CA3031540A1 - Anti-histone therapy in acute kidney injury - Google Patents
Anti-histone therapy in acute kidney injury Download PDFInfo
- Publication number
- CA3031540A1 CA3031540A1 CA3031540A CA3031540A CA3031540A1 CA 3031540 A1 CA3031540 A1 CA 3031540A1 CA 3031540 A CA3031540 A CA 3031540A CA 3031540 A CA3031540 A CA 3031540A CA 3031540 A1 CA3031540 A1 CA 3031540A1
- Authority
- CA
- Canada
- Prior art keywords
- histone
- antibody
- cells
- antibodies
- nets
- 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.)
- Pending
Links
- 230000002583 anti-histone Effects 0.000 title claims abstract description 91
- 201000011040 acute kidney failure Diseases 0.000 title claims abstract description 56
- 208000009304 Acute Kidney Injury Diseases 0.000 title claims abstract description 55
- 208000033626 Renal failure acute Diseases 0.000 title claims abstract description 55
- 238000002560 therapeutic procedure Methods 0.000 title description 12
- 108010033040 Histones Proteins 0.000 claims abstract description 215
- 102000006947 Histones Human genes 0.000 claims abstract description 140
- 239000003112 inhibitor Substances 0.000 claims abstract description 113
- 230000006378 damage Effects 0.000 claims abstract description 77
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 65
- 208000014674 injury Diseases 0.000 claims abstract description 63
- 208000027418 Wounds and injury Diseases 0.000 claims abstract description 61
- 210000000056 organ Anatomy 0.000 claims abstract description 52
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 46
- 239000003814 drug Substances 0.000 claims abstract description 38
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 29
- 206010038540 Renal tubular necrosis Diseases 0.000 claims abstract description 25
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims abstract description 22
- 229960002897 heparin Drugs 0.000 claims abstract description 22
- 229920000669 heparin Polymers 0.000 claims abstract description 22
- 210000004072 lung Anatomy 0.000 claims abstract description 22
- 101800004937 Protein C Proteins 0.000 claims abstract description 16
- 101800001700 Saposin-D Proteins 0.000 claims abstract description 16
- 229960000856 protein c Drugs 0.000 claims abstract description 16
- 210000002216 heart Anatomy 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 123
- 108091007433 antigens Proteins 0.000 claims description 57
- 102000036639 antigens Human genes 0.000 claims description 57
- 239000000427 antigen Substances 0.000 claims description 56
- 230000027455 binding Effects 0.000 claims description 48
- 102000008394 Immunoglobulin Fragments Human genes 0.000 claims description 34
- 239000012634 fragment Substances 0.000 claims description 34
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 34
- 108010021625 Immunoglobulin Fragments Proteins 0.000 claims description 33
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 33
- 230000000694 effects Effects 0.000 claims description 32
- 102000004127 Cytokines Human genes 0.000 claims description 31
- 108090000695 Cytokines Proteins 0.000 claims description 31
- 102000002262 Thromboplastin Human genes 0.000 claims description 19
- 108010000499 Thromboplastin Proteins 0.000 claims description 19
- -1 IL-14 Proteins 0.000 claims description 17
- 230000000295 complement effect Effects 0.000 claims description 16
- 239000012636 effector Substances 0.000 claims description 16
- 102000017975 Protein C Human genes 0.000 claims description 15
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 claims description 13
- 102100039360 Toll-like receptor 4 Human genes 0.000 claims description 13
- 239000002158 endotoxin Substances 0.000 claims description 13
- 210000005007 innate immune system Anatomy 0.000 claims description 13
- 102000003390 tumor necrosis factor Human genes 0.000 claims description 13
- 208000004852 Lung Injury Diseases 0.000 claims description 12
- 206010069363 Traumatic lung injury Diseases 0.000 claims description 12
- 108020001507 fusion proteins Proteins 0.000 claims description 12
- 102000037865 fusion proteins Human genes 0.000 claims description 12
- 231100000515 lung injury Toxicity 0.000 claims description 12
- 239000002955 immunomodulating agent Substances 0.000 claims description 11
- 229940121354 immunomodulator Drugs 0.000 claims description 11
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 10
- 108090001005 Interleukin-6 Proteins 0.000 claims description 10
- 102000015696 Interleukins Human genes 0.000 claims description 10
- 108010063738 Interleukins Proteins 0.000 claims description 10
- 229920006008 lipopolysaccharide Polymers 0.000 claims description 10
- 102000004889 Interleukin-6 Human genes 0.000 claims description 9
- 108010079274 Thrombomodulin Proteins 0.000 claims description 9
- 102100026966 Thrombomodulin Human genes 0.000 claims description 9
- 229940079593 drug Drugs 0.000 claims description 9
- 108010071942 Colony-Stimulating Factors Proteins 0.000 claims description 8
- 102000000589 Interleukin-1 Human genes 0.000 claims description 8
- 108010002352 Interleukin-1 Proteins 0.000 claims description 8
- 230000002584 immunomodulator Effects 0.000 claims description 8
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 102000003951 Erythropoietin Human genes 0.000 claims description 7
- 108090000394 Erythropoietin Proteins 0.000 claims description 7
- 239000004365 Protease Substances 0.000 claims description 7
- 102000036693 Thrombopoietin Human genes 0.000 claims description 7
- 108010041111 Thrombopoietin Proteins 0.000 claims description 7
- 229940088598 enzyme Drugs 0.000 claims description 7
- 229940105423 erythropoietin Drugs 0.000 claims description 7
- OXCMYAYHXIHQOA-UHFFFAOYSA-N potassium;[2-butyl-5-chloro-3-[[4-[2-(1,2,4-triaza-3-azanidacyclopenta-1,4-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol Chemical compound [K+].CCCCC1=NC(Cl)=C(CO)N1CC1=CC=C(C=2C(=CC=CC=2)C2=N[N-]N=N2)C=C1 OXCMYAYHXIHQOA-UHFFFAOYSA-N 0.000 claims description 7
- 230000000770 proinflammatory effect Effects 0.000 claims description 7
- 108010012236 Chemokines Proteins 0.000 claims description 6
- 102000019034 Chemokines Human genes 0.000 claims description 6
- 101710103773 Histone H2B Proteins 0.000 claims description 6
- 102100021639 Histone H2B type 1-K Human genes 0.000 claims description 6
- 102000004083 Lymphotoxin-alpha Human genes 0.000 claims description 6
- 108090000542 Lymphotoxin-alpha Proteins 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 6
- 108090000190 Thrombin Proteins 0.000 claims description 6
- 229940088597 hormone Drugs 0.000 claims description 6
- 239000005556 hormone Substances 0.000 claims description 6
- 230000008085 renal dysfunction Effects 0.000 claims description 6
- 229960004072 thrombin Drugs 0.000 claims description 6
- 239000003053 toxin Substances 0.000 claims description 6
- 231100000765 toxin Toxicity 0.000 claims description 6
- 108700012359 toxins Proteins 0.000 claims description 6
- 108010003723 Single-Domain Antibodies Proteins 0.000 claims description 5
- 108020004459 Small interfering RNA Proteins 0.000 claims description 5
- 102000015081 Blood Coagulation Factors Human genes 0.000 claims description 4
- 108010039209 Blood Coagulation Factors Proteins 0.000 claims description 4
- 102100032528 C-type lectin domain family 11 member A Human genes 0.000 claims description 4
- 101710167766 C-type lectin domain family 11 member A Proteins 0.000 claims description 4
- 102100025680 Complement decay-accelerating factor Human genes 0.000 claims description 4
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 claims description 4
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 claims description 4
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 claims description 4
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 claims description 4
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 4
- 102000001398 Granzyme Human genes 0.000 claims description 4
- 108060005986 Granzyme Proteins 0.000 claims description 4
- 101000856022 Homo sapiens Complement decay-accelerating factor Proteins 0.000 claims description 4
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 4
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 4
- 239000000854 Human Growth Hormone Substances 0.000 claims description 4
- 108010065805 Interleukin-12 Proteins 0.000 claims description 4
- 102000013462 Interleukin-12 Human genes 0.000 claims description 4
- 108090000172 Interleukin-15 Proteins 0.000 claims description 4
- 102000003812 Interleukin-15 Human genes 0.000 claims description 4
- 108050003558 Interleukin-17 Proteins 0.000 claims description 4
- 102000013691 Interleukin-17 Human genes 0.000 claims description 4
- 102000003810 Interleukin-18 Human genes 0.000 claims description 4
- 108090000171 Interleukin-18 Proteins 0.000 claims description 4
- 108010002386 Interleukin-3 Proteins 0.000 claims description 4
- 102000000646 Interleukin-3 Human genes 0.000 claims description 4
- 102000004388 Interleukin-4 Human genes 0.000 claims description 4
- 108090000978 Interleukin-4 Proteins 0.000 claims description 4
- 108010002616 Interleukin-5 Proteins 0.000 claims description 4
- 102100039897 Interleukin-5 Human genes 0.000 claims description 4
- 102000004890 Interleukin-8 Human genes 0.000 claims description 4
- 108090001007 Interleukin-8 Proteins 0.000 claims description 4
- 102000009151 Luteinizing Hormone Human genes 0.000 claims description 4
- 108010073521 Luteinizing Hormone Proteins 0.000 claims description 4
- 102000007079 Peptide Fragments Human genes 0.000 claims description 4
- 108010033276 Peptide Fragments Proteins 0.000 claims description 4
- 102000011923 Thyrotropin Human genes 0.000 claims description 4
- 108010061174 Thyrotropin Proteins 0.000 claims description 4
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 4
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 4
- 239000003114 blood coagulation factor Substances 0.000 claims description 4
- 229940028334 follicle stimulating hormone Drugs 0.000 claims description 4
- 239000003102 growth factor Substances 0.000 claims description 4
- 230000003394 haemopoietic effect Effects 0.000 claims description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 4
- 229940040129 luteinizing hormone Drugs 0.000 claims description 4
- 230000000861 pro-apoptotic effect Effects 0.000 claims description 4
- FSPQCTGGIANIJZ-UHFFFAOYSA-N 2-[[(3,4-dimethoxyphenyl)-oxomethyl]amino]-4,5,6,7-tetrahydro-1-benzothiophene-3-carboxamide Chemical compound C1=C(OC)C(OC)=CC=C1C(=O)NC1=C(C(N)=O)C(CCCC2)=C2S1 FSPQCTGGIANIJZ-UHFFFAOYSA-N 0.000 claims description 3
- 102400000068 Angiostatin Human genes 0.000 claims description 3
- 108010079709 Angiostatins Proteins 0.000 claims description 3
- 102100032912 CD44 antigen Human genes 0.000 claims description 3
- 108010079505 Endostatins Proteins 0.000 claims description 3
- 102000017286 Histone H2A Human genes 0.000 claims description 3
- 108050005231 Histone H2A Proteins 0.000 claims description 3
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 claims description 3
- 101000853002 Homo sapiens Interleukin-25 Proteins 0.000 claims description 3
- 101001128431 Homo sapiens Myeloid-derived growth factor Proteins 0.000 claims description 3
- 102000003814 Interleukin-10 Human genes 0.000 claims description 3
- 108090000174 Interleukin-10 Proteins 0.000 claims description 3
- 108090000177 Interleukin-11 Proteins 0.000 claims description 3
- 102000003815 Interleukin-11 Human genes 0.000 claims description 3
- 102000003816 Interleukin-13 Human genes 0.000 claims description 3
- 108090000176 Interleukin-13 Proteins 0.000 claims description 3
- 101800003050 Interleukin-16 Proteins 0.000 claims description 3
- 102000049772 Interleukin-16 Human genes 0.000 claims description 3
- 102000000588 Interleukin-2 Human genes 0.000 claims description 3
- 108010002350 Interleukin-2 Proteins 0.000 claims description 3
- 102100030703 Interleukin-22 Human genes 0.000 claims description 3
- 102100021592 Interleukin-7 Human genes 0.000 claims description 3
- 108010002586 Interleukin-7 Proteins 0.000 claims description 3
- 108010002335 Interleukin-9 Proteins 0.000 claims description 3
- 102000000585 Interleukin-9 Human genes 0.000 claims description 3
- 102100032352 Leukemia inhibitory factor Human genes 0.000 claims description 3
- 108090000581 Leukemia inhibitory factor Proteins 0.000 claims description 3
- 102100031789 Myeloid-derived growth factor Human genes 0.000 claims description 3
- 101710151245 Receptor-type tyrosine-protein kinase FLT3 Proteins 0.000 claims description 3
- 102100020718 Receptor-type tyrosine-protein kinase FLT3 Human genes 0.000 claims description 3
- 108060008245 Thrombospondin Proteins 0.000 claims description 3
- 102000002938 Thrombospondin Human genes 0.000 claims description 3
- 206010054094 Tumour necrosis Diseases 0.000 claims description 3
- 239000004037 angiogenesis inhibitor Substances 0.000 claims description 3
- FZCSTZYAHCUGEM-UHFFFAOYSA-N aspergillomarasmine B Natural products OC(=O)CNC(C(O)=O)CNC(C(O)=O)CC(O)=O FZCSTZYAHCUGEM-UHFFFAOYSA-N 0.000 claims description 3
- 102000034356 gene-regulatory proteins Human genes 0.000 claims description 3
- 108091006104 gene-regulatory proteins Proteins 0.000 claims description 3
- 102000006495 integrins Human genes 0.000 claims description 3
- 108010044426 integrins Proteins 0.000 claims description 3
- 108010074108 interleukin-21 Proteins 0.000 claims description 3
- 108010059616 Activins Proteins 0.000 claims description 2
- 108010005853 Anti-Mullerian Hormone Proteins 0.000 claims description 2
- 102100032367 C-C motif chemokine 5 Human genes 0.000 claims description 2
- 102100025248 C-X-C motif chemokine 10 Human genes 0.000 claims description 2
- 101710098275 C-X-C motif chemokine 10 Proteins 0.000 claims description 2
- 102100022002 CD59 glycoprotein Human genes 0.000 claims description 2
- 108010055166 Chemokine CCL5 Proteins 0.000 claims description 2
- 102100021809 Chorionic somatomammotropin hormone 1 Human genes 0.000 claims description 2
- XUIIKFGFIJCVMT-GFCCVEGCSA-N D-thyroxine Chemical compound IC1=CC(C[C@@H](N)C(O)=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-GFCCVEGCSA-N 0.000 claims description 2
- 108010088842 Fibrinolysin Proteins 0.000 claims description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 claims description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 claims description 2
- 108010086677 Gonadotropins Proteins 0.000 claims description 2
- 102000006771 Gonadotropins Human genes 0.000 claims description 2
- 102100030595 HLA class II histocompatibility antigen gamma chain Human genes 0.000 claims description 2
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 claims description 2
- 101001082627 Homo sapiens HLA class II histocompatibility antigen gamma chain Proteins 0.000 claims description 2
- 101000961414 Homo sapiens Membrane cofactor protein Proteins 0.000 claims description 2
- 102100026818 Inhibin beta E chain Human genes 0.000 claims description 2
- 108010004250 Inhibins Proteins 0.000 claims description 2
- 102000002746 Inhibins Human genes 0.000 claims description 2
- 102000004877 Insulin Human genes 0.000 claims description 2
- 108090001061 Insulin Proteins 0.000 claims description 2
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 2
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 claims description 2
- 102100037852 Insulin-like growth factor I Human genes 0.000 claims description 2
- 102000016267 Leptin Human genes 0.000 claims description 2
- 108010092277 Leptin Proteins 0.000 claims description 2
- 102100039373 Membrane cofactor protein Human genes 0.000 claims description 2
- 102000003982 Parathyroid hormone Human genes 0.000 claims description 2
- 108090000445 Parathyroid hormone Proteins 0.000 claims description 2
- 108010003044 Placental Lactogen Proteins 0.000 claims description 2
- 239000000381 Placental Lactogen Substances 0.000 claims description 2
- 108010076181 Proinsulin Proteins 0.000 claims description 2
- 102000003946 Prolactin Human genes 0.000 claims description 2
- 108010057464 Prolactin Proteins 0.000 claims description 2
- 108090000103 Relaxin Proteins 0.000 claims description 2
- 102000003743 Relaxin Human genes 0.000 claims description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 2
- 239000000488 activin Substances 0.000 claims description 2
- 239000000868 anti-mullerian hormone Substances 0.000 claims description 2
- 108010006025 bovine growth hormone Proteins 0.000 claims description 2
- 229940126864 fibroblast growth factor Drugs 0.000 claims description 2
- 239000002622 gonadotropin Substances 0.000 claims description 2
- 230000002440 hepatic effect Effects 0.000 claims description 2
- 239000000893 inhibin Substances 0.000 claims description 2
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 claims description 2
- 229940125396 insulin Drugs 0.000 claims description 2
- 230000002138 osteoinductive effect Effects 0.000 claims description 2
- 229960001319 parathyroid hormone Drugs 0.000 claims description 2
- 239000000199 parathyroid hormone Substances 0.000 claims description 2
- 229940012957 plasmin Drugs 0.000 claims description 2
- 229940097325 prolactin Drugs 0.000 claims description 2
- 108010087851 prorelaxin Proteins 0.000 claims description 2
- 150000003180 prostaglandins Chemical class 0.000 claims description 2
- 229940034208 thyroxine Drugs 0.000 claims description 2
- XUIIKFGFIJCVMT-UHFFFAOYSA-N thyroxine-binding globulin Natural products IC1=CC(CC([NH3+])C([O-])=O)=CC(I)=C1OC1=CC(I)=C(O)C(I)=C1 XUIIKFGFIJCVMT-UHFFFAOYSA-N 0.000 claims description 2
- 102000008228 Toll-like receptor 2 Human genes 0.000 claims 4
- 108010060888 Toll-like receptor 2 Proteins 0.000 claims 4
- 102400001047 Endostatin Human genes 0.000 claims 2
- 108010065637 Interleukin-23 Proteins 0.000 claims 2
- 102000013264 Interleukin-23 Human genes 0.000 claims 2
- 102100036842 C-C motif chemokine 19 Human genes 0.000 claims 1
- 102100036846 C-C motif chemokine 21 Human genes 0.000 claims 1
- 102100036150 C-X-C motif chemokine 5 Human genes 0.000 claims 1
- 108010029697 CD40 Ligand Proteins 0.000 claims 1
- 102100032937 CD40 ligand Human genes 0.000 claims 1
- 102000006354 HLA-DR Antigens Human genes 0.000 claims 1
- 108010058597 HLA-DR Antigens Proteins 0.000 claims 1
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 claims 1
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 claims 1
- 101000947186 Homo sapiens C-X-C motif chemokine 5 Proteins 0.000 claims 1
- 102100025947 Insulin-like growth factor II Human genes 0.000 claims 1
- 102100020793 Interleukin-13 receptor subunit alpha-2 Human genes 0.000 claims 1
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 claims 1
- 102000034655 MIF Human genes 0.000 claims 1
- 108060004872 MIF Proteins 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 108040003607 interleukin-13 receptor activity proteins Proteins 0.000 claims 1
- 108040002039 interleukin-15 receptor activity proteins Proteins 0.000 claims 1
- 102000008616 interleukin-15 receptor activity proteins Human genes 0.000 claims 1
- 108040001304 interleukin-17 receptor activity proteins Proteins 0.000 claims 1
- 102000053460 interleukin-17 receptor activity proteins Human genes 0.000 claims 1
- 108040002014 interleukin-18 receptor activity proteins Proteins 0.000 claims 1
- 102000008625 interleukin-18 receptor activity proteins Human genes 0.000 claims 1
- 108040006852 interleukin-4 receptor activity proteins Proteins 0.000 claims 1
- 108040006858 interleukin-6 receptor activity proteins Proteins 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 124
- 210000000440 neutrophil Anatomy 0.000 abstract description 112
- 210000003734 kidney Anatomy 0.000 abstract description 104
- 230000017074 necrotic cell death Effects 0.000 abstract description 81
- 230000005764 inhibitory process Effects 0.000 abstract description 28
- 230000001681 protective effect Effects 0.000 abstract description 18
- 210000001519 tissue Anatomy 0.000 abstract description 13
- 230000034994 death Effects 0.000 abstract description 10
- 231100000517 death Toxicity 0.000 abstract description 10
- 230000009977 dual effect Effects 0.000 abstract description 5
- 230000000996 additive effect Effects 0.000 abstract description 4
- 210000005084 renal tissue Anatomy 0.000 abstract description 4
- 239000000654 additive Substances 0.000 abstract description 3
- 102100036546 Salivary acidic proline-rich phosphoprotein 1/2 Human genes 0.000 abstract 1
- 206010028851 Necrosis Diseases 0.000 description 79
- 102000001235 protein arginine deiminase Human genes 0.000 description 68
- 108060006632 protein arginine deiminase Proteins 0.000 description 68
- 241000699670 Mus sp. Species 0.000 description 65
- 229940027941 immunoglobulin g Drugs 0.000 description 53
- 208000028867 ischemia Diseases 0.000 description 45
- 108090000623 proteins and genes Proteins 0.000 description 44
- 230000002146 bilateral effect Effects 0.000 description 43
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 42
- 230000010410 reperfusion Effects 0.000 description 41
- 238000007912 intraperitoneal administration Methods 0.000 description 33
- 102000004169 proteins and genes Human genes 0.000 description 30
- 206010040047 Sepsis Diseases 0.000 description 29
- 235000001014 amino acid Nutrition 0.000 description 29
- 238000000338 in vitro Methods 0.000 description 29
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical group COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 28
- 230000001225 therapeutic effect Effects 0.000 description 28
- 230000014509 gene expression Effects 0.000 description 27
- 230000001434 glomerular Effects 0.000 description 26
- 238000001727 in vivo Methods 0.000 description 26
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 25
- 235000018102 proteins Nutrition 0.000 description 25
- 238000010186 staining Methods 0.000 description 25
- 201000010099 disease Diseases 0.000 description 24
- 238000011282 treatment Methods 0.000 description 24
- 101150044980 Akap1 gene Proteins 0.000 description 23
- 101000831567 Homo sapiens Toll-like receptor 2 Proteins 0.000 description 23
- 102100024333 Toll-like receptor 2 Human genes 0.000 description 23
- 230000002829 reductive effect Effects 0.000 description 23
- 230000004913 activation Effects 0.000 description 21
- 239000000203 mixture Substances 0.000 description 21
- 238000006467 substitution reaction Methods 0.000 description 21
- 238000001356 surgical procedure Methods 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 20
- 102000001253 Protein Kinase Human genes 0.000 description 20
- 102100040247 Tumor necrosis factor Human genes 0.000 description 20
- 125000003275 alpha amino acid group Chemical group 0.000 description 20
- 108060006633 protein kinase Proteins 0.000 description 20
- 229950000815 veltuzumab Drugs 0.000 description 20
- 238000002347 injection Methods 0.000 description 19
- 239000007924 injection Substances 0.000 description 19
- 210000002540 macrophage Anatomy 0.000 description 19
- 230000001338 necrotic effect Effects 0.000 description 19
- BPWATVWOHQZVRP-NSHDSACASA-N Cl-Amidine Chemical compound ClCC(=N)NCCC[C@@H](C(=O)N)NC(=O)C1=CC=CC=C1 BPWATVWOHQZVRP-NSHDSACASA-N 0.000 description 18
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 18
- 210000001744 T-lymphocyte Anatomy 0.000 description 18
- 230000003472 neutralizing effect Effects 0.000 description 18
- 108010028275 Leukocyte Elastase Proteins 0.000 description 17
- 241001529936 Murinae Species 0.000 description 17
- 102100033174 Neutrophil elastase Human genes 0.000 description 17
- 210000003719 b-lymphocyte Anatomy 0.000 description 17
- 239000006228 supernatant Substances 0.000 description 17
- 108010047041 Complementarity Determining Regions Proteins 0.000 description 16
- 206010061218 Inflammation Diseases 0.000 description 16
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 16
- 230000004054 inflammatory process Effects 0.000 description 16
- 150000001413 amino acids Chemical class 0.000 description 15
- 210000000987 immune system Anatomy 0.000 description 15
- 238000012744 immunostaining Methods 0.000 description 15
- 230000008569 process Effects 0.000 description 15
- 230000001105 regulatory effect Effects 0.000 description 15
- 206010018364 Glomerulonephritis Diseases 0.000 description 14
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 14
- 230000001154 acute effect Effects 0.000 description 14
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 14
- 229940024606 amino acid Drugs 0.000 description 14
- 210000001707 glomerular endothelial cell Anatomy 0.000 description 14
- 210000004408 hybridoma Anatomy 0.000 description 14
- 238000010172 mouse model Methods 0.000 description 14
- 210000000557 podocyte Anatomy 0.000 description 14
- 102000004196 processed proteins & peptides Human genes 0.000 description 14
- 230000001988 toxicity Effects 0.000 description 14
- 231100000419 toxicity Toxicity 0.000 description 14
- PHEDXBVPIONUQT-UHFFFAOYSA-N Cocarcinogen A1 Natural products CCCCCCCCCCCCCC(=O)OC1C(C)C2(O)C3C=C(C)C(=O)C3(O)CC(CO)=CC2C2C1(OC(C)=O)C2(C)C PHEDXBVPIONUQT-UHFFFAOYSA-N 0.000 description 13
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 13
- 102000014150 Interferons Human genes 0.000 description 13
- 108010050904 Interferons Proteins 0.000 description 13
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 13
- 210000004369 blood Anatomy 0.000 description 13
- 239000008280 blood Substances 0.000 description 13
- 210000002889 endothelial cell Anatomy 0.000 description 13
- 125000005647 linker group Chemical group 0.000 description 13
- PHEDXBVPIONUQT-RGYGYFBISA-N phorbol 13-acetate 12-myristate Chemical compound C([C@]1(O)C(=O)C(C)=C[C@H]1[C@@]1(O)[C@H](C)[C@H]2OC(=O)CCCCCCCCCCCCC)C(CO)=C[C@H]1[C@H]1[C@]2(OC(C)=O)C1(C)C PHEDXBVPIONUQT-RGYGYFBISA-N 0.000 description 13
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 12
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 12
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 12
- 231100000433 cytotoxic Toxicity 0.000 description 12
- 230000001472 cytotoxic effect Effects 0.000 description 12
- 239000011159 matrix material Substances 0.000 description 12
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 11
- 206010021143 Hypoxia Diseases 0.000 description 11
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 11
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 11
- 230000030833 cell death Effects 0.000 description 11
- 239000000032 diagnostic agent Substances 0.000 description 11
- 229940039227 diagnostic agent Drugs 0.000 description 11
- 208000005017 glioblastoma Diseases 0.000 description 11
- 230000003993 interaction Effects 0.000 description 11
- 238000002823 phage display Methods 0.000 description 11
- 231100000331 toxic Toxicity 0.000 description 11
- 230000002588 toxic effect Effects 0.000 description 11
- TXUWMXQFNYDOEZ-UHFFFAOYSA-N 5-(1H-indol-3-ylmethyl)-3-methyl-2-sulfanylidene-4-imidazolidinone Chemical compound O=C1N(C)C(=S)NC1CC1=CNC2=CC=CC=C12 TXUWMXQFNYDOEZ-UHFFFAOYSA-N 0.000 description 10
- 208000003918 Acute Kidney Tubular Necrosis Diseases 0.000 description 10
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 10
- 108010036949 Cyclosporine Proteins 0.000 description 10
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 10
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 10
- 229960001265 ciclosporin Drugs 0.000 description 10
- 230000007954 hypoxia Effects 0.000 description 10
- 208000015181 infectious disease Diseases 0.000 description 10
- 238000006386 neutralization reaction Methods 0.000 description 10
- 229960004641 rituximab Drugs 0.000 description 10
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 9
- 108010077544 Chromatin Proteins 0.000 description 9
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 9
- 125000000539 amino acid group Chemical group 0.000 description 9
- 210000003483 chromatin Anatomy 0.000 description 9
- 229930182912 cyclosporin Natural products 0.000 description 9
- UJHBVMHOBZBWMX-UHFFFAOYSA-N ferrostatin-1 Chemical compound NC1=CC(C(=O)OCC)=CC=C1NC1CCCCC1 UJHBVMHOBZBWMX-UHFFFAOYSA-N 0.000 description 9
- 230000028993 immune response Effects 0.000 description 9
- 210000000265 leukocyte Anatomy 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000007246 mechanism Effects 0.000 description 9
- 229910052757 nitrogen Inorganic materials 0.000 description 9
- 230000004044 response Effects 0.000 description 9
- 238000001262 western blot Methods 0.000 description 9
- 208000023275 Autoimmune disease Diseases 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 8
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 8
- 210000005006 adaptive immune system Anatomy 0.000 description 8
- 150000001720 carbohydrates Chemical class 0.000 description 8
- 229940109239 creatinine Drugs 0.000 description 8
- 230000003013 cytotoxicity Effects 0.000 description 8
- 231100000135 cytotoxicity Toxicity 0.000 description 8
- 230000001419 dependent effect Effects 0.000 description 8
- 229950009760 epratuzumab Drugs 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 230000006870 function Effects 0.000 description 8
- 210000003904 glomerular cell Anatomy 0.000 description 8
- 230000005847 immunogenicity Effects 0.000 description 8
- 238000011534 incubation Methods 0.000 description 8
- 230000006698 induction Effects 0.000 description 8
- 229940047124 interferons Drugs 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 230000037361 pathway Effects 0.000 description 8
- 210000001539 phagocyte Anatomy 0.000 description 8
- 102000005962 receptors Human genes 0.000 description 8
- 108020003175 receptors Proteins 0.000 description 8
- 230000009467 reduction Effects 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- 230000010024 tubular injury Effects 0.000 description 8
- 208000037978 tubular injury Diseases 0.000 description 8
- 239000004475 Arginine Substances 0.000 description 7
- 238000002965 ELISA Methods 0.000 description 7
- 108010049003 Fibrinogen Proteins 0.000 description 7
- 102000008946 Fibrinogen Human genes 0.000 description 7
- 241000282412 Homo Species 0.000 description 7
- 108060003951 Immunoglobulin Proteins 0.000 description 7
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 7
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 7
- 241001465754 Metazoa Species 0.000 description 7
- 208000013901 Nephropathies and tubular disease Diseases 0.000 description 7
- 230000003213 activating effect Effects 0.000 description 7
- 235000004279 alanine Nutrition 0.000 description 7
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 7
- 210000004556 brain Anatomy 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- 239000003636 conditioned culture medium Substances 0.000 description 7
- 230000021615 conjugation Effects 0.000 description 7
- 210000004443 dendritic cell Anatomy 0.000 description 7
- 229940012952 fibrinogen Drugs 0.000 description 7
- 230000036039 immunity Effects 0.000 description 7
- 238000003125 immunofluorescent labeling Methods 0.000 description 7
- 102000018358 immunoglobulin Human genes 0.000 description 7
- 230000001976 improved effect Effects 0.000 description 7
- 230000015788 innate immune response Effects 0.000 description 7
- 238000011862 kidney biopsy Methods 0.000 description 7
- 239000001301 oxygen Substances 0.000 description 7
- 229910052760 oxygen Inorganic materials 0.000 description 7
- 244000052769 pathogen Species 0.000 description 7
- 238000011002 quantification Methods 0.000 description 7
- 238000013042 tunel staining Methods 0.000 description 7
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 6
- 102100035360 Cerebellar degeneration-related antigen 1 Human genes 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical class CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 6
- 101000737793 Homo sapiens Cerebellar degeneration-related antigen 1 Proteins 0.000 description 6
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 6
- 231100000416 LDH assay Toxicity 0.000 description 6
- 108010029485 Protein Isoforms Proteins 0.000 description 6
- 102000001708 Protein Isoforms Human genes 0.000 description 6
- 102000002689 Toll-like receptor Human genes 0.000 description 6
- 108020000411 Toll-like receptor Proteins 0.000 description 6
- 230000000903 blocking effect Effects 0.000 description 6
- 230000006329 citrullination Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 239000000539 dimer Substances 0.000 description 6
- 210000005086 glomerual capillary Anatomy 0.000 description 6
- 230000000302 ischemic effect Effects 0.000 description 6
- 238000002843 lactate dehydrogenase assay Methods 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 6
- 229910052751 metal Inorganic materials 0.000 description 6
- 239000002184 metal Substances 0.000 description 6
- 229930182817 methionine Natural products 0.000 description 6
- 230000002956 necrotizing effect Effects 0.000 description 6
- 230000007170 pathology Effects 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 101100406797 Arabidopsis thaliana PAD4 gene Proteins 0.000 description 5
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 5
- 101150094373 Padi4 gene Proteins 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 5
- 102100035731 Protein-arginine deiminase type-4 Human genes 0.000 description 5
- 101100182713 Rattus norvegicus Ly6b gene Proteins 0.000 description 5
- 206010040070 Septic Shock Diseases 0.000 description 5
- 208000024248 Vascular System injury Diseases 0.000 description 5
- 208000012339 Vascular injury Diseases 0.000 description 5
- 241001416177 Vicugna pacos Species 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000001143 conditioned effect Effects 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 238000006471 dimerization reaction Methods 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 238000011156 evaluation Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 238000003364 immunohistochemistry Methods 0.000 description 5
- 230000002757 inflammatory effect Effects 0.000 description 5
- 229940079322 interferon Drugs 0.000 description 5
- 229940047122 interleukins Drugs 0.000 description 5
- 230000002147 killing effect Effects 0.000 description 5
- 230000003902 lesion Effects 0.000 description 5
- 239000003446 ligand Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000003032 molecular docking Methods 0.000 description 5
- 210000004940 nucleus Anatomy 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 201000001474 proteinuria Diseases 0.000 description 5
- 230000007115 recruitment Effects 0.000 description 5
- 238000012552 review Methods 0.000 description 5
- 230000036303 septic shock Effects 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 239000004575 stone Substances 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 125000003396 thiol group Chemical group [H]S* 0.000 description 5
- 230000001960 triggered effect Effects 0.000 description 5
- 239000004474 valine Substances 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- XGWFJBFNAQHLEF-UHFFFAOYSA-N 9-anthroic acid Chemical compound C1=CC=C2C(C(=O)O)=C(C=CC=C3)C3=CC2=C1 XGWFJBFNAQHLEF-UHFFFAOYSA-N 0.000 description 4
- 102000004506 Blood Proteins Human genes 0.000 description 4
- 108010017384 Blood Proteins Proteins 0.000 description 4
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 101150013553 CD40 gene Proteins 0.000 description 4
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 239000004472 Lysine Substances 0.000 description 4
- 102000043129 MHC class I family Human genes 0.000 description 4
- 108091054437 MHC class I family Proteins 0.000 description 4
- 102000043131 MHC class II family Human genes 0.000 description 4
- 108091054438 MHC class II family Proteins 0.000 description 4
- 108700018351 Major Histocompatibility Complex Proteins 0.000 description 4
- 241000699660 Mus musculus Species 0.000 description 4
- 102000003896 Myeloperoxidases Human genes 0.000 description 4
- 108090000235 Myeloperoxidases Proteins 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 206010061481 Renal injury Diseases 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- 230000024932 T cell mediated immunity Effects 0.000 description 4
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 4
- 230000004721 adaptive immunity Effects 0.000 description 4
- 230000033115 angiogenesis Effects 0.000 description 4
- 210000004979 bone marrow derived macrophage Anatomy 0.000 description 4
- 210000001736 capillary Anatomy 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 4
- 239000002738 chelating agent Substances 0.000 description 4
- 230000015271 coagulation Effects 0.000 description 4
- 238000005345 coagulation Methods 0.000 description 4
- 238000011109 contamination Methods 0.000 description 4
- 230000007123 defense Effects 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 230000003111 delayed effect Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 208000009190 disseminated intravascular coagulation Diseases 0.000 description 4
- 230000003511 endothelial effect Effects 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- 235000013922 glutamic acid Nutrition 0.000 description 4
- 239000004220 glutamic acid Substances 0.000 description 4
- 210000002865 immune cell Anatomy 0.000 description 4
- 229940127121 immunoconjugate Drugs 0.000 description 4
- 230000002163 immunogen Effects 0.000 description 4
- 230000003308 immunostimulating effect Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 208000037806 kidney injury Diseases 0.000 description 4
- 230000000670 limiting effect Effects 0.000 description 4
- 150000002739 metals Chemical class 0.000 description 4
- 230000000813 microbial effect Effects 0.000 description 4
- 229950003734 milatuzumab Drugs 0.000 description 4
- 210000001616 monocyte Anatomy 0.000 description 4
- 230000021597 necroptosis Effects 0.000 description 4
- 210000000496 pancreas Anatomy 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000001717 pathogenic effect Effects 0.000 description 4
- 210000004180 plasmocyte Anatomy 0.000 description 4
- 206010039073 rheumatoid arthritis Diseases 0.000 description 4
- 239000004055 small Interfering RNA Substances 0.000 description 4
- 238000007619 statistical method Methods 0.000 description 4
- PVYJZLYGTZKPJE-UHFFFAOYSA-N streptonigrin Chemical compound C=1C=C2C(=O)C(OC)=C(N)C(=O)C2=NC=1C(C=1N)=NC(C(O)=O)=C(C)C=1C1=CC=C(OC)C(OC)=C1O PVYJZLYGTZKPJE-UHFFFAOYSA-N 0.000 description 4
- 230000020382 suppression by virus of host antigen processing and presentation of peptide antigen via MHC class I Effects 0.000 description 4
- 230000009885 systemic effect Effects 0.000 description 4
- 238000011830 transgenic mouse model Methods 0.000 description 4
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 4
- 230000002792 vascular Effects 0.000 description 4
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 241000024188 Andala Species 0.000 description 3
- 102100029470 Apolipoprotein E Human genes 0.000 description 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 3
- 206010053567 Coagulopathies Diseases 0.000 description 3
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 3
- 102000008130 Cyclic AMP-Dependent Protein Kinases Human genes 0.000 description 3
- 108010049894 Cyclic AMP-Dependent Protein Kinases Proteins 0.000 description 3
- 238000000116 DAPI staining Methods 0.000 description 3
- 102000053602 DNA Human genes 0.000 description 3
- 108010073385 Fibrin Proteins 0.000 description 3
- 102000009123 Fibrin Human genes 0.000 description 3
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 3
- 102000003886 Glycoproteins Human genes 0.000 description 3
- 108090000288 Glycoproteins Proteins 0.000 description 3
- 101000771674 Homo sapiens Apolipoprotein E Proteins 0.000 description 3
- 101000935587 Homo sapiens Flavin reductase (NADPH) Proteins 0.000 description 3
- 101000914484 Homo sapiens T-lymphocyte activation antigen CD80 Proteins 0.000 description 3
- 108010044467 Isoenzymes Proteins 0.000 description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 3
- 208000034486 Multi-organ failure Diseases 0.000 description 3
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 241001494479 Pecora Species 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
- 102000003992 Peroxidases Human genes 0.000 description 3
- 206010057249 Phagocytosis Diseases 0.000 description 3
- 102000000874 Pyrin Domain-Containing 3 Protein NLR Family Human genes 0.000 description 3
- 108010001946 Pyrin Domain-Containing 3 Protein NLR Family Proteins 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- 108010083644 Ribonucleases Proteins 0.000 description 3
- 102000006382 Ribonucleases Human genes 0.000 description 3
- 239000002262 Schiff base Substances 0.000 description 3
- 150000004753 Schiff bases Chemical class 0.000 description 3
- 102100027222 T-lymphocyte activation antigen CD80 Human genes 0.000 description 3
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 description 3
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 3
- 239000004473 Threonine Substances 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 230000003044 adaptive effect Effects 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 230000003042 antagnostic effect Effects 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 230000000890 antigenic effect Effects 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 235000009582 asparagine Nutrition 0.000 description 3
- 229960001230 asparagine Drugs 0.000 description 3
- 229940009098 aspartate Drugs 0.000 description 3
- 125000004429 atom Chemical group 0.000 description 3
- 229920001222 biopolymer Polymers 0.000 description 3
- 244000309466 calf Species 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 239000013522 chelant Substances 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 3
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 3
- 210000003162 effector t lymphocyte Anatomy 0.000 description 3
- 230000001747 exhibiting effect Effects 0.000 description 3
- 210000001723 extracellular space Anatomy 0.000 description 3
- 229950003499 fibrin Drugs 0.000 description 3
- 239000012737 fresh medium Substances 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 210000001282 glomerular podocyte Anatomy 0.000 description 3
- 229930195712 glutamate Natural products 0.000 description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 3
- 210000002443 helper t lymphocyte Anatomy 0.000 description 3
- 230000028996 humoral immune response Effects 0.000 description 3
- 206010020718 hyperplasia Diseases 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 229960000310 isoleucine Drugs 0.000 description 3
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 3
- 208000005430 kidney cortex necrosis Diseases 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 3
- 230000002297 mitogenic effect Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 210000002864 mononuclear phagocyte Anatomy 0.000 description 3
- 208000029744 multiple organ dysfunction syndrome Diseases 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 230000008816 organ damage Effects 0.000 description 3
- 229910000489 osmium tetroxide Inorganic materials 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 210000003658 parietal epithelial cell Anatomy 0.000 description 3
- 229960003330 pentetic acid Drugs 0.000 description 3
- 229940111202 pepsin Drugs 0.000 description 3
- 108040007629 peroxidase activity proteins Proteins 0.000 description 3
- 230000008782 phagocytosis Effects 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 230000002285 radioactive effect Effects 0.000 description 3
- 238000003753 real-time PCR Methods 0.000 description 3
- 150000003335 secondary amines Chemical class 0.000 description 3
- 230000003248 secreting effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 210000001541 thymus gland Anatomy 0.000 description 3
- 208000037816 tissue injury Diseases 0.000 description 3
- 230000009261 transgenic effect Effects 0.000 description 3
- 230000008733 trauma Effects 0.000 description 3
- 210000005239 tubule Anatomy 0.000 description 3
- 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 2
- FYGDTMLNYKFZSV-URKRLVJHSA-N (2s,3r,4s,5s,6r)-2-[(2r,4r,5r,6s)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2r,4r,5r,6s)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1[C@@H](CO)O[C@@H](OC2[C@H](O[C@H](O)[C@H](O)[C@H]2O)CO)[C@H](O)[C@H]1O FYGDTMLNYKFZSV-URKRLVJHSA-N 0.000 description 2
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 description 2
- 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 2
- GRMMWMKNOJVAQP-MERQFXBCSA-N 2-[[(2s)-1-amino-5-[(1-amino-2-fluoroethylidene)amino]-1-oxopentan-2-yl]carbamoyl]benzoic acid;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.FCC(=N)NCCC[C@@H](C(=O)N)NC(=O)C1=CC=CC=C1C(O)=O GRMMWMKNOJVAQP-MERQFXBCSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 206010001580 Albuminuria Diseases 0.000 description 2
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 2
- 101100339431 Arabidopsis thaliana HMGB2 gene Proteins 0.000 description 2
- 229920002498 Beta-glucan Polymers 0.000 description 2
- VYLJAYXZTOTZRR-BTPDVQIOSA-N CC(C)(O)[C@H]1CC[C@@]2(C)[C@H]1CC[C@]1(C)[C@@H]2CC[C@@H]2[C@@]3(C)CCCC(C)(C)[C@@H]3[C@@H](O)[C@H](O)[C@@]12C Chemical compound CC(C)(O)[C@H]1CC[C@@]2(C)[C@H]1CC[C@]1(C)[C@@H]2CC[C@@H]2[C@@]3(C)CCCC(C)(C)[C@@H]3[C@@H](O)[C@H](O)[C@@]12C VYLJAYXZTOTZRR-BTPDVQIOSA-N 0.000 description 2
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 206010009944 Colon cancer Diseases 0.000 description 2
- 102000007644 Colony-Stimulating Factors Human genes 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 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 2
- 108700039887 Essential Genes Proteins 0.000 description 2
- 108010054265 Factor VIIa Proteins 0.000 description 2
- 208000022461 Glomerular disease Diseases 0.000 description 2
- 108700010013 HMGB1 Proteins 0.000 description 2
- 101150021904 HMGB1 gene Proteins 0.000 description 2
- 102100037907 High mobility group protein B1 Human genes 0.000 description 2
- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 description 2
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 2
- 101001067880 Homo sapiens Histone H4 Proteins 0.000 description 2
- 101001046686 Homo sapiens Integrin alpha-M Proteins 0.000 description 2
- 101000599852 Homo sapiens Intercellular adhesion molecule 1 Proteins 0.000 description 2
- 101001089266 Homo sapiens Receptor-interacting serine/threonine-protein kinase 3 Proteins 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 208000029462 Immunodeficiency disease Diseases 0.000 description 2
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 2
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 2
- 102100022338 Integrin alpha-M Human genes 0.000 description 2
- 102100037877 Intercellular adhesion molecule 1 Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 2
- 206010067125 Liver injury Diseases 0.000 description 2
- 231100000002 MTT assay Toxicity 0.000 description 2
- 238000000134 MTT assay Methods 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
- 208000010718 Multiple Organ Failure Diseases 0.000 description 2
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 2
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 2
- 102100023195 Nephrin Human genes 0.000 description 2
- 241001181114 Neta Species 0.000 description 2
- 102000043276 Oncogene Human genes 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 2
- 108090000526 Papain Proteins 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 2
- 108010094028 Prothrombin Proteins 0.000 description 2
- 102100027378 Prothrombin Human genes 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 102100022501 Receptor-interacting serine/threonine-protein kinase 1 Human genes 0.000 description 2
- 102100033729 Receptor-interacting serine/threonine-protein kinase 3 Human genes 0.000 description 2
- 208000035415 Reinfection Diseases 0.000 description 2
- 241000219061 Rheum Species 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 238000000692 Student's t-test Methods 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 208000034841 Thrombotic Microangiopathies Diseases 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 206010052779 Transplant rejections Diseases 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000012452 Xenomouse strains Methods 0.000 description 2
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 2
- 230000008649 adaptation response Effects 0.000 description 2
- 230000033289 adaptive immune response Effects 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 125000003277 amino group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000003172 anti-dna Effects 0.000 description 2
- 230000000690 anti-lymphoma Effects 0.000 description 2
- 239000000074 antisense oligonucleotide Substances 0.000 description 2
- 238000012230 antisense oligonucleotides Methods 0.000 description 2
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 2
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 208000006673 asthma Diseases 0.000 description 2
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 210000002665 bowman capsule Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 150000001718 carbodiimides Chemical class 0.000 description 2
- 229960004562 carboplatin Drugs 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 230000005779 cell damage Effects 0.000 description 2
- 208000037887 cell injury Diseases 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000005482 chemotactic factor Substances 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 229960002173 citrulline Drugs 0.000 description 2
- 235000013477 citrulline Nutrition 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 229940047120 colony stimulating factors Drugs 0.000 description 2
- 230000024203 complement activation Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 239000002872 contrast media Substances 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000003246 corticosteroid Substances 0.000 description 2
- 229960001334 corticosteroids Drugs 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 108010057085 cytokine receptors Proteins 0.000 description 2
- 102000003675 cytokine receptors Human genes 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 229940127089 cytotoxic agent Drugs 0.000 description 2
- 239000002254 cytotoxic agent Substances 0.000 description 2
- 231100000599 cytotoxic agent Toxicity 0.000 description 2
- 238000002784 cytotoxicity assay Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000001493 electron microscopy Methods 0.000 description 2
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 229940012414 factor viia Drugs 0.000 description 2
- 230000004806 ferroptosis Effects 0.000 description 2
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 231100000852 glomerular disease Toxicity 0.000 description 2
- 231100000853 glomerular lesion Toxicity 0.000 description 2
- 230000010247 heart contraction Effects 0.000 description 2
- 210000003630 histaminocyte Anatomy 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- VYLJAYXZTOTZRR-UHFFFAOYSA-N hopane-6alpha,7beta,22-triol Natural products C12CCC3C4(C)CCCC(C)(C)C4C(O)C(O)C3(C)C1(C)CCC1C2(C)CCC1C(C)(O)C VYLJAYXZTOTZRR-UHFFFAOYSA-N 0.000 description 2
- 230000004727 humoral immunity Effects 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 125000004435 hydrogen atom Chemical class [H]* 0.000 description 2
- 150000002466 imines Chemical class 0.000 description 2
- 208000026278 immune system disease Diseases 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000001506 immunosuppresive effect Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 208000027866 inflammatory disease Diseases 0.000 description 2
- 229960000598 infliximab Drugs 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000001361 intraarterial administration Methods 0.000 description 2
- 238000010253 intravenous injection Methods 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 230000003907 kidney function Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000004324 lymphatic system Anatomy 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 239000012516 mab select resin Substances 0.000 description 2
- 238000002595 magnetic resonance imaging Methods 0.000 description 2
- 229960001428 mercaptopurine Drugs 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
- 230000009456 molecular mechanism Effects 0.000 description 2
- 201000006417 multiple sclerosis Diseases 0.000 description 2
- 238000013059 nephrectomy Methods 0.000 description 2
- 108010027531 nephrin Proteins 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000012285 osmium tetroxide Substances 0.000 description 2
- 229960001592 paclitaxel Drugs 0.000 description 2
- 229940055729 papain Drugs 0.000 description 2
- 235000019834 papain Nutrition 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 230000005298 paramagnetic effect Effects 0.000 description 2
- 230000007310 pathophysiology Effects 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 235000019419 proteases Nutrition 0.000 description 2
- 108010061338 ranpirnase Proteins 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000006894 reductive elimination reaction Methods 0.000 description 2
- 210000002254 renal artery Anatomy 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 238000004626 scanning electron microscopy Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 208000002491 severe combined immunodeficiency Diseases 0.000 description 2
- 230000019491 signal transduction Effects 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 239000002356 single layer Substances 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000010561 standard procedure Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 2
- 238000012353 t test Methods 0.000 description 2
- 230000008685 targeting Effects 0.000 description 2
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 description 2
- 230000000451 tissue damage Effects 0.000 description 2
- 231100000827 tissue damage Toxicity 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000003151 transfection method Methods 0.000 description 2
- 230000014616 translation Effects 0.000 description 2
- 238000002054 transplantation Methods 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- 210000004926 tubular epithelial cell Anatomy 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-N 0.000 description 1
- PXOMSWXCVZBBIV-PQKSKRJKSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4S,6R)-4-amino-2-methyl-6-[[(1S,3S)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1H-tetracen-1-yl]oxy]oxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound C[C@H]1[C@@H]([C@H](C[C@@H](O1)O[C@H]2C[C@@](CC3=C2C(=C4C(=C3O)C(=O)C5=C(C4=O)C(=CC=C5)OC)O)(C(=O)CO)O)N)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)C(=O)O)O)O)O PXOMSWXCVZBBIV-PQKSKRJKSA-N 0.000 description 1
- APOKYMYZOKIMLM-LUMVZWMBSA-N (2s,3s,4s,5r,6s)-3,4,5-trihydroxy-6-[4-[[(2s,3s,4s,6r)-3-hydroxy-2-methyl-6-[[(1s,3s)-3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-2,4-dihydro-1h-tetracen-1-yl]oxy]oxan-4-yl]carbamoyloxymethyl]-2-nitrophenoxy]oxane-2-carboxylic acid Chemical compound N([C@H]1C[C@@H](O[C@@H](C)[C@H]1O)O[C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)C(=O)OCC(C=C1[N+]([O-])=O)=CC=C1O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O APOKYMYZOKIMLM-LUMVZWMBSA-N 0.000 description 1
- URCVASXWNJQAEH-HDWVWLDDSA-N (2s,3s,4s,5r,6s)-6-[4-[(5s,5ar,8ar,9r)-5-[[(2r,4ar,6r,7r,8r,8as)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-8-oxo-5a,6,8a,9-tetrahydro-5h-[2]benzofuro[5,6-f][1,3]benzodioxol-9-yl]-2,6-dimethoxyphenoxy]-3,4,5-trihydrox Chemical compound COC1=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=CC(OC)=C1O[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O URCVASXWNJQAEH-HDWVWLDDSA-N 0.000 description 1
- ONJZYZYZIKTIEG-CFBQITSMSA-N (3s,6s,9r,10r,11s,12s,13e,15e,18s,21s)-18-[(2e,4e,8s,9s)-10-[(2s,3r,4s,5s,6r,9s,11s)-9-ethyl-4-hydroxy-3,5,11-trimethyl-8-oxo-1-oxa-7-azaspiro[5.5]undecan-2-yl]-9-hydroxy-8-methyldeca-2,4-dien-2-yl]-10,12-dihydroxy-3-[(3-hydroxyphenyl)methyl]-11-methyl-9- Chemical compound N1C(=O)[C@@H](CC)C[C@H](C)[C@]21[C@@H](C)[C@@H](O)[C@@H](C)[C@H](C[C@H](O)[C@@H](C)CC\C=C\C=C(/C)[C@H]1OC(=O)[C@@H]3CCCN(N3)C(=O)[C@H](CC=3C=C(O)C=CC=3)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CCC(C)=O)[C@H](O)[C@@H](C)[C@@H](O)/C=C/C=C/C1)O2 ONJZYZYZIKTIEG-CFBQITSMSA-N 0.000 description 1
- YJGVMLPVUAXIQN-LGWHJFRWSA-N (5s,5ar,8ar,9r)-5-hydroxy-9-(3,4,5-trimethoxyphenyl)-5a,6,8a,9-tetrahydro-5h-[2]benzofuro[5,6-f][1,3]benzodioxol-8-one Chemical compound COC1=C(OC)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O)[C@@H]3[C@@H]2C(OC3)=O)=C1 YJGVMLPVUAXIQN-LGWHJFRWSA-N 0.000 description 1
- FPVKHBSQESCIEP-UHFFFAOYSA-N (8S)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol Natural products C1C(O)C(CO)OC1N1C(NC=NCC2O)=C2N=C1 FPVKHBSQESCIEP-UHFFFAOYSA-N 0.000 description 1
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 1
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- TUSDEZXZIZRFGC-UHFFFAOYSA-N 1-O-galloyl-3,6-(R)-HHDP-beta-D-glucose Natural products OC1C(O2)COC(=O)C3=CC(O)=C(O)C(O)=C3C3=C(O)C(O)=C(O)C=C3C(=O)OC1C(O)C2OC(=O)C1=CC(O)=C(O)C(O)=C1 TUSDEZXZIZRFGC-UHFFFAOYSA-N 0.000 description 1
- XQUPVDVFXZDTLT-UHFFFAOYSA-N 1-[4-[[4-(2,5-dioxopyrrol-1-yl)phenyl]methyl]phenyl]pyrrole-2,5-dione Chemical compound O=C1C=CC(=O)N1C(C=C1)=CC=C1CC1=CC=C(N2C(C=CC2=O)=O)C=C1 XQUPVDVFXZDTLT-UHFFFAOYSA-N 0.000 description 1
- 102100025573 1-alkyl-2-acetylglycerophosphocholine esterase Human genes 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- HAWSQZCWOQZXHI-FQEVSTJZSA-N 10-Hydroxycamptothecin Chemical compound C1=C(O)C=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 HAWSQZCWOQZXHI-FQEVSTJZSA-N 0.000 description 1
- MISZALMBODQYFT-URVXVIKDSA-N 125-69-9 Chemical compound Br.C([C@@H]12)CCC[C@]11CCN(C)[C@H]2CC2=CC=C(OC)C=C21 MISZALMBODQYFT-URVXVIKDSA-N 0.000 description 1
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- VXVVJOJJOSRVDG-MERQFXBCSA-N 2-[[(2s)-1-amino-5-[(1-amino-2-chloroethylidene)amino]-1-oxopentan-2-yl]carbamoyl]benzoic acid;2,2,2-trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.ClCC(=N)NCCC[C@@H](C(=O)N)NC(=O)C1=CC=CC=C1C(O)=O VXVVJOJJOSRVDG-MERQFXBCSA-N 0.000 description 1
- RTQWWZBSTRGEAV-PKHIMPSTSA-N 2-[[(2s)-2-[bis(carboxymethyl)amino]-3-[4-(methylcarbamoylamino)phenyl]propyl]-[2-[bis(carboxymethyl)amino]propyl]amino]acetic acid Chemical compound CNC(=O)NC1=CC=C(C[C@@H](CN(CC(C)N(CC(O)=O)CC(O)=O)CC(O)=O)N(CC(O)=O)CC(O)=O)C=C1 RTQWWZBSTRGEAV-PKHIMPSTSA-N 0.000 description 1
- FZDFGHZZPBUTGP-UHFFFAOYSA-N 2-[[2-[bis(carboxymethyl)amino]-3-(4-isothiocyanatophenyl)propyl]-[2-[bis(carboxymethyl)amino]propyl]amino]acetic acid Chemical compound OC(=O)CN(CC(O)=O)C(C)CN(CC(O)=O)CC(N(CC(O)=O)CC(O)=O)CC1=CC=C(N=C=S)C=C1 FZDFGHZZPBUTGP-UHFFFAOYSA-N 0.000 description 1
- BUOYTFVLNZIELF-UHFFFAOYSA-N 2-phenyl-1h-indole-4,6-dicarboximidamide Chemical compound N1C2=CC(C(=N)N)=CC(C(N)=N)=C2C=C1C1=CC=CC=C1 BUOYTFVLNZIELF-UHFFFAOYSA-N 0.000 description 1
- YIMDLWDNDGKDTJ-QLKYHASDSA-N 3'-deamino-3'-(3-cyanomorpholin-4-yl)doxorubicin Chemical compound N1([C@H]2C[C@@H](O[C@@H](C)[C@H]2O)O[C@H]2C[C@@](O)(CC=3C(O)=C4C(=O)C=5C=CC=C(C=5C(=O)C4=C(O)C=32)OC)C(=O)CO)CCOCC1C#N YIMDLWDNDGKDTJ-QLKYHASDSA-N 0.000 description 1
- JDXQWYKOKYUQDN-UHFFFAOYSA-N 3-hydroxypyrrolidine-2,5-dione Chemical class OC1CC(=O)NC1=O JDXQWYKOKYUQDN-UHFFFAOYSA-N 0.000 description 1
- IDPUKCWIGUEADI-UHFFFAOYSA-N 5-[bis(2-chloroethyl)amino]uracil Chemical compound ClCCN(CCCl)C1=CNC(=O)NC1=O IDPUKCWIGUEADI-UHFFFAOYSA-N 0.000 description 1
- WYWHKKSPHMUBEB-UHFFFAOYSA-N 6-Mercaptoguanine Natural products N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 1
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 1
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 1
- FJHBVJOVLFPMQE-QFIPXVFZSA-N 7-Ethyl-10-Hydroxy-Camptothecin Chemical compound C1=C(O)C=C2C(CC)=C(CN3C(C4=C([C@@](C(=O)OC4)(O)CC)C=C33)=O)C3=NC2=C1 FJHBVJOVLFPMQE-QFIPXVFZSA-N 0.000 description 1
- 102100024049 A-kinase anchor protein 13 Human genes 0.000 description 1
- 108010066676 Abrin Proteins 0.000 description 1
- 208000010444 Acidosis Diseases 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 101710092462 Alpha-hemolysin Proteins 0.000 description 1
- 101710197219 Alpha-toxin Proteins 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 108010089414 Anaphylatoxins Proteins 0.000 description 1
- 235000002198 Annona diversifolia Nutrition 0.000 description 1
- 101710099705 Anti-lipopolysaccharide factor Proteins 0.000 description 1
- 108010024976 Asparaginase Proteins 0.000 description 1
- 101710192393 Attachment protein G3P Proteins 0.000 description 1
- 102100022005 B-lymphocyte antigen CD20 Human genes 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 235000018185 Betula X alpestris Nutrition 0.000 description 1
- 235000018212 Betula X uliginosa Nutrition 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 description 1
- 102100021943 C-C motif chemokine 2 Human genes 0.000 description 1
- 102100039398 C-X-C motif chemokine 2 Human genes 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 238000011814 C57BL/6N mouse Methods 0.000 description 1
- HAWSQZCWOQZXHI-UHFFFAOYSA-N CPT-OH Natural products C1=C(O)C=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 HAWSQZCWOQZXHI-UHFFFAOYSA-N 0.000 description 1
- FVLVBPDQNARYJU-XAHDHGMMSA-N C[C@H]1CCC(CC1)NC(=O)N(CCCl)N=O Chemical compound C[C@H]1CCC(CC1)NC(=O)N(CCCl)N=O FVLVBPDQNARYJU-XAHDHGMMSA-N 0.000 description 1
- 206010006895 Cachexia Diseases 0.000 description 1
- 102000004631 Calcineurin Human genes 0.000 description 1
- 108010042955 Calcineurin Proteins 0.000 description 1
- 241000282832 Camelidae Species 0.000 description 1
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710169873 Capsid protein G8P Proteins 0.000 description 1
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- JWBOIMRXGHLCPP-UHFFFAOYSA-N Chloditan Chemical compound C=1C=CC=C(Cl)C=1C(C(Cl)Cl)C1=CC=C(Cl)C=C1 JWBOIMRXGHLCPP-UHFFFAOYSA-N 0.000 description 1
- PTOAARAWEBMLNO-KVQBGUIXSA-N Cladribine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 PTOAARAWEBMLNO-KVQBGUIXSA-N 0.000 description 1
- 102000002029 Claudin Human genes 0.000 description 1
- 108050009302 Claudin Proteins 0.000 description 1
- 102100040836 Claudin-1 Human genes 0.000 description 1
- 108090000600 Claudin-1 Proteins 0.000 description 1
- 102100022641 Coagulation factor IX Human genes 0.000 description 1
- 102100023804 Coagulation factor VII Human genes 0.000 description 1
- 102100031162 Collagen alpha-1(XVIII) chain Human genes 0.000 description 1
- 208000001333 Colorectal Neoplasms Diseases 0.000 description 1
- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010078546 Complement C5a Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 239000004971 Cross linker Substances 0.000 description 1
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 108010092160 Dactinomycin Proteins 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- AHCYMLUZIRLXAA-SHYZEUOFSA-N Deoxyuridine 5'-triphosphate Chemical compound O1[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C[C@@H]1N1C(=O)NC(=O)C=C1 AHCYMLUZIRLXAA-SHYZEUOFSA-N 0.000 description 1
- 108010053187 Diphtheria Toxin Proteins 0.000 description 1
- 102000016607 Diphtheria Toxin Human genes 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 208000037487 Endotoxemia Diseases 0.000 description 1
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- 239000001263 FEMA 3042 Substances 0.000 description 1
- 108010076282 Factor IX Proteins 0.000 description 1
- 108010023321 Factor VII Proteins 0.000 description 1
- 108010014173 Factor X Proteins 0.000 description 1
- 206010053172 Fatal outcomes Diseases 0.000 description 1
- 108091006020 Fc-tagged proteins Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000724791 Filamentous phage Species 0.000 description 1
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 229910052688 Gadolinium Inorganic materials 0.000 description 1
- GYHNNYVSQQEPJS-UHFFFAOYSA-N Gallium Chemical compound [Ga] GYHNNYVSQQEPJS-UHFFFAOYSA-N 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
- 208000024869 Goodpasture syndrome Diseases 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 206010018691 Granuloma Diseases 0.000 description 1
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 1
- 208000002250 Hematologic Neoplasms Diseases 0.000 description 1
- 241000545744 Hirudinea Species 0.000 description 1
- 108010025076 Holoenzymes Proteins 0.000 description 1
- 101000833679 Homo sapiens A-kinase anchor protein 13 Proteins 0.000 description 1
- 101000897405 Homo sapiens B-lymphocyte antigen CD20 Proteins 0.000 description 1
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 description 1
- 101000889128 Homo sapiens C-X-C motif chemokine 2 Proteins 0.000 description 1
- 101000777370 Homo sapiens Coiled-coil domain-containing protein 6 Proteins 0.000 description 1
- 101001109145 Homo sapiens Receptor-interacting serine/threonine-protein kinase 1 Proteins 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 101001059454 Homo sapiens Serine/threonine-protein kinase MARK2 Proteins 0.000 description 1
- 101000635804 Homo sapiens Tissue factor Proteins 0.000 description 1
- 101000801228 Homo sapiens Tumor necrosis factor receptor superfamily member 1A Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 102000026633 IL6 Human genes 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- 208000028622 Immune thrombocytopenia Diseases 0.000 description 1
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 1
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 1
- 108700005091 Immunoglobulin Genes Proteins 0.000 description 1
- 102400000022 Insulin-like growth factor II Human genes 0.000 description 1
- 208000008839 Kidney Neoplasms Diseases 0.000 description 1
- 102100020880 Kit ligand Human genes 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N L-cystine Chemical compound [O-]C(=O)[C@@H]([NH3+])CSSC[C@H]([NH3+])C([O-])=O LEVWYRKDKASIDU-IMJSIDKUSA-N 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
- 125000000510 L-tryptophano group Chemical group [H]C1=C([H])C([H])=C2N([H])C([H])=C(C([H])([H])[C@@]([H])(C(O[H])=O)N([H])[*])C2=C1[H] 0.000 description 1
- 241000282838 Lama Species 0.000 description 1
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 description 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 101150058224 MIF gene Proteins 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710156564 Major tail protein Gp23 Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 206010027417 Metabolic acidosis Diseases 0.000 description 1
- 208000009857 Microaneurysm Diseases 0.000 description 1
- 102000029749 Microtubule Human genes 0.000 description 1
- 108091022875 Microtubule Proteins 0.000 description 1
- 108010020004 Microtubule-Associated Proteins Proteins 0.000 description 1
- 102000009664 Microtubule-Associated Proteins Human genes 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 102000013967 Monokines Human genes 0.000 description 1
- 108010050619 Monokines Proteins 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- 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 1
- YDOAWJHYHGBQFI-QHCPKHFHSA-N N-[(1S)-4-[(1-amino-2-chloroethylidene)amino]-1-(1H-benzimidazol-2-yl)butyl]-4-phenylbenzamide Chemical compound N1C(=NC2=C1C=CC=C2)[C@H](CCCNC(CCl)=N)NC(=O)C1=CC=C(C=C1)C1=CC=CC=C1 YDOAWJHYHGBQFI-QHCPKHFHSA-N 0.000 description 1
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 description 1
- 108010057466 NF-kappa B Proteins 0.000 description 1
- 102000003945 NF-kappa B Human genes 0.000 description 1
- 108010025020 Nerve Growth Factor Proteins 0.000 description 1
- 102000007072 Nerve Growth Factors Human genes 0.000 description 1
- 102000007999 Nuclear Proteins Human genes 0.000 description 1
- 108010089610 Nuclear Proteins Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108700022034 Opsonin Proteins Proteins 0.000 description 1
- 206010053159 Organ failure Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 1
- 208000030852 Parasitic disease Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000034038 Pathologic Neovascularization Diseases 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
- LRBQNJMCXXYXIU-PPKXGCFTSA-N Penta-digallate-beta-D-glucose Natural products OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-PPKXGCFTSA-N 0.000 description 1
- 229940083963 Peptide antagonist Drugs 0.000 description 1
- 102100034943 Peptidyl-prolyl cis-trans isomerase F, mitochondrial Human genes 0.000 description 1
- 101710191011 Peptidyl-prolyl cis-trans isomerase F, mitochondrial Proteins 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- 101710124951 Phospholipase C Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- ZYFVNVRFVHJEIU-UHFFFAOYSA-N PicoGreen Chemical compound CN(C)CCCN(CCCN(C)C)C1=CC(=CC2=[N+](C3=CC=CC=C3S2)C)C2=CC=CC=C2N1C1=CC=CC=C1 ZYFVNVRFVHJEIU-UHFFFAOYSA-N 0.000 description 1
- 241000404883 Pisa Species 0.000 description 1
- 241000276498 Pollachius virens Species 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- GOOHAUXETOMSMM-UHFFFAOYSA-N Propylene oxide Chemical compound CC1CO1 GOOHAUXETOMSMM-UHFFFAOYSA-N 0.000 description 1
- 108090000315 Protein Kinase C Proteins 0.000 description 1
- 102000003923 Protein Kinase C Human genes 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 101000762949 Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1) Exotoxin A Proteins 0.000 description 1
- 206010037394 Pulmonary haemorrhage Diseases 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 108010045108 Receptor for Advanced Glycation End Products Proteins 0.000 description 1
- 102000005622 Receptor for Advanced Glycation End Products Human genes 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 206010038546 Renal vasculitis Diseases 0.000 description 1
- 108090000829 Ribosome Inactivating Proteins Proteins 0.000 description 1
- 108010039491 Ricin Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- ONJZYZYZIKTIEG-UHFFFAOYSA-N Sanglifehrin A Natural products N1C(=O)C(CC)CC(C)C21C(C)C(O)C(C)C(CC(O)C(C)CCC=CC=C(C)C1OC(=O)C3CCCN(N3)C(=O)C(CC=3C=C(O)C=CC=3)NC(=O)C(C(C)C)NC(=O)C(CCC(C)=O)C(O)C(C)C(O)C=CC=CC1)O2 ONJZYZYZIKTIEG-UHFFFAOYSA-N 0.000 description 1
- 108010084592 Saporins Proteins 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 102000012479 Serine Proteases Human genes 0.000 description 1
- 108010022999 Serine Proteases Proteins 0.000 description 1
- 102100028904 Serine/threonine-protein kinase MARK2 Human genes 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241001479493 Sousa Species 0.000 description 1
- 108010039445 Stem Cell Factor Proteins 0.000 description 1
- 208000006011 Stroke Diseases 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
- 230000006044 T cell activation Effects 0.000 description 1
- LJTFFORYSFGNCT-UHFFFAOYSA-N Thiocarbohydrazide Chemical compound NNC(=S)NN LJTFFORYSFGNCT-UHFFFAOYSA-N 0.000 description 1
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 1
- 208000031981 Thrombocytopenic Idiopathic Purpura Diseases 0.000 description 1
- 102100030859 Tissue factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 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
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 1
- COQLPRJCUIATTQ-UHFFFAOYSA-N Uranyl acetate Chemical compound O.O.O=[U]=O.CC(O)=O.CC(O)=O COQLPRJCUIATTQ-UHFFFAOYSA-N 0.000 description 1
- 206010047115 Vasculitis Diseases 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- CESGKXMBHGUQTB-VONOSFMSSA-N [(1S,2S,6R,10S,11R,13S,14R,15R)-1,6,14-trihydroxy-8-(hydroxymethyl)-4,12,12,15-tetramethyl-5-oxo-13-tetracyclo[8.5.0.02,6.011,13]pentadeca-3,8-dienyl] tetradecanoate Chemical compound C1=C(CO)C[C@]2(O)C(=O)C(C)=C[C@H]2[C@@]2(O)[C@H](C)[C@@H](O)[C@@]3(OC(=O)CCCCCCCCCCCCC)C(C)(C)[C@H]3[C@@H]21 CESGKXMBHGUQTB-VONOSFMSSA-N 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 1
- 206010069351 acute lung injury Diseases 0.000 description 1
- 229960002964 adalimumab Drugs 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 201000009628 adenosine deaminase deficiency Diseases 0.000 description 1
- 229960000548 alemtuzumab Drugs 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- 239000002776 alpha toxin Substances 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 229960002932 anastrozole Drugs 0.000 description 1
- YBBLVLTVTVSKRW-UHFFFAOYSA-N anastrozole Chemical compound N#CC(C)(C)C1=CC(C(C)(C#N)C)=CC(CN2N=CN=C2)=C1 YBBLVLTVTVSKRW-UHFFFAOYSA-N 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 201000008244 anti-basement membrane glomerulonephritis Diseases 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000002927 anti-mitotic effect Effects 0.000 description 1
- 230000002946 anti-pancreatic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000006023 anti-tumor response Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000010056 antibody-dependent cellular cytotoxicity Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- YZXBAPSDXZZRGB-DOFZRALJSA-N arachidonic acid Chemical class CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(O)=O YZXBAPSDXZZRGB-DOFZRALJSA-N 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 239000005667 attractant Substances 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 208000036556 autosomal recessive T cell-negative B cell-negative NK cell-negative due to adenosine deaminase deficiency severe combined immunodeficiency Diseases 0.000 description 1
- QQOBRRFOVWGIMD-OJAKKHQRSA-N azaribine Chemical compound CC(=O)O[C@@H]1[C@H](OC(C)=O)[C@@H](COC(=O)C)O[C@H]1N1C(=O)NC(=O)C=N1 QQOBRRFOVWGIMD-OJAKKHQRSA-N 0.000 description 1
- 229950010054 azaribine Drugs 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 229960004669 basiliximab Drugs 0.000 description 1
- 229960002707 bendamustine Drugs 0.000 description 1
- YTKUWDBFDASYHO-UHFFFAOYSA-N bendamustine Chemical compound ClCCN(CCCl)C1=CC=C2N(C)C(CCCC(O)=O)=NC2=C1 YTKUWDBFDASYHO-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 108010027090 biotin-streptavidin complex Proteins 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 208000015294 blood coagulation disease Diseases 0.000 description 1
- 239000003130 blood coagulation factor inhibitor Substances 0.000 description 1
- 229940019700 blood coagulation factors Drugs 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 150000001639 boron compounds Chemical class 0.000 description 1
- 229960001467 bortezomib Drugs 0.000 description 1
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 description 1
- 230000003925 brain function Effects 0.000 description 1
- 229960005539 bryostatin 1 Drugs 0.000 description 1
- MJQUEDHRCUIRLF-TVIXENOKSA-N bryostatin 1 Chemical compound C([C@@H]1CC(/[C@@H]([C@@](C(C)(C)/C=C/2)(O)O1)OC(=O)/C=C/C=C/CCC)=C\C(=O)OC)[C@H]([C@@H](C)O)OC(=O)C[C@H](O)C[C@@H](O1)C[C@H](OC(C)=O)C(C)(C)[C@]1(O)C[C@@H]1C\C(=C\C(=O)OC)C[C@H]\2O1 MJQUEDHRCUIRLF-TVIXENOKSA-N 0.000 description 1
- 229960002092 busulfan Drugs 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 229940127093 camptothecin Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- JLQUFIHWVLZVTJ-UHFFFAOYSA-N carbosulfan Chemical compound CCCCN(CCCC)SN(C)C(=O)OC1=CC=CC2=C1OC(C)(C)C2 JLQUFIHWVLZVTJ-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 229960005243 carmustine Drugs 0.000 description 1
- 229960000590 celecoxib Drugs 0.000 description 1
- RZEKVGVHFLEQIL-UHFFFAOYSA-N celecoxib Chemical compound C1=CC(C)=CC=C1C1=CC(C(F)(F)F)=NN1C1=CC=C(S(N)(=O)=O)C=C1 RZEKVGVHFLEQIL-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000010822 cell death assay Methods 0.000 description 1
- 230000006721 cell death pathway Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000007541 cellular toxicity Effects 0.000 description 1
- 229960003115 certolizumab pegol Drugs 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000013000 chemical inhibitor Substances 0.000 description 1
- 230000031902 chemoattractant activity Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 239000002820 chemotaxin Substances 0.000 description 1
- 229960004630 chlorambucil Drugs 0.000 description 1
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 description 1
- 230000001684 chronic effect Effects 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
- 229960002436 cladribine Drugs 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 238000002648 combination therapy Methods 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000003184 complementary RNA Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 229940111134 coxibs Drugs 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 150000003983 crown ethers Chemical class 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000000113 cyclohexyl group Chemical group [H]C1([H])C([H])([H])C([H])([H])C([H])(*)C([H])([H])C1([H])[H] 0.000 description 1
- 239000003255 cyclooxygenase 2 inhibitor Substances 0.000 description 1
- 229960004397 cyclophosphamide Drugs 0.000 description 1
- 229960003067 cystine Drugs 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 210000004292 cytoskeleton Anatomy 0.000 description 1
- 210000000172 cytosol Anatomy 0.000 description 1
- 229960003901 dacarbazine Drugs 0.000 description 1
- 229960002806 daclizumab Drugs 0.000 description 1
- 229960000640 dactinomycin Drugs 0.000 description 1
- 229960000975 daunorubicin Drugs 0.000 description 1
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- CFCUWKMKBJTWLW-UHFFFAOYSA-N deoliosyl-3C-alpha-L-digitoxosyl-MTM Natural products CC=1C(O)=C2C(O)=C3C(=O)C(OC4OC(C)C(O)C(OC5OC(C)C(O)C(OC6OC(C)C(O)C(C)(O)C6)C5)C4)C(C(OC)C(=O)C(O)C(C)O)CC3=CC2=CC=1OC(OC(C)C1O)CC1OC1CC(O)C(O)C(C)O1 CFCUWKMKBJTWLW-UHFFFAOYSA-N 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- JXSJBGJIGXNWCI-UHFFFAOYSA-N diethyl 2-[(dimethoxyphosphorothioyl)thio]succinate Chemical compound CCOC(=O)CC(SP(=S)(OC)OC)C(=O)OCC JXSJBGJIGXNWCI-UHFFFAOYSA-N 0.000 description 1
- 238000002050 diffraction method Methods 0.000 description 1
- 230000010339 dilation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- OGGXGZAMXPVRFZ-UHFFFAOYSA-M dimethylarsinate Chemical compound C[As](C)([O-])=O OGGXGZAMXPVRFZ-UHFFFAOYSA-M 0.000 description 1
- 229940042396 direct acting antivirals thiosemicarbazones Drugs 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229960003668 docetaxel Drugs 0.000 description 1
- 208000036552 dowling-degos disease 3 Diseases 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000008482 dysregulation Effects 0.000 description 1
- 229960000284 efalizumab Drugs 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 231100001135 endothelial toxicity Toxicity 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- YJGVMLPVUAXIQN-UHFFFAOYSA-N epipodophyllotoxin Natural products COC1=C(OC)C(OC)=CC(C2C3=CC=4OCOC=4C=C3C(O)C3C2C(OC3)=O)=C1 YJGVMLPVUAXIQN-UHFFFAOYSA-N 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 description 1
- 229960000752 etoposide phosphate Drugs 0.000 description 1
- LIQODXNTTZAGID-OCBXBXKTSA-N etoposide phosphate Chemical compound COC1=C(OP(O)(O)=O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 LIQODXNTTZAGID-OCBXBXKTSA-N 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 229960004222 factor ix Drugs 0.000 description 1
- 229940012413 factor vii Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 229960002949 fluorouracil Drugs 0.000 description 1
- 229960002074 flutamide Drugs 0.000 description 1
- MKXKFYHWDHIYRV-UHFFFAOYSA-N flutamide Chemical compound CC(C)C(=O)NC1=CC=C([N+]([O-])=O)C(C(F)(F)F)=C1 MKXKFYHWDHIYRV-UHFFFAOYSA-N 0.000 description 1
- VVIAGPKUTFNRDU-ABLWVSNPSA-N folinic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-ABLWVSNPSA-N 0.000 description 1
- 235000008191 folinic acid Nutrition 0.000 description 1
- 239000011672 folinic acid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 229910052733 gallium Inorganic materials 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 229960005277 gemcitabine Drugs 0.000 description 1
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 description 1
- 229960000578 gemtuzumab Drugs 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 206010061989 glomerulosclerosis Diseases 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 210000003714 granulocyte Anatomy 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000000122 growth hormone Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 201000005787 hematologic cancer Diseases 0.000 description 1
- 208000024200 hematopoietic and lymphoid system neoplasm Diseases 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000002439 hemostatic effect Effects 0.000 description 1
- 231100000753 hepatic injury Toxicity 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 229960001340 histamine Drugs 0.000 description 1
- 239000003667 hormone antagonist Substances 0.000 description 1
- 102000044579 human CCDC6 Human genes 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000005661 hydrophobic surface Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000001146 hypoxic effect Effects 0.000 description 1
- 229960001001 ibritumomab tiuxetan Drugs 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 229940127130 immunocytokine Drugs 0.000 description 1
- 238000009177 immunoglobulin therapy Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 238000011532 immunohistochemical staining Methods 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 239000000367 immunologic factor Substances 0.000 description 1
- 230000001024 immunotherapeutic effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000006662 intracellular pathway Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 229960004768 irinotecan Drugs 0.000 description 1
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 201000010982 kidney cancer Diseases 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 229960001691 leucovorin Drugs 0.000 description 1
- 230000021633 leukocyte mediated immunity Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 230000007056 liver toxicity Effects 0.000 description 1
- 229960002247 lomustine Drugs 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 230000001926 lymphatic effect Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 201000004792 malaria Diseases 0.000 description 1
- 230000007257 malfunction Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical compound ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
- 229960004961 mechlorethamine Drugs 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 description 1
- 229960001924 melphalan Drugs 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 108020004084 membrane receptors Proteins 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000011235 metanalysis Methods 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 206010062198 microangiopathy Diseases 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 210000004688 microtubule Anatomy 0.000 description 1
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- CFCUWKMKBJTWLW-BKHRDMLASA-N mithramycin Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@H](O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@@H](O)[C@H](O)[C@@H](C)O1 CFCUWKMKBJTWLW-BKHRDMLASA-N 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 229960000350 mitotane Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229960003816 muromonab-cd3 Drugs 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 210000004296 naive t lymphocyte Anatomy 0.000 description 1
- 229960005027 natalizumab Drugs 0.000 description 1
- 229940086322 navelbine Drugs 0.000 description 1
- 201000008383 nephritis Diseases 0.000 description 1
- 210000000885 nephron Anatomy 0.000 description 1
- 231100000637 nephrotoxin Toxicity 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 230000003448 neutrophilic effect Effects 0.000 description 1
- OSTGTTZJOCZWJG-UHFFFAOYSA-N nitrosourea Chemical compound NC(=O)N=NO OSTGTTZJOCZWJG-UHFFFAOYSA-N 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 238000007899 nucleic acid hybridization Methods 0.000 description 1
- 238000001668 nucleic acid synthesis Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 229960000470 omalizumab Drugs 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 239000011022 opal Substances 0.000 description 1
- 230000014207 opsonization Effects 0.000 description 1
- 230000003571 opsonizing effect Effects 0.000 description 1
- 210000004789 organ system Anatomy 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000007248 oxidative elimination reaction Methods 0.000 description 1
- 229910052763 palladium Inorganic materials 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 208000008443 pancreatic carcinoma Diseases 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 230000009963 pathologic angiogenesis Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229960002340 pentostatin Drugs 0.000 description 1
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 230000037050 permeability transition Effects 0.000 description 1
- 238000009521 phase II clinical trial Methods 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 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
- 108010055837 phosphocarrier protein HPr Proteins 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000000906 photoactive agent Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 238000002616 plasmapheresis Methods 0.000 description 1
- 229910052697 platinum Inorganic materials 0.000 description 1
- 229960003171 plicamycin Drugs 0.000 description 1
- 229950008499 plitidepsin Drugs 0.000 description 1
- UUSZLLQJYRSZIS-LXNNNBEUSA-N plitidepsin Chemical compound CN([C@H](CC(C)C)C(=O)N[C@@H]1C(=O)N[C@@H]([C@H](CC(=O)O[C@H](C(=O)[C@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N2CCC[C@H]2C(=O)N(C)[C@@H](CC=2C=CC(OC)=CC=2)C(=O)O[C@@H]1C)C(C)C)O)[C@@H](C)CC)C(=O)[C@@H]1CCCN1C(=O)C(C)=O UUSZLLQJYRSZIS-LXNNNBEUSA-N 0.000 description 1
- 108010049948 plitidepsin Proteins 0.000 description 1
- 108700028325 pokeweed antiviral Proteins 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920000447 polyanionic polymer Polymers 0.000 description 1
- 229920000570 polyether Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004032 porphyrins Chemical class 0.000 description 1
- 235000020004 porter Nutrition 0.000 description 1
- 230000031915 positive regulation of coagulation Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 229940071643 prefilled syringe Drugs 0.000 description 1
- 230000007112 pro inflammatory response Effects 0.000 description 1
- 229960000624 procarbazine Drugs 0.000 description 1
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229940002612 prodrug Drugs 0.000 description 1
- 239000000651 prodrug Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 239000003528 protein farnesyltransferase inhibitor Substances 0.000 description 1
- 238000000164 protein isolation Methods 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 230000003331 prothrombotic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- GZUITABIAKMVPG-UHFFFAOYSA-N raloxifene Chemical compound C1=CC(O)=CC=C1C1=C(C(=O)C=2C=CC(OCCN3CCCCC3)=CC=2)C2=CC=C(O)C=C2S1 GZUITABIAKMVPG-UHFFFAOYSA-N 0.000 description 1
- 229960004622 raloxifene Drugs 0.000 description 1
- 201000008158 rapidly progressive glomerulonephritis Diseases 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000012959 renal replacement therapy Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000019254 respiratory burst Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 230000008593 response to virus Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000011808 rodent model Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 229960003440 semustine Drugs 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000011272 standard treatment Methods 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229960001052 streptozocin Drugs 0.000 description 1
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 229920002258 tannic acid Polymers 0.000 description 1
- LRBQNJMCXXYXIU-NRMVVENXSA-N tannic acid Chemical compound OC1=C(O)C(O)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(=O)OC[C@@H]2[C@H]([C@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)[C@@H](OC(=O)C=3C=C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)C(O)=C(O)C=3)O2)OC(=O)C=2C=C(OC(=O)C=3C=C(O)C(O)=C(O)C=3)C(O)=C(O)C=2)O)=C1 LRBQNJMCXXYXIU-NRMVVENXSA-N 0.000 description 1
- 229940033123 tannic acid Drugs 0.000 description 1
- 235000015523 tannic acid Nutrition 0.000 description 1
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 description 1
- 229960001278 teniposide Drugs 0.000 description 1
- 238000012349 terminal deoxynucleotidyl transferase dUTP nick-end labeling Methods 0.000 description 1
- 229960003433 thalidomide Drugs 0.000 description 1
- 230000004797 therapeutic response Effects 0.000 description 1
- CNHYKKNIIGEXAY-UHFFFAOYSA-N thiolan-2-imine Chemical compound N=C1CCCS1 CNHYKKNIIGEXAY-UHFFFAOYSA-N 0.000 description 1
- ATGUDZODTABURZ-UHFFFAOYSA-N thiolan-2-ylideneazanium;chloride Chemical compound Cl.N=C1CCCS1 ATGUDZODTABURZ-UHFFFAOYSA-N 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 229960001196 thiotepa Drugs 0.000 description 1
- 230000002885 thrombogenetic effect Effects 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- MNRILEROXIRVNJ-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=NC=N[C]21 MNRILEROXIRVNJ-UHFFFAOYSA-N 0.000 description 1
- 229960003989 tocilizumab Drugs 0.000 description 1
- 229960000303 topotecan Drugs 0.000 description 1
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 description 1
- 229960005267 tositumomab Drugs 0.000 description 1
- 231100000816 toxic dose Toxicity 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 230000005909 tumor killing Effects 0.000 description 1
- 230000005951 type IV hypersensitivity Effects 0.000 description 1
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229960001055 uracil mustard Drugs 0.000 description 1
- 239000002441 uremic toxin Substances 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000008728 vascular permeability Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 230000008189 vertebrate development Effects 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- GBABOYUKABKIAF-IELIFDKJSA-N vinorelbine Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-IELIFDKJSA-N 0.000 description 1
- 229960002066 vinorelbine Drugs 0.000 description 1
- CILBMBUYJCWATM-PYGJLNRPSA-N vinorelbine ditartrate Chemical compound OC(=O)[C@H](O)[C@@H](O)C(O)=O.OC(=O)[C@H](O)[C@@H](O)C(O)=O.C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC CILBMBUYJCWATM-PYGJLNRPSA-N 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- 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
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Public Health (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Urology & Nephrology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Acute kidney injury (AKI) is often associated with damage to remote organs, such as lungs or heart. AKI induces kidney tubular necrosis as well as NETosis, programmed neutrophil death leading to neutrophil extracellular traps (NETs). Histones released during NETosis induces further formation of NETs, which is damaging to renal tissues and remote organs. Circulating trap-forming neutrophils directly injured the lung, while other dead tissue releases contributed to injury in other organs. Suppressing renal necroinflammation using inhibitors of NET formation, tubular cell necrosis or extracellular histones prevented kidney as well as remote organ injuries. Dual inhibition of neutrophil trap formation together with tubular cell necrosis had an additive protective effect. Preferably, damage to remote organs induced by AKI may be treated and/or prevented using anti-histone agents such as anti-histone IgG, recombinant activated protein C, or heparin, alone or in combination with other therapeutic agents, such as PAD inhibitors.
Description
ANTI-HISTONE THERAPY IN ACUTE KIDNEY INJURY
Related Applications [01] This application claims the benefit under 35 U.S.C. 119(e) of provisional U.S. Patent Application Serial No. 62/394,529, filed September 14, 2016. The present application is a continuation-in-part of U.S. Patent Application Serial No. 15/402,585, filed January 10, 2017, which was a divisional of U.S. Patent Application Serial No. 14/746,997 (now issued U.S.
Patent 9,580,495), filed June 23, 2015, which claimed the benefit under 35 U.S.C. 119(e) of provisional U.S. Patent Application Serial No. 62/016,277, filed June 24, 2014. The text of each priority application is incorporated herein by reference in its entirety.
Sequence Listing
Related Applications [01] This application claims the benefit under 35 U.S.C. 119(e) of provisional U.S. Patent Application Serial No. 62/394,529, filed September 14, 2016. The present application is a continuation-in-part of U.S. Patent Application Serial No. 15/402,585, filed January 10, 2017, which was a divisional of U.S. Patent Application Serial No. 14/746,997 (now issued U.S.
Patent 9,580,495), filed June 23, 2015, which claimed the benefit under 35 U.S.C. 119(e) of provisional U.S. Patent Application Serial No. 62/016,277, filed June 24, 2014. The text of each priority application is incorporated herein by reference in its entirety.
Sequence Listing
[02] The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on September 13, 2017 is named IMM346W03 SL.txt and is 22,703 bytes in size.
FIELD OF THE INVENTION
FIELD OF THE INVENTION
[03] The invention relates to compositions and methods of use of histone-neutralizing agents, such as anti-histone IgG, activated protein C, and heparin, or PAD
inhibitors for treatment of remote organ injury induced by acute kidney injury. Remote organ injury refers to injury to nonrenal organs, for example the lungs and heart. In certain preferred embodiments, the histone-neutralizing agent is an anti-histone antibody or antigen-binding fragment thereof, such as the BWA-3 anti-H4 antibody. In other embodiments, the anti-histone antibodies bind to human histones H2B, H3 or H4. More particular embodiments may concern chimeric or more preferably humanized forms of anti-histone antibodies. However, any other known histone-neutralizing agent may be utilized for treating remote organ injury induced by acute kidney injury. In alternative embodiments, a PAD inhibitor may be Cl amidine, although other known PAD inhibitors such as o-F-amidine, o-Cl-amidine, TDFA
(Thr-Asp-F-amidine), YW3-56 or streptonigrin (Bicker & Thompson, 2013, Biopolymers 99:155-63) may be utilized.
BACKGROUND
inhibitors for treatment of remote organ injury induced by acute kidney injury. Remote organ injury refers to injury to nonrenal organs, for example the lungs and heart. In certain preferred embodiments, the histone-neutralizing agent is an anti-histone antibody or antigen-binding fragment thereof, such as the BWA-3 anti-H4 antibody. In other embodiments, the anti-histone antibodies bind to human histones H2B, H3 or H4. More particular embodiments may concern chimeric or more preferably humanized forms of anti-histone antibodies. However, any other known histone-neutralizing agent may be utilized for treating remote organ injury induced by acute kidney injury. In alternative embodiments, a PAD inhibitor may be Cl amidine, although other known PAD inhibitors such as o-F-amidine, o-Cl-amidine, TDFA
(Thr-Asp-F-amidine), YW3-56 or streptonigrin (Bicker & Thompson, 2013, Biopolymers 99:155-63) may be utilized.
BACKGROUND
[04] Acute kidney injury (AKI) is a common problem in both tertiary care centers as well as in the developing world. AKI often arises from injuries such as trauma, severe infection, sepsis, medications and contrast agents, or following major surgery. AKI is common among patients requiring intensive care on hospital admission, often requiring the use of dialysis.
Renal failure is often associated with damage to remote (nonrenal) organs, particularly the lungs, with increased pulmonary vascular permeability and pulmonary hemorrhage (Kramer et al., 1999, Kidney Int. 55:2362-7).
Renal failure is often associated with damage to remote (nonrenal) organs, particularly the lungs, with increased pulmonary vascular permeability and pulmonary hemorrhage (Kramer et al., 1999, Kidney Int. 55:2362-7).
[05] Acute kidney injury involves cell necrosis as well as NETosis, a programmed neutrophil death leading to expulsion of nuclear chromatin leading to neutrophil extracellular traps (NETs). ETosis is a programmed form of cell death of mostly neutrophils (referred to as NETosis) and other granulocytes (Brinkmann et al., 2004, Science 303:1532).
NETosis causes an explosion-like directed expulsion of chromatin generating a meshwork called neutrophil extracellular traps (NETs), which immobilize and kill bacteria during infections (Brinkmann et at., 2004, Science 303:1532). Cytokine-induced NETosis also drives sterile injury including necrotizing GN (Kessenbrock et al., 2009, Nat Med 15:623;
Kambas et al., 2013, Ann Rheum Dis 73:1854; Nakazawa et al., 2012, Front Immunol 3:333;
Tsuboi et at., 2002, J Immunol 169:2026). Many cytosolic or chromatin-related components could account for the toxic and pro-inflammatory effect of NETs, such as proteolytic enzymes or intracellular molecules with immunostimulatory effects, referred to as danger-associated molecular patterns (DAMPs) (Rock et al., 2010, Annual Review of Immunotogy 28:321).
NETosis causes an explosion-like directed expulsion of chromatin generating a meshwork called neutrophil extracellular traps (NETs), which immobilize and kill bacteria during infections (Brinkmann et at., 2004, Science 303:1532). Cytokine-induced NETosis also drives sterile injury including necrotizing GN (Kessenbrock et al., 2009, Nat Med 15:623;
Kambas et al., 2013, Ann Rheum Dis 73:1854; Nakazawa et al., 2012, Front Immunol 3:333;
Tsuboi et at., 2002, J Immunol 169:2026). Many cytosolic or chromatin-related components could account for the toxic and pro-inflammatory effect of NETs, such as proteolytic enzymes or intracellular molecules with immunostimulatory effects, referred to as danger-associated molecular patterns (DAMPs) (Rock et al., 2010, Annual Review of Immunotogy 28:321).
[06] Histones are nuclear proteins that wind up the double-stranded DNA to form chromatin. Dynamic modifications of histone residues regulate gene transcription by determining the accessibility of transcription factors to their DNA binding sites (Helin &
Dhanak, 2013, Nature 502:480). When cell necrosis releases histones into the extracellular space they display significant cytotoxic effects (Hirsch, 1958, J Exp Med 108:925; Xu et al., 2009, Nat Med 15:1318; Chaput & Zychlinsky, 2009, Nat Med 15:1245; Allam et al., 2014, J
Mot Med 92:465). Histones contribute to fatal outcomes in murine endotoxinemia caused by microvascular injury and activation of coagulation (Xu et at., 2009, Nat Med 15:1318;
Abrams et at., 2013, Am J Respir Crit Care Med 187:160; Saffarzadeh et at., 2012, PLoS One
Dhanak, 2013, Nature 502:480). When cell necrosis releases histones into the extracellular space they display significant cytotoxic effects (Hirsch, 1958, J Exp Med 108:925; Xu et al., 2009, Nat Med 15:1318; Chaput & Zychlinsky, 2009, Nat Med 15:1245; Allam et al., 2014, J
Mot Med 92:465). Histones contribute to fatal outcomes in murine endotoxinemia caused by microvascular injury and activation of coagulation (Xu et at., 2009, Nat Med 15:1318;
Abrams et at., 2013, Am J Respir Crit Care Med 187:160; Saffarzadeh et at., 2012, PLoS One
7:e32366; Semeraro et al., 2011, Blood 118:1952). Dying renal cells release extracellular histones that promote septic and post-ischemic acute kidney injury (Allam et at., 2012, J Am Soc Nephrol 23:1375). Further, histones act as DAMPs by activating Toll-like receptor (TLR)-2 and -4 as well as NLRP3 (Allam et at., 2012, J Am Soc Nephrol 23:1375;
Allam et al., 2013, Eur J Immunol 43:3336; Semeraro et at., 2011, Blood 118:1952; Huang et at., 2013, J Immunol 191:2665; Xu et al., 2011, J Immunol 187:2626). TLR2/-4-mediated pathology is an essential mechanism of crescentic GN (Brown et al., 2006, J
Immunol 177:1925; Brown et al., 2007, J Am Soc Nephrol 18:1732).
[07] A need exists for improved methods and compositions for treatment of remote organ injury induced by acute kidney injury, preferably using histone-neutralizing agents such as anti-histone antibodies or fragments thereof, or agents that inhibit post-translational modification of histones, such as PAD (protein arginine deiminase) inhibitors (Bicker &
Thompson, 2013, Biopolymers 99:155-63).
SUMMARY
Allam et al., 2013, Eur J Immunol 43:3336; Semeraro et at., 2011, Blood 118:1952; Huang et at., 2013, J Immunol 191:2665; Xu et al., 2011, J Immunol 187:2626). TLR2/-4-mediated pathology is an essential mechanism of crescentic GN (Brown et al., 2006, J
Immunol 177:1925; Brown et al., 2007, J Am Soc Nephrol 18:1732).
[07] A need exists for improved methods and compositions for treatment of remote organ injury induced by acute kidney injury, preferably using histone-neutralizing agents such as anti-histone antibodies or fragments thereof, or agents that inhibit post-translational modification of histones, such as PAD (protein arginine deiminase) inhibitors (Bicker &
Thompson, 2013, Biopolymers 99:155-63).
SUMMARY
[08] The present invention concerns compositions and methods of anti-histone therapy for remote organ injury induced by acute kidney injury. Preferably the anti-histone therapy may involve use of agents such as activated protein C, heparin, or anti-histone antibodies, such as antibodies against histone H2B, H3 or H4. In more preferred embodiments, the anti-histone antibody may be a BWA-3 anti-H4 antibody (see, e.g., U.S. Patent Application Serial No.
14/620,315, the Examples section and Figures of which are incorporated herein by reference).
In alternative embodiments, PAD (peptidyl arginine deiminase) inhibitors may be utilized alone or in combination with other histone inhibitors.
14/620,315, the Examples section and Figures of which are incorporated herein by reference).
In alternative embodiments, PAD (peptidyl arginine deiminase) inhibitors may be utilized alone or in combination with other histone inhibitors.
[09] Preferably, the anti-histone antibodies or fragments thereof may be chimeric, humanized or human. The antibody can be of various isotypes, preferably human IgGl, IgG2, IgG3 or IgG4, more preferably comprising human IgG1 hinge and constant region sequences.
Most preferably, the antibody or fragment thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when the immunoconjugate is administered to a human subject.
Preferred allotypes for administration include a non-Glml allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selected from the group consisting of the nGlml, G1m3, nG1m1,2 and Km3 allotypes. Exemplary humanized anti-histone antibodies are disclosed in U.S. Patent Application Serial No.
14/620,315, the Figures and Examples section of which are incorporated herein by reference.
Most preferably, the antibody or fragment thereof may be designed or selected to comprise human constant region sequences that belong to specific allotypes, which may result in reduced immunogenicity when the immunoconjugate is administered to a human subject.
Preferred allotypes for administration include a non-Glml allotype (nG1m1), such as G1m3, G1m3,1, G1m3,2 or G1m3,1,2. More preferably, the allotype is selected from the group consisting of the nGlml, G1m3, nG1m1,2 and Km3 allotypes. Exemplary humanized anti-histone antibodies are disclosed in U.S. Patent Application Serial No.
14/620,315, the Figures and Examples section of which are incorporated herein by reference.
[010] In certain preferred embodiments, a combination of anti-histone antibodies may be used. Antibodies against human histones H1, H2A, H2B, H3 or H4 may be used in any combination. Other non-antibody therapeutic agents targeted against either histones or downstream effectors of a histone-mediated pathway may also be utilized in combination with anti-histone antibodies or fragments thereof, administered either before, simultaneously with, or following administration of one or more anti-histone antibodies or fragments thereof Various therapeutic agents of use in treating histone-associated diseases are known in the art, such as activated protein C (APC), thrombomodulin, a peptide fragment of histone H1, H2A, H2B, H3 or H4, granzyme A, granzyme B, plasmin, Factor 7-activating protease, heparin, Cl-amidine and any such known agent may be utilized in combination with anti-histone antibodies or antibody fragments. A human histone H4 peptide may comprise residues 50-67 or 40-78 of human H4 (see, e.g., U.S. Publ. No. 20090117099). Depending on the underlying etiology, the anti-histone agents may also be utilized in combination with one or more standard treatments for acute kidney injury, such as corticosteroids, immune-suppressing drugs or plasmapheresis.
BRIEF DESCRIPTION OF THE DRAWINGS
BRIEF DESCRIPTION OF THE DRAWINGS
[011] The following drawings are provided to illustrate preferred embodiments of the invention. However, the claimed subject matter is in no way limited by the illustrative embodiments disclosed in the drawings.
[012] FIG. 1A. NETs evidence in 2 patients with acute tubular necrosis (ATN) after kidney transplantation. Representative NETs immunostaining in 2 human kidney biopsy samples with severe ATN. Neutrophil elastase (NE): Green, citrullinated histone 3(CitH3):
Red, DAPI staining: Blue and overlay with phase contrast. The staining by isotype control IgG for NE and CitH3 was conducted (Right figure). NE/CitH3 positive NETs are detected in tubular-interstitium space (Upper figures: Case 1, Lower figures: Case 2).
Scale Bar: 25 p.m.
Red, DAPI staining: Blue and overlay with phase contrast. The staining by isotype control IgG for NE and CitH3 was conducted (Right figure). NE/CitH3 positive NETs are detected in tubular-interstitium space (Upper figures: Case 1, Lower figures: Case 2).
Scale Bar: 25 p.m.
[013] FIG. 1B. NETs evidence in 2 patients with acute tubular necrosis (ATN) after kidney transplantation. Leukocytes are infiltrating surrounding tubular ducts in RE staining (Case 1: left, Case 2: right). Scale Bar: 25 p.m.
[014] FIG. 2A. NETs initiate the loop of necroinflammation in vitro. The media of human induced tubular epithelial cells (iTECs) treated with normal oxygen, hypoxia (1% 02) or 10 mM H202 for 24 hours, were applied to healthy human neutrophils. After 4 hours of incubation, the NETs were detected by immunofluorescence staining. NE: Green, CitH3:
Red, DAPI staining: Blue. Scale Bar: 50 p.m.
Red, DAPI staining: Blue. Scale Bar: 50 p.m.
[015] FIG. 2B. NETs initiate the loop of necroinflammation in vitro. The graph shows the ratio of CitH3/DAPI positive area of iTECs.
[016] FIG. 2C. NETs initiate the loop of necroinflammation in vitro. The media of HK2 cells treated with normal oxygen, hypoxia (1% 02) or 10 mM H202 for 24 hours, were applied to healthy human neutrophils. After 4 hours of incubation, the NETs were detected by immunofluorescence staining. NE: Green, CitH3: Red, DAPI staining: Blue. Scale Bar: 50
[017] FIG. 2D. NETs initiate the loop of necroinflammation in vitro. The graph shows the ratio of CitH3/DAPI positive area of HK2 cells.
[018] FIG. 2E. NETs initiate the loop of necroinflammation in vitro.
Neutrophils were treated with 25 nM PMA in the presence or absence of PAD inhibitor (200 M) and the degree of NETs was evaluated by MPO-DNA complex of supernatants.
Neutrophils were treated with 25 nM PMA in the presence or absence of PAD inhibitor (200 M) and the degree of NETs was evaluated by MPO-DNA complex of supernatants.
[019] FIG. 2F. NETs initiate the loop of necroinflammation in vitro.
Neutrophils were treated with necrotic TCs media in the presence or absence of PAD inhibitor (200 M) and the degree of NETs was evaluated by MPO-DNA complex of supernatants.
Neutrophils were treated with necrotic TCs media in the presence or absence of PAD inhibitor (200 M) and the degree of NETs was evaluated by MPO-DNA complex of supernatants.
[020] FIG. 2G. NETs initiate the loop of necroinflammation in vitro. The neutrophil supernatants treated with PMA were applied to TCs, and the cytotoxicity of TCs was evaluated by LDH assay 20 hours after addition. The conditioned NETs media was prepared by replacing to fresh media to avoid the contamination of PMA as previously described (McParland et al., 2015, J Vis Exp 98:e52684).
[021] FIG. 211. NETs initiate the loop of necroinflammation in vitro. The neutrophil supernatants treated with necrotic TCs media were applied to TCs, and the cytotoxicity of TCs was evaluated by LDH assay 20 hours after addition. The conditioned NETs media was prepared by replacing to fresh media to avoid the contamination of TC necrotic media as previously described (McParland et al., 2015, J Vis Exp 98:e52684).
[022] FIG. 21. NETs initiate the loop of necroinflammation in vitro. The expression of CitH3 of neutrophils treated with normal oxygen or hypoxia condition, or different TCs necrotic media was detected by western blot with 13-actin as a loading control. As a positive control of CitH3 expression, neutrophils were treated with 25 nM PMA. Data represent the means SEM of 3-6 independent experiments, and were analyzed using Student's t-test.
*P<0.05, ** P<0.01 versus respective control. DAPI: 4,6-diamidino-2-phenylindole. PMA:
Phorbol 12-myristate 13-acetate.
*P<0.05, ** P<0.01 versus respective control. DAPI: 4,6-diamidino-2-phenylindole. PMA:
Phorbol 12-myristate 13-acetate.
[023] FIG. 3A. In vivo evidence for NETs in acute phase of IRI kidney.
Representative NETs staining in outer medulla lesion of unilateral MI kidney (ischemia 35min, reperfusion 24hous). Co-localization of CitH3 (Red), Ly6b(Green), and swelled nuclei (Blue) surrounding tubular duct indicates the NETs formation. Scale Bar: 50 p.m.
Representative NETs staining in outer medulla lesion of unilateral MI kidney (ischemia 35min, reperfusion 24hous). Co-localization of CitH3 (Red), Ly6b(Green), and swelled nuclei (Blue) surrounding tubular duct indicates the NETs formation. Scale Bar: 50 p.m.
[024] FIG. 3B. In vivo evidence for NETs in acute phase of IRI kidney. NETs staining of unilateral IRI kidney at different time point after reperfusion and different ischemia time.
Upper figures show Ly6b (Green) staining and lower figures show CitH3 staining (Red).
Scale Bar: 100 p.m.
Upper figures show Ly6b (Green) staining and lower figures show CitH3 staining (Red).
Scale Bar: 100 p.m.
[025] FIG. 3C. In vivo evidence for NETs in acute phase of IRI kidney. Ly6b positive area.
[026] FIG. 3D. In vivo evidence for NETs in acute phase of IRI kidney. CitH3 positive area in IF staining.
[027] FIG. 3E. In vivo evidence for NETs in acute phase of IRI kidney. CitH3 expression of IRI kidney by western blot.
[028] FIG. 3F. In vivo evidence for NETs in acute phase of IRI kidney. Tubular necrosis was determined by TUNEL staining. Scale Bar: 200 p.m.
[029] FIG. 3G. In vivo evidence for NETs in acute phase of IRI kidney. The positive area (tubular injury score) at different time was quantified. Scale Bar: 200 p.m.
[030] FIG. 311. In vivo evidence for NETs in acute phase of IRI kidney.
Histological evaluation was conducted by PAS staining. Representative image of tubular injury at different reperfusion time. Scale Bar: 500 p.m.
Histological evaluation was conducted by PAS staining. Representative image of tubular injury at different reperfusion time. Scale Bar: 500 p.m.
[031] FIG. 31. In vivo evidence for NETs in acute phase of IRI kidney.
Histological evaluation was conducted by PAS staining. Injury score in time course of reperfusion.
ischemia (K). Data are means SEM from five mice in each group. TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.
Histological evaluation was conducted by PAS staining. Injury score in time course of reperfusion.
ischemia (K). Data are means SEM from five mice in each group. TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling.
[032] FIG. 3J. In vivo evidence for NETs in acute phase of IRI kidney.
Histological evaluation was conducted by PAS staining. Representative image of tubular injury at different ischemic time. Scale Bar: 500 p.m.
Histological evaluation was conducted by PAS staining. Representative image of tubular injury at different ischemic time. Scale Bar: 500 p.m.
[033] FIG. 3K. In vivo evidence for NETs in acute phase of IRI kidney.
Histological evaluation was conducted by PAS staining. Injury score in time course of ischemia. Data are means SEM from five mice in each group.
Histological evaluation was conducted by PAS staining. Injury score in time course of ischemia. Data are means SEM from five mice in each group.
[034] FIG. 4A. NET inhibitor ameliorates bilateral IRI kidney. Bilateral IRI
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Representative overlay figure of NETs staining in each group. NE: Green, CitH3: Red, DAPI: Blue. Scale Bar: 100 p.m.
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Representative overlay figure of NETs staining in each group. NE: Green, CitH3: Red, DAPI: Blue. Scale Bar: 100 p.m.
[035] FIG. 4B. NET inhibitor ameliorates bilateral IRI kidney. Bilateral IRI
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Upper figures and graph show NE staining and the ratio of NE positive area. Lower figures and graph show CitH3 staining and the ratio of CitH3 positive area in different treatment group. Scale Bar: 100 p.m.
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Upper figures and graph show NE staining and the ratio of NE positive area. Lower figures and graph show CitH3 staining and the ratio of CitH3 positive area in different treatment group. Scale Bar: 100 p.m.
[036] FIG. 4C. NET inhibitor ameliorates bilateral IRI kidney. Bilateral IRI
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Plasma creatinine levels in each group.
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Plasma creatinine levels in each group.
[037] FIG. 4D. NET inhibitor ameliorates bilateral IRI kidney. Bilateral IRI
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Plasma urea levels in each group.
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Plasma urea levels in each group.
[038] FIG. 4E. NET inhibitor ameliorates bilateral IRI kidney. Bilateral IRI
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Circulating NETs in each group.
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20 mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Circulating NETs in each group.
[039] FIG. 4F. NET inhibitor ameliorates bilateral IRI kidney. Bilateral IRI
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Histological findings and TUNEL staining. Upper figures and graph shows representative PAS staining and tubular necrosis area, respectively (Scale bar: 500um).
Lower figures and graph show representative TUNEL staining and the ratio of TUNEL
positive area, respectively (Scale Bar: 200um). Data are means SEM from five mice in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
kidney model mice (Ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO
in PBS), PAD inhibitor (Cl-amidine 20mg/kg, i.p.), and neutrophil depletion by injection of anti-Ly6G monoclonal antibody (50011g anti-Ly6G IgGs (1A8) or control IgGs 24 and 2 hours before ischemia) before the surgery. Sham operated mice were prepared as a control (each group, N=5). Histological findings and TUNEL staining. Upper figures and graph shows representative PAS staining and tubular necrosis area, respectively (Scale bar: 500um).
Lower figures and graph show representative TUNEL staining and the ratio of TUNEL
positive area, respectively (Scale Bar: 200um). Data are means SEM from five mice in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
[040] FIG. 5A. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Representative NETs staining in IRI kidney treated with vehicle, necrosis inhibitor (Nec In) and the combination Nec In and PAD inhibitor (PAD
In). NE:
Green, CitH3: Red, DAPI: Blue. Scale Bar: 200 p.m.
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Representative NETs staining in IRI kidney treated with vehicle, necrosis inhibitor (Nec In) and the combination Nec In and PAD inhibitor (PAD
In). NE:
Green, CitH3: Red, DAPI: Blue. Scale Bar: 200 p.m.
[041] FIG. 5B. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Representative protein expression of CitH3 in IRI
kidney treated with different inhibitors.
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Representative protein expression of CitH3 in IRI
kidney treated with different inhibitors.
[042] FIG. 5C. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Left graph show NE positive area and right graph show CitH3 positive area.
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Left graph show NE positive area and right graph show CitH3 positive area.
[043] FIG. 5D. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Quantification of protein expression, normalized to 13-actin expression.
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Quantification of protein expression, normalized to 13-actin expression.
[044] FIG. 5E. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Representative PAS (upper figures, Scale Bar: 500um) and TUNEL
staining (lower figures, Scale Bar: 200 p.m).
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Representative PAS (upper figures, Scale Bar: 500um) and TUNEL
staining (lower figures, Scale Bar: 200 p.m).
[045] FIG. 5F. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. The quantification of necrotic area in PAS staining (upper graph) and TUNEL positive area (lower graph).
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. The quantification of necrotic area in PAS staining (upper graph) and TUNEL positive area (lower graph).
[046] FIG. 5G. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Plasma creatinine and (H) plasma urea in each group.
Data are means SEM from at least five mice in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Plasma creatinine and (H) plasma urea in each group.
Data are means SEM from at least five mice in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
[047] FIG. 511. NET inhibition had additional protective effect on necrosis inhibition in IRI kidney. Bilateral IRI kidney model mice (ischemia 35 min, reperfusion 24 hours) were treated with vehicle (20% DMSO in PBS, N=14), necrosis inhibitor cocktail (Necrostatin-1;
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Plasma urea in each group.
1.65 mg/kg i.p.), Ferrostatin-1 (2 mg/kg i.p.), Cyclosporine (10 mg/kg, i.v, N=5) and the combination of necrosis inhibitor cocktail and PAD inhibitor (Cl-amidine 20 mg/kg, i.p., N=5) before the surgery. Plasma urea in each group.
[048] FIG. 6A. Histones are central key players of necroinflammation including NETosis. Histone concentration of the supernatant in HK2 cells treated with 1mM H202 and PBS for 24 hours was measured by histone ELISA detection kit. Data represent the means SEM of 4 independent experiments. *P<0.05, versus respective control.
[049] FIG. 6B. Histones are central key players of necroinflammation including NETosis. Representative NETs staining of histone stimulated neutrophils using CitH3 (Red) and DAPI (Blue). Scale Bar: 50 p.m.
[050] FIG. 6C. Histones are central key players of necroinflammation including NETosis. Representative Scanning Electron microscopy images of unstimulated neutrophils (upper) and histone-stimulated neutrophils (lower). Scale Bar: 20 i.tm.
[051] FIG. 6D. Histones are central key players of necroinflammation including NETosis. Neutrophils were treated with exogenous histones (50 [tg/m1) in the presence of neutralizing histone abs (aHisAbs) (10Oug/m1) and control Abs (100 pg/m1) and the ratio of CitH3 positive cells was quantified.
[052] FIG. 6E. Histones are central key players of necroinflammation including NETosis. The supernatants of histone-stimulated neutrophils were applied to HK2 cells and the cytotoxicity was determined by LDH assay. The conditioned media was prepared as previously described to avoid the contamination of exogenous histones. Data represent the means SEM of 4 independent experiments. *P<0.05, **P<0.01, versus respective control.
[053] FIG. 6F. Histones are central key players of necroinflammation including NETosis. Representative image of NETs (left), PAS (middle), and TUNEL (right) staining in IRI kidney treated with control (upper) and aHisAbs (lower, aHisAbs 20 mg/kg, i.p, N=5) NE: Green, CitH3: Red, DAPI: Blue. Scale Bar: 200 p.m.
[054] FIG. 6G. Histones are central key players of necroinflammation including NETosis. (G) The quantification of CitH3 positive NETs area.
[055] FIG. 611. Histones are central key players of necroinflammation including NETosis. Histological necrotic area.
[056] FIG. 61. Histones are central key players of necroinflammation including NETosis. TUNEL positive area.
[057] FIG. 6J. Histones are central key players of necroinflammation including NETosis. Plasma creatinine in IRI kidney mice treated with control and aHisAbs. Data show the means SEM from at least five mice in each group. *P<0.05, versus respective control.
[058] FIG. 7A. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Plasma TNF-a in sham operated mice and bilateral IRI
kidney mice (ischemia 35min, reperfusion 24h) was measured by ELISA.
kidney mice (ischemia 35min, reperfusion 24h) was measured by ELISA.
[059] FIG. 7B. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Plasma IL-6 in sham operated mice and bilateral IRI
kidney mice (ischemia 35min, reperfusion 24h) was measured by ELISA
kidney mice (ischemia 35min, reperfusion 24h) was measured by ELISA
[060] FIG. 7C. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Plasma hi stone 3 content in sham operated mice and bilateral IRI
kidney mice (ischemia 35min, reperfusion 24h) was measured by western blotting. As a positive control for plasma histone, the plasma of LPS-induced sepsis mice was used.
kidney mice (ischemia 35min, reperfusion 24h) was measured by western blotting. As a positive control for plasma histone, the plasma of LPS-induced sepsis mice was used.
[061] FIG. 7D. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Tissue injury in multi-organ (kidney, lung, liver, brain, heart and pancreas) of sham and bilateral IRI (ischemia 35min, reperfusion 24h) kidney mice was evaluated by TUNEL staining. Scale Bar: 100 p.m.
[062] FIG. 7E. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Neutrophil infiltration in multi-organ (kidney, lung, liver, brain, heart and pancreas) of sham and bilateral IRI (ischemia 35min, reperfusion 24h) kidney mice was evaluated by Ly6b-immunostaining. Scale Bar: 100 p.m.
[063] FIG. 7F. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. NETs expression in multi-organ (kidney, lung, liver, brain, heart and pancreas) of sham and bilateral IRI (ischemia 35min, reperfusion 24h) kidney mice was evaluated by western blotting.
[064] FIG. 7G. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs was evaluated by NETs immunostaining (upper figures, NE: Green, CitH3: Red, DAPI: Blue) and TUNEL
staining (lower figures). Scale Bar: 100 p.m.
staining (lower figures). Scale Bar: 100 p.m.
[065] FIG. 711. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. The graph shows NETs area (H) in lung. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
[066] FIG. 71. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. The graph shows TUNEL
positive area in lung. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
positive area in lung. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
[067] FIG. 7J. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. The cell number in bronchoalveolar lavage (BAL) of these groups was counted. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
[068] FIG. 7K. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. The quantification of TUNEL
positive area in liver in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
#p<0.05, compared to aHisAbs group.
positive area in liver in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
#p<0.05, compared to aHisAbs group.
[069] FIG. 7L. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. The quantification of TUNEL
positive area in heart in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
#p<0.05, compared to aHisAbs group.
positive area in heart in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control.
#p<0.05, compared to aHisAbs group.
[070] FIG. 7M. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. The quantification of TUNEL
positive area in brain in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
positive area in brain in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
[071] FIG. 7N. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. Plasma TNF-a in different treated mice was measured by ELISA method. Data show the means SEM from at least five mice in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
[072] FIG. 70. AM-related remote organ injury is caused by circulating NETs and DAMPs such as histones. Lung injury followed bilateral IRI kidney (ischemia 35min, reperfusion 24h) treated with vehicle, PAD inhibitor, neutrophil depletion, Necrosis inhibitor, Necrosis inhibitor+PAD inhibitor, neutralizing aHisAbs. IL6 in different treated mice was measured by ELISA method. Data show the means SEM from at least five mice in each group. *P<0.05, **P<0.01, ***P<0.01 versus respective control. #p<0.05, compared to aHisAbs group.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[073] In the description that follows, a number of terms are used and the following definitions are provided to facilitate understanding of the claimed subject matter. Terms that are not expressly defined herein are used in accordance with their plain and ordinary meanings.
[074] Unless otherwise specified, "a" or "an" means "one or more".
[075] As used herein, the terms "and" and "or" may be used to mean either the conjunctive or disjunctive. That is, both terms should be understood as equivalent to "and/or" unless otherwise stated.
[076] A "therapeutic agent" is an atom, molecule, or compound that is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, peptides, drugs, toxins, enzymes, nucleases, hormones, immunomodulators, antisense oligonucleotides, small interfering RNA (siRNA), chelators, boron compounds, photoactive agents, dyes, and radioisotopes.
[077] A "diagnostic agent" is an atom, molecule, or compound that is useful in diagnosing a disease. Useful diagnostic agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI).
[078] An "antibody" as used herein refers to a full-length (i.e., naturally occurring or formed by normal immunoglobulin gene fragment recombinatorial processes) immunoglobulin molecule (e.g., an IgG antibody). An "antibody" includes monoclonal, polyclonal, bispecific, multispecific, murine, chimeric, humanized and human antibodies.
[079] A "naked antibody" is an antibody or antigen binding fragment thereof that is not attached to a therapeutic or diagnostic agent. The Fc portion of an intact naked antibody can provide effector functions, such as complement fixation and ADCC (see, e.g., Markrides, Pharmacol Rev 50:59-87, 1998). Other mechanisms by which naked antibodies induce cell death may include apoptosis. (Vaswani and Hamilton, Ann Allergy Asthma Immunol 81: 105-119, 1998.)
[080] An "antibody fragment" is a portion of an intact antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, sFv, scFv, dAb and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the full-length antibody. For example, antibody fragments include isolated fragments consisting of the variable regions, such as the "Fv"
fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"). "Single-chain antibodies", often abbreviated as "scFv"
consist of a polypeptide chain that comprises both a V. and a V, domain which interact to form an antigen- binding site. The VH and VL domains are usually linked by a peptide of 1 to 25 amino acid residues. Antibody fragments also include diabodies, triabodies and single domain antibodies (dAb). Fragments of antibodies that do not bind to the same antigen as the intact antibody, such as the Fc fragment, are not included within the scope of an "antibody fragment" as used herein.
fragments consisting of the variable regions of the heavy and light chains or recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins"). "Single-chain antibodies", often abbreviated as "scFv"
consist of a polypeptide chain that comprises both a V. and a V, domain which interact to form an antigen- binding site. The VH and VL domains are usually linked by a peptide of 1 to 25 amino acid residues. Antibody fragments also include diabodies, triabodies and single domain antibodies (dAb). Fragments of antibodies that do not bind to the same antigen as the intact antibody, such as the Fc fragment, are not included within the scope of an "antibody fragment" as used herein.
[081] A "chimeric antibody" is a recombinant protein that contains the variable domains of both the heavy and light antibody chains, including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, more preferably a murine antibody, while the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of other species, such as a primate, cat or dog.
[082] A "humanized antibody" is a recombinant protein in which the CDRs from an antibody from one species; e.g., a murine antibody, are transferred from the heavy and light variable chains of the murine antibody into human heavy and light variable domains (framework regions). The constant domains of the antibody molecule are derived from those of a human antibody. In some cases, specific residues of the framework region of the humanized antibody, particularly those that are touching or close to the CDR
sequences, may be modified, for example replaced with the corresponding residues from the original murine, rodent, subhuman primate, or other antibody.
sequences, may be modified, for example replaced with the corresponding residues from the original murine, rodent, subhuman primate, or other antibody.
[083] A "human antibody" is an antibody obtained, for example, from transgenic mice that have been "engineered" to produce human antibodies in response to antigenic challenge. In this technique, elements of the human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci. The transgenic mice can synthesize human antibodies specific for various antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described by Green et at., Nature Genet. 7:13 (1994), Lonberg et at., Nature 368:856 (1994), and Taylor et at., Int. Immun. 6:579 (1994). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art. See for example, McCafferty et at., Nature 348:552-553 (1990) for the production of human antibodies and fragments thereof in vitro, from immunoglobulin variable domain gene repertoires from unimmunized donors.
In this technique, human antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B
cell. Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may also be generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, the Examples section of each of which is incorporated herein by reference.
In this technique, human antibody variable domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B
cell. Phage display can be performed in a variety of formats, for their review, see e.g. Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571 (1993). Human antibodies may also be generated by in vitro activated B cells. See U.S. Patent Nos. 5,567,610 and 5,229,275, the Examples section of each of which is incorporated herein by reference.
[084] An "immunoconjugate" is an antibody, antigen-binding antibody fragment, antibody complex or antibody fusion protein that is conjugated to a therapeutic agent.
Conjugation may be covalent or non-covalent. Preferably, conjugation is covalent.
Conjugation may be covalent or non-covalent. Preferably, conjugation is covalent.
[085] As used herein, the term "antibody fusion protein" is a recombinantly-produced antigen-binding molecule in which one or more natural antibodies, single-chain antibodies or antibody fragments are linked to another moiety, such as a protein or peptide, a toxin, a cytokine, a hormone, etc. In certain preferred embodiments, the fusion protein may comprise two or more of the same or different antibodies, antibody fragments or single-chain antibodies fused together, which may bind to the same epitope, different epitopes on the same antigen, or different antigens.
[086] An "immunomodulator" is a therapeutic agent that when present, alters, suppresses or stimulates the body's immune system. Typically, an immunomodulator of use stimulates immune cells to proliferate or become activated in an immune response cascade, such as macrophages, dendritic cells, B-cells, and/or T-cells. However, in some cases an immunomodulator may suppress proliferation or activation of immune cells. An example of an immunomodulator as described herein is a cytokine, which is a soluble small protein of approximately 5-20 kDa that is released by one cell population (e.g., primed T-lymphocytes) on contact with specific antigens, and which acts as an intercellular mediator between cells.
As the skilled artisan will understand, examples of cytokines include lymphokines, monokines, interleukins, and several related signaling molecules, such as tumor necrosis factor (TNF) and interferons. Chemokines are a subset of cytokines. Certain interleukins and interferons are examples of cytokines that stimulate T cell or other immune cell proliferation.
Exemplary interferons include interferon-a, interferon-0, interferon-y and interferon-k.
As the skilled artisan will understand, examples of cytokines include lymphokines, monokines, interleukins, and several related signaling molecules, such as tumor necrosis factor (TNF) and interferons. Chemokines are a subset of cytokines. Certain interleukins and interferons are examples of cytokines that stimulate T cell or other immune cell proliferation.
Exemplary interferons include interferon-a, interferon-0, interferon-y and interferon-k.
[087] An anti-histone antibody or antibody fragment, or a composition described herein, is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. In particular embodiments, an antibody preparation is physiologically significant if its presence invokes an antitumor response or mitigates the signs and symptoms of an autoimmune disease state. A
physiologically significant effect could also be the evocation of a humoral and/or cellular immune response in the recipient subject leading to growth inhibition or death of target cells.
Anti-Histone Antibodies
physiologically significant effect could also be the evocation of a humoral and/or cellular immune response in the recipient subject leading to growth inhibition or death of target cells.
Anti-Histone Antibodies
[088] Various anti-histone antibodies and/or antigen-binding fragments thereof may be of use. The murine BWA-3 (anti-H4), LG2-1 (anti-H3) and LG2-2 (anti-H2B) hybridomas were reported by Monestier et al. (1993, Mol. Immunol 30:1069-75). However, murine antibodies are generally not appropriate for human therapeutic use, due to the formation of human anti-mouse antibodies (HAMA) that can neutralize these antibodies and thus make them less active.
[089] In preferred embodiments, a humanized or chimeric anti-histone H4 antibody is one that comprises the heavy chain complementarity-determining region (CDR) sequences CDR1 (DDYLH, SEQ ID NO:1), CDR2 (WIGWIDPENGDTEYASKFQG, SEQ ID NO:2) and CDR3 (PLVHLRTFAY, SEQ ID NO:3) and the light chain CDR sequences CDR1 (RASESVDSYDNSLH, SEQ ID NO:4), CDR2 (LASNLES, SEQ ID NO:5) and CDR3 (QQNNEDPWT, SEQ ID NO:6). (See, e.g., U.S. Patent No. 8,987,421, the Figures and Examples section of which are incorporated herein by reference.)
[090] In other preferred embodiments, a humanized or chimeric anti-histone H3 antibody is one that comprises the heavy chain CDR sequences CDR1 (SYWN41-1, SEQ ID NO:7), (NIDPSDSETHYNQKFKD, SEQ ID NO:8) and CDR3 (EKITDDYNYFDY, SEQ ID NO:9) and the light chain CDR sequences CDR1 (RASESVDSYGNSFMH, SEQ ID NO:10), CDR2 (HASNLES, SEQ ID NO:11) and CDR3 (QQNNEDPLT, SEQ ID NO:12) (see, e.g., U.S.
Patent No. 8,987,421).
Patent No. 8,987,421).
[091] In still other preferred embodiments, a humanized or chimeric anti-histone H2B
antibody is one that comprises the heavy chain CDR sequences CDR1 (SYVMY, SEQ
ID
NO:13), CDR2 (YINPYNDGTKYNEKFKG, SEQ ID NO:14) and CDR3 (PGDGYPFDY, SEQ ID NO:15) and the light chain CDR sequences CDR1 (RSSQSIVHSNGNTYLE, SEQ
ID NO:16), CDR2 (KVSNRFS, SEQ ID NO:17) and CDR3 (FQGSHVPYT, SEQ ID NO:18) (see, e.g., U.S. Patent No. 8,987,421).
General Techniques for Antibodies and Antibody Fragments
antibody is one that comprises the heavy chain CDR sequences CDR1 (SYVMY, SEQ
ID
NO:13), CDR2 (YINPYNDGTKYNEKFKG, SEQ ID NO:14) and CDR3 (PGDGYPFDY, SEQ ID NO:15) and the light chain CDR sequences CDR1 (RSSQSIVHSNGNTYLE, SEQ
ID NO:16), CDR2 (KVSNRFS, SEQ ID NO:17) and CDR3 (FQGSHVPYT, SEQ ID NO:18) (see, e.g., U.S. Patent No. 8,987,421).
General Techniques for Antibodies and Antibody Fragments
[092] Techniques for preparing monoclonal antibodies against virtually any target antigen are well known in the art. See, for example, Kohler and Milstein, Nature 256:
495 (1975), and Coligan et at. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). The person of ordinary skill may readily produce antibodies against any known and characterized target antigen, using only routine experimentation. Known antigens that may be targeted include, but are not limited to, human histone H4 (e.g., NCBI Ref. No. NP 778224.1), human histone H3 (e.g., GenBank Ref No.
CAB02546.1) or human histone H2B (e.g., GenBank Ref No. CAB02542.1)
495 (1975), and Coligan et at. (eds.), CURRENT PROTOCOLS IN IMMUNOLOGY, VOL. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991). The person of ordinary skill may readily produce antibodies against any known and characterized target antigen, using only routine experimentation. Known antigens that may be targeted include, but are not limited to, human histone H4 (e.g., NCBI Ref. No. NP 778224.1), human histone H3 (e.g., GenBank Ref No.
CAB02546.1) or human histone H2B (e.g., GenBank Ref No. CAB02542.1)
[093] Briefly, monoclonal antibodies can be obtained by injecting mice with a composition comprising an antigen, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
[094] MAbs can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography.
See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et at., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR
BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).
See, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3. Also, see Baines et at., "Purification of Immunoglobulin G (IgG)," in METHODS IN MOLECULAR
BIOLOGY, VOL. 10, pages 79-104 (The Humana Press, Inc. 1992).
[095] After the initial raising of antibodies to the immunogen, the antibodies can be sequenced and subsequently prepared by recombinant techniques. Humanization and chimerization of murine antibodies and antibody fragments are well known to those skilled in the art. The use of antibody components derived from humanized, chimeric or human antibodies obviates potential problems associated with the immunogenicity of murine constant regions.
Chimeric Antibodies
Chimeric Antibodies
[096] A chimeric antibody is a recombinant protein in which the variable regions of a human antibody have been replaced by the variable regions of, for example, a mouse antibody, including the complementarity-determining regions (CDRs) of the mouse antibody.
Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject. General techniques for cloning murine immunoglobulin variable domains are disclosed, for example, in Orlandi et al., Proc. Nat'l Acad. Sci.
USA 86: 3833 (1989). Techniques for constructing chimeric antibodies are well known to those of skill in the art. As an example, Leung et at., Hybridoma /3:469 (1994), produced an LL2 chimera by combining DNA sequences encoding the VK and VH domains of murine LL2, an anti-monoclonal antibody, with respective human lc and IgGi constant region domains.
Humanized Antibodies
Chimeric antibodies exhibit decreased immunogenicity and increased stability when administered to a subject. General techniques for cloning murine immunoglobulin variable domains are disclosed, for example, in Orlandi et al., Proc. Nat'l Acad. Sci.
USA 86: 3833 (1989). Techniques for constructing chimeric antibodies are well known to those of skill in the art. As an example, Leung et at., Hybridoma /3:469 (1994), produced an LL2 chimera by combining DNA sequences encoding the VK and VH domains of murine LL2, an anti-monoclonal antibody, with respective human lc and IgGi constant region domains.
Humanized Antibodies
[097] Techniques for producing humanized MAbs are well known in the art (see, e.g., Jones et at., Nature 321: 522 (1986), Riechmann et at., Nature 332: 323 (1988), Verhoeyen et at., Science 239: 1534 (1988), Carter et at., Proc. Nat'l Acad. Sci. USA 89: 4285 (1992), Sandhu, Crit. Rev. Biotech. 12: 437 (1992), and Singer et al., I Immun. 150: 2844 (1993)). A
chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the corresponding variable domains of a human antibody. The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences. As simply transferring mouse CDRs into human FRs often results in a reduction or even loss of antibody affinity, additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding affinity to its epitope. See, for example, Tempest et at., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534 (1988). Generally, those human FR amino acid residues that differ from their murine counterparts and are located close to or touching one or more CDR amino acid residues would be candidates for substitution.
Human Antibodies
chimeric or murine monoclonal antibody may be humanized by transferring the mouse CDRs from the heavy and light variable chains of the mouse immunoglobulin into the corresponding variable domains of a human antibody. The mouse framework regions (FR) in the chimeric monoclonal antibody are also replaced with human FR sequences. As simply transferring mouse CDRs into human FRs often results in a reduction or even loss of antibody affinity, additional modification might be required in order to restore the original affinity of the murine antibody. This can be accomplished by the replacement of one or more human residues in the FR regions with their murine counterparts to obtain an antibody that possesses good binding affinity to its epitope. See, for example, Tempest et at., Biotechnology 9:266 (1991) and Verhoeyen et al., Science 239: 1534 (1988). Generally, those human FR amino acid residues that differ from their murine counterparts and are located close to or touching one or more CDR amino acid residues would be candidates for substitution.
Human Antibodies
[098] Methods for producing fully human antibodies using either combinatorial approaches or transgenic animals transformed with human immunoglobulin loci are known in the art (e.g., Mancini et al., 2004, New Microbiol. 27:315-28; Conrad and Scheller, 2005, Comb.
Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin.
Phamacol.
3:544-50). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art.
See for example, McCafferty et at., Nature 348:552-553 (1990). Such fully human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and to function in vivo as essentially endogenous human antibodies.
In certain embodiments, the claimed methods and procedures may utilize human antibodies produced by such techniques.
Chem. High Throughput Screen. 8:117-26; Brekke and Loset, 2003, Curr. Opin.
Phamacol.
3:544-50). A fully human antibody also can be constructed by genetic or chromosomal transfection methods, as well as phage display technology, all of which are known in the art.
See for example, McCafferty et at., Nature 348:552-553 (1990). Such fully human antibodies are expected to exhibit even fewer side effects than chimeric or humanized antibodies and to function in vivo as essentially endogenous human antibodies.
In certain embodiments, the claimed methods and procedures may utilize human antibodies produced by such techniques.
[099] In one alternative, the phage display technique may be used to generate human antibodies (e.g., Dantas-Barbosa et al., 2005, Genet. Mot. Res. 4:126-40).
Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005). The advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
Human antibodies may be generated from normal humans or from humans that exhibit a particular disease state, such as cancer (Dantas-Barbosa et al., 2005). The advantage to constructing human antibodies from a diseased individual is that the circulating antibody repertoire may be biased towards antibodies against disease-associated antigens.
[0100] In one non-limiting example of this methodology, Dantas-Barbosa et al.
(2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Generally, total RNA was obtained from circulating blood lymphocytes (Id.).
Recombinant Fab were cloned from the II., y and lc chain antibody repertoires and inserted into a phage display library (Id.). RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, 1 Mol. Biol. 222:581-97). Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 14 edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 9.1 to 9.22). The final Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library. Such libraries may be screened by standard phage display methods, as known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159-162).
(2005) constructed a phage display library of human Fab antibody fragments from osteosarcoma patients. Generally, total RNA was obtained from circulating blood lymphocytes (Id.).
Recombinant Fab were cloned from the II., y and lc chain antibody repertoires and inserted into a phage display library (Id.). RNAs were converted to cDNAs and used to make Fab cDNA libraries using specific primers against the heavy and light chain immunoglobulin sequences (Marks et al., 1991, 1 Mol. Biol. 222:581-97). Library construction was performed according to Andris-Widhopf et al. (2000, In: Phage Display Laboratory Manual, Barbas et al. (eds), 14 edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY pp. 9.1 to 9.22). The final Fab fragments were digested with restriction endonucleases and inserted into the bacteriophage genome to make the phage display library. Such libraries may be screened by standard phage display methods, as known in the art (see, e.g., Pasqualini and Ruoslahti, 1996, Nature 380:364-366; Pasqualini, 1999, The Quart. J. Nucl. Med. 43:159-162).
[0101] Phage display can be performed in a variety of formats, for their review, see e.g.
Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571(1993).
Human antibodies may also be generated by in vitro activated B cells. See U.S.
Patent Nos.
5,567,610 and 5,229,275, incorporated herein by reference in their entirety.
The skilled artisan will realize that these techniques are exemplary and any known method for making and screening human antibodies or antibody fragments may be utilized.
Johnson and Chiswell, Current Opinion in Structural Biology 3:5564-571(1993).
Human antibodies may also be generated by in vitro activated B cells. See U.S.
Patent Nos.
5,567,610 and 5,229,275, incorporated herein by reference in their entirety.
The skilled artisan will realize that these techniques are exemplary and any known method for making and screening human antibodies or antibody fragments may be utilized.
[0102] In another alternative, transgenic animals that have been genetically engineered to produce human antibodies may be used to generate antibodies against essentially any immunogenic target, using standard immunization protocols. Methods for obtaining human antibodies from transgenic mice are disclosed by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example of such a system is the XenoMouseg (e.g., Green et al., 1999, 1 Immunol.
Methods 231:11-23) from Abgenix (Fremont, CA). In the XenoMouseg and similar animals, the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et al., Int. Immun. 6:579 (1994). A non-limiting example of such a system is the XenoMouseg (e.g., Green et al., 1999, 1 Immunol.
Methods 231:11-23) from Abgenix (Fremont, CA). In the XenoMouseg and similar animals, the mouse antibody genes have been inactivated and replaced by functional human antibody genes, while the remainder of the mouse immune system remains intact.
[0103] The XenoMouseg was transformed with germline-configured YACs (yeast artificial chromosomes) that contained portions of the human IgH and Igkappa loci, including the majority of the variable region sequences, along accessory genes and regulatory sequences.
The human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques. A XenoMouseg immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above. A
variety of strains of XenoMouse are available, each of which is capable of producing a different class of antibody. Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). The skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
Antibody Fragments
The human variable region repertoire may be used to generate antibody producing B cells, which may be processed into hybridomas by known techniques. A XenoMouseg immunized with a target antigen will produce human antibodies by the normal immune response, which may be harvested and/or produced by standard techniques discussed above. A
variety of strains of XenoMouse are available, each of which is capable of producing a different class of antibody. Transgenically produced human antibodies have been shown to have therapeutic potential, while retaining the pharmacokinetic properties of normal human antibodies (Green et al., 1999). The skilled artisan will realize that the claimed compositions and methods are not limited to use of the XenoMouse system but may utilize any transgenic animal that has been genetically engineered to produce human antibodies.
Antibody Fragments
[0104] Antibody fragments which recognize specific epitopes can be generated by known techniques. Antibody fragments are antigen binding portions of an antibody, such as F(ab)2, Fab', F(ab)2, Fab, Fv, sFy and the like. F(ab')2fragments can be produced by pepsin digestion of the antibody molecule and Fab' fragments can be generated by reducing disulfide bridges of the F(ab')2fragments. Alternatively, Fab' expression libraries can be constructed (Huse et at., 1989, Science, 246:1274-1281) to allow rapid and easy identification of monoclonal Fab' fragments with the desired specificity. F(ab)2fragments may be generated by papain digestion of an antibody.
[0105] A single chain Fv molecule (scFv) comprises a VL domain and a VH
domain. The VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L). Methods for making scFv molecules and designing suitable peptide linkers are described in US Patent No. 4,704,692, US Patent No. 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol 9:73-80 (1995) and R.E.
Bird and B.W. Walker, "Single Chain Antibody Variable Regions," TIBTECH, Vol 9: 132-137 (1991).
domain. The VL and VH domains associate to form a target binding site. These two domains are further covalently linked by a peptide linker (L). Methods for making scFv molecules and designing suitable peptide linkers are described in US Patent No. 4,704,692, US Patent No. 4,946,778, R. Raag and M. Whitlow, "Single Chain Fvs." FASEB Vol 9:73-80 (1995) and R.E.
Bird and B.W. Walker, "Single Chain Antibody Variable Regions," TIBTECH, Vol 9: 132-137 (1991).
[0106] Techniques for producing single domain antibodies are also known in the art, as disclosed for example in Cossins et al. (2006, Prot Express Purif 51:253-259), incorporated herein by reference. Single domain antibodies (VHH) may be obtained, for example, from camels, alpacas or llamas by standard immunization techniques. (See, e.g., Muyldermans et al., TIBS 26:230-235, 2001; Yau et al., J Immunol Methods 281:161-75, 2003;
Maass et al., J
Immunol Methods 324:13-25, 2007). The VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inacessible to conventional VH-VL pairs.
(Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized with known antigens, such as TNF-a, and VHHs can be isolated that bind to and neutralize the target antigen (Maass et al., 2007). PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al., 2007). In certain embodiments, anti-pancreatic cancer VHH antibody fragments may be utilized in the claimed compositions and methods.
Maass et al., J
Immunol Methods 324:13-25, 2007). The VHH may have potent antigen-binding capacity and can interact with novel epitopes that are inacessible to conventional VH-VL pairs.
(Muyldermans et al., 2001). Alpaca serum IgG contains about 50% camelid heavy chain only IgG antibodies (HCAbs) (Maass et al., 2007). Alpacas may be immunized with known antigens, such as TNF-a, and VHHs can be isolated that bind to and neutralize the target antigen (Maass et al., 2007). PCR primers that amplify virtually all alpaca VHH coding sequences have been identified and may be used to construct alpaca VHH phage display libraries, which can be used for antibody fragment isolation by standard biopanning techniques well known in the art (Maass et al., 2007). In certain embodiments, anti-pancreatic cancer VHH antibody fragments may be utilized in the claimed compositions and methods.
[0107] An antibody fragment can be prepared by proteolytic hydrolysis of the full length antibody or by expression in E. coil or another host of the DNA coding for the fragment. An antibody fragment can be obtained by pepsin or papain digestion of full length antibodies by conventional methods. These methods are described, for example, by Goldenberg, U.S.
Patent Nos. 4,036,945 and 4,331,647 and references contained therein. Also, see Nisonoff et at., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. 1 73: 119 (1959), Edelman et at., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
Known Antibodies
Patent Nos. 4,036,945 and 4,331,647 and references contained therein. Also, see Nisonoff et at., Arch Biochem. Biophys. 89: 230 (1960); Porter, Biochem. 1 73: 119 (1959), Edelman et at., in METHODS IN ENZYMOLOGY VOL. 1, page 422 (Academic Press 1967), and Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
Known Antibodies
[0108] In various embodiments, the claimed methods and compositions may utilize any of a variety of antibodies known in the art. Antibodies of use may be commercially obtained from a number of known sources. For example, a variety of antibody secreting hybridoma lines are available from the American Type Culture Collection (ATCC, Manassas, VA). A
large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions.
See, e.g., U.S.
Patent Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;
7,049,060;
7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133;
7,001,598;
6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854;
6,962,981;
6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;
6,921,645;
6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879;
6,893,625;
6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568;
6,867,006;
6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;
6,824,780;
6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688;
6,764,681;
6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966; 6,709,653;
6,693,176;
6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;
6,673,344;
6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441;
6,605,279;
6,596,852; 6,592,868; 6,576,745; 6,572;856; 6,566,076; 6,562,618; 6,545,130;
6,544,749;
6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;
6,488,930;
6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823;
6,458,356;
6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402;
6,419,928;
6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;
6,383,759;
6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245;
6,355,244;
6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868;
6,187,287;
6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;
5,814,440;
5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459;
5,443,953, 5,525,338, the Examples section of each of which is incorporated herein by reference. These are exemplary only and a wide variety of other antibodies and their hybridomas are known in the art. The skilled artisan will realize that antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest. The antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art (see, e.g., U.S. Patent Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples section of each of which is incorporated herein by reference).
large number of antibodies against various disease targets, including but not limited to tumor-associated antigens, have been deposited at the ATCC and/or have published variable region sequences and are available for use in the claimed methods and compositions.
See, e.g., U.S.
Patent Nos. 7,312,318; 7,282,567; 7,151,164; 7,074,403; 7,060,802; 7,056,509;
7,049,060;
7,045,132; 7,041,803; 7,041,802; 7,041,293; 7,038,018; 7,037,498; 7,012,133;
7,001,598;
6,998,468; 6,994,976; 6,994,852; 6,989,241; 6,974,863; 6,965,018; 6,964,854;
6,962,981;
6,962,813; 6,956,107; 6,951,924; 6,949,244; 6,946,129; 6,943,020; 6,939,547;
6,921,645;
6,921,645; 6,921,533; 6,919,433; 6,919,078; 6,916,475; 6,905,681; 6,899,879;
6,893,625;
6,887,468; 6,887,466; 6,884,594; 6,881,405; 6,878,812; 6,875,580; 6,872,568;
6,867,006;
6,864,062; 6,861,511; 6,861,227; 6,861,226; 6,838,282; 6,835,549; 6,835,370;
6,824,780;
6,824,778; 6,812,206; 6,793,924; 6,783,758; 6,770,450; 6,767,711; 6,764,688;
6,764,681;
6,764,679; 6,743,898; 6,733,981; 6,730,307; 6,720,155; 6,716,966; 6,709,653;
6,693,176;
6,692,908; 6,689,607; 6,689,362; 6,689,355; 6,682,737; 6,682,736; 6,682,734;
6,673,344;
6,653,104; 6,652,852; 6,635,482; 6,630,144; 6,610,833; 6,610,294; 6,605,441;
6,605,279;
6,596,852; 6,592,868; 6,576,745; 6,572;856; 6,566,076; 6,562,618; 6,545,130;
6,544,749;
6,534,058; 6,528,625; 6,528,269; 6,521,227; 6,518,404; 6,511,665; 6,491,915;
6,488,930;
6,482,598; 6,482,408; 6,479,247; 6,468,531; 6,468,529; 6,465,173; 6,461,823;
6,458,356;
6,455,044; 6,455,040, 6,451,310; 6,444,206; 6,441,143; 6,432,404; 6,432,402;
6,419,928;
6,413,726; 6,406,694; 6,403,770; 6,403,091; 6,395,276; 6,395,274; 6,387,350;
6,383,759;
6,383,484; 6,376,654; 6,372,215; 6,359,126; 6,355,481; 6,355,444; 6,355,245;
6,355,244;
6,346,246; 6,344,198; 6,340,571; 6,340,459; 6,331,175; 6,306,393; 6,254,868;
6,187,287;
6,183,744; 6,129,914; 6,120,767; 6,096,289; 6,077,499; 5,922,302; 5,874,540;
5,814,440;
5,798,229; 5,789,554; 5,776,456; 5,736,119; 5,716,595; 5,677,136; 5,587,459;
5,443,953, 5,525,338, the Examples section of each of which is incorporated herein by reference. These are exemplary only and a wide variety of other antibodies and their hybridomas are known in the art. The skilled artisan will realize that antibody sequences or antibody-secreting hybridomas against almost any disease-associated antigen may be obtained by a simple search of the ATCC, NCBI and/or USPTO databases for antibodies against a selected disease-associated target of interest. The antigen binding domains of the cloned antibodies may be amplified, excised, ligated into an expression vector, transfected into an adapted host cell and used for protein production, using standard techniques well known in the art (see, e.g., U.S. Patent Nos. 7,531,327; 7,537,930; 7,608,425 and 7,785,880, the Examples section of each of which is incorporated herein by reference).
[0109] Particular antibodies that may be of use include, but are not limited to, LL1 (anti-CD74), LL2 and RFB4 (anti-CD22), MN-14 (anti-carcinoembryonic antigen (CEA, also known as CD66e), hL243 (anti-HLA-DR), alemtuzumab (anti-CD52), gemtuzumab (anti-CD33), ibritumomab tiuxetan (anti-CD20); rituximab (anti-CD20); tositumomab (anti-CD20); and GA101 (anti-CD20). Such antibodies are known in the art (e.g., U.S.
Patent Nos.
5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730,300;
6,899,864;
6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004;
7,282,567;
7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. Patent Application Publ.
No. 20040202666 (now abandoned); 20050271671; and 20060193865; the Examples section of each incorporated herein by reference.) Specific known antibodies of use include hA20 (U.S. Patent No. 7,151,164), hA19 (U.S. Patent No. 7,109,304), hLL1 (U.S.
Patent No.
7,312,318, ), hLL2 (U.S. Patent No. 5,789,554), hL243 (U.S. Patent No.
7,612,180), hMN-14 (U.S. Patent No. 6,676,924), hMN-15 (U.S. Patent No. 8,287,865), and hMN-3 (U.S. Patent No. 7,541,440), the text of each recited patent or application is incorporated herein by reference with respect to the Figures and Examples sections.
Patent Nos.
5,686,072; 5,874,540; 6,107,090; 6,183,744; 6,306,393; 6,653,104; 6,730,300;
6,899,864;
6,926,893; 6,962,702; 7,074,403; 7,230,084; 7,238,785; 7,238,786; 7,256,004;
7,282,567;
7,300,655; 7,312,318; 7,585,491; 7,612,180; 7,642,239; and U.S. Patent Application Publ.
No. 20040202666 (now abandoned); 20050271671; and 20060193865; the Examples section of each incorporated herein by reference.) Specific known antibodies of use include hA20 (U.S. Patent No. 7,151,164), hA19 (U.S. Patent No. 7,109,304), hLL1 (U.S.
Patent No.
7,312,318, ), hLL2 (U.S. Patent No. 5,789,554), hL243 (U.S. Patent No.
7,612,180), hMN-14 (U.S. Patent No. 6,676,924), hMN-15 (U.S. Patent No. 8,287,865), and hMN-3 (U.S. Patent No. 7,541,440), the text of each recited patent or application is incorporated herein by reference with respect to the Figures and Examples sections.
[0110] Anti-TNF-a antibodies are known in the art and may be of use to treat immune diseases, such as autoimmune disease, immune dysfunction (e.g., graft-versus-host disease, organ transplant rejection) or diabetes. Known antibodies against TNF-a include the human antibody CDP571 (Ofei et al., 2011, Diabetes 45:881-85); murine antibodies MTNFAI, M2TNFAI, M3TNFAI, M3TNFABI, M302B and M303 (Thermo Scientific, Rockford, IL);
infliximab (Centocor, Malvern, PA); certolizumab pegol (UCB, Brussels, Belgium); and adalimumab (Abbott, Abbott Park, IL). These and many other known anti-TNF-a antibodies may be used in the claimed methods and compositions. Other antibodies of use for therapy of immune dysregulatory or autoimmune disease include, but are not limited to, anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25); daclizumab (anti-CD25); efalizumab (anti-CD11a);
muromonab-CD3 (anti-CD3 receptor); anti-CD4OL (UCB, Brussels, Belgium);
natalizumab (anti-a4 integrin) and omalizumab (anti-IgE).
infliximab (Centocor, Malvern, PA); certolizumab pegol (UCB, Brussels, Belgium); and adalimumab (Abbott, Abbott Park, IL). These and many other known anti-TNF-a antibodies may be used in the claimed methods and compositions. Other antibodies of use for therapy of immune dysregulatory or autoimmune disease include, but are not limited to, anti-B-cell antibodies such as veltuzumab, epratuzumab, milatuzumab or hL243; tocilizumab (anti-IL-6 receptor); basiliximab (anti-CD25); daclizumab (anti-CD25); efalizumab (anti-CD11a);
muromonab-CD3 (anti-CD3 receptor); anti-CD4OL (UCB, Brussels, Belgium);
natalizumab (anti-a4 integrin) and omalizumab (anti-IgE).
[0111] Macrophage migration inhibitory factor (MIF) is an important regulator of innate and adaptive immunity and apoptosis. It has been reported that CD74 is the endogenous receptor for MIF (Leng et al., 2003, J Exp Med 197:1467-76). The therapeutic effect of antagonistic anti-CD74 antibodies on MIF-mediated intracellular pathways may be of use for treatment of a broad range of disease states, such as autoimmune diseases like rheumatoid arthritis and systemic lupus erythematosus (Morand & Leech, 2005, Front Biosci 10:12-22;
Shachar &
Haran, 2011, Leuk Lymphoma 52:1446-54); kidney diseases such as renal allograft rejection (Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and numerous inflammatory diseases (Meyer-Siegler et al., 2009, Mediators Inflamm epub March 22, 2009; Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of therapeutic use for treatment of MIF-mediated diseases.
Bispecific and Multispecific Antibodies
Shachar &
Haran, 2011, Leuk Lymphoma 52:1446-54); kidney diseases such as renal allograft rejection (Lan, 2008, Nephron Exp Nephrol. 109:e79-83); and numerous inflammatory diseases (Meyer-Siegler et al., 2009, Mediators Inflamm epub March 22, 2009; Takahashi et al., 2009, Respir Res 10:33; Milatuzumab (hLL1) is an exemplary anti-CD74 antibody of therapeutic use for treatment of MIF-mediated diseases.
Bispecific and Multispecific Antibodies
[0112] Bispecific or multispecific antibodies can be prepared by a variety of procedures, ranging from glutaraldehyde linkage to more specific linkages between functional groups.
The antibodies and/or antibody fragments are preferably covalently bound to one another, directly or through a linker moiety, through one or more functional groups on the antibody or fragment, e. g., amine, carboxyl, phenyl, thiol, or hydroxyl groups. Various conventional linkers in addition to glutaraldehyde can be used, e. g., disiocyanates, diiosothiocyanates, bis (hydroxysuccinimide) esters, carbodiimides, maleimidehydroxy-succinimde esters, and the like. The optimal length of the linker may vary according to the type of target cell.
The antibodies and/or antibody fragments are preferably covalently bound to one another, directly or through a linker moiety, through one or more functional groups on the antibody or fragment, e. g., amine, carboxyl, phenyl, thiol, or hydroxyl groups. Various conventional linkers in addition to glutaraldehyde can be used, e. g., disiocyanates, diiosothiocyanates, bis (hydroxysuccinimide) esters, carbodiimides, maleimidehydroxy-succinimde esters, and the like. The optimal length of the linker may vary according to the type of target cell.
[0113] A simple method to produce multivalent antibodies is to mix the antibodies or fragments in the presence of glutaraldehyde. The initial Schiff base linkages can be stabilized, e. g., by borohydride reduction to secondary amines. A
diiosothiocyanate or carbodiimide can be used in place of glutaraldehyde as a non-site-specific linker.
diiosothiocyanate or carbodiimide can be used in place of glutaraldehyde as a non-site-specific linker.
[0114] The simplest form of a multivalent, multispecific antibody is a bispecific antibody.
Bispecific antibodies can be made by a variety of conventional methods, e. g., disulfide cleavage and reformation of mixtures of whole IgG or, preferably F (ab')2 fragments, fusions of more than one hybridoma to form polyomas that produce antibodies having more than one specificity, and by genetic engineering. Bispecific antibodies have been prepared by oxidative cleavage of Fab' fragments resulting from reductive cleavage of different antibodies. This is advantageously carried out by mixing two different F
(ab')2 fragments produced by pepsin digestion of two different antibodies, reductive cleavage to form a mixture of Fab' fragments, followed by oxidative reformation of the disulfide linkages to produce a mixture of F (ab')2 fragments including bispecific antibodies containing a Fab' portion specific to each of the original epitopes.
Bispecific antibodies can be made by a variety of conventional methods, e. g., disulfide cleavage and reformation of mixtures of whole IgG or, preferably F (ab')2 fragments, fusions of more than one hybridoma to form polyomas that produce antibodies having more than one specificity, and by genetic engineering. Bispecific antibodies have been prepared by oxidative cleavage of Fab' fragments resulting from reductive cleavage of different antibodies. This is advantageously carried out by mixing two different F
(ab')2 fragments produced by pepsin digestion of two different antibodies, reductive cleavage to form a mixture of Fab' fragments, followed by oxidative reformation of the disulfide linkages to produce a mixture of F (ab')2 fragments including bispecific antibodies containing a Fab' portion specific to each of the original epitopes.
[0115] General techniques for the preparation of multivalent antibodies may be found, for example, in Nisonhoff et al., Arch Biochem. Biophys. 93: 470 (1961), Hammerling et al., J.
Exp. Med. 128: 1461 (1968), and U. S. Patent No. 4,331,647.
Exp. Med. 128: 1461 (1968), and U. S. Patent No. 4,331,647.
[0116] More selective linkage can be achieved by using a heterobifunctional linker such as maleimide-hydroxysuccinimide ester. Reaction of the ester with an antibody or fragment will derivatize amine groups on the antibody or fragment, and the derivative can then be reacted with, e. g., an antibody Fab fragment having free sulfhydryl groups (or, a larger fragment or intact antibody with sulfhydryl groups appended thereto by, e. g., Traut's Reagent. Such a linker is less likely to crosslink groups in the same antibody and improves the selectivity of the linkage.
[0117] It is advantageous to link the antibodies or fragments at sites remote from the antigen binding sites. This can be accomplished by, e. g., linkage to cleaved interchain sulfydryl groups, as noted above. Another method involves reacting an antibody having an oxidized carbohydrate portion with another antibody which has at least one free amine function. This results in an initial Schiff base (imine) linkage, which is preferably stabilized by reduction to a secondary amine, e. g., by borohydride reduction, to form the final product.
Such site-specific linkages are disclosed, for small molecules, in U. S. Patent No.
4,671,958, and for larger addends in U. S. Patent No. 4,699,784.
Such site-specific linkages are disclosed, for small molecules, in U. S. Patent No.
4,671,958, and for larger addends in U. S. Patent No. 4,699,784.
[0118] Alternatively, such bispecific antibodies can be produced by fusing two hybridoma cell lines that produce appropriate Mabs. Techniques for producing tetradomas are described, for example, by Milstein et al., Nature 305: 537 (1983) and Pohl et al., Int.
I Cancer 54: 418 (1993).
I Cancer 54: 418 (1993).
[0119] Alternatively, chimeric genes can be designed that encode both binding domains.
General techniques for producing bispecific antibodies by genetic engineering are described, for example, by Songsivilai et al., Biochem Biophys Res. Commun 164: 271 (1989);
Traunecker et al., EMBO 1 10: 3655 (1991); and Weiner et al., I Immunol. 147:
(1991).
General techniques for producing bispecific antibodies by genetic engineering are described, for example, by Songsivilai et al., Biochem Biophys Res. Commun 164: 271 (1989);
Traunecker et al., EMBO 1 10: 3655 (1991); and Weiner et al., I Immunol. 147:
(1991).
[0120] A higher order multivalent, multispecific molecule can be obtained by adding various antibody components to a bispecific antibody, produced as above. For example, a bispecific antibody can be reacted with 2-iminothiolane to introduce one or more sulfhydryl groups for use in coupling the bispecific antibody to a further antibody derivative that binds an the same or a different epitope of the target antigen, using the bis-maleimide activation procedure described above. These techniques for producing multivalent antibodies are well known to those of skill in the art. See, for example, U. S. Patent No. 4,925,648, and Goldenberg, international publication No. WO 92/19273, which are incorporated by reference.
DOCK-AND-LOCK (DNLO)
DOCK-AND-LOCK (DNLO)
[0121] In preferred embodiments, a bispecific or multispecific antibody is formed as a DOCK-AND-LOCK (DNL4D) complex (see, e.g., U.S. Patent Nos. 7,521,056;
7,527,787;
7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004;
5: 959).
The DDD and AD peptides may be attached to any protein, peptide or other molecule.
Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.
7,527,787;
7,534,866; 7,550,143 and 7,666,400, the Examples section of each of which is incorporated herein by reference.) Generally, the technique takes advantage of the specific and high-affinity binding interactions that occur between a dimerization and docking domain (DDD) sequence of the regulatory (R) subunits of cAMP-dependent protein kinase (PKA) and an anchor domain (AD) sequence derived from any of a variety of AKAP proteins (Baillie et al., FEBS Letters. 2005; 579: 3264. Wong and Scott, Nat. Rev. Mol. Cell Biol. 2004;
5: 959).
The DDD and AD peptides may be attached to any protein, peptide or other molecule.
Because the DDD sequences spontaneously dimerize and bind to the AD sequence, the technique allows the formation of complexes between any selected molecules that may be attached to DDD or AD sequences.
[0122] Although the standard DNL complex comprises a trimer with two DDD-linked molecules attached to one AD-linked molecule, variations in complex structure allow the formation of dimers, trimers, tetramers, pentamers, hexamers and other multimers. In some embodiments, the DNL complex may comprise two or more antibodies, antibody fragments or fusion proteins which bind to the same antigenic determinant or to two or more different antigens. The DNL complex may also comprise one or more other effectors, such as proteins, peptides, immunomodulators, cytokines, interleukins, interferons, binding proteins, peptide ligands, carrier proteins, toxins, ribonucleases such as onconase, inhibitory oligonucleotides such as siRNA, antigens or xenoantigens, polymers such as PEG, enzymes, therapeutic agents, hormones, cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents or any other molecule or aggregate.
[0123] PKA, which plays a central role in one of the best studied signal transduction pathways triggered by the binding of the second messenger cAMP to the R
subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al.,J. Biol. Chem.
1968;243:3763).
The structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, I Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RI), and each type has a and I isoforms (Scott, Pharmacol.
Ther. . 1991;50:123). Thus, the four isoforms of PKA regulatory subunits are Ma, RI13, RIIa and RII13. The R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues of RIIa (Newlon et at., Nat. Struct. Biol. 1999; 6:222). As discussed below, similar portions of the amino acid sequences of other regulatory subunits are involved in dimerization and docking, each located near the N-terminal end of the regulatory subunit. Binding of cAMP to the R
subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et at., I Biol. Chem. 1990;265;21561)
subunits, was first isolated from rabbit skeletal muscle in 1968 (Walsh et al.,J. Biol. Chem.
1968;243:3763).
The structure of the holoenzyme consists of two catalytic subunits held in an inactive form by the R subunits (Taylor, I Biol. Chem. 1989;264:8443). Isozymes of PKA are found with two types of R subunits (RI and RI), and each type has a and I isoforms (Scott, Pharmacol.
Ther. . 1991;50:123). Thus, the four isoforms of PKA regulatory subunits are Ma, RI13, RIIa and RII13. The R subunits have been isolated only as stable dimers and the dimerization domain has been shown to consist of the first 44 amino-terminal residues of RIIa (Newlon et at., Nat. Struct. Biol. 1999; 6:222). As discussed below, similar portions of the amino acid sequences of other regulatory subunits are involved in dimerization and docking, each located near the N-terminal end of the regulatory subunit. Binding of cAMP to the R
subunits leads to the release of active catalytic subunits for a broad spectrum of serine/threonine kinase activities, which are oriented toward selected substrates through the compartmentalization of PKA via its docking with AKAPs (Scott et at., I Biol. Chem. 1990;265;21561)
[0124] Since the first AKAP, microtubule-associated protein-2, was characterized in 1984 (Lohmann et al., Proc. Natl. Acad. Sci USA. 1984; 81:6723), more than 50 AKAPs that localize to various sub-cellular sites, including plasma membrane, actin cytoskeleton, nucleus, mitochondria, and endoplasmic reticulum, have been identified with diverse structures in species ranging from yeast to humans (Wong and Scott, Nat. Rev.
Mot. Cell Biol. 2004;5:959). The AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr et at., I Biol. Chem. 1991;266:14188). The amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII
dimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445).
AKAPs will only bind to dimeric R subunits. For human RIIa, the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;
6:216). Thus, the dimerization domain and AKAP binding domain of human RIIa are both located within the same N-terminal 44 amino acid sequence (Newlon et at., Nat. Struct. Biol.
1999;6:222;
Newlon et al ., EMBO 1 2001;20:1651), which is termed the DDD herein.
Mot. Cell Biol. 2004;5:959). The AD of AKAPs for PKA is an amphipathic helix of 14-18 residues (Carr et at., I Biol. Chem. 1991;266:14188). The amino acid sequences of the AD are quite varied among individual AKAPs, with the binding affinities reported for RII
dimers ranging from 2 to 90 nM (Alto et al., Proc. Natl. Acad. Sci. USA. 2003;100:4445).
AKAPs will only bind to dimeric R subunits. For human RIIa, the AD binds to a hydrophobic surface formed by the 23 amino-terminal residues (Colledge and Scott, Trends Cell Biol. 1999;
6:216). Thus, the dimerization domain and AKAP binding domain of human RIIa are both located within the same N-terminal 44 amino acid sequence (Newlon et at., Nat. Struct. Biol.
1999;6:222;
Newlon et al ., EMBO 1 2001;20:1651), which is termed the DDD herein.
[0125] We have developed a platform technology to utilize the DDD of human PKA
regulatory subunits and the AD of AKAP as an excellent pair of linker modules for docking any two entities, referred to hereafter as A and B, into a noncovalent complex, which could be further locked into a DNL complex through the introduction of cysteine residues into both the DDD and AD at strategic positions to facilitate the formation of disulfide bonds. The general methodology of the approach is as follows. Entity A is constructed by linking a DDD
sequence to a precursor of A, resulting in a first component hereafter referred to as a.
Because the DDD sequence would effect the spontaneous formation of a dimer, A
would thus be composed of az. Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b. The dimeric motif of DDD
contained in a2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a2 and b to form a binary, trimeric complex composed of a2b. This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et at., Proc. Natl.
Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations of linkers, adaptor modules and precursors, a wide variety of DNL constructs of different stoichiometry may be produced and used (see, e.g., U.S. Nos. 7,550,143;
7,521,056;
7,534,866; 7,527,787 and 7,666,400.)
regulatory subunits and the AD of AKAP as an excellent pair of linker modules for docking any two entities, referred to hereafter as A and B, into a noncovalent complex, which could be further locked into a DNL complex through the introduction of cysteine residues into both the DDD and AD at strategic positions to facilitate the formation of disulfide bonds. The general methodology of the approach is as follows. Entity A is constructed by linking a DDD
sequence to a precursor of A, resulting in a first component hereafter referred to as a.
Because the DDD sequence would effect the spontaneous formation of a dimer, A
would thus be composed of az. Entity B is constructed by linking an AD sequence to a precursor of B, resulting in a second component hereafter referred to as b. The dimeric motif of DDD
contained in a2 will create a docking site for binding to the AD sequence contained in b, thus facilitating a ready association of a2 and b to form a binary, trimeric complex composed of a2b. This binding event is made irreversible with a subsequent reaction to covalently secure the two entities via disulfide bridges, which occurs very efficiently based on the principle of effective local concentration because the initial binding interactions should bring the reactive thiol groups placed onto both the DDD and AD into proximity (Chmura et at., Proc. Natl.
Acad. Sci. USA. 2001;98:8480) to ligate site-specifically. Using various combinations of linkers, adaptor modules and precursors, a wide variety of DNL constructs of different stoichiometry may be produced and used (see, e.g., U.S. Nos. 7,550,143;
7,521,056;
7,534,866; 7,527,787 and 7,666,400.)
[0126] By attaching the DDD and AD away from the functional groups of the two precursors, such site-specific ligations are also expected to preserve the original activities of the two precursors. This approach is modular in nature and potentially can be applied to link, site-specifically and covalently, a wide range of substances, including peptides, proteins, antibodies, antibody fragments, and other effector moieties with a wide range of activities.
Utilizing the fusion protein method of constructing AD and DDD conjugated effectors described in the Examples below, virtually any protein or peptide may be incorporated into a DNL construct. However, the technique is not limiting and other methods of conjugation may be utilized.
Utilizing the fusion protein method of constructing AD and DDD conjugated effectors described in the Examples below, virtually any protein or peptide may be incorporated into a DNL construct. However, the technique is not limiting and other methods of conjugation may be utilized.
[0127] A variety of methods are known for making fusion proteins, including nucleic acid synthesis, hybridization and/or amplification to produce a synthetic double-stranded nucleic acid encoding a fusion protein of interest. Such double-stranded nucleic acids may be inserted into expression vectors for fusion protein production by standard molecular biology techniques (see, e.g. Sambrook et al., Molecular Cloning, A laboratory manual, 2' Ed, 1989).
In such preferred embodiments, the AD and/or DDD moiety may be attached to either the N-terminal or C-terminal end of an effector protein or peptide. However, the skilled artisan will realize that the site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity. Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
Structure-Function Relationships in AD and DDD Moieties
In such preferred embodiments, the AD and/or DDD moiety may be attached to either the N-terminal or C-terminal end of an effector protein or peptide. However, the skilled artisan will realize that the site of attachment of an AD or DDD moiety to an effector moiety may vary, depending on the chemical nature of the effector moiety and the part(s) of the effector moiety involved in its physiological activity. Site-specific attachment of a variety of effector moieties may be performed using techniques known in the art, such as the use of bivalent cross-linking reagents and/or other chemical conjugation techniques.
Structure-Function Relationships in AD and DDD Moieties
[0128] For different types of DNL constructs, different AD or DDD sequences may be utilized. Exemplary DDD and AD sequences are provided below.
DDD/
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:19) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:20) AD/
QIEYLAKQIVDNAIQQA (SEQ ID NO:21) CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:22)
DDD/
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:19) CGHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID
NO:20) AD/
QIEYLAKQIVDNAIQQA (SEQ ID NO:21) CGQIEYLAKQIVDNAIQQAGC (SEQ ID NO:22)
[0129] The skilled artisan will realize that DDD1 and DDD2 are based on the DDD sequence of the human RIIa isoform of protein kinase A. However, in alternative embodiments, the DDD and AD moieties may be based on the DDD sequence of the human Ma form of protein kinase A and a corresponding AKAP sequence, as exemplified in DDD3, and AD3 below.
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK
(SEQ ID NO:23) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEE
AK (SEQ ID NO:24) CGFEELAWKIAKMIWSDVFQQGC (SEQ ID NO:25)
SLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEEAK
(SEQ ID NO:23) MSCGGSLRECELYVQKHNIQALLKDSIVQLCTARPERPMAFLREYFERLEKEE
AK (SEQ ID NO:24) CGFEELAWKIAKMIWSDVFQQGC (SEQ ID NO:25)
[0130] In other alternative embodiments, other sequence variants of AD and/or DDD
moieties may be utilized in construction of the DNL complexes. For example, there are only four variants of human PKA DDD sequences, corresponding to the DDD
moieties of PKA Ma, RITa, RI(3 and RII(3. The Mkt DDD sequence is the basis of DDD1 and disclosed above. The four human PKA DDD sequences are shown below. The DDD
sequence represents residues 1-44 of Mx, 1-44 of RII13, 12-61 of RIa and 13-66 of RIP.
(Note that the sequence of DDD1 is modified slightly from the human PKA RIIa DDD
moiety.) PKA Rla SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK (SEQ ID
NO:26) PKA RI,8 SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA (SEQ
ID NO:27) PKA Rila SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ (SEQ ID NO:28) PKA
SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER (SEQ ID NO :29)
moieties may be utilized in construction of the DNL complexes. For example, there are only four variants of human PKA DDD sequences, corresponding to the DDD
moieties of PKA Ma, RITa, RI(3 and RII(3. The Mkt DDD sequence is the basis of DDD1 and disclosed above. The four human PKA DDD sequences are shown below. The DDD
sequence represents residues 1-44 of Mx, 1-44 of RII13, 12-61 of RIa and 13-66 of RIP.
(Note that the sequence of DDD1 is modified slightly from the human PKA RIIa DDD
moiety.) PKA Rla SLRECELYVQKHNIQALLKDVSIVQLCTARPERPMAFLREYFEKLEKEEAK (SEQ ID
NO:26) PKA RI,8 SLKGCELYVQLHGIQQVLKDCIVHLCISKPERPMKFLREHFEKLEKEENRQILA (SEQ
ID NO:27) PKA Rila SHIQIPPGLTELLQGYTVEVGQQPPDLVDFAVEYFTRLREARRQ (SEQ ID NO:28) PKA
SIEIPAGLTELLQGFTVEVLRHQPADLLEFALQHFTRLQQENER (SEQ ID NO :29)
[0131] The structure-function relationships of the AD and DDD domains have been the subject of investigation. (See, e.g., Burns-Hamuro et al., 2005, Protein Sci 14:2982-92; Can et al., 2001, J Blot Chem 276:17332-38; Alto et al., 2003, Proc Natl Acad Sci USA 100:4445-50; Hundsrucker et al., 2006, Biochem J396:297-306; Stokka et al., 2006, Biochem J
400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408, the entire text of each of which is incorporated herein by reference.)
400:493-99; Gold et al., 2006, Mol Cell 24:383-95; Kinderman et al., 2006, Mol Cell 24:397-408, the entire text of each of which is incorporated herein by reference.)
[0132] For example, Kinderman et al. (2006, Mol Cell 24:397-408) examined the crystal structure of the AD-DDD binding interaction and concluded that the human DDD
sequence contained a number of conserved amino acid residues that were important in either dimer formation or AKAP binding, underlined in SEQ ID NO:19 below. (See Figure 1 of Kinderman et al., 2006, incorporated herein by reference.) The skilled artisan will realize that in designing sequence variants of the DDD sequence, one would desirably avoid changing any of the underlined residues, while conservative amino acid substitutions might be made for residues that are less critical for dimerization and AKAP binding.
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:19)
sequence contained a number of conserved amino acid residues that were important in either dimer formation or AKAP binding, underlined in SEQ ID NO:19 below. (See Figure 1 of Kinderman et al., 2006, incorporated herein by reference.) The skilled artisan will realize that in designing sequence variants of the DDD sequence, one would desirably avoid changing any of the underlined residues, while conservative amino acid substitutions might be made for residues that are less critical for dimerization and AKAP binding.
SHIQIPPGLTELLQGYTVEVLRQQPPDLVEFAVEYFTRLREARA (SEQ ID NO:19)
[0133] Alto et al. (2003, Proc Natl Acad Sci USA 100:4445-50) performed a bioinformatic analysis of the AD sequence of various AKAP proteins to design an Rh selective AD
sequence called AKAP-IS (SEQ ID NO:21), with a binding constant for DDD of 0.4 nM.
The AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA.
Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD
are underlined in SEQ ID NO:21 below. The skilled artisan will realize that in designing sequence variants of the AD sequence, one would desirably avoid changing any of the underlined residues, while conservative amino acid substitutions might be made for residues that are less critical for DDD binding.
AKAP-IS
QIEYLAKQIVDNAIQQA (SEQ ID NO:21)
sequence called AKAP-IS (SEQ ID NO:21), with a binding constant for DDD of 0.4 nM.
The AKAP-IS sequence was designed as a peptide antagonist of AKAP binding to PKA.
Residues in the AKAP-IS sequence where substitutions tended to decrease binding to DDD
are underlined in SEQ ID NO:21 below. The skilled artisan will realize that in designing sequence variants of the AD sequence, one would desirably avoid changing any of the underlined residues, while conservative amino acid substitutions might be made for residues that are less critical for DDD binding.
AKAP-IS
QIEYLAKQIVDNAIQQA (SEQ ID NO:21)
[0134] Gold et al. (2006, Mol Cell 24:383-95) utilized crystallography and peptide screening to develop a SuperAKAP-IS sequence (SEQ ID NO:30), exhibiting a five order of magnitude higher selectivity for the Rh isoform of PKA compared with the RI isoform.
Underlined residues indicate the positions of amino acid substitutions, relative to the AKAP-IS sequence, which increased binding to the DDD moiety of RIIa. In this sequence, the N-terminal Q
residue is numbered as residue number 4 and the C-terminal A residue is residue number 20.
Residues where substitutions could be made to affect the affinity for RIIa were residues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It is contemplated that in certain alternative embodiments, the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD
moiety sequence to prepare DNL constructs. Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins.
SuperAKAP-IS
QIEYVAKQIVDYAIHQA (SEQ ID NO:30)
Underlined residues indicate the positions of amino acid substitutions, relative to the AKAP-IS sequence, which increased binding to the DDD moiety of RIIa. In this sequence, the N-terminal Q
residue is numbered as residue number 4 and the C-terminal A residue is residue number 20.
Residues where substitutions could be made to affect the affinity for RIIa were residues 8, 11, 15, 16, 18, 19 and 20 (Gold et al., 2006). It is contemplated that in certain alternative embodiments, the SuperAKAP-IS sequence may be substituted for the AKAP-IS AD
moiety sequence to prepare DNL constructs. Figure 2 of Gold et al. disclosed additional DDD-binding sequences from a variety of AKAP proteins.
SuperAKAP-IS
QIEYVAKQIVDYAIHQA (SEQ ID NO:30)
[0135] Stokka et al. (2006, Biochem J 400:493-99) also developed peptide competitors of AKAP binding to PKA. The peptide antagonists were designated as Ht31, RIAD and PV-38.
The Ht-31 peptide exhibited a greater affinity for the MI isoform of PKA, while the MAD
and PV-38 showed higher affinity for RI.
The Ht-31 peptide exhibited a greater affinity for the MI isoform of PKA, while the MAD
and PV-38 showed higher affinity for RI.
[0136] Hundsrucker et al. (2006, Biochem J396:297-306) developed still other peptide competitors for AKAP binding to PKA, with a binding constant as low as 0.4 nM
to the DDD
of the MI form of PKA. The sequences of various AKAP antagonistic peptides are provided in Table 1 of Hundsrucker et al.
to the DDD
of the MI form of PKA. The sequences of various AKAP antagonistic peptides are provided in Table 1 of Hundsrucker et al.
[0137] Residues that were highly conserved among the AD domains of different AKAP
proteins are indicated below by underlining with reference to the AKAP IS
sequence (SEQ
ID NO:21). The residues are the same as observed by Alto et al. (2003), with the addition of the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated herein by reference.) The sequences of peptide antagonists with particularly high affinities for the RII DDD sequence were those of AKAP-IS, AKAP76-wt-pep, AKAP76-L304T-pep and AKAP76-L308D-pep.
AKAP -IS
QIEYLAKQIVDNAIQQA (SEQ ID NO:21)
proteins are indicated below by underlining with reference to the AKAP IS
sequence (SEQ
ID NO:21). The residues are the same as observed by Alto et al. (2003), with the addition of the C-terminal alanine residue. (See FIG. 4 of Hundsrucker et al. (2006), incorporated herein by reference.) The sequences of peptide antagonists with particularly high affinities for the RII DDD sequence were those of AKAP-IS, AKAP76-wt-pep, AKAP76-L304T-pep and AKAP76-L308D-pep.
AKAP -IS
QIEYLAKQIVDNAIQQA (SEQ ID NO:21)
[0138] Can et al. (2001, J Biol Chem 276:17332-38) examined the degree of sequence homology between different AKAP-binding DDD sequences from human and non-human proteins and identified residues in the DDD sequences that appeared to be the most highly conserved among different DDD moieties. These are indicated below by underlining with reference to the human PKA RIIa DDD sequence of SEQ ID NO:19. Residues that were particularly conserved are further indicated by italics. The residues overlap with, but are not identical to those suggested by Kinderman et al. (2006) to be important for binding to AKAP
proteins. The skilled artisan will realize that in designing sequence variants of DDD, it would be most preferred to avoid changing the most conserved residues (italicized), and it would be preferred to also avoid changing the conserved residues (underlined), while conservative amino acid substitutions may be considered for residues that are neither underlined nor italicized.
SHIQ/PP GLTELL QGY TV EVLRQOPPDLVEFA VEYF1RLREARA (SEQ ID NO:19)
proteins. The skilled artisan will realize that in designing sequence variants of DDD, it would be most preferred to avoid changing the most conserved residues (italicized), and it would be preferred to also avoid changing the conserved residues (underlined), while conservative amino acid substitutions may be considered for residues that are neither underlined nor italicized.
SHIQ/PP GLTELL QGY TV EVLRQOPPDLVEFA VEYF1RLREARA (SEQ ID NO:19)
[0139] The skilled artisan will realize that these and other amino acid substitutions in the DDD or AD amino acid sequences may be utilized to produce alternative species within the genus of AD or DDD moieties, using techniques that are standard in the field and only routine experimentation.
Alternative DNLO Structures
Alternative DNLO Structures
[0140] In certain alternative embodiments, DNL constructs may be formed using alternatively constructed antibodies or antibody fragments, in which an AD
moiety may be attached at the C-terminal end of the kappa light chain (Ck), instead of the C-terminal end of the Fc on the heavy chain. The alternatively formed DNL constructs may be prepared as disclosed in U.S. Patent No. 9,446,123, the entire text of which is incorporated herein by reference. The light chain conjugated DNL constructs exhibit enhanced Fc-effector function activity in vitro and improved pharmacokinetics, stability and anti-lymphoma activity in vivo (Rossi et al., 2013, Bioconjug Chem 24:63-71).
moiety may be attached at the C-terminal end of the kappa light chain (Ck), instead of the C-terminal end of the Fc on the heavy chain. The alternatively formed DNL constructs may be prepared as disclosed in U.S. Patent No. 9,446,123, the entire text of which is incorporated herein by reference. The light chain conjugated DNL constructs exhibit enhanced Fc-effector function activity in vitro and improved pharmacokinetics, stability and anti-lymphoma activity in vivo (Rossi et al., 2013, Bioconjug Chem 24:63-71).
[0141] Ck-conjugated DNL constructs may be prepared as disclosed in U.S.
Patent No.
9,446,123. Briefly, Ck-AD2-IgG, was generated by recombinant engineering, whereby the AD2 peptide was fused to the C-terminal end of the kappa light chain. Because the natural C-terminus of CK is a cysteine residue, which forms a disulfide bridge to CHL a 16-amino acid residue "hinge" linker was used to space the AD2 from the CK-VH1 disulfide bridge. The mammalian expression vectors for Ck-AD2-IgG-veltuzumab and Ck-AD2-IgG-epratuzumab were constructed using the pdHL2 vector, which was used previously for expression of the homologous CH3-AD2-IgG modules. A 2208-bp nucleotide sequence was synthesized comprising the pdHL2 vector sequence ranging from the Barn HI restriction site within the VK/CK intron to the Xho I restriction site 3' of the Ck intron, with the insertion of the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ ID NO:31) and AD2, in frame at the 3' end of the coding sequence for CK. This synthetic sequence was inserted into the IgG-pdHL2 expression vectors for veltuzumab and epratuzumab via Barn HI and Xho I
restriction sites. Generation of production clones with SpESFX-10 were performed as described for the CH3-AD2-IgG modules. Ck-AD2-IgG-veltuzumab and Ck-AD2-IgG-epratuzumab were produced by stably-transfected production clones in batch roller bottle culture, and purified from the supernatant fluid in a single step using Mab Select (GE
Healthcare) Protein A affinity chromatography.
Patent No.
9,446,123. Briefly, Ck-AD2-IgG, was generated by recombinant engineering, whereby the AD2 peptide was fused to the C-terminal end of the kappa light chain. Because the natural C-terminus of CK is a cysteine residue, which forms a disulfide bridge to CHL a 16-amino acid residue "hinge" linker was used to space the AD2 from the CK-VH1 disulfide bridge. The mammalian expression vectors for Ck-AD2-IgG-veltuzumab and Ck-AD2-IgG-epratuzumab were constructed using the pdHL2 vector, which was used previously for expression of the homologous CH3-AD2-IgG modules. A 2208-bp nucleotide sequence was synthesized comprising the pdHL2 vector sequence ranging from the Barn HI restriction site within the VK/CK intron to the Xho I restriction site 3' of the Ck intron, with the insertion of the coding sequence for the hinge linker (EFPKPSTPPGSSGGAP, SEQ ID NO:31) and AD2, in frame at the 3' end of the coding sequence for CK. This synthetic sequence was inserted into the IgG-pdHL2 expression vectors for veltuzumab and epratuzumab via Barn HI and Xho I
restriction sites. Generation of production clones with SpESFX-10 were performed as described for the CH3-AD2-IgG modules. Ck-AD2-IgG-veltuzumab and Ck-AD2-IgG-epratuzumab were produced by stably-transfected production clones in batch roller bottle culture, and purified from the supernatant fluid in a single step using Mab Select (GE
Healthcare) Protein A affinity chromatography.
[0142] Following the same DNL process described previously for 22-(20)-(20) (Rossi et al., 2009, Blood 113:6161-71), Ck-AD2-IgG-epratuzumab was conjugated with CH1-Fab-veltuzumab, a Fab-based module derived from veltuzumab, to generate the bsHexAb 22*-(20)-(20), where the 22* indicates the Ck-AD2 module of epratuzumab and each (20) symbolizes a stabilized dimer of veltuzumab Fab. The properties of 22*-(20)-(20) were compared with those of 22-(20)-(20), the homologous Fc-bsHexAb comprising CH3-IgG-epratuzumab, which has similar composition and molecular size, but a different architecture.
[0143] Following the same DNL process described previously for 20-2b (Rossi et al., 2009, Blood 114:3864-71), Ck-AD2-IgG-veltuzumab, was conjugated with IFNa2b-DDD2, a module of IFNa2b with a DDD2 peptide fused at its C-terminal end, to generate 20*-2b, which comprises veltuzumab with a dimeric IFNa2b fused to each light chain.
The properties of 20*-2b were compared with those of 20-2b, which is the homologous Fc-IgG-IFNa.
The properties of 20*-2b were compared with those of 20-2b, which is the homologous Fc-IgG-IFNa.
[0144] Each of the bsHexAbs and IgG-IFNa were isolated from the DNL reaction mixture by MabSelect affinity chromatography. The two Ck-derived prototypes, an anti-bispecific hexavalent antibody, comprising epratuzumab (anti-CD22) and four Fabs of veltuzumab (anti-CD20), and a CD20-targeting immunocytokine, comprising veltuzumab and four molecules of interferon-a2b, displayed enhanced Fc-effector functions in vitro, as well as improved pharmacokinetics, stability and anti-lymphoma activity in vivo, compared to their Fc-derived counterparts.
Amino Acid Substitutions
Amino Acid Substitutions
[0145] In alternative embodiments, the disclosed methods and compositions may involve production and use of proteins or peptides with one or more substituted amino acid residues.
For example, the DDD and/or AD sequences used to make DNL constructs may be modified as discussed above.
For example, the DDD and/or AD sequences used to make DNL constructs may be modified as discussed above.
[0146] The skilled artisan will be aware that, in general, amino acid substitutions typically involve the replacement of an amino acid with another amino acid of relatively similar properties (i.e., conservative amino acid substitutions). The properties of the various amino acids and effect of amino acid substitution on protein structure and function have been the subject of extensive study and knowledge in the art.
[0147] For example, the hydropathic index of amino acids may be considered (Kyte &
Doolittle, 1982,1 Mol Biol., 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte &
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9);
and arginine (-4.5).
In making conservative substitutions, the use of amino acids whose hydropathic indices are within 2 is preferred, within 1 are more preferred, and within 0.5 are even more preferred.
Doolittle, 1982,1 Mol Biol., 157:105-132). The relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics (Kyte &
Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6);
histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9);
and arginine (-4.5).
In making conservative substitutions, the use of amino acids whose hydropathic indices are within 2 is preferred, within 1 are more preferred, and within 0.5 are even more preferred.
[0148] Amino acid substitution may also take into account the hydrophilicity of the amino acid residue (e.g., U.S. Pat. No. 4,554,101). Hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0);
glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5 ±1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
Replacement of amino acids with others of similar hydrophilicity is preferred.
glutamate (+3.0); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4);
proline (-0.5 ±1);
alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8);
isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
Replacement of amino acids with others of similar hydrophilicity is preferred.
[0149] Other considerations include the size of the amino acid side chain. For example, it would generally not be preferred to replace an amino acid with a compact side chain, such as glycine or serine, with an amino acid with a bulky side chain, e.g., tryptophan or tyrosine.
The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse turn secondary structure has been determined and is known in the art (see, e.g., Chou &
Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys.
26:367-384).
The effect of various amino acid residues on protein secondary structure is also a consideration. Through empirical study, the effect of different amino acid residues on the tendency of protein domains to adopt an alpha-helical, beta-sheet or reverse turn secondary structure has been determined and is known in the art (see, e.g., Chou &
Fasman, 1974, Biochemistry, 13:222-245; 1978, Ann. Rev. Biochem., 47: 251-276; 1979, Biophys.
26:367-384).
[0150] Based on such considerations and extensive empirical study, tables of conservative amino acid substitutions have been constructed and are known in the art. For example:
arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine;
and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gln, asn, lys;
Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser;
Val (V) ile, leu, met, phe, ala.
arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine;
and valine, leucine and isoleucine. Alternatively: Ala (A) leu, ile, val; Arg (R) gln, asn, lys;
Asn (N) his, asp, lys, arg, gln; Asp (D) asn, glu; Cys (C) ala, ser; Gln (Q) glu, asn; Glu (E) gln, asp; Gly (G) ala; His (H) asn, gln, lys, arg; Ile (I) val, met, ala, phe, leu; Leu (L) val, met, ala, phe, ile; Lys (K) gln, asn, arg; Met (M) phe, ile, leu; Phe (F) leu, val, ile, ala, tyr; Pro (P) ala; Ser (S), thr; Thr (T) ser; Trp (W) phe, tyr; Tyr (Y) trp, phe, thr, ser;
Val (V) ile, leu, met, phe, ala.
[0151] Other considerations for amino acid substitutions include whether or not the residue is located in the interior of a protein or is solvent exposed. For interior residues, conservative substitutions would include: Asp and Asn; Ser and Thr; Ser and Ala; Thr and Ala; Ala and Gly; Ile and Val; Val and Leu; Leu and Ile; Leu and Met; Phe and Tyr; Tyr and Trp. (See, e.g., PROWL website at rockefeller.edu) For solvent exposed residues, conservative substitutions would include: Asp and Asn; Asp and Glu; Glu and Gln; Glu and Ala; Gly and Asn; Ala and Pro; Ala and Gly; Ala and Ser; Ala and Lys; Ser and Thr; Lys and Arg; Val and Leu; Leu and Ile; Ile and Val; Phe and Tyr. (Id.) Various matrices have been constructed to assist in selection of amino acid substitutions, such as the PAM250 scoring matrix, Dayhoff matrix, Grantham matrix, McLachlan matrix, Doolittle matrix, Henikoff matrix, Miyata matrix, Fitch matrix, Jones matrix, Rao matrix, Levin matrix and Risler matrix (Idem.)
[0152] In determining amino acid substitutions, one may also consider the existence of intermolecular or intramolecular bonds, such as formation of ionic bonds (salt bridges) between positively charged residues (e.g., His, Arg, Lys) and negatively charged residues (e.g., Asp, Glu) or disulfide bonds between nearby cysteine residues.
[0153] Methods of substituting any amino acid for any other amino acid in an encoded protein sequence are well known and a matter of routine experimentation for the skilled artisan, for example by the technique of site-directed mutagenesis or by synthesis and assembly of oligonucleotides encoding an amino acid substitution and splicing into an expression vector construct.
Antibody Allotypes
Antibody Allotypes
[0154] Immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and decreased duration of therapeutic response (Baert et al., 2003, N Engl J Med 348:602-08). The extent to which therapeutic antibodies induce an immune response in the host may be determined in part by the allotype of the antibody (Stickler et al., 2011, Genes and Immunity 12:213-21). Antibody allotype is related to amino acid sequence variations at specific locations in the constant region sequences of the antibody. The allotypes of IgG antibodies containing a heavy chain y-type constant region are designated as Gm allotypes (1976, J
Immunol 117:1056-59).
Immunol 117:1056-59).
[0155] For the common IgG1 human antibodies, the most prevalent allotype is Glml (Stickler et al., 2011, Genes and Immunity 12:213-21). However, the G1m3 allotype also occurs frequently in Caucasians (Id.). It has been reported that Glml antibodies contain allotypic sequences that tend to induce an immune response when administered to non-Glml (nGlml) recipients, such as G1m3 patients (Id.). Non-Glml allotype antibodies are not as immunogenic when administered to Glml patients (Id.).
[0156] The human Glml allotype comprises the amino acids aspartic acid at Kabat position 356 and leucine at Kabat position 358 in the CH3 sequence of the heavy chain IgGl. The nGlml allotype comprises the amino acids glutamic acid at Kabat position 356 and methionine at Kabat position 358. Both Glml and nGlml allotypes comprise a glutamic acid residue at Kabat position 357 and the allotypes are sometimes referred to as DEL and EEM
allotypes. A
non-limiting example of the heavy chain constant region sequences for Glml and nGlml allotype antibodies is shown for the exemplary antibodies rituximab (SEQ ID
NO:32) and veltuzumab (SEQ ID NO:33).
Rituximab heavy chain variable region sequence (SEQ ID NO:32) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Veltuzumab heavy chain variable region (SEQ ID NO:33) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
allotypes. A
non-limiting example of the heavy chain constant region sequences for Glml and nGlml allotype antibodies is shown for the exemplary antibodies rituximab (SEQ ID
NO:32) and veltuzumab (SEQ ID NO:33).
Rituximab heavy chain variable region sequence (SEQ ID NO:32) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Veltuzumab heavy chain variable region (SEQ ID NO:33) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
[0157] Jefferis and Lefranc (2009, mAbs 1:1-7) reviewed sequence variations characteristic of IgG allotypes and their effect on immunogenicity. They reported that the G1m3 allotype is characterized by an arginine residue at Kabat position 214, compared to a lysine residue at Kabat 214 in the G1m17 allotype. The nG1m1,2 allotype was characterized by glutamic acid at Kabat position 356, methionine at Kabat position 358 and alanine at Kabat position 431. The G1m1,2 allotype was characterized by aspartic acid at Kabat position 356, leucine at Kabat position 358 and glycine at Kabat position 431. In addition to heavy chain constant region sequence variants, Jefferis and Lefranc (2009) reported allotypic variants in the kappa light chain constant region, with the Km1 allotype characterized by valine at Kabat position 153 and leucine at Kabat position 191, the Km1,2 allotype by alanine at Kabat position 153 and leucine at Kabat position 191, and the Km3 allotypoe characterized by alanine at Kabat position 153 and valine at Kabat position 191.
[0158] With regard to therapeutic antibodies, veltuzumab and rituximab are, respectively, humanized and chimeric IgG1 antibodies against CD20, of use for therapy of a wide variety of hematological malignancies and/or autoimmune diseases. Table 1 compares the allotype sequences of rituximab vs. veltuzumab. As shown in Table 1, rituximab (G1m17,1) is a DEL
allotype IgGl, with an additional sequence variation at Kabat position 214 (heavy chain CH1) of lysine in rituximab vs. arginine in veltuzumab. It has been reported that veltuzumab is less immunogenic in subjects than rituximab (see, e.g., Morchhauser et al., 2009, J
Clin Oncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed to the difference between humanized and chimeric antibodies. However, the difference in allotypes between the EEM and DEL
allotypes likely also accounts for the lower immunogenicity of veltuzumab.
Table 1. Allotypes of Rituximab vs. Veltuzumab Heavy chain position and associated allotypes Complete allotype 214 (allotype) 356/358 (allotype) 431 (allotype) Rituximab G1m17,1 K 17 D/L 1 A
Veltuzumab G1m3 R 3 E/M A
allotype IgGl, with an additional sequence variation at Kabat position 214 (heavy chain CH1) of lysine in rituximab vs. arginine in veltuzumab. It has been reported that veltuzumab is less immunogenic in subjects than rituximab (see, e.g., Morchhauser et al., 2009, J
Clin Oncol 27:3346-53; Goldenberg et al., 2009, Blood 113:1062-70; Robak & Robak, 2011, BioDrugs 25:13-25), an effect that has been attributed to the difference between humanized and chimeric antibodies. However, the difference in allotypes between the EEM and DEL
allotypes likely also accounts for the lower immunogenicity of veltuzumab.
Table 1. Allotypes of Rituximab vs. Veltuzumab Heavy chain position and associated allotypes Complete allotype 214 (allotype) 356/358 (allotype) 431 (allotype) Rituximab G1m17,1 K 17 D/L 1 A
Veltuzumab G1m3 R 3 E/M A
[0159] In order to reduce the immunogenicity of therapeutic antibodies in individuals of nGlml genotype, it is desirable to select the allotype of the antibody to correspond to the G1m3 allotype, characterized by arginine at Kabat 214, and the nG1m1,2 null-allotype, characterized by glutamic acid at Kabat position 356, methionine at Kabat position 358 and alanine at Kabat position 431. Surprisingly, it was found that repeated subcutaneous administration of G1m3 antibodies over a long period of time did not result in a significant immune response. In alternative embodiments, the human IgG4 heavy chain in common with the G1m3 allotype has arginine at Kabat 214, glutamic acid at Kabat 356, methionine at Kabat 359 and alanine at Kabat 431. Since immunogenicity appears to relate at least in part to the residues at those locations, use of the human IgG4 heavy chain constant region sequence for therapeutic antibodies is also a preferred embodiment. Combinations of G1m3 IgG1 antibodies with IgG4 antibodies may also be of use for therapeutic administration.
[0160] Exemplary antibody constant region sequences of use in the chimeric and humanized anti-histone antibodies are disclosed in SEQ ID NO:34 and SEQ ID NO:35 below.
Exemplary human heavy chain constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO :34) Exemplary human light chain constant region TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO: 35) Immunoconjugates
Exemplary human heavy chain constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLG
GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR
EEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVY
TLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO :34) Exemplary human light chain constant region TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ SGNSQESVTE
QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO: 35) Immunoconjugates
[0161] In certain embodiments, the antibodies or fragments thereof may be conjugated to one or more therapeutic or diagnostic agents. The therapeutic agents do not need to be the same but can be different, e.g. a drug and a radioisotope. For example, 131I can be incorporated into a tyrosine of an antibody or fusion protein and a drug attached to an epsilon amino group of a lysine residue. Therapeutic and diagnostic agents also can be attached, for example to reduced SH groups and/or to carbohydrate side chains. Many methods for making covalent or non-covalent conjugates of therapeutic or diagnostic agents with antibodies or fusion proteins are known in the art and any such known method may be utilized.
[0162] A therapeutic or diagnostic agent can be attached at the hinge region of a reduced antibody component via disulfide bond formation. Alternatively, such agents can be attached using a heterobifunctional cross-linker, such as N-succinyl 3-(2-pyridyldithio)propionate (SPDP). Yu et at., Int. I Cancer 56: 244 (1994). General techniques for such conjugation are well-known in the art. See, for example, Wong, CHEMISTRY OF PROTEIN
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et at., "Modification of Antibodies by Chemical Methods," in MONOCLONAL ANTIBODIES:
PRINCIPLES AND APPLICATIONS, Birch et at. (eds.), pages 187-230 (Wiley-Liss, Inc.
1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et at. (eds.), pages 60-84 (Cambridge University Press 1995).
Alternatively, the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
CONJUGATION AND CROSS-LINKING (CRC Press 1991); Upeslacis et at., "Modification of Antibodies by Chemical Methods," in MONOCLONAL ANTIBODIES:
PRINCIPLES AND APPLICATIONS, Birch et at. (eds.), pages 187-230 (Wiley-Liss, Inc.
1995); Price, "Production and Characterization of Synthetic Peptide-Derived Antibodies," in MONOCLONAL ANTIBODIES: PRODUCTION, ENGINEERING AND CLINICAL
APPLICATION, Ritter et at. (eds.), pages 60-84 (Cambridge University Press 1995).
Alternatively, the therapeutic or diagnostic agent can be conjugated via a carbohydrate moiety in the Fc region of the antibody. The carbohydrate group can be used to increase the loading of the same agent that is bound to a thiol group, or the carbohydrate moiety can be used to bind a different therapeutic or diagnostic agent.
[0163] Methods for conjugating peptides to antibody components via an antibody carbohydrate moiety are well-known to those of skill in the art. See, for example, Shih et at., Int.' Cancer 41: 832 (1988); Shih et al., Int.' Cancer 46: 1101 (1990); and Shih et al.,U U.S.
Patent No. 5,057,313, incorporated herein in their entirety by reference. The general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
Patent No. 5,057,313, incorporated herein in their entirety by reference. The general method involves reacting an antibody component having an oxidized carbohydrate portion with a carrier polymer that has at least one free amine function. This reaction results in an initial Schiff base (imine) linkage, which can be stabilized by reduction to a secondary amine to form the final conjugate.
[0164] The Fc region may be absent if the antibody used as the antibody component of the immunoconjugate is an antibody fragment. However, it is possible to introduce a carbohydrate moiety into the light chain variable region of a full length antibody or antibody fragment.
See, for example, Leung et al., I Immunol. 154: 5919 (1995); Hansen et al.,U
U.S. Patent No.
5,443,953 (1995), Leung et al.,U U.S. patent No. 6,254,868, incorporated herein by reference in their entirety. The engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
See, for example, Leung et al., I Immunol. 154: 5919 (1995); Hansen et al.,U
U.S. Patent No.
5,443,953 (1995), Leung et al.,U U.S. patent No. 6,254,868, incorporated herein by reference in their entirety. The engineered carbohydrate moiety is used to attach the therapeutic or diagnostic agent.
[0165] In some embodiments, a chelating agent may be attached to an antibody, antibody fragment or fusion protein and used to chelate a therapeutic or diagnostic agent, such as a radionuclide. Exemplary chelators include but are not limited to DTPA (such as Mx-DTPA), DOTA, TETA, NETA or NOTA. Methods of conjugation and use of chelating agents to attach metals or other ligands to proteins are well known in the art (see, e.g., U.S.
Patent No.
7,563,433, the Examples section of which is incorporated herein by reference).
Patent No.
7,563,433, the Examples section of which is incorporated herein by reference).
[0166] In certain embodiments, radioactive metals or paramagnetic ions may be attached to proteins or peptides by reaction with a reagent having a long tail, to which may be attached a multiplicity of chelating groups for binding ions. Such a tail can be a polymer such as a polylysine, polysaccharide, or other derivatized or derivatizable chains having pendant groups to which can be bound chelating groups such as, e.g., ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), porphyrins, polyamines, crown ethers, bis-thiosemicarbazones, polyoximes, and like groups known to be useful for this purpose.
[0167] Chelates may be directly linked to antibodies or peptides, for example as disclosed in U.S. Patent 4,824,659, incorporated herein in its entirety by reference.
Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 1251, 1311, 1231, 1241, 62cti, 64cti, 18F, "In, 67Ga, 68-a, G 99mTc, 94mTc, "C, 13N, 150, 76Br , for radioimaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223Ra for RAIT are encompassed.
Particularly useful metal-chelate combinations include 2-benzyl-DTPA and its monomethyl and cyclohexyl analogs, used with diagnostic isotopes in the general energy range of 60 to 4,000 keV, such as 1251, 1311, 1231, 1241, 62cti, 64cti, 18F, "In, 67Ga, 68-a, G 99mTc, 94mTc, "C, 13N, 150, 76Br , for radioimaging. The same chelates, when complexed with non-radioactive metals, such as manganese, iron and gadolinium are useful for MRI. Macrocyclic chelates such as NOTA, DOTA, and TETA are of use with a variety of metals and radiometals, most particularly with radionuclides of gallium, yttrium and copper, respectively. Such metal-chelate complexes can be made very stable by tailoring the ring size to the metal of interest. Other ring-type chelates such as macrocyclic polyethers, which are of interest for stably binding nuclides, such as 223Ra for RAIT are encompassed.
[0168] More recently, methods of 18F-labeling of use in PET scanning techniques have been disclosed, for example by reaction of F-18 with a metal or other atom, such as aluminum. The 18F-Al conjugate may be complexed with chelating groups, such as DOTA, NOTA or NETA
that are attached directly to antibodies or used to label targetable constructs in pre-targeting methods. Such F-18 labeling techniques are disclosed in U.S. Patent No.
7,563,433, the Examples section of which is incorporated herein by reference.
PAD Inhibitors
that are attached directly to antibodies or used to label targetable constructs in pre-targeting methods. Such F-18 labeling techniques are disclosed in U.S. Patent No.
7,563,433, the Examples section of which is incorporated herein by reference.
PAD Inhibitors
[0169] In certain preferred embodiments, a therapeutic agent for use either alone or in combination with or conjugated to an anti-histone agent, such as an anti-histone antibody, may be a PAD inhibitor. PADs (protein arginine deiminases) catalyze the post-translational citrullination of histones and other proteins, in which arginine amino acid residues are converted to citrulline. Overexpression of PADs has been observed in various disease states, such as rheumatoid arthritis, Alzheimer's disease, multiple sclerosis, systemic lupus, Parkinson's disease and cancer (Bicker & Thompson, 2013, 99:155-63). In humans, PADs comprise five calcium-dependent isozymes (PADs 1-4 and 6), which share about 50%
sequence homology (Bicker & Thompson, 2013, 99:155-63). While the various isozymes are found in different tissues throughout the body, only PAD4 has been confirmed to play a role in histone citrullination (Bicker & Thompson, 2013, 99:155-63).
sequence homology (Bicker & Thompson, 2013, 99:155-63). While the various isozymes are found in different tissues throughout the body, only PAD4 has been confirmed to play a role in histone citrullination (Bicker & Thompson, 2013, 99:155-63).
[0170] PADs have also been identified as playing crucial roles in the generation of neutrophil extracellular traps (NETs) (Bicker & Thompson, 2013, 99:155-63). PAD4 plays a regulatory role in NET formation by mediating chromatin decondensation through histone citrullination (Bicker & Thompson, 2013, 99:155-63). In particular, citrullinated histone H3 appears to be important in NET formation (Bicker & Thompson, 2013, 99:155-63). The pan-PAD
inhibitor Cl-amidine has been commonly used in studies of disease therapy, such as rheumatoid arthritis treatment, and may be of use in the instant claimed methods.
However, other known PAD inhibitors, such as BB-Cl-amidine, YW3-56, (see, e.g., Wang et al., 2012, J Biol Chem 287:25941-53; Knight et al., 2015, Ann Rheum Dis 74:2199-206), o-F-amidine, o-amidine, TDFA (Thr-Asp-F-amidine) or streptonigrin (Bicker & Thompson, 2013, Biopolymers 99:155-63) may also be used in the methods and compositions.
Therapeutic Agents
inhibitor Cl-amidine has been commonly used in studies of disease therapy, such as rheumatoid arthritis treatment, and may be of use in the instant claimed methods.
However, other known PAD inhibitors, such as BB-Cl-amidine, YW3-56, (see, e.g., Wang et al., 2012, J Biol Chem 287:25941-53; Knight et al., 2015, Ann Rheum Dis 74:2199-206), o-F-amidine, o-amidine, TDFA (Thr-Asp-F-amidine) or streptonigrin (Bicker & Thompson, 2013, Biopolymers 99:155-63) may also be used in the methods and compositions.
Therapeutic Agents
[0171] In alternative embodiments, therapeutic agents such as cytotoxic agents, anti-angiogenic agents, pro-apoptotic agents, antibiotics, hormones, hormone antagonists, chemokines, drugs, prodrugs, toxins, enzymes or other agents may be used, either conjugated to the subject anti-histone antibodies or other histone-neutralizing agents, or else separately administered before, simultaneously with, or after the histone-neutralizing agent. Drugs of use may possess a pharmaceutical property selected from the group consisting of antimitotic, antikinase (e.g., anti-tyrosine kinase), alkylating, antimetabolite, antibiotic, alkaloid, anti-angiogenic, pro-apoptotic agents, immune modulators, and combinations thereof
[0172] Exemplary drugs of use may include 5-fluorouracil, aplidin, azaribine, anastrozole, anthracyclines, bendamustine, bleomycin, bortezomib, bryostatin-1, busulfan, calicheamycin, camptothecin, carboplatin, 10-hydroxycamptothecin, carmustine, celecoxib, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-11), SN-38, carboplatin, cladribine, camptothecans, cyclophosphamide, cytarabine, dacarbazine, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano-morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, estramustine, epipodophyllotoxin, estrogen receptor binding agents, etoposide (VP16), etoposide glucuronide, etoposide phosphate, floxuridine (FUdR), 3',5'-0-dioleoyl-FudR
(FUdR-d0), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.
(FUdR-d0), fludarabine, flutamide, farnesyl-protein transferase inhibitors, gemcitabine, hydroxyurea, idarubicin, ifosfamide, L-asparaginase, lenolidamide, leucovorin, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, nitrosourea, plicomycin, procarbazine, paclitaxel, pentostatin, PSI-341, raloxifene, semustine, streptozocin, tamoxifen, taxol, temazolomide, transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vinorelbine, vinblastine, vincristine and vinca alkaloids.
[0173] Toxins of use may include ricin, abrin, alpha toxin, saporin, ribonuclease (RNase), e.g., onconase, DNase I, Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin, diphtheria toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin.
[0174] Chemokines of use may include RANTES, MCAF, MIP1-alpha, MIP1-Beta and IP-10.
[0175] Immunomodulators of use may be selected from a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interferon (IFN), erythropoietin, thrombopoietin and a combination thereof Specifically useful are lymphotoxins such as tumor necrosis factor (TNF), hematopoietic factors, such as interleukin (IL), colony stimulating factor, such as granulocyte-colony stimulating factor (G-CSF) or granulocyte macrophage-colony stimulating factor (GM-C SF), interferon, such as interferons-a, -0 or -y, and stem cell growth factor, such as that designated "51 factor".
Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor;
prolactin; placental lactogen, OB protein; tumor necrosis factor-a and - B; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TP0); nerve growth factors such as NGF-B; platelet-growth factor;
transforming growth factors (TGFs) such as TGF- a and TGF- B; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -13, and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. Lenolidamide is yet another immunomodulator that has shown activity in controlling certain cancers, such as multiple myeloma and hematopoietic tumors.
Included among the cytokines are growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH); hepatic growth factor; prostaglandin, fibroblast growth factor;
prolactin; placental lactogen, OB protein; tumor necrosis factor-a and - B; mullerian-inhibiting substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factor;
integrin; thrombopoietin (TP0); nerve growth factors such as NGF-B; platelet-growth factor;
transforming growth factors (TGFs) such as TGF- a and TGF- B; insulin-like growth factor-I
and -II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-a, -13, and -y; colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
interleukins (ILs) such as IL-1, IL-la, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, kit-ligand or FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and LT. Lenolidamide is yet another immunomodulator that has shown activity in controlling certain cancers, such as multiple myeloma and hematopoietic tumors.
[0176] Other useful therapeutic agents may comprise oligonucleotides, especially antisense oligonucleotides that preferably are directed against oncogenes and oncogene products, such as bc1-2. A preferred form of therapeutic oligonucleotide is siRNA.
Immune Dysregulatory Disease
Immune Dysregulatory Disease
[0177] In various embodiments, the hi stone-neutralizing agents are of use to treat immune-dysregulatory diseases, such as acute kidney injury. In certain preferred embodiments, the therapy may utilize either a combination of two or more hi stone-neutralizing agents.
[0178] Additional therapeutic agents that may be added in combination include a cytokine, a chemokine, a coagulation inhibitor, an anti-T cell or anti B-cell antibody or antibody fragment, an immunomodulator, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor, an interferon, erythropoietin or thrombopoietin. An optional therapeutic agent may include activated protein C, heparin or thrombomodulin, as mentioned above.
Combinations of anti-histone antibodies or fragments thereof with other hi stone neutralizing agents, including but not limited to antibodies or antibody fragments against additional immune system target antigens, as discussed below, may be utilized in certain embodiments.
Combinations of anti-histone antibodies or fragments thereof with other hi stone neutralizing agents, including but not limited to antibodies or antibody fragments against additional immune system target antigens, as discussed below, may be utilized in certain embodiments.
[0179] The immune system comprises both the innate immune system and the adaptive or acquired immune system. Many host cells participate in the processes of innate and adaptive immunity, such as neutrophils, T- and B-lymphocytes, macrophages and monocytes, dendritic cells, and plasma cells. They usually act in concert, affecting one another, particularly in the regulation of certain factors and cytokines that contribute to the recognition and processing of innate and external noxients, and these systems have evolved over the millions of years of the development of vertebrate, mammalian, and human organisms.
[0180] A major goal of immunotherapy is to exploit or enhance a patient's immune system against an innate or foreign noxient, such as a malignant cell or an invading microorganism.
The immune system has been studied more in relation to recognizing and responding to exogenous noxients, such as microbial organisms, than it has in relation to indigenous malfunctions, such as cancer and certain autoimmune and immune-dysregulatory diseases, particularly since the latter may have both genetic as well as environmental components. The defenses against microbial organisms, such as bacteria, fungi, parasites, and viruses, are innate to the particular organism, with the immune system being programmed to recognize biochemical patterns of these microorganisms and to respond to attack them without requiring prior exposure to the microorganism. This innate immune system includes, for example, neutrophils, natural killer cells and monocytes/macrophages that can eradicate the invading microorganisms by direct engulfment and destruction.
The immune system has been studied more in relation to recognizing and responding to exogenous noxients, such as microbial organisms, than it has in relation to indigenous malfunctions, such as cancer and certain autoimmune and immune-dysregulatory diseases, particularly since the latter may have both genetic as well as environmental components. The defenses against microbial organisms, such as bacteria, fungi, parasites, and viruses, are innate to the particular organism, with the immune system being programmed to recognize biochemical patterns of these microorganisms and to respond to attack them without requiring prior exposure to the microorganism. This innate immune system includes, for example, neutrophils, natural killer cells and monocytes/macrophages that can eradicate the invading microorganisms by direct engulfment and destruction.
[0181] The innate immune response is often referred to as a nonspecific one that controls an invading external noxient until the more specific adaptive immune system can marshal specific antibodies and T cells (cf. Modlin et al., N Engl J Med 1999, 340:1834-1835; Das, Cra. Care 2000; 4:290-296). The nonspecific immune responses involve the lymphatic system and phagocytes. The lymphatic system includes the lymphocytes and macrophages.
Macrophages can engulf, kill and dispose of foreign particles. Phagocytes include neutrophils and macrophages, which again ingest, degrade and dispose of debris, and have receptors for complement and antibody. In summary, the innate immune system provides a line of defense again certain antigens because of inherited characteristics.
Macrophages can engulf, kill and dispose of foreign particles. Phagocytes include neutrophils and macrophages, which again ingest, degrade and dispose of debris, and have receptors for complement and antibody. In summary, the innate immune system provides a line of defense again certain antigens because of inherited characteristics.
[0182] In contrast, the adaptive, or acquired, immune system is highly evolved and very specific in its responses. It is called an adaptive system because it occurs during the lifetime of an individual as an adaptation to infection with a pathogen. Adaptive immunity can be artificially acquired in response to a vaccine (antigens) or by administering antibodies, or can be naturally acquired by infection. The acquired immunity can be active, if an antibody was produced, or it can be passive, if exogenous antibody made from another source is injected.
[0183] The adaptive immune system produces antibodies specific to a given antigen. The simplest and most direct way in which antibodies provide protection is by binding to them and thereby blocking their access to cells that they may infect or destroy.
This is known as neutralization. Binding by antibodies, however, is not sufficient to arrest the replication of bacteria that multiply outside cells. In this case, one role of antibody is to enable a phagocytic cell to ingest and destroy the bacterium. This is known as opsonization. The third function of antibodies is to activate a system of plasma proteins, known as complement. In many cases, the adaptive immune system confers lifelong protective immunity to re-infection with the same pathogen, because the adaptive immune system has a 'memory' of the antigens presented to it.
This is known as neutralization. Binding by antibodies, however, is not sufficient to arrest the replication of bacteria that multiply outside cells. In this case, one role of antibody is to enable a phagocytic cell to ingest and destroy the bacterium. This is known as opsonization. The third function of antibodies is to activate a system of plasma proteins, known as complement. In many cases, the adaptive immune system confers lifelong protective immunity to re-infection with the same pathogen, because the adaptive immune system has a 'memory' of the antigens presented to it.
[0184] Antibody-mediated immunity is called humoral immunity and is regulated by B cells and the antibodies they produce. Cell-mediated immunity is controlled by T
cells. Both humoral and cell-mediated immunity participate in protecting the host from invading organisms. This interplay can result in an effective killing or control of foreign organisms.
Occasionally, however, the interplay can become erratic. In these cases, there is a dysregulation that can cause disease. Sometimes the disease is life-threatening, such as with septic shock and certain autoimmune disorders.
cells. Both humoral and cell-mediated immunity participate in protecting the host from invading organisms. This interplay can result in an effective killing or control of foreign organisms.
Occasionally, however, the interplay can become erratic. In these cases, there is a dysregulation that can cause disease. Sometimes the disease is life-threatening, such as with septic shock and certain autoimmune disorders.
[0185] The B and T lymphocytes are critical components of a specific immune response. B
cells are activated by antigen to engender clones of antigen-specific cells that mediate adaptive immunity. Most clones differentiate to plasma cells that secrete antibody, while a few clones form memory cells that revert to plasma cells. Upon subsequent re-infection, memory cells produce a higher level of antibody in a shorter period than in the primary response. Antibodies secreted by the plasma cells can play multiple roles in immunity, such as binding and neutralizing a foreign agent, acting as opsonins (IgG) to promote phagocytosis, directly affecting metabolism and growth of some organisms, engaging in antigen-antibody reactions that activate complement, causing phagocytosis and membrane attack complex, and/or engaging in antigen-antibody reactions that activate T
cells and other killer cells.
cells are activated by antigen to engender clones of antigen-specific cells that mediate adaptive immunity. Most clones differentiate to plasma cells that secrete antibody, while a few clones form memory cells that revert to plasma cells. Upon subsequent re-infection, memory cells produce a higher level of antibody in a shorter period than in the primary response. Antibodies secreted by the plasma cells can play multiple roles in immunity, such as binding and neutralizing a foreign agent, acting as opsonins (IgG) to promote phagocytosis, directly affecting metabolism and growth of some organisms, engaging in antigen-antibody reactions that activate complement, causing phagocytosis and membrane attack complex, and/or engaging in antigen-antibody reactions that activate T
cells and other killer cells.
[0186] T lymphocytes function as both helper cells and suppressor cells.
Helper T cells induce antigen-specific B cells and effector T cells to proliferate and differentiate. Suppressor T cells interact with helper T cells to prevent an immune response or to suppress an ongoing one, or to regulate effector T cells. Cytotoxic T cells destroy antigen by binding to target cells. In a delayed-type hypersensitivity reaction, the T cells do not destroy antigen, but attract macrophages, neutrophils and other cells to destroy and dispose of the antigen.
Helper T cells induce antigen-specific B cells and effector T cells to proliferate and differentiate. Suppressor T cells interact with helper T cells to prevent an immune response or to suppress an ongoing one, or to regulate effector T cells. Cytotoxic T cells destroy antigen by binding to target cells. In a delayed-type hypersensitivity reaction, the T cells do not destroy antigen, but attract macrophages, neutrophils and other cells to destroy and dispose of the antigen.
[0187] T cells can detect the presence of intracellular pathogens because infected cells display on their surface peptide fragments derived from the pathogens' proteins. These foreign peptides are delivered to the cell surface by specialized host-cell glycoproteins, termed Major Histocompatibility Complex (MHC) molecules. The recognition of antigen as a small peptide fragment bound to a MHC molecule and displayed at the cell surface is one of the most distinctive features of T cells. There are two different classes of MHC molecules, known as MHC class I and MHC class II, that deliver peptides from different cellular compartments to the surface of the infected cell. Peptides from the cytosol are bound to MHC
class I molecules which are expressed on the majority of nucleated cells and are recognized by CD8+ T cells. MHC class II molecules, in contrast, traffic to lysosomes for sampling endocytosed protein antigens which are presented to the CD4+ T cells (Bryant and Ploegh, Curr Opin Immunol 2004; 16:96-102).
class I molecules which are expressed on the majority of nucleated cells and are recognized by CD8+ T cells. MHC class II molecules, in contrast, traffic to lysosomes for sampling endocytosed protein antigens which are presented to the CD4+ T cells (Bryant and Ploegh, Curr Opin Immunol 2004; 16:96-102).
[0188] CD8+ T cells differentiate into cytotoxic T cells, and kill the target cell. CD4+ T cells differentiate into two types of effector T cells. Pathogens that accumulate in large numbers inside macrophage vesicles tend to stimulate the differentiation of TH1 cells which activate macrophages and induce B cells to make IgG antibodies that are effective in opsonizing extracellular pathogens for uptake by phagocytes. Extracellular antigens tend to stimulate the production of TH2 cells which initiate the humoral immune response by activating naive antigen-specific B cells to produce IgM antibodies, inter alia.
[0189] The innate and adaptive immune systems interact, in that the cells of the innate immune system can express various molecules that can interact with or trigger the adaptive immune system by activating certain cells capable of producing immune factors, such as by activating T and B cells of the lymphatic series of leukocytes. The early induced but non-adaptive responses are important for two main reasons. First, they can repel a pathogen or, more often, control it until an adaptive immune response can be mounted.
Second, these early responses influence the adaptive response in several ways. For example, the innate immune response produces cytokines and other inflammatory mediators that have profound effects on subsequent events, including the recruitment of new phagocytic cells to local sites of infection. Another effect of these mediators is to induce the expression of adhesion molecules on the endothelial cells of the local blood vessels, which bind to the surface of circulating monocytes and neutrophils and greatly increase their rate of migration of these cells out of the blood and into the tissues. These events all are included under the term inflammation, which is a feature of the innate immune system that forms part of the protective response at a localized site to isolate, destroy and remove a foreign material. This is followed by repair.
Inflammation is divided into acute and chronic forms.
Second, these early responses influence the adaptive response in several ways. For example, the innate immune response produces cytokines and other inflammatory mediators that have profound effects on subsequent events, including the recruitment of new phagocytic cells to local sites of infection. Another effect of these mediators is to induce the expression of adhesion molecules on the endothelial cells of the local blood vessels, which bind to the surface of circulating monocytes and neutrophils and greatly increase their rate of migration of these cells out of the blood and into the tissues. These events all are included under the term inflammation, which is a feature of the innate immune system that forms part of the protective response at a localized site to isolate, destroy and remove a foreign material. This is followed by repair.
Inflammation is divided into acute and chronic forms.
[0190] The immune system communicates via nonspecific tissue resistance factors. These include the interferons, which are proteins produced in response to viruses, endotoxins and certain bacteria. Interferons inhibit viral replication and activate certain host-defense responses. Infected cells produce interferon that binds the infected cells to other, neighboring cells, causing them to produce antiviral proteins and enzymes that interfere with viral gene transcription and proteins synthesis. Interferons can also affect normal cell growth and suppress cell-mediated immunity.
[0191] Complement is another nonspecific tissue resistance factor, and comprises plasma proteins and membrane proteins that mediate specific and non-specific defenses.
Complement has two pathways, the classical pathway associated with specific defense, and the alternative pathway that is activated in the absence of specific antibody, and is thus non-specific. In the classical pathway, antigen-antibody complexes are recognized when Cl interacts with the Fc of the antibody, such as IgM and to some extent, IgG, ultimately causing mast cells to release chemotactic factors, vascular mediators and a respiratory burst in phagocytes, as one of many mechanisms. The key complement factors include C3a and C5a, which cause mast cells to release chemotactic factors such as histamine and serotonin that attract phagocytes, antibodies and complement, etc. Other key complement factors are C3b and C5b, which enhance phagocytosis of foreign cells, and C8 and C9, which induce lysis of foreign cells (membrane attack complex).
Complement has two pathways, the classical pathway associated with specific defense, and the alternative pathway that is activated in the absence of specific antibody, and is thus non-specific. In the classical pathway, antigen-antibody complexes are recognized when Cl interacts with the Fc of the antibody, such as IgM and to some extent, IgG, ultimately causing mast cells to release chemotactic factors, vascular mediators and a respiratory burst in phagocytes, as one of many mechanisms. The key complement factors include C3a and C5a, which cause mast cells to release chemotactic factors such as histamine and serotonin that attract phagocytes, antibodies and complement, etc. Other key complement factors are C3b and C5b, which enhance phagocytosis of foreign cells, and C8 and C9, which induce lysis of foreign cells (membrane attack complex).
[0192] Gelderman et al. (Mol Immunol 2003; 40:13-23) reported that membrane-bound complement regulatory proteins (mCRP) inhibit complement activation by an immunotherapeutic mAb in a syngeneic rat colorectal cancer model. While the use of mAb against tumor antigens and mCRP overcame an observed effect of mCRP on tumor cells, there has been no direct evidence to support this approach. Still other attempts to use bispecific antibodies against CD55 and against a tumor antigen (G250 or EpCAM) have been suggested by Gelderman et al. (Lab Invest 2002; 82:483-493; Eur Jlmmunol 2002;
32:128-135) based on in vitro studies that showed a 2-13-fold increase in C3 deposition compared to use of the parental antitumor antibody. However, no results involving enhanced cell killing were reported. Jurianz et al. (Immunopharmacology 1999; 42:209-218) also suggested that combining treatment of a tumor with anti-HER2 antibodies in vitro could be enhanced by prior treatment with antibody-neutralization of membrane-complement-regulatory protein, but again no in vivo results were provided. Sier et al. (Int J Cancer 2004;
109:900-908) recently reported that a bispecific antibody made against an antigen expressed on renal cell carcinoma (Mab G250) and CD55 enhanced killing of renal cancer cells in spheroids when beta-glucan was added, suggesting that the presence of CR3-priming beta-glucan was obligatory.
32:128-135) based on in vitro studies that showed a 2-13-fold increase in C3 deposition compared to use of the parental antitumor antibody. However, no results involving enhanced cell killing were reported. Jurianz et al. (Immunopharmacology 1999; 42:209-218) also suggested that combining treatment of a tumor with anti-HER2 antibodies in vitro could be enhanced by prior treatment with antibody-neutralization of membrane-complement-regulatory protein, but again no in vivo results were provided. Sier et al. (Int J Cancer 2004;
109:900-908) recently reported that a bispecific antibody made against an antigen expressed on renal cell carcinoma (Mab G250) and CD55 enhanced killing of renal cancer cells in spheroids when beta-glucan was added, suggesting that the presence of CR3-priming beta-glucan was obligatory.
[0193] Neutrophils, another cell involved in innate immune response, also ingest, degrade and dispose of debris. Neutrophils have receptors for complement and antibody.
By means of complement-receptor bridges and antibody, the foreign noxients can be captured and presented to phagocytes for engulfment and killing.
By means of complement-receptor bridges and antibody, the foreign noxients can be captured and presented to phagocytes for engulfment and killing.
[0194] Macrophages are white blood cells that are part of the innate system that continually search for foreign antigenic substances. As part of the innate immune response, macrophages engulf, kill and dispose of foreign particles. However, they also process antigens for presentation to B and T cells, invoking humoral or cell-mediated immune responses.
[0195] The dendritic cell is one of the major means by which innate and adaptive immune systems communicate (Reis e Sousa, Curr Opin Immunol 2004; 16:21-25). It is believed that these cells shape the adaptive immune response by the reactions to microbial molecules or signals. Dendritic cells capture, process and present antigens, thus activating CD4+ and CD8+ naive T lymphocytes, leading to the induction of primary immune responses, and derive their stimulatory potency from expression of MHC class I, MHC class II, and accessory molecules, such as CD40, CD54, CD80, CD86, and T-cell activating cytokines (Steinman, J Exp Hematol 1996; 24:859-862; Banchereau and Steinman, Nature 1998;
392:245-252). These properties have made dendritic cells candidates for immunotherapy of cancers and infectious diseases (Nestle, Oncogene 2000; 19:673-679; Fong and Engleman, Annu Rev Immunol 2000; 18:245-273; Lindquist and Pisa, Med Oncol 2002; 19:197-211), and have been shown to induce antigen-specific cytotoxic T cells that result in strong immunity to viruses and tumors (Kono et al., Clin Cancer Res 2002; 8:394-40).
392:245-252). These properties have made dendritic cells candidates for immunotherapy of cancers and infectious diseases (Nestle, Oncogene 2000; 19:673-679; Fong and Engleman, Annu Rev Immunol 2000; 18:245-273; Lindquist and Pisa, Med Oncol 2002; 19:197-211), and have been shown to induce antigen-specific cytotoxic T cells that result in strong immunity to viruses and tumors (Kono et al., Clin Cancer Res 2002; 8:394-40).
[0196] Also important for interaction of the innate and adaptive immune systems is the NK
cell, which appears as a lymphocyte but behaves like a part of the innate immune system. NK
cells have been implicated in the killing of tumor cells as well as essential in the response to viral infections (Lanier, Curr Opin Immunol 2003; 15:308-314; Carayannopoulos and Yokoyama, Curr Opin Immunol 2004; 16:26-33). Yet another important mechanism of the innate immune system is the activation of cytokine mediators that alert other cells of the mammalian host to the presence of infection, of which a key component is the transcription factor NF-KB (Li and Verna, Nat Rev Immunol 2002; 2:725-734).
cell, which appears as a lymphocyte but behaves like a part of the innate immune system. NK
cells have been implicated in the killing of tumor cells as well as essential in the response to viral infections (Lanier, Curr Opin Immunol 2003; 15:308-314; Carayannopoulos and Yokoyama, Curr Opin Immunol 2004; 16:26-33). Yet another important mechanism of the innate immune system is the activation of cytokine mediators that alert other cells of the mammalian host to the presence of infection, of which a key component is the transcription factor NF-KB (Li and Verna, Nat Rev Immunol 2002; 2:725-734).
[0197] As mentioned earlier, the immune system can overreact, resulting in allergies or autoimmune diseases. It can also be suppressed, absent, or destroyed, resulting in disease and death. When the immune system cannot distinguish between "self' and "nonself,"
it can attach and destroy cells and tissues of the body, producing autoimmune diseases, e.g., juvenile diabetes, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, and immune thrombocytopenic purpura. Immunodeficiency disease results from the lack or failure of one or more parts of the immune system, and makes the individuals susceptible to diseases that usually do not affect individuals with a normal immune system. Examples of immunodeficiency disease are severe combined immunodeficiency disease (SCID) and acquired immunodeficiency disease (AIDS).
The latter results from human immunodeficiency virus (HIV) and the former from enzyme or other inherited defects, such as adenosine deaminase deficiency.
it can attach and destroy cells and tissues of the body, producing autoimmune diseases, e.g., juvenile diabetes, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, rheumatoid arthritis, and immune thrombocytopenic purpura. Immunodeficiency disease results from the lack or failure of one or more parts of the immune system, and makes the individuals susceptible to diseases that usually do not affect individuals with a normal immune system. Examples of immunodeficiency disease are severe combined immunodeficiency disease (SCID) and acquired immunodeficiency disease (AIDS).
The latter results from human immunodeficiency virus (HIV) and the former from enzyme or other inherited defects, such as adenosine deaminase deficiency.
[0198] Numerous and diverse methods of immunosuppression or of neutralizing proinflammatory cytokines have proven to be unsuccessful clinically in patients with sepsis and septic shock anti-inflammatory strategies. (Riedmann, et al., cited above;
Van Amersfoort et al. (Clin Microbial Rev 2003; 16:379-414), such as general immunosuppression, use of nonsteroidal anti-inflammatory drugs, TNF-a antibody (infliximab) or a TNF-R:Fc fusion protein (etanercept), IL-1 (interleukin-1) receptor antagonist, or high doses of corticosteroids. However, a success in the treatment of sepsis in adults was the PROWESS study (Human Activated Protein C Worldwide Evaluation in Severe Sepsis (Bernard et al., N Engl J Med 2001; 344:699-709)), showing a lower mortality (24.7%) than in the placebo group (30.8%). This activated protein C (APC) agent probably inhibits both thrombosis and inflammation, whereas fibrinolysis is fostered.
Friggeri et al.
(2012, Mal Med 18:825-33) reported that APC degrades histones H3 and H4, which block uptake and clearance of apoptotic cells by macrophages and thereby contribute to organ system dysfunction and mortality in acute inflammatory states. Van Amersfoort et al. state, in their review (ibid.) that: "Although the blocking or modulation of a number of other targets including complement and coagulation factors, neutrophil adherence, and NO
release, are promising in animals, it remains to be determined whether these therapeutic approaches will be effective in humans." This is further emphasized in a review by Abraham, "Why immunomodulatory therapies have not worked in sepsis" (Intensive Care Med 1999; 25:556-566). In general, although many rodent models of inflammation and sepsis have shown encouraging results with diverse agents over the past decade or more, most agents translated to the clinic failed to reproduce in humans what was observed in these animal models, so that there remains a need to provide new agents that can control the complex presentations and multiple-organ involvement of various diseases involving sepsis, coagulopathy, and certain neurodegenerative conditions having inflammatory or immune dysregulatory components.
Van Amersfoort et al. (Clin Microbial Rev 2003; 16:379-414), such as general immunosuppression, use of nonsteroidal anti-inflammatory drugs, TNF-a antibody (infliximab) or a TNF-R:Fc fusion protein (etanercept), IL-1 (interleukin-1) receptor antagonist, or high doses of corticosteroids. However, a success in the treatment of sepsis in adults was the PROWESS study (Human Activated Protein C Worldwide Evaluation in Severe Sepsis (Bernard et al., N Engl J Med 2001; 344:699-709)), showing a lower mortality (24.7%) than in the placebo group (30.8%). This activated protein C (APC) agent probably inhibits both thrombosis and inflammation, whereas fibrinolysis is fostered.
Friggeri et al.
(2012, Mal Med 18:825-33) reported that APC degrades histones H3 and H4, which block uptake and clearance of apoptotic cells by macrophages and thereby contribute to organ system dysfunction and mortality in acute inflammatory states. Van Amersfoort et al. state, in their review (ibid.) that: "Although the blocking or modulation of a number of other targets including complement and coagulation factors, neutrophil adherence, and NO
release, are promising in animals, it remains to be determined whether these therapeutic approaches will be effective in humans." This is further emphasized in a review by Abraham, "Why immunomodulatory therapies have not worked in sepsis" (Intensive Care Med 1999; 25:556-566). In general, although many rodent models of inflammation and sepsis have shown encouraging results with diverse agents over the past decade or more, most agents translated to the clinic failed to reproduce in humans what was observed in these animal models, so that there remains a need to provide new agents that can control the complex presentations and multiple-organ involvement of various diseases involving sepsis, coagulopathy, and certain neurodegenerative conditions having inflammatory or immune dysregulatory components.
[0199] More recent work on immunoglobulins in sepsis or septic shock has been reported.
For example, Toussaint and Gerlach (2012, Curr Infect Dis Rep 14:522-29) summarized the use of ivIG as an adjunct therapy in sepsis. The metanalysis failed to show any strong correlation between general immunoglobulin therapy and outcome. LaRosa and Opal (2012, Curr Infect Dis Rep 14:474-83) reported on new therapeutic agents of potential use in sepsis.
Among other agents, anti-TNF antibodies are in current clinical trials for sepsis, while complement antagonists have shown promising results in preclinical models of sepsis.
Nalesso etal. (2012, Curr Infect Dis Rep 14:462-73) suggested that combination therapies with multiple agents may prove more effective for sepsis treatment. The immunopathogenesis of sepsis has been summarized by Cohen (2002, Nature 420:885-91).
For example, Toussaint and Gerlach (2012, Curr Infect Dis Rep 14:522-29) summarized the use of ivIG as an adjunct therapy in sepsis. The metanalysis failed to show any strong correlation between general immunoglobulin therapy and outcome. LaRosa and Opal (2012, Curr Infect Dis Rep 14:474-83) reported on new therapeutic agents of potential use in sepsis.
Among other agents, anti-TNF antibodies are in current clinical trials for sepsis, while complement antagonists have shown promising results in preclinical models of sepsis.
Nalesso etal. (2012, Curr Infect Dis Rep 14:462-73) suggested that combination therapies with multiple agents may prove more effective for sepsis treatment. The immunopathogenesis of sepsis has been summarized by Cohen (2002, Nature 420:885-91).
[0200] The immune system in sepsis is believed to have an early intense proinflammatory response after infection or trauma, leading to organ damage, but it is also believed that the innate immune system often fails to effectively kill invading microorganisms (Riedmann and Ward, Expert Opin Blot Ther 2003; 3:339-350). There have been some studies of macrophage migration inhibitory factor (MIF) in connection with sepsis that have shown some promise. For example, blockage of MIF or targeted disruption of the MIF
gene significantly improved survival in a model of septic shock in mice (Calandra et al., Nature Med 2000; 6:164-170), and several lines of evidence have pointed to MIF as a potential target for therapeutic intervention in septic patients (Riedmann et al., cited above). Bucala et al.
(U.S. Pat. No. 6,645,493 B1) have claimed that an anti-MIF antibody can be effective therapeutically for treating a condition or disease caused by cytokine-mediated toxicity, including different forms of sepsis, inflammatory diseases, acute respiratory disease syndrome, granulomatous diseases, chronic infections, transplant rejection, cachexia, asthma, viral infections, parasitic infections, malaria, and bacterial infections, which is incorporated herein in its entirety, including references. The use of anti-LPS
(lipopolysaccharide) antibodies alone similarly has had mixed results in the treatment of patients with septic shock (Astiz and Rackow, Lancet 1998; 351:1501-1505; Van Amersfoort etal., Clin Microbiol Rev 2003; 16:379-414.
gene significantly improved survival in a model of septic shock in mice (Calandra et al., Nature Med 2000; 6:164-170), and several lines of evidence have pointed to MIF as a potential target for therapeutic intervention in septic patients (Riedmann et al., cited above). Bucala et al.
(U.S. Pat. No. 6,645,493 B1) have claimed that an anti-MIF antibody can be effective therapeutically for treating a condition or disease caused by cytokine-mediated toxicity, including different forms of sepsis, inflammatory diseases, acute respiratory disease syndrome, granulomatous diseases, chronic infections, transplant rejection, cachexia, asthma, viral infections, parasitic infections, malaria, and bacterial infections, which is incorporated herein in its entirety, including references. The use of anti-LPS
(lipopolysaccharide) antibodies alone similarly has had mixed results in the treatment of patients with septic shock (Astiz and Rackow, Lancet 1998; 351:1501-1505; Van Amersfoort etal., Clin Microbiol Rev 2003; 16:379-414.
[0201] Complement C5a, like C3a, is an anaphylatoxin. It mediates inflammation and is a chemotactic attractant for induction of neutrophilic release of antimicrobial proteases and oxygen radicals. Therefore, C5a and its predecessor C5 are particularly preferred targets. By targeting C5, not only is C5a affected, but also C5b, which initiates assembly of the membrane-attack complex. Thus, C5 is another preferred target. C3b, and its predecessor C3, also are preferred targets, as both the classical and alternate complement pathways depend upon C3b. Three proteins affect the levels of this factor, Cl inhibitor, protein H and Factor I, and these are also preferred targets according to the invention. Complement regulatory proteins, such as CD46, CD55, and CD59, may be targets to which the multispecific antibodies bind.
[0202] Coagulation factors also are preferred targets according to the invention, particularly tissue factor (TF), thrombomodulin, and thrombin. TF is also known also as tissue thromboplastin, CD142, coagulation factor III, or factor III. TF is an integral membrane receptor glycoprotein and a member of the cytokine receptor superfamily. The ligand binding extracellular domain of TF consists of two structural modules with features that are consistent with the classification of TF as a member of type-2 cytokine receptors. TF is involved in the blood coagulation protease cascade and initiates both the extrinsic and intrinsic blood coagulation cascades by forming high affinity complexes between the extracellular domain of TF and the circulating blood coagulation factors, serine proteases factor VII
or factor VIIa.
These enzymatically active complexes then activate factor IX and factor X, leading to thrombin generation and clot formation.
or factor VIIa.
These enzymatically active complexes then activate factor IX and factor X, leading to thrombin generation and clot formation.
[0203] TF is expressed by various cell types, including monocytes, macrophages and vascular endothelial cells, and is induced by IL-I, TNF-a or bacterial lipopolysaccharides.
Protein kinase C is involved in cytokine activation of endothelial cell TF
expression.
Induction of TF by endotoxin and cytokines is an important mechanism for initiation of disseminated intravascular coagulation seen in patients with Gram-negative sepsis. TF also appears to be involved in a variety of non-hemostatic functions including inflammation, cancer, brain function, immune response, and tumor-associated angiogenesis.
Thus, multispecific antibodies that target TF are useful not only in the treatment of coagulopathies, but also in the treatment of sepsis, cancer, pathologic angiogenesis, and other immune and inflammatory dysregulatory diseases according to the invention. A complex interaction between the coagulation pathway and the cytokine network is suggested by the ability of several cytokines to influence TF expression in a variety of cells and by the effects of ligand binding to the receptor. Ligand binding (factor VIIa) has been reported to give an intracellular calcium signal, thus indicating that TF is a true receptor.
Protein kinase C is involved in cytokine activation of endothelial cell TF
expression.
Induction of TF by endotoxin and cytokines is an important mechanism for initiation of disseminated intravascular coagulation seen in patients with Gram-negative sepsis. TF also appears to be involved in a variety of non-hemostatic functions including inflammation, cancer, brain function, immune response, and tumor-associated angiogenesis.
Thus, multispecific antibodies that target TF are useful not only in the treatment of coagulopathies, but also in the treatment of sepsis, cancer, pathologic angiogenesis, and other immune and inflammatory dysregulatory diseases according to the invention. A complex interaction between the coagulation pathway and the cytokine network is suggested by the ability of several cytokines to influence TF expression in a variety of cells and by the effects of ligand binding to the receptor. Ligand binding (factor VIIa) has been reported to give an intracellular calcium signal, thus indicating that TF is a true receptor.
[0204] Thrombin is the activated form of coagulation factor II (prothrombin);
it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin for macrophages, and can alter their production of cytokines and arachidonic acid metabolites. It is of particular importance in the coagulopathies that accompany sepsis. Numerous studies have documented the activation of the coagulation system either in septic patients or following LPS
administration in animal models. Despite more than thirty years of research, the mechanisms of LPS-induced liver toxicity remain poorly understood. It is now clear that they involve a complex and sequential series of interactions between cellular and humoral mediators. In the same period of time, gram-negative systemic sepsis and its sequellae have become a major health concern, attempts to use monoclonal antibodies directed against LPS or various inflammatory mediators have yielded only therapeutic failures, as noted elsewhere herein.
Multispecific antibodies according to the invention that target both thrombin and at least one other target address the clinical failures in sepsis treatment.
it converts fibrinogen to fibrin. Thrombin is a potent chemotaxin for macrophages, and can alter their production of cytokines and arachidonic acid metabolites. It is of particular importance in the coagulopathies that accompany sepsis. Numerous studies have documented the activation of the coagulation system either in septic patients or following LPS
administration in animal models. Despite more than thirty years of research, the mechanisms of LPS-induced liver toxicity remain poorly understood. It is now clear that they involve a complex and sequential series of interactions between cellular and humoral mediators. In the same period of time, gram-negative systemic sepsis and its sequellae have become a major health concern, attempts to use monoclonal antibodies directed against LPS or various inflammatory mediators have yielded only therapeutic failures, as noted elsewhere herein.
Multispecific antibodies according to the invention that target both thrombin and at least one other target address the clinical failures in sepsis treatment.
[0205] A recombinant form of thrombomodulin has been approved for treatment of disseminated intravascular coagulation (DIC) and is in phase II clinical trials for DIC
associated with sepsis (Okamoto et al., 2012, Crit Care Res Pract, Epub 2012 Feb 28).
Thrombomodulin has a pivotal role in the protein C system that is important in the pathogensis of sepsis (Levi and Van der Poll, Minerva Anestesiol Epub Dec 17, 2012).
Downregulation of thrombomodulin in sepsis causes impaired activation of protein C that is central in the modulation of coagulation and inflammation (Levi and Van der Poll, Minerva Anestesiol Epub Dec 17, 2012). Administration of recombinant thrombomodulin is reported to have a beneficial effect on restoration of coagulation and improvement of organ failure (Levi and Van der Poll, Minerva Anestesiol Epub Dec 17, 2012). A recent retrospective study confirmed that treatment with recombinant thrombomodulin was associated with reduced mortality in hospitalized patients with sepsis-induced DIC (Yamakawa et al., 2013, Intensive Care Med, Epub January 30, 2013).
associated with sepsis (Okamoto et al., 2012, Crit Care Res Pract, Epub 2012 Feb 28).
Thrombomodulin has a pivotal role in the protein C system that is important in the pathogensis of sepsis (Levi and Van der Poll, Minerva Anestesiol Epub Dec 17, 2012).
Downregulation of thrombomodulin in sepsis causes impaired activation of protein C that is central in the modulation of coagulation and inflammation (Levi and Van der Poll, Minerva Anestesiol Epub Dec 17, 2012). Administration of recombinant thrombomodulin is reported to have a beneficial effect on restoration of coagulation and improvement of organ failure (Levi and Van der Poll, Minerva Anestesiol Epub Dec 17, 2012). A recent retrospective study confirmed that treatment with recombinant thrombomodulin was associated with reduced mortality in hospitalized patients with sepsis-induced DIC (Yamakawa et al., 2013, Intensive Care Med, Epub January 30, 2013).
[0206] In other embodiments, the multispecific antibodies bind to a MHC class I, MHC class II or accessory molecule, such as CD40, CD54, CD80 or CD86. The multispecific antibody also may bind to a T-cell activation cytokine, or to a cytokine mediator, such as NF-KB.
Kits
Kits
[0207] Various embodiments may concern kits containing components suitable for treating or diagnosing acute kidney injury in a patient. Exemplary kits may contain one or more histone-neutralizing agents, such as the anti-histone antibodies described herein. If the composition containing components for administration is not formulated for delivery via the alimentary canal, such as by oral delivery, a device capable of delivering the kit components through some other route may be included. One type of device, for applications such as parenteral delivery, is a syringe that is used to inject the composition into the body of a subject.
Inhalation devices may also be used. In certain embodiments, a therapeutic agent may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation.
Inhalation devices may also be used. In certain embodiments, a therapeutic agent may be provided in the form of a prefilled syringe or autoinjection pen containing a sterile, liquid formulation or lyophilized preparation.
[0208] The kit components may be packaged together or separated into two or more containers. In some embodiments, the containers may be vials that contain sterile, lyophilized formulations of a composition that are suitable for reconstitution. A kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents. Other containers that may be used include, but are not limited to, a pouch, tray, box, tube, or the like. Kit components may be packaged and maintained sterilely within the containers.
Another component that can be included is instructions to a person using a kit for its use.
EXAMPLES
Example!. Role of Histones in Inducing Remote Organ Injury and Further Inducing Tubular Necrosis in Acute Kidney Injury Introduction
Another component that can be included is instructions to a person using a kit for its use.
EXAMPLES
Example!. Role of Histones in Inducing Remote Organ Injury and Further Inducing Tubular Necrosis in Acute Kidney Injury Introduction
[0209] Acute kidney injury (AKI) causes renal dysfunction and has potential life-threatening complications, such as the accumulation of uremic toxins, imbalance of fluid volume, mineral disorders, and metabolic acidosis (2015, Clin J Am Soc Nephrol 10:21-8). Acute tubular necrosis triggered by ischemia-reperfusion (IR) or nephrotoxins is a major cause of severe AKI (Basile et al., 2012, Compr Physiol 2:1303-53). The mortality rate of AKI
patients has not improved despite advances in technologies such as renal replacement therapy.
patients has not improved despite advances in technologies such as renal replacement therapy.
[0210] The molecular mechanisms of AKI have been well studied in terms of epithelial and endothelial cell physiology and cell-to-cell interactions using ischemic AKI
model animals (Sharfuddin & Molitoris, 2011, Nat Rev Nephrol 7:189-200). Recently, we introduced the concept of necroinflammation, where renal cell necrosis via the release of damage-associated molecular patterns (DAMPs) from necrotic cells drives intrarenal inflammation and the injury of other cells in an auto-amplification loop (Mulay et al., 2015, J Am Soc Nephrol 27:27-39).
Regulated forms of necrosis include necroptosis, ferroptosis, and mitochondrial permeability transition-mediated regulated necrosis (MPT-RN) (Linkermann et al., 2014, Nat Rev Immunol 14:759-67). All of these release DAMPs that alert the innate immune system (Braza et al., 2016, Nat Rev Nephrol 12:281-90). While this process can help to control infections, it accelerates unnecessary tissue damage such as IR injury (IRI).
model animals (Sharfuddin & Molitoris, 2011, Nat Rev Nephrol 7:189-200). Recently, we introduced the concept of necroinflammation, where renal cell necrosis via the release of damage-associated molecular patterns (DAMPs) from necrotic cells drives intrarenal inflammation and the injury of other cells in an auto-amplification loop (Mulay et al., 2015, J Am Soc Nephrol 27:27-39).
Regulated forms of necrosis include necroptosis, ferroptosis, and mitochondrial permeability transition-mediated regulated necrosis (MPT-RN) (Linkermann et al., 2014, Nat Rev Immunol 14:759-67). All of these release DAMPs that alert the innate immune system (Braza et al., 2016, Nat Rev Nephrol 12:281-90). While this process can help to control infections, it accelerates unnecessary tissue damage such as IR injury (IRI).
[0211] Among DAMPs, histones have a particular role as they elicit direct cytotoxic effects, hence histones are cytotoxic DAMPs (Kumar et al., 2015, J Am Soc Nephrol 26:2399-413).
Especially, neutrophils infiltrate the kidney during the early injury phase (Matthijsen et al., 2007, Am J Pathol 171:1743-52; Lech et al., 2013, Kidney Int 83:647-61) and contribute to organ damage (Hayama et al., 2006, Transplant Proc 38:2201-2), but the mechanisms are not clear. Zychlinsky, et at. discovered the phenomenon of neutrophil extracellular trap (NET) formation as a previously unknown mode of bacterial killing. (Brinkmann et al., 2004, Science 303:1532-1535). NET formation is usually associated with neutrophil death, a process named NETosis, and that is morphologically distinguished from apoptosis and necrosis (Fuchs et al., 2007, J Cell Biol 176:231-41).
Especially, neutrophils infiltrate the kidney during the early injury phase (Matthijsen et al., 2007, Am J Pathol 171:1743-52; Lech et al., 2013, Kidney Int 83:647-61) and contribute to organ damage (Hayama et al., 2006, Transplant Proc 38:2201-2), but the mechanisms are not clear. Zychlinsky, et at. discovered the phenomenon of neutrophil extracellular trap (NET) formation as a previously unknown mode of bacterial killing. (Brinkmann et al., 2004, Science 303:1532-1535). NET formation is usually associated with neutrophil death, a process named NETosis, and that is morphologically distinguished from apoptosis and necrosis (Fuchs et al., 2007, J Cell Biol 176:231-41).
[0212] NET formation depends on the activation of peptidyl arginine deiminase (PAD) enzymes, which convert arginine residues of histones to citrulline (Li et al., 2010, J Exp Med 207:1853-62). Hi stone citrullination neutralizes DNA-hi stone interactions, resulting in chromatin de-condensation and NET release (Remijsen et al., 2011, Cell Death Differ 18:581-8). We speculated on a role of NETs in AKI. In addition, mortality of AKI relates also to multiple organ failure (Scheel et al., 2008, Kidney Int 74:849-51), but the mechanistic link between kidney injury and multiple organ failure could not yet be defined (Klein et al., 2008, Kidney Int 74:901-9; Yap & Lee, 2012, Anesthesiology 116:1139-48). We hypothesized that neutrophils infiltrating the kidney during AKI release cytotoxic histones while undergoing NET formation and that such these histones contribute accelerated AKI as well as to AKI-related remote organ damage. We further hypothesized that anti-histone agents, such as anti-histone antibodies, could ameliorate the toxic effects of AKI, and that PAD inhibitors might further enhance the beneficial effects of histone inhibition.
Materials and Methods
Materials and Methods
[0213] Animal studies. C57BL/6N male mice were procured from Charles River Laboratories (Sulzfeld, Germany). 6-8 week-old mice (N=5-14) were anesthetized before renal pedicle clamping (unilateral: 15, 25, 35, or 45 min) with a microaneurysm clamp via flank incisions (Medicon, Tuttlingen, Germany). Body temperature was maintained at between 37 C and 38 C throughout the procedure by placing the mice on a heating pad. After clamp removal, recovery of blood flow to the kidney was confirmed as evidenced by returning to its original color, before closing the wound. To maintain fluid balance, all mice were supplemented with 0.5 ml of 0.9% NaCl administered peritoneally. Mice were killed after 0 min, 15 min, 30 min, 6 h, 12 h, 15 h, 24 h, 2 d, 3 d, 7 d and 10 days after surgery.
Bilateral IRI mice (ischemia 35 min, reperfusion 24 h) were treated with PAD
inhibitor (20 mg/kg, intraperitoneal injection; i.p: CALBIOCHEM ), anti-histone IgGs (20 mg/kg, i.p;
clone BWA3; IMMUNOMEDICS , Morris Plains, NJ), necrosis inhibitor combination (Necrostatini /Enzo 1.65 mg/kg i.p, Ferrostatin/CALBIOCHEM , 2mg/kg i.p, Ciclosporin/Novartis 10 mg/kg, i.v) 2 hours before IRI surgery. Neutrophil depletion was performed as described previously (Huang et al., 2015, Hepatology 62:600-14) with an i.p.
injection of 500 tg anti-Ly6G IgGs (1A8) (BIOXCELL ) or control IgGs 24 and 2 hours before IRI surgery.
Bilateral IRI mice (ischemia 35 min, reperfusion 24 h) were treated with PAD
inhibitor (20 mg/kg, intraperitoneal injection; i.p: CALBIOCHEM ), anti-histone IgGs (20 mg/kg, i.p;
clone BWA3; IMMUNOMEDICS , Morris Plains, NJ), necrosis inhibitor combination (Necrostatini /Enzo 1.65 mg/kg i.p, Ferrostatin/CALBIOCHEM , 2mg/kg i.p, Ciclosporin/Novartis 10 mg/kg, i.v) 2 hours before IRI surgery. Neutrophil depletion was performed as described previously (Huang et al., 2015, Hepatology 62:600-14) with an i.p.
injection of 500 tg anti-Ly6G IgGs (1A8) (BIOXCELL ) or control IgGs 24 and 2 hours before IRI surgery.
[0214] Assessment of Tissue Injury and Inflammation. Tissues were embedded in paraffin, and 3-mm sections were used for RE (hematoxylin and eosin staining), Periodic acid-Schiff (PAS), and IHC (immunohistochemistry) staining. The sections for IHC were deparaffinized, and rehydrated in a graded ethanol series. Endogenous peroxidase was inhibited using 0.3%
H202 in PBS for 30 min. Sections were then heated in 97 C sodium citrate buffer (10 mM, pH 6) for 40 min for antigen retrieval. Sections were treated with the Vector Blocking kit (Vector Laboratories, Burlingame, CA) for endogenous biotin inhibition.
H202 in PBS for 30 min. Sections were then heated in 97 C sodium citrate buffer (10 mM, pH 6) for 40 min for antigen retrieval. Sections were treated with the Vector Blocking kit (Vector Laboratories, Burlingame, CA) for endogenous biotin inhibition.
[0215] For immunofluorescence, the tissues after sacrifice were fixed with 4%
paraformaldehyde for 2 hours, dehydrated with 30% sucrose for 24 hours at 4 C, frozen in OCT (TISSUE-TEK , OCT compounds, SAKURAg) and cryosectioned (51.tm thick). The CitH3 positive NETs and TUNEL positive necrotic cells were quantified using ImageJ
software. Tubular injury in PAS staining was scored by assessing the percentage of tubules in the cortex and outer-medullary lesion that displayed tubular cell necrosis, tubular dilation, luminal cast formation and inflammatory cells infiltration.
paraformaldehyde for 2 hours, dehydrated with 30% sucrose for 24 hours at 4 C, frozen in OCT (TISSUE-TEK , OCT compounds, SAKURAg) and cryosectioned (51.tm thick). The CitH3 positive NETs and TUNEL positive necrotic cells were quantified using ImageJ
software. Tubular injury in PAS staining was scored by assessing the percentage of tubules in the cortex and outer-medullary lesion that displayed tubular cell necrosis, tubular dilation, luminal cast formation and inflammatory cells infiltration.
[0216] For immunostaining, rat anti-mouse Ly6b (neutrophils) ab (AbD Serotec, Oxford, United Kingdom), rabbit citrullinated histone 3 ab (Abcam, Cambridge, United Kingdom), or goat anti neutrophil elastase ab (Santa Cruz Biotechnology, USA) were used. To count the positive cells, 3 high-power fields (100x) were analyzed. BUN and creatinine were measured using urea or creatinine FS kits (DiaSys Diagnostic Systems, Holzheim, Germany) according to the manufacturer's protocols. Mice plasma was analysed for IL-6 and TNF
cytokine secretion by ELISA (BD Pharmingen, San Diego, CA). Plasma histone was analyzed by western blotting. To prepare a control model with high concentration of histone, lipopolysaccharide (LPS, 20 mg/kg, i.p) was injected into WT mice (8 weeks age, male), and blood samples were taken 6 hours after LPS injection (Li et al., 2011, Surgery 150:442-51).
Bronchoalveolar lavage fluid (BAL) in IRI mice was performed as previously described (Sayah et al., 2015, Am J Respir Crit Care Med 191:455-63). The BAL cell number and supernatant NETs were evaluated by cell counter and NE-DNA complexes ELISA, respectively.
cytokine secretion by ELISA (BD Pharmingen, San Diego, CA). Plasma histone was analyzed by western blotting. To prepare a control model with high concentration of histone, lipopolysaccharide (LPS, 20 mg/kg, i.p) was injected into WT mice (8 weeks age, male), and blood samples were taken 6 hours after LPS injection (Li et al., 2011, Surgery 150:442-51).
Bronchoalveolar lavage fluid (BAL) in IRI mice was performed as previously described (Sayah et al., 2015, Am J Respir Crit Care Med 191:455-63). The BAL cell number and supernatant NETs were evaluated by cell counter and NE-DNA complexes ELISA, respectively.
[0217] Immunohistofluorescence staining in Human Tissues. Fresh frozen sections of renal biopsies from two patients with acute tubular necrosis caused by ischemic condition after renal transplant were provided from the Institute of Pathology at the Sapporo city general hospital. Informed consent was obtained from the patients. The samples were used for RE
stain and NETs-immunostaining as previously described.
stain and NETs-immunostaining as previously described.
[0218] RNA Preparation and Real-Time RT-PCR. Total renal RNA was isolated using a Qiagen RNA extraction kit (Qiagen, Germany) as previously described (Kumar et al., 2015, J
Am Soc Nephrol 26:2399-413). From isolated RNA, complementary DNA was prepared using reverse transcriptase (SUPERSCRIPTTm II; INVITROGENTm, USA). The SYBR
Green Dye detection system was used for quantitative real-time PCR on a LIGHTCYCLER
480 (Roche). All gene expression values were normalized using 18s RNA as a house keeping gene.
Am Soc Nephrol 26:2399-413). From isolated RNA, complementary DNA was prepared using reverse transcriptase (SUPERSCRIPTTm II; INVITROGENTm, USA). The SYBR
Green Dye detection system was used for quantitative real-time PCR on a LIGHTCYCLER
480 (Roche). All gene expression values were normalized using 18s RNA as a house keeping gene.
[0219] Cell culture and treatment. The HK-2 (human) and iTEC (human) proximal tubular cell lines were cultured in DMEM medium supplemented with 10% fetal calf serum (FCS) and 1% penicillin¨streptomycin until the cells were 80-90% confluent. Before the experiments these cells media were exchanged to DMEM in the absence of FCS/glucose, and placed in normal oxygen (02:20%) or hypoxia chamber (02:1%), or hydrogen peroxidase (H202: 1mM) for 24 hours. HK-2 cells were originally purchased from ATCC, and were generously provided by B. Luckow and P.J. Nelson, respectively. iTEC cells were prepared from human renal progenitor cells and tubulogenic differentiation was induced with REGMTm (Lonza Ltd) as described previously (Sayah et al., 2009, J Am Soc Nephrol 20:322-32).
[0220] Neutrophil isolation and In vitro NET formation. Neutrophils were isolated from human healthy volunteers using standard dextran sedimentation followed by Ficoll-Hypaque density centrifugation procedures (Brinkmann et al., 2004, Science 303:1532-1535). Blood donors provided written informed consent forms approved by the local ethical committee.
Neutrophils were suspended in RPMI medium (2x105-2x106cell/m1) and seeded into 8-well microns slides or 12-96-well plates (Ibidi, Martinsried, Germany) in a 5%
carbon dioxide atmosphere at 37 C for 30 minute before stimulation. The neutrophils were placed in normal or hypoxia condition (1% oxygen) for 3,6, or 24 hours, or stimulated by Phorbol 12-myristate 13-acetate (PMA, 25 nM, Sigma-Aldrich), total calf thymus histones (10,50, or 100 [tg/m1), and necrotic conditioned media from TCs (control, hypoxia, H202 stimulation) for 3-4 hours. The same groups of neutrophils were pre-treated with PAD
inhibitor (C1-amidine 200 [tM), aHisAbs (100 [tg/m1) or control IgGs (100 [tg/m1).
Neutrophils were suspended in RPMI medium (2x105-2x106cell/m1) and seeded into 8-well microns slides or 12-96-well plates (Ibidi, Martinsried, Germany) in a 5%
carbon dioxide atmosphere at 37 C for 30 minute before stimulation. The neutrophils were placed in normal or hypoxia condition (1% oxygen) for 3,6, or 24 hours, or stimulated by Phorbol 12-myristate 13-acetate (PMA, 25 nM, Sigma-Aldrich), total calf thymus histones (10,50, or 100 [tg/m1), and necrotic conditioned media from TCs (control, hypoxia, H202 stimulation) for 3-4 hours. The same groups of neutrophils were pre-treated with PAD
inhibitor (C1-amidine 200 [tM), aHisAbs (100 [tg/m1) or control IgGs (100 [tg/m1).
[0221] NETs cytotoxicity to TCs and fresh neutrophils. Neutrophils were stimulated by PMA, histones, or necrotic TCs derived conditioned media and 3 hours after incubation the supernatant was replaced with fresh media (RPMI), and the bottom NETs were collected to avoid the contamination of the media with exogenous PMA or necrotic TCs or histones as previously described (Najmeh et al., 2015, J Vis Exp 16:98). After centrifugation (1200 rpm, min), the supernatants were applied to TCs (1:1). At 20 hours after addition of conditioned NETs media, TCs injury was evaluated by LDH assay. To investigate whether the formed NETs affect fresh neutrophils, conditioned NETs media was applied to fresh neutrophils and 4 h after incubation, additional NET formation in fresh neutrophils was evaluated by immunostaining.
[0222] NETs quantification assay. In human neutrophil experiments, NETs were quantified by the MPO-DNA sandwich ELISA method using anti-DNA abs (Roche, Mannheim, Germany) and anti-human MPO abs (AbD Serotec) as described (Nakazawa, 2014, J
Am Soc Nephrol 25:990-7). In mouse neutrophils experiments, BAL NETs (undiluted) and plasma NETs (x2 diluted in PBS) were evaluated by NE-DNA complex using anti-DNA abs and anti-mouse NE abs (Santa Cruz) as described (Sayah et al., 2015, Am J Respir Crit Care Med 191:455-63). The CitH3 positive NETs were quantified using ImageJ software.
Am Soc Nephrol 25:990-7). In mouse neutrophils experiments, BAL NETs (undiluted) and plasma NETs (x2 diluted in PBS) were evaluated by NE-DNA complex using anti-DNA abs and anti-mouse NE abs (Santa Cruz) as described (Sayah et al., 2015, Am J Respir Crit Care Med 191:455-63). The CitH3 positive NETs were quantified using ImageJ software.
[0223] Scanning electron microscopy. Cells were fixed with 2.5%
glutaraldehyde, post fixed using repeated incubations with 1% osmium tetroxide /1% tannic acid, dehydrated with a graded ethanol series, critical-point dried and coated with 2 nm platinum.
Since the NETs are fragile, each step was done with minimal disturbance of the media to preserve the structures.
glutaraldehyde, post fixed using repeated incubations with 1% osmium tetroxide /1% tannic acid, dehydrated with a graded ethanol series, critical-point dried and coated with 2 nm platinum.
Since the NETs are fragile, each step was done with minimal disturbance of the media to preserve the structures.
[0224] Cell death assay. A cell death detection (TUNEL) kit (Roche) was used to quantify dead cells in accordance with the manufacturer's instructions. Systemic dead cell derived DNAs in plasma were quantified by PICOGREEN dsDNA assay kit (Thermo Fisher Scientific). In vitro, cytotoxicity was evaluated by LDH assay (Roche).
[0225] Western Blotting. Mouse plasma or tissue extracts were analyzed by standard immunoblot technique as described elsewhere (Allam et al., 2011, J Immunol 186:2714-8).
Anti¨histone H3 antibodies, anti-f3-actin, and anti-citrullinated histone3 antibodies were purchased from Cell Signaling Technology (Danvers, MA) and Abcam. The expression of protein was quantified using ImageJ software.
Anti¨histone H3 antibodies, anti-f3-actin, and anti-citrullinated histone3 antibodies were purchased from Cell Signaling Technology (Danvers, MA) and Abcam. The expression of protein was quantified using ImageJ software.
[0226] Statistical Analyses. Data were expressed as the mean SEM. Comparison between groups was performed by the two-tailed t test or One-way ANOVA (nonparametric tests). A
P-value less than 0.05 indicated statistical significance. All statistical analyses were calculated using GraphPad Prism software (GraphPad).
Results
P-value less than 0.05 indicated statistical significance. All statistical analyses were calculated using GraphPad Prism software (GraphPad).
Results
[0227] NETs expression in severe human acute tubular necrosis. NETs have been observed at sites of sterile inflammation in humans (Kessenbrock et al., 2009, Nat Med 15:623-5). Hence we first questioned whether NETs develop also in human post-ischemic tubular necrosis. We performed immunofluorescence staining on two kidney biopsies obtained from patients with post-transplant acute tubular necrosis related to long cold ischemia times (Donor of case 1; A
68 year old, male, non-heart-beating donor. Ischemic time=30minutes. Donor of case 2; A 69 year old, male, Heart-beating donor). Immunostaining in both cases showed the presence of neutrophil elastase/Citrullinated histone 3 (CitH3) positive cells, which implies NET
formation, surrounding necrotic tubular cells (FIG. 1A). Haematoxylin and eosin (RE) staining showed that infiltrating leukocytes were present in damaged tubular ducts and the surrounding interstitial space (FIG. 1B).
68 year old, male, non-heart-beating donor. Ischemic time=30minutes. Donor of case 2; A 69 year old, male, Heart-beating donor). Immunostaining in both cases showed the presence of neutrophil elastase/Citrullinated histone 3 (CitH3) positive cells, which implies NET
formation, surrounding necrotic tubular cells (FIG. 1A). Haematoxylin and eosin (RE) staining showed that infiltrating leukocytes were present in damaged tubular ducts and the surrounding interstitial space (FIG. 1B).
[0228] The formation of NETs and tubular cell necrosis enhance each other. To evaluate whether hypoxia-induced necrosis of tubular cells activates neutrophils, conditioned media from human induced tubular epithelial cells (iTECs) treated with hypoxia (02;
1%) or hydrogen peroxidase (H202) (10 mM) were applied to human neutrophils. After 4 h of incubation, NET formation was confirmed by fluorescence microscopy (FIG. 2A
and 2B).
Similarly, conditioned media from necrotic Human Kidney (HK)-2 cells triggered neutrophils to undergo NETs (FIG. 2C, FIG. 2D) via the activation of citrullinated histones (FIG. 21) and the neutrophil supernatants were positive for extracellular DNA-myeloperoxidase complexes (FIG. 2F), which implies NET formation. To test whether hypoxia can trigger NET formation directly, neutrophils were incubated under 20% 02 or hypoxic conditions for 24 h but no difference in NET formation was observed (FIG. 21). These results indicate that only factors released from ischemic tubular cells, but not ischemia itself, triggers NET
formation.
1%) or hydrogen peroxidase (H202) (10 mM) were applied to human neutrophils. After 4 h of incubation, NET formation was confirmed by fluorescence microscopy (FIG. 2A
and 2B).
Similarly, conditioned media from necrotic Human Kidney (HK)-2 cells triggered neutrophils to undergo NETs (FIG. 2C, FIG. 2D) via the activation of citrullinated histones (FIG. 21) and the neutrophil supernatants were positive for extracellular DNA-myeloperoxidase complexes (FIG. 2F), which implies NET formation. To test whether hypoxia can trigger NET formation directly, neutrophils were incubated under 20% 02 or hypoxic conditions for 24 h but no difference in NET formation was observed (FIG. 21). These results indicate that only factors released from ischemic tubular cells, but not ischemia itself, triggers NET
formation.
[0229] To examine whether NETs affect tubular cells, conditioned media containing NETs was applied to HK-2 cells. After 20 h of incubation, the NET-containing media (FIG. 2E and 2F) induced HK-2 injury compared to conditioned media obtained from intact neutrophils (FIG. 2G and 211). In addition, the NET-containing media activated fresh neutrophils to undergo further NET formation (not shown). PAD inhibitor abrogated induced NET
formation in neutrophils and subsequent tubular cell injury (FIG. 2E-211).
Together these data imply that neutrophils undergoing NETosis release factors that kill tubular cells and that induce further NET formation. In turn, necrotic tubular cells induce neutrophils to undergo NETosis. Thus, tubular and neutrophil death could enhance each other.
formation in neutrophils and subsequent tubular cell injury (FIG. 2E-211).
Together these data imply that neutrophils undergoing NETosis release factors that kill tubular cells and that induce further NET formation. In turn, necrotic tubular cells induce neutrophils to undergo NETosis. Thus, tubular and neutrophil death could enhance each other.
[0230] Postischemic tubular necrosis is associated with formation of NETs. We hypothesized that infiltrating neutrophils form NETs in the post-ischemic kidney, which would imply release of more histones and other DAMPs that further accelerate AKI.
Postischemic AKI
was induced in wild type mice by unilateral clamping of the renal pedicle for 35 minutes followed by reperfusion for 0 h-72 h. Postischemic kidneys (unilateral ischemia time 35 min, reperfusion 24 h) displayed positivity for CitH3 and Ly6b co-localizing with NETs in areas of S3 segment tubular necrosis of the outer stripe of the outer medulla as assessed by immunofluorescence (FIG. 3A) and immunohistochemistry staining (not shown).
The number of NETs in the outer medulla increased between 1511-24 h after reperfusion and decreased thereafter as determined by immunofluorescence staining (FIG. 3B-3D) and western blotting (FIG. 3E). Similarly, circulating NET components in plasma were detected 15-24 h after reperfusion (not shown). Interestingly, renal cell death in the outer stripe of the outer medulla preceded NET formation, between 111-6 h after reperfusion as demonstrated by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL)-positivity in that area (FIG. 3F and 3G) as well as by detection of free DNA in plasma (not shown).
Histological analysis showed that tubule necrosis persisted for a long time after the peak of TUNEL positivity and NETs abundance (FIG. 31I-3K). These results show that necrosis of some tubular cells is an early event upon IRI followed by neutrophils undergoing NETosis, which is associated with ongoing tubular injury
Postischemic AKI
was induced in wild type mice by unilateral clamping of the renal pedicle for 35 minutes followed by reperfusion for 0 h-72 h. Postischemic kidneys (unilateral ischemia time 35 min, reperfusion 24 h) displayed positivity for CitH3 and Ly6b co-localizing with NETs in areas of S3 segment tubular necrosis of the outer stripe of the outer medulla as assessed by immunofluorescence (FIG. 3A) and immunohistochemistry staining (not shown).
The number of NETs in the outer medulla increased between 1511-24 h after reperfusion and decreased thereafter as determined by immunofluorescence staining (FIG. 3B-3D) and western blotting (FIG. 3E). Similarly, circulating NET components in plasma were detected 15-24 h after reperfusion (not shown). Interestingly, renal cell death in the outer stripe of the outer medulla preceded NET formation, between 111-6 h after reperfusion as demonstrated by terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL)-positivity in that area (FIG. 3F and 3G) as well as by detection of free DNA in plasma (not shown).
Histological analysis showed that tubule necrosis persisted for a long time after the peak of TUNEL positivity and NETs abundance (FIG. 31I-3K). These results show that necrosis of some tubular cells is an early event upon IRI followed by neutrophils undergoing NETosis, which is associated with ongoing tubular injury
[0231] NET formation contributes to post-ischemic tubular necrosis in vivo. To determine whether NET formation drives ongoing tubular injury, bilateral IRI kidney mice (ischemia 35 min, reperfusion 24 h) underwent either treatment with a PAD inhibitor or with neutrophil depletion by injection of anti-Ly6G monoclonal antibody. The PAD inhibitor substantially reduced the abundance of NETs in the post-ischemic kidney (FIG. 4A, 4B) as well as NET
components in plasma (FIG. 3C). Furthermore, renal function (FIG. 4D and 4E), tissue necrotic area (FIG. 4F), and the expression of AKI marker genes (not shown) were significantly reduced in the mice treated with the PAD inhibitor as well as neutrophil depletion. We conclude that neutrophils contribute to post-ischemic AKI by forming NETs.
components in plasma (FIG. 3C). Furthermore, renal function (FIG. 4D and 4E), tissue necrotic area (FIG. 4F), and the expression of AKI marker genes (not shown) were significantly reduced in the mice treated with the PAD inhibitor as well as neutrophil depletion. We conclude that neutrophils contribute to post-ischemic AKI by forming NETs.
[0232] Dual inhibition of NET formation and cell necrosis has additive protective effects on post-ischemic AM. To study the contribution of regulated necrosis and NETosis to renal necroinflammation we tested dual NET and necrosis inhibition in mice with post-ischemic AKI. Necrosis inhibition reduced the abundance of NETs (FIG. 5A-5D), necrotic area (FIG.
5E and 5F), and AKI-related gene expression (not shown) and improved renal function (FIG.
5G and 511). Adding a PAD inhibitor to necrosis inhibition abrogated intrarenal NET
formation, further improved renal dysfunction, reduced tubular necrosis (FIG.
5A-5E and 5G) and decreased induction of injury-related genes compared to necrosis inhibition alone (not shown). Together, these results show that NET formation is an accelerating element of the crescendo of necroinflammation in the post-ischemic kidney.
5E and 5F), and AKI-related gene expression (not shown) and improved renal function (FIG.
5G and 511). Adding a PAD inhibitor to necrosis inhibition abrogated intrarenal NET
formation, further improved renal dysfunction, reduced tubular necrosis (FIG.
5A-5E and 5G) and decreased induction of injury-related genes compared to necrosis inhibition alone (not shown). Together, these results show that NET formation is an accelerating element of the crescendo of necroinflammation in the post-ischemic kidney.
[0233] Histones are central mediators of necroinflammation in AKI. To examine which components of necrotic cells induce NETs and which components of NETs injure tubular cells, we focused on histones because histones have unique cytotoxic DAMP
effects (Allam et al., 2014, J Mol Med (Berlin) 92:465-72). Necrotic HK-2 cells treated with H202 released histones into the supernatant (FIG. 6A). To test if histones are sufficient to induce neutrophils to undergo NET formation, we exposed neutrophils from healthy human donors to extracellular histones. 3h later, the neutrophils had formed aggregated NETs and showed CitH3 positivity (FIG. 6B and 6C). Histone-induced NET formation could be inhibited with a histone-neutralizing BWA3 antibody (aHisAbs) (FIG. 6D). To examine the toxicity of histone-induced NETs to tubular cells, the supernatants of NETs were applied to HK-2 cells.
At 20h of incubation, NETs supernatants induced HK-2 cell death, a process suppressed by adding aHisAbs, which suggests that histones are a central element of NET-related tissue injury (FIG. 6E).
effects (Allam et al., 2014, J Mol Med (Berlin) 92:465-72). Necrotic HK-2 cells treated with H202 released histones into the supernatant (FIG. 6A). To test if histones are sufficient to induce neutrophils to undergo NET formation, we exposed neutrophils from healthy human donors to extracellular histones. 3h later, the neutrophils had formed aggregated NETs and showed CitH3 positivity (FIG. 6B and 6C). Histone-induced NET formation could be inhibited with a histone-neutralizing BWA3 antibody (aHisAbs) (FIG. 6D). To examine the toxicity of histone-induced NETs to tubular cells, the supernatants of NETs were applied to HK-2 cells.
At 20h of incubation, NETs supernatants induced HK-2 cell death, a process suppressed by adding aHisAbs, which suggests that histones are a central element of NET-related tissue injury (FIG. 6E).
[0234] To examine whether aHisAbs could suppress also the vicious cycle of tubular necrosis and NET formation, bilateral IRI kidney mice were treated with aHisAbs before the surgery.
aHisAbs reduced the abundance of NETs as assessed by immunofluorescence staining (FIG.
6F and 6G) and western blotting (not shown). Consistent with the decreased abundance of NETs, tubular necrosis, renal dysfunction, and the expression of kidney injury marker genes were suppressed (FIG. 6F, 61I-6J). Together, histones released from dying tubular cells and from NETs contributed to renal necroinflammation in AKI.
aHisAbs reduced the abundance of NETs as assessed by immunofluorescence staining (FIG.
6F and 6G) and western blotting (not shown). Consistent with the decreased abundance of NETs, tubular necrosis, renal dysfunction, and the expression of kidney injury marker genes were suppressed (FIG. 6F, 61I-6J). Together, histones released from dying tubular cells and from NETs contributed to renal necroinflammation in AKI.
[0235] Upon AKI circulating NETs and histones promote remote organ injury. The molecular mechanisms underlying AKI-related remote organ injury were previously unknown. Since we had found that post-ischemic AKI is associated with NETs and DAMPs in the plasma we speculated that the injured kidney releases NETs and their cytotoxic components into the circulation, promoting injury in other organs. Indeed, mice with bilateral IRI displayed not only NETs but also increased plasma concentrations of histones, tumor necrosis factor (TNF)-a and IL-6 (FIG. 7A-7C). To assess remote organ injury, TUNEL
staining and neutrophil immunostaining were performed in lung, liver, brain, heart, and pancreas after bilateral IRI surgery. TUNEL-positive cells and neutrophil infiltration were identified in multiple organs (lung, liver, brain, heart) (FIG. 7D, 7E) but western blotting of lysates from these organs detected histone citrullination only in kidney and lung (FIG. 7F).
Furthermore, NETs were detected in lung by immunofluorescence staining (FIG.
7G), but not in other organs (data not shown). The area of NETs in lung correlated with the TUNEL-positive area and the number of cells in bronchoalveolar lavage fluid (BAL) (FIG. 711, 71, 7J). NET area and BAL cell number peaked between 6 h-24 h after reperfusion in the unilateral IRI kidney model (not shown) and NETs components were detected in BAL
supernatant of bilateral IRI mice (not shown). Meanwhile, NETs and necrosis inhibition reduced these markers of lung injury and also reduced remote organ injury to liver, heart, and brain (FIG. 7K-7M) and systemic inflammation (FIG. 7N and 70). aHisAbs were especially effective for preventing remote organ injury (FIG. 71, 7K-7M). These results show that circulating histones promote lung injury and remote organ injury following renal IRI .
Discussion
staining and neutrophil immunostaining were performed in lung, liver, brain, heart, and pancreas after bilateral IRI surgery. TUNEL-positive cells and neutrophil infiltration were identified in multiple organs (lung, liver, brain, heart) (FIG. 7D, 7E) but western blotting of lysates from these organs detected histone citrullination only in kidney and lung (FIG. 7F).
Furthermore, NETs were detected in lung by immunofluorescence staining (FIG.
7G), but not in other organs (data not shown). The area of NETs in lung correlated with the TUNEL-positive area and the number of cells in bronchoalveolar lavage fluid (BAL) (FIG. 711, 71, 7J). NET area and BAL cell number peaked between 6 h-24 h after reperfusion in the unilateral IRI kidney model (not shown) and NETs components were detected in BAL
supernatant of bilateral IRI mice (not shown). Meanwhile, NETs and necrosis inhibition reduced these markers of lung injury and also reduced remote organ injury to liver, heart, and brain (FIG. 7K-7M) and systemic inflammation (FIG. 7N and 70). aHisAbs were especially effective for preventing remote organ injury (FIG. 71, 7K-7M). These results show that circulating histones promote lung injury and remote organ injury following renal IRI .
Discussion
[0236] We hypothesized that several of the infiltrating neutrophils during AKI
undergo NETosis, leading to the release of cytotoxic DAMPs such as histones, which exacerbates kidney tubular injury and interstitial inflammation. Furthermore, we speculated that such NET components enter the circulation and contribute to remote organ injury that is often associated with AKI, e.g. in multi-organ failure. Our study revealed that post-ischemic tubular necrosis involves DAMP release, promoting NET formation as a second event, a process that results in further renal and extrarenal injury.
undergo NETosis, leading to the release of cytotoxic DAMPs such as histones, which exacerbates kidney tubular injury and interstitial inflammation. Furthermore, we speculated that such NET components enter the circulation and contribute to remote organ injury that is often associated with AKI, e.g. in multi-organ failure. Our study revealed that post-ischemic tubular necrosis involves DAMP release, promoting NET formation as a second event, a process that results in further renal and extrarenal injury.
[0237] The pathophysiology of AKI involves regulated cell death and inflammation (Linkermann et al., 2014, Proc Natl Acad Sci USA 111:16836-41). In particular, necroptosis, ferroptosis, and MPT-RN of TCs result in the release of DAMPs, leading to the recruitment of inflammatory cells and further injury (Linkermann et al., 2013, Proc Natl Acad Sci USA
110:12024-9). Numerous neutrophils were detected in the early phase of ischemic AKI (Lech et al., 2013, Kidney Int 83:647-61) and neutrophil depletion prevented renal dysfunction, indicating the contribution of neutrophils to AKI (Hayama et al., 2006, Transplant Proc 38:2201-2).
110:12024-9). Numerous neutrophils were detected in the early phase of ischemic AKI (Lech et al., 2013, Kidney Int 83:647-61) and neutrophil depletion prevented renal dysfunction, indicating the contribution of neutrophils to AKI (Hayama et al., 2006, Transplant Proc 38:2201-2).
[0238] We found the presence of NETs in kidney biopsies of patients with acute tubular necrosis, which is consistent with data from non-infectious human kidney diseases such as ANCA vasculitis (Kessenblock 19448636, Kumar et al., 2015, J Am Soc Nephrol 26:2399-413). Based on these findings, we speculated that hypoxia-induced necrotic TCs activate neutrophils to promote NET formation, which induced further TC injury and further NETs formation. These indicate that tubular necrosis and NETosis could enhance the inflammation and surrounding tissue damage induced by each other, i.e. renal necroinflammation. Indeed, the IRI kidney was protected by treatment with an inhibitor of NET formation, which is consistent with previous findings in glomerular disease (Kumar et al., 2015, J
Am Soc Nephrol 26:2399-413, 23722903).
Am Soc Nephrol 26:2399-413, 23722903).
[0239] Cl-amidine is a pan-PAD enzyme inhibitor that can inhibit all types of PAD in other cells. PAD4 is located inside neutrophil nuclei, where it facilitates citrullination of histones.
Other PAD enzymes are mostly absent from the kidney (Wong et al., 2015, Nat Med 21:815-9). Although PAD4 is expressed in tubular cells (Ham et al., 2014, Am J
Physiol Renal Physiol 307:F1052-62), the precise pathophysiology in injured TCs remains unclear. Our in vitro data show that conditioned NET media pretreated with PAD inhibitor reduced the damage of TCs. This shows that the PAD inhibitor in our study mainly inhibited NETosis by affecting PAD4 activity in neutrophils. Furthermore, we demonstrated that dual inhibition of NET formation and cell necrosis has additive protective effects on IRI kidney.
Other PAD enzymes are mostly absent from the kidney (Wong et al., 2015, Nat Med 21:815-9). Although PAD4 is expressed in tubular cells (Ham et al., 2014, Am J
Physiol Renal Physiol 307:F1052-62), the precise pathophysiology in injured TCs remains unclear. Our in vitro data show that conditioned NET media pretreated with PAD inhibitor reduced the damage of TCs. This shows that the PAD inhibitor in our study mainly inhibited NETosis by affecting PAD4 activity in neutrophils. Furthermore, we demonstrated that dual inhibition of NET formation and cell necrosis has additive protective effects on IRI kidney.
[0240] Although RIPK3/CypD double knockout mice (Linkermann et al., 2013, Proc Natl Acad Sci USA 110:12024-9), which have defects in necroptosis and MPT-RN, are prevented from IRI-induced kidney damage, chemical inhibitors of these processes such as necrostatin 1, ferrostatin 1, and sanglifehrin A did not completely prevent kidney injury.
Thus, our data indicate that although these inhibitors prevented some TCs death, the amount of DAMPs that was released from the fewer TCs undergoing necrosis was still sufficient to induce NETs formation, and the NETs could be inhibited by PAD inhibitor. Nevertheless, the additive effect of dual therapy was not large compared to necrosis inhibitor alone, which raises the possibility that in the necrosis inhibitor cocktail, 1) necrostatin 1 could inhibit neutrophil necroptosis and NET formation via suppressing the RIPK1/RIPK3/pMLKL signalling pathway (Desai et al. 2016, Eur J Immunol 46:223-9), and 2) cyclosporine, which was used for blocking MPT-RN, could react with calcineurin to inhibit NET induction (Gupta et al., 2014, PLoS One 9:e97088). Consequently, these agents could inhibit the formation of NETs.
However, it is unclear whether these inhibitors could affect the PAD4-citrullinated histone pathway in neutrophils.
Thus, our data indicate that although these inhibitors prevented some TCs death, the amount of DAMPs that was released from the fewer TCs undergoing necrosis was still sufficient to induce NETs formation, and the NETs could be inhibited by PAD inhibitor. Nevertheless, the additive effect of dual therapy was not large compared to necrosis inhibitor alone, which raises the possibility that in the necrosis inhibitor cocktail, 1) necrostatin 1 could inhibit neutrophil necroptosis and NET formation via suppressing the RIPK1/RIPK3/pMLKL signalling pathway (Desai et al. 2016, Eur J Immunol 46:223-9), and 2) cyclosporine, which was used for blocking MPT-RN, could react with calcineurin to inhibit NET induction (Gupta et al., 2014, PLoS One 9:e97088). Consequently, these agents could inhibit the formation of NETs.
However, it is unclear whether these inhibitors could affect the PAD4-citrullinated histone pathway in neutrophils.
[0241] Our study indicated that remote organ injury following AKI at least partially relates to formation of NETs. In particular, the lungs are the most common site of remote injury, since the permeability of lung capillaries increases during AKI (Ware & Matthay, 2000, N Engl J
Med. 342:1334-49). In this study, we demonstrated that NETs also form in the lungs in association with IRI kidney inducing acute lung injury (ALT).
Med. 342:1334-49). In this study, we demonstrated that NETs also form in the lungs in association with IRI kidney inducing acute lung injury (ALT).
[0242] One possible mechanism why NETs mostly occurred in lungs as a remote organ seems to be that pre-NET-forming neutrophils, which passed through the injured kidney and were stimulated by highly concentrated DAMPs, would anatomically return to the lung and could easily become trapped in vessels because capillaries in lung have a narrower bore compared with those in other organs (Bathe et al., 2002, Biophys J 83:1917-33). Doi et al.
(2014, Kidney Int 86:316-26) revealed that in bilateral nephrectomy mice, HMGB1 among DAMPs affected ALT via TLR4 signalling, but in bilateral IRI kidney mice, it induced ALT
independent from TLR4. These findings seem to be compatible with those of our study, since IRI could induce necroinflammation including NETs formation to activate various receptors as well as TLR4. For example, histones can activate TLRs 2, 4, and 9, while HMGB1 can activate TLRs 2, 4, 9 and receptor for advanced glycation end-products (Chen et al. 2016, Acta Pharm Sin B 6:183-8).
(2014, Kidney Int 86:316-26) revealed that in bilateral nephrectomy mice, HMGB1 among DAMPs affected ALT via TLR4 signalling, but in bilateral IRI kidney mice, it induced ALT
independent from TLR4. These findings seem to be compatible with those of our study, since IRI could induce necroinflammation including NETs formation to activate various receptors as well as TLR4. For example, histones can activate TLRs 2, 4, and 9, while HMGB1 can activate TLRs 2, 4, 9 and receptor for advanced glycation end-products (Chen et al. 2016, Acta Pharm Sin B 6:183-8).
[0243] The inhibitors of NETs and necrosis also protected against other remote organ injuries via blocking circulating histones, cytotoxic cytokines, and circulating NETs components.
Among these treatments, aHisAbs were the most effective intervention to protect distant organs against damage, which indicates that histones are one of the most important DAMPs mediating AKI-related remote organ injury. Although inhibitors of NETs and necrosis are supposed to prevent remote organ injury via regulating the cell death pathway caused by DAMPs, neutralizing histones could possibly contribute to inhibiting the direct toxicity of histones (Chen et al., 2014, Cell Death Dis 5:e1370) as well as TLR
activation, which could not be inhibited by NETs and necrosis inhibitors. The direct cytotoxicity is known to be due to the high positive charge, which affects the cell membrane to induce a damage of cells (Gillrie et al., 2012, Am J Pathol 180:1028-39). Conversely, inhibitors of NETs and necrosis showed a trend towards a better protective effect against kidney damage during IRI compared with that of aHisAbs. These findings indicate that the site of necroinflammation might be more effectively spatiotemporally regulated by blocking intracellular signaling arising from IRI than by neutralizing locally produced histones.
Among these treatments, aHisAbs were the most effective intervention to protect distant organs against damage, which indicates that histones are one of the most important DAMPs mediating AKI-related remote organ injury. Although inhibitors of NETs and necrosis are supposed to prevent remote organ injury via regulating the cell death pathway caused by DAMPs, neutralizing histones could possibly contribute to inhibiting the direct toxicity of histones (Chen et al., 2014, Cell Death Dis 5:e1370) as well as TLR
activation, which could not be inhibited by NETs and necrosis inhibitors. The direct cytotoxicity is known to be due to the high positive charge, which affects the cell membrane to induce a damage of cells (Gillrie et al., 2012, Am J Pathol 180:1028-39). Conversely, inhibitors of NETs and necrosis showed a trend towards a better protective effect against kidney damage during IRI compared with that of aHisAbs. These findings indicate that the site of necroinflammation might be more effectively spatiotemporally regulated by blocking intracellular signaling arising from IRI than by neutralizing locally produced histones.
[0244] In conclusion, during IRI of kidney, the necrosis of TCs appears to be an initial event that leads to the release of DAMPs and the induction of NETosis. NETs and NETs-derived DAMPs such as histones and DNA act as mediators of necroinflammation to induce further injury of TCs and further NETs formation. The cycle of TC deaths and NETs formation exacerbates kidney injury and induces remote organ injury. Conversely, use of anti-histone agents, such as anti-histone antibodies, alone or in combination with PAD
inhibitors, can ameliorate the remote organ injury consequent to AKI.
Example 2. Effect of Histone-Neutralizing Agents on Vascular Necrosis in Severe Glomerulonephritis
inhibitors, can ameliorate the remote organ injury consequent to AKI.
Example 2. Effect of Histone-Neutralizing Agents on Vascular Necrosis in Severe Glomerulonephritis
[0245] Severe glomerulonephritis involves cell necrosis as well as NETosis, a programmed neutrophil death leading to expulsion of nuclear chromatin leading to neutrophil extracellular traps (NETs). We speculated on a role of the dying cell' s and NET s histone component in necrotizing glomerulonephritis. Histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. As discussed below, this effect was prevented by histone-neutralizing agents such as anti-histone IgG, activated protein C, or heparin.
[0246] Histone toxicity on glomeruli ex vivo was TLR2/4-dependent. Lack of attenuated intra-arterial histone injection-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-GBM glomerulonephritis involved NET
formation and vascular necrosis. Pre-emptive anti-histone IgG administration significantly reduced all aspects of glomerulonephritis, i.e. vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment and activation of glomerular leukocytes as well as glomerular crescent formation.
formation and vascular necrosis. Pre-emptive anti-histone IgG administration significantly reduced all aspects of glomerulonephritis, i.e. vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment and activation of glomerular leukocytes as well as glomerular crescent formation.
[0247] To evaluate the therapeutic potential of histone neutralization we treated mice with established glomerulonephritis with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin all abrogated severe glomerulonephritis, suggesting that histone-mediated glomerular pathology is not an initial but rather a subsequent event in necrotizing glomerulonephritis. Together, histone release during glomerulonephritis elicits cytotoxic and immunostimulatory effects.
Neutralizing extracellular histones is therapeutic in severe experimental glomerulonephritis.
Materials and methods
Neutralizing extracellular histones is therapeutic in severe experimental glomerulonephritis.
Materials and methods
[0248] Mice and anti-GBM nephritis model - C57BL/6 mice were procured from Charles River (Sulzfeld, Germany). 6-8 week old mice received an intravenous injection of 100 11.1 of anti-GBM serum (sheep anti-rat glomeruli basement membrane serum procured from Probetex INC, PTX-001). Urine samples were collected at different time points after antiserum injection to evaluate the functional parameters of kidney damage. On day 7 the mice were sacrificed by cervical dislocation to collect plasma and kidney tissue. Kidneys were kept at -80 C for protein isolation and in RNALATER solution at -20 C
for RNA
isolation. A part of the kidney was also kept in formalin to be embedded in paraffin for histological analysis (Teixeira et al., 2005, Kidney Int 67:514B). We treated groups of mice either with 20mg/kg, i.p. control IgG or anti-histone antibody (clone BWA-3) to neutralize the effects of extracellular hi stones.
for RNA
isolation. A part of the kidney was also kept in formalin to be embedded in paraffin for histological analysis (Teixeira et al., 2005, Kidney Int 67:514B). We treated groups of mice either with 20mg/kg, i.p. control IgG or anti-histone antibody (clone BWA-3) to neutralize the effects of extracellular hi stones.
[0249] Assessment of renal pathology - Renal sections of 2 i_tm were stained with periodic acid-Schiff reagent. Glomerular abnormalities were scored in 50 glomeruli per section by a blinded observer. The following criteria were assessed in each of the 50 glomeruli and scored as segmental or global lesions if less or more than 50% of the glomerular tuft were affected by focal necrosis and capsule adhesions. Cellular crescents were assessed separately when more than a single layer of PECs were present around the inner circumference of Bowman's capsule. Immunostaining was performed as described using the following primary antibodies:
for WT-1/nephrin, neutrophils (Serotec, Oxford, UK), Mac-2 (Cedarlane, Ontario, Canada), TNF-a (Abeam, Cambridge, UK) and fibrinogen (Abeam, Cambridge, UK). Stained glomerular cells were quantified in 50 glomeruli per section.
for WT-1/nephrin, neutrophils (Serotec, Oxford, UK), Mac-2 (Cedarlane, Ontario, Canada), TNF-a (Abeam, Cambridge, UK) and fibrinogen (Abeam, Cambridge, UK). Stained glomerular cells were quantified in 50 glomeruli per section.
[0250] Electron microscopy - Kidney tissues and endothelial cell monolayers were fixed in 2.0% paraformaldehyde/ 2.0% glutaraldehyde, in 0.1M sodium phosphate buffer, pH 7.4 for 24h, followed by 3 washes x15 min in 0.1m sodium phosphate buffer, pH 7.4 and distilled water. For transmission EM kidneys were post-fixed, in phosphate cacodylate-buffered 2%
0s04 for lh, dehydrated in graded ethanols with a final dehydration in propylene oxide and embedded in Embed-812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (-90-nm thick) were stained with uranyl acetate and Venable's lead citrate.
For scanning EM, after rinsing in distilled H20, cells on coverslips were treated with 1%
thiocarbohydrazide, post-fixed with 0.1% osmium tetroxide, dehydrated in ethanol, mounted on stubs with silver paste and critical-point dried before being sputter coated with gold/palladium. Specimens were viewed with a JEOL model 1200EX electron microscope (JEOL, Tokyo, Japan).
0s04 for lh, dehydrated in graded ethanols with a final dehydration in propylene oxide and embedded in Embed-812 (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections (-90-nm thick) were stained with uranyl acetate and Venable's lead citrate.
For scanning EM, after rinsing in distilled H20, cells on coverslips were treated with 1%
thiocarbohydrazide, post-fixed with 0.1% osmium tetroxide, dehydrated in ethanol, mounted on stubs with silver paste and critical-point dried before being sputter coated with gold/palladium. Specimens were viewed with a JEOL model 1200EX electron microscope (JEOL, Tokyo, Japan).
[0251] Immunohistochemistry of human tissues - Formalin-fixed paraffin-embedded sections of renal biopsies from five subjects with ANCA-positive RPGN, newly diagnosed in 2013, were drawn from the files of the Institute of Pathology at the Ludwig-Maximilians-University of Munich. The renal biopsies were fixed in 4 % PBS-buffered formalin solution and embedded in paraffin. Biopsies contained normal glomeruli and glomeruli exhibiting cellular, fibrocellular or fibrous crescents. Controls consisted of normal kidney tissue from tumor nephrectomies. TLR2 and TLR4 expression was assessed by using specific antibodies (TLR2-LS Bio, Seattle, WA, TLR4- Novus, Littleton, CO).
In-vitro models
In-vitro models
[0252] Cytotoxicity assay - Mouse glomerular endothelial cells (GEnC (46)), podocytes (47)), and parietal epithelial cells (PECs,(48)) were cultured in 96 well plates with RPMI
media without FCS and PS and allowed to adhere overnight. The cells were stimulated with the different concentrations of total calf thymus histones (10, 20, 30, 40, 50 and 100 g/m1) with or without histone antibody for another 18-20h. Cytotoxicity assay was performed using Promega CELLTITER 96 non-radioactive cell proliferation assay (MTT Assay Kit, Mannheim, Germany). Glomerular cells were also incubated with histones with or without anti-histone IgG, heparin and/or aPC. LDH assay using cytotoxicity detection kit (Roche Diagnostics, Mannheim, Germany) was used to assess cell death.
media without FCS and PS and allowed to adhere overnight. The cells were stimulated with the different concentrations of total calf thymus histones (10, 20, 30, 40, 50 and 100 g/m1) with or without histone antibody for another 18-20h. Cytotoxicity assay was performed using Promega CELLTITER 96 non-radioactive cell proliferation assay (MTT Assay Kit, Mannheim, Germany). Glomerular cells were also incubated with histones with or without anti-histone IgG, heparin and/or aPC. LDH assay using cytotoxicity detection kit (Roche Diagnostics, Mannheim, Germany) was used to assess cell death.
[0253] Podocyte detachment assay - Podocytes were grown at 33 C using modified RPMI
media in the presence of IFN-y in collagen coated 10 cm dishes and 8x104 cells were seeded and allowed to differentiate as podocytes at 37 C for two weeks in collagen plates without IFN-y. Once the monolayers of podocytes were differentiated, the cells were treated with either histones or GBM antiserum with or without histone antibody and allowed to sit for 18h. Detached cells which are present in supernatant were manually counted using an hemocytometer. Adhered cells were trypsinised and counted manually to calculate the percentage of cells detached.
media in the presence of IFN-y in collagen coated 10 cm dishes and 8x104 cells were seeded and allowed to differentiate as podocytes at 37 C for two weeks in collagen plates without IFN-y. Once the monolayers of podocytes were differentiated, the cells were treated with either histones or GBM antiserum with or without histone antibody and allowed to sit for 18h. Detached cells which are present in supernatant were manually counted using an hemocytometer. Adhered cells were trypsinised and counted manually to calculate the percentage of cells detached.
[0254] In-vitro tube formation assay - Matrigel was thawed overnight at 4 C to make it liquid. After 10 ill per well of IJ-slide angiogenesis (MIDI, Munich, Germany) was added, the gel was allowed to solidify at 37 C. GEnCs were seeded at 1x104 cells/well and stimulated with VEGF and b-FGF as positive control or with histones with or without anti-histone antibody. Tube formation as a marker of angiogenesis was assessed by light microscopy by taking a series of pictures at 0 h, 4h 8h and 24h (49).
[0255] NETosis assay - Neutrophils were isolated from healthy mice by dextran sedimentation and hypotonic lysis of RBCs. Neutrophil extracellular traps (NETs) were induced in-vitro by adding TNF-a (Immunotools, Friesoythe, Germany) or phorbol myristate 13-acetate (PMA, Sigma-Aldrich, MO, USA) for 12 h in with or without anti-histone antibody. Endothelial cell death was assessed by MTT assay and immunofluorescence staining for histones (BWA-3 clone), neutrophil elastase (ABCAM , Cambridge, UK) and 4',6-Diamidin-2-phenylindol (DAPI, Vector labs, Burlingame, CA) after fixing with acetone.
[0256] BMDCs and J774 macrophages - Bone marrow cells were isolated from healthy mouse and plated at lx106 cells per well and differentiated into BMDCs in the presence of GM-CSF (Immunotools). J774 macrophage cells were grown in RPMI media, plated at 1x106 cells per well, and stimulated with different doses of histones with or without anit-histone antibody for 18 h. Supernatants were collected for TNF-a (Bio Legend, San Diego, CA) and IL-6 Elisa (BD Biosciences, San Diego, CA) determination. Flow cytometry for the activation markers MI-IC-II, CD40, CD103 and CD86 (BD) was also performed.
[0257] Flow cytometry - Flow cytometric analysis of cultured and renal immune cells was performed on a FACSCALIBUIRTM flow cytometer (BD) as described (Lech et at., 2009, J
Immunol 183:4109). Every isolate was incubated with binding buffer containing either anti-mouse CD11 c, CD11b, CD103, F4/80, and CD45 antibodies (BD) for 45 min at 4 C
in the dark were used to detect renal mononuclear phagocyte populations. Anti-CD86 (BD) was used as an activation marker. Anti-CD3 and CD4 (BD) were used to identify the respective T-cell populations.
Immunol 183:4109). Every isolate was incubated with binding buffer containing either anti-mouse CD11 c, CD11b, CD103, F4/80, and CD45 antibodies (BD) for 45 min at 4 C
in the dark were used to detect renal mononuclear phagocyte populations. Anti-CD86 (BD) was used as an activation marker. Anti-CD3 and CD4 (BD) were used to identify the respective T-cell populations.
[0258] RNA preparation and real-time RT-PCR - Reverse transcription and real time RT-PCR from total renal RNA was prepared as described (Patole et at., 2007, J
Autoimmun 29:52). SYBR Green Dye detection system was used for quantitative real-time PCR on a Light Cycler 480 (Roche, Mannheim, Germany). Gene-specific primers (300 nM, Metabion, Martinsried, Germany) were used as follows: Reverse and forward primers respectively 18s:
AGGGCCTCACTAAACCATCC (SEQ ID NO:36) and GCAATTATTCCCCATGAACG
(SEQ ID NO:37), TNF-a: CCACCACGCTCTTCTGTCTAC (SEQ ID NO:38) and AGGGTCTGGGCCATAGAACT (SEQ ID NO:39), Fibrinogen (FGL-2):
AGGGGTAACTCTGTAGGCCC (SEQ ID NO:40) and GAACACATGCAGTCACAGCC
(SEQ ID NO:41), WT-1: CATCCCTCGTCTCCCATTTA (SEQ ID NO:42) and TATCCGAGTTGGGGAAATCA (SEQ ID NO:43), CD44:
AGCGGCAGGTTACATTCAAA (SEQ ID NO:44) and CAAGTTTTGGTGGCACACAG
(SEQ ID NO:45). Controls consisting of ddH20 were negative for target and housekeeping genes.
Statistical analysis
Autoimmun 29:52). SYBR Green Dye detection system was used for quantitative real-time PCR on a Light Cycler 480 (Roche, Mannheim, Germany). Gene-specific primers (300 nM, Metabion, Martinsried, Germany) were used as follows: Reverse and forward primers respectively 18s:
AGGGCCTCACTAAACCATCC (SEQ ID NO:36) and GCAATTATTCCCCATGAACG
(SEQ ID NO:37), TNF-a: CCACCACGCTCTTCTGTCTAC (SEQ ID NO:38) and AGGGTCTGGGCCATAGAACT (SEQ ID NO:39), Fibrinogen (FGL-2):
AGGGGTAACTCTGTAGGCCC (SEQ ID NO:40) and GAACACATGCAGTCACAGCC
(SEQ ID NO:41), WT-1: CATCCCTCGTCTCCCATTTA (SEQ ID NO:42) and TATCCGAGTTGGGGAAATCA (SEQ ID NO:43), CD44:
AGCGGCAGGTTACATTCAAA (SEQ ID NO:44) and CAAGTTTTGGTGGCACACAG
(SEQ ID NO:45). Controls consisting of ddH20 were negative for target and housekeeping genes.
Statistical analysis
[0259] Data were expressed as mean standard error of the mean (SEM).
Comparison between groups was performed by two-tailed t-test or ANOVA. A value of p<0.05 was considered to be statistically significant. All statistical analyses were calculated using Graph Pad Prism (GraphPad).
Example 3. Glomerular TLR2 and TLR4 expression in severe human glomerulonephritis
Comparison between groups was performed by two-tailed t-test or ANOVA. A value of p<0.05 was considered to be statistically significant. All statistical analyses were calculated using Graph Pad Prism (GraphPad).
Example 3. Glomerular TLR2 and TLR4 expression in severe human glomerulonephritis
[0260] We first asked whether the TLR2 and TLR4 (Allam et al., 2012, J Am Soc Nephrol 23:1375) extracellular histones were expressed in the healthy and diseased glomeruli. TLR2/4 immunostaining of normal human kidney showed a weak granular positivity in all glomerular cells. TLR4 positivity was clearly noted in glomerular endothelial cells (not shown). In addition, TLR2 was strongly positive in the cytoplasm of epithelial cells of the proximal and distal tubule, while this was less prominent for TLR4 (not shown).
Immunostaining of kidney biopsies of patients with ANCA-associated necrotizing and crescentic GN
revealed prominent positivity also in PECs along the inner aspect of Bowman' s capsule (not shown).
As glomerular crescents are largely formed by PECs (Smeets et at., 2009, J Am Soc Nephrol 20:2593; Smeets et at., 2009, J Am Soc Nephrol 20:2604), glomerular crescents displayed TLR2 and TLR4 positivity (not shown). Thus, the cells of the normal glomerulus express TLR2/4 and PECs induce these TLRs in crescentic GN.
Example 4. Anti-histone IgG prevents histone toxicity on glomerular cells
Immunostaining of kidney biopsies of patients with ANCA-associated necrotizing and crescentic GN
revealed prominent positivity also in PECs along the inner aspect of Bowman' s capsule (not shown).
As glomerular crescents are largely formed by PECs (Smeets et at., 2009, J Am Soc Nephrol 20:2593; Smeets et at., 2009, J Am Soc Nephrol 20:2604), glomerular crescents displayed TLR2 and TLR4 positivity (not shown). Thus, the cells of the normal glomerulus express TLR2/4 and PECs induce these TLRs in crescentic GN.
Example 4. Anti-histone IgG prevents histone toxicity on glomerular cells
[0261] Histones were previously shown to be toxic to pulmonary endothelial cells in vitro and in vivo (Xu et at., 2009, Nat Med 15:1318; Abrams et at., 2013, Am J
Respir Crit Care Med 187:160). We tested this effect on cultured glomerular endothelial cells and found that a total histone preparation was cytotoxic in a dose-dependent manner. Anti-histone IgG derived from the BWA-3 hybridoma is known to neutralize the toxic and immunostimulatory effect of extracellular histones (Xu et at., 2009, Nat Med 15:1318; Xu et at., 2011, J Immunol 187:2626; Monestier et at., 1993, Mot Immunol 30:1069. Anti-histone IgG almost entirely prevented histone toxicity on glomerular endothelial cells up to a histone concentration of 30 [tg/m1 (not shown). Anti-histone IgG also prevented histone-induced GEnC
microtubule destruction in angiogenesis assays (not shown). Histone-induced toxicity was also evident in cultured podocytes and PECs albeit at much higher histone concentrations compared to the toxic dose required to kill endothelial cells (not shown). Anti-histone-IgG
also significantly reduced histone-induced detachment of cultured podocytes (not shown). Thus, extracellular histones are toxic to glomerular cells, which toxicity can be blocked by anti-histone IgG.
Example 5. Neutrophil extracellular traps kill glomerular endothelial cells through histone release
Respir Crit Care Med 187:160). We tested this effect on cultured glomerular endothelial cells and found that a total histone preparation was cytotoxic in a dose-dependent manner. Anti-histone IgG derived from the BWA-3 hybridoma is known to neutralize the toxic and immunostimulatory effect of extracellular histones (Xu et at., 2009, Nat Med 15:1318; Xu et at., 2011, J Immunol 187:2626; Monestier et at., 1993, Mot Immunol 30:1069. Anti-histone IgG almost entirely prevented histone toxicity on glomerular endothelial cells up to a histone concentration of 30 [tg/m1 (not shown). Anti-histone IgG also prevented histone-induced GEnC
microtubule destruction in angiogenesis assays (not shown). Histone-induced toxicity was also evident in cultured podocytes and PECs albeit at much higher histone concentrations compared to the toxic dose required to kill endothelial cells (not shown). Anti-histone-IgG
also significantly reduced histone-induced detachment of cultured podocytes (not shown). Thus, extracellular histones are toxic to glomerular cells, which toxicity can be blocked by anti-histone IgG.
Example 5. Neutrophil extracellular traps kill glomerular endothelial cells through histone release
[0262] In severe GN neutrophils undergo NETosis, which deposits nuclear chromatin within the glomerular capillaries (Kessenbrock et al., 2009, Nat Med 15:623).
Immunohistochemical staining showed nuclear chromatin release from netting neutrophils, including the spread of histones outside the dying cells (not shown). Neutrophils undergoing TNF-a- or PMA-induced NETosis on monolayers of glomerular endothelial cells destroyed this monolayer by inducing endothelial cell death, while TNF or PMA alone did not (not shown).
This NETosis-related endothelial cell toxicity was entirely prevented by anti-histone IgG
(not shown). We conclude that netting neutrophils damage glomerular endothelial cells via the release of histones.
Example 6. Histones need TLR2/4 to trigger glomerular necrosis and microangiopathy
Immunohistochemical staining showed nuclear chromatin release from netting neutrophils, including the spread of histones outside the dying cells (not shown). Neutrophils undergoing TNF-a- or PMA-induced NETosis on monolayers of glomerular endothelial cells destroyed this monolayer by inducing endothelial cell death, while TNF or PMA alone did not (not shown).
This NETosis-related endothelial cell toxicity was entirely prevented by anti-histone IgG
(not shown). We conclude that netting neutrophils damage glomerular endothelial cells via the release of histones.
Example 6. Histones need TLR2/4 to trigger glomerular necrosis and microangiopathy
[0263] Whether glomerular toxicity of extracellular histones is TLR2/4-dependent is not clear. To answer this question we exposed glomeruli isolated from wild type and Tlr2/4-deficient mice to histones ex vivo. Histones exposure was cytotoxic to glomeruli, a process that was entirely prevented using glomeruli from T/r2/4-deficient mice (not shown). Lack of TLR2/4 also prevented IL-6 and TNF expression in histone-exposed glomeruli (not shown).
We also studied the effects of extracellular histones on glomeruli in vivo.
Because intravenous histone injection kills mice immediately by pulmonary microvascular injury (Xu et al., 2009, Nat Med 15:1318), we injected histones directly into the left renal artery in anaesthetized mice. Unilateral histone injection caused glomerular lesions within 24 hours ranging from minor endothelial fibrinogen positivity to thrombotic microangiopathy and global glomerular necrosis (not shown). The contralateral kidney remained unaffected (not shown). Histone injection into the renal artery of T/r2/4-deficient mice showed significantly reduced glomerular lesions and fibrinogen positivity (not shown). These results demonstrate that extracellular histones induce glomerular injury in a TLR2/4-dependent manner.
Example 7. Extracellular histones contribute to severe glomerulonephritis
We also studied the effects of extracellular histones on glomeruli in vivo.
Because intravenous histone injection kills mice immediately by pulmonary microvascular injury (Xu et al., 2009, Nat Med 15:1318), we injected histones directly into the left renal artery in anaesthetized mice. Unilateral histone injection caused glomerular lesions within 24 hours ranging from minor endothelial fibrinogen positivity to thrombotic microangiopathy and global glomerular necrosis (not shown). The contralateral kidney remained unaffected (not shown). Histone injection into the renal artery of T/r2/4-deficient mice showed significantly reduced glomerular lesions and fibrinogen positivity (not shown). These results demonstrate that extracellular histones induce glomerular injury in a TLR2/4-dependent manner.
Example 7. Extracellular histones contribute to severe glomerulonephritis
[0264] Based on these results we speculated that intrinsic histone release may also contribute to severe GN in vivo. To address this question we applied the same neutralizing anti-histone IgG as used in vitro that demonstrated the functional contribution of extracellular histones in lethal endotoxemia (Xu et al., 2009, Nat Med 15:1318). Mice were injected i.p.
with 20 mg/kg anti-histone IgG or with 20 mg/kg control IgG 24 hours before the intravenous injection of a GBM antiserum raised in sheep. At the end of the study at day 7 only sheep IgG
but no mouse IgG deposits were found in glomeruli, excluding any autologous anti-sheep IgG
response contributing to glomerulonephritis (not shown). Anti-histone IgG
significantly reduced blood urea nitrogen (BUN) and serum creatinine levels following GBM
antiserum injections (not shown). This was associated with a significant reduction in crescent formation and global glomerular pathology with less severe lesions 7 days after antiserum injection (not shown). Myeloperoxidase (MPO) immunostaining visualized NETs inside glomeruli, which was associated with focal loss of endothelial CD31 positivity as a marker of glomerular vascular injury (not shown). Anti-histone IgG did not affect extracellular positivity but maintained CD31+ vasculature (not shown), indicating a protective effect on NET-related vascular injury.
with 20 mg/kg anti-histone IgG or with 20 mg/kg control IgG 24 hours before the intravenous injection of a GBM antiserum raised in sheep. At the end of the study at day 7 only sheep IgG
but no mouse IgG deposits were found in glomeruli, excluding any autologous anti-sheep IgG
response contributing to glomerulonephritis (not shown). Anti-histone IgG
significantly reduced blood urea nitrogen (BUN) and serum creatinine levels following GBM
antiserum injections (not shown). This was associated with a significant reduction in crescent formation and global glomerular pathology with less severe lesions 7 days after antiserum injection (not shown). Myeloperoxidase (MPO) immunostaining visualized NETs inside glomeruli, which was associated with focal loss of endothelial CD31 positivity as a marker of glomerular vascular injury (not shown). Anti-histone IgG did not affect extracellular positivity but maintained CD31+ vasculature (not shown), indicating a protective effect on NET-related vascular injury.
[0265] Because histones were toxic to glomerular endothelial cells and podocytes in vitro, we assessed the glomerular capillary ultrastructure by transmission electron microscopy. In control mice with crescentic glomeruli there was severe glomerular damage with fibrin deposits replacing large glomerular segments (fibrinoid necrosis). The capillary loops showed extensive GBM splitting and thinning, prominent endothelial cell nuclei, massive subendothelial edema with closure of the endothelial fenestrae, and obliteration of the capillary lumina. Subendothelial transudates (leaked serum proteins) and luminal platelets and neutrophils were also noted. Severe podocyte injury with diffuse foot process effacement, reactive cytoplasmic changes and detachment from the GBM were apparent (not shown).
[0266] In contrast, glomeruli of mice injected with anti-histone IgG showed restored endothelial fenestrations, flat appearing endothelial cells and preserved podocytes with intact foot processes (not shown). WT-1/nephrin co-immunostaining revealed that anti-histone IgG
largely prevented podocyte loss in antiserum-induced GN (not shown). This was consistent with significant reduction of albuminuria on day 7 following antiserum injection as compared to control IgG-treated mice (not shown). These results demonstrate that extracellular histones induce severe GN by causing glomerular vascular injury and podocyte loss, accompanied by proteinuria. They also demonstrate the efficacy of anti-histone antibody in preventing glomerular damage in glomerulonephritis.
Example 8. Extracellular histones drive glomerular leukocyte recruitment and activation
largely prevented podocyte loss in antiserum-induced GN (not shown). This was consistent with significant reduction of albuminuria on day 7 following antiserum injection as compared to control IgG-treated mice (not shown). These results demonstrate that extracellular histones induce severe GN by causing glomerular vascular injury and podocyte loss, accompanied by proteinuria. They also demonstrate the efficacy of anti-histone antibody in preventing glomerular damage in glomerulonephritis.
Example 8. Extracellular histones drive glomerular leukocyte recruitment and activation
[0267] Infiltrating leukocytes are not only a documented source of extracellular histones in severe GN (Kessenbrock et at., 2009, Nat Med 15:623) but also important effector cells (Kurts et al., 2013, Nat Rev Immunol 13:738). For example, in GBM antiserum-exposed glomerular endothelial cells, histone exposure triggered CXCL2 expression (not shown). In vivo, anti-histone IgG significantly reduced the numbers of glomerular neutrophils and macrophages as quantified by immunostaining (not shown). Flow cytometry of renal cell suspensions allowed us to better distinguish renal mononuclear phagocyte populations. Anti-histone IgG significantly reduced the numbers of activated (MEW II+) F4/80+
cells as well as of activated (CD86+) CD11b/CD103+ cells, and of CD4+ dendritic cells (not shown). In fact, histones dose-dependently induced activation markers like MEICII, CD40, CD80, and CD86 also in cultured bone marrow derived macrophages (BMDCs), which was entirely prevented with anti-histone IgG (not shown). Taken together, extracellular histones trigger glomerular leukocyte recruitment and activation, which can be blocked with anti-histone IgG in vitro and in vivo.
Example 9. Extracellular histones trigger intraglomerular TNF-a release and thrombosis
cells as well as of activated (CD86+) CD11b/CD103+ cells, and of CD4+ dendritic cells (not shown). In fact, histones dose-dependently induced activation markers like MEICII, CD40, CD80, and CD86 also in cultured bone marrow derived macrophages (BMDCs), which was entirely prevented with anti-histone IgG (not shown). Taken together, extracellular histones trigger glomerular leukocyte recruitment and activation, which can be blocked with anti-histone IgG in vitro and in vivo.
Example 9. Extracellular histones trigger intraglomerular TNF-a release and thrombosis
[0268] Activated mononuclear phagocytes are also an important source of pro-inflammatory cytokines in glomerular disease. Among these, TNF-a particularly contributes to podocyte loss, proteinuria, and glomerulosclerosis (Ryu et al., 2012, J Pathol 226:120). Because anti-histone IgG entirely prevented histone-induced TNF-a secretion in cultured macrophages and dendritic cells (not shown), we next assessed glomerular TNF-a expression.
Immunostaining displayed robust TNF-a positivity within the glomerular tuft, which not only localized in infiltrating cells but also in inner and outer aspect of the glomerular capillaries (not shown).
Anti-histone IgG strongly reduced glomerular TNF-a positivity, which was consistent with the corresponding renal mRNA expression levels (not shown). TNF-a is not only an inducer of NETosis but also triggers a prothrombotic activation of (glomerular) endothelial cells and intravascular fibrin formation (32-34). Our GN model displayed global fibrinogen positivity within glomerular capillaries, which was almost entirely prevented with anti-histone IgG (not shown). Also fibrinogen mRNA levels were reduced in the anti-histone IgG group (not shown). These results show that extracellular histones trigger intraglomerular TNF-a production and microthrombi formation within glomerular capillaries.
Example 10. Extracellular histones activate parietal epithelial cells via
Immunostaining displayed robust TNF-a positivity within the glomerular tuft, which not only localized in infiltrating cells but also in inner and outer aspect of the glomerular capillaries (not shown).
Anti-histone IgG strongly reduced glomerular TNF-a positivity, which was consistent with the corresponding renal mRNA expression levels (not shown). TNF-a is not only an inducer of NETosis but also triggers a prothrombotic activation of (glomerular) endothelial cells and intravascular fibrin formation (32-34). Our GN model displayed global fibrinogen positivity within glomerular capillaries, which was almost entirely prevented with anti-histone IgG (not shown). Also fibrinogen mRNA levels were reduced in the anti-histone IgG group (not shown). These results show that extracellular histones trigger intraglomerular TNF-a production and microthrombi formation within glomerular capillaries.
Example 10. Extracellular histones activate parietal epithelial cells via
[0269] Mitogenic plasma proteins leaking from injured glomerular capillaries cause PEC
hyperplasia and glomerular crescent formation (Ryu et al., 2012, J Pathol 228:482; Smeets et at., 2009, J Am Soc Nephrol 20:2593; Smeets et al., 2009, J Am Soc Nephrol 20:2604). In fact, in antiserum-induced GN glomerular crescents were positive for claudin-positive cells (not shown), where claudin-1 identifies PECs and WT-1 marks PEC
activation (Shankland et at., 2013, Curr Opin Nephrol Hypertens 22:302. PECs cultured in 10% serum started proliferating upon histone exposure (not shown). Having shown that TLR2 and -4 are upregulated in PECs during severe human GN, we questioned whether extracellular histones drive PEC activation in a TLR2/4-dependent manner.
hyperplasia and glomerular crescent formation (Ryu et al., 2012, J Pathol 228:482; Smeets et at., 2009, J Am Soc Nephrol 20:2593; Smeets et al., 2009, J Am Soc Nephrol 20:2604). In fact, in antiserum-induced GN glomerular crescents were positive for claudin-positive cells (not shown), where claudin-1 identifies PECs and WT-1 marks PEC
activation (Shankland et at., 2013, Curr Opin Nephrol Hypertens 22:302. PECs cultured in 10% serum started proliferating upon histone exposure (not shown). Having shown that TLR2 and -4 are upregulated in PECs during severe human GN, we questioned whether extracellular histones drive PEC activation in a TLR2/4-dependent manner.
[0270] The mitogenic effect of histones to serum exposed PECs was entirely blocked by TLR2/4 inhibition (not shown). TLR2/4 inhibition also blocked histone-induced expression of CD44 and WT-1 in PECs (not shown). Previous reports documented that heparin and recombinant activated protein C (aPC) also block histone toxicity (Xu et at., 2009, Nat Med 15:1318; Wildhagen et al., 2013, Blood 123:1098). As such, the protective effect on PEC
activation was shared by anti-histone IgG, heparin or activated protein C
(aPC) (not shown), the latter two suppressing histone cytotoxicity on glomerular endothelial cells just like anti-histone IgG (not shown). Thus, extracellular histones activate PECs in a TLR2/4-dependent manner, a process that may act synergistically with other triggers of PEC
hyperplasia during crescent formation and that can be blocked by anti-histone IgG, aPC or heparin.
Example 11. Delayed onset of histone neutralization still improves severe GN
activation was shared by anti-histone IgG, heparin or activated protein C
(aPC) (not shown), the latter two suppressing histone cytotoxicity on glomerular endothelial cells just like anti-histone IgG (not shown). Thus, extracellular histones activate PECs in a TLR2/4-dependent manner, a process that may act synergistically with other triggers of PEC
hyperplasia during crescent formation and that can be blocked by anti-histone IgG, aPC or heparin.
Example 11. Delayed onset of histone neutralization still improves severe GN
[0271] The results of pre-emptive histone neutralization proved their pathogenic contribution to severe GN. We mext examined whether histone neutralization could be therapeutic in established disease. Anti-histone IgG, heparin, and aPC all completely blocked histone toxicity on glomeruli ex vivo (not shown). In another series of experiments we initiated anti-histone IgG, heparin, and aPC treatments 24 hours after GBM antiserum injection, a time point where massive proteinuria and elevated BUN were already established (not shown). All these treatments consistently and significantly reduced plasma creatinine levels, proteinuria, and podocyte loss at day 7 (not shown). Histone blockade also significantly reduced the percentage of glomeruli with global lesions or halted damage (not shown).
Glomerular crescents were reduced by 80% (not shown) and so were features of secondary tubular injury (not shown). This was associated with less glomerular neutrophil and macrophage infiltrates as well as a significant reduction of intrarenal leukocyte subpopulations as well as their activation, as identified by flow cytometry (not shown). Thus, delayed onset of histone blockade with anti-histone IgG, heparin or aPC protects from renal dysfunction and structural injury during severe GN.
Example 12. Summary of effects of histone neutralization on necrosis in glomerulonephritis
Glomerular crescents were reduced by 80% (not shown) and so were features of secondary tubular injury (not shown). This was associated with less glomerular neutrophil and macrophage infiltrates as well as a significant reduction of intrarenal leukocyte subpopulations as well as their activation, as identified by flow cytometry (not shown). Thus, delayed onset of histone blockade with anti-histone IgG, heparin or aPC protects from renal dysfunction and structural injury during severe GN.
Example 12. Summary of effects of histone neutralization on necrosis in glomerulonephritis
[0272] We hypothesized that extracellular histones elicit toxic and immunostimulatory effects on glomerular cells during necrotizing and crescentic GN. The data reported in the Examples above confirm this concept and also demonstrate that histone neutralization continues to be protective when it commences after disease onset, which implies a potential therapeutic use of histone neutralizing agents in severe GN.
[0273] Necrotizing and crescentic GN, such as seen in ANCA-associated renal vasculitis or anti-GBM disease, is associated with neutrophil-induced glomerular injury.
First discovered in 2004, NETosis is a regulated form of neutrophil death that supports killing of extracellular bacteria (Brinkmann et al., 2004, Science 303:1532). NETosis is not limited to antibacterial host defence but also occurs in sterile forms of inflammation, because it can be triggered by pro-inflammatory cytokines such as TNF-a. Our in vitro studies show that TNF-a is a sufficient stimulus to trigger NETosis-driven injury of glomerular endothelial cells. NETosis releases many aggressive proteases, oxygen radicals, and potential DAMPs into the extracellular space that have the potential to drive vascular injury in the glomerulus.
First discovered in 2004, NETosis is a regulated form of neutrophil death that supports killing of extracellular bacteria (Brinkmann et al., 2004, Science 303:1532). NETosis is not limited to antibacterial host defence but also occurs in sterile forms of inflammation, because it can be triggered by pro-inflammatory cytokines such as TNF-a. Our in vitro studies show that TNF-a is a sufficient stimulus to trigger NETosis-driven injury of glomerular endothelial cells. NETosis releases many aggressive proteases, oxygen radicals, and potential DAMPs into the extracellular space that have the potential to drive vascular injury in the glomerulus.
[0274] Our data demonstrate an essential role of histones in this context. The endothelial toxicity of extracellular histones was first described in a seminal paper on sepsis, where early lethality was due to microvascular endothelial cell injury in the lung (Xu et at., 2009, Nat Med 15:1318). Subsequent reports further explored the thrombogenic potential of extracellular histones via direct activation of endothelial cells as well as of platelets (Abrams et at., 2013, Am J Respir Crit Care Med 187:160; Saffarzadeh et at., 2012, PLoS One 7:e32366; Semeraro et al., 2011, Blood 118:1952; Ammollo et al., 2011, J
Thromb Haemost 9:1795; Fuchs et al., 2011, Blood 118:3708;
Fuchs et al., 2010. Proc Natl Acad Sci USA 107:15880).
Thromb Haemost 9:1795; Fuchs et al., 2011, Blood 118:3708;
Fuchs et al., 2010. Proc Natl Acad Sci USA 107:15880).
[0275] In infection and sepsis models, NETosis is the most likely source of extracellular histones. However, in mechanical trauma, toxic liver injury, cerebral stroke, and post-ischemic renal tubular necrosis histones are also released from dying tissue cells (Allam et al., 2014, J Mot Med, 92:465; Allam et al., 2012, J Am Soc Nephrol 23:1375).
The source of extracellular histones in our in vivo model could be dying glomerular cells as well as netting neutrophils, which we identified by MPO staining in situ. Histone blockade had no effect on NETosis per se but rather worked on the related vascular injury inside the glomerulus.
The source of extracellular histones in our in vivo model could be dying glomerular cells as well as netting neutrophils, which we identified by MPO staining in situ. Histone blockade had no effect on NETosis per se but rather worked on the related vascular injury inside the glomerulus.
[0276] Our in vitro and in vivo data clearly demonstrate that extracellular histones are toxic to glomerular cells and promote glomerular injury in healthy mice upon intra-arterial injection or during severe antiserum-induced GN. The mechanisms of histone toxicity are not entirely clear but are thought to be due to their strong basic charge (Gillrie et al., 2012, Am J
Pathol 180:1028). While histones basic charge is needed inside the nucleus to neutralize acidic residues of the DNA, outside the cell, it has the capacity to damage cell membranes (Gillrie et al., 2012, Am J Pathol 180:1028). The polyanion heparin blocks this charge effect of histones, which may explain its antagonistic effect on histone toxicity in vitro and in vivo.
However, we and others discovered that histones elicit also DAMP-like activity by activating TLR2, TLR4, and NLRP3 (Semeraro et al., 2011, Blood 118:1952; Allam et al., 2012, J Am Soc Nephrol 23:1375; Allam et al., 2013, Eur J Immunol 43:3336; Huang et al., 2013,J
Immunol 191:2665; Xu et al., 2011, J Immunol 187:2626), which is another pathway of how extracellular histones trigger sterile inflammation.
Pathol 180:1028). While histones basic charge is needed inside the nucleus to neutralize acidic residues of the DNA, outside the cell, it has the capacity to damage cell membranes (Gillrie et al., 2012, Am J Pathol 180:1028). The polyanion heparin blocks this charge effect of histones, which may explain its antagonistic effect on histone toxicity in vitro and in vivo.
However, we and others discovered that histones elicit also DAMP-like activity by activating TLR2, TLR4, and NLRP3 (Semeraro et al., 2011, Blood 118:1952; Allam et al., 2012, J Am Soc Nephrol 23:1375; Allam et al., 2013, Eur J Immunol 43:3336; Huang et al., 2013,J
Immunol 191:2665; Xu et al., 2011, J Immunol 187:2626), which is another pathway of how extracellular histones trigger sterile inflammation.
[0277] Because TLR2 and TLR4 (but not NLRP3) are known to induce glomerular injury in the heterologous anti-GBM GN model (Brown et al., 2006, J Immunol 177:1925;
Brown et al., 2007, J Am Soc Nephrol 18:1732; Lichtnekert et al., 2011, PLoS One 6:e26778;
Lichtnekert et al., 2009, Am J Physiol Renal Physiol 296:F867), we further explored the histone-TLR2/4 axis. Tlr2/ 4-deficient glomeruli were protected from histone-induced injury ex vivo and in vivo, implying that the histone-related glomerular injury relates to the TLR2/4-dependent DAMP effect. In particular the presence of serum turned the cytotoxic effect of histones on PECs into PEC proliferation, which was entirely TLR2/4 dependent.
Although PEC necrosis can be followed by excessive PEC recovery leading to PEC
hyperplasia and crescent formation (Sicking et al., 2012, J Am Soc Nephrol 23:629), concomitant plasma leakage and histone release provide additional mitogenic stimuli during severe GN (Ryu et at., 2012, J Pathol 228:382).
Brown et al., 2007, J Am Soc Nephrol 18:1732; Lichtnekert et al., 2011, PLoS One 6:e26778;
Lichtnekert et al., 2009, Am J Physiol Renal Physiol 296:F867), we further explored the histone-TLR2/4 axis. Tlr2/ 4-deficient glomeruli were protected from histone-induced injury ex vivo and in vivo, implying that the histone-related glomerular injury relates to the TLR2/4-dependent DAMP effect. In particular the presence of serum turned the cytotoxic effect of histones on PECs into PEC proliferation, which was entirely TLR2/4 dependent.
Although PEC necrosis can be followed by excessive PEC recovery leading to PEC
hyperplasia and crescent formation (Sicking et al., 2012, J Am Soc Nephrol 23:629), concomitant plasma leakage and histone release provide additional mitogenic stimuli during severe GN (Ryu et at., 2012, J Pathol 228:382).
[0278] Our proof-of-concept experiments were based on pre-emptive histone neutralization with anti-histone IgG. To explore a potential efficacy of histone blockade in severe GN we also applied three different modes of histone inactivation following GN
induction. Delayed onset of anti-histone IgG was equally protective as pre-emptive therapy in terms of glomerular injury, proteinuria, and serum creatinine levels. The same applies to heparin treatment, which confirms previously published results in GN models (Floege et at., 1993, Kidney Int 43:369). Our data clearly show that heparin inhibits the direct toxic effects of histones on glomerular endothelial cells, which is consistent with the results of other investigators in other cell types (Hirsch, 1958, J Exp Med 108:925; Ammollo et al., J Thromb Haemost 9:1795; Fuchs et al., 2010, Proc Natl Acad Sci USA 107:15880). As previously reported aPC degrades extracellular histones (Xu et at., 2009, Nat Med 15:1318. In the current studies it was equally effective as anti-histone IgG and heparin in abrogating extracellular histone toxicity in vitro and severe GN in vivo.
induction. Delayed onset of anti-histone IgG was equally protective as pre-emptive therapy in terms of glomerular injury, proteinuria, and serum creatinine levels. The same applies to heparin treatment, which confirms previously published results in GN models (Floege et at., 1993, Kidney Int 43:369). Our data clearly show that heparin inhibits the direct toxic effects of histones on glomerular endothelial cells, which is consistent with the results of other investigators in other cell types (Hirsch, 1958, J Exp Med 108:925; Ammollo et al., J Thromb Haemost 9:1795; Fuchs et al., 2010, Proc Natl Acad Sci USA 107:15880). As previously reported aPC degrades extracellular histones (Xu et at., 2009, Nat Med 15:1318. In the current studies it was equally effective as anti-histone IgG and heparin in abrogating extracellular histone toxicity in vitro and severe GN in vivo.
[0279] Together, NETosis releases histones into the extracellular space where they have toxic effects on glomerular endothelial cells and podocytes. Extracellular histone-induced glomerular injury is partially due to TLR2/4. Pre-emptive as well as delayed onset of histone neutralization either by anti-histone IgG, recombinant aPC or heparin abrogates all aspects of GBM antiserum-induced severe GN. We conclude that extracellular histones represent a novel therapeutic target in severe GN.
[0280] One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the invention.
Claims (26)
1. A method of treating remote organ injury induced by acute kidney injury, comprising administering to a subject with acute kidney injury at least one anti-histone agent.
2. The method of claim 1, wherein the agent is selected from the group consisting of an anti-histone antibody or antigen-binding fragment thereof, activated protein C
(APC), thrombomodulin, a peptide fragment of human histone H1, H2A, H2B, H3 or H4, granzyme A, granzyme B, plasmin, Factor 7-activating protease, and heparin.
(APC), thrombomodulin, a peptide fragment of human histone H1, H2A, H2B, H3 or H4, granzyme A, granzyme B, plasmin, Factor 7-activating protease, and heparin.
3. The method of claim 1, wherein the agent is selected from the group consisting of an anti-histone antibody, activated protein C and heparin.
4. The method of claim 2, wherein the anti-histone antibody binds to a human histone selected from the group consisting of histone H1/H5, histone H2A, histone H2B, histone H3 and histone H4.
5. The method of claim 4, wherein the anti-histone antibody is an anti-histone H4 antibody.
6. The method of claim 4, wherein the anti-histone antibody is selected from the group consisting of BWA-3, LG2-1 and LG2-2.
7. The method of claim 1, wherein the anti-histone agent inhibits activity of toll-like receptor 2 (TLR-2) and TLR-4.
8. The method of claim 2, wherein administration of anti-histone antibody reduces post-ischemic tubular necrosis, renal dysfunction, NET formation and lung injury induced by AKI.
9. The method of claim 2, wherein the anti-histone antibody is a chimeric, humanized or human antibody.
10. The method of claim 2, wherein the antibody fragment is selected from the group consisting of F(ab)2, Fab', F(ab)2, Fab, Fv, sFv, scFv and single domain antibody (nanobody).
11. The method of claim 1, further comprising administering two or more anti-histone agents to the subject.
12. The method of claim 13, wherein the two or more agents are selected from the group consisting of an anti-histone antibody, activated protein C and heparin.
13. The method of claim 1, wherein the subject is a human subject.
14. The method of claim 1, wherein administration of the anti-histone agent is effective to prevent remote organ injury in subjects with acute kidney injury.
15. The method of claim 1, further comprising administering to the subject an anti-TNF-.alpha.
antibody.
antibody.
16. The method of claim 1, further comprising administering to the subject a PAD inhibitor.
17. The method of claim 1, further comprising administering to the subject an antibody against toll-like receptor 2 (TLR-2) or TLR-4.
18. The method of claim 2, wherein the anti-histone antibody or fragment thereof is not conjugated to a therapeutic agent.
19. The method of claim 2, wherein the anti-histone antibody or fragment thereof is conjugated to at least one therapeutic agent.
20. The method of claim 18, wherein the therapeutic agent is selected from the group consisting of a second antibody, a second antibody fragment, a radionuclide, an immunomodulator, an anti-angiogenic agent, a pro-apoptotic agent, a cytokine, a chemokine, a drug, a toxin, a hormone, an siRNA and an enzyme.
21. The method of claim 1, further comprising administering to the subject an immunomodulator selected from the group consisting of a cytokine, a stem cell growth factor, a lymphotoxin, a hematopoietic factor, a colony stimulating factor (CSF), an interleukin (IL), erythropoietin, thrombopoietin, tumor necrosis factor (TNF), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon-a, interferon-I3, interferon-y, interferon-k, TGF-a, TGF-13, interleukin-1 (IL-1), IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-23, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin and lymphotoxin.
22. The method of claim 21, wherein the cytokine is selected from the group consisting of human growth hormone, N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), luteinizing hormone (LH), hepatic growth factor, prostaglandin, fibroblast growth factor, prolactin, placental lactogen, OB protein, tumor necrosis factor-.alpha., tumor necrosis factor-.beta., mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor, integrin, thrombopoietin (TPO), NGF-.beta., platelet-growth factor, TGF-.alpha., TGF-.beta., insulin-like growth factor-I, insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors, interferon-.alpha., interferon-.beta., interferon-.gamma., macrophage-CSF
(M-CSF), IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and lymphotoxin.
(M-CSF), IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, IL-25, LIF, FLT-3, angiostatin, thrombospondin, endostatin, tumor necrosis factor and lymphotoxin.
23. The method of claim 2, further comprising administering to the subject a second antibody or antigen-binding fragment thereof, where the second antibody binds to an antigen selected from the group consisting of histone H2B, histone H3, histone H4, a proinflammatory effector of the innate immune system, a proinflammatory effector cytokine, a proinflammatory effector chemokine, TNF-.alpha., MIF, CD74, HLA-DR, IL-1, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, CD40L, CD44, CD46, CD55, CD59, CCL19, CCL21, mCRP, MCP-19, MIP-1A, MIP-1B, RANTES, ENA-78, IP-10, GRO-.beta., lipopolysaccharide, lymphotoxin, HMGB-1, tissue factor, a complement regulatory protein, a coagulation factor, thrombin, a complement factor, C3, C3a, C3b, C4a, C4b, C5, C5a, C5b, F1t-1 and VEGF.
24. The method of claim 2, wherein the antibody is a bispecific antibody comprising a first binding site for a histone and a second binding site for a non-histone antigen.
25. The method of claim 2, wherein the anti-histone antibody or fragment thereof is a fusion protein.
26. The method of claim 1, wherein the remote organ is lung or heart.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201662394529P | 2016-09-14 | 2016-09-14 | |
US62/394,529 | 2016-09-14 | ||
US15/402,585 US20170114126A1 (en) | 2014-06-24 | 2017-01-10 | Anti-histone therapy for vascular necrosis in severe glomerulonephritis |
US15/402,585 | 2017-01-10 | ||
PCT/US2017/051485 WO2018053080A1 (en) | 2016-09-14 | 2017-09-14 | Anti-histone therapy in acute kidney injury |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3031540A1 true CA3031540A1 (en) | 2018-03-22 |
Family
ID=61619282
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3031540A Pending CA3031540A1 (en) | 2016-09-14 | 2017-09-14 | Anti-histone therapy in acute kidney injury |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP3512552A4 (en) |
CA (1) | CA3031540A1 (en) |
WO (1) | WO2018053080A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2020225549A1 (en) * | 2019-05-06 | 2020-11-12 | The Francis Crick Institute Limited | Method for preventing inflammation |
CN113122538A (en) * | 2021-04-15 | 2021-07-16 | 遵义医科大学附属医院 | shRNA expressed by targeted knockdown Rip3 gene, recombinant vector and application thereof |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012166611A2 (en) * | 2011-05-27 | 2012-12-06 | Immune Disease Institute, Inc. | Methods for treating and preventing neutrophil-derived net toxicity and thrombosis |
CA2953567C (en) * | 2014-06-24 | 2023-09-05 | Immunomedics, Inc. | Anti-histone therapy for vascular necrosis in severe glomerulonephritis |
-
2017
- 2017-09-14 EP EP17851497.2A patent/EP3512552A4/en not_active Withdrawn
- 2017-09-14 CA CA3031540A patent/CA3031540A1/en active Pending
- 2017-09-14 WO PCT/US2017/051485 patent/WO2018053080A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
EP3512552A1 (en) | 2019-07-24 |
WO2018053080A1 (en) | 2018-03-22 |
EP3512552A4 (en) | 2020-05-06 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10160801B2 (en) | Nucleic acids encoding chimeric and humanized anti-histone antibodies | |
US10385139B2 (en) | Murine, chimeric, humanized or human anti-TNF-alpha antibodies | |
US20170088619A1 (en) | Antibody-Based Depletion of Antigen-Presenting Cells and Dendritic Cells | |
US8591892B2 (en) | FTY720 increases CD74 expression and sensitizes cancer cells to anti-CD74 antibody-mediated cell death | |
US10351621B2 (en) | Anti-histone therapy in acute kidney injury | |
US20170114126A1 (en) | Anti-histone therapy for vascular necrosis in severe glomerulonephritis | |
CA3031540A1 (en) | Anti-histone therapy in acute kidney injury | |
CHANG et al. | Patent 2794499 Summary |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20220912 |
|
EEER | Examination request |
Effective date: 20220912 |
|
EEER | Examination request |
Effective date: 20220912 |
|
EEER | Examination request |
Effective date: 20220912 |