SE545694C2 - Dna construct comprising nucleotide sequences encoding amino acid sequences of certolizumab and pelb signal peptides - Google Patents
Dna construct comprising nucleotide sequences encoding amino acid sequences of certolizumab and pelb signal peptidesInfo
- Publication number
- SE545694C2 SE545694C2 SE2130264A SE2130264A SE545694C2 SE 545694 C2 SE545694 C2 SE 545694C2 SE 2130264 A SE2130264 A SE 2130264A SE 2130264 A SE2130264 A SE 2130264A SE 545694 C2 SE545694 C2 SE 545694C2
- Authority
- SE
- Sweden
- Prior art keywords
- nucleotide sequence
- seq
- certolizumab
- host cell
- dna construct
- Prior art date
Links
- 229960003115 certolizumab pegol Drugs 0.000 title claims abstract description 98
- 108010076504 Protein Sorting Signals Proteins 0.000 title claims abstract description 58
- 108020004414 DNA Proteins 0.000 title claims description 78
- 108091028043 Nucleic acid sequence Proteins 0.000 title claims description 40
- 125000003275 alpha amino acid group Chemical group 0.000 title claims 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 165
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 165
- 239000013604 expression vector Substances 0.000 claims abstract description 51
- 210000004027 cell Anatomy 0.000 claims description 87
- 241000588724 Escherichia coli Species 0.000 claims description 37
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims description 23
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims description 23
- 230000035772 mutation Effects 0.000 claims description 22
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims description 21
- 101150018266 degP gene Proteins 0.000 claims description 19
- 238000012217 deletion Methods 0.000 claims description 18
- 230000037430 deletion Effects 0.000 claims description 18
- 108010041974 Rhamnulokinase Proteins 0.000 claims description 17
- 230000001580 bacterial effect Effects 0.000 claims description 15
- 101100178822 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) htrA1 gene Proteins 0.000 claims description 14
- 101100277437 Rhizobium meliloti (strain 1021) degP1 gene Proteins 0.000 claims description 14
- 210000000349 chromosome Anatomy 0.000 claims description 14
- 230000004060 metabolic process Effects 0.000 claims description 11
- 241001302584 Escherichia coli str. K-12 substr. W3110 Species 0.000 claims description 10
- 230000037433 frameshift Effects 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 7
- 230000004048 modification Effects 0.000 claims description 7
- 101150032455 rhaB gene Proteins 0.000 claims description 6
- 231100000221 frame shift mutation induction Toxicity 0.000 claims description 4
- 238000013518 transcription Methods 0.000 description 33
- 230000035897 transcription Effects 0.000 description 33
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 31
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 31
- 108090000623 proteins and genes Proteins 0.000 description 29
- 239000003550 marker Substances 0.000 description 27
- 239000012190 activator Substances 0.000 description 25
- 239000012634 fragment Substances 0.000 description 21
- 238000000034 method Methods 0.000 description 21
- 230000033228 biological regulation Effects 0.000 description 20
- 108010087558 pectate lyase Proteins 0.000 description 20
- 101150036535 prc gene Proteins 0.000 description 20
- 230000003115 biocidal effect Effects 0.000 description 18
- 238000004519 manufacturing process Methods 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 18
- 229960000106 biosimilars Drugs 0.000 description 16
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 15
- 239000002253 acid Substances 0.000 description 13
- 238000011144 upstream manufacturing Methods 0.000 description 13
- 230000000694 effects Effects 0.000 description 12
- 108020004705 Codon Proteins 0.000 description 11
- 150000001413 amino acids Chemical group 0.000 description 10
- 229930027917 kanamycin Natural products 0.000 description 10
- 229960000318 kanamycin Drugs 0.000 description 10
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 10
- 229930182823 kanamycin A Natural products 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 239000013612 plasmid Substances 0.000 description 10
- 101150081864 Spr gene Proteins 0.000 description 9
- 239000002202 Polyethylene glycol Substances 0.000 description 8
- 108091081024 Start codon Proteins 0.000 description 8
- 229920001223 polyethylene glycol Polymers 0.000 description 8
- 230000010076 replication Effects 0.000 description 8
- 239000013598 vector Substances 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 101710154303 Cyclic AMP receptor protein Proteins 0.000 description 6
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 238000010828 elution Methods 0.000 description 6
- 125000003827 glycol group Chemical group 0.000 description 6
- 108020004707 nucleic acids Proteins 0.000 description 6
- 102000039446 nucleic acids Human genes 0.000 description 6
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 5
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 5
- 108700009124 Transcription Initiation Site Proteins 0.000 description 5
- 238000010276 construction Methods 0.000 description 5
- 201000010099 disease Diseases 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 5
- 230000001105 regulatory effect Effects 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 238000003780 insertion Methods 0.000 description 4
- 230000037431 insertion Effects 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 3
- 108010030718 DegP protease Proteins 0.000 description 3
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 3
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 3
- PNNNRSAQSRJVSB-BXKVDMCESA-N aldehydo-L-rhamnose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O PNNNRSAQSRJVSB-BXKVDMCESA-N 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 229940090100 cimzia Drugs 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 101150078160 rhaR gene Proteins 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 231100000241 scar Toxicity 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 230000004543 DNA replication Effects 0.000 description 2
- 108010001469 L-rhamnose isomerase Proteins 0.000 description 2
- 108010011945 N-carbamoyl-L-amino-acid hydrolase Proteins 0.000 description 2
- 239000004235 Orange GGN Substances 0.000 description 2
- 239000004695 Polyether sulfone Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000008033 biological extinction Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- GNGACRATGGDKBX-UHFFFAOYSA-N dihydroxyacetone phosphate Chemical compound OCC(=O)COP(O)(O)=O GNGACRATGGDKBX-UHFFFAOYSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010195 expression analysis Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000003119 immunoblot Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 229920006393 polyether sulfone Polymers 0.000 description 2
- 230000017854 proteolysis Effects 0.000 description 2
- 101150091078 rhaA gene Proteins 0.000 description 2
- 101150052254 rhaD gene Proteins 0.000 description 2
- 239000012723 sample buffer Substances 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 2
- BSABBBMNWQWLLU-VKHMYHEASA-N (S)-lactaldehyde Chemical compound C[C@H](O)C=O BSABBBMNWQWLLU-VKHMYHEASA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- QZNPNKJXABGCRC-OTWZMJIISA-N 6-deoxy-L-sorbose Chemical compound C[C@H](O)[C@@H](O)[C@H](O)C(=O)CO QZNPNKJXABGCRC-OTWZMJIISA-N 0.000 description 1
- 101100228725 Acidaminococcus fermentans (strain ATCC 25085 / DSM 20731 / CCUG 9996 / CIP 106432 / VR4) gctA gene Proteins 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108020004256 Beta-lactamase Proteins 0.000 description 1
- 101100314454 Caenorhabditis elegans tra-1 gene Proteins 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 102100026816 DNA-dependent metalloprotease SPRTN Human genes 0.000 description 1
- 101710175461 DNA-dependent metalloprotease SPRTN Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000001390 Fructose-Bisphosphate Aldolase Human genes 0.000 description 1
- 108010068561 Fructose-Bisphosphate Aldolase Proteins 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 108091029795 Intergenic region Proteins 0.000 description 1
- QZNPNKJXABGCRC-FUTKDDECSA-N L-rhamnulose Chemical compound C[C@H](O)[C@H](O)[C@@H](O)C(=O)CO QZNPNKJXABGCRC-FUTKDDECSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 101100471831 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) rpl-5 gene Proteins 0.000 description 1
- 101100092791 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) rps-14 gene Proteins 0.000 description 1
- 101100362789 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) rps-17 gene Proteins 0.000 description 1
- 108010079246 OMPA outer membrane proteins Proteins 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101710118538 Protease Proteins 0.000 description 1
- 108010032060 RNA polymerase alpha subunit Proteins 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 206010054094 Tumour necrosis Diseases 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 102000006635 beta-lactamase Human genes 0.000 description 1
- 230000001925 catabolic effect Effects 0.000 description 1
- 238000013354 cell banking Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 210000001322 periplasm Anatomy 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 101150040968 rhaS gene Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 101150025220 sacB gene Proteins 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- -1 tetracyc1ine Chemical compound 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
- C12N15/625—DNA sequences coding for fusion proteins containing a sequence coding for a signal sequence
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates a DNA construct for expressing Certolizumab wherein the DNA construct comprises at least one nucleotide sequence for expressing a PelB signal peptide. The invention further relates to an expression vector and host cell which comprise said DNA construct.
Description
TECHNICAL FIELD
The present invention relates to DNA construct suitable for expressing recombinant proteins in a bacterial host cell. The present invention further relates to a vector and bacterial host cell comprising the DNA construct as Well as a method of producing said recombinant protein by exposing said bacterial host cell to rhamnose and thereby inducing expression of said
recombinant protein.
BACKGRUND OF THE INVENTION
The metabolism of rhamnose involves L-rhamnose being taken up into cells via the perrnease RhaT and then isomerized into L-rhamnulose by L-rhamnose isomerase (RhaA), and L- rhamnulose is then phosphorylated further by rhamnulokinase (RhaB) and finally hydrolyzed by rhanmulose-l-phosphate aldolase (RhaD) to give dihydroxyacetonephosphate and L- lactaldehyde [l]. The genes rhaA, rhaB and rhaD form an operon referred to as rhaBAD and are transcribed With the aid of the rhaBAD promoter [l]. In comparison With other systems, the rhamnose metabolism pathWay is distinguished by the fact that tWo transcription activators known as RhaS and RhaR are required for regulation as explained below [l].
The rhaBAD operon is a positively regulated catabolic operon Which transcribes above mentioned rhaB, rhaA and rhaD genes divergently from the rhaSR operon With approximately 240 bp of DNA separating their respective transcription start sites [l]. The rhaSR operon encodes RhaS and RhaR Wherein each monomer of the dimeric RhaS and RhaR proteins contains tWo helix-tum-helix motifs and contacts tWo major grooves of DNA. RhaR regulates transcription of rhaSR by binding promoter DNA spanning -32 to -82 bases relative to the rhaSR transcription start site [l]. Subsequent to rhaSR expression, RhaS bind DNA upstream of the rhaBAD operon at -32 to -8l bases relative to the transcription start site to increase rhaBAD expression [l]. Furthermore, the rhaSR-rhaBAD intergenic region contains CRP binding sites at positions -92.5 (CRP l) relative to the transcription start site of the rhaBAD operon and CRP binding sites at positions -92.5 (CRP 2), -l 15.5 (CRP 3) and -l 16.5 (CRP 4) relative to the transcription start site of the rhaSR operon [l]. The cyclic AMP
receptor protein (CRP) regulates the expression of more than 100 promoters in Escheríchía
coli.
DNA constructs comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter are known in the art. US8l38324 discloses pTACO- and pLEMO-derived plasmids (i.e. DNA constructs) comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter. However, US8l38324 is silent about using host cells which have a disabled rhamnose
metabolism.
DNA constructs based on pRha-derived plasmids comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter are also known in the art, for example from Giacalone et al. [5] or Hjelm et al. [2]. Giacalone et al. describe for example the plasmids pRha67A and pRhal09A whereas Hjelm et al. disclose the plasmid pRha67K.
Although DNA constructs comprising DNA sequences encoding RhaS, RhaR and the rhaBAD promoter are known in the art there are still many challenges, especially in industrial scale production of recombinant proteins, in particular monoclonal antibodies or fragments thereof. The main challenges are:
(i) the host cells being subject to stress which results in damage to cellular
macromolecules such as membranes, proteins and nucleic acids; (ii) poor growth of the host cells; (iii) poor activity of the produced recombinant proteins; and/or
(iv) obtaining recombinant proteins in low yields.
Hence, there is a need for improved DNA constructs as well as a host cell and method suitable for the efficient production of recombinant proteins, such as monoclonal antibodies or
fragments thereof in a high yield.
In particular, there is a need for improved DNA constructs as well as a host cell for the efficient production of Certolizumab which is a humanized Fab' fragment (from an IgG l isotype) of an anti-tumor necrosis factor (TNF) monoclonal antibody with aff1nity for TNF- alpha. The conjugation of Certolizumab with an approximately 40kDa polyethylene glycol (PEG) results in Certolizumab pegol which is a pharrnaceutical marketed by UCB as Cimzia® and which is administered by subcutaneous injection for the treatment of Crohn°s disease,
rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis.
Patents such as EP1287140 and US7012135 disclose DNA constructs for the production of Certolizumab. HoWever, these DNA constructs, Which comprise Lan-evolved translation initiation regions (TIRs) and Which furtherrnore appear to lack nucleotide sequences encoding
PelB signal peptide, are not optimal for producing Certolizumab With high yields.
Typically, When generating recombinant expression vectors such as in the ones disclosed in EP1287140 and US7012135, the TIR is forrned by fusing the 5'UTR (i.e. untranslated region upstream of the ATG start codon) from the expression vector With the coding sequence of a signal peptide. Each time a different signal peptide is used, a different TIR is generated. Such TIRs are referred to as Lan-evolved as they Were forrned by ad hoc genetic fusion rather than the synthetically evolved TIRs described in the present invention as Well as in US10696963 and WO
Patents such as US6828121 and EP1341899 relate to host cells for the production of Various types of antibodies and antibody fragments such as humanized Fab” fragments. Some specific examples of antibodies Which can be produced by these host cells are anti-IgE, anti-IgG, anti- Her-2, anti-CD1 la, anti-CD18, anti-CD20 and anti-VEGF. An example of a host cell disclosed in US6828121 and EP1341899 is an E. coli strain def1cient in chromosomal degP and prc encoding protease DegP and Prc, respectively, and harboring a mutant spr gene, Wherein the product of the mutant spr gene is characterized by the tryptophan at position 148 being changed to arginine. HoWever, US6828121 and EP1341899 are both silent about (a) mutations of the host cell relating to the metabolism of rhamnose, and (b) production of a
specific Fab” fragment such as Certolizumab.
Intemational patent application WO21158163 relates to DNA constructs comprising synthetically evolved TIRs for regulating the performance of signal peptides in the production of recombinant proteins. WO21158163 clearly shows that synthetically evolved TIRs have technical advantages over Lan-evolved TIRs. HoWever, WO21158163 is silent about
synthetically evolved TIRs specifically developed for the optimal expression of Certolizumab.
WO21158163 further relates to nucleotide sequences for the expression of the Pelb signal peptide. HoWever, WO21158163 is silent about nucleotide sequences specifically developed
for the optimal expression of Certolizumab.
Hence, there is a need for optimization of DNA constructs, host cells, TIRs and signal
peptides for the expression of recon1binant proteins such as Certolizuniab.
OBJECTS OF THE INVENTION
The object of the present invention is to provide advantageous technical effect of DNA
constructs. A further object of the present invention is to provide advantageous technical effect of TIRs.
A further object of the present invention is to provide advantageous technical effect of the
host cells.
A further object of the present invention is to provide advantageous technical effect of signal
peptide nucleotide sequences.
A further object of the present invention is to provide advantageous methods for the efficient
production of recon1binant proteins.
SUMMARY OF THE INVENTION
The objects of the invention have been attained by any one or more of the below disclosed
aspects of the invention.
A first aspect of the invention relates to a DNA construct suitable for expressing Certolizuniab in a host cell, Wherein Certolizuniab con1prises (i) a light chain con1prising the an1ino acid sequence of SEQ ID 3, and (ii) a heavy chain coniprising the an1ino acid sequence of SEQ ID 4, Wherein said DNA construct con1prises a nucleotide sequence encoding Certolizuniab, Wherein said DNA construct further con1prises at least one nucleotide sequence encoding a signal peptide Which is operably linked in the direction of transcription to the nucleotide sequence encoding the light chain of Certolizumab and/or the heavy chain of Certolizuniab, Wherein said DNA construct further con1prises nucleotide sequences encoding:
- a promoter,
- RhaR transcription activator,
- RhaS transcription activator,
an antibiotic resistance marker, at least one terrninator, and
an origin of replication.
In a preferred embodiment, the DNA construct is characterized by:
a promoter,
RhaR transcription activator,
RhaS transcription activator,
an antibiotic resistance marker,
a promoter operably linked to the nucleotide sequence encoding the antibiotic resistance marker,
at least one terrninator, and
an origin of replication.
In a preferred embodiment, the DNA construct is characterized by:
rhaBAD promoter,
RhaR transcription activator,
RhaS transcription activator,
an antibiotic resistance marker,
a promoter operably linked to the nucleotide sequence encoding the antibiotic resistance marker,
rrnB Tl terrninator,
rrnB T2 terrninator, and
an pMBl origin of replication.
In a preferred embodiment, the DNA construct is characterized by:
the antibiotic resistance marker is a kanamycin resistance marker, preferably a
kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12 or
a sequence With at least 90 % sequence identity thereto;
the promoter operably linked to the nucleotide sequence encoding the antibiotic
resistance marker is an AmpR promoter, preferably an AmpR promoter
comprising the nucleotide sequence of SEQ ID 13 or a sequence With at least% sequence identity thereto;
the rrnB T1 terrninator comprising the nucleotide sequence of SEQ ID 14 or a
sequence With at least 90 % sequence identity thereto;the rrnB T2 terrninator comprising the nucleotide sequence of SEQ ID 15 or a sequence With at 1east 90 % sequence identity thereto; and/or the pMB1 origin of rep1ication comprising the nucleotide sequence of SEQ IDor a sequence With at 1east 90 % sequence identity thereto.
In a preferred embodiment, the DNA construct is characterized by:
the antibiotic resistance marker being a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12;
the promoter operab1y linked to the nucleotide sequence encoding the antibiotic resistance marker is an AmpR promoter comprising the nucleotide sequence of SEQ ID 13;
the rrnB T1 terrninator comprising the nucleotide sequence of SEQ ID 14;
the rrnB T2 terrninator comprising the nucleotide sequence of SEQ ID 15; and/or
the pMB1 origin of rep1ication comprising the nucleotide sequence of SEQ ID
In a preferred embodiment, the DNA construct is characterized by:
- the rhaBAD promoter comprising the nucleotide sequence of SEQ ID 8 or a sequence With at 1east 90 % sequence identity thereto;
- the RhaR transcription activator comprising the nucleotide sequence of SEQ ID 9 or a sequence With at 1east 90 % sequence identity thereto;
- the RhaS transcription activator comprising the nucleotide sequence of SEQ ID 11 or a sequence With at 1east 90 % sequence identity thereto;
- the antibiotic resistance marker is a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12 or a sequence With at 1east 90 % sequence identity thereto;
- the AmpR promoter comprising the nucleotide sequence of SEQ ID 13 or a sequence With at 1east 90 % sequence identity thereto;
- the rrnB T1 terrninator comprising the nucleotide sequence of SEQ ID 14 or a sequence With at 1east 90 % sequence identity thereto;
- the rrnB T2 terrninator comprising the nucleotide sequence of SEQ ID 15 or a sequence With at 1east 90 % sequence identity thereto; and
- the pMB1 origin of rep1ication comprising the nucleotide sequence of SEQ ID
16 or a sequence With at 1east 90 % sequence identity thereto.
In a preferred embodiment, the DNA construct is characterized by:
the rhaBAD promoter comprising the nucleotide sequence of SEQ ID 8;
the RhaR transcription activator comprising the nucleotide sequence of SEQ ID 9; - the RhaS transcription activator comprising the nucleotide sequence of SEQ ID 1 1; - the antibiotic resistance marker is a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12; - the AmpR promoter comprising the nucleotide sequence of SEQ ID 13; - the rrnB T1 terrninator comprising the nucleotide sequence of SEQ ID 14; - the rrnB T2 terrninator comprising the nucleotide sequence of SEQ ID 15; and - the pMB1 origin of replication comprising the nucleotide sequence of SEQ ID 1
In a preferred embodiment, the DNA construct may comprise one or more restriction sites cleavable by restriction enzymes such as EcoRI, NdeI, NotI, XhoI, PspXI, PaeR71, BbsI, StyI, AVrII, BanI, Acc65I, KpnI, Eco53kI, SacI, BamHI, XbaI, SalI, AccI, PstI, SbfI, SphI and/or HindIII.
In a preferred embodiment, the DNA construct further comprises a nucleotide sequence encoding said recombinant protein operably linked to the rhaBAD promoter, Wherein said recombinant protein is a monoclonal antibody or fragment thereof, preferably said recombinant protein is Certolizumab. More preferably said recombinant protein is Certolizumab comprising (i) a light chain comprising the amino acid sequence of SEQ ID 3,
and/or (ii) a heavy chain comprising the amino acid sequence of SEQ ID
In a preferred embodiment, the DNA construct comprises a nucleotide sequence encoding the recombinant protein operably linked to the rhaBAD promoter comprising (i) a nucleotide sequence encoding for the light chain of Certolizumab comprising the sequence of SEQ ID 5 or a sequence With at least 90 % sequence identity thereto, and/or (ii) a nucleotide sequence encoding for the heavy chain of Certolizumab comprising the sequence of SEQ ID 6 or a sequence With at least 90 % sequence identity thereto; preferably said nucleotide sequence encoding the recombinant protein comprises (i) a nucleotide sequence encoding for the light
chain of Certolizumab comprising the sequence of SEQ ID 5, and/or (ii) a nucleotide
sequence encoding for the heavy chain of Certolizuniab coniprising the sequence of SEQ ID
In an enibodinient, the DNA construct further con1prises a nucleotide sequence encoding the reconibinant protein operably linked to the rhaBAD pronioter con1prising at least one nucleotide sequence encoding a signal peptide Which is operably linked in the direction of transcription to either one or both of the nucleotide sequence of SEQ ID 5 and SEQ ID 6, preferably the signal peptide is a PelB (pectate lyase B) signal peptide.
The nucleotide sequence encoding the PelB signal peptide Which is operably linked in the direction of transcription to the nucleotide sequence of the light chain of Certolizuniab is in the present invention referred to as PelB1. The nucleotide sequence of PelB1 con1prises a
sequence of SEQ ID 18 or a sequence With at least 90 % sequence identity thereto.
The nucleotide sequence encoding the PelB signal peptide Which is operably linked in the direction of transcription to the nucleotide sequence of the heavy chain of Certolizuniab is in the present invention referred to as PelB2. The nucleotide sequence of PelB2 con1prises a
sequence of SEQ ID 19 or a sequence With at least 90 % sequence identity thereto.
The resulting PelB signal peptide con1prises an aniino acid sequence of SEQ ID 7 [6]: MKYLLPTAAAGLLLLAAQPAMA.
In an enibodinient, the DNA construct con1prises a TIR having a nucleotide sequence of SEQ ID 20, Wherein said sequence of SEQ ID 20 con1prises at least the first 9 nucleotides of the nucleotide sequence of PelB1, i.e. the first 9 nucleotides of SEQ ID 18. This particular TIR is
in the present invention also referred to as TIR-LC.
In an enibodinient, the DNA construct con1prises a TIR having a sequence of SEQ ID 21, Wherein said sequence of SEQ ID 21 con1prises at least the first 9 nucleotides of the nucleotide sequence of PelB2, i.e. the first 9 nucleotides of SEQ ID 19. This particular TIR is
in the present invention also referred to as TIR-HC.
In a preferred enibodinient, the DNA construct con1prises the sequence of SEQ ID 17 or a sequence With at least 90 % sequence identity thereto, preferably con1prises the sequence of
SEQ 1D
A second aspect of the invention relates to a DNA construct for expressing a reconibinant protein, Wherein the DNA construct coniprises: - at least one of the nucleotide sequence of SEQ ID No 20 and 21, Wherein a nucleotide sequence of SEQ ID 20 and 21 is a TIR sequence; and - a nucleotide sequence Which encodes a signal peptide; and Wherein a nucleotide sequence of SEQ ID No 20 and 21 coniprises at least the firstnucleotides of said signal peptide encoding sequence.
In an enibodinient, the DNA construct coniprises a TIR having a nucleotide sequence of SEQ ID 20, Wherein said sequence of SEQ ID 20 coniprises at least the first 9 nucleotides of the nucleotide sequence of PelBl, i.e. the first 9 nucleotides of SEQ ID 18. This particular TIR is
in the present invention also referred to as TIR-LC.
In an enibodinient, the DNA construct coniprises a TIR having a sequence of SEQ ID 21, Wherein said sequence of SEQ ID 21 coniprises at least the first 9 nucleotides of the nucleotide sequence of PelB2, i.e. the first 9 nucleotides of SEQ ID 19. This particular TIR is
in the present invention also referred to as TIR-HC.
In an enibodinient, the nucleotide sequence Which encodes a signal peptide is operably linked to: - the first nucleotide sequence Which encodes the light chain of an antibody; and/or
- the second nucleotide sequence Which encodes the heavy chain of an antibody.
In an enibodinient, the DNA construct coniprises a Shine-Dalgarno sequence. The Shine- Dalgarno sequence is located upstreani fron1 the ATG start codon of the nucleotide sequence Which encodes a signal peptide. In an enibodinient, said Shine-Dalgarno sequence is located upstreani froni the ATG start codon of the nucleotide sequence Which encodes a signal peptide Which is operably linked to the light and/or heavy chain of an antibody. In an enibodinient, said Shine-Dalgarno sequence coniprises nucleotide sequence AGGAGGAA and/or GAGGAGAA in the direction of transcription. Preferably, AGGAGGAA is upstreani of nucleotide sequence coding for the light chain of an antibody. Preferably, GAGGAGAA is upstreani of nucleotide sequence coding for the heavy chain of an antibody. More preferably, AGGAGGAA is upstreani of TIR-LC. More preferably, GAGGAGAA is upstreani of TIR- HC.
In an enibodinient, the first nucleotide sequence Which encodes a signal peptide (e.g. PelB1) is operably linked to the first nucleotide sequence Which encodes the light chain of an
antibody.
In an enibodinient, the second nucleotide sequence Which encodes a signal peptide (e.g. PelB2) is operably linked to the second nucleotide sequence Which encodes the heavy chain of an antibody.
In an enibodinient, the first and second nucleotide sequences Which encode for the light and heavy chains of an antibody, respectively, encode an1ino acid sequences of SEQ ID No 3 and
SEQ ID No 4, respectively.
In an enibodinient, the first and second nucleotide sequence Which encode the light and heavy chains of an antibody, respectively, coniprise nucleotide sequences of SEQ ID No 5 and SEQ ID No 6, respectively.
A third aspect of the invention relates to a DNA construct for expressing a signal peptide, Wherein the DNA construct coniprises a nucleotide sequence Which encodes a PelB signal peptide, Wherein the nucleotide sequence Which encodes said PelB signal peptide coniprises at least one of the nucleotide sequences of SEQ ID No 18 and 19. In an enibodinient, the DNA construct coniprises both of the nucleotide sequences SEQ ID No 18 and
A fourth aspect of the invention relates to DNA construct coniprising nucleotide sequence encoding an1ino acid sequences, Wherein the an1ino acid sequences coniprise: a. the an1ino acid sequence for Certolizuniab; b. a first signal peptide of an1ino acid sequence of SEQ ID No 7 fused to the N- terrninus of light chain an1ino acid sequence of Certolizuniab; and c. a second signal peptide of an1ino acid sequence of SEQ ID NO 7 fused to the N-
terrninus of heavy chain an1ino acid sequence of Certolizuniab.
In an enibodinient, the nucleotide sequences Which encodes said first and second signal
peptides coniprise nucleotide sequences of SEQ ID No 18 and 19, respectively.A f1fth aspect of the inVention relates to an expression Vector comprising any of the DNA
constructs according to the first, second, third and/or fourth aspects of the invention.
A sixth aspect of the inVention relates to a host cell characterized by a chromosome comprising: a. a mutation in the nucleotide sequence encoding RhaB Which disables rhamnose metabolism; b. a mutation in the degP gene Which disables (i) expression of DegP protease and/or (ii) activity of DegP protease; c. a mutation in prc gene Which disables (i) expression of Prc protease, and/or (ii) activity of Prc protease; and
d. a mutation in the spr gene.
In an embodiment, a mutation is selected from the group consisting of frameshift, deletion,
substitution and insertion.
In an embodiment, said mutation in the nucleotide sequence encoding RhaB Which disables
rhamnose metabolism is a frame shift-mutation in the nucleotide sequence encoding RhaB. In an embodiment, said mutation in the degP gene is a degP deletion. In an embodiment, said mutation in the prc gene is a prc deletion.
In an embodiment, said mutation in the spr gene is a spr WI 48R mutation characterized by
substitution in the spr gene resulting in tryptophan at position 148 being changed to arginine.
In an embodiment, said host cell characterized by a chromosome comprising: a. a mutation in the nucleotide sequence encoding RhaB Which disables rhamnose metabolism; b. a mutation in the degP gene Which disables expression of DegP protease; c. a mutation in prc gene Which disables expression of Prc protease; and
d. a mutation in the spr gene.
In an embodiment, said host cell is a bacterial cell, more preferably E. coli, most preferably E.
coli W3I I
SIn an embodiment, said host cell is an E. coli WS l 10, comprising a chromosome Which comprises a frame shift-mutation in the nucleotide sequence encoding RhaB. This particular
host cell is in the present invention referred to as E. coli WS l 10 rhaBfi as Well as XBl
In an embodiment, said host cell is E. coli WS l 10 rhaBfi further comprising a chromosome Which comprises a clegP deletion. This particular host cell is in the present inVention referred
to as E. coli WS l 10 rhaBfi ADegP as Well as XB8S.
In an embodiment, said host cell is E. coli WS l l0 rhaBfi ADegP further comprising a chromosome Which comprises a prc deletion. This particular host cell is in the present
inVention referred to as E. coli WS l l0 rhaBfi AdegP Aprc as Well as XBl
In an embodiment, said host cell is E. coli WS l l0 rhaBfi AclegP Aprc further comprising a chromosome Which comprises a spr WI 48R mutation. This particular host cell is in the present
inVention referred to as E. coli WS l l0 rhaBfi ADegP Aprc sprWI48R as Well as XBl
A seVenth aspect of the inVention relates to a host cell according to the sixth aspect of the inVention comprising a DNA construct according to the first, second, third and/or fourth
aspects of the inVention.
An eighth aspect of the inVention relates to a method of producing a recombinant protein comprising the step of exposing the host cell according to the seVenth aspect of the inVention to rhamnose, thereby inducing expression of said recombinant protein. In a preferred embodiment, the method further comprises the step of recovering the recombinant protein from the bacterial host cell; and optionally further comprises one or more step(s) of purifying
the recovered recombinant protein, preferably by one or more chromatography steps.
A ninth aspect of the inVention relates to a method of producing a recombinant protein, comprising the step of introducing the DNA construct according to the first, second, third and/or fourth aspects of the inVention into a host cell according to the sixth aspect of the
inVention.
In an embodiment, the method further comprises the step of exposing the host cell to
rhamnose, thereby inducing expression of the recombinant protein.In an embodiment, the method further comprises the step of recovering the recombinant protein from the host cell; and optionally further comprises one or more step(s) of purifying
the recovered recombinant protein, preferably by one or more chromatography steps.
In an embodiment, the method further comprises the step of deriVatizing the purif1ed recombinant protein, preferably With a polyethylene glycol moiety, more preferably With an
about 40 kDa polyethylene glycol moiety.
A tenth aspect of the inVention relates to a recombinant protein obtainable by a method according to the ninth aspect of the inVention. The recombinant protein is preferably an antibody or a fragment thereof, more preferably a Fab” fragment antibody, most preferably
Certolizumab.
An eleventh aspect of the inVention relates to a Certolizumab biosimilar obtainable by a method according to the ninth aspect of the inVention. Preferably, the Certolizumab biosimilar comprises a polyethylene glycol moiety such as an about 40 kDa polyethylene glycol moiety. The Certolizumab biosimilar is here disclosed as a product-by-process in order to satisfactorily protect the molecular structure of the Certolizumab biosimilar. A biosimilar is a highly similar to the reference product, i.e. the Certolizumab biosimilar Will have highly similar molecular structure and bioactiVity as Certolizumab pegol (Cimzia®) Which is produced by the reference product sponsor. Moreover, a biosimilar has no clinically meaningful differences from a reference product and the clinical trials that are conducted on biosimilars assess pharmacokinetics and immunogenicity. NeVertheless, the minor structural differences between a Certolizumab biosimilar according to the present inVention and Certolizumab pegol of the reference product sponsor Will partially be due to the method
according to the ninth aspect of the inVention (as Well as the eight aspect of the inVention).
A tWelfth aspect of the inVention relates to a Certolizumab biosimilar, or a deriVatiVe thereof, for use as medicament, preferably for use in the treatment of Crohn°s disease, rheumatoid arthritis, psoriatic arthritis and ankylosing spondylitis. Said deriVatiVe preferably comprises a polyethylene glycol moiety such as an about 40 kDa polyethylene glycol moiety. An
embodiment of the inVention relates to a method of treating a disease by using a Certolizumabbiosimilar. The disease may be Crohn°s disease, rheumatoid arthritis, psoriatic arthritis and
ankylosing spondylitis
A thirteenth aspect of the invention relates to a method of producing a signal peptide, comprising the step of introducing the DNA construct according to the first, second, third and/or fourth aspects of the invention into a host cell according to the sixth aspect of the
invention.
One or more of the above indicated SEQ IDs 1-21 of the various aspects of the invention (and embodiments thereof) may in some embodiments be replaced by a sequence With at least 90 % sequence identity thereto. The term "sequence identity" as used herein is used With regard to amino acid or nucleotide sequences and the sequence identity is over the entire length of the specified sequence. A sequence may thus be at least 90 percent, at least 92 percent, at least 95 percent, at least 96 percent, at least 97 percent, at least 98 percent or at least 99 percent, identical in sequence to the amino acid or nucleotide sequence specified. Such sequences of the invention thus include single or multiple nucleotide or amino acid alterations (additions, substitutions, insertions or deletions) to the sequences of the invention. At the amino acid level preferred sequences With the above defined sequence identity contain up to 5, e. g. only l, 2, 3, 4 or 5, preferably l, 2 or 3, more preferably l or 2, altered amino acids in the
sequences of the invention.
BRIEF DESCRIPTION OF DRAWINGS
Figure l - Plasmid map for KTXHIS
Figure 2 - The nucleotide sequence of the multiple cloning site (MCS) of KTXHIS Figure 3 - Plasmid map of KTXHIS-Cert-PelBl-LC-PelB2-HC
Figure 4 - Example of TIR library selection and isolation of an evolved TIR therefrom Figure 5 - Westem-blot analysis of media fraction
Figure 6 - Westem-blot analysis of total fraction
Figures 7 - NanoDropTM and ÄKTA chromatography performed to detect yield and titer
differences between expression systems XB62 (XB17 host cell containing expression vector
D37 having un-evolved TIRs) and XB102 (XB17 host cell containing expression Vector E83 having synthetically evolved TIR for the regulation of the Certolizumab heavy chain
expression).
Figure 8 - A comparative expression analysis through periplasmic extraction followed by Affinity-HPLC: E83 expression vector expressed in XBl7 host cell versus E83 vector expressed in XBl66 host cell
Figure 9 - A comparative expression analysis through periplasmic extraction followed by Affinity-HPLC: E83 expression vector expressed in XBl66 host cell versus El ll vector expressed in XBl66 host cell
DETAILED DESCRIPTION
A specific embodiment of the present invention relates to DNA constructs for the expression of antibody Wherein said DNA construct comprises an improved TIR of SEQ ID 20: TTGCTCATGAAGTAT
Another specific embodiment relates to DNA constructs for the expression of antibody Wherein said DNA construct comprises an improved TIR of SEQ ID 21: TGTTAAATGAAGTAT
The TIRs of SEQ ID 20 and 2l may be comprised in the same DNA construct. An example of such an embodiment is that the TIR of SEQ ID 20 is upstream of the nucleotide sequence expressing a light chain of an antibody or a fragment thereof (such as Certolizumab) While the TIR of SEQ ID 2l is upstream of the nucleotide sequence expressing a heavy chain of an antibody or a fragment thereof (such as Certolizumab).
A specific embodiment of the invention relates to an improved nucleotide sequence of SEQ ID l8 encoding a PelB signal peptide Which is operably linked to the nucleotide sequences encoding a chain of an antibody: ATGAAGTATCTTCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGC CTGCAATGGCAAnother specific embodiment relates to an improved nucleotide sequence of SEQ ID 19 encoding a Pe1B signal peptide Which is operably linked to the nucleotide sequences encoding
a chain of an antibody:
ATGAAGTATCTGTTGCCGACTGCTGCAGCGGGACTGCTGCTGTTAGCGGCACAAC CGGCGATGGCG
The Pe1B nucleotide sequence of SEQ ID 18 and 19 may be comprised in the same DNA construct. An example of such an DNA construct is When the Pe1B nucleotide sequence of SEQ ID 18 is operably linked to the nucleotide sequences encoding the light chain of an antibody or fragment thereof (such as Certolizumab) While the Pe1B nucleotide sequence of SEQ ID 19 is operably linked to the nucleotide sequences encoding the heavy chain of an antibody or fragment thereof (such as Certolizumab).
Yet in other specific embodiments, the above described sequences of SEQ ID 18-21 may be comprised in the same DNA construct. In such embodiments, a TIR nucleotide sequence of SEQ ID No 20 and 21 Will comprises at least the first 9 nucleotides of a signal peptide nucleotide sequence of SEQ ID 18 and
Other specific embodiments of present inVention may relate to regulating the L-rhamnose rhaBAD promoter-based production of recombinant proteins such as Certolizumab. In other Words, Certolizumab may be produced by:
a. cloning a nucleotide sequence encoding Certolizumab into a DNA construct such that the nucleotide sequence is operably linked to a rhaBAD promoter, and
b. introducing the resulting nucleotide sequence into a bacterial host cell comprising a chromosome Which comprises a mutation or modification Which
disables rhanmose metabolism.
In an embodiment, the nucleotide sequence of the rhaBAD promoter comprises the sequence of SEQ ID 8 (and Wherein the sequence is referred to as “rhaBAD” in figures 1 and
The DNA construct may comprise a nucleotide sequence encoding the RhaR transcription activator. In an embodiment of the inVention, the nucleotide sequence of the RhaR transcription activator comprises a sequence of SEQ ID 9 (and Wherein the sequence is referred to as “rhaR” in figures 1 and 3):
The DNA construct may further comprise a nucleotide sequence encoding an extension of the RhaR transcription activator Which is in frame With RhaR because of a missing stop codon. In an embodiment of the invention, the nucleotide sequence of the extension of the RhaR transcription activator comprises the sequence of SEQ ID 10 (and Wherein the sequence is referred to as “rhaR extended” in figures 1 and 3): AGACGAAAGGGCCTCGTGATACGCCTATTTTTATAG.The DNA construct may comprise a nuc1eotide sequence encoding the RhaS transcription activator. In an embodiment of the invention, the nuc1eotide sequence of the RhaS transcription activator comprises the sequence of SEQ ID 11 (and Wherein the sequence is referred to as “rhaS” in figures 1 and 3):
The DNA construct may comprise a nucleotide sequence encoding an "antibiotic resistance marker" or "selection marker". Such a marker is a fragment of DNA that contains a gene whose product confers resistance to an antibiotic (e.g., ch1oramphenico1, ampici11in, gentamycin, streptomycin, tetracyc1ine, kanamycin, neomycin) or the ability to grow on se1ective media (e.g., ura (uraci1), 1eu (1eucine), trp (tryptophan), his (histidine)). Usua11y, p1asmids contain antibiotic resistance marker to force the bacteria1 ce11 to maintain the plasmid. In an embodiment of the inVention, the DNA construct may comprise a nucleotide sequence of a kanamycin resistance marker. In a specific embodiment of the inVention, the nucleotide sequence for conferring kanamycin resistance comprises the sequence of SEQ ID 12 (and Wherein the sequence is referred to as “KanR” in figures 1 and 3): The DNA construct may comprise a nucleotide sequence encoding for both of the rrnB T1 terrninator and the rrnB T2 terrninator. The rrnB T1 and T2 terrninators are both efficient transcription terrninators in isolated forms, however, When used together, rrnB T1 and Tterrninators may more eff1cient1y terrninate transcription.
In an embodiment, the nucleotide sequence of the rrnB T1 terrninator comprises the sequence
of SEQ ID 14:
In an embodiment, the nucleotide sequence of the rrnB T2 terrninator comprises the sequence of SEQ ID 15: AGAAGGCCATCCTGACGGATGGCCTTTT
The DNA construct may further comprise an origin of replication Which is a particular nucleotide sequence at Which DNA replication is initiated. DNA replication may proceed from this point bidirectionally or unidirectionally. Some commonly used origins of replication are ColEl, pMBl, pSCl0l, R6K, pBR322, R6K, pl5A, and pUC. In an embodiment of the inVention, the origin of replication is pMBl or deriVatiVes thereof In a specific embodiment of the inVention, the nucleic acid sequence of pMB1comprises the sequence of SEQ ID 16: TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAG AGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGC TCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTT TCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCC CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACAC GACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTAT GTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAA GGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGT TGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTT GCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA
In an embodiment, the DNA construct is an expression Vector Which comprises a nucleotide sequence encoding one or more of:
- rhaBAD promoter,
- RhaR transcription actiVator,
- RhaS transcription actiVator,
- an antibiotic resistance marker,
- a promoter operably linked to the nucleic acid encoding the antibiotic resistance marker,
- at least one terrninator, and- an origin of rep1ication.
In an embodiment, the expression Vector comprises a nucleotide sequence encoding:
- rhaBAD promoter,
- RhaR transcription actiVator,
- RhaS transcription actiVator,
- an antibiotic resistance marker,
- a promoter operably linked to the nucleic acid sequence encoding the antibiotic resistance marker,
- rrnB T1 terrninator,
- rrnB T2 terrninator, and
- an origin of rep1ication.
In an embodiment, the expression Vector comprises a nucleotide sequence encoding: - rhaBAD promoter,
- RhaR transcription actiVator,
- RhaS transcription actiVator,
- a kanamycin resistance marker,
- AmpR promoter,
- rrnB T1 terrninator,
- rrnB T2 terrninator, and
- pMB1 origin of rep1ication.
In an embodiment, the expression Vector comprises nucleotide sequences encoding: - rhaBAD promoter comprising the nucleotide sequence of SEQ ID 8,
- RhaR transcription activator comprising the nucleotide sequence of SEQ ID 9,
- RhaS transcription activator comprising the nucleotide sequence of SEQ ID 11,
- a kanamycin resistance marker comprising the nucleotide sequence of SEQ ID 12, - AmpR promoter comprising the nucleotide sequence of SEQ ID 13,
- rrnB T1 terrninator comprising the nucleotide sequence of SEQ ID 14,
- rrnB T2 terrninator comprising the nucleotide sequence of SEQ ID 15, and
- pMB1 origin of rep1ication comprising the nucleotide sequence of SEQ IDThe nucleotide sequence encoding a recombinant protein Which is to be cloned into the DNA constructs described above may comprise a nucleic acid encoding a monoclonal antibody or fragment thereof, preferably Certolizumab. The nucleic acid encoding Certolizumab
comprises a nucleic acid encoding the light and heavy chains of Certolizumab.
In an embodiment, the nucleotide sequence encoding the light chain of Certolizumab comprises the sequence of SEQ ID 5: gatattcagatgactcagagcccaagttcgctgagcgcttctgttggcgatcgtgtgaccattacatgcaaagcctcacagaacgttggt accaatgtcgcctggtatcagcagaaacctggaaaagcgcccaaagcgctcatctactcagcgagcttcctgtattcaggcgtgccgta tcgctttagcggctctggttccggtacagactttaccctcacgatttcgtccttacaaccggaagatttcgccacgtactattgccagcaat acaacatctatccgctgacctttggacaaggcaccaaagtggagatcaaacgcactgttgctgcaccgagtgtgttcatctttccaccgt ctgatgagcagctgaagtctggtacagcaagtgttgtgtgtctgctgaacaacttctatccgcgtgaagctaaagtacagtggaaagtcg acaatgccttgcaatccgggaatagccaggaaagcgtgactgaacaggacagcaaggattcgacctacagtctgagcagtaccttaa ccttgtcgaaagcggattacgagaaacacaaggtctatgcctgtgaagtcacgcaTCAAGGCCTGTCATCGCCTGT TACTAAATCATTTAATAGAGGAGAATGTTAA
In an embodiment of the invention, the nucleotide sequence encoding the heavy chain of Certolizumab comprises the sequence of SEQ ID 6: gaagtgcagcttgtggagtctggaggtggcttagtccagccaggtggttccctgcgcttgtcctgtgcagcgagcgggtatgtAttcac agattatggcatgaactgggttcggcaagcaccaggcaaaggcctcgaatggatggggtggatcaacacgtatattggggaaccgatt tatgcggatagcgtcaaaggtcgcttcacgttcagtctggataccagcaaatcaaccgcgtatctccagatgaatagcctccgtgctgaa gatactgccgtgtactactgtgcgcgtggttatcgcagttatgcgatggattactggggccaaggcaccttagtcaccgttagttctgcct ccaccaaaggcccatcagtgtttccgctggccccttcgtctaaatcgacgagtggtggcacagccgcactgggatgcctggtcaaaga ctactttcccgaacctgtaaccgtaagctggaatagtggtgctttgacctcaggcgtgcatacgtttccggctgtcctgcagtcatccggt ctgtactcgctttcgagcgttgttactgtaccctctagctccctgggcacccagacgtacatctgcaatgtgaaccataagccgtcgaaca
ccaaagtggacaagaaagttgagccgaaaagctgcgacaaaacgcacacatgtgccgccTAA
In an embodiment, the nucleic acid encoding the light and heavy chains of Certolizumab further comprises a nucleotide sequence encoding a signal peptide operably linked to either one or both of the nucleotide sequences encoding the heavy and light chains of Certolizumab.
The signal peptide is preferably selected from the group consisting of MalE, OmpA, PhoA,DsbA and Pelb. The nucleic acid sequence encoding the signal peptide is preferably a nucleotide sequence encoding the PelB signal peptide.
In an enibodinient, the nucleotide sequence encoding the PelB signal peptide Which is operably linked to the nucleotide sequences encoding the light chain of Certolizuniab con1prises the sequence of SEQ ID 18 and is in the present invention also referred to as PelB signal sequence 1 (see Figure 3) and abbreviated PelBl:
ATGAAGTATCTtCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGC CTGCAATGGCA
In an enibodinient of the invention, the nucleotide sequence encoding the PelB signal peptide Which is operably linked to the nucleotide sequences encoding the heavy chain of Certolizuniab con1prises the sequence of SEQ ID 19 and is in the present invention also
referred to as PelB signal sequence 2 (see Figure 3) and abbreviated PelB2:
ATGAAGTATCTGTTGCCGACTGCTGCAGCGGGACTGCTGCTGTTAGCGGCACAAC CGGCGATGGCG
In an enibodinient, the DNA constructs con1prises an improved TIR of SEQ ID 20: TTGCTCATGAAGTAT
In an enibodinient, the DNA constructs con1prises an improved TIR of SEQ ID 21: TGTTAAATGAAGTAT
In an enibodinient, the DNA construct con1prises TIRs of nucleotide sequence SEQ ID No 20 and 21 and Wherein these TIR nucleotide sequences Will con1prises at least the first 9 nucleotides of a signal peptide nucleotide sequence of SEQ ID 18 and 19, respectively.
In a specific enibodinient of the invention, DNA construct con1prises the nucleotide sequence of SEQ ID 17 Which is also referred to as KTXHIS-Cert-PelB1-LC-PelB2-HC in the present invention. This DNA construct is preferably an expression vector.The bacterial host cell to be used for producing the recombinant protein comprises a chromosome having a mutation or modif1cation Which disables rhamnose metabolism. The bacterial host cell may be an E. coli cell. In a preferred embodiment, the bacterial host cell is an E. coli K-l2 cell, more preferably the bacterial host cell is an E. coli WS l l0 cell. The disabled rhamnose metabolism is achieved by a mutation in the nucleotide sequence encoding RhaB Which renders RhaB inactive. Altematively, the disabled rhamnose metabolism is achieved by using a bacterial host cell having a chromosome in Which the nucleotide sequence encoding RhaB is deleted; this can e.g. be achieved by deleting the nucleotide sequence encoding RhaB. Preferably, the chromosome of the bacterial host cell comprises the
nucleic acid sequence encoding RhaT, i.e. the RhaT gene is intact.
In a specific embodiment of the invention, the bacterial host cell is E. coli WS l l0 rhaBfi
ADegP Aprc sprWI48R.
In a specific embodiment of the invention, the bacterial host cell E. coli WS l l0 rhaBfi ADegP Aprc sprWI48R comprises the KTXHIS-Cert-PelBl-LC-PelB2-HC expression vector and is used in a method of expressing Certolizumab Which can be used in the production of a
Certolizumab biosimilar.
A biosimilar is a highly similar to the reference product, i.e. a Certolizumab biosimilar has highly similar molecular structure and function (i.e. bioactivity) as Certolizumab pegol (Cimzia®) Which is produced by the reference product sponsor (i.e. the originator). Moreover, a biosimilar has no clinically meaningful differences from a reference product and the clinical trials that are conducted on biosimilars assess pharmacokinetics and immunogenicity. Most importantly, biosimilars: a) meet medical agency standards of approval, (b) are manufactured in medical agency licensed facilities, and (c) are tracked as part of post-market surveillance to
ensure continued safety (as indicated in https://WvvW.fda.gov/media/l08905/download).
The present invention can be exemplified as disclosed in the examples l-9 in the beloW non-
limiting EXAMPLES section.
It should be understood that these examples, relating to the XB166 host cell and the KTXHIS- Cert-PelB1-LC-PelB2-HC expression vector, as well as their combined use, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above disclosed embodiments of the invention and the following examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various types of therapeutic antibodies and immunoglobulins. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. An example of such a modification is that one or more of the above indicated SEQ IDs 1-21 may be replaced by a sequence with at least 90 % sequence identity thereto.
EXAMPLES
Example 1 relates to the construction of the XB166 host cell which is in the present invention
also referred to as E. coli W3110 rhaBfi ADegP Aprc sprWI48R.
Example 2 relates to the construction of the KTXHIS-Cert-PelB1-LC-PelB2-HC expression
vector.
Example 3 relates to the light and the heavy chains of Certolizumab expressed by the
expression vector in disclosed in Example
Example 4 shows that Certolizumab produced according to the present invention is free of
bacteriophages. Example 5 relates to TIR library selection and the isolation of evolved TIRs.
Example 6 relates to Westem-blot analysis of media fraction expression -the results show that the E83 expression vector which comprised the synthetically evolved TIR for the regulation of Certolizumab heavy chain expression resulted in the highest levels of
Certolizumab.
Example 7 relates to Westem-blot analysis of total fraction expression -the results show that the E83 expression vector which comprised the synthetically evolved TIR for the regulation
of Certolizumab heavy chain expression resulted in the highest levels of Certolizumab.Example 8 relates to NanoDrop® and ÄKTA chromatography which was performed to detect yield and titer differences between expression systems XB62 (XB17 host cell containing D37 expression vector having un-evolved TIRs and a wild-type nucleotide sequence for the PelB signal peptide upstream of each of the nucleotide sequences for the light and heavy chains of Certolizumab) and XBl02 (XB17 host cell containing expression vector E83 having synthetically engineered TIR for the regulation of the Certolizumab heavy chain expression) - the results show that the expression system XBl02 which comprises the expression vector E83 containing the synthetically engineered TIR (for the regulation of the Certolizumab
heavy chain expression) results in highest yields and titer of Certolizumab.
Example 9 relates to the comparison of Certolizumab expression in host cells XBl7 and XBl66 -the results show that the use of the XBl66 host cell (E. coli W3 l 10 rhaBfi ADegP Aprc sprWI48R) in combination with El ll (KTXHIS-Cert-Pelbl-LC-Pelb2-HC) results in highest relative yields of Certolizumab.
Example l - Strain development
The XBl66 host cell is an E. coli W33 10 derivative which was genetically engineered for the production of recombinant proteins such as antibodies and antibody fragments. Moreover, the XBl66 host cell was designed as a strain for a rhamnose inducible system where the nucleotide sequence encoding the recombinant protein of interest is cloned into the KTXHIS
plasmid and expressed under the control of a rhamnose inducible promoter.
The XB l 66 host cell was developed from the parental E. coli strain W3 l 10 which was obtained from the E. coli Genetic Stock Center (CGSC), Yale University (New Haven, USA), Catalog No.: 4474. Genotype: F-, /l-, INÛfrnD-rrnEfl, rph-I .
XBl66 was customized for efficient induction from a rhamnose-dependent promoter by deactivating the rhaB gene [7]. Moreover, three genomic modifications useful for the
production of antibody fragments [8], were introduced as described below subsections in detail.
In summary, the method for developing the XBl66 host cell involved the following listed modifications which will be discussed in detail in the below subsections: a) RhaB frame shift mutation; b) clegP deletion;
c) prc deletion; andd) sprWI 48R mutation.
Step (a) - Construction of the RhaB frame shift mutant.
The parental E. coli strain WS l 10 Was engineered to generate a derivative With a frameshift in the chromosomal copy of rhaB making it unable to utilize rhamnose as a carbon source. To this end, cells Were genetically engineered using the gene replacement plasmid pMAK705-
rhaBfs [9].
The engineered strain Was phenotypically tested to Verify that rhamnose cannot be utilized as carbon source anymore. Furthermore, the chromosomal fragment containing the rhaB frameshift Was PCR amplified and the PCR product Was sequenced to confirm the correct
insertion of two bases (see underlined CG bases in below disclosed SEQ ID NO 22):
tgtggcagcaactgattcagcccggcgagaaactgaaatcgatccggcgagcgatacagcacattggtcagacacagattatcggtat gttcatacagatgccgatcatgatcgcgtacgaaacagaccgtgccaccggtgatggtatagggctgcccattaaacacatgaataccc gtgccatgttcgacaatcacaatttcatgaaaatcatgatgatgttcaggaaaatccgcctgcgggagccggggttctatcgccacggac gcgttaccagacggaaaaaaatccacactatgtaatacggtcatactggcctcctgatgtcgtcaacacggcgaaatagtaatcacgag gtcaggttcttaccttaaattttcgacggaaaaccacgtaaaaaacgtcgatttttcaagatacagcgtgaattttcaggaaatgcggtgag catcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaag gtcgcgaattcaggcgctttttagactggtcgtaatgaaattcagcaggatcacattatgacctttcgcaattgtgtcgccgtcgatctcgg
cgcatccagtgggcgcgtgatgctggcgcgttacgagcgtgaatgccgcagcctgacgctgcgcgaaatccatcgttttaacaatggg ctgcatagtcagaacggctatgtcacctgggatgtggatagcctGgaaagtgccattcgccttggattaaacaaggtgtgcgaggaag
ggattcgtatcgïatagcattgggattgatacctggggcgtggactttgtgctgctcgaccaacagggtcagcgtgtgggcctgccc gttgcttatcgcgatagccgcaccaatggcctaatggcgcaggcacaacaacaactcggcaaacgcgatatttatcaacgtagcggca tccagtttctgcccttcaatacgctttatcagttgcgtgcgctgacggagcaacaacctgaacttattccacacattgctcacgctctgctga tgccggattacttcagttatcgcctgaccggcaagatgaactgggaatataccaacgccacgaccacgcaactggtcaatatcaatagc
gacgactgggacgagtcgctactggcgtggagcggggccaacaaagcctggtttggtcgcccgacgcatccgggtaatgtcataggt cactggatttgcccgcagggtaatgagattccagtggtcgccgttgccagccatgataccgccagcgcggttatcgcctcgccgttaaa cggctcacgtgctgcttatctctcttctggcacctggtcattgatgggcttcgaaagccagacgccatttaccaatgacacggcactggc
agccaacatcaccaatgaaggcggggcggaaggtcgctatcgggtgctgaaaaatattatgggcttatggctgcttcagcgagtgcttc aggagcagcaaatcaacgatcttccggcgcttatctccgcgacacaggcacttccggcttgccgcttcattatcaatcccaatgacgatc
gctA single colony was picked and grown in LB Vegitone to prepare a glycerol Stock. The resulting strain E. coli W3110 rhaBfi which was designated as XB17 served as starting strain
for the following genetic n1odif1cations.
Step (b) - degP deletion
The XB17 (E. coli W3110 rhaBfi) strain was further engineered to be equipped with three key n1odif1cations (degP prc spr) in order to create a “triple-niutant” host strain allowing high-level accuniulation of reconibinant antibody fragments due to reduced proteolytic
degradation of the light chain in the periplasni [8].
To this end, the genoniic copy of the gene coding for the periplasniic serine endoprotease DegP was knocked-out using the gene replacen1ent plasn1id pMAK705-sacB-DegP, in which a fragment honiologous to the degP upstream region is fused to a fragment honiologous to the degP downstream region. In order to avoid polar effects, the gene replacen1ent cassette was designed to preserve the degP start codon as well as the last 7 degP codons. Next to a temperature-sensitive origin of replication, this gene replacen1ent plasn1id also carries the
sacB gene for counterselection, thus facilitating plasn1id curing [10] after strain construction.
Deletion of the chron1oson1al degP gene was confirrned by PCR an1plif1cation of the degP “scar” region and sequencing of the PCR product (see below SEQ ID 23 wherein the underlined codon is the degP start codon and the codons in bold letters are the last 7 codons
of the degP gene):
atataaaaatgtcgctgtaaaacatgtgtttagccatccagatgtcgagcggcttgaattgcagggctatcgggtcattagcggattattag agatttatcgtcctttattaagcctgtcgttatcagactttactgaactggtagaaaaagaacgggtgaaacgtttccctattgaatcgcgctt attccacaaactctcgacgcgccatcggctggcctatgtcgaggctgtcagtaaattaccgtcagattctcctgagtttccgctatgggaa tattattaccgttgccgcctgctgcaggattatatcagcggtatgaccgacctctatgcgtgggatgaataccgacgtctgatggccgtag aacaataaccaggcttttgtaaagacgaacaataaatttttaccttttgcagaaactttagttcggaacttcaggctataaaacgaatctgaa gaacacagcaattttgcgttatctgttaatcgagactgaaatacíatctacctgttaatgcagTAAtctccctcaaccccttcctg aaaacgggaaggggttctccttacaatctgtgaacttcaccacaactccatacatcttcatcatcctttaggcatttgcacaatgccgtacg ttacgtacttccttatgctaagccgtgcataacggaggacttatggctggctggcatcttgataccaaaatggcgcaggatatcgtggcac gtaccatgcgcatcatcgataccaatatcaacgtaatggatgcccgtgggcgaattatcggcagcggcgatcgtgagcgtattggtgaa ttgcacgaaggtgcattgctggtactttcacagggacgagtcgtcgatatcgatgacgcggtagcacgtcatctgcacggtgtgcggcaggggattaatctaccgttacggctggaaggtgaaattgtcggcgtaattggcctgacaggtgaaccagagaatctgcgtaaatatggcg aactggtctgcatgacggc
A single colony of the engineered strain Was picked and groWn in LB Vegitone to prepare a glycerol stock. The resulting strain E. coli W3110 rhaBfi ADegP Which Was designated as
XB83 served as starting strain for the following genetic n1odif1cations.
Step (c) - prc deletion
With E. coli W3110 rhaBfi ADegP (XB83) as starting strain, the prc gene Was knocked-out using the gene replacen1entplasn1id pMAK705-sacB-prc in analogy to the above described degP deletion. Deletion of the chron1oson1al prc gene Was confirmed by PCR aniplification of the prc scar region and sequencing of the PCR product (see below SEQ ID 24 Wherein the underlined codon is the prc start codon and the codons in bold letters are the last 7 codons of
the prc gene):
tttacggtgttaaacccggcgcaacgcgtgtcgatcttgacggcaacccatgcggtgagctggacgagcaacatgtagagcatgctcg caagcagcttgaagaagcgaaagcgcgtgttcaggcacagcgtgctgaacagcaagcgaaaaaacgcgaagctgccgcaactgct ggtgagaaagaagacgcaccgcgccgcgaacgcaagccacgtccgactacgccacgccgcaaagaaggcgctgaacgtaaacct cgtgcgcaaaagccggtagagaaagcgccaaaaacagtaaaagcacctcgcgaagaacagcacaccccggtttctgacatttcagct ctgactgtcggacaagccctgaaggtgaaagcgggtcaaaacgcgatggatgccaccgtattagaaatcaccaaagacggcgtccg cgtccagctgaattcgggtatgtctttgattgtgcgcgcagaacacctggtgttctgaaacggaggccgggccaggcícaaccc gctcccgtcaagTAAtatcaatcaggcacaagaaattgtgcctgattttttaacagcgacaagatgccgtaaatcagatgctacaaaa tgtaaagttgtgtctttctggtgacttacgcactatccagacttgaaaatagtcgcgtaacccatacgatgtgggtatcgcatattgcgttttg ttaaactgaggtaaaaagaaaattatgatgcgaatcgcgctcttcctgctaacgaacctggccgtaatggtcgttttcgggctggtactga gcctgacagggatacagtcgagcagcgttcaggggctgatgatcatggccttgctgttcggttttggtggttccttcgtttcgcttctgatg tccaaatggatggcattacgatctgttggcggggaagtgatcgagcaaccgcgtaacgaaagggaacgttggctggtcaatactgtag caacccaggctcgtcaggcggggatcgctatgccgcaagtggctatctacc
A single colony of the engineered strain Was picked and groWn in LB Vegitone to prepare a glycerol stock. The resulting strain E. coli W3110 rhaBfi AdegP Aprc designated here as
XB152 served as starting strain for final genetic engineering step.
Step (d) - sprWI48R
The final step of engineering the expression host Was devoted to coniplenient the triple n1utant genotype by the introduction of the sprWI 48R niutation leading to an an1ino acid substitution in the spr gene. While the deletion of degP and prc is expected to result in a strain With reduced proteolytic degradation of the light chain of the antibody fragn1ents, the spr niutation
is described to produce higher aniounts of reconibinant protein [8].
Using E. coli WS l l0 rhaBfi ADegP Aprc (XBl52) as starting strain, the genon1ic copy of the spr gene Was engineered using the gene replacenient plasn1id pMAK705-sacB-sprWl48R containing the n1utant spr fragment [l0]. The chron1oson1al fragment containing the
spr WI 48R niutation Was PCR an1plif1ed and the PCR product Was sequenced to confirrn that the genon1ic spr gene Was correctly replaced by the n1utant allele (see below SEQ ID 25; the T to A niutation is highlighted in bold):
aacaaacaacatggtcaaatctcaaccgattttgagatatatcttgcgcgggattcccgcgattgcagtagcggttctgctttctgcatgta gtgcaaataacaccgcaaagaatatgcatcctgagacacgtgcagtgggtagtgaaacatcatcactgcaagcttctcaggatgaattt
gaaaacctggttcgtaatgtcgacgtaaaatcgcgaattatggatcagtatgctgactggaaaggcgtacgttatcgtctgggcggcag
cactaaaaaaggtatcgattgttctggtttcgtacagcgtacattccgtgagcaatttggcttagaacttccgcgttcgacttacgaacagc aggaaatgggtaaatctgtttcccgcagtaatttgcgtacgggtgatttagttctgttccgtgccggttcaacgggacgccatgtcggtatt tatatcggcaacaatcagtttgtccatgcttccaccagcagtggtgttattatttccagcatgaatgaaccgtacAggaagaagcgttaca acgaagcacgccgggttctcagccgcagctaataaaccgtttggatgcaatcccttggctatcctgacgagttaactgaaagcactgctt aggcagtgcttttttgttttcattcatcagagaaaatgatgtttccgcgtcttgatccaggctatagtccggtcattgttatcttttaaatgttgtc gtaatttcaggaaattaacggaatcatgttcatacgcgctcccaattttggacgtaagctcctgcttacctgcattgttgcaggcgtaatgat tgcgatactggtgagttgccttcagtttttagtggcctggcataagcacgaagtcaaatacgacacactgattaccgacgtacaaaagtat ctcgatacctattttgccgacctgaaatccactactgaccggctccagccgctgaccttagatacctgccagcaagctaaccccgaactg
accgcccgcgcagcgtttagcatgaatgtccgaacgtttgtgctggtgaaagataaaa
A single colony of the engineered strain Was picked and groWn in LB Vegitone to prepare a glycerol stock. The resulting strain E. coli W3 l 10 rhaBfi ADegP Aprc sprWI48R Was designated as XB l
The advantageous technical effects of the XBl66 is shown and discussed in detail in Exaniple
8 and FigureExaniple 2 - KTXHIS-Cert-PelBl-LC-PelB2-HC
The first construct of Certolizuniab that Was n1ade Was With signal peptides On1pA-LC, PelB- HC (gene synthesized) and cloned into the KTXHIS plasn1id Via EcoRI and HindIII sites. From this first construct, the signal peptide(s) Was then exchanged With the use of hon1ologous reconibination of PCR fragnients in E coli. I°ve made several versions of PelBLC, PelBHC in KTXHIS vector Where, even though the resulting an1ino acid sequence of PelB Would be the same, the codons Were different. I”n1 not sure Which one Was used as the
starting plasniid by Kiavash to be honest.
The n1other construct for the expression of Certolizuniab Was n1ade Was With signal peptides On1pA (upstreani of nucleotide sequence encoding light chain) and PelB (upstreani of nucleotide sequence encoding the heavy chain) and cloned into the KTXHIS plasniid (see Seq ID l) via EcoRI and HindIII sites (see SEQ ID 2). From this n1other construct, the existing signal peptide nucleotide sequences Were then swapped With signal peptide nucleotide sequences of SEQ ID 18 and 19 by using honiologous reconibination of PCR fragnients in E. coli yielding expression vector D37. Post synthetic construction of both TIRs regulating the expression of light and heavy chains of Certolizuniab (as explained in the beloW Exaniple 5- 10), an expression vector coniprising SEQ ID l7 Was generated.
The expression plasniid KTXHIS has previously been described in European patent application EP2020l096.3. The resulting nucleotide sequence con1prises a nucleotide sequence of SEQ ID l7 and is referred to as KTXHIS-Cert-PelBl-LC-PelB2-HC in the present invention. The plasniid n1ap of KTXHIS-Cert-PelBl-LC-PelB2-HC is illustrated in figure 3:
GGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAAC GCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCC CACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGT GGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGG CTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTC CTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCC GGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTT CTAAATACATTCAAATATGTATCCGCTCATGAGACTAGGCTTCCGCGCCCTCATCC GAAAGGGCGTATTCATATATGCGGTGÛAAATACCGCACAGATGCGTAAGGAGAA AATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTC GTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA CAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAG
GCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTYÜÛCCATAGGCTCCGCCCCC CTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAG GACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTT CCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGC GCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGG TAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTÛÉ
TTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTYÜÜTGTTTGCAAGCAGCAGATTACGCGC
AGAA$uM&&AGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTC AGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGA
TCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACT GCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGT AATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTAT CGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTC AAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGA GAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTA
CGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGC CTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAAT CGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAA TCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGA
GTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCA TAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACG CTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCG ATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTG AATATGGCTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATT GTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGT TCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATC ATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTATCTTTCTG CGAATTGAGATGACGCCACTGGCTGGGCGTCATCCCGGTTTCCCGGGTAAACACC ACCGAAAAATAGTTACTATCTTCAAAGCCACATTCGGTCGAAATATCACTGATTA ACAGGCGGCTATGCTGGAGAAGATATTGCGCATGACACACTCTGACCTGTCGCAG ATATTGATTGATGGTCATTCCAGTCTGCTGGCGAAATTGCTGACGCAAAACGCGC TCACTGCACGATGCCTCATCACAAAATTTATCCAGCGCAAAGGGACTTTTCAGGC TAGCCGCCAGCCGGGTAATCAGCTTATCCAGCAACGTTTCGCTGGATGTTGGCGG CAACGAATCACTGGTGTAACGATGGCGATTCAGCAACATCACCAACTGCCCGAAC AGCAACTCAGCCATTTCGTTAGCAAACGGCACATGCTGACTACTTTCATGCTCAA GCTGACCAATAACCTGCCGCGCCTGCGCCATCCCCATGCTACCTAAGCGCCAGTG TGGTTGCCCTGCGCTGGCGTTAAATCCCGGAATCGCCCCCTGCCAGTCAAGATTC AGCTTCAGACGCTCCGGGCAATAAATAATATTCTGCAAAACCAGATCGTTAACGG AAGCGTAGGAGTGTTTATCATCAGCATGAATGTAAAAGAGATCGCCACGGGTAA TGCGATAAGGGCGATCGTTGAGTACATGCAGGCCATTACCGCGCCAGACAATCAC CAGCTCACAAAAATCATGTGTATGTTCAGCAAAGACATCTTGCGGATAACGGTCA GCCACAGCGACTGCCTGCTGGTCGCTGGCAAAAAAATCATCTTTGAGAAGTTTTA ACTGATGCGCCACCGTGGCTACCTCGGCCAGAGAACGAAGTTGATTATTCGCAAT ATGGCGTACAAATACGTTGAGAAGATTCGCGTTATTGCAGAAAGCCATCCCGTCC CTGGCGAATATCACGCGGTGACCAGTTAAACTCTCGGCGAAAAAGCGTCGAAAA GTGGTTACTGTCGCTGAATCCACAGCGATAGGCGATGTCAGTAACGCTGGCCTCG CTGTGGCGTAGCAGATGTCGGGCTTTCATCAGTCGCAGGCGGTTCAGGTATCGCT GAGGCGTCAGTCCCGTTTGCTGCTTAAGCTGCCGATGTAGCGTACGCAGTGAAAG AGAAAATTGATCCGCCACGGCATCCCAATTCACCTCATCGGCAAAATGGTCCTCC AGCCAGGCCAGAAGCAAGTTGAGACGTGATGCGCTGTTTTCCAGGTTCTCCTGCA AACTGCTTTTACGCAGCAAGAGCAGTAATTGCATAAACAAGATCTCGCGACTGGC GGTCGAGGGTAAATCATTTTCCCCTTCCTGCTGTTCCATCTGTGCAACCAGCTGTC GCACCTGCTGCAATACGCTGTGGTTAACGCGCCAGTGAGACGGATACTGCCCATC CAGCTCTTGTGGCAGCAACTGATTCAGCCCGGCGAGAAACTGAAATCGATCCGGC GAGCGATACAGCACATTGGTCAGACACAGATTATCGGTATGTTCATACAGATGCC GATCATGATCGCGTACGAAACAGACCGTGCCACCGGTGATGGTATAGGGCTGCCCATTAAACACATGAATACCCGTGCCATGTTCGACAATCACAATTTCATGAAAATCA TGATGATGTTCAGGAAAATCCGCCTGCGGGAGCCGGGGTTCTATCGCCACGGACG CGTTACCAGACGGAAAAAAATCCACACTATGTAATACGGTCATACTGGCCTCCTG ATGTCGTCAACACGGCGAAATAGTAATCACGAGGTCAGGTTCTTACCTTAAATTT TCGACGGAAAACCAC GTAAAAAAC GTCGATTTTTCAAGATACAGC GTGAATTTTC AGGAAATGCGGTGAGCATCACATCACCACAATTCAGCAAATTGTGAACATCATCA CGTTCATCTTTCCCTGGTTGCCAATGGCCCATTTTCTTGTCAGTAACGAGAAGGTC GCGAATCCAGGCGCTTTTTAGACTGGTCGTAATGAAATTCAGGAGGAAtTgctcATG AAGTATCTtCTGCCGACCGCAGCAGCGGGTCTGCTGCTGCTGGCAGCACAGCCTG CAATGGCAgatattcagatgactcagagcccaagttcgctgagcgcttctgttggcgategtgtgaccattacatgcaaagcctc acagaacgttggtaccaatgtcgcctggtatcagcagaaacctggaaaagcgcccaaagcgctcatctactcagcgagcttcctgtatt caggcgtgccgtatcgctttagcggctctggttccggtacagactttaccctcacgatttcgtccttacaaccggaagatttcgccacgta ctattgccageaatacaacatctatccgctgacctttggacaaggcaccaaagtggagatcaaacgcactgttgctgcaccgagtgtgtt catctttccaccgtctgatgagcagctgaagtctggtacageaagtgttgtgtgtctgctgaacaacttctatccgcgtgaagctaaagtac agtggaaagtcgacaatgccttgcaatccgggaatagccaggaaagcgtgactgaacaggacagcaaggattcgacctacagtctga gcagtaccttaaccttgtcgaaagcggattacgagaaacacaaggtctatgcctgtgaagtcacgcaTCAAGGCCTGTCAT CGCCTGTTACTAAATCATTTAATAGAGGAGAATGTTAAATGAAGTATCTGTTGCC GACTGCTGCAGCGGGACTGCTGCTGTTAGCGGCACAACCGGCGATGGCGgaagtgcag cttgtggagtctggaggtggcttagtccagccaggtggttccctgcgcttgtcctgtgcagegagegggtatgtAttcacagattatggc atgaactgggttcggcaagcaccaggcaaaggcctcgaatggatggggtggatcaacacgtatattggggaaccgatttatgcggata gcgtcaaaggtcgcttcacgttcagtctggataccagcaaatcaaccgcgtatctccagatgaatagcctccgtgctgaagatactgcc gtgtactactgtgcgcgtggttatcgcagttatgcgatggattactggggccaaggcaccttagtcaccgttagttctgcctccaccaaag gcccatcagtgtttccgctggccccttcgtctaaatcgacgagtggtggcacagecgcactgggatgcctggtcaaagactactttccc gaacctgtaaccgtaagctggaatagtggtgctttgacctcaggcgtgcatacgtttccggctgtcctgcagtcatccggtctgtactcgc tttcgagcgttgttactgtaccctctagctccctgggcacccagacgtacatctgcaatgtgaaccataagccgtcgaacaccaaagtgg acaagaaagttgagcegaaaagctgcgacaaaacgcacacatgtgccgccTAATAAaagctt
Example 3 - Recornbinant protein - Certolizurnab
The amino acid sequence of the light chain of Certolizumab expressed by the expression vector KTXHIS-Cert-PeIB1-LC-Pe1B2-HC cornprises the sequence of SEQ ID 3: DIQMTQSPSSLSASVGDRVTITCKASQNVGTNVAWYQQKPGKAPKALIYSASFLYSG VPYRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNIYPLTFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LS STLTLSKADYEKHKVYACEVTHQGLS SPVTKSFNRGEC
The amino acid sequence of the heavy chain of Certolizumab expressed by the expression vector KTXHIS-cert-PelB1-LC-PelB2-HC comprises the sequence of SEQ ID 4: EVQLVESGGGLVQPGGSLRLSCAASGYVFTDYGMNWVRQAPGKGLEWMGWINTYI GEPIYADSVKGRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCARGYRSYAMDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT CAA
Moreover, the resulting PelB signal peptide comprises an amino acid sequence of SEQ ID 3 [6]: MKYLLPTAAAGLLLLAAQPAMA
Example 4 - Cell banking The XB166 Was transforrned With KTXHIS-Cert-Pelb1-LC-Pelb2-HC.
An RCB Was prepared by picking a single colony and overnight growth in Defined Bioreactor Medium (DBM). The following day, 20% (final concentration) glycerol Was aseptically added, the culture Was aliquoted and stored in -80 °C.
All materials used in the manufacture of the RCB are of non-animal and non-human origin.
Example 5 - TIR library selection and the isolation of eVolVed TIRs
Synthetic evolution Was used to select TIRs that Were more compatible With the host cell ribosomes by utilizing the methods disclosed in US10696963 and Mirzadeh et al 2015 [11] and briefly described in this example (see also WO21158163 for synthetical evolution of similar TIRs). A TIR library Was created With the design that meant the six nucleotides immediately upstream from the ATG start codon Were completely randomized, and the six nucleotides immediately doWnstream from the ATG start codon Were randomized With synonymous codon changes only. Each TIR library theoretically contained >18,000 expression plasmids With a different TIR. In this experiment, the expression plasmids comprising the expression cassette PelBss-Certolizumab-LC- PelBss-Certolizumab-HC (Wherein “ss” is abbreViation for signal sequence) Which in tum Was fused to ß-lactamaseWere subjected to the described TIR library generation. For each certolizumab chain in separate experiments, a TIR library Was transforrned into bacteria and plated onto LB agar plates containing a fixed amount of rhamnose and increasing concentrations of ampicillin (Figure 4). Colonies from the TIR library plates (upper panel/roW in Figure 4) that Were resistant to a high concentration of ampicillin, or Were visually bigger than a corresponding colony from the un-evolved expression cassette plates (lower panel/row in Figure 4), Were isolated and the TIRs Were sequenced. The identified TIRs (i.e. synthetically evolved TIRs) Were then back-engineered into plasmids devoid of the ß-lactamase in order to assess their
effect on Certolizumab expression in larger ferrnentation scales .
A specific example of a TIR that resulted by using the above described synthetic evolution method is in the present invention referred to as TIR-LC and has SEQ ID 20 and this TIR-LC regulates the expression of the light chain of Certolizumab . The TIR-LC of SEQ ID 20 is the
TIR comprised in the El ll expression Vector discussed in Example
Example 6 - Westem-blot analysis of media fraction after 20 hours of induced expression in DASbox® ferrnenters (i.e. DASbox® Mini Bioreactor Systems).
Certolizumab Was expressed by using:
i. an expression Vector referred to as D36 Which comprises a codon optimized version of the Wild-type PelB signal peptide nucleotide sequence (of SEQ ID 19) upstream of the nucleotide sequence of the heavy chain of Certolizumab, and
ii. expression vectors E8 l, E82 and E83 Which differ from the D36 vector in that the E8 l, E82 and E83 expression vectors each comprise synthetically engineered modif1cations of the TIRs responsible for the regulation of the Certolizumab heavy chain expression (i.e. the TIRs upstream of the nucleotide sequence of the
Certolizumab heavy chain).
The synthetically engineered TIR (for the regulation of the Certolizumab heavy chain expression) comprised in the E83 expression vector has a nucleotide sequence of SEQ ID 2l. The nucleotide sequences of the TIRs upstream of the heavy chains of E8l and E82 (and other expression vectors that Were researched, developed and tested) are not shown in the present invention. The aim of the synthetic engineering of the TIRs upstream of the heavy chain nucleotide sequence Was to test the effect of nucleotide substitutions on the expression
of Certolizumab.A Volume of 100 ul of each Certolizumab containing sample was spun down at 14000 X g for 5 minutes after which the supematant was separated and toped up with H20 to a total of 100 ul. Next, 100 ul of 2x sample buffer was added to the sample before it was boiled for 5 minutes at 95 °C, after which an equal amount of volume was loaded in each well and
separated by a 12% SDS-PAGE. Protein levels were visualized by immuno-blotting with
antisera to Certolizumab. The results are shown in Figure 5 which illustrates from left to right:
D36, E81, E82 and E83 expression vectors.
As illustrated in the Westem-blots in Figure 5, the E83 expression Vector resulted in the highest levels of Certolizumab (indicated as fragment antibody Fab” in the figure) in the media fraction. There was also signs of Fab” dimers and free light chain (LC) and heavy chain
(HC) present in the sample.
Example 7 - Westem-blot analysis of total (media and cell) fraction after 20 hours of induced
expression in DASbox® ferrnenters Certolizumab was expressed by using the D36, E81, E82, E83 expression vectors.
A volume of 5 ul of each sample was added to 100 ul of 2x Sample buffer before it was boiled for 5 minutes at 95 °C, and an equal amount of volume was loaded in each well and
separated by a 12% SDS-PAGE. Protein levels were visualized by immuno-blotting with
antisera to Certolizumab. The results are shown in Figure 6 which illustrates from left to right:
D36, E81, E82 and E83 expression vectors.
As illustrated in the Westem-blots in Figure 6, the E83 expression vector (which comprised the synthetically evolved TIR for the regulation of Certolizumab heavy chain expression) resulted in the highest levels of Certolizumab as indicated as fragment antibody Fab” in the
figure).
Example 8 - NanoDrop® and ÄKTA chromatography
Certolizumab was expressed by using the XB17 host cell (E. coli W3110 rhaBfi) by using (i) the D37 expression vector having an un-evolved TIR and a wild-type nucleotide sequence for
the PelB signal peptide upstream of each of the nucleotide sequences for the light and heavychains of Certolizumab , and (ii) expression Vector E83 comprising synthetically engineered
TIR for the regulation of the Certolizumab heavy chain expression.
After 20 hours of expression in DASbox® ferrnenters, Certolizumab purif1cation runs were perforrned with frozen clarif1ed lysate from expression systems XBl02 and XB62. Said clarif1ed lysate was prepared by (l) homogenization for three passages at about 800-900 Bar, (2) centrifugation, and (3) f1ltering of the supematant with 0.45 PES (polyethersulfone) filter. More specifically, 9.5 mL of the clarified lysate was loaded onto a CaptureSelectTM CHl-XL column (aff1nity resin having selectivity for the CHl domain) coupled to an ÄKTA chromatography system for each purif1cation run and 28 mL of the elution phase, which
contained the majority of the assembled Certolizumab, was collected from each run.
The protein concentration in the collected elution pool was measured by using a NanoDrop® microvolume UV-VIS spectrophotometer at 280 nm wavelength with 1.6 set as the extinction coefficient (see Figure 7, column titled “Nanodrop” for the results). The total amount of protein in the elution pool could then be calculated by multiplying 28 mL elution with the concentration measured. The total amount of protein in the collected elution pool was then divided by the volume sample loaded onto the column to calculate the yield target protein in
the clarified lysate, which was compared between the two batches.
In addition, the two runs were also evaluated by calculating the amount of protein throughout the whole elution phase and strip phase by using Unicom intemal evaluation software (of the
ÄKTA system) with 1.6 set as the extinction coefficient (see Figure 7, column titled “ÄKTA” for the results). The titer value is 2/3 of the yield value, due to volume of cell mass in the
harvest which is lost during clarification prior the purif1cation on column.
The results from both the NanoDrop® and ÄKTA analyses summarized in Figure 7 clearly show that the expression system XBl02 which comprises the expression vector E83 containing the synthetically engineered TIR (for the regulation of the Certolizumab heavy chain expression) results in highest yields and titer of Certolizumab (when compared to expression vector D37 which lacks the synthetically engineered TIR for the regulation of the
Certolizumab heavy chain expression).
Example 9 - Comparison of Certolizumab expression in host cells XBl7 and XBlCertolizumab expression levels were analyzed when using the E83 expression vector (which comprises synthetically engineered TIR for the regulation of the Certolizumab heavy chain expression) in the following host cells:
- XB17 (E. coli W3110 rhaBfi), and
- XB166 (E. coli W3110 rhaBfi ADegP Aprc sprWI48R).
After 20 hours of induced expression in the DASbox® ferrnenter, l ml sample was centrifuged at 13500 rpm for 20 min at 4 °C. Pellet and media fraction were separated and the pellet was resuspended with 0.5ml of 100 mM Tris HCl/ 10 mM EDTA, pH 7.4. Next, the resuspension was vortexed thoroughly before incubated at 60 °C for 16 hours. The pellet samples were then clarif1ed by centrifugation at 13500 rpm for 20 minutes at 4 °C before the supematant (extracted periplasmic sample) was collected and treated with DNase (final concentration of 0.02 mg/ml). Samples were f1ltered with low protein binding syringe filters (0.2 um, Spartan 13, GE Healthcare). After periplasmic extraction, samples were centrifuged at 14000 X g for 5 minutes after which 20 ul supematant was directly analyzed using aff1nity column (CHl-XL) - HPLC. Protein concentrations were compared to a standard curve using
purif1ed Certolizumab with known concentrations.
The results illustrated in Figure 8 show that the use of the XB166 host cell results in higher
relative yields of Certolizumab when compared with the expression in host cell XB
Example 10 - Comparison of Certolizumab expression in host cell XB166 by using E83 and
El l l expression vectors
Due to the above described advantageous effects of the E83 expression Vector (see Examples 6-9), the E83 expression vector was used as a template to synthetically evolve the light chain TIR (according to the general method described in Example 5). The resulting vector with best technical comprised the synthetically evolved the TIR upstream of the light chain having SEQ ID 20 (TIR-LC). Said vector is in the present invention referred to as El 1
In other words, the El l l expression Vector comprises: - a synthetically evolved TIR of SEQ ID 20 (TIR-LC) for the regulation of the expression of the light chain of Certolizumab; and - a synthetically engineered TIR of SEQ ID 21 (TIR-HC) for the regulation of the
expression of the heavy chain of Certolizumab
This example is similar to Example 9 but differs in that the difference of Certolizumab expression Was instead compared between:
- XB166 host cell comprising the E83 expression vector (having the synthetically engineered TIR of SEQ ID NO 21 for the regulation of only the heavy chain of Certolizumab)and Wherein the combination of host cell and vector is referred to as XB174 in Figure 9; and
- XB166 host cell comprising the expression vector El 11 having the synthetically constructed TIRs of SEQ ID 20 and SEQ ID NO 21 for the regulation of the
expression of light and heavy chains of Certolizumab, respectively.
The results illustrated in Figure 9 show that the use of the expression vector E111 results in higher relative yields of Certolizumab When compared With E83. In other Words, the E111 expression vector Which comprises:
- a synthetically engineered TIR for the regulation of the heavy chain; and
- synthetically evolved TIR for the regulation of the light chain, results in higher yields of Certolizumab When compared to E83 Which does not have
synthetically evolved TIR for the regulation of the light chain expression.REFERENCES
Holcroft CC et al. Interdependence of activation at rhaSR by cyclic AMP receptor protein, the RNA polymerase alpha subunit C-terrninal domain, and rhaR. J Bacteriol. 2000;182(23): 6774-Hjelm, A. et al. Tailoring Escherichia coli for the l-Rhamnose PBAD Promoter-Based Production of Membrane and Secretory Proteins. ACS synthetic biology 2017; 6 6: 985-
. Wilms, B. et al. High-cell-density ferrnentation for production of L-N-carbamoylase using
an expression system based on the Escherichia coli rhaBAD promoter. Biotechnol.
Bioeng. 2001; 73: 95-. Kumar, D. et al. QbD Based Media Development for the Production of Fab Fragments in
E. coli. Bioengineering 2019; 6,
Giacalone MJ et al. “Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. Biotechniques. 2006; 40(3): 355-364. Choi, J .H. et al. Secretory and extracellular production of recombinant proteins using Escherichia coli. Appl Microbiol Biotechnol 2004; 64, 625-Wilms B, Hauck A, Reuss M, Syldatk C, Mattes R, Siemann M, Altenbuchner J. High- cell-density ferrnentation for production of L-N-carbamoylase using an expression system based on the Escherichia coli rhaBAD promoter. Biotechnol Bioeng. 2001 Apr 20;73(2):95-
Chen C, Snedecor B, Nishihara JC, Joly JC, McFarland N, Andersen DC, Battersby JE, Champion KM. High-level accumulation of a recombinant antibody fragment in the periplasm of Escherichia coli requires a triple-mutant (degP prc spr) host strain. Biotechnol Bioeng. 2004 Mar 5;85(5):463-
Hamilton CM, Aldea M, Washbum BK, Babitzke P, Kushner SR. New method for generating deletions and gene replacements in Escherichia coli. Joumal of Bacteriology. 1989;171(9):4617-
Link AJ, Phillips D, Church GM. Methods for generating precise deletions and insertions in the genome of Wild-type Escherichia coli: application to open reading frame characterization. J Bacteriol. 1997 Oct;179(20):6228-Mirzadeh K, Martínez V, Toddo S, Guntur S, Herrgård MJ, Elofsson A, Nørholm MH, Daley DO. Enhanced Protein Production in Escherichia coli by Optimization of Cloning Scars at the Vector-Coding Sequence Junction. ACS Synth Biol. 2015 Sep 18;4(9):959-65
Claims (10)
1. DNA construct comprising nucleotide sequences encoding amino acid sequences, Wherein the amino acid sequences comprise: the amino acid sequence for Certolizumab; b. a first signal peptide of amino acid sequence of SEQ ID No 7 fused to the N- terrninus of light chain amino acid sequence of Certolizumab; and c. a second signal peptide of amino acid sequence of SEQ ID NO 7 fused to the N- terrninus of heavy chain amino acid sequence of Certolizumab, Wherein either the nucleotide sequence Which encodes the first signal peptide comprises a nucleotide sequences of SEQ ID No 18 or Wherein the nucleotide sequence Which encodes said second signal peptide comprises nucleotide sequences of SEQ ID No
2. DNA construct according to claim 1, Wherein the nucleotide sequence Which encodes said first signal peptide comprises nucleotide sequences of SEQ ID No
3. DNA construct according to claim 1, Wherein the nucleotide sequence Which encodes said second signal peptide comprises nucleotide sequences of SEQ ID No
4. Expression vector Which comprises the DNA construct according to any one of the previous claims 1-
5. Host cell Which comprises a DNA construct according to any one of the previous claims 1-3, Wherein said host cell is preferably a bacterial cell, more preferably E. coli, most preferably E. coli comprising a chromosome Which comprises a mutation or modification Which disables rhamnose metabolism.
6. Host cell according to claim 5, Wherein said host cell either comprises (i) a chromosome Which comprises a mutation in the nucleotide sequence of the rhaB gene Which renders RhaB inactive, or (ii) a chromosome in Which the nucleotide sequence encoding RhaB is deleted.
7. Host cell according to claim 5 or 6, Wherein said host cell is an E. coli W3110, preferably comprising a chromosome Which comprises a frame shift-mutation in the nucleotide sequence encoding RhaB.
8. Host cell according to any one of clainis 5-7, Wherein said host cell coniprises a chron1oson1e coniprising: a. a frame shift-niutation in the nucleotide sequence encoding RhaB; b. a degP deletion; c. a prc deletion; and d. a sprWI 48R niutation.
9. Host cell according to any one of the previous clainis 5-8, Wherein said host cell is E. coli WS l 10 rhaBfi ADegP Aprc sprWI48R.
l0. RNA expressed by a DNA construct according to any one of the previous clainis 1-3.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE2130264A SE545694C2 (en) | 2021-09-24 | 2021-09-24 | Dna construct comprising nucleotide sequences encoding amino acid sequences of certolizumab and pelb signal peptides |
| CA3232722A CA3232722A1 (en) | 2021-09-24 | 2022-09-23 | Dna constructs and host cells for expressing recombinant protein |
| AU2022349814A AU2022349814A1 (en) | 2021-09-24 | 2022-09-23 | Dna constructs and host cells for expressing recombinant protein |
| PCT/EP2022/076591 WO2023046930A1 (en) | 2021-09-24 | 2022-09-23 | Dna constructs and host cells for expressing recombinant protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE2130264A SE545694C2 (en) | 2021-09-24 | 2021-09-24 | Dna construct comprising nucleotide sequences encoding amino acid sequences of certolizumab and pelb signal peptides |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| SE2130264A1 SE2130264A1 (en) | 2023-03-25 |
| SE545694C2 true SE545694C2 (en) | 2023-12-05 |
Family
ID=86052935
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| SE2130264A SE545694C2 (en) | 2021-09-24 | 2021-09-24 | Dna construct comprising nucleotide sequences encoding amino acid sequences of certolizumab and pelb signal peptides |
Country Status (1)
| Country | Link |
|---|---|
| SE (1) | SE545694C2 (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001094585A1 (en) * | 2000-06-06 | 2001-12-13 | Celltech R & D Limited | Antibody molecules having specificity for human tumor necrosis factor alpha, and use thereof |
| WO2002048376A2 (en) * | 2000-12-14 | 2002-06-20 | Genentech, Inc. | Bacterial host strains |
| WO2011086136A1 (en) * | 2010-01-14 | 2011-07-21 | Ucb Pharma S.A. | Bacterial host strain comprising a mutant spr gene and having reduced tsp activity |
| WO2014058389A1 (en) * | 2012-10-12 | 2014-04-17 | Agency For Science, Technology And Research | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
| WO2019035058A1 (en) * | 2017-08-17 | 2019-02-21 | National Research Council Of Canada | Systems and methods for the production of diphtheria toxin polypeptides |
| WO2021158163A1 (en) * | 2020-02-07 | 2021-08-12 | Xbrane Biopharma Ab | Synthetically evolved dna constructs for regulating signal peptide performance as well as vectors, host cells and recombinant proteins thereof |
-
2021
- 2021-09-24 SE SE2130264A patent/SE545694C2/en unknown
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001094585A1 (en) * | 2000-06-06 | 2001-12-13 | Celltech R & D Limited | Antibody molecules having specificity for human tumor necrosis factor alpha, and use thereof |
| WO2002048376A2 (en) * | 2000-12-14 | 2002-06-20 | Genentech, Inc. | Bacterial host strains |
| WO2011086136A1 (en) * | 2010-01-14 | 2011-07-21 | Ucb Pharma S.A. | Bacterial host strain comprising a mutant spr gene and having reduced tsp activity |
| WO2014058389A1 (en) * | 2012-10-12 | 2014-04-17 | Agency For Science, Technology And Research | Optimised heavy chain and light chain signal peptides for the production of recombinant antibody therapeutics |
| WO2019035058A1 (en) * | 2017-08-17 | 2019-02-21 | National Research Council Of Canada | Systems and methods for the production of diphtheria toxin polypeptides |
| WO2021158163A1 (en) * | 2020-02-07 | 2021-08-12 | Xbrane Biopharma Ab | Synthetically evolved dna constructs for regulating signal peptide performance as well as vectors, host cells and recombinant proteins thereof |
Non-Patent Citations (2)
| Title |
|---|
| Low K. O. et al., "Optimisation of signal peptide for recombinant protein secretion in bacterial hosts", Appl Microbiol Biotechnol, 2013, Vol. 97, pages 3811-3826 * |
| Zheng M. K. et al., "Insights on the use of biosimilars in the treatment of inflammatory bowel disease", World J Gastroenterology, 2017, Vol. 23, pages 1932-1943 * |
Also Published As
| Publication number | Publication date |
|---|---|
| SE2130264A1 (en) | 2023-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7062626B2 (en) | Bacterial host strain expressing recombinant DsbC | |
| CA2940316C (en) | Enhanced production of recombinant crm197 in e. coli | |
| KR101737152B1 (en) | Bacterial host strain comprising a mutant spr gene and having reduced tsp activity | |
| KR101741865B1 (en) | Bacterial host strain comprising a mutant spr gene and a wild-type tsp gene | |
| KR101729361B1 (en) | Bacterial host strain expressing recombinant dsbc and having reduced tsp activity | |
| NZ621527A (en) | A genetically modified yeast cell and method of production of recombinant protein or polypeptide thereof | |
| CN111394343A (en) | L-threonine aldolase mutant R318L and application thereof | |
| JPH0376580A (en) | Escherichia coli manifestation vector and production of antiviral protein using the same | |
| US20080233614A1 (en) | Production of recombinant collagenases colg and colh in escherichia coli | |
| SE545694C2 (en) | Dna construct comprising nucleotide sequences encoding amino acid sequences of certolizumab and pelb signal peptides | |
| SE545714C2 (en) | Dna contructs for producing a pelb signal peptide | |
| SE545715C2 (en) | Dna contructs comprising tirs for producing recombinant proteins | |
| WO2023046930A1 (en) | Dna constructs and host cells for expressing recombinant protein | |
| WO1988008430A1 (en) | Peptide production by protein engineering | |
| US20040175705A1 (en) | Systems for expression of heterologous proteins in m capsulatus | |
| JP5846523B2 (en) | Escherichia coli expressing β-glucosidase |