WO2015092335A2 - Proteines recombinantes possedant une activite de facteur h - Google Patents
Proteines recombinantes possedant une activite de facteur h Download PDFInfo
- Publication number
- WO2015092335A2 WO2015092335A2 PCT/FR2014/053484 FR2014053484W WO2015092335A2 WO 2015092335 A2 WO2015092335 A2 WO 2015092335A2 FR 2014053484 W FR2014053484 W FR 2014053484W WO 2015092335 A2 WO2015092335 A2 WO 2015092335A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- factor
- sequence
- domains
- scr20
- scr1
- Prior art date
Links
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 title claims abstract description 85
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 85
- 230000000694 effects Effects 0.000 title claims abstract description 34
- 102000016550 Complement Factor H Human genes 0.000 claims abstract description 142
- 108010053085 Complement Factor H Proteins 0.000 claims abstract description 142
- 101100365087 Arabidopsis thaliana SCRA gene Proteins 0.000 claims description 152
- 101150105073 SCR1 gene Proteins 0.000 claims description 152
- 101100134054 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NTG1 gene Proteins 0.000 claims description 152
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 77
- 108090000623 proteins and genes Proteins 0.000 claims description 73
- 102000004169 proteins and genes Human genes 0.000 claims description 67
- 239000013598 vector Substances 0.000 claims description 57
- 150000007523 nucleic acids Chemical class 0.000 claims description 47
- 108020004707 nucleic acids Proteins 0.000 claims description 44
- 102000039446 nucleic acids Human genes 0.000 claims description 44
- NEEVCWPRIZJJRJ-LWRDCAMISA-N 5-(benzylideneamino)-6-[(e)-benzylideneamino]-2-sulfanylidene-1h-pyrimidin-4-one Chemical compound C=1C=CC=CC=1C=NC=1C(=O)NC(=S)NC=1\N=C\C1=CC=CC=C1 NEEVCWPRIZJJRJ-LWRDCAMISA-N 0.000 claims description 38
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 37
- 102100030386 Granzyme A Human genes 0.000 claims description 33
- 101001009599 Homo sapiens Granzyme A Proteins 0.000 claims description 33
- 102100034088 40S ribosomal protein S4, X isoform Human genes 0.000 claims description 24
- 101000732165 Homo sapiens 40S ribosomal protein S4, X isoform Proteins 0.000 claims description 24
- 241000282414 Homo sapiens Species 0.000 claims description 16
- 230000009261 transgenic effect Effects 0.000 claims description 5
- 235000018102 proteins Nutrition 0.000 description 66
- 239000012634 fragment Substances 0.000 description 51
- 210000004027 cell Anatomy 0.000 description 43
- 235000001014 amino acid Nutrition 0.000 description 35
- 150000001413 amino acids Chemical class 0.000 description 33
- 101000737574 Homo sapiens Complement factor H Proteins 0.000 description 27
- 102000045512 human CFH Human genes 0.000 description 26
- 101000633613 Homo sapiens Probable threonine protease PRSS50 Proteins 0.000 description 25
- 102100029523 Probable threonine protease PRSS50 Human genes 0.000 description 25
- 101000668165 Homo sapiens RNA-binding motif, single-stranded-interacting protein 1 Proteins 0.000 description 19
- 101000668170 Homo sapiens RNA-binding motif, single-stranded-interacting protein 2 Proteins 0.000 description 19
- 102100039692 RNA-binding motif, single-stranded-interacting protein 1 Human genes 0.000 description 19
- 102100039690 RNA-binding motif, single-stranded-interacting protein 2 Human genes 0.000 description 19
- 230000029087 digestion Effects 0.000 description 16
- 238000010276 construction Methods 0.000 description 15
- 108010067641 Complement C3-C5 Convertases Proteins 0.000 description 14
- 102000016574 Complement C3-C5 Convertases Human genes 0.000 description 14
- 238000000034 method Methods 0.000 description 14
- 238000000746 purification Methods 0.000 description 13
- 230000009089 cytolysis Effects 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 10
- 230000014509 gene expression Effects 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 230000004071 biological effect Effects 0.000 description 9
- 201000010099 disease Diseases 0.000 description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 9
- 210000003743 erythrocyte Anatomy 0.000 description 9
- 241001494479 Pecora Species 0.000 description 7
- 239000000872 buffer Substances 0.000 description 7
- 230000024203 complement activation Effects 0.000 description 7
- 238000010494 dissociation reaction Methods 0.000 description 7
- 230000005593 dissociations Effects 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 102100022133 Complement C3 Human genes 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 6
- 101000901154 Homo sapiens Complement C3 Proteins 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 238000004440 column chromatography Methods 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- 238000007792 addition Methods 0.000 description 5
- 239000000853 adhesive Substances 0.000 description 5
- 230000001070 adhesive effect Effects 0.000 description 5
- 210000004899 c-terminal region Anatomy 0.000 description 5
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 206010064930 age-related macular degeneration Diseases 0.000 description 4
- 230000033228 biological regulation Effects 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 208000002780 macular degeneration Diseases 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 230000028327 secretion Effects 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- 108090000056 Complement factor B Proteins 0.000 description 3
- 102000003712 Complement factor B Human genes 0.000 description 3
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 3
- 108060003951 Immunoglobulin Proteins 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 241000124008 Mammalia Species 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 108020004566 Transfer RNA Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000001133 acceleration Effects 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 102000018358 immunoglobulin Human genes 0.000 description 3
- 229940072221 immunoglobulins Drugs 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 230000008520 organization Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000003146 transient transfection Methods 0.000 description 3
- JXYWFNAQESKDNC-BTJKTKAUSA-N (z)-4-hydroxy-4-oxobut-2-enoate;2-[(4-methoxyphenyl)methyl-pyridin-2-ylamino]ethyl-dimethylazanium Chemical compound OC(=O)\C=C/C(O)=O.C1=CC(OC)=CC=C1CN(CCN(C)C)C1=CC=CC=N1 JXYWFNAQESKDNC-BTJKTKAUSA-N 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- 108091026890 Coding region Proteins 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
- 108010023321 Factor VII Proteins 0.000 description 2
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 101150102573 PCR1 gene Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000005349 anion exchange Methods 0.000 description 2
- 230000002391 anti-complement effect Effects 0.000 description 2
- 108010008730 anticomplement Proteins 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004900 c-terminal fragment Anatomy 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- 230000006037 cell lysis Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000009109 curative therapy Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000013024 dilution buffer Substances 0.000 description 2
- 239000000539 dimer Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- 239000003446 ligand Substances 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000004898 n-terminal fragment Anatomy 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000008707 rearrangement Effects 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- JARGNLJYKBUKSJ-KGZKBUQUSA-N (2r)-2-amino-5-[[(2r)-1-(carboxymethylamino)-3-hydroxy-1-oxopropan-2-yl]amino]-5-oxopentanoic acid;hydrobromide Chemical compound Br.OC(=O)[C@H](N)CCC(=O)N[C@H](CO)C(=O)NCC(O)=O JARGNLJYKBUKSJ-KGZKBUQUSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108010003529 Alternative Pathway Complement C3 Convertase Proteins 0.000 description 1
- 101100519158 Arabidopsis thaliana PCR2 gene Proteins 0.000 description 1
- 208000035913 Atypical hemolytic uremic syndrome Diseases 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- 101100275770 Caenorhabditis elegans cri-3 gene Proteins 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 102000011632 Caseins Human genes 0.000 description 1
- 108010076119 Caseins Proteins 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 108010078015 Complement C3b Proteins 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102100036745 Epididymal secretory glutathione peroxidase Human genes 0.000 description 1
- 101710147929 Epididymal secretory glutathione peroxidase Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 102000017177 Fibromodulin Human genes 0.000 description 1
- 108010013996 Fibromodulin Proteins 0.000 description 1
- 206010018370 Glomerulonephritis membranoproliferative Diseases 0.000 description 1
- 206010018372 Glomerulonephritis membranous Diseases 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 208000032759 Hemolytic-Uremic Syndrome Diseases 0.000 description 1
- 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 1
- 241000282412 Homo Species 0.000 description 1
- 101100335629 Homo sapiens FH gene Proteins 0.000 description 1
- 101001082142 Homo sapiens Pentraxin-related protein PTX3 Proteins 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100023915 Insulin Human genes 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 102000008192 Lactoglobulins Human genes 0.000 description 1
- 108010060630 Lactoglobulins Proteins 0.000 description 1
- 208000005777 Lupus Nephritis Diseases 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000588650 Neisseria meningitidis Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- BFHAYPLBUQVNNJ-UHFFFAOYSA-N Pectenotoxin 3 Natural products OC1C(C)CCOC1(O)C1OC2C=CC(C)=CC(C)CC(C)(O3)CCC3C(O3)(O4)CCC3(C=O)CC4C(O3)C(=O)CC3(C)C(O)C(O3)CCC3(O3)CCCC3C(C)C(=O)OC2C1 BFHAYPLBUQVNNJ-UHFFFAOYSA-N 0.000 description 1
- 102100027351 Pentraxin-related protein PTX3 Human genes 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 206010063897 Renal ischaemia Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101000767160 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Intracellular protein transport protein USO1 Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108010027252 Trypsinogen Proteins 0.000 description 1
- 102000018690 Trypsinogen Human genes 0.000 description 1
- 101710087237 Whey acidic protein Proteins 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000002587 anti-hemolytic effect Effects 0.000 description 1
- 238000007845 assembly PCR Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 201000001981 dermatomyositis Diseases 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000011026 diafiltration Methods 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 230000005714 functional activity Effects 0.000 description 1
- 108010044804 gamma-glutamyl-seryl-glycine Proteins 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000008588 hemolysis Effects 0.000 description 1
- 230000002008 hemorrhagic effect Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 206010025135 lupus erythematosus Diseases 0.000 description 1
- 229940118019 malondialdehyde Drugs 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 239000003471 mutagenic agent Substances 0.000 description 1
- 231100000707 mutagenic chemical Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 244000045947 parasite Species 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- LQRJAEQXMSMEDP-XCHBZYMASA-N peptide a Chemical compound N([C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](C)C(=O)NCCCC[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C/C=1C=CC=CC=1)C(N)=O)C(=O)C(\NC(=O)[C@@H](CCCCN)NC(=O)CNC(C)=O)=C\C1=CC=CC=C1 LQRJAEQXMSMEDP-XCHBZYMASA-N 0.000 description 1
- 201000001245 periodontitis Diseases 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 230000003285 pharmacodynamic effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108020001580 protein domains Proteins 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000010474 transient expression Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- -1 ΓΕΡΟ Substances 0.000 description 1
- 235000021247 β-casein Nutrition 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/472—Complement proteins, e.g. anaphylatoxin, C3a, C5a
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
Definitions
- the invention relates to a recombinant protein having an H factor activity.
- Factor H is a 155 kDa plasma protein whose main function is the regulation of complement activity induced by the alternative pathway.
- Factor H is composed of 20 short repeat consensus (SCR) domains (also called CCP or SHUSHI) of about 60 amino acids linked together by a short linker sequence of 3 to 8 amino acids.
- SCR1-4 domains carry the activity of accelerating the dissociation of C3 and C5 convertases and the factor I regulatory activity which allows the inactivation of the C3b protein.
- SCR1-4 domains carry the activity of accelerating the dissociation of C3 and C5 convertases and the factor I regulatory activity which allows the inactivation of the C3b protein.
- These N-terminal domains are sufficient to regulate the activity of C3 convertase in the liquid phase, but the SCR19-20 domains are necessary for the activity of factor H on the cell surface.
- the other Factor H SCR domains may also contribute more or less directly to the activity of the factor H, some containers of the binding sites for other molecules such as heparan sulfates and glycosaminoglycans, pentraxins (CRP, PTX3), fibromodulin or malondialdehyde. Some of these domains also contain glycosylation sites that may contribute to the half-life of the molecule and the efficiency of its production in recombinant form, others may have an important role for the three-dimensional conformation of factor H, as for example, SCR12, SCR13, SCR14 domains which confer on the H factor a particular hairpin-like folding (Schmidt et al., J. Mol Biol., 2010 January 08; 395 (1): 105-122).
- Factor H is a promising therapeutic factor for the treatment of many diseases related to dense deposits of C3 or complement activation or uncontrolled inflammation.
- AMD age-related macular degeneration
- type 2 GNMP Glomerulonephritis Membrano -Proliferate
- atypical HUS Uremic Hemorrhagic Syndrome
- certain autoimmune diseases the use of Factor H could be limited due to some disadvantages: bioavailability, pharmacokinetics and pharmacodynamics, immunogenicity, heparan sulfate binding, binding to unidentified ligands, binding to pathogens allowing immune escape (S. pneumoniae, N.meningitidis, ...) and These disadvantages are related to the molecular properties of the factor H and thus to the organization of the SCR domains of the factor H.
- a factor H derivative combining the CRI to SCR4 S domains with the S CRI 9 and SCR20 H factor domains has been proposed.
- this factor H derivative constructed to direct the activity of factor H on the cell surface the two domains of complement regulation and surface recognition are linked together by the 6 naturally occurring amino acids found between the fourth domain cysteine. SCR4 and the first cysteine of the SCR19 domain.
- the Applicant here responds to this need by proposing recombinant proteins derived from factor H having a rearrangement of the number and organization of SCR domains of factor H which retain an activity of factor H.
- the recombinant proteins according to the invention comprise at least one the SCR1-4 domains (SCR1, SCR2, SCR3 and SCR4, in that order), SCR19 and SCR20 of the H factor.
- An object of the invention therefore relates to a recombinant protein having a factor H activity, comprising, from the N-terminus to the C-terminus, a first amino acid sequence comprising at least the domains SCR1 to SCR4 of factor H, and a second amino acid sequence comprising at least the domains SCR19 and SCR20 of factor H, said recombinant protein not being a natural factor H, and provided that if the first sequence consists of domains SCR1 to SCR4 and the second sequence consists of domains SCR19 and SCR20, the linker is a synthetic linker.
- the subject of the invention is also a nucleic acid encoding a recombinant protein according to the invention, a vector comprising such a nucleic acid, a host cell comprising such a nucleic acid or such a vector and a transgenic animal whose genome comprises such a nucleic acid.
- the invention also relates to a recombinant protein according to the invention, for the treatment of diseases due to uncontrolled inflammation or deposition of uncontrolled C3 convertase. More generally, the recombinant protein according to the invention may be useful for the treatment of any disease for which anti-complement factor H activity is beneficial.
- the present invention relates to a factor H-derived recombinant protein having a rearrangement of the SCR domains, both in terms of their number and their organization, said recombinant protein comprising, in this order, a first sequence of acids. amino acids comprising the H-factor SCR1-4 domains and a second amino acid sequence comprising the F-factor SCR19 and SCR20 domains. The first and / or second amino acid sequence may further comprise one or more other factor SCR domains. H rearranged
- one or more SCR domains of the factor H may be present in multiple copies.
- a recombinant protein according to the invention may include one, two, three, or more than three copies of the same SCR, for example one, two, three or more than three copies of the domain SCR1, SCR2, SCR3, SCR4 , SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and / SCR20 of the factor H.
- the recombinant protein according to The invention comprises one, two, three or more than three copies of the SCR7 domain of factor H, in particular the SCR7 domain of the Y402 variant of factor H.
- Another variant includes a recombinant protein comprising multiple copies of a set of SCR domains.
- the recombinant protein according to the invention may comprise one, two, three or more repetitions of SCR1 to SCR4 domains, i.e. the protein comprises the (SCR1-SCR4) n motif, n being an integer equal to 1, 2, 3 or greater than 3, in particular n being equal to 1, 2 or 3.
- the domain or set of domains present in multiple copies are not repetitions domains or set of domains, but may be present in the recombinant protein separated by other SCR domains.
- the invention relates in particular to recombinant proteins comprising, in this order, the domains SCR1-SCR4, SCR7, SCR7 and SCR19-SCR20, or the SCR1-SCR4, SCR7, SCR1-SCR4, and SCR19-20 domains, or any other possible combination of these domains.
- the factor H from which the protein according to the invention is derived can be any factor H whose activity is that of the natural factor H.
- H factor is understood to mean any protein having the amino acid sequence of human native H factor or from another species (for example bovine, porcine, canine or murine).
- the recombinant protein is derived from human factor H.
- the term also includes any recombinant, derivative or mutant having a sequence substantially homologous to native H-factor.
- substantially homologous sequence includes any sequence subject to one or more substitutions, additions and / or deletions, preferably conservative.
- substitutions, additions and / or deletions express any replacement, addition or deletion of amino acid residue by another, without major alteration of the general conformation and / or biological activity of factor H.
- Conservative substitution includes , but not limited to, replacement with an amino acid having similar properties (such as, for example, shape, polarity, hydrogen bonding potential, acidity, basicity, hydrophobicity and the like). Amino acids with similar properties are well known in the art.
- H factor further includes natural allelic variations and / or factor H isoforms found naturally in individuals of the same species, and any form or degree of glycosylation or other post-translational modification.
- H-factor also included in the term "H-factor" are homologues or derivatives of factor H which exhibit the same activity, or a higher biological activity with respect to the activity of the wild-type form and / or which have a sequence identity of from at least 80%, preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 98%, more preferably at least 99%.
- the factor H variant used to design the recombinant protein of the present invention may in particular be a variant mentioned in the website http://www.uniprot.org/uniprot/P08603.
- Factor H complies with factor B for C3b binding and accelerates the dissociation of the already formed alternating C3 convertase (C3bBb). It acts as a cofactor of Factor I in the proteolysis of C3b, free or bound to the cell surface, which leads to the inactive form C3bi.
- the immune complexes composed of an antigen-antibody complex associated with the complement component C3b or the activating factors of the alternative complement pathway (bacterial surfaces, infected cells, yeasts, parasites, lipopolysaccharides, endotoxins) can no longer activate the subsequent cascade of complement (C5-C9 components).
- the term "biological activity" of factor H thus includes here the ability to inhibit C3 convertase and / or to act as a factor I co-factor, resulting in the inhibition of activation of the complement cascade.
- the recombinant protein retains the biological activity of a plasma human factor H.
- the biological activity of human plasma H-factor includes regulation of Factor I activity, inhibition of alternating C3 convertase formation and acceleration of C3 convertase dissociation.
- said recombinant protein retains the biological activity of a plasma human factor H to accelerate the dissociation of C3 convertase.
- the amount of dissociated C3 convertase can be determined as a function of the concentration of recombinant protein added.
- the IC50 is determined from the equation calculated for a variable slope sigmoid.
- said recombinant protein retains the biological activity of a plasma H factor to regulate factor I activity.
- factor H can be evaluated by measuring the protection activity of red blood cells against lysis by complement according to procedures well known in the state of the art.
- the sequence SEQ ID NO: 1 represents the amino acid sequence of the Y402 variant of human factor H in which the amino acid at position 402 is a tyrosine. This sequence does not include a signal peptide.
- the sequence SEQ ID NO: 2 represents the amino acid sequence of the human factor H402 variant in which the amino acid at position 402 is a histidine. This sequence does not include a signal peptide.
- one or more SCR domains of the recombinant protein according to the invention are derived from one or the other of these two variants.
- the recombinant protein comprises the SCR7 domain of the Y402 variant of the factor H.
- the first sequence of the recombinant protein comprises the S domains CRI to SCR7, the S domains CRI to SCR8 or S domains CRI to SCR9 of the factor H, the domain SCR7 in this first sequence being the domain SCR7 of the variant Y402 of the factor H.
- the other domains SCR can be derived from variant Y402 of factor H or another variant of factor H.
- the first sequence can in particular be combined with a second amino acid sequence comprising domains SCR19 and SCR20, SCR18 to SCR20, SCR17 to SCR20 or SCR16 to SCR20 of factor H, the second amino acid sequence being in particular derived from the Y402 variant or the H402 factor H variant.
- These recombinant proteins include the Y402 polymorphism present in SCR7.
- the recombinant protein according to the invention comprises, in this order, the domains SCR1, SCR2, SCR3, SCR4, SCR19 and SCR20 of the factor H.
- the domains SCR4 and SCR19 are linked between them by an unnatural (or synthetic) linker consisting of a sequence comprising between 2 and 20 amino acids.
- the term "non-natural (or synthetic) linker” refers in particular to a linker that does not correspond to the sequence found between the fourth cysteine of the SCR4 domain and the first cysteine of the SCR19 domain.
- the amino acid sequence of the linker is chosen such that it is encoded by a nucleic acid which, when introduced into a cloning and / or expression vector, comprises a unique restriction site ( see below).
- the linker may be of GASG sequence (SEQ ID NO: 3).
- the first or second amino acid sequence of the recombinant protein according to the invention comprises one or more additional SCR domains of the factor H chosen from among the domains SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR11, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17 and SCR18.
- the order of these domains in the recombinant protein may correspond to the order of the same domains in the natural factor H. Alternatively, the order of the domains may be different from that of the natural H factor.
- the amino acid sequence of the SCR1 domain (amino acids 21 to 80) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 4 (CNELPPRRNTEILTGSWSDQTYPEGTQAIYKCRPGYRSLGNVIMVCRKGEWVALNPL R C).
- the sequence of the SCR1 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, especially at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 4.
- the sequence of the SCR1 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 4.
- the amino acid sequence of the SCR2 domain (amino acids 85 to 141) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 5 (CGHPGDTPFGTFTLTGGNVFEYGVKAVYTCNEGYQLLGEINYRECDTDGWTNDIPIC ).
- the sequence of the SCR2 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99% identity with the sequence SEQ ID NO: 5.
- the sequence of the SCR2 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 5.
- the amino acid sequence of the SCR3 domain (amino acids 146 to 205) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 6 (CLPVTAPENGKIVSSAMEPDREYHFGQAVRFVCNSGYKIEGDEEMHCSDDGFWSKE KPKC).
- the sequence of the SCR3 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 6.
- the sequence of the SCR3 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 6.
- amino acid sequence of the SCR4 domain (amino acids 210 to 262) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 7 (CKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSC).
- sequence SEQ ID NO: 7 CKSPDVINGSPISQKIIYKENERFQYKCNMGYEYSERGDAVCTESGWRPLPSC.
- sequence of the SCR4 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99% identity with the sequence SEQ ID NO: 7.
- the sequence of the SCR4 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 7.
- the amino acid sequence of the SCR5 domain (amino acids 266 to 320) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 8 (CDNPYIPNGDYSPLRIKHRTGDEITYQCRNGFYPATRGNTAKCTSTGWIPAPRC).
- the sequence of the SCR5 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 8.
- the sequence of the SCR5 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 8.
- amino acids 325 to 385 of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 9 (CDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGW SPAVPC).
- sequence SEQ ID NO: 9 CDYPDIKHGGLYHENMRRPYFPVAVGKYYSYYCDEHFETPSGSYWDHIHCTQDGW SPAVPC.
- the sequence of the SCR6 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99%. % identity with the sequence SEQ ID NO: 9.
- the sequence of the SCR6 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 9.
- amino acids 389 to 442 of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 10 (CYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRC).
- sequence SEQ ID NO: 10 CYFPYLENGYNQNYGRKFVQGKSIDVACHPGYALPKAQTTVTCMENGWSPTPRC.
- the sequence of the SCR7 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, especially at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 10.
- the sequence of the SCR7 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 10.
- amino acids 448 to 505 of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 11 (CSKSSIDIENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTC).
- sequence SEQ ID NO: 11 CSKSSIDIENGFISESQYTYALKEKAKYQCKLGYVTADGETSGSITCGKDGWSAQPTC.
- the sequence of the SCR8 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 1.
- the sequence of the SCR8 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 11.
- the amino acid sequence of the SCR9 domain (amino acids 509 to 564) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 12 (CDIPVFMNARTKNDFTWFKLNDTLDYECHDGYESNTGSTTGSIVCGYNGWSDLPIC).
- the sequence of the SCR9 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 12.
- the sequence of the SCR9 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 12.
- amino acids 569 to 623 of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 13 (CELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPIC).
- sequence SEQ ID NO: 13 CELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPIC.
- sequence SEQ ID NO: 13 CELPKIDVHLVPDRKKDQYKVGEVLKFSCKPGFTIVGPNSVQCYHFGLSPDLPIC.
- the sequence of the SCR10 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 13.
- the sequence of the domain SCR10 introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 13.
- the amino acid sequence of the SCR11 domain (amino acids 630 to 674) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 14 (CGPPPELLNGNVKEKTKEEYGHSEVVEYYCNPRFLMKGPNKIQCVDGEWTTLPVC).
- the sequence of the SCR11 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 14.
- the sequence of the SCR11 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 14.
- amino acid sequence of the SCR12 domain (amino acids 691 to 744) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 15 (CGDIPELEHGWAQLSSPPYYYGDSVEFNCSESFTMIGHRSITCIHGVWTQLPQC).
- sequence SEQ ID NO: 15 CCDIPELEHGWAQLSSPPYYYGDSVEFNCSESFTMIGHRSITCIHGVWTQLPQC.
- the sequence of the SCR12 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 15.
- the sequence of the SCR12 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 15.
- amino acids 753 to 803 of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 16 (CKSSNLIILEEHLKNKKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPEVNC).
- sequence SEQ ID NO: 16 CKSSNLIILEEHLKNKKEFDHNSNIRYRCRGKEGWIHTVCINGRWDPEVNC.
- the sequence of the SCR13 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 16.
- the sequence of the SCR13 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 16.
- the amino acid sequence of domain SCR14 (amino acids 811 to 864) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 17 (CPPPPQIPNSHNMTTTLNYRDGEKVSVLCQENYLIQEGEEITCKDGRWQSIPLC).
- the sequence of the SCR14 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 17.
- the sequence of the SCR14 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 17.
- the amino acid sequence of domain SCR15 (amino acids 869 to 926) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 18 (CSQPPQIEHGTINSSRSSQESYAHGTKLSYTCEGGFRISEENETTCYMGKWSSPPQC).
- the sequence of the SCR15 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 18.
- the sequence of the SCR15 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 18.
- amino acid sequence of domain SCR16 (amino acids 931 to 984) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 19 (CKSPPEISHGVVAHMSDSYQYGEEVTYKCFEGFGIDGPAIAKCLGEKWSHPPSC).
- sequence SEQ ID NO: 19 CKSPPEISHGVVAHMSDSYQYGEEVTYKCFEGFGIDGPAIAKCLGEKWSHPPSC.
- the sequence of the SCR16 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 19.
- the sequence of the SCR16 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 19.
- amino acid sequence of domain SCR17 (amino acids 989 to 1043) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 20 (CLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRWTGRPTC).
- sequence SEQ ID NO: 20 CLSLPSFENAIPMGEKKDVYKAGEQVTYTCATYYKMDGASNVTCINSRWTGRPTC.
- sequence of the SCR17 domain introduced into the protein according to the invention has at least 85%, especially at least 90%, especially at least 95%, especially at least 99% identity with the sequence SEQ ID NO: 20.
- the sequence of the SCR17 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 20.
- the amino acid sequence of the SCR18 domain (amino acids 1048 to 1102) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 21 (CVNPPTVQNAYIVSRQMSKYPSGERVRYQCRSPYEMFGDEEVMCLNGNWTEPPQC).
- the sequence of the SCR18 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, especially at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 21.
- the sequence of the SCR18 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 21.
- the amino acid sequence of domain SCR19 (amino acids 1109 to 1163) of the human factor H variant Y402 is represented by the sequence SEQ ID NO: 22 (CGPPPPPIDNGDITSFPLSVYAPASSVEYQCQNLYQLEGNKRITCR GQWSEPPKC).
- the sequence of the SCR19 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 22.
- the sequence of the SCR18 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 22.
- amino acid sequence of domain SCR20 (amino acids 1167 to 1231) of human factor H variant Y402 is represented by the sequence SEQ ID NO: 23 (CVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKL EYPTCAKR).
- sequence SEQ ID NO: 23 CVISREIMENYNIALRWTAKQKLYSRTGESVEFVCKRGYRLSSRSHTLRTTCWDGKL EYPTCAKR.
- sequence of the SCR18 domain introduced into the protein according to the invention has at least 85%, in particular at least 90%, in particular at least 95%, in particular at least 99% identity with the sequence SEQ ID NO: 23.
- the sequence of the SCR20 domain introduced into the protein according to the invention is that represented in the sequence SEQ ID NO: 23.
- the first or second amino acid sequence comprises at least the SCR12, SCR13, and SCR14 domains of the H factor. In another embodiment, none of the SCR12, S CRI3, and S CRI4 domains are is present in the recombinant protein according to the invention.
- An embodiment variant of the present invention relates to a recombinant protein whose SCR domains consist, in this order, of domains SCR1, SCR2, SCR3, SCR4, SCR12, SCR13, SCR14, SCR19 and SCR20. Such a protein is represented in the sequence SEQ ID NO: 142.
- Each SCR domain included in the first or second amino acid sequence may be linked to the contiguous SCR (s) by means of the natural linker found between said SCR domains, as appropriate.
- the linker between each SCR domain of the recombinant protein may be a non-natural linker.
- the unnatural linker may consist of a sequence comprising between 2 and 20 amino acids, in particular between 3 and 8 amino acids.
- the link between the first and the second amino acid sequence can be achieved by means of a natural or synthetic linker present in the C-terminal position of the first amino acid sequence or in the N-terminal position of the second sequence. in amino acids.
- the first and second polypeptide may be linked by a GASG sequence linker (SEQ ID NO: 3).
- first and second amino acid sequences may be linked together by a linker combining the natural linker sequence according to the last domain of the first sequence (ie the C-terminal domain of the first sequence) and a linker. synthetic such as the linker GASG.
- the Natural linkers found at the C-terminal position of each of the SCR domains of the factor H are, for example: QKRP (SEQ ID NO: 143) for the SCR1 domain; EVVK (SEQ ID NO: 144) for the SCR2 domain; VEIS (SEQ ID NO: 145) for the SCR3 domain; EEKS (SEQ ID NO: 146) for the SCR4 domain; TLKP (SEQ ID NO: 147) for the SCR5 domain; LRK for the SCR6 domain; IRVKT (SEQ ID NO: 148) for the SCR7 domain; IKS for the SCR8 domain; YERE (SEQ ID NO: 149) for the SCR9 domain; KEQVQS (SEQ ID NO: 150) for the SCR10 domain; IVEEST (SEQ ID NO: 151) for the SCR1 domain l; VAIDKLKK (SEQ ID NO: 152) for the SCR12 domain; SMAQIQL (SEQ ID NO: 153) for the domain S
- the first amino acid sequence comprises the domains SCR1, SCR2, SCR3 and SCR4 of the factor H.
- the first amino acid sequence comprises the domains SCR1, SCR2, SCR3, SCR4 and SCR5 of the factor H.
- the first amino acid sequence comprises the domains SCR1, SCR2, SCR3, SCR4, SCR5 and SCR6 of the factor H.
- the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6 and SCR7 domains of the factor H.
- the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7 and SCR8 domains of the H factor.
- the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8 and SCR9 domains of the H factor.
- the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9 and SCR10 domains of the H factor.
- the first amino acid sequence comprises the domains SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9, SCR10 and SCR1, factor H.
- the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR1 and SCR12 H-factor domains.
- the first amino acid sequence comprises the SCR1, SCR2, SCR3, SCR4, SCR5, SCR6, SCR7, SCR8, SCR9, SCR10, SCR1, SCR12 and SCR13 H-factor domains.
- the second amino acid sequence comprises the domains SCR19 and SCR20 of the factor H.
- the second amino acid sequence comprises the domains SCR18, SCR19 and SCR20 of the factor H.
- the second amino acid sequence comprises the domains SCR17, SCR18, SCR19 and SCR20 of the factor H.
- the second amino acid sequence comprises the domains SCR16, SCR17, SCR18, SCR19 and SCR20 of the factor H.
- the second amino acid sequence comprises the domains SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 of the factor H.
- the second amino acid sequence comprises the domains
- the second amino acid sequence comprises the domains
- the second amino acid sequence comprises the domains
- the second amino acid sequence comprises the domains
- the second amino acid sequence comprises the SCR10, SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 domains of the H factor.
- the second amino acid sequence comprises the SCR9, SCR10, SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 domains of the H factor.
- the second amino acid sequence comprises the SCR8, SCR9, SCR10, SCR1, SCR12, SCR13, SCR14, SCR15, SCR16, SCR17, SCR18, SCR19 and SCR20 domains of the H factor.
- the recombinant protein having a factor H activity comprises the first and second amino acid sequences listed in Table 1 below. below, these sequences being separated by a linker, in particular an artificial linker, in particular the GASG linker (SEQ ID NO: 3).
- the recombinant protein having a factor H activity comprises the first and second amino acid sequences listed in Table 2 below, these sequences being separated by a linker, in particular an artificial linker, in particular the linker G AS G (SEQ ID NO: 3).
- the first sequence comprises the domains SCR1 to SCR7 of factor H
- the second sequence is chosen from the group consisting of: domains SCR9 to SCR20,
- the first sequence comprises the domains SCR1 to SCR8 and the second sequence comprises the domains SCR10 to SCR20.
- the first sequence comprises the domains SCR1 to SCR4 of the factor H and the second sequence comprises the domains SCR16 to SCR20 of the factor H, a third sequence comprising the domain SCR7 of the factor 7 being between the first and the second sequence, and being more particularly separated from these by linkers such as those described above.
- the first amino acid sequence comprises a signal peptide (PS) in the N-terminal position.
- the signal peptide can be the natural signal peptide of factor H (MRLLAKIICLMLWAICVA - SEQ ID NO: 24), the signal peptide of a protein different from factor H, or a signal peptide described in application PCT / 2001/050544, especially the peptide MRWSWIFLLLLSITSANA (SEQ ID NO: 25, or otherwise referred to as PS-MB7 thereafter).
- the natural signal peptide of a protein different from human factor H may be a signal peptide chosen from the signal peptides of all the proteins which are secreted in eukaryotes and in particular in mammals and more particularly in humans, such as those of immunoglobulins. growth factors such as ⁇ , hormones such as insulin, enzymes such as trypsinogen, coagulation factors such as prothrombin.
- growth factors such as ⁇ , hormones such as insulin, enzymes such as trypsinogen, coagulation factors such as prothrombin.
- the invention relates to one of the peptides of Table 1 in which the first amino acid sequence comprises at the N-terminal such a signal peptide, in particular the natural peptide of the factor H of sequence SEQ ID NO: 24 or the signal peptide PS-MB7 of sequence SEQ ID NO: 25.
- the recombinant protein according to the invention is chosen from one of the proteins of Table 1, the first and second sequences being separated by a linker, including a GASG linker (SEQ ID NO: 3).
- the recombinant protein according to the invention is chosen from one of the proteins listed in Table 2 below in which the first amino acid sequence comprises a signal peptide of sequence SEQ ID NO: 25 and the first and second sequences are separated by a GASG sequence linker (SEQ ID NO: 3).
- the recombinant protein according to the invention contains a first sequence comprising the domains SCR1 to SCR4 of factor H, and a second sequence chosen from the group consisting of:
- the recombinant protein according to the invention contains a first sequence comprising the domains SCR1 to SCR7 of factor H, and a second sequence comprising the SCR1 6 to SCR20 domains of the factor H, said second sequence preferably being chosen from the group consisting of:
- the invention relates to a recombinant protein as described above, the first sequence comprising the domains SCR1 to SCR8 of the factor H, and the second sequence comprising the domains SCR1 6 to SCR20 of the factor H, said second sequence preferably comprising the domains SCR10 to SCR20 of the factor H.
- the recombinant protein according to the invention contains a first sequence comprising the domains SCR1 to SCR4 of the factor H and a second sequence comprising the domains SCR1 6 to SCR20 of the factor H, a third sequence comprising the domain SCR7 of the factor H being between the first and the second sequence.
- the first sequence comprises a signal peptide of sequence SEQ ID NO: 25 and domains SCR1 to SCR7 of factor H, and the second sequence is chosen from the group consisting of:
- the first and the second sequences are separated by a GASG sequence linker.
- the first sequence comprises a signal peptide of sequence SEQ ID NO: 25 and domains SCR1 to SCR8 and the second sequence comprises domains SCR10 to SCR20.
- the first and the second sequences are separated by a GASG sequence linker.
- the first sequence comprises a signal peptide of sequence SEQ ID NO: 25 and domains SCR1 to SCR4 of factor H and the second sequence comprises domains SCR16 to SCR20 of factor H, a third sequence comprising domain SCR7 factor 7 being between the first and the second sequence, and being separated therefrom by linkers such as those described above.
- the first and the third sequences, and the third and the second sequences are separated by a GASG sequence linker.
- a sequence is represented in SEQ ID NO: 159.
- the present invention also relates to a pharmaceutical composition comprising a recombinant protein according to the invention.
- the invention also relates to a nucleic acid construct encoding a recombinant protein having a factor H activity as described above.
- nucleic acids encoding the recombinant proteins according to the invention have undergone codon optimization.
- Codon optimization aims to replace natural codons by codons whose transfer RNA (tRNA) carrying the amino acids are most common in the cell type considered.
- tRNA transfer RNA
- the mobilization of frequently encountered tRNAs has the major advantage of increasing the translation speed of the messenger RNAs (mRNA) and therefore of increasing the final titre (JM Carton et al., Protein Expr Purif, 2007).
- Sequence optimization also plays on the prediction of mRNA secondary structures that could slow down reading by the ribosomal complex. Sequence optimization also has an impact on the percentage of G / C that is directly related to the half-life of the mRNAs and therefore to their potential to be translated (Chechetkin, J. of Theoretical Biology 242, 2006 922-934 ).
- Codon optimization can be done by substitution of natural codons using codon frequency (Codon Usage Table) tables for mammals and more specifically for Homo sapiens.
- codon frequency Codon Usage Table
- a codon-optimized sequence is represented by the sequence SEQ ID NO: 85. This sequence comprises the natural signal peptide of the factor H.
- the nucleic acids according to the invention may comprise a unique restriction site between the two nucleic acid sequences encoding the first and the second acid sequence. amines of the recombinant protein according to the invention. This unique restriction site may especially correspond to the Nhe I site present in the portion encoding the GASG linker (nucleic sequence: GCCGCTAGCGCC (SEQ ID NO: 86), the underlined portion corresponding to the NheI site which corresponds to the amino acids AS mentioned above.
- This unique restriction site makes it possible to envisage an improvement in the recombinant proteins produced, in particular by facilitated introduction of one or more additional SCR domains at the level of the protein sequence corresponding to said restriction site, or by introduction of a sequence into Amino acids other than an SCR domain may be, for example, another protein domain not belonging to the natural factor H, or a linker of different size and sequence (especially a longer linker).
- nucleic acids encoding the recombinant protein according to the invention may be constructed according to any method known to those skilled in the field of molecular biology.
- said nucleic acid is constructed in two stages according to a method peculiar to the present invention.
- a nucleic acid library cloned into cloning or expression vectors may be constructed.
- Each vector of the library contains a sequence encoding one of the first or second amino acid sequences comprising the recombinant protein according to the invention.
- a vector comprising the sequences coding a first amino acid sequence comprising at least the sequences of the SCR1 to SCR4 domains of factor H (or N-Ter vector) is described.
- a vector comprising the sequences encoding a second amino acid sequence comprising at least the sequences of the domains SCR19 and SCR20 of the factor H (or C-Ter vector).
- the N-Ter and C-Ter vectors are designed to include unique restriction sites useful for excising and then assembling nucleic acid fragments encoding each of the portions of the recombinant protein into a single vector.
- the constructs allowing the expression of the proteins of Table 2 above can thus be produced from 8 N-Ter vectors and 10 C-Ter vectors. This strategy is described in the examples below.
- the nucleic acids according to the invention may also comprise any useful sequence, in particular any sequence which makes it possible to optimize the expression or the secretion of the recombinant protein or to facilitate the cloning and subcloning of the nucleic acids of the invention.
- the nucleic acid may comprise at least one restriction site, a coding sequence and / or a signal peptide coding sequence.
- it may in particular be useful to introduce a sequence encoding an amino acid unit useful for the labeling or purification of the recombinant protein, for example a histidine tag, and one or more restriction sites.
- the nucleic acid construct according to the invention comprises a sequence encoding a signal peptide chosen from:
- nucleic acid represented by the sequence SEQ ID NO: 87 and coding the natural signal peptide of the factor H, or
- nucleic acid represented by the sequence SEQ ID NO: 88 or by a sequence having at least 85%, in particular 90%, especially 95% of sequence identity with the sequence SEQ ID NO: 88, and coding the signal peptide factor H, (optimized natural PS) or
- nucleic acid encoding a natural signal peptide of a protein different from the factor H, or
- nucleic acid encoding the signal peptide encoded by the sequence SEQ ID NO: 89 (described in application PCT / FR2011 / 050544) or by a sequence having at least 85%, in particular 90%), particularly 95% identity of sequence with the sequence SEQ ID NO: 89.
- sequence SEQ ID NO: 88 or a sequence having at least 85%, especially 90%>, especially 95% of sequence identity with the sequence SEQ ID NO: 88 is a sequence obtained by codon optimization from the sequence SEQ ID NO: 87.
- nucleic acid construct encoding the recombinant protein according to the invention comprises, or consists of:
- nucleic acid encoding a signal peptide in particular a nucleic acid represented by the sequence SEQ ID NO: 87 and encoding the natural signal peptide of factor H, or a nucleic acid represented by the sequence SEQ ID NO: 88 or by a sequence having at least 85%), especially 90%, particularly 95% of sequence identity with the sequence SEQ ID NO: 88, and coding the signal peptide of factor H, (optimized natural PS) or a nucleic acid encoding a natural signal peptide a protein different from the factor H, or a nucleic acid encoding the signal peptide encoded by the sequence SEQ ID NO: 89 (described in application PCT / FR2011 / 050544) or by a sequence having at least 85%, in particular 90% especially 95% sequence identity with the sequence SEQ ID NO: 89;
- nucleic acid encoding at least the domains SCR1, SCR2, SCR3 and SCR4 of factor H; a nucleic acid encoding a linker, in particular a GASG linker (SEQ ID NO: 3), said nucleic acid encoding a linker comprising a unique restriction site, in particular an NheI site;
- nucleic acid encoding at least the domains SCR19 and SCR20 of the factor H.
- the nucleic acid construct allows the expression of a recombinant protein selected from one of the sequences SEQ ID NO: 26 to SEQ ID NO: 84.
- the invention also relates to an expression vector comprising the nucleic acid construct described above, operably linked to sequences for controlling the expression of said nucleic acid.
- the control sequences may in particular comprise a promoter (in particular a CMV promoter), an enhancer, and any sequence known to those skilled in the art useful for allowing the expression of the recombinant protein in a eukaryotic cell, in particular a mammalian cell.
- the invention also relates to a recombinant eukaryotic cell, in particular a mammalian cell, more particularly a non-human (eg a CHO cell) or human cell, transformed by means of the nucleic acid construct or the vector. expression according to the invention.
- the recombinant protein as described above is produced in the PER.C6® cell line or a HEK cell line, in particular the HEK 293F cell line.
- the PER.C6® cell line is derived from human primary retinal cells in which an Ad5 adenoviral DNA fragment that contains both the E1A gene and the ElB gene is inserted into the cells by a vector.
- This adenoviral DNA fragment makes it possible to impart immortality to the cells in which it is inserted, via the ElB protein which inhibits the p53 protein.
- the El A protein has a tropism for the hCMV viral promoter and allows its transactivation and the potentiation of the gene sequence which will be inserted 3 'of the latter and which may be the recombinant protein according to the invention.
- the present invention particularly relates to the use of the cell line PER.C6® for the implementation of a method for preparing a recombinant protein having a factor H activity, in particular one of the proteins of sequence SEQ ID NO: 26 to SEQ ID NO: 84.
- the present invention also particularly relates to the use of the HEK 293F cell line for implementing a method for preparing a recombinant protein according to the invention, in particular one of the recombinant proteins represented by one of the SEQ sequences. ID NO: 26 to SEQ ID NO: 84.
- the invention also relates to a process for the production of a recombinant protein having an activity of factor H.
- the method according to the invention in particular one of the proteins represented by the sequences SEQ ID NO: 26 to 84, said method comprising the culture of a recombinant cell according to the invention transformed by a vector comprising the nucleic acid construct according to the invention coding for said recombinant protein.
- the vector comprising such a nucleic acid may be any expression vector for eukaryotic cell lines known to those skilled in the art.
- the transformation of the cell line can be carried out by electroporation, AMAXA type nucleofection, a "gene gun” (gun gene) or else using a transfection agent known to man of the art, such as cationic agents, liposomes or polymers such as fectin or PEI agent.
- a transfection agent known to man of the art, such as cationic agents, liposomes or polymers such as fectin or PEI agent.
- the method according to the present invention comprises the following steps:
- the expression vector may contain an antibiotic resistance gene to allow selection of transfected cells upon establishment of cells stably producing the protein of interest.
- the recombinant protein according to the invention is produced at a concentration greater than or equal to 10 mg / L as detected by ELISA assay of culture supernatant, after 7 days of production in batch mode.
- the purification of the recombinant protein can be carried out by chromatographic techniques in one, two or more steps.
- the one-step purification may be an ion exchange column or an affinity column (heparin, factor H ligand or anti-factor H antibody).
- the purification in two steps may be a cation exchange column chromatography step followed by an anion exchange column chromatography step or an anion exchange column chromatography step followed by a column chromatography step cation exchange or an ion exchange column chromatography step followed by an affinity column chromatography step or an affinity column chromatography step followed by an ion exchange column chromatography step.
- the purification in addition to two steps can be performed by a combination of these different chromatographies.
- a step of diafiltration, ultrafiltration or gel filtration can be carried out in addition.
- the purity of a product after such purification can reach 99% purified product.
- the proteins according to the invention can also be produced in the milk of non-human transgenic animals, such as a goat, a rabbit, the sheep, the cow or a pig.
- the secretion of proteins by the mammary glands involves the control of the expression of the proteins according to the invention in a tissue-dependent manner.
- Such control methods are well known to those skilled in the art.
- the control of the expression is carried out by means of sequences allowing the expression of the protein towards a particular tissue of the animal. These include the promoter sequences WAP, beta-casein, beta-lactoglobulin and signal peptide sequences.
- the process for extracting proteins of interest from the milk of transgenic animals is described in patent EP 0 264 166.
- Another aspect of the invention also relates to the use of a recombinant protein according to the invention as a medicament.
- the invention also relates to the use of a recombinant protein according to the invention for the manufacture of a medicament for the treatment of a disease involving an undesirable or inappropriate complement activity, in particular for the treatment of diseases due to inflammation uncontrolled or an uncontrolled C3b deposit.
- the invention also relates to a method of treating a disease involving an undesirable or inappropriate complement activity, comprising administering a recombinant protein according to the invention to a patient in need of such treatment.
- treatment includes both curative and prophylactic treatment of the disease.
- a curative treatment is defined as a treatment resulting in a cure or treatment that alleviates, improves and / or eliminates, reduces and / or stabilizes the symptoms of an illness or the suffering it causes.
- Prophylactic treatment includes both treatment leading to disease prevention and treatment that reduces and / or delays the incidence or risk of disease.
- the invention aims in particular at the treatment of a disease selected from the group consisting of age-related macular degeneration (AMD), periodontitis, lupus erythematosus, lupus nephritis, dermatomyositis, myasthenia gravis, glomerulonephritis membranoproliferative (GNMP), psoriasis, multiple sclerosis and lesions of renal ischemia / reperfusion.
- ASD age-related macular degeneration
- GNMP glomerulonephritis membranoproliferative
- psoriasis multiple sclerosis and lesions of renal ischemia / reperfusion.
- FIG. 1 is a diagram representing the construction strategy of the N-terminal fragments of the recombinant proteins according to the invention.
- FIG. 2 is a diagram showing the construction strategy of the C-terminal fragments of the recombinant proteins according to the invention.
- Figures 3A, 3B and 3C are graphs showing the acceleration activity of C3 convertase dissociation for H-factor fragments having the highest productivity.
- Example 1 Construction of the Factor H N-ter and C-ter Fragments in the pCEP4 Plasmid
- the purpose is to subclone in different forms the N-terminal and C-terminal fragments of the factor H variant Y402 in an optimized version in the pCEP4 expression vector.
- the fragments will both be completed 5 'of a NotI site, the kozak sequence and a signal peptide, and at 3' of a His-TAG followed by the BamHI site, the difference will be made by a NheI site. located at 3 'for the N-ter fragments and at 5' for the C-ter fragments.
- Explanatory diagrams will be described in the protocol below. 1 / Vector construction pCEP4-N-ter
- the N-ter fragments are constructed by PCR from the pCDNA2001neo-MD3Y vector.
- This vector corresponds to the vector pCDNA2001neo containing the nucleic acid represented by the sequence SEQ ID NO: 90 which is an optimized sequence encoding the Y402 variant of factor H comprising an artificial signal peptide PS-MB7.
- the construction strategy is shown in Figure 1.
- the primers used are the following:
- antisense primers or P2-NT-X primers:
- Each of these primers contains, in this order of 5 'to 3': a BamHI site, 2 stop codons, a hexahistidine label, a linker (GS), a NheI site and a factor-specific sequence. These elements are represented on Figure 1.
- P2-NT-4 (SEQ ID NO: 92)
- P2-NT-7 (SEQ ID NO: 93)
- P2-NT-8 (SEQ ID NO: 94)
- P2-NT-12 (SEQ ID NO: 98)
- CMV1 and SV40-3 'UTR primers are used for sequencing all vectors. Only the pCEP4-lNTer1, pCEP4-lNTer1 2 and pCEP4-INTer1 3 vectors have additional sequencing with the MD2-3 primer.
- the C-ter fragments are constructed by PCR from the pCDNA2001neo-MD3Y vector. This vector corresponds to the vector pCDNA2001neo containing the nucleic acid represented by the sequence SEQ ID NO: 90.
- the construction strategy is shown in Figure 2.
- the primers used are the following:
- Each of these primers contains, in this order from 5 'to 3': a NotI site, a Kozack (SK) sequence, a signal peptide (PS), a NheI site and a factor H specific sequence. These elements are represented in Figure 2.
- Pl-CT-9 (SEQ ID NO: 120) CTCTAGCGGCCGCGCGCCACCATGCGATGGTCTTGGATTTTTCTGCTGCTGCTGTC TATCACTTCTGCTAACGCTGCTAGCGGCTGTGACATTCCAGTGTTTATGAACG
- This primer contains, in the 5 'to 3' orientation: a BamHI site, 2 stop codons, a hexahistidine tag, a linker (GGSG) and a specific H factor sequence.
- the series of Pl-CT-X primer (X, variable) and the P2-CT-FCTH primer are obtained by assembly PCR.
- PCR1 1-P 1 -CT-FCTH 1 (SEQ ID NO: 125)
- PCR1 Fragment P1-CT-FCTH-PCR1 (SEQ ID NO: 127)
- 59 plasmid constructs which correspond to the 59 combinations 1NTX-XCT20 of the fragments FH. These sequences are present in the pCDNA2001neo vector which allows stable expression of these molecules in the PERC6 cell line.
- the FH 1NTX-XCT20 fragments are extracted from the pCDNA2001neo vector by NotI / BamHI digestion and cloned into the pCEP4 vector which allows transient expression in HEK293F cells.
- NotI / BamHI and NheI / BamHI control digestion are notI / BamHI and NheI / BamHI control digestion.
- the 1NTX / XCT20 fragments are then extracted by NotI / BamHI digestion and reintroduced into the pCEP4 vector.
- the HEK 293F cells are subcultured at a cell concentration of 7 E 5 vc / ml.
- the cell density and viability of HEK 293F cells are determined on the day of transfection.
- the culture volume corresponding to 30 E 6 cv / ml is centrifuged.
- the supernatant is removed and the cell pellet is taken up in 28 ml of F17 culture medium (Invitrogen), transferred to a 250 ml Erlenmeyer flask and incubated at 37 ° C.
- the transfection agent and the DNA corresponding to the vector pCEP4 containing one of the sequences of the FH fragments are prepared in OptiMEM medium (Invitrogen) as follows:
- the cells are kept in culture for 7 days without adding or renewing culture medium. The 7th day, the cells were centrifuged at 3000g for 15 minutes. The cell pellets are removed and the cell supernatants containing the recombinant FH fragments are filtered in 0.22 ⁇ ⁇ frozen at -20 ° C. 3.2: Assaying human FH fragments in the culture medium by ELISA
- Antibody coating antibody human anti-Human H mutagen (The Binding Site) diluted extemporaneously in PBS buffer pH 7.4 to obtain a concentration of between 3.5 and 5.5 ⁇ g / ml.
- Dilution buffer PBS buffer - Tween-20 0.1% (vol / vol) - 0.1% BSA (w / vol)
- Samples the samples are prediluted so as to obtain a concentration close to that of the first point of range. Then dilute 1 ⁇ 2 to 1 ⁇ 2.
- Monoclonal Antibody Anti Factor H Monoclonal Immunoglobulins from Purified Mouse Anti Human Factor H (SEROTEC)
- Revealing antibody Peroxidase-labeled mouse anti-mouse IgG goat immunoglobulins (Jackson Immuno Research Laboratories).
- Stop Solution 4 N or 2 M sulfuric acid (Fisher Scientif ⁇ c).
- HRP Conjugate Marking HorseRadish Peroxidase in English, or Horseradish Peroxidase:
- TMB containing the substrate 100 ⁇ per well of TMB containing the substrate is dispensed at a regular time interval and away from any intense light. Then incubate at room temperature between 5 - 15 minutes. This time must be identical for all dosing points.
- Example 4 Characterization by SDS-PAGE of the recombinant FH fragments present in the culture supernatant (FIGS. 6A, 6B and 7).
- a volume corresponding to 1 g of recombinant factor H is diluted to 1 ⁇ 2 in 2x Laemmli buffer, heated at 95 ° C. for 5 minutes and then deposited on a linear gel. 10% polyacrylamide. After migration, the proteins contained in the gel are stained with Coomassie blue. Analysis of the polyacrylamide gels shows that the recombinant FH fragments migrate according to the apparent molecular masses expected.
- the recombinant FH fragments are diluted to a final concentration of 20 ⁇ g / ml. From this tube a range is achieved by the following successive dilutions: 1/2; 1/10; 1/4; 1/4; 1/4; 1/40. This decreasing concentration range of FH fragments is added to the C3 convertase complex formed in the wells of a 96-well plate which is then incubated 32-34 minutes at + 34 ° C. After several washes, the factor B remaining complexed with the immobilized C3 molecule at the bottom of the well is then assayed by an immunoenzymatic reaction of the ELISA type. Absorbance values obtained as a function of the concentration of added FH fragments are then processed in a nonlinear modeled system (variable slope sigmoid) to determine the IC50 value of each sample. The calculation equation of this model is as follows:
- X is the logarithm of the concentration (g / ml).
- the activity determined in the cell supernatant is shown in Figures 3A, 3B and 3C for the fragments having the highest productivity.
- the FH fragments were purified via the hexahistidine tag which was added to the c-terminus of the proteins.
- the purification was carried out in an affinity chromatography step using HisTALON (Clontech) columns which contain a resin having high affinity and specificity for polyshistidins.
- HisTALON Click-ontech
- the purification was carried out according to the supplier's recommendations (HisTALON Gravity Column Purification Kit User Manual). After purification the samples are dialyzed against PBS buffer and stored at
- the concentration of the purified recombinant FH fragments is determined by measuring the OD at 280 nm.
- the purified recombinant FH fragments are diluted to a final concentration of 150 nM and then from this tube a range is achieved by the following successive dilutions: 1/2; 1/10; 1/4; 1/4; 1/4; 1/4; 1/40.
- This decreasing concentration range of FH fragments is added to the C3 convertase complex formed in the wells of a 96-well plate which is then incubated 32-34 minutes at + 34 ° C.
- the factor B remaining complexed with the immobilized C3 molecule at the bottom of the well is then assayed by an ELISA-type immunoenzymatic reaction, using TMB (3,3 ', 5,5'-tetramethylbenzidine) or OPD (ortho-phenylenediamine) as reaction substrate.
- Absorbance values obtained as a function of the concentration of added FH fragments are then processed in a nonlinear modeled system (variable slope sigmoid) to determine the IC50 value of each sample.
- the calculation equation of this model is as follows:
- X is the logarithm of the concentration (g / ml).
- pCEP4- 1NT4-19CT20 0.0916 73% pCEP4- 1NT7- 8CT20 0.5419 72% pCEP 4-lNT9-l l CT20 0.1716 69% pCEP 4-lNT9-10CT20 0.5517 64% pCEP 4-lNT9-14 CT20 0.5698 64% pCEP4- 1NT8-14CT20 0.6454 61% pCEP4- 1NT7-10CT20 0.6015 59% pCEP4- 1NT10- 11CT20 0.6118 58% pCEP4- 1NT8- 15CT20 0.6279 58% pCEP4- 1NT4- 15CT20 0.2061 58% pCEP 4-lNT9-13 CT20 0.1663 56% pCEP4- 1NT7-15CT20 0.6393 56% pCEP 4-lNT9-19 CT20 0.2166 49% pCEP4- 1NT13- 14CT20 0.7765 45% pCEP4- 1NT4- 8CT20
- Example 7 Determination of the protection activity of lysis of red blood cells of sheep by modified Sanchez-C orrai test.
- This test makes it possible to measure the functional activity of the C-terminal portion of the purified human recombinant H factor during the development of therapeutic batches by evaluating its ability to protect sheep red blood cells from lysis induced by a depleted or factor-deficient serum. H functional.
- This test is adapted from the "Sanchez-Corral” method to measure the anti-hemolytic activity of factor H present in the plasma of patients with hemolytic uremic syndrome. (P. Sanchez-Corral, C. Gonzalez-Rubio, S. Rodriguez of Cordoba and M. Lopez-Trascasa, Molecular Immunology 41 (2004) 81-84).
- H factor depleted serum and a human plasma pool is produced in equal proportion to create the conditions for a specific lysis.
- the addition of purified human recombinant H factor ensures the protection of sheep red blood cells (lack of cell lysis) against complement-induced lysis.
- reaction buffer 10 mM Hepes, 144 mM NaCl, 7 mM MgCl 2, 10 mM EGTA, pH 7.2.
- variable volume of FH fragment or whole FH (0-30 ⁇ 1) corresponding to concentrations of 0 to 44.74pM, then 9 ⁇ of a pool of human plasma followed by 9 ⁇ of human plasma depleted in FH.
- the reaction volume is completed with PBS buffer to obtain a final volume of 1 ⁇ .
- HBS-EDTA buffer (10 mM Hepes, 144 mM NaCl, 2 mM EDTA, pH 7.2) were used to stop the reaction. After centrifugation for 5 min at 1730 g, 200 ⁇ l of supernatant are removed and deposited in a microtiter plate in order to measure the absorbance at 414 nm.
- the percentage of lysis is determined according to the formula:
- the "100% lysis" control corresponds to the maximum lysis of sheep red blood cells observed in the presence of water.
- the blank control comprises the + 50mM EDTA reaction buffer and corresponds to the spontaneous lysis of sheep red blood cells.
- the various fragments of factor H according to the invention are very active with an inhibition of specific lysis greater than the entire rFH from the lowest concentration tested.
- 1NT9-16CT20 and 1NT7-16CT20 a profile closer to the plasma H factor LP03 is very interestingly observed.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016541495A JP2017500049A (ja) | 2013-12-20 | 2014-12-19 | H因子活性を有する組換えタンパク質 |
US15/105,829 US20170190753A1 (en) | 2013-12-20 | 2014-12-19 | Recombinant proteins having factor h activity |
EP14830995.8A EP3083664A2 (fr) | 2013-12-20 | 2014-12-19 | Proteines recombinantes possedant une activite de facteur h |
CA2934558A CA2934558A1 (fr) | 2013-12-20 | 2014-12-19 | Proteines recombinantes possedant une activite de facteur h |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR1363351 | 2013-12-20 | ||
FR1363351A FR3015484A1 (fr) | 2013-12-20 | 2013-12-20 | Proteines recombinantes possedant une activite de facteur h |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2015092335A2 true WO2015092335A2 (fr) | 2015-06-25 |
WO2015092335A3 WO2015092335A3 (fr) | 2015-12-03 |
Family
ID=50489269
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2014/053484 WO2015092335A2 (fr) | 2013-12-20 | 2014-12-19 | Proteines recombinantes possedant une activite de facteur h |
Country Status (6)
Country | Link |
---|---|
US (1) | US20170190753A1 (fr) |
EP (1) | EP3083664A2 (fr) |
JP (2) | JP2017500049A (fr) |
CA (1) | CA2934558A1 (fr) |
FR (1) | FR3015484A1 (fr) |
WO (1) | WO2015092335A2 (fr) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20180055872A (ko) * | 2015-09-24 | 2018-05-25 | 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 | 보체 매개 질환 치료용 조성물 및 방법 |
CN108699121A (zh) * | 2015-12-23 | 2018-10-23 | 格林诺瓦森生物技术股份有限公司 | 用于抑制补体激活的多肽 |
WO2019243586A1 (fr) * | 2018-06-22 | 2019-12-26 | Universität Ulm | Inhibiteurs du complément et utilisations de ceux-ci |
WO2020128516A3 (fr) * | 2018-12-21 | 2020-08-06 | Gyroscope Therapeutics Limited | Facteur i de complément optimisé en termes de codons |
RU2824048C2 (ru) * | 2018-12-21 | 2024-08-01 | Джайроскоуп Терапьютикс Лимитед | Кодон-оптимизированный фактор комплемента i |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2811526T3 (es) | 2010-12-30 | 2021-03-12 | Lab Francais Du Fractionnement | Glicoles como agentes de inactivación de patógenos |
AR094778A1 (es) | 2013-02-13 | 2015-08-26 | Laboratoire Français Du Fractionnement Et Des Biotechnologies | Proteínas con glicosilación modificada y métodos para producirlas |
EP3594230A1 (fr) | 2013-02-13 | 2020-01-15 | Laboratoire Français du Fractionnement et des Biotechnologies | Anticorps anti-tnf alpha hautement galactosylés et leurs utilisations |
CN105358228A (zh) | 2013-07-05 | 2016-02-24 | 法国血液分割暨生化制品实验室 | 亲和层析基质 |
FR3038517B1 (fr) | 2015-07-06 | 2020-02-28 | Laboratoire Francais Du Fractionnement Et Des Biotechnologies | Utilisation de fragments fc modifies en immunotherapie |
GB201800620D0 (en) * | 2018-01-15 | 2018-02-28 | Univ Manchester | C3b Binding Polypeptide |
WO2021081395A1 (fr) * | 2019-10-23 | 2021-04-29 | Gemini Therapeutics Inc. | Méthodes de traitement de patients présentant des mutations de cfh avec des protéines de cfh de recombinaison |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP2044111B1 (fr) * | 2006-06-21 | 2014-08-13 | MUSC Foundation For Research Development | Ciblage du facteur h du système complémentaire destiné au traitement de maladies |
GB0922659D0 (en) * | 2009-12-24 | 2010-02-10 | Univ Edinburgh | Factor H |
US9540626B2 (en) * | 2012-03-19 | 2017-01-10 | The Trustees Of The University Of Pennsylvania | Regulator of complement activation and uses thereof |
-
2013
- 2013-12-20 FR FR1363351A patent/FR3015484A1/fr not_active Withdrawn
-
2014
- 2014-12-19 EP EP14830995.8A patent/EP3083664A2/fr not_active Withdrawn
- 2014-12-19 CA CA2934558A patent/CA2934558A1/fr not_active Abandoned
- 2014-12-19 WO PCT/FR2014/053484 patent/WO2015092335A2/fr active Application Filing
- 2014-12-19 JP JP2016541495A patent/JP2017500049A/ja active Pending
- 2014-12-19 US US15/105,829 patent/US20170190753A1/en not_active Abandoned
-
2019
- 2019-09-11 JP JP2019165435A patent/JP2020014468A/ja active Pending
Cited By (27)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR102773865B1 (ko) * | 2015-09-24 | 2025-03-04 | 더 트러스티스 오브 더 유니버시티 오브 펜실베니아 | 보체 매개 질환 치료용 조성물 및 방법 |
US10988519B2 (en) | 2015-09-24 | 2021-04-27 | The Trustees Of The University Of Pennsylvania | Composition and method for treating complement-mediated disease |
JP2018527941A (ja) * | 2015-09-24 | 2018-09-27 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | 補体媒介性疾患を処置するための組成物及び方法 |
JP7324253B2 (ja) | 2015-09-24 | 2023-08-09 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | 補体媒介性疾患を処置するための組成物及び方法 |
JP7261583B2 (ja) | 2015-09-24 | 2023-04-20 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | 補体媒介性疾患を処置するための組成物及び方法 |
EP3359663A4 (fr) * | 2015-09-24 | 2019-08-14 | The Trustees of The University of Pennsylvania | Composition et méthode de traitement d'une maladie à médiation par le complément |
KR20180055872A (ko) * | 2015-09-24 | 2018-05-25 | 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 | 보체 매개 질환 치료용 조성물 및 방법 |
IL296929B2 (en) * | 2015-09-24 | 2025-07-01 | Univ Pennsylvania | Composition and method for treating complement-mediated disease |
CN115976105A (zh) * | 2015-09-24 | 2023-04-18 | 宾夕法尼亚州大学信托人 | 用于治疗补体介导的疾病的组合物和方法 |
IL296929B1 (en) * | 2015-09-24 | 2025-03-01 | Univ Pennsylvania | Composition and method for treating complement-mediated disease |
CN108291216A (zh) * | 2015-09-24 | 2018-07-17 | 宾夕法尼亚州大学信托人 | 用于治疗补体介导的疾病的组合物和方法 |
US12180254B1 (en) | 2015-09-24 | 2024-12-31 | The Trustees Of The University Of Pennsylvania | Factor H variants for treatment of disease |
RU2727411C2 (ru) * | 2015-09-24 | 2020-07-21 | Дзе Трастиз Оф Дзе Юниверсити Оф Пенсильвания | Композиция и способ для лечения заболевания, опосредованного комплементом |
JP2022000017A (ja) * | 2015-09-24 | 2022-01-04 | ザ・トラステイーズ・オブ・ザ・ユニバーシテイ・オブ・ペンシルベニア | 補体媒介性疾患を処置するための組成物及び方法 |
IL258024B (en) * | 2015-09-24 | 2022-11-01 | Univ Pennsylvania | Composition and method for treating complement-mediated disease |
AU2016326627B2 (en) * | 2015-09-24 | 2022-12-15 | The Trustees Of The University Of Pennsylvania | Composition and method for treating complement-mediated disease |
IL258024B2 (en) * | 2015-09-24 | 2023-03-01 | Univ Pennsylvania | A preparation and method for the treatment of a complement-mediated disease |
US11591378B2 (en) | 2015-12-23 | 2023-02-28 | eleva GmbH | Polypeptides for inhibiting complement activation |
US10640540B2 (en) | 2015-12-23 | 2020-05-05 | Greenovation Biotech Gmbh | Polypeptides for inhibiting complement activation |
JP2019508022A (ja) * | 2015-12-23 | 2019-03-28 | グリーンオヴェイション・バイオテック・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツングGreenovation Biotech Gmbh | 補体活性化を抑制するポリペプチド |
CN108699121B (zh) * | 2015-12-23 | 2023-07-04 | 艾丽法有限责任公司 | 用于抑制补体激活的多肽 |
CN108699121A (zh) * | 2015-12-23 | 2018-10-23 | 格林诺瓦森生物技术股份有限公司 | 用于抑制补体激活的多肽 |
WO2019243586A1 (fr) * | 2018-06-22 | 2019-12-26 | Universität Ulm | Inhibiteurs du complément et utilisations de ceux-ci |
CN113365648A (zh) * | 2018-06-22 | 2021-09-07 | 乌尔姆大学 | 补体抑制因子及其用途 |
EP3586860A1 (fr) * | 2018-06-22 | 2020-01-01 | Universität Ulm | Inhibiteurs de complément et leurs utilisations |
RU2824048C2 (ru) * | 2018-12-21 | 2024-08-01 | Джайроскоуп Терапьютикс Лимитед | Кодон-оптимизированный фактор комплемента i |
WO2020128516A3 (fr) * | 2018-12-21 | 2020-08-06 | Gyroscope Therapeutics Limited | Facteur i de complément optimisé en termes de codons |
Also Published As
Publication number | Publication date |
---|---|
US20170190753A1 (en) | 2017-07-06 |
CA2934558A1 (fr) | 2015-06-25 |
JP2017500049A (ja) | 2017-01-05 |
WO2015092335A3 (fr) | 2015-12-03 |
EP3083664A2 (fr) | 2016-10-26 |
JP2020014468A (ja) | 2020-01-30 |
FR3015484A1 (fr) | 2015-06-26 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2015092335A2 (fr) | Proteines recombinantes possedant une activite de facteur h | |
Cheng et al. | Identification and characterization of VPO1, a new animal heme-containing peroxidase | |
Ulucan et al. | Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy | |
Knudsen et al. | The function of acyl-CoA-binding protein (ACBP)/diazepam binding inhibitor (DBI) | |
Hansen et al. | CL-46, a novel collectin highly expressed in bovine thymus and liver | |
EP2611833B1 (fr) | Anticorps se liant a l'adrenomedulline et aux recepteurs de l'adrenomedulline et leurs utilisations comme medicament | |
EP0637334B1 (fr) | Peptides ayant une activite de facteur d'echange du gdp, sequences d'acides nucleiques codant pour ces peptides, preparation et utilisation | |
BRPI0913577A2 (pt) | polipeptídeos cd83 solúveis, formulações e métodos de uso | |
EP0652952A1 (fr) | Peptides inhibant l'activite des proteines ras, preparation et utilisation | |
EP0980427B1 (fr) | Utilisation des proteines ulip dans le diagnostic et la therapie des cancers et des syndromes neurologiques paraneoplasiques | |
EP0793721A1 (fr) | Peptides capables de se lier au domaine sh3 de la proteine gap, sequences nucleotidiques codant pour ces peptides, leur preparation et utilisation | |
CA2169938A1 (fr) | Gene grb3-3, ses variants et leurs utilisations | |
RU2409590C2 (ru) | Новый пептид, участвующий в энергетическом гомеостазе | |
EP2780457B1 (fr) | Utilisation de la fibromoduline et du lumican pour augmenter la masse musculaire | |
CN117180404A (zh) | Cholesin在调节胆固醇稳态中的用途 | |
Ueki et al. | Isolation of cDNAs encoding subunits A and B of the vacuolar-type ATPase from the vanadium-rich ascidian, Ascidia sydneiensis samea | |
EP3655014B1 (fr) | Clusterine pour son utilisation dans le traitement des micro-angiopathies thrombotiques | |
CZ178396A3 (en) | Novel tumor-suppressor gene | |
WO2002000878A2 (fr) | Dynamine mitochondriale humaine msp1, ses isoformes msp1-x, et leur utilisation en therapeutique | |
EP2771354A1 (fr) | Procede de preparation du facteur h humain | |
Furutani et al. | Androgen-dependent expression, gene structure, and molecular evolution of guinea pig caltrin II, a WAP-motif protein | |
EP0686194A1 (fr) | Facteurs de croissance de la famille de l'harp, procede d'obtention et applications | |
EP1257642A1 (fr) | Partenaires du domaine ptb1 de fe65, preparation et utilisations | |
CN114787361A (zh) | 微生物胆固醇催化基因的靶向表达降低过量脂质 | |
EP1194552A1 (fr) | Acides nucleiques codant pour des peptides possedant l'activite biologique de la sorbine |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
REEP | Request for entry into the european phase |
Ref document number: 2014830995 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2014830995 Country of ref document: EP |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14830995 Country of ref document: EP Kind code of ref document: A2 |
|
ENP | Entry into the national phase in: |
Ref document number: 2934558 Country of ref document: CA Ref document number: 2016541495 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase in: |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 15105829 Country of ref document: US |