CN108699121B - 用于抑制补体激活的多肽 - Google Patents
用于抑制补体激活的多肽 Download PDFInfo
- Publication number
- CN108699121B CN108699121B CN201680075659.0A CN201680075659A CN108699121B CN 108699121 B CN108699121 B CN 108699121B CN 201680075659 A CN201680075659 A CN 201680075659A CN 108699121 B CN108699121 B CN 108699121B
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- complement
- convertase
- mfhr1
- fhr1
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 75
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 71
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 71
- 230000002401 inhibitory effect Effects 0.000 title claims abstract description 39
- 230000024203 complement activation Effects 0.000 title description 35
- 238000006471 dimerization reaction Methods 0.000 claims abstract description 9
- 108010053085 Complement Factor H Proteins 0.000 claims description 69
- 102000016550 Complement Factor H Human genes 0.000 claims description 69
- 102100040132 Complement factor H-related protein 1 Human genes 0.000 claims description 41
- 101000890732 Homo sapiens Complement factor H-related protein 1 Proteins 0.000 claims description 41
- 210000004027 cell Anatomy 0.000 claims description 41
- 230000027455 binding Effects 0.000 claims description 31
- 208000029574 C3 glomerulopathy Diseases 0.000 claims description 29
- 208000027134 non-immunoglobulin-mediated membranoproliferative glomerulonephritis Diseases 0.000 claims description 29
- 230000015572 biosynthetic process Effects 0.000 claims description 25
- 208000035913 Atypical hemolytic uremic syndrome Diseases 0.000 claims description 24
- 208000000733 Paroxysmal Hemoglobinuria Diseases 0.000 claims description 15
- 102100036050 Phosphatidylinositol N-acetylglucosaminyltransferase subunit A Human genes 0.000 claims description 15
- 201000003045 paroxysmal nocturnal hemoglobinuria Diseases 0.000 claims description 15
- 238000011282 treatment Methods 0.000 claims description 14
- 206010064930 age-related macular degeneration Diseases 0.000 claims description 12
- 208000002780 macular degeneration Diseases 0.000 claims description 12
- 101100365087 Arabidopsis thaliana SCRA gene Proteins 0.000 claims description 11
- 101150105073 SCR1 gene Proteins 0.000 claims description 11
- 101100134054 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NTG1 gene Proteins 0.000 claims description 11
- 108020004707 nucleic acids Proteins 0.000 claims description 10
- 102000039446 nucleic acids Human genes 0.000 claims description 10
- 150000007523 nucleic acids Chemical class 0.000 claims description 10
- 208000034841 Thrombotic Microangiopathies Diseases 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 8
- 239000013598 vector Substances 0.000 claims description 7
- 101000668165 Homo sapiens RNA-binding motif, single-stranded-interacting protein 1 Proteins 0.000 claims description 6
- 102100039692 RNA-binding motif, single-stranded-interacting protein 1 Human genes 0.000 claims description 6
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 6
- 208000035473 Communicable disease Diseases 0.000 claims description 5
- 206010052779 Transplant rejections Diseases 0.000 claims description 5
- 208000014674 injury Diseases 0.000 claims description 5
- 208000028867 ischemia Diseases 0.000 claims description 5
- 201000008383 nephritis Diseases 0.000 claims description 5
- 239000013612 plasmid Substances 0.000 claims description 5
- 208000010159 IgA glomerulonephritis Diseases 0.000 claims description 4
- 206010021263 IgA nephropathy Diseases 0.000 claims description 4
- 206010040047 Sepsis Diseases 0.000 claims description 4
- 208000015181 infectious disease Diseases 0.000 claims description 4
- 206010063837 Reperfusion injury Diseases 0.000 claims description 3
- 206010051379 Systemic Inflammatory Response Syndrome Diseases 0.000 claims description 3
- 239000003814 drug Substances 0.000 claims description 3
- 208000010125 myocardial infarction Diseases 0.000 claims description 3
- 230000008736 traumatic injury Effects 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 2
- 108010071253 factor H-related protein 1 Proteins 0.000 claims description 2
- 238000011321 prophylaxis Methods 0.000 claims description 2
- 108010067641 Complement C3-C5 Convertases Proteins 0.000 abstract description 78
- 102000016574 Complement C3-C5 Convertases Human genes 0.000 abstract description 78
- 239000012636 effector Substances 0.000 abstract description 46
- 230000001225 therapeutic effect Effects 0.000 abstract description 9
- 102100022133 Complement C3 Human genes 0.000 description 42
- 101000901154 Homo sapiens Complement C3 Proteins 0.000 description 42
- 230000000295 complement effect Effects 0.000 description 31
- 210000002966 serum Anatomy 0.000 description 25
- 230000000694 effects Effects 0.000 description 24
- 230000004913 activation Effects 0.000 description 23
- 108090000623 proteins and genes Proteins 0.000 description 23
- 108010034753 Complement Membrane Attack Complex Proteins 0.000 description 21
- 125000003275 alpha amino acid group Chemical group 0.000 description 21
- 102000004169 proteins and genes Human genes 0.000 description 20
- 238000003776 cleavage reaction Methods 0.000 description 19
- 230000007017 scission Effects 0.000 description 19
- 229960002224 eculizumab Drugs 0.000 description 18
- 102100035326 Complement factor H-related protein 2 Human genes 0.000 description 15
- 101000878135 Homo sapiens Complement factor H-related protein 2 Proteins 0.000 description 15
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 15
- 108090000056 Complement factor B Proteins 0.000 description 14
- 230000005764 inhibitory process Effects 0.000 description 14
- 102100035325 Complement factor H-related protein 5 Human genes 0.000 description 13
- 101000878134 Homo sapiens Complement factor H-related protein 5 Proteins 0.000 description 13
- 150000001413 amino acids Chemical class 0.000 description 13
- 230000001105 regulatory effect Effects 0.000 description 13
- 230000001404 mediated effect Effects 0.000 description 12
- 230000037361 pathway Effects 0.000 description 12
- 210000002381 plasma Anatomy 0.000 description 12
- 230000008021 deposition Effects 0.000 description 11
- 238000000151 deposition Methods 0.000 description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- 102000003712 Complement factor B Human genes 0.000 description 10
- 206010018910 Haemolysis Diseases 0.000 description 10
- 229940098773 bovine serum albumin Drugs 0.000 description 10
- 230000008588 hemolysis Effects 0.000 description 10
- 238000003556 assay Methods 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 238000002965 ELISA Methods 0.000 description 8
- 230000021917 activation of membrane attack complex Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 6
- 239000000306 component Substances 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 6
- 230000001434 glomerular Effects 0.000 description 6
- 244000052769 pathogen Species 0.000 description 6
- 230000002829 reductive effect Effects 0.000 description 6
- 108010089414 Anaphylatoxins Proteins 0.000 description 5
- 238000013459 approach Methods 0.000 description 5
- 230000000903 blocking effect Effects 0.000 description 5
- 230000004154 complement system Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 239000008194 pharmaceutical composition Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 108090000955 Complement C2 Proteins 0.000 description 4
- 102100034622 Complement factor B Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 241001494479 Pecora Species 0.000 description 4
- 230000001133 acceleration Effects 0.000 description 4
- 210000004899 c-terminal region Anatomy 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 239000003112 inhibitor Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000001573 invertase Substances 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 210000004379 membrane Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000001742 protein purification Methods 0.000 description 4
- 238000001542 size-exclusion chromatography Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 101710172562 Cobra venom factor Proteins 0.000 description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 230000003831 deregulation Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000008482 dysregulation Effects 0.000 description 3
- 210000000585 glomerular basement membrane Anatomy 0.000 description 3
- 238000007912 intraperitoneal administration Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 229910052709 silver Inorganic materials 0.000 description 3
- 239000004332 silver Substances 0.000 description 3
- 230000000451 tissue damage Effects 0.000 description 3
- 231100000827 tissue damage Toxicity 0.000 description 3
- RDTRHBCZFDCUPW-KWICJJCGSA-N 2-[(4r,7s,10s,13s,19s,22s,25s,28s,31s,34r)-4-[[(2s,3r)-1-amino-3-hydroxy-1-oxobutan-2-yl]carbamoyl]-34-[[(2s,3s)-2-amino-3-methylpentanoyl]amino]-25-(3-amino-3-oxopropyl)-7-[3-(diaminomethylideneamino)propyl]-10,13-bis(1h-imidazol-5-ylmethyl)-19-(1h-indol Chemical compound C([C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CSSC[C@@H](C(N[C@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)NCC(=O)N[C@@H](CC=2NC=NC=2)C(=O)N1)C(C)C)C(C)C)=O)NC(=O)[C@@H](N)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(N)=O)C1=CN=CN1 RDTRHBCZFDCUPW-KWICJJCGSA-N 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 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 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 101710121996 Hexon protein p72 Proteins 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 108010052285 Membrane Proteins Proteins 0.000 description 2
- 102100039373 Membrane cofactor protein Human genes 0.000 description 2
- 208000001647 Renal Insufficiency Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 108010051210 beta-Fructofuranosidase Proteins 0.000 description 2
- 230000015861 cell surface binding Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 108010074405 complement C5b-6 complex Proteins 0.000 description 2
- 230000009918 complex formation Effects 0.000 description 2
- 108010027437 compstatin Proteins 0.000 description 2
- 230000002596 correlated effect Effects 0.000 description 2
- 230000009089 cytolysis Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000004023 fresh frozen plasma Substances 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 238000007913 intrathecal administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 235000011073 invertase Nutrition 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 201000006370 kidney failure Diseases 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000001543 one-way ANOVA Methods 0.000 description 2
- 238000007911 parenteral administration Methods 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 238000011533 pre-incubation Methods 0.000 description 2
- 230000009711 regulatory function Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 206010039073 rheumatoid arthritis Diseases 0.000 description 2
- 238000005063 solubilization Methods 0.000 description 2
- 230000007928 solubilization Effects 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 238000011269 treatment regimen Methods 0.000 description 2
- 230000001960 triggered effect Effects 0.000 description 2
- 239000013638 trimer Substances 0.000 description 2
- YTPUIQCGRWDPTM-UHFFFAOYSA-N 2-acetyloxybenzoic acid;5-(2-methylpropyl)-5-prop-2-enyl-1,3-diazinane-2,4,6-trione;1,3,7-trimethylpurine-2,6-dione Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O.CN1C(=O)N(C)C(=O)C2=C1N=CN2C.CC(C)CC1(CC=C)C(=O)NC(=O)NC1=O YTPUIQCGRWDPTM-UHFFFAOYSA-N 0.000 description 1
- 239000011475 Accrington brick Substances 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 108010003529 Alternative Pathway Complement C3 Convertase Proteins 0.000 description 1
- 244000105975 Antidesma platyphyllum Species 0.000 description 1
- 101100390700 Arabidopsis thaliana FH20 gene Proteins 0.000 description 1
- 108010009575 CD55 Antigens Proteins 0.000 description 1
- 102100022002 CD59 glycoprotein Human genes 0.000 description 1
- 208000031229 Cardiomyopathies Diseases 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- 102000003780 Clusterin Human genes 0.000 description 1
- 108090000197 Clusterin Proteins 0.000 description 1
- 108010078015 Complement C3b Proteins 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 102100035321 Complement factor H-related protein 3 Human genes 0.000 description 1
- 102100035324 Complement factor H-related protein 4 Human genes 0.000 description 1
- 229940124073 Complement inhibitor Drugs 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- 102000010911 Enzyme Precursors Human genes 0.000 description 1
- 108010062466 Enzyme Precursors Proteins 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101100440311 Homo sapiens C5 gene Proteins 0.000 description 1
- 101000897400 Homo sapiens CD59 glycoprotein Proteins 0.000 description 1
- 101000878136 Homo sapiens Complement factor H-related protein 3 Proteins 0.000 description 1
- 101000878133 Homo sapiens Complement factor H-related protein 4 Proteins 0.000 description 1
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 102000050019 Membrane Cofactor Human genes 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 101710146216 Membrane cofactor protein Proteins 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 208000034762 Meningococcal Infections Diseases 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 208000031662 Noncommunicable disease Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000195888 Physcomitrella Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 241000206608 Pyropia tenera Species 0.000 description 1
- 101150039984 RCA gene Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 125000000837 carbohydrate group Chemical group 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 208000037976 chronic inflammation Diseases 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 239000004074 complement inhibitor Substances 0.000 description 1
- 102000006834 complement receptors Human genes 0.000 description 1
- 108010047295 complement receptors Proteins 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000008358 core component Substances 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000000326 densiometry Methods 0.000 description 1
- 230000002074 deregulated effect Effects 0.000 description 1
- 230000001687 destabilization Effects 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 230000005750 disease progression Effects 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000002222 downregulating effect Effects 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 210000001707 glomerular endothelial cell Anatomy 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 235000009424 haa Nutrition 0.000 description 1
- 238000001631 haemodialysis Methods 0.000 description 1
- 230000000322 hemodialysis Effects 0.000 description 1
- 238000011102 hetero oligomerization reaction Methods 0.000 description 1
- 210000003917 human chromosome Anatomy 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 239000012642 immune effector Substances 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 229940121354 immunomodulator Drugs 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007917 intracranial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007919 intrasynovial administration Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 238000011862 kidney biopsy Methods 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 231100000255 pathogenic effect Toxicity 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000004963 pathophysiological condition Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 238000002616 plasmapheresis Methods 0.000 description 1
- 102000054765 polymorphisms of proteins Human genes 0.000 description 1
- 229940071643 prefilled syringe Drugs 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000028646 regulation of complement activation Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000002626 targeted therapy Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126622 therapeutic monoclonal antibody Drugs 0.000 description 1
- 150000007970 thio esters Chemical class 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/66—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid the modifying agent being a pre-targeting system involving a peptide or protein for targeting specific cells
-
- 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
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Transplantation (AREA)
- Diabetes (AREA)
- Dermatology (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Ophthalmology & Optometry (AREA)
- Hematology (AREA)
- Vascular Medicine (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明涉及包含C3转化酶效应结构域、C5转化酶效应结构域和任选的末端复合抑制性效应结构域的多肽及其治疗应用,所述多肽对由生理FHR‑蛋白引起的失调具有抗性并具有二聚化基序。
Description
发明背景
补体系统是天然免疫的组成部分,并且有助于识别并消除病原体,清除凋亡细胞或免疫复合物,以及调节适应性免疫应答(Ricklin等人,2010,Carroll等人,2011)。补体由多种血浆蛋白(主要由肝脏产生)构成,通常以酶原或膜蛋白形式循环并在血浆、组织或细胞内起作用。三种补体途径,经典途径(CP)、凝集素途径(LP)或旁路途径(AP)的激活,各自导致C3转化酶(在CP、LP中为C4b2a,或者在AP中为C3bBb)的形成,该酶催化补体系统的核心组分C3裂解成激活产物C3b和过敏肽C3a。随后并且在级联状触发的反应中,标记病原体以将其破坏,并且诱导一系列炎症响应,招募免疫细胞对抗感染并保持稳态(参见(Merle等人,2015))。补体的无效激活或无效调控均可引起或促进多种感染或非传染性疾病,包括免疫缺陷、自身免疫、慢性炎症、血栓性微血管病、移植排斥以及肾和视网膜疾病,诸如非典型溶血尿毒症综合征(aHUS)、C3肾小球病(C3G)和年龄相关性黄斑变性(AMD) (Holers,2008)。
旁路途径(AP)
虽然CP和LP由某些识别分子触发,但AP以低水平永久激活。这通过称为“tick-over”的机制发生,其由C3b样分子C3(H2O)的内部C3硫酯的水解引发,形成其生物活性形式。与裂解C3(H20)结合的FB的可溶性因子B(FB)和因子D(FD)一起,液相C3转化酶复合物(C3b(H20)Bb)生成,并且天然C3分子被裂解和激活。激活的C3b经由包含硫酯的结构域(TED或C3d结构域)共价结合任何邻近表面的羟基。在病原体上,紧邻其产生位置的C3b积聚并且另外的C3转化酶(C3bBb)形成,从而放大补体激活。第二C3b与C3转化酶的结合导致C5转化酶(C3bBbC3b)的产生,其引发C5裂解成有效的免疫效应分子C5a和C5b。C5b募集补体组分C6、C7、C8和C9,形成C5b-9末端膜攻击复合物(MAC),其导致病原体的溶解(Ricklin等人,2010,Carroll等人,2011)。
AP的调节
必须精确调控AP以允许快速消除调理的细胞和病原体,以及最小化可能引起宿主组织损伤的无限制的AP激活。健康的宿主细胞通常受到补体激活(RCA)家族的调节剂的多个膜结合蛋白或可溶性蛋白的保护而免于补体介导的攻击,所述蛋白作用于级联的不同激活水平,其中一些具有重叠功能,而其它蛋白具有独特的补体调控特性(Zipfel等人,2009)。
新C3b的产生受到用作用于因子I (FI)介导的C3b向iC3b的不可逆降解的辅因子的RCA蛋白的严格控制,或者受到C3bBb转化酶复合物的失稳的严格控制(衰变加速活性,DAA)。此外,RCA能够干扰C5转化酶活性,从而控制C5裂解成它的激活产物C5a和C5b,或者通过结合末端补体复合物(TCC)化合物的因子,从而阻止MAC复合物插入膜中。与膜辅因子蛋白(MCP或CD46)、补体受体1 (CR1或CD35)、活性溶解的衰变加速因子(DAF或CD55)膜抑制物(MIRL,CD59)和可溶性因子玻璃粘连蛋白与簇集蛋白及其它蛋白一起,因子H/FHR蛋白家族的成员(尤其是FH)支持循环中和补体特异性结合的表面上的补体调控。
因子H/FHR蛋白家族
因子H/FHR蛋白家族包括一组高度相关的血浆蛋白,它们包括五种补体因子H相关蛋白(FHR),FHR1、FHR2、FHR3、FHR4、FHR5,因子H (FH)和剪切变体因子H-样蛋白1 (FHL-1)。家族成员的每个单个基因位于人染色体1q32上的不同片段中,在RCA基因簇内(Skerka等人,2013)。尽管FH是旁路途径的主要调节剂,但FHR的功能尚未完全了解。已经提出FHR蛋白的相互作用调节细胞表面上的补体调控活性(Jozsi等人,2015)。此外,它们用于同源寡聚化和异源寡聚化的能力可提高FHR1、FHR2和FHR5对于它们的配体的亲合力。这已经作为补体激活的识别和调控中的微调样机制而被提出(Jozsi等人,2015)。然而,还已经针对FHR1和FHR2描述了一些FH非依赖性和特有的补体调控特性。因为FH、FHR1和FHR2能够下调补体激活,它们已被提出作为用于在病理条件下调节补体激活的有希望的候选(Licht等人,2005,Licht等人,2006,Skerka等人,2013,Haffner等人,2015)。
在FH/FHR蛋白家族中,FH是最丰富的补体蛋白,它以~350–600µg/ml的浓度在血浆中循环。具有155kDa的分子量,单体糖蛋白FH调控AP和补体途径的放大环路。FH由20个重复的短同源重复序列(SCR)结构域组成并调控液相中及细胞表面上的C3转化酶激活。N-末端结构域SCR1-4包含该蛋白的补体调控区。通过与FB竞争结合C3b,FH SCR1-4结合C3b并因此阻止C3和C5转化酶的形成,并且有利于已经形成的转化酶的分解(Weiler等人,1976)。另外,这些结构域与FH相关,用作FI介导的C3b失活的辅因子(Gordon等人,1995,Rodriguezde Cordoba等人,2004,Alexander等人,2007,de Cordoba等人,2008)。FH的C-末端(SCR19-20)主要构成与C3b、C3d、正五聚蛋白、细胞外基质和细胞表面相互作用的结合识别结构域(Jarva等人,1999,Oppermann等人,2006,Hebecker等人,2013)。FH结合到细胞表面或生物膜上受到聚阴离子结构样糖胺聚糖(GAG)(例如肝素)或唾液酸的介导,并且调控内源细胞,诸如肾小球内皮细胞或肾小球基底膜(GBM)上的局部补体激活(Jozsi等人,2004,Ferreira等人,2006,Jozsi等人,2007,Blaum等人,2015)。
FHR1由五个SCR构成(Skerka等人,1991)并具有两种同工型。具有一个或两个碳水化合物侧链的两种糖基化形式(FHR1α ~41和FHR1β ~37kDa)在人血浆中循环,浓度为约100µg/ml。FHR1与FH具有高C-末端序列同源性,并且C-末端SCR1和SCR2与FHR2的SCR1和SCR2具有高氨基酸同一性(分别是97%和100%),以及与FHR5的SCR1和SCR2具有高氨基酸同一性(分别是91%和83%)。FHR1调控C5-转化酶活性并抑制补体激活,但是C3-转化酶活性不受影响。N-末端SCR1-2结合C5和C5b6,而C-末端SCR3-5结合C3b、C3d和肝素。据推测,FHR通过SCR1-2与C5的结合抑制C5激活和裂解成C5a和C5b。此外,FHR1是末端途径调节剂,并且据推测通过SCR1-2与C5b6复合物的结合抑制MAC的组装(Heinen等人,2009)。
FHR2由四个SCR组成(Skerka等人,1992),表现出与FH的SCR 6-7和19-20的氨基酸同一性。FHR2的N-末端SCR1几乎与FHR1和FHR5相同,并且允许与FHR1形成同源二聚体和异源二聚体,但是与FHR5不会形成同源二聚体和异源二聚体。FHR2以约50µg/ml的浓度在人血浆中循环。据推测经由其中FHR2结合的C3转化酶不裂解底物C3的机制,FHR2调控补体激活。令人感兴趣的是,FHR2几乎不与因子H竞争C3b。在生理浓度下,FHR2不与FH竞争结合C3b(Goicoechea de Jorge等人,2013)。
FHR1样FHR2和FHR5各自包含由位于SCR1中的残基Tyr34、Ser36和Tyr39形成的保守二聚界面,在组装中起到关键作用并有利于同源二聚体和异源二聚体的形成,其通过N-末端的牢固反向平行结合来传递。除它的调控功能之外,FHR还在某些条件下阻止FH结合(或与FH竞争) C3b或宿主/病原体细胞表面,引起补体的失调(激活)(Jozsi等人,2015)。多聚FHR复合物的形成可提高局部浓度,从而提高将由FHR调控的与其底物或与表面的亲合力和/或亲和力。已经提出,在病理生理条件下,失调可被加强,例如由于C3肾小球病中FHR的异常多聚化导致,其增强配体结合和FH竞争(Goicoechea de Jorge等人,2013)。
AP相关的疾病
突变引起的AP失调、补体组分和调节剂(如FH或促进AP激活的抗体)中的功能异常多态性与以下疾病高度相关:诸如非典型溶血尿毒症综合征(aHUS)(Noris等人,2009)或C3肾小球病(C3G)(Barbour等人,2013)年龄相关性黄斑变性(AMD)(Kawa等人,2014)或阵发性夜间血红蛋白尿(PNH)(Holers,2008)。除这些之外,还对于IgA-肾病(Maillard等人,2015)、系统性红斑狼疮(SLE)(Wilson等人,1976),局部缺血-再灌注(IR)损伤或移植排斥、类风湿性关节炎(RA)以及多种其它疾病(Holers,2008)(Ricklin等人,2013)显示或推测AP的病原性作用。
非典型HUS和C3G是AP相关疾病的主模型。在aHUS中,AP组分或其调节剂(C3、FB、FI、FH、FHR1或MCP)或抗-FH抗体的突变导致不受控的补体激活和最终的C5b-9形成以及内皮细胞损伤。这伴随着肾小球血栓性微血管病和急性肾衰竭,它们过去导致超过60%的患者死亡或最终肾衰竭。在十年内在超过50%的患者中发展成最终肾衰竭的C3G中,发现补体沉积在肾小球基底膜中或膜上。C3G中的AP激活也由补体基因的突变引起,尤其是FH的突变,或者由影响C3转化酶的自身抗体(C3肾炎因子)引起(Loirat等人,2011,Sethi等人,2012)。
补体相关疾病的治疗选项
在aHUS或C3G的治疗中建立的治疗选项是有限的,并且包括血浆去除术或用新鲜冷冻血浆(FFP)置换、免疫抑制治疗和肾移植,它们具有潜在疾病的高复发风险(Braun等人,2005,Lu等人,2012,Cataland等人,2014,Masani等人,2014)。目前,正在研究多种补体靶向疗法,并且未来可能提供治疗选项(Wagner等人,2010,Ricklin等人,2013)。其中,依库珠单抗(eculizumab),一种人源单克隆抗-C5抗体,阻断TCC形成并且近来已被批准用于治疗aHUS (Zimmerhackl等人,2010)。对于C3G,尚未建立治疗方案(Masani等人,2014)。依库珠单抗用于C3G患者仅导致某些患者的部分响应(Bomback等人,2012)。
如果防止C5转化酶裂解C5,则能够阻断补体的晚期效应功能。治疗性单克隆抗体依库珠单抗结合人C5并抑制其由C5转化酶活化成有效的过敏毒素C5a和末端补体途径的引发物C5b (Parker等人,2007)。因此,依库珠单抗阻断由MAC形成导致的炎性信号转导和细胞溶解,但是留下C3转化酶和不受控的C3a生成不受影响。依库珠单抗显示临床结果的显著改善并已被接受用于治疗补体介导的疾病,包括阵发性夜间血红蛋白尿(PNH)(Hillmen等人,2006)和aHUS(Zuber等人,2012)。
在补体级联中C3的中心位置使其成为用于治疗干预的有吸引力的目标,因此已经设计了在C3水平起作用的抑制剂。compstatin是一种13个氨基酸的小肽,它正在进行临床前测试(Mastellos等人,2015)。结构研究已经表明compstatin结合C3的β-链和C3b并阻断与C3转化酶的相互作用。因此,compstatin抑制C3的激活并进一步抑制级联放大,并且阻止补体效应物的下游形成。compstatin不阻止由蛋白酶如凝血酶导致的C3裂解,也不阻止C3经由“Tick-Over”“激活”成C3(H2O)(Ricklin等人,2015),并且已经在体外及动物模型中(包括体外循环、败血病和PNH)显示补体阻断功效。另一种方法是利用一组天然的RCA,例如FH或可溶性CR1,或者通过组合选择的结构域模块改善它们的功效(Ricklin等人,2013)。设计包含FH SCR1–5和CR2 SCR1–4的TT30以在已经遭受补体介导的攻击的位点处优先积聚。TT30同时与C3b和C3d相互作用,显示液相FH结合C3b的功能以及宿主细胞表面上CR2与C3d的相互作用。在AMD、局部缺血/再灌注损伤和PNH模型中,TT30显示显著的改善(Merle等人,2015)。包含SCR1–4或SCR 1-5和SCR19–20的Mini-FH分子以高亲和力结合C3b和C3d,并且在aHUS和PNH的体外模型中显示比天然FH更好的功效(Hebecker等人,2013,Schmidt等人,2013)。然而,在显示C3G样表型的FH缺陷型小鼠中,mini-FH和TT30的鼠类似物对血浆旁路途径控制的治疗效果是比较适度的,结合有短的半衰期(Nichols等人,2015,Ruseva等人,2015)。
批准依库珠单抗用于aHUS和PNH。它非特异性地阻断C5转化酶裂解C5。接受依库珠单抗治疗的患者具有发展出严重传染病的风险,尤其是脑膜炎球菌感染如脑膜炎。另一方面,TCC形成的阻断不影响C3转化酶激活。这可能导致受治疗患者持续产生过敏毒素C3a以及C3裂解产物在肾中的连续沉积,这可见于接受依库珠单抗治疗的C3G患者。另外,依库珠单抗抗体的沉积可见于接受依库珠单抗治疗的患者的肾活组织检查(Herlitz等人,2012)。直到目前,未研究过这些沉积的长期效应。
在所有AP相关疾病中,仅阻断C5转化酶可能也是不够的。对于依库珠单抗对C3G患者的治疗效果的单病例报告仅在一些患者中显示益处,大多数是部分减轻(Bomback等人,2012,Legendre等人,2013)。
新的补体调节剂,例如compstatin、mini-FH或TT30,试图在C3转化酶水平上影响补体激活。这些开发处于临床前阶段的研究中。依库珠单抗和目前处于开发阶段的其它调节剂靶向C5转化酶或C3转化酶。
发明内容
本专利申请的发明人发现通过同时作用于补体级联中的多个效应位点(抑制C3/C5-转化酶、抑制C5裂解和TCC形成),能够有效地阻断补体激活。这导致对AP(及CP)活性的更有效的调控,允许在AP(CP)下调和阻止过敏毒素释放之间微调,从而可能减少非期望的副作用。
此外,本发明人发现在调节剂内由选择的结构域模块形成的二聚化基序,1)通过形成多聚复合物提高调控活性,以及2)对FHR介导的失调具有抗性。
因此,本发明涉及以下实施方案(1)至(24)
(1) 一种多肽,所述多肽包含抑制性C3转化酶效应结构域和抑制性C5转化酶(C5结合)效应结构域。
(2) 根据项1所述的多肽,其中所述抑制性C3转化酶效应结构域通过衰变加速和辅因子活性抑制C3转化酶。
(3) 根据项1或2所述的多肽,其中所述多肽具有衰变加速和辅因子活性。
(4) 根据项1至3中任一项所述的多肽,其中所述抑制性C3转化酶效应结构域是因子H (FH)的片段。
(5) 根据前述项中任一项所述的多肽,其中所述抑制性C3转化酶效应结构域包含FH的短同源重复序列(SCR)1至4或由它们组成。
(6) 根据项1至3中任一项所述的多肽,其中所述抑制性C3转化酶效应结构域包含FHR2的SCR 1-4或由它们组成。
(7) 根据前述项中任一项所述的多肽,其中所述抑制性C5转化酶效应结构域是因子H相关蛋白1(FHR1)的片段。
(8) 根据前述项中任一项所述的多肽,其中所述抑制性C5转化酶效应结构域包含FHR1的SCR1和SCR2或由它们组成。
(9) 根据前述项中任一项所述的多肽,其中C5激活并裂解成C5a和C5b受到所述多肽与C5结合的抑制。
(10) 根据前述项中任一项所述的多肽,所述多肽还包含能够结合细胞表面的结构域。
(11) 根据前述项中任一项所述的多肽,其中所述能够结合细胞表面的结构域包含FH的SCR19和SCR20。
(12) 根据前述项中任一项所述的多肽,其中所述多肽是多聚体。
(13) 根据项12所述的多肽,所述多肽包含来自FHR1的SCR1的至少一个二聚化基序。
(14) 根据前述项中任一项所述的多肽,其中所述多肽能够抑制TCC(C5b-9)形成。
(15) 根据项14所述的多肽,其中所述TCC形成受到所述多肽与C5b-6结合的抑制。
(16) 具有结构A-B-C的多肽,其中A是根据前述项中任一项所定义的抑制性C5转化酶效应结构域,B是根据前述项中任一项所定义的抑制性C3转化酶效应结构域,并且C不存在或者是根据前述项中任一项所定义的能够结合细胞表面的结构域。
(17) 根据项16所述的多肽,其中A和B直接融合或者经由接头融合。
(18) 根据项16或17所述的多肽,其中B和C直接融合或者经由接头融合。
(19) 根据前述项中任一项所定义的多肽,用于治疗或预防与补体系统相关或关联的病症。
(20) 根据项19使用的多肽,其中所述与补体系统相关或关联的病症选自非典型溶血尿毒症综合征(aHUS)、血栓性微血管病(TMA)、C3肾小球病(C3G)、IgA肾病、系统性红斑狼疮肾炎、移植排斥、阵发性夜间血红蛋白尿(PNH)、年龄相关性黄斑变性(AMD)、传染病、败血病、SIRS、创伤性损伤、局部缺血/再灌注损伤以及心肌梗塞。
(21) 一种核酸,所述核酸编码根据项1至18中任一项所述的多肽。
(22) 一种质粒或载体,所述质粒或载体包含根据项21所述的核酸。
(23) 一种细胞,所述细胞包含根据项21所述的核酸或根据项22所述的质粒或载体。
(24) 一种制备根据项1至18中任一项所述的多肽的方法,所述方法包括在允许表达所述多肽的条件下,在培养基中培养根据项23所述的细胞,和从所述细胞或所述培养基回收所述多肽。
附图简述
图1:MFHR1的结构和表征。A) MFHR1的结构装配。MFHR1是一种融合蛋白,包含FHR1的SCR1-2 (FHR1 1-2),其N-末端连接hFH的SCR1-4和SCR19-20,分别称为FH1-4和FH19-20。五-组氨酸标签(N-末端)用作纯化工具。FHR1-2包含二聚化基序,抑制C5转化酶活性和MAC装配。FH具有衰变加速和辅因子活性(FH1-4)以及C3b和细胞表面的结合位点(FH19-20)。B)经由Ni-亲和色谱和尺寸排阻色谱,从杆状病毒感染的SF9细胞的上清液中纯化的MFHR1的SDS-PAGE和考马斯(左)或银(右)染色。MFHR1在还原条件下表现出58.65kDa的计算分子量。MFHR1在非还原条件下更快的移动性(考马斯染色,右道)指示二硫键的存在。C) 利用多克隆抗-人FH、抗-FH1-4或单克隆抗-C18和抗-FHR1-2抗体的免疫检测指示FHR1-2、FH1-4和FH20在重组MFHR1(I.)中的正确完整性。重组FH1-4;19-20(II.)、全长FHR1(III.)、FH1-4(IV.)或人血浆来源的hFH(V.)用作对照。
图2:MFHR1结合C3b,展示辅因子活性并解离C3转化酶。A) 通过ELISA分析MFHR1与C3b的相互作用。将C3b固定在微量滴定板上并加入连续稀释的MFHR1或hFH,并且利用抗-FH一抗和HRP-标记的抗-山羊二抗检测结合。经BSA涂覆的孔用作对照。数据为来自n=3次实验的平均值 ±SD。hFH与C3b的最大结合设为100%相对结合。B) MFHR1有效地解离C3bBb(C3转化酶)复合物。转化酶在C3b、因子B(FB)和因子D(FD)的存在下,在微量滴定板上装配,并且加入hFH或MFHR1并在37℃下孵育。完整的C3bBb以及这些复合物的解离通过FB的相对量进行测量。对照孔(C3b+FB+FD,无调节剂)的OD450值设为100%。不加入FD制备阴性对照。数据为来自n=3次实验的平均值 ±SD。C) MFHR1显示辅因子活性。通过在37℃下用递增浓度的MFHR1或hFH(5-250nM)孵育C3b和因子I (FI) 30分钟,进行辅因子测试。使用SDS-PAGE和考马斯蓝染色可视化α-链裂解和α`68、α`43和α`46片段。D) 为了定量辅因子活性,通过密度测定法测定C3b α-链的条带强度,并且完整的C3b α-链对β-链进行归一化并设为100%。数据表示为来自n=5次实验的平均值 ±SD。
图3:MFHR1结合C5并通过抑制C5转化酶/C5裂解和MAC形成来调控末端途径激活。A) MFHR1结合C5,这通过ELISA来测定。将等摩尔量的MFHR1、全长FHR1、hFH或BSA(133nM)固定在Nunc板上并与递增浓度(10、20、40µg/ml)的C5一起孵育。结合利用单克隆C5抗体和HRP-标记的二抗进行检测。数据为来自n=3次实验的平均值 ±SD。B) MFHR1通过阻断C5裂解来抑制AP激活。在加入因子B(FB)和因子D(FD)后,在绵羊红细胞(sE)上生成眼镜蛇蛇毒因子(CVF)C5转化酶。在加入C5、C6、C7、C8和C9组分后诱导溶血并在414nm下检测。C5与FHR1、MFHR1或依库珠单抗的预孵育显著抑制溶血,而hFH或BSA显示无抑制。无CVF转化酶(-FB/FD)或C5的对照不诱导溶血。C) MFHR1抑制sE上的膜攻击复合物(MAC)的形成。sE上的MAC形成通过用C5b6、C7、C8和C9组分孵育来诱导,并且通过细胞溶血进行检测。与hFH或BSA相比,C5b6与MFHR1、FHR1或依库珠单抗的预孵育以显著水平抑制溶血。无C9的样品不诱导溶血。在B和C中,无调节剂(w/o)的对照设为100%。所有数据表示为3次单独实验的平均值±SD。***P<0.001,对比w/o对照,单因素ANOVA及Bonferroni多重比较检验。
图4:MFHR1抑制补体旁路途径(AP)和经典途径(CP)激活并以临床相关水平减少人血清中的过敏毒素释放。将MFHR1、hFH、FHR1或依库珠单抗加入到人血清中,并且通过LPS,利用特定缓冲条件激活旁路途径(AP)后,测量A) C3b沉积或B) C5b-9复合物形成。A)MFHR1以比hFH更高的效率减少表面C3b沉积,这通过ELISA,利用抗-人C3和HRP-标记的二抗进行测量。正如预期,依库珠单抗不抑制C3b沉积。B) MFHR1以比hFH或依库珠单抗更高的效率抑制C5b-9,这利用补体AP-ELISA(WIESLAB®)来测定。对于每个单独实验,将未经处理的对照血清设为100%。数据表示来自n=4次测定 ±SEM的单个值,所述测定使用4名单独的健康供体的血清。此外,MFHR1有效地阻断C) C3a和D) C5a的产生,这利用特异性C3a、C5aELISA(Quidel),在AP激活的血清的上清液中进行测定(n=3次测定±SEM)。E) MFHR1对经典途径(CP)的抑制利用补体CP-ELISA(WIESLAB®)进行评估。对于每个单独实验,将未经处理的对照血清的AP活性设为100%。数据表示来自n=3次测定 ±SEM的单个值,所述测定使用3名单独的健康供体的血清。计算的IC50值参见表1。
图5:多聚复合物提高人血清中MFHR1的AP调控活性。A)牛血清白蛋白(BSA)、血清血浆来源的FH(hFH)和MFHR1的尺寸排阻色谱(Superdex 200 10/300 GL)。BSA混合物的三种组分基于分子量具有不同的保留体积,它们是I. BSA三聚体(198kDa,10.35ml),II.BSA二聚体(132kDa,11.45ml)和III.BSA单体(66kDa,13.4ml)。在相同条件下,hFH(9.3ml)显示蛋白物质的峰作为二聚蛋白迁移至大约300kDa。MFHR1显示保留体积为10ml的峰(I.),指示MFHR1在液相中主要以多聚状态迁移。理论三聚体(II.10.7)、二聚体(III.11.4)、中间体(IV.-V.)或单体(IV.13.5) MFHR1在洗脱特征图中示出。B)按A进行从SEC柱中洗脱后,分析MFHR1。将来自各级分的20µl蛋白加载到10% SDS-PAGE上并进行银染。C)随后,将单独或汇集的MFHR1 SEC级分以10nM加入人血清中,并且利用AP特异性缓冲条件,通过在LPS涂覆的孔中孵育进行AP激活。MFHR1级分的调控效率通过测量C5b-9复合物形成进行分析。将未经处理的对照血清的AP活性设为100%。
图6:MFHR1对由FHR1和FHR5引起的失调具有抗性。A) MFHR1与C3b的结合未受到FHR1或FHR5的竞争性抑制。将MFHR1(三角形)或hFH(正方形)(称为测试蛋白)单独地或与递增浓度的FHR1(黑线)或FHR5(灰线) (范围从等摩尔量至过量100倍)一起加到C3b涂覆的微量滴定板上。MFHR1或hFH与C3b的结合利用特异性抗体进行检测。示出3次独立测定的平均值及SD。B) FHR5不减弱MFHR1介导的使绵羊红细胞(sE)免于血清诱导的AP-激活的保护,而hFH的AP调控功能被FHR5剂量依赖性失调。使用将FH-消耗的血清诱导的sE溶解减少到50%的浓度的MFHR1或hFH,并且加入范围从等摩尔量至过量100倍的递增浓度的FHR5。在414nm处测定溶血,并且将数据表示为与其中未加入FHR5的样品相比的相对提高。数据表示来自n=3次测定 ±SEM的单个值。
图7:在非典型溶血尿毒症综合征(aHUS)和C3肾小球病(C3G)的建模治疗中,MFHR1减少不受控的补体激活。A) FH中的突变可引起家族性aHUS,和这些患者的血清中的补体调控丧失,如果加入绵羊红细胞(sE)中则介导溶血。加入aHUS患者(pat.#1 FH R1215Q(Gerber等人,2003))的血清的MFHR1保护sE免于补体介导的MAC形成和溶解,其效率高于依库珠单抗。数据表示为来自n=3次实验的平均值 ±SD。B)由于过量的补体激活,C3G中的血清C3普遍提高,并且抑制不受控的补体活性可提供C3G的备选治疗选项。加入C3Neph阳性患者的血清中的MFHR1有效地抑制LPS刺激后的AP激活和C5b-9形成。
图8:MFHR1显示针对C3肾小球病(C3G)体内不受控的补体激活的显著治疗益处。A-C)由于AP过度激活,FH-缺陷型小鼠(FH-/-)发展出异常的肾小球C3积聚和低血清C3水平,从而提供一种有用的C3G模型,用于测试补体靶向药的治疗功效。A)在腹膜内注射0.5mgMFHR1后的指示时间点,MFHR1显著提高C3血清水平至可与hFH相比的水平。平均值显示为棒,具有绘制的单个数据点。B) MFHR1显著减少异常的肾小球C3积聚。在给予MFHR1、hFH或PBS治疗的FH-/-小鼠后24小时,测定肾小球C3荧光强度。使用未经治疗的野生型小鼠的切片作为阴性对照。平均值显示为棒± SD,具有绘制的单个数据点,用任意荧光单位(AFU)表示。C)肾小球C3沉积的代表性图像。比例尺;50µm。***P<0.001,对比PBS组,单因素ANOVA及Bonferroni检验。
详细描述
本发明涉及一种多肽,其包含抑制性C3转化酶效应结构域、抑制性C5转化酶效应结构域(C5结合结构域)、以及任选的抑制性TCC形成结构域和/或二聚化基序。
与本发明有关的“抑制性C3转化酶效应结构域”是指能够抑制C3转化酶,即,抑制C3转化酶形成的氨基酸序列。在补体旁路途径激活后,相对于对照如果C3b的沉积受到抑制或者如果C3a的形成减少,则通常存在C3转化酶的抑制。C3b和C3a的形成可用分别如图4A或图4C所示的测定法进行测定。在另一个实施方案中,抑制性C3转化酶效应结构域具有C3转化酶衰变加速活性和辅因子活性。C3转化酶衰变加速活性可用如图2B所示的测定法进行测定。辅因子活性可用如图2C-2D所示的测定法进行测定。
在某些实施方案中,抑制性C3转化酶效应结构域是FH的片段。根据本发明,FH代表与如SEQ ID NO:1所示的氨基酸序列具有至少70%的序列同一性的蛋白。优选地,FH与SEQID NO:1所示的氨基酸序列具有至少80%,更优选地至少90%,更优选地至少95%的氨基酸序列同一性。最优选地,FH包含SEQ ID NO:1所示的氨基酸序列或由其组成。
两个氨基酸序列之间的序列比较和同一性百分比(以及相似性百分比)测定可利用任何合适的程序实现,例如程序“BLAST 2 SEQUENCES(blastp)”(Tatusova等人,(1999)FEMS Microbiol.Lett.174,247-250),使用以下参数:矩阵BLOSUM62;开放空位11和延伸空位1罚分;空位x_dropoff50;期望值10.0字长3;过滤器:无。
C3转化酶效应结构域优选地包含FH的SCR1-4或由其组成。在一个实施方案中,C3转化酶效应结构域包含SEQ ID NO:1的氨基酸19-264或由其组成。
在另一个实施方案中,抑制性C3转化酶效应结构域是FHR2的片段。根据本发明,FHR2代表与SEQ ID NO:3所示的氨基酸序列具有至少70%的序列同一性的蛋白。优选地,FHR2的氨基酸序列与SEQ ID NO:3所示的氨基酸序列具有至少80%,更优选地至少90%,更优选地至少95%的序列同一性。最优选地,FHR2包含SEQ ID NO:3所示的氨基酸序列或由其组成。
C3转化酶效应结构域可包含FHR2的SCR1-4或由其组成。在一个实施方案中,C3转化酶效应结构域包含SEQ ID NO:3的氨基酸22-268或由其组成。
与本发明有关的“抑制性C5转化酶效应结构域”是指能够抑制C5转化酶的氨基酸序列。如果C5a的形成减少,则通常存在C5转化酶的抑制(阻止C5裂解)。C5a的形成可用如图4D所示的测定法进行测定。在备选的实验方法中,示出抑制性效应结构域与C5的结合(图3A)。这阻止通过实验性C5转化酶将C5裂解成C5a和C5b,并且抑制MAC形成及细胞溶解,如(图3B)所示。
在某些实施方案中,抑制性C5转化酶效应结构域是FHR1的片段。根据本发明,FHR1代表与SEQ ID NO:2所示的氨基酸序列具有至少70%的序列同一性的蛋白。优选地,FHR1的氨基酸序列与SEQ ID NO:2所示的氨基酸序列具有至少80%,更优选地至少90%,更优选地至少95%的序列同一性。最优选地,FHR1包含SEQ ID NO:2所示的氨基酸序列或由其组成。除结合C5并防止由C5转化酶引起的C5裂解之外,来自FHR1的SCR 1-2还包含二聚化基序,其导致MFHR1的多聚化,放大了该多肽的功能,如图5所示。除C5转化酶抑制之外,来自FHR1的SCR1-2还通过结合C5b-6抑制MAC形成(图3C)。
抑制性C5转化酶效应结构域优选地包含FHR1的SCR1和SCR2或由它们组成。在一个实施方案中,抑制性C5转化酶效应结构域包含SEQ ID NO:2的氨基酸22-142或由其组成。
抑制性C3转化酶效应结构域、抑制性C5转化酶和抑制性MAC形成效应结构域可直接融合或经由接头融合。接头可为肽接头或非肽接头。优选地,接头由1至100个氨基酸,更优选地1至50个氨基酸,更优选地1至20个氨基酸,更优选地1至10个氨基酸,最优选地1至5个氨基酸组成。接头序列通常与C3转化酶效应结构域的氨基酸序列和抑制性C5转化酶效应结构域的氨基酸序列异源。
本发明的多肽可具有结构A-B,其中A是如本文所定义的C3转化酶效应结构域,并且B是如本文所定义的C5转化酶效应结构域。在另一个实施方案中,本发明的多肽可具有结构B-A,其中A是如本文所定义的C3转化酶效应结构域,并且B是如本文所定义的C5转化酶效应结构域。
在一个具体实施方案中,本发明的多肽包含选自SEQ ID NO:4、SEQ ID NO:5、SEQID NO:6和SEQ ID NO:7的氨基酸序列或由其组成。
在某些实施方案中,本发明的多肽包含第三结构域,所述第三结构域具有细胞表面结合特性。优选地,所述第三结构域包含FH的片段或由其组成。更优选地,第三结构域包含FH的SCR19和SCR20或由它们组成。在一个实施方案中,第三结构域包含SEQ ID NO:1的氨基酸1107-1230或由其组成。
本发明的多肽可具有结构A-B-C,其中A是如本文所定义的C5转化酶效应结构域,B是如本文所定义的C3转化酶效应结构域,并且C是如本文所定义的第三结构域。在另一个实施方案中,本发明的多肽可具有结构A-C-B、B-A-C、B-C-A、C-A-B或C-B-A,其中A、B和C的含义如本文所定义。字母A、B和C的顺序指示从多肽的N-末端至C-末端的序列,例如在A-B-C中,结构域A位于N-末端,并且结构域C位于C-末端。
第三结构域可与C3转化酶效应结构域和/或C5效应结构域以及MAC效应结构域直接融合,或者经由如上文所定义的接头来融合。
在一个具体实施方案中,本发明的多肽包含如SEQ ID NO:8所示的氨基酸序列或由其组成。在另一个实施方案中,本发明的多肽包含如SEQ ID NO:9所示的氨基酸序列或由其组成。
本发明的多肽可用于治疗或预防与补体系统相关和/或关联的病症。这些病症包括但不限于非典型溶血尿毒症综合征(aHUS)、血栓性微血管病(TMA)、C3肾小球病(C3G)、IgA肾病、系统性红斑狼疮肾炎、肾移植患者的体液排斥反应、局部缺血-再灌注事件后(如肾移植后)的组织损伤、过度补体激活、组织损伤(如受血液透析影响)、阵发性夜间血红蛋白尿(PNH)、年龄相关性黄斑变性(AMD)、传染病、败血病、SIRS、创伤性损伤、心肌梗塞以及其中补体激活是另外的局部或全身损伤的原因的疾病或病况。
优选地,待治疗或预防的病症选自aHUS、TMA、C3G、IgA肾病、系统性红斑狼疮肾炎、PNH和AMD。
本发明还提供编码本发明的多肽的核酸,其可插入合适的质粒和载体中,用于在宿主细胞中表达。
编码待表达的多肽的核酸可根据本领域已知的方法进行制备。基于FH (NCBI参考序列:NM_000186.3)、FHR1 (NCBI参考序列:NM_002113.2)和FHR2 (NCBI参考序列:NP_005657.1)的cDNA序列,可设计并生成编码上文提及的多肽的重组DNA。
其中cDNA包含以正确取向插入表达质粒的整个开放阅读框的构建体可用于蛋白表达。典型的表达载体包含启动子,其指导在携带质粒的细胞中对应于插入核酸的大量mRNA的合成。它们还可包括允许它们在宿主生物体中自主复制的复制起点序列、以及提高合成的mRNA翻译的效率的序列。稳定的长期载体可利用例如病毒的调控元件(例如,来自爱泼斯坦巴尔病毒基因组的OriP序列),以自由复制实体的形式保持。也可制备细胞系,其已经将载体整合进基因组DNA,并且以此方式连续生产基因产物。通常待提供的细胞通过将编码所述多肽的核酸引入合适的宿主细胞而获得。
根据本发明可利用易于进行细胞培养并表达多肽的任何宿主细胞。在某些实施方案中,宿主细胞是哺乳动物细胞。在其它实施方案中,宿主细胞是昆虫细胞(例如Sf9细胞),或者小立碗藓属(physcomitrella)细胞(例如小立碗藓(physcomitrella patens))。另选地,宿主细胞可为细菌细胞。
一般来讲,通常将期望分离和/或纯化根据本发明表达的糖蛋白。在某些实施方案中,表达的糖蛋白被分泌到培养基中,因此可例如通过离心或过滤除去细胞和其它固体,作为纯化过程的第一步。
可通过标准方法分离并纯化表达的多肽,所述方法包括但不限于色谱法(例如离子交换色谱、亲和色谱、尺寸排除色谱和羟磷灰石色谱)、凝胶过滤、离心或差别溶解、乙醇沉淀和/或用于蛋白纯化的任何其它可用技术(参见例如Scopes,Protein PurificationPrinciples and Practice,第2版,Springer-Verlag,New York,1987;Higgins,S. J.和Hames,B. D.(编辑),Protein Expression:A Practical Approach,Oxford Univ Press,1999;以及Deutscher,M. P.,Simon,M. I.,Abelson,J. N.(编辑),Guide to ProteinPurification:Methods in Enzymology (Methods in Enzymology Series,Vol. 182),Academic Press,1997,它们每个均以引用方式并入本文)。
本领域的普通技术人员将会知道,根椐待纯化的多肽的特性、表达多肽的细胞的特性和/或培养细胞的培养基的组成,准确的纯化技术将会不同。
本发明的另一方面是包含本发明的多肽和药学上可接受的赋形剂或载体的药物组合物。药物组合物可包含有效治疗或预防受试者的补体相关病症的量的多肽。
可通过混合具有期望纯度的糖蛋白和本领域通常使用的任选的药学上可接受的载体、赋形剂或稳定剂(本文它们均称为“载体”,即,缓冲剂、稳定剂、防腐剂、等渗剂、非离子洗涤剂、抗氧化剂以及其它各种添加剂)来制备适于本文所述方法的本发明多肽的治疗制剂,其可以冻干制剂或水溶液的形式储存。参见Remington's PharmaceuticalSciences,第16版(Osol编辑,1980年)。此类添加剂必须是在使用的剂量和浓度下对接受者无毒的。
可配制本发明的药物组合物用于口服、舌下、鼻内、眼内、直肠、透皮、粘膜、局部或胃肠外给药。胃肠外给药可包括皮内、皮下、肌内(i.m.)、静脉内(i.v.)、腹膜内(i.p.)、动脉内、髓内、心内、关节内(关节)、滑膜内、颅内、椎管内和鞘内(脊髓液)注射或输注,优选在小鼠中腹膜内(i.p.)注射和在人中静脉内(i.v.)。适用于肠胃外注射或输注药物制剂的任何装置可用于此类给药。例如,药物组合物可包含在无菌的预填充注射器内。
本发明的可溶性多肽的有效剂量、剂量总数和治疗时长的确定在本领域技术人员的能力范围内,并且可利用标准剂量递增研究来测定。本发明的可溶性多肽的给药剂量将根据具体的可溶性多肽、受试者、和疾病的性质及严重度、受试者的身体状况、治疗方案(例如是否使用第二治疗剂)、以及所选的给药途径而不同;本领域的技术人员能够容易地确定合适的剂量。
氨基酸序列汇总:
实施例
MFHR1由N-末端FH调控活性结构域SCR1-4(C3/C5转化酶衰变加速和辅因子活性)与FH的C-末端表面识别结构域(SCR19-20)以及FHR1的N-末端结构域(SCR1-2)组成[图1]。FHR1是仅有的已知C5转化酶内源补体调节剂。FHR1 SCR1-2结合C5并从而抑制C5裂解成C5a和C5b。另外,FHR1 SCR1-2通过结合C5b6来抑制末端补体途径。通过这些结构域的组合,补体激活在级联的多个效应位点受到抑制(C3b降解、抑制C3/C5转化酶、抑制C5裂解和C5b-9形成)。这也导致过敏毒素C3a和C5a的抑制和形成,认为它们促进AP相关疾病的疾病进展。
为了制备MFHR1,包含上文提及的FH和FHR1结构域的请求序列的cDNA片段通过PCR扩增并随后通过自启动重叠PCR进行装配。将DNA克隆进pFastbac gp67-10xHis杆状病毒表达载体,并且MFHR1在SF9昆虫细胞中表达。MFHR1的氨基酸序列如SEQ ID NO:8所示。蛋白纯化通过亲和色谱法和尺寸排阻色谱法进行。SDS-PAGE和考马斯或银染凝胶显示单个58.65kDa条带,其对应于MFHR1的计算分子量[图1B]。FHR1 SCR1-2和FH SCR 1-4以及SCR19-20的成功融合通过免疫检测进行确认,该免疫检测使用用于检测FHR1来源的结构域SCR1-2的特异性抗体、用于检测FH来源的结构域1-4的抗-FH1-4、用于检测FH来源的结构域20的单克隆抗-C18以及多克隆FH抗体[图1C]。
MFHR1结合C3b [图2A],解离C3转化酶[图2B]并展示辅因子活性[图2C- 2D]。与血浆纯化的FH(FHplasma)形成对比,FHR1和MFHR1显示由来自FHR1的SCR1-2介导的结合C5的能力[图3A]。FHR1和MFHR1通过抑制C5转化酶/ C5裂解[图3B]和MAC形成[图3C]来调控末端途径激活。这表明新融合蛋白MFHR1不仅保留FH[图2]和FHR1[图3]的补体调控活性,而且此外还组合以上二者的特性。MFHR1比依库珠单抗更有效地抑制aHUS血清诱导的绵羊红细胞溶血,表明除其调控活性之外,在MFHR1中还保留了来源于FH结构域19-20的表面识别[图7A]。MFHR1的高效能还突出表现在基于ELISA的补体活性测定中。在激活添加有MFHR1的人血清中的补体级联后,它的AP调控活性通过测量C3b沉积[图4A]或C5b6-9[图4B]进行测定,并且它的CP调控效率通过利用WIESLAB® CP-ELISA测量C5b6-9进行测定[图4E]。将MFHR1加入正常人血清(NHS)或来自C3G患者(复发的、C3Nef自身抗体阳性的患者)的血清以剂量依赖的方式有效地抑制补体激活[图4A-B、4E、7B]。在溶血分析中,MFHR1显示比hFH(表1)、依库珠单抗和处于临床前阶段研究中的其它相关抑制剂更高的抑制效率(以摩尔计)(IC50值汇总在表1中)。此外,我们表明MFHR1比依库珠单抗更有效地抑制人血清中C3a[图4C]和C5a[图4D]的生成。
使用二聚化基序(由FHR1的SCR1介导)有利于MFHR1的多聚复合物的产生。我们表明多聚复合物具有更高的调控活性,据推测通过提高调节剂的局部浓度而实现[图5]。此外,MFHR1对FHR介导的失调具有抗性[图6A-B]。
MFHR1的治疗价值在两种体外建模治疗方法和一种体内C3G鼠模型中得到证明。加入MFHR1有效地减少aHUS[图7A]和C3G[图7B]患者血浆中不受控的补体激活。在显示C3G-样表型的FH-/-缺陷型小鼠中,给予MFHR1导致血浆C3水平快速正常化[图8A],并解决肾小球C3沉积[图8 B-C]。
表1:人血清中抑制活性的估计IC50值汇总
值以µM表示。IC50值利用log(抑制剂) vs.响应来计算--利用GraphPad Prism软件将X数据转化成Log X后的变斜率方程,n.t.= 未检测,n.e.= 无效。粗体字母,AG Häffner,*Schmidt等人,2013年,**Hareli等人,2011年,***Schmidt等人,2015年,#Gorham等人,2013年。
总之,结果表明为了改善补体激活的调节剂,用于在补体级联的多个效应位点上同时阻止补体激活(C3b降解、抑制C3/C5转化酶、抑制C5裂解和TCC形成)并通过其多聚的能力(或分别两者)的组合方法与“常规”方法相比具有若干有利的优点。包含来自FH和FHR1的不同功能特性的MFHR1在C3和C5转化酶的水平上并通过阻断末端补体途径来调节补体活性,并且可能比FH或依库珠单抗或其它公布的临床相关补体抑制剂活性更强。值得注意的是,MFHR1对由FHR1和FHR5引起的失调尤其具有抗性,并且多聚复合物可提高它的调控效率。
参考文献
Alexander, J. J.和R. J. Quigg (2007)."The simple design of complementfactor H:Looks can be deceiving."Mol Immunol 44(1-3):123-132.
Barbour, T. D., M. C. Pickering和H. T. Cook (2013)."Recent insightsinto C3 glomerulopathy."Nephrol Dial Transplant 28(7):1685-1693.
Blaum, B. S., J. P. Hannan, A. P. Herbert, D. Kavanagh, D. Uhrin和T.Stehle (2015)."Structural basis for sialic acid-mediated self-recognition bycomplement factor H." Nature chemical biology 11(1):77-82.
Bomback, A. S., R. J. Smith, G. R. Barile, Y. Zhang, E. C. Heher, L.Herlitz, M. B. Stokes, G. S. Markowitz, V. D. D'Agati, P. A. Canetta, J.Radhakrishnan和G. B. Appel (2012)."Eculizumab for dense deposit disease andC3 glomerulonephritis."Clinical journal of the American Society of Nephrology:CJASN 7(5):748-756.
Braun, M. C., D. M. Stablein, L. A. Hamiwka, L. Bell, S. M. Bartosh和C. F. Strife (2005)."Recurrence of membranoproliferative glomerulonephritistype II in renal allografts:The North American Pediatric Renal TransplantCooperative Study experience."J Am Soc Nephrol 16(7):2225-2233.
Carroll, M. V.和R. B. Sim (2011)."Complement in health and disease."Adv Drug Deliv Rev 63(12):965-975.
Cataland, S. R.和H. M. Wu (2014)."Diagnosis and management ofcomplement mediated thrombotic microangiopathies."Blood reviews 28(2):67-74.
de Cordoba, S. R.和E. G. de Jorge (2008)."Translational mini-reviewseries on complement factor H: genetics and disease associations of humancomplement factor H." Clin Exp Immunol 151(1):1-13.
Ferreira, V. P., A. P. Herbert, H. G. Hocking, P. N. Barlow和M. K.Pangburn (2006)."Critical role of the C-terminal domains of factor H inregulating complement activation at cell surfaces."J Immunol 177(9):6308-6316.
Goicoechea de Jorge, E., J. J. Caesar, T. H. Malik, M. Patel, M.Colledge, S. Johnson, S. Hakobyan, B. P. Morgan, C. L. Harris, M. C.Pickering和S. M. Lea (2013)."Dimerization of complement factor H-relatedproteins modulates complement activation in vivo."Proc Natl Acad Sci U S A110(12):4685-4690.
Gordon, D. L., R. M. Kaufman, T. K. Blackmore, J. Kwong和D. M. Lublin(1995)."Identification of complement regulatory domains in human factor H." JImmunol 155(1):348-356.
Haffner, K., S. Michelfelder和M. Pohl (2015)."Successful therapy ofC3Nef-positive C3 glomerulopathy with plasma therapy and immunosuppression."Pediatric nephrology.
Hebecker, M., M. Alba-Dominguez, L. T. Roumenina, S. Reuter, S.Hyvarinen, M. A. Dragon-Durey, T. S. Jokiranta, P. Sanchez-Corral和M. Jozsi(2013)."An engineered construct combining complement regulatory and surface-recognition domains represents a minimal-size functional factor H." J Immunol191(2):912-921.
Herlitz, L. C., A. S. Bomback, G. S. Markowitz, M. B. Stokes, R. N.Smith, R. B. Colvin, G. B. Appel和V. D. D'Agati (2012)."Pathology aftereculizumab in dense deposit disease and C3 GN."J Am Soc Nephrol 23(7):1229-1237.
Hillmen, P., N. S. Young, J. Schubert, R. A. Brodsky, G. Socie, P.Muus, A. Roth, J. Szer, M. O. Elebute, R. Nakamura, P. Browne, A. M.Risitano, A. Hill, H. Schrezenmeier, C. L. Fu, J. Maciejewski, S. A. Rollins,C. F. Mojcik, R. P. Rother和L. Luzzatto (2006)."The complement inhibitoreculizumab in paroxysmal nocturnal hemoglobinuria."The New England journal ofmedicine 355(12):1233-1243.
Holers, V. M. (2008)."The spectrum of complement alternative pathway-mediated diseases."Immunological reviews 223:300-316.
Jarva, H., T. S. Jokiranta, J. Hellwage, P. F. Zipfel和S. Meri(1999)."Regulation of complement activation by C-reactive protein: targetingthe complement inhibitory activity of factor H by an interaction with shortconsensus repeat domains 7 and 8-11."J Immunol 163(7):3957-3962.
Jozsi, M., T. Manuelian, S. Heinen, M. Oppermann和P. F. Zipfel(2004)."Attachment of the soluble complement regulator factor H to cell andtissue surfaces: relevance for pathology."Histol Histopathol 19(1):251-258.
Jozsi, M., M. Oppermann, J. D. Lambris和P. F. Zipfel (2007)."The C-terminus of complement factor H is essential for host cell protection."MolImmunol 44(10):2697-2706.
Jozsi, M., A. Tortajada, B. Uzonyi, E. Goicoechea de Jorge和S.Rodriguez de Cordoba (2015)."Factor H-related proteins determine complement-activating surfaces."Trends in immunology 36(6):374-384.
Kawa, M. P., A. Machalinska, D. Roginska和B. Machalinski (2014)."Complement system in pathogenesis of AMD: dual player in degeneration andprotection of retinal tissue."Journal of immunology research 2014:483960.
Legendre, C. M., C. Licht, P. Muus, L. A. Greenbaum, S. Babu, C.Bedrosian, C. Bingham, D. J. Cohen, Y. Delmas, K. Douglas, F. Eitner, T.Feldkamp, D. Fouque, R. R. Furman, O. Gaber, M. Herthelius, M. Hourmant, D.Karpman, Y. Lebranchu, C. Mariat, J. Menne, B. Moulin, J. Nurnberger, M.Ogawa, G. Remuzzi, T. Richard, R. Sberro-Soussan, B. Severino, N. S. Sheerin,A. Trivelli, L. B. Zimmerhackl, T. Goodship和C. Loirat (2013)."Terminalcomplement inhibitor eculizumab in atypical hemolytic-uremic syndrome."TheNew England journal of medicine 368(23):2169-2181.
Licht, C., S. Heinen, M. Jozsi, I. Loschmann, R. E. Saunders, S. J.Perkins, R. Waldherr, C. Skerka, M. Kirschfink, B. Hoppe和P. F. Zipfel(2006)."Deletion of Lys224 in regulatory domain 4 of Factor H reveals a novelpathomechanism for dense deposit disease (MPGN II)."Kidney Int 70(1):42-50.
Licht, C., A. Weyersberg, S. Heinen, L. Stapenhorst, J. Devenge, B.Beck, R. Waldherr, M. Kirschfink, P. F. Zipfel和B. Hoppe (2005)."Successfulplasma therapy for atypical hemolytic uremic syndrome caused by factor Hdeficiency owing to a novel mutation in the complement cofactor proteindomain 15."American journal of kidney diseases : the official journal of theNational Kidney Foundation 45(2):415-421.
Loirat, C.和V. Fremeaux-Bacchi (2011)."Atypical hemolytic uremicsyndrome."Orphanet J Rare Dis 6:60.
Lu, D. F., M. Moon, L. D. Lanning, A. M. McCarthy和R. J. Smith(2012)."Clinical features and outcomes of 98 children and adults with densedeposit disease."Pediatric nephrology 27(5):773-781.
Maillard, N., R. J. Wyatt, B. A. Julian, K. Kiryluk, A. Gharavi, V.Fremeaux-Bacchi和J. Novak (2015)."Current Understanding of the Role ofComplement in IgA Nephropathy."J Am Soc Nephrol 26(7):1503-1512.
Manuelian, T., J. Hellwage, S. Meri, J. Caprioli, M. Noris, S.Heinen, M. Jozsi, H. P. Neumann, G. Remuzzi和P. F. Zipfel (2003)."Mutationsin factor H reduce binding affinity to C3b and heparin and surface attachmentto endothelial cells in hemolytic uremic syndrome."J Clin Invest 111(8):1181-1190.
Masani, N., K. D. Jhaveri和S. Fishbane (2014)."Update onmembranoproliferative GN."Clinical journal of the American Society ofNephrology :CJASN 9(3):600-608.
Mastellos, D. C., D. Yancopoulou, P. Kokkinos, M. Huber-Lang, G.Hajishengallis, A. R. Biglarnia, F. Lupu, B. Nilsson, A. M. Risitano, D.Ricklin和J. D. Lambris (2015)."Compstatin: a C3-targeted complement inhibitorreaching its prime for bedside intervention."European journal of clinicalinvestigation 45(4):423-440.
Merle, N. S., S. E. Church, V. Fremeaux-Bacchi和L. T. Roumenina(2015)."Complement System Part I - Molecular Mechanisms of Activation andRegulation."Frontiers in immunology 6:262.
Nichols, E. M., T. D. Barbour, I. Y. Pappworth, E. K. Wong, J. M.Palmer, N. S. Sheerin, M. C. Pickering和K. J. Marchbank (2015)."An extendedmini-complement factor H molecule ameliorates experimental C3glomerulopathy."Kidney Int.
Noris, M.和G. Remuzzi (2009)."Atypical hemolytic-uremic syndrome."TheNew England journal of medicine 361(17):1676-1687.
Oppermann, M., T. Manuelian, M. Jozsi, E. Brandt, T. S. Jokiranta, S.Heinen, S. Meri, C. Skerka, O. Gotze和P. F. Zipfel (2006)."The C-terminus ofcomplement regulator Factor H mediates target recognition: evidence for acompact conformation of the native protein."Clin Exp Immunol 144(2):342-352.
Parker, C. J., S. Kar和P. Kirkpatrick (2007)."Eculizumab."Naturereviews.Drug discovery 6(7):515-516.
Ricklin, D., G. Hajishengallis, K. Yang和J. D. Lambris (2010)."Complement: a key system for immune surveillance and homeostasis."Natureimmunology 11(9):785-797.
Ricklin, D.和J. D. Lambris (2013)."Progress and Trends in ComplementTherapeutics."Advances in experimental medicine and biology 735:1-22.
Ricklin, D.和J. D. Lambris (2015)."Therapeutic control of complementactivation at the level of the central component C3."Immunobiology.
Rodriguez de Cordoba, S., J. Esparza-Gordillo, E. Goicoechea deJorge, M. Lopez-Trascasa和P. Sanchez-Corral (2004)."The human complementfactor H: functional roles, genetic variations and disease associations."MolImmunol 41(4):355-367.
Ruseva, M. M., T. Peng, M. A. Lasaro, K. Bouchard, S. Liu-Chen, F.Sun, Z. X. Yu, A. Marozsan, Y. Wang和M. C. Pickering (2015)."Efficacy ofTargeted Complement Inhibition in Experimental C3 Glomerulopathy."J Am SocNephrol.
Schmidt, C. Q., H. Bai, Z. Lin, A. M. Risitano, P. N. Barlow, D.Ricklin和J. D. Lambris (2013)."Rational engineering of a minimized immuneinhibitor with unique triple-targeting properties."J Immunol 190(11):5712-5721.
Sethi, S.和F. C. Fervenza (2012)."Membranoproliferativeglomerulonephritis--a new look at an old entity."The New England journal ofmedicine 366(12):1119-1131.
Skerka, C., Q. Chen, V. Fremeaux-Bacchi和L. T. Roumenina (2013)."Complement factor H related proteins (CFHRs)."Mol Immunol 56(3):170-180.
Skerka, C., R. D. Horstmann和P. F. Zipfel (1991)."Molecular cloningof a human serum protein structurally related to complement factor H." J BiolChem 266(18):12015-12020.
Skerka, C., C. Timmann, R. D. Horstmann和P. F. Zipfel (1992)."Twoadditional human serum proteins structurally related to complement factor H.Evidence for a family of factor H-related genes."J Immunol 148(10):3313-3318.
Wagner, E.和M. M. Frank (2010)."Therapeutic potential of complementmodulation."Nature reviews.Drug discovery 9(1):43-56.
Weiler, J. M., M. R. Daha, K. F. Austen和D. T. Fearon (1976)."Controlof the amplification convertase of complement by the plasma protein beta1H."Proc Natl Acad Sci U S A 73(9):3268-3272.
Wilson, M. R., C. M. Arroyave, R. M. Nakamura, J. H. Vaughan和E. M.Tan (1976)."Activation of the alternative complement pathway in systemiclupus erythematosus."Clin Exp Immunol 26(1):11-20.
Zimmerhackl, L. B., J. Hofer, G. Cortina, W. Mark, R. Wurzner, T. C.Jungraithmayr, G. Khursigara, K. O. Kliche和W. Radauer (2010)."Prophylacticeculizumab after renal transplantation in atypical hemolytic-uremicsyndrome."The New England journal of medicine 362(18):1746-1748.
Zipfel, P. F.和C. Skerka (2009)."Complement regulators and inhibitoryproteins."Nat Rev Immunol 9(10):729-740.
Zuber, J., F. Fakhouri, L. T. Roumenina, C. Loirat, V. Fremeaux-Bacchi和H. C. G. French Study Group for a (2012)."Use of eculizumab foratypical haemolytic uraemic syndrome and C3 glomerulopathies."Naturereviews.Nephrology 8(11):643-657。
Claims (10)
1.一种结构A-B-C的多肽,其中A是因子H相关蛋白1 (FHR1)的SCR1和SCR2,B是因子H(FH)的短同源重复序列(SCR) 1至4,并且C是能够结合细胞表面的结构域,其中所述能够结合细胞表面的结构域是FH的SCR19和SCR20,其中所述结构为FHR1 (SCR1-2) –FH (SCR1-4)–FH (SCR19-20),其中A和B直接融合或者经由接头融合,B和C直接融合或者经由接头融合,并且所述FHR1和FH都是人源的。
2.根据权利要求1所述的多肽,其中所述多肽是多聚体。
3.根据权利要求2所述的多肽,所述多肽包含来自FHR1的SCR1的至少一个二聚化基序。
4.根据权利要求1所述的多肽,其中所述多肽能够抑制TCC(C5b-9)形成。
5.根据权利要求4所述的多肽,其中所述TCC形成受到所述多肽与C5b-6结合的抑制。
6.根据权利要求1-5中任一项所定义的多肽在制备用于治疗或预防非典型溶血尿毒症综合征(aHUS)、血栓性微血管病(TMA)、C3肾小球病(C3G)、IgA肾病、系统性红斑狼疮肾炎、移植排斥、阵发性夜间血红蛋白尿(PNH)、年龄相关性黄斑变性(AMD)、传染病、败血病、SIRS、创伤性损伤、局部缺血/再灌注损伤和心肌梗塞的药物中的用途。
7.一种核酸,所述核酸编码根据权利要求1至5中任一项所述的多肽。
8.一种质粒或载体,所述质粒或载体包含根据权利要求7所述的核酸。
9.一种细胞,所述细胞包含根据权利要求7所述的核酸或根据权利要求8所述的质粒或载体。
10.一种制备根据权利要求1至5中任一项所述的多肽的方法,所述方法包括在允许表达所述多肽的条件下,在培养基中培养根据权利要求9所述的细胞,和从所述细胞或所述培养基回收所述多肽。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102015016665.4 | 2015-12-23 | ||
DE102015016665 | 2015-12-23 | ||
PCT/EP2016/082614 WO2017109208A1 (en) | 2015-12-23 | 2016-12-23 | Polypeptides for inhibiting complement activation |
Publications (2)
Publication Number | Publication Date |
---|---|
CN108699121A CN108699121A (zh) | 2018-10-23 |
CN108699121B true CN108699121B (zh) | 2023-07-04 |
Family
ID=57838331
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201680075659.0A Active CN108699121B (zh) | 2015-12-23 | 2016-12-23 | 用于抑制补体激活的多肽 |
Country Status (19)
Country | Link |
---|---|
US (2) | US10640540B2 (zh) |
EP (1) | EP3394089B1 (zh) |
JP (2) | JP6771568B2 (zh) |
KR (1) | KR20180091097A (zh) |
CN (1) | CN108699121B (zh) |
AU (1) | AU2016375183B2 (zh) |
BR (1) | BR112018012844A2 (zh) |
CA (1) | CA3009393C (zh) |
DK (1) | DK3394089T3 (zh) |
ES (1) | ES2895256T3 (zh) |
HR (1) | HRP20211608T1 (zh) |
HU (1) | HUE056019T2 (zh) |
IL (1) | IL260182B2 (zh) |
LT (1) | LT3394089T (zh) |
MX (1) | MX2018007392A (zh) |
PL (1) | PL3394089T3 (zh) |
PT (1) | PT3394089T (zh) |
SI (1) | SI3394089T1 (zh) |
WO (1) | WO2017109208A1 (zh) |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2019043609A1 (en) | 2017-08-31 | 2019-03-07 | Novartis Ag | NEW USES OF PIPERIDINYL-INDOLE DERIVATIVES |
EP3682006A1 (en) | 2017-09-11 | 2020-07-22 | Insideoutbio, Inc. | Methods and compositions to enhance the immunogenicity of tumors |
EP3788038B1 (en) | 2018-05-04 | 2023-10-11 | Amgen Inc. | Kras g12c inhibitors and methods of using the same |
EP3586860A1 (en) * | 2018-06-22 | 2020-01-01 | Universität Ulm | Complement inhibitors and uses thereof |
DE102018120016B4 (de) * | 2018-08-16 | 2020-09-03 | Leibniz-Institut für Naturstoff-Forschung und Infektionsbiologie e.V. -Hans-Knöll-Institut- | Modulatoren der Funktion von Faktor H-verwandtem Protein 1 und deren Verwendungen in der Kontrolle von Entzündung |
Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0097440A1 (en) * | 1982-06-14 | 1984-01-04 | The Upjohn Company | Method and kit for removing and assaying complement system fragments |
CN1138863A (zh) * | 1993-09-24 | 1996-12-25 | 赛托医学有限公司 | 封阻补体激活的嵌合蛋白 |
CN101443050A (zh) * | 2006-03-08 | 2009-05-27 | 阿切埃米克斯有限公司 | 治疗视觉失调中使用的补体结合适体和抗-c5药物 |
CN101754980A (zh) * | 2007-05-23 | 2010-06-23 | 健泰科生物技术公司 | 补体相关眼疾的预防和治疗 |
CN101848937A (zh) * | 2007-11-02 | 2010-09-29 | 诺瓦提斯公司 | 调节补体组分的分子和方法 |
CN102271703A (zh) * | 2008-11-10 | 2011-12-07 | 阿雷克森制药公司 | 用于治疗补体相关障碍的方法和组合物 |
CN102481348A (zh) * | 2009-08-31 | 2012-05-30 | Ibc药品公司 | 双特异性免疫细胞因子停靠-和-加锁(dnl)复合物及其治疗性用途 |
CN102978204A (zh) * | 2011-11-11 | 2013-03-20 | 张康 | Cfhr1基因型、高密度脂蛋白上cfhr1及氧化磷酸胆碱的测定试剂盒及测定方法 |
WO2013142362A1 (en) * | 2012-03-19 | 2013-09-26 | The Trustees Of The University Of Pennsylvania | Regulator of complement activation and uses thereof |
CN103781492A (zh) * | 2011-04-08 | 2014-05-07 | 莱斯特大学 | 用于治疗与masp-2依赖性补体活化相关的状况的方法 |
WO2015092335A2 (fr) * | 2013-12-20 | 2015-06-25 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Proteines recombinantes possedant une activite de facteur h |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2802017A1 (en) * | 2010-06-09 | 2011-12-15 | Zymogenetics, Inc. | Dimeric vstm3 fusion proteins and related compositions and methods |
US20220009979A1 (en) * | 2018-08-22 | 2022-01-13 | Alexion Pharmaceuticals, Inc. | Fusion proteins and methods of treating complement dysregulation using the same |
GB2583560A (en) * | 2018-12-11 | 2020-11-04 | Admirx Inc | Fusion protein constructs for complement associated disease |
US20220220169A1 (en) * | 2019-05-15 | 2022-07-14 | Insideoutbio, Inc. | Modular Therapeutics for the Treatment of Inflammatory Diseases and Cancer |
US20210371534A1 (en) * | 2020-06-01 | 2021-12-02 | Musc Foundation For Research Development | Recombinant Fusion Proteins Targeting P-selectin, and Methods of Use Thereof for Treating Diseases and Disorders |
-
2016
- 2016-12-23 LT LTEPPCT/EP2016/082614T patent/LT3394089T/lt unknown
- 2016-12-23 AU AU2016375183A patent/AU2016375183B2/en active Active
- 2016-12-23 CN CN201680075659.0A patent/CN108699121B/zh active Active
- 2016-12-23 DK DK16828743.1T patent/DK3394089T3/da active
- 2016-12-23 SI SI201631377T patent/SI3394089T1/sl unknown
- 2016-12-23 JP JP2018533148A patent/JP6771568B2/ja active Active
- 2016-12-23 CA CA3009393A patent/CA3009393C/en active Active
- 2016-12-23 PT PT168287431T patent/PT3394089T/pt unknown
- 2016-12-23 BR BR112018012844A patent/BR112018012844A2/pt active Search and Examination
- 2016-12-23 HR HRP20211608TT patent/HRP20211608T1/hr unknown
- 2016-12-23 WO PCT/EP2016/082614 patent/WO2017109208A1/en active Application Filing
- 2016-12-23 ES ES16828743T patent/ES2895256T3/es active Active
- 2016-12-23 US US16/065,272 patent/US10640540B2/en active Active
- 2016-12-23 MX MX2018007392A patent/MX2018007392A/es unknown
- 2016-12-23 IL IL260182A patent/IL260182B2/en unknown
- 2016-12-23 EP EP16828743.1A patent/EP3394089B1/en active Active
- 2016-12-23 KR KR1020187020929A patent/KR20180091097A/ko not_active Application Discontinuation
- 2016-12-23 HU HUE16828743A patent/HUE056019T2/hu unknown
- 2016-12-23 PL PL16828743T patent/PL3394089T3/pl unknown
-
2020
- 2020-03-20 US US16/825,711 patent/US11591378B2/en active Active
- 2020-05-01 JP JP2020081070A patent/JP2020167999A/ja not_active Withdrawn
Patent Citations (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0097440A1 (en) * | 1982-06-14 | 1984-01-04 | The Upjohn Company | Method and kit for removing and assaying complement system fragments |
CN1138863A (zh) * | 1993-09-24 | 1996-12-25 | 赛托医学有限公司 | 封阻补体激活的嵌合蛋白 |
CN101443050A (zh) * | 2006-03-08 | 2009-05-27 | 阿切埃米克斯有限公司 | 治疗视觉失调中使用的补体结合适体和抗-c5药物 |
CN101754980A (zh) * | 2007-05-23 | 2010-06-23 | 健泰科生物技术公司 | 补体相关眼疾的预防和治疗 |
CN101848937A (zh) * | 2007-11-02 | 2010-09-29 | 诺瓦提斯公司 | 调节补体组分的分子和方法 |
CN102271703A (zh) * | 2008-11-10 | 2011-12-07 | 阿雷克森制药公司 | 用于治疗补体相关障碍的方法和组合物 |
CN102481348A (zh) * | 2009-08-31 | 2012-05-30 | Ibc药品公司 | 双特异性免疫细胞因子停靠-和-加锁(dnl)复合物及其治疗性用途 |
CN103781492A (zh) * | 2011-04-08 | 2014-05-07 | 莱斯特大学 | 用于治疗与masp-2依赖性补体活化相关的状况的方法 |
CN102978204A (zh) * | 2011-11-11 | 2013-03-20 | 张康 | Cfhr1基因型、高密度脂蛋白上cfhr1及氧化磷酸胆碱的测定试剂盒及测定方法 |
WO2013142362A1 (en) * | 2012-03-19 | 2013-09-26 | The Trustees Of The University Of Pennsylvania | Regulator of complement activation and uses thereof |
WO2015092335A2 (fr) * | 2013-12-20 | 2015-06-25 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Proteines recombinantes possedant une activite de facteur h |
Non-Patent Citations (1)
Title |
---|
Factor H–related protein 1 (CFHR-1) inhibits complement C5 convertase activity and terminal complex formation;Stefan Heinen等;《BLOOD》;20091231;第114卷(第12期);摘要部分,第2444页第4段,第2445页右栏第2段,第2446页左栏第2-5段,右栏第1-2段 * |
Also Published As
Publication number | Publication date |
---|---|
EP3394089A1 (en) | 2018-10-31 |
LT3394089T (lt) | 2021-11-25 |
US20200277347A1 (en) | 2020-09-03 |
MX2018007392A (es) | 2018-08-15 |
IL260182A (en) | 2018-07-31 |
PL3394089T3 (pl) | 2022-01-17 |
DK3394089T3 (da) | 2021-11-01 |
IL260182B2 (en) | 2023-12-01 |
JP2020167999A (ja) | 2020-10-15 |
CN108699121A (zh) | 2018-10-23 |
WO2017109208A1 (en) | 2017-06-29 |
ES2895256T3 (es) | 2022-02-18 |
PT3394089T (pt) | 2021-11-03 |
CA3009393C (en) | 2023-08-22 |
JP2019508022A (ja) | 2019-03-28 |
KR20180091097A (ko) | 2018-08-14 |
US20190300589A1 (en) | 2019-10-03 |
AU2016375183B2 (en) | 2021-01-21 |
HRP20211608T1 (hr) | 2022-01-21 |
IL260182B1 (en) | 2023-08-01 |
BR112018012844A2 (pt) | 2018-12-04 |
US11591378B2 (en) | 2023-02-28 |
JP6771568B2 (ja) | 2020-10-21 |
AU2016375183A1 (en) | 2018-07-19 |
EP3394089B1 (en) | 2021-07-28 |
HUE056019T2 (hu) | 2022-01-28 |
US10640540B2 (en) | 2020-05-05 |
CA3009393A1 (en) | 2017-06-29 |
SI3394089T1 (sl) | 2021-12-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN108699121B (zh) | 用于抑制补体激活的多肽 | |
Mastellos et al. | Compstatin: a C3‐targeted complement inhibitor reaching its prime for bedside intervention | |
Durey et al. | Anti-complement-factor H-associated glomerulopathies | |
Parente et al. | Complement factor H in host defense and immune evasion | |
US6846649B1 (en) | Recombinant human mannan-binding lectin | |
Tegla et al. | Membrane attack by complement: the assembly and biology of terminal complement complexes | |
Pazgier et al. | Human defensins: synthesis and structural properties | |
Lesher et al. | Properdin in complement activation and tissue injury | |
Ricklin et al. | Therapeutic control of complement activation at the level of the central component C3 | |
S Laursen et al. | Structure, function and control of complement C5 and its proteolytic fragments | |
US9540626B2 (en) | Regulator of complement activation and uses thereof | |
Emlen et al. | Therapeutic complement inhibition: new developments | |
JP2019523238A (ja) | 補体阻害剤及びその使用 | |
US20150031621A1 (en) | Method for purification of complement factor h | |
CA2811291C (en) | Treatment of chronic nephropathies using soluble complement receptor type i (scr1) | |
JP2021527439A (ja) | 補体阻害剤及びその使用 | |
Serrano et al. | The hidden side of complement regulator C4BP: dissection and evaluation of its immunomodulatory activity | |
Hardy et al. | Soluble complement receptor 1 therapeutics | |
Li et al. | Defensins: Key Molecules in Ocular Surface Protection | |
Roumeninaa et al. | Molecular characterization and clinical history of a homozygous functional C1q abnormality | |
EP3154639A1 (en) | Uses of humanized cobra venom factor for reducing or preventing immunogenicity |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
CB02 | Change of applicant information |
Address after: Freiburg, Bryce highland, Germany Applicant after: Ailifa Co.,Ltd. Address before: Freiburg, Bryce highland, Germany Applicant before: GREENOVATION BIOTECH GmbH |
|
CB02 | Change of applicant information | ||
GR01 | Patent grant | ||
GR01 | Patent grant |