | 2002-03-05 | 2012-10-09 | Transtech Pharma, Inc. | Mono-and bicyclic azole derivatives that inhibit the interaction of ligands with rage |
CA2491321A1 (en) | 2002-07-03 | 2004-01-15 | Fondazione Centro San Raffaele Del Monte Tabor | Use of hmgb1 in the treatment of tissue damage and/or to promote tissue repair |
GB0226251D0 (en) | 2002-11-11 | 2002-12-18 | San Raffaele Centro Fond | Acetylated protein |
US20040141948A1 (en) * | 2002-11-20 | 2004-07-22 | Critical Therapeutics, Inc. | Use of HMGB fragments as anti-inflammatory agents |
AU2003295653B2 (en) | 2002-11-20 | 2007-08-16 | The Feinstein Institute Of Medical Research | Use of HMGB polypeptides for increasing immune responses |
JP2006510619A (en) | 2002-11-20 | 2006-03-30 | クリティカル セラピューティクス,インコーポレイテッド | Use of HMGB fragments as anti-inflammatory agents |
US7696169B2 (en) | 2003-06-06 | 2010-04-13 | The Feinstein Institute For Medical Research | Inhibitors of the interaction between HMGB polypeptides and toll-like receptor 2 as anti-inflammatory agents |
US7288250B2 (en) | 2003-09-11 | 2007-10-30 | Critical Therapeutics, Inc. | Monoclonal antibodies against HMGB1 |
US7807696B2 (en) | 2003-10-07 | 2010-10-05 | The Feinstein Institute For Medical Research | Isoxazole and isothiazole compounds useful in the treatment of inflammation |
WO2006083301A2 (en) | 2004-06-17 | 2006-08-10 | Medimmune, Inc. | Immunogenic compositions comprising hmgb1 polypeptides |
US7470521B2 (en) | 2004-07-20 | 2008-12-30 | Critical Therapeutics, Inc. | RAGE protein derivatives |
WO2006008779A1 (en) | 2004-07-20 | 2006-01-26 | Provincia Italiana Della Congregazione Dei Figli Dell'immacolata Concezione-Istituto Dermopatico Dell'immacolata | Use of hmgb1 for wound healing |
EP1781700B1 (en) | 2004-08-03 | 2014-03-19 | TransTech Pharma, LLC | Rage fusion proteins and methods of use |
WO2006024547A2 (en) | 2004-09-03 | 2006-03-09 | Creabilis Therapeutics S.P.A. | Protease resistant human and non-human hmgb1 box-a mutants and their therapeutic/diagnostic use |
EP1812065A4 (en) | 2004-10-22 | 2009-09-02 | Medimmune Inc | High affinity antibodies against hmgb1 and methods of use thereof |
US8129130B2 (en) | 2004-10-22 | 2012-03-06 | The Feinstein Institute For Medical Research | High affinity antibodies against HMGB1 and methods of use thereof |
US20100040608A1 (en) * | 2005-07-18 | 2010-02-18 | Marie Wahren-Herlenius | Use of HMGB1 antagonists for the treatment of inflammatory skin conditions |
WO2007054090A1 (en) | 2005-11-09 | 2007-05-18 | Pharmexa A/S | Therapeutic vaccines targeting hmgb1 |
AU2006330807A1 (en) | 2005-11-28 | 2007-07-05 | Medimmune, Llc | Antagonists of HMBG1 and/or rage and methods of use thereof |
MY188368A (en) * | 2006-09-08 | 2021-12-06 | Abbott Lab | Interleukin-13 binding proteins |
WO2008076758A2 (en) | 2006-12-13 | 2008-06-26 | William Marsh Rice University | Nerolidol, terpene, and terpene derivative synthesis |
WO2008154638A2 (en) | 2007-06-12 | 2008-12-18 | Board Of Regents, The University Of Texas System | Antagonists of the receptor for advanced glycation end-products (rage) |
-
2004
- 2004-09-10 US US10/938,992 patent/US7288250B2/en not_active Expired - Fee Related
- 2004-09-10 JP JP2006526305A patent/JP4792392B2/en not_active Expired - Fee Related
- 2004-09-10 CA CA2538763A patent/CA2538763C/en active Active
- 2004-09-10 CA CA2882022A patent/CA2882022A1/en not_active Abandoned
- 2004-09-10 EP EP04788671A patent/EP1668035A2/en not_active Withdrawn
- 2004-09-10 AU AU2004272607A patent/AU2004272607B2/en not_active Ceased
- 2004-09-10 CN CNA2004800332177A patent/CN1878793A/en active Pending
- 2004-09-10 WO PCT/US2004/029527 patent/WO2005026209A2/en active Application Filing
-
2007
- 2007-09-20 US US11/903,129 patent/US7632500B2/en active Active
- 2007-10-15 US US11/974,923 patent/US20090148453A1/en not_active Abandoned
-
2010
- 2010-10-04 US US12/897,453 patent/US8846047B2/en not_active Expired - Fee Related
-
2011
- 2011-05-27 JP JP2011118559A patent/JP5513441B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CA2538763A1 (en) | 2005-03-24 |
US7632500B2 (en) | 2009-12-15 |
WO2005026209A3 (en) | 2005-09-15 |
CN1878793A (en) | 2006-12-13 |
JP2011241219A (en) | 2011-12-01 |
US8846047B2 (en) | 2014-09-30 |
WO2005026209A2 (en) | 2005-03-24 |
US20090148453A1 (en) | 2009-06-11 |
US7288250B2 (en) | 2007-10-30 |
JP2007527406A (en) | 2007-09-27 |
AU2004272607A1 (en) | 2005-03-24 |
JP5513441B2 (en) | 2014-06-04 |
US20080113385A1 (en) | 2008-05-15 |
AU2004272607B2 (en) | 2008-11-06 |
US20050152903A1 (en) | 2005-07-14 |
EP1668035A2 (en) | 2006-06-14 |
US20110217292A1 (en) | 2011-09-08 |
JP4792392B2 (en) | 2011-10-12 |
CA2538763C (en) | 2015-05-05 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2538763C (en) | Monoclonal antibodies against hmgb1 | |
US11680100B2 (en) | B7-H3 antibody, antigen-binding fragment thereof and medical use thereof | |
TWI655209B (en) | Antibody against human plan death (PROGRAMMED DEATH) ligand 1 (PD-L1) | |
US20130115217A1 (en) | Antibodies against hmgb1 and fragments thereof | |
CA2802631A1 (en) | S100a4 antibodies and therapeutic uses thereof | |
US20230331847A1 (en) | Anti-phosphotyrosinylated programmed death 1 (pd-1) monoclonal antibodies, methods of making and methods of using thereof | |
KR20100023869A (en) | Cancer remedy containing antibody against peptide encoded by exon-17 of periostin | |
IL294814A (en) | Anti-αvβ8 integrin antibodies for use in treating kidney disease | |
KR20100103675A (en) | Antibody directed against pcrv | |
EP2088159B1 (en) | Antibody recognizing c-domain of midkine | |
JP2023508277A (en) | Novel DDR1 antibodies and uses thereof | |
CN111094354B (en) | Thrombin antibody, antigen binding fragment thereof and medical application | |
KR102662387B1 (en) | B7-H3 antibody, antigen-binding fragment thereof and medical uses thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20150217 |
|
FZDE | Discontinued |
Effective date: 20170809 |