US20220010294A1 - Novel recombinant botulinum neurotoxins with increased duration of effect - Google Patents
Novel recombinant botulinum neurotoxins with increased duration of effect Download PDFInfo
- Publication number
- US20220010294A1 US20220010294A1 US17/482,953 US202117482953A US2022010294A1 US 20220010294 A1 US20220010294 A1 US 20220010294A1 US 202117482953 A US202117482953 A US 202117482953A US 2022010294 A1 US2022010294 A1 US 2022010294A1
- Authority
- US
- United States
- Prior art keywords
- neurotoxin
- amino acid
- exosite
- recombinant
- botulinum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108030001720 Bontoxilysin Proteins 0.000 title claims abstract description 170
- 231100001103 botulinum neurotoxin Toxicity 0.000 title claims abstract description 154
- 230000000694 effects Effects 0.000 title claims abstract description 45
- 239000002581 neurotoxin Substances 0.000 claims abstract description 84
- 231100000618 neurotoxin Toxicity 0.000 claims abstract description 84
- 101710138657 Neurotoxin Proteins 0.000 claims abstract description 67
- 150000001413 amino acids Chemical class 0.000 claims abstract description 64
- 230000004048 modification Effects 0.000 claims abstract description 39
- 238000012986 modification Methods 0.000 claims abstract description 39
- 235000001014 amino acid Nutrition 0.000 claims abstract description 38
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 28
- 230000004071 biological effect Effects 0.000 claims abstract description 22
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims abstract description 21
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims abstract description 20
- 235000004279 alanine Nutrition 0.000 claims abstract description 20
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims abstract description 9
- 238000000034 method Methods 0.000 claims description 53
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 13
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 11
- 239000004473 Threonine Substances 0.000 claims description 11
- 239000002537 cosmetic Substances 0.000 claims description 9
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 8
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 8
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 8
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 7
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 claims description 7
- 206010040954 Skin wrinkling Diseases 0.000 claims description 6
- 230000009467 reduction Effects 0.000 claims description 6
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 239000003937 drug carrier Substances 0.000 claims description 5
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 5
- 206010044074 Torticollis Diseases 0.000 claims description 4
- 230000001684 chronic effect Effects 0.000 claims description 3
- 210000001352 masseter muscle Anatomy 0.000 claims description 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 208000000289 Esophageal Achalasia Diseases 0.000 claims description 2
- 206010068737 Facial asymmetry Diseases 0.000 claims description 2
- 208000027109 Headache disease Diseases 0.000 claims description 2
- 206010020880 Hypertrophy Diseases 0.000 claims description 2
- 208000008930 Low Back Pain Diseases 0.000 claims description 2
- 208000019695 Migraine disease Diseases 0.000 claims description 2
- 206010030136 Oesophageal achalasia Diseases 0.000 claims description 2
- 201000000621 achalasia Diseases 0.000 claims description 2
- 230000036617 axillary hyperhidrosis Effects 0.000 claims description 2
- 206010005159 blepharospasm Diseases 0.000 claims description 2
- 230000000744 blepharospasm Effects 0.000 claims description 2
- 244000309466 calf Species 0.000 claims description 2
- 201000002866 cervical dystonia Diseases 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 238000002347 injection Methods 0.000 claims description 2
- 208000005877 painful neuropathy Diseases 0.000 claims description 2
- 210000002307 prostate Anatomy 0.000 claims description 2
- 230000037303 wrinkles Effects 0.000 claims description 2
- 239000002243 precursor Substances 0.000 abstract description 37
- 239000000203 mixture Substances 0.000 abstract description 11
- 230000001747 exhibiting effect Effects 0.000 abstract description 6
- 150000007523 nucleic acids Chemical group 0.000 description 36
- 108091028043 Nucleic acid sequence Proteins 0.000 description 33
- 108090000623 proteins and genes Proteins 0.000 description 32
- 101710117542 Botulinum neurotoxin type A Proteins 0.000 description 28
- 102000004169 proteins and genes Human genes 0.000 description 28
- 229940024606 amino acid Drugs 0.000 description 25
- 235000018102 proteins Nutrition 0.000 description 24
- 210000004027 cell Anatomy 0.000 description 22
- 239000004365 Protease Substances 0.000 description 20
- 102000035195 Peptidases Human genes 0.000 description 18
- 108091005804 Peptidases Proteins 0.000 description 18
- 238000003776 cleavage reaction Methods 0.000 description 18
- 241001112695 Clostridiales Species 0.000 description 15
- 235000019419 proteases Nutrition 0.000 description 15
- 230000007017 scission Effects 0.000 description 15
- 238000003556 assay Methods 0.000 description 14
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 206010033799 Paralysis Diseases 0.000 description 13
- 101710118538 Protease Proteins 0.000 description 13
- 229940053031 botulinum toxin Drugs 0.000 description 13
- 210000002569 neuron Anatomy 0.000 description 13
- 230000000875 corresponding effect Effects 0.000 description 12
- 238000000746 purification Methods 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 11
- 108010055044 Tetanus Toxin Proteins 0.000 description 11
- 230000004913 activation Effects 0.000 description 11
- 108010059378 Endopeptidases Proteins 0.000 description 10
- 102000005593 Endopeptidases Human genes 0.000 description 10
- 108010057722 Synaptosomal-Associated Protein 25 Proteins 0.000 description 10
- 102000004183 Synaptosomal-Associated Protein 25 Human genes 0.000 description 10
- 108700012359 toxins Proteins 0.000 description 10
- 108090000190 Thrombin Proteins 0.000 description 9
- 210000003205 muscle Anatomy 0.000 description 9
- 229960004072 thrombin Drugs 0.000 description 9
- 239000003053 toxin Substances 0.000 description 9
- 231100000765 toxin Toxicity 0.000 description 9
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000008194 pharmaceutical composition Substances 0.000 description 8
- 108010076504 Protein Sorting Signals Proteins 0.000 description 7
- 230000009471 action Effects 0.000 description 7
- 239000012528 membrane Substances 0.000 description 7
- 108090000765 processed proteins & peptides Proteins 0.000 description 7
- 241000193403 Clostridium Species 0.000 description 6
- 241000193155 Clostridium botulinum Species 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 108010057266 Type A Botulinum Toxins Proteins 0.000 description 6
- 210000000170 cell membrane Anatomy 0.000 description 6
- 238000000855 fermentation Methods 0.000 description 6
- 230000004151 fermentation Effects 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 230000002887 neurotoxic effect Effects 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 239000007983 Tris buffer Substances 0.000 description 5
- 230000003247 decreasing effect Effects 0.000 description 5
- 230000002638 denervation Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 230000035772 mutation Effects 0.000 description 5
- 231100000189 neurotoxic Toxicity 0.000 description 5
- 239000002858 neurotransmitter agent Substances 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000006337 proteolytic cleavage Effects 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 102000005917 R-SNARE Proteins Human genes 0.000 description 4
- 108010005730 R-SNARE Proteins Proteins 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 238000001042 affinity chromatography Methods 0.000 description 4
- 239000000969 carrier Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 108091008146 restriction endonucleases Proteins 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- RAXXELZNTBOGNW-UHFFFAOYSA-N 1H-imidazole Chemical compound C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000004475 Arginine Substances 0.000 description 3
- 101710117515 Botulinum neurotoxin type E Proteins 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 3
- 241000282414 Homo sapiens Species 0.000 description 3
- 206010021639 Incontinence Diseases 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- 108090000526 Papain Proteins 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 230000008827 biological function Effects 0.000 description 3
- 229940094657 botulinum toxin type a Drugs 0.000 description 3
- 230000015556 catabolic process Effects 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 231100001102 clostridial toxin Toxicity 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 3
- 108010024001 incobotulinumtoxinA Proteins 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 229930182817 methionine Natural products 0.000 description 3
- 230000002232 neuromuscular Effects 0.000 description 3
- 230000003957 neurotransmitter release Effects 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 229940055729 papain Drugs 0.000 description 3
- 235000019834 papain Nutrition 0.000 description 3
- 230000002688 persistence Effects 0.000 description 3
- 238000001742 protein purification Methods 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229940118376 tetanus toxin Drugs 0.000 description 3
- 230000005945 translocation Effects 0.000 description 3
- 229940018272 xeomin Drugs 0.000 description 3
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 2
- GHKCSRZBNZQHKW-UWTATZPHSA-N (1R)-1-sulfanylethanol Chemical compound C[C@H](O)S GHKCSRZBNZQHKW-UWTATZPHSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101000933563 Clostridium botulinum Botulinum neurotoxin type G Proteins 0.000 description 2
- 108010013369 Enteropeptidase Proteins 0.000 description 2
- 102100029727 Enteropeptidase Human genes 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 101710175625 Maltose/maltodextrin-binding periplasmic protein Proteins 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 208000005392 Spasm Diseases 0.000 description 2
- 108700005078 Synthetic Genes Proteins 0.000 description 2
- 108030001722 Tentoxilysin Proteins 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 210000003484 anatomy Anatomy 0.000 description 2
- 239000012148 binding buffer Substances 0.000 description 2
- 238000000423 cell based assay Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000001186 cumulative effect Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009792 diffusion process Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000005342 ion exchange Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 238000005304 joining Methods 0.000 description 2
- 230000002045 lasting effect Effects 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 229940124272 protein stabilizer Drugs 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000002864 sequence alignment Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000001542 size-exclusion chromatography Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 230000004960 subcellular localization Effects 0.000 description 2
- 210000000225 synapse Anatomy 0.000 description 2
- 230000000946 synaptic effect Effects 0.000 description 2
- 210000001738 temporomandibular joint Anatomy 0.000 description 2
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- 108010091324 3C proteases Proteins 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 206010002153 Anal fissure Diseases 0.000 description 1
- 208000016583 Anus disease Diseases 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 101710117524 Botulinum neurotoxin type B Proteins 0.000 description 1
- 101710117520 Botulinum neurotoxin type F Proteins 0.000 description 1
- 208000003508 Botulism Diseases 0.000 description 1
- 102100021935 C-C motif chemokine 26 Human genes 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 102000003952 Caspase 3 Human genes 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 101000985023 Clostridium botulinum C phage Botulinum neurotoxin type C Proteins 0.000 description 1
- 101000985020 Clostridium botulinum D phage Botulinum neurotoxin type D Proteins 0.000 description 1
- 241000193449 Clostridium tetani Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 208000032131 Diabetic Neuropathies Diseases 0.000 description 1
- 101100364969 Dictyostelium discoideum scai gene Proteins 0.000 description 1
- 208000014094 Dystonic disease Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 241000672609 Escherichia coli BL21 Species 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 208000009531 Fissure in Ano Diseases 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 description 1
- 101000994149 Homo sapiens Iron-sulfur cluster assembly 2 homolog, mitochondrial Proteins 0.000 description 1
- 101000582320 Homo sapiens Neurogenic differentiation factor 6 Proteins 0.000 description 1
- 241001207270 Human enterovirus Species 0.000 description 1
- 241000430519 Human rhinovirus sp. Species 0.000 description 1
- 208000008454 Hyperhidrosis Diseases 0.000 description 1
- 206010020853 Hypertonic bladder Diseases 0.000 description 1
- XQFRJNBWHJMXHO-RRKCRQDMSA-N IDUR Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(I)=C1 XQFRJNBWHJMXHO-RRKCRQDMSA-N 0.000 description 1
- 102100031428 Iron-sulfur cluster assembly 2 homolog, mitochondrial Human genes 0.000 description 1
- 208000002260 Keloid Diseases 0.000 description 1
- 206010023330 Keloid scar Diseases 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- 240000007472 Leucaena leucocephala Species 0.000 description 1
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108010085220 Multiprotein Complexes Proteins 0.000 description 1
- 102000007474 Multiprotein Complexes Human genes 0.000 description 1
- 101100364971 Mus musculus Scai gene Proteins 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 206010028391 Musculoskeletal Pain Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000000693 Neurogenic Urinary Bladder Diseases 0.000 description 1
- 206010029279 Neurogenic bladder Diseases 0.000 description 1
- 102100030589 Neurogenic differentiation factor 6 Human genes 0.000 description 1
- 241001452677 Ogataea methanolica Species 0.000 description 1
- 208000009722 Overactive Urinary Bladder Diseases 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 208000033952 Paralysis flaccid Diseases 0.000 description 1
- 208000018737 Parkinson disease Diseases 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 206010037211 Psychomotor hyperactivity Diseases 0.000 description 1
- 108010010469 Qa-SNARE Proteins Proteins 0.000 description 1
- 102000000583 SNARE Proteins Human genes 0.000 description 1
- 108010041948 SNARE Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 206010039424 Salivary hypersecretion Diseases 0.000 description 1
- 241001274197 Scatophagus argus Species 0.000 description 1
- 206010041235 Snoring Diseases 0.000 description 1
- 208000027520 Somatoform disease Diseases 0.000 description 1
- 235000021355 Stearic acid Nutrition 0.000 description 1
- 208000006011 Stroke Diseases 0.000 description 1
- 108090000787 Subtilisin Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000012505 Superdex™ Substances 0.000 description 1
- 108010076818 TEV protease Proteins 0.000 description 1
- 206010043376 Tetanus Diseases 0.000 description 1
- 241000723790 Tobacco vein mottling virus Species 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 208000000697 Vocal Cord Dysfunction Diseases 0.000 description 1
- 208000013154 Vocal cord disease Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 108010079650 abobotulinumtoxinA Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 1
- 229960004373 acetylcholine Drugs 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940089093 botox Drugs 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 208000015114 central nervous system disease Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 206010008129 cerebral palsy Diseases 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 239000012504 chromatography matrix Substances 0.000 description 1
- 206010009259 cleft lip Diseases 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000009709 cytosolic degradation Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 229940098753 dysport Drugs 0.000 description 1
- 208000010118 dystonia Diseases 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000009144 enzymatic modification Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000000367 exoproteolytic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 208000028331 flaccid paralysis Diseases 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000030136 gastric emptying Effects 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000037315 hyperhidrosis Effects 0.000 description 1
- 229960004716 idoxuridine Drugs 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 229940127121 immunoconjugate Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000005462 in vivo assay Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000035987 intoxication Effects 0.000 description 1
- 231100000566 intoxication Toxicity 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 210000001847 jaw Anatomy 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 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 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 210000001117 keloid Anatomy 0.000 description 1
- 208000024765 knee pain Diseases 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 231100000636 lethal dose Toxicity 0.000 description 1
- 230000029226 lipidation Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000012433 multimodal chromatography Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 208000004296 neuralgia Diseases 0.000 description 1
- 208000018360 neuromuscular disease Diseases 0.000 description 1
- 210000000715 neuromuscular junction Anatomy 0.000 description 1
- 208000021722 neuropathic pain Diseases 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 208000020629 overactive bladder Diseases 0.000 description 1
- 208000027753 pain disease Diseases 0.000 description 1
- 230000001769 paralizing effect Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000001814 pectin Substances 0.000 description 1
- 235000010987 pectin Nutrition 0.000 description 1
- 229920001277 pectin Polymers 0.000 description 1
- 230000006320 pegylation Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000010837 receptor-mediated endocytosis Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 208000026451 salivation Diseases 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 230000001148 spastic effect Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 238000012799 strong cation exchange Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 108010093253 tentoxin Proteins 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960003766 thrombin (human) Drugs 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
- 230000032258 transport Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 206010044652 trigeminal neuralgia Diseases 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000001260 vocal cord Anatomy 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Images
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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- This invention relates to novel recombinant botulinum neurotoxins serotype A exhibiting both (i) an increased duration of effect and (ii) a high specific biological activity.
- the invention also relates to methods for the manufacture of such recombinant botulinum neurotoxins.
- These novel recombinant botulinum neurotoxins comprise at least two additional domains and least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- the invention further relates to pharmaceutical compositions comprising said recombinant neurotoxins.
- the invention further relates to novel recombinant single-chain precursor botulinum neurotoxins and nucleic acid sequences encoding such recombinant single-chain precursor botulinum neurotoxins.
- Clostridium is a genus of anaerobe gram-positive bacteria, belonging to the Firmicutes. Clostridium consists of around 100 species that include common free-living bacteria as well as important pathogens, such as Clostridium botulinum and Clostridium tetani . Both species produce neurotoxins, botulinum toxin and tetanus toxin, respectively. These neurotoxins are potent inhibitors of calcium-dependent neurotransmitter secretion of neuronal cells and are among the strongest toxins known to man. The lethal dose in humans lies between 0.1 ng and 1 ng per kilogram of body weight.
- botulism which is characterised by paralysis of various muscles. Paralysis of the breathing muscles can cause death of the affected individual.
- botulinum neurotoxin BoNT
- tetanus neurotoxin TxNT
- the botulinum toxin acts at the neuromuscular junction and other cholinergic synapses in the peripheral nervous system, inhibiting the release of the neurotransmitter acetylcholine and thereby causing flaccid paralysis
- the tetanus toxin acts mainly in the central nervous system, preventing the release of the inhibitory neurotransmitters GABA (gamma-aminobutyric acid) and glycine by degrading the protein synaptobrevin.
- GABA gamma-aminobutyric acid
- glycine gamma-aminobutyric acid
- the consequent overactivity in the muscles results in generalized contractions of the agonist and antagonist musculature, termed a tetanic spasm (rigid paralysis).
- BoNT/A the immunogenic type of tetanus neurotoxin
- BoNT/G the botulinum neurotoxins are known to occur in seven different immunogenic types, termed BoNT/A through BoNT/G. Most Clostridium botulinum strains produce one type of neurotoxin, but strains producing multiple toxins have also been described.
- Botulinum and tetanus neurotoxins have highly homologous amino acid sequences and show a similar domain structure.
- Their biologically active form comprises two peptide chains, a light chain of about 50 kDa and a heavy chain of about 100 kDa, linked by a disulfide bond.
- a linker or loop region whose length varies among different clostridial toxins, is located between the two cysteine residues forming the disulfide bond. This loop region is proteolytically cleaved by an unknown clostridial endoprotease to obtain the biologically active toxin.
- the light chain can then selectively cleave so called SNARE-proteins, which are essential for different steps of neurotransmitter release into the synaptic cleft, e.g. recognition, docking and fusion of neurotransmitter-containing vesicles with the plasma membrane.
- TeNT, BoNT/B, BoNT/D, BoNT/F, and BoNT/G cause proteolytic cleavage of synaptobrevin or VAMP (vesicle-associated membrane protein), BoNT/A and BoNT/E cleave the plasma membrane-associated protein SNAP-25, and BoNT/C cleaves the integral plasma membrane protein syntaxin and SNAP-25.
- Clostridial neurotoxins display variable durations of action that are serotype specific.
- the clinical therapeutic effect of BoNT/A lasts approximately 3 months for neuromuscular disorders and 6 to 12 months for hyperhidrosis.
- the effect of BoNT/E on the other hand, lasts less than 4 weeks.
- the longer lasting therapeutic effect of BoNT/A makes it preferable for certain clinical use compared to the other serotypes, for example serotypes B, C 1 , D, E, F, G.
- One possible explanation for the divergent durations of action might be the distinct subcellular localizations of BoNT serotypes.
- the protease domain of BoNT/A light chain localizes in a punctate manner to the plasma membrane of neuronal cells, co-localizing with its substrate SNAP-25.
- the short-duration BoNT/E serotype is cytoplasmic. Membrane association might protect BoNT/A from cytosolic degradation mechanisms allowing for prolonged persistence of BoNT/A in the neuronal cell.
- botulinum toxin is formed as a protein complex comprising the neurotoxic component and non-toxic proteins.
- the accessory proteins embed the neurotoxic component thereby protecting it from degradation by digestive enzymes in the gastrointestinal tract.
- botulinum neurotoxins of most serotypes are orally toxic.
- Complexes with, for example, 450 kDa or with 900 kDa are obtainable from cultures of Clostridium botulinum.
- botulinum neurotoxins have been used as therapeutic agents in the treatment of dystonias and spasms.
- Preparations comprising botulinum toxin complexes are commercially available, e.g. from Ipsen Ltd (Dysport®) or Allergan Inc. (Botox®).
- a high purity neurotoxic component, free of any complexing proteins, is for example available from Merz Pharmaceuticals GmbH, Frankfurt (Xeomin®).
- Clostridial neurotoxins are usually injected into the affected muscle tissue, bringing the agent close to the neuromuscular end plate, i.e. close to the cellular receptor mediating its uptake into the nerve cell controlling said affected muscle.
- Various degrees of neurotoxin spread have been observed. The neurotoxin spread is thought to depend on the injected amount and the particular neurotoxin preparation. It can result in adverse side effects such as paralysis in nearby muscle tissue, which can largely be avoided by reducing the injected doses to the therapeutically relevant level. Overdosing can also trigger the immune system to generate neutralizing antibodies that inactivate the neurotoxin preventing it from relieving the involuntary muscle activity. Immunologic tolerance to botulinum toxin has been shown to correlate with cumulative doses.
- clostridial neurotoxins are still predominantly produced by fermentation processes using appropriate Clostridium strains.
- industrial production of clostridial neurotoxin from anaerobic Clostridium culture is a cumbersome and time-consuming process. Due to the high toxicity of the final product, the procedure must be performed under strict containment.
- the single-chain precursors are proteolytically cleaved by an unknown clostridial protease to obtain the biologically active di-chain clostridial neurotoxin.
- the degree of neurotoxin activation by proteolytic cleavage varies between different strains and neurotoxin serotypes, which is a major consideration for the manufacture due to the requirement of neurotoxin preparations with a well-defined biological activity.
- the clostridial neurotoxins are produced as protein complexes, in which the neurotoxic component is embedded by accessory proteins. These accessory proteins have no beneficial effect on biological activity or duration of effect. They can however trigger an immune reaction in the patient, resulting in immunity against the clostridial neurotoxin. Manufacture of recombinant clostridial neurotoxins, which are not embedded by auxiliary proteins, might therefore be advantageous.
- clostridial neurotoxins have been expressed in eukaryotic expression systems, such as in Pichia pastoris, Pichia methanolica, Saccharomyces cerevisiae , insect cells and mammalian cells (see WO 2006/017749).
- botulinum neurotoxins may be expressed as single-chain precursors, which subsequently have to be proteolytically cleaved to obtain the final biologically active botulinum neurotoxin.
- botulinum neurotoxins may be expressed in high yield in rapidly-growing bacteria as relatively non-toxic single-chain polypeptides.
- botulinum neurotoxin characteristics regarding biological activity, cell specificity, antigenic potential and duration of effect by genetic engineering to obtain recombinant neurotoxins with new therapeutic properties in specific clinical areas.
- Genetic modification of botulinum neurotoxins might allow altering the mode of action or expanding the range of therapeutic targets.
- Botulinum toxin variants exhibiting an increased duration of effect and a high specific biological activity in neuromuscular tissue than naturally occurring botulinum toxins would be very advantageous in order to reduce administration frequency and the incidence of neutralizing antibody generation since immunologic tolerance to botulinum toxin is correlated with cumulative doses.
- US 2002/0127247 describes clostridial neurotoxins comprising modifications in secondary modification sites and exhibiting altered biological persistence.
- botulinum neurotoxins serotype A with an increased duration of effect and with improved properties, in order to allow for exploitation of the therapeutic potential of BoNT serotype A, which have so far been considered impractical for certain clinical application.
- the increased duration of effect of a particular botulinum neurotoxin serotype A could be adjusted in a tailor-made fashion in order to address any particular features and demands of a given indication, such as the amount of neurotoxin being administered, frequency of administration etc.
- botulinum neurotoxins serotype A which exhibit an increased duration of effect than naturally occurring botulinum toxins serotype A with a high specific biological activity. To date, such aspects have not been solved satisfactorily.
- Such a method and novel precursor botulinum neurotoxins used in such methods would serve to satisfy the great need for recombinant botulinum neurotoxins exhibiting an increased duration of effect with a high specific biological activity.
- BoNT/A exhibits the longest persistence and was shown to localize in the vicinity of the plasma membrane of neuronal cells.
- additional factors such as degradation, spread or diffusion, and/or translocation rates might have a decisive impact on the differences in the duration for the individual botulinum toxin serotypes.
- these neurotoxins comprise at least two additional domains consisting of proline, alanine and an additional amino acid residue.
- these neurotoxins according to the invention comprise at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- the present invention relates to a recombinant botulinum neurotoxin serotype A comprising at least two domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- the present invention relates to the use of the composition of the present invention for cosmetic treatment.
- the present invention relates to a recombinant single-chain precursor botulinum neurotoxin serotype A comprising at least two additional domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, and a modified alpha-exosite and/or beta-exosite of the light chain.
- the present invention relates to a nucleic acid sequence encoding the recombinant single-chain precursor botulinum neurotoxin serotype A of the present invention.
- the present invention relates to a method for obtaining the nucleic acid sequence of the present invention, comprising the step of adding nucleic acid sequences to the heavy and the light chain and in addition modifying a nucleic acid sequence encoding the alpha-exosite and/or at the beta-exosite of the light chain of the parental botulinum neurotoxin serotype A.
- the present invention relates to a vector comprising the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention.
- the present invention relates to a recombinant host cell comprising the nucleic acid sequence of the present invention, the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention.
- the present invention relates to a method for producing the recombinant single-chain precursor botulinum neurotoxin of the present invention, comprising the step of expressing the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention in a recombinant host cell, or cultivating the recombinant host cell of the present invention under conditions that result in the expression of said nucleic acid sequence.
- a sequence listing containing SEQ ID NOs: 1-5, created Nov. 8, 2019, 48 KB is provided herewith in a computer-readable nucleotide/amino acid .txt file and is specifically incorporated by reference.
- FIG. 2 SDS ⁇ PAGE of purified BoNT/A MaJ007 (PAS100-BoNT/A-ISA2-PAS100).
- Lane 1 Molecular weight marker. Prior to applying the samples to the gel, R-mercaptoethanol was added.
- Lane “v.A.” (before activation) purified, non-activated single-chain PAS100-BoNT/A-ISA2-PAS100.
- Lanes “n.A.” (after activation) and “n.R.” (after purification) show light chain (LC) and heavy chain (HC) obtained after activation by thrombin under reducing conditions.
- LC light chain
- HC heavy chain
- both the light chain (LC) and the heavy chain (HC) each comprise an additional amino acid sequence consisting of 100 amino acid residues, wherein said amino acid sequence consists of proline, alanine and serine residues (PAS) and where
- FIG. 4 SDS ⁇ PAGE of purified BoNT/A MaJ024 (PAS100-BoNT/A-ISA5-PAS(100).
- Lane 1 Molecular weight marker. Prior to applying the samples to the gel, R-mercaptoethanol was added.
- Lane “v.A.” (before activation) purified, non-activated single-chain PAS100-BoNT/A-ISA5-PAS(100.
- Lanes “n.A.” (after activation) and “n.R.” (after purification) show light chain (LC) and heavy chain (HC) obtained after activation by thrombin under reducing conditions.
- LC light chain
- HC heavy chain
- FIG. 5 Mouse running assay with BoNT/A PAS100-BoNT/A-ISA5-PAS100 (MaJ024)
- the present invention relates to a recombinant botulinum neurotoxin serotype A comprising at least two domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- botulinum neurotoxin serotype A refers to a natural neurotoxin serotype A obtainable from bacteria Clostridium botulinum , or to a neurotoxin obtainable from alternative sources, including from recombinant technologies or from genetic or chemical modification.
- the botulinum neurotoxins have a specific biological activity, i.e. endopeptidase activity.
- Botulinum neurotoxins are produced as single-chain precursors that are proteolytically cleaved by an unknown clostridial endoprotease within the loop region to obtain the biologically active disulfide-linked di-chain form of the neurotoxin, which comprises two chain elements, a functionally active light chain and a functionally active heavy chain, where one end of the light chain is linked to one end of the heavy chain not via a peptide bond, but via a disulfide bond.
- botulinum neurotoxin light chain refers to that part of a botulinum neurotoxin serotype A that comprises an endopeptidase activity responsible for cleaving one or more proteins that is/are part of the so-called SNARE-complex involved in the process resulting in the release of neurotransmitter into the synaptic cleft:
- the light chain has a molecular weight of approx. 50 kDa.
- alpha-exosite of the light chain refers to four LC alpha-helices (102-113, 310-321, 335-348, and 351-358) that interface a helical motif of SNAP-25 that is approximately 30-50 amino acids away from the cleavage site of SNAP-25 which interacts with the substrate SNAP25 (see Xue S, Javor S, Hixon M S, Janda K D, Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity. Biochemistry. 2014; 53(43):6820-4).
- beta-exosite of the light chain refers to a beta-sheet region located in a beta-sheet close to the active site which interacts with the substrate SNAP25 comprising AS 162-254 (see Breidenbach MA1, Brunger A T. Substrate recognition strategy for botulinum neurotoxin serotype A, Nature. 2004, 432(7019):925-9).
- the term “functionally active botulinum neurotoxin chain” refers to a recombinant botulinum neurotoxin serotype A chain able to perform the biological functions of a naturally occurring botulinum neurotoxin serotype A chain to at least about 50%, particularly to at least about 60%, to at least about 70%, to at least about 80%, and most particularly to at least about 90%, where the biological functions of botulinum neurotoxin chains include, but are not limited to, binding of the heavy chain to the neuronal cell, entry of the neurotoxin into a neuronal cell, release of the light chain from the di-chain neurotoxin, and endopeptidase activity of the light chain.
- WO 95/32738 describes the reconstitution of separately obtained light and heavy chains of tetanus toxin and botulinum toxin. Also cell-based assay methods as described for example in WO2009/114748, WO 2013/049508 and WO2014/207109.
- the term “about” or “approximately” means within 20%, alternatively within 10%, including within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e. an order of magnitude), including within a factor of two of a given value.
- the term “recombinant botulinum neurotoxin” refers to a composition comprising a botulinum neurotoxin serotype A that is obtained by expression of the neurotoxin in a heterologous cell such as E. coli , and including, but not limited to, the raw material obtained from a fermentation process (supernatant, composition after cell lysis), a fraction comprising a botulinum neurotoxin serotype A obtained from separating the ingredients of such a raw material in a purification process, an isolated and essentially pure protein, and a formulation for pharmaceutical and/or aesthetic use comprising a botulinum neurotoxin serotype A and additionally pharmaceutically acceptable solvents and/or excipients.
- the recombinant botulinum neurotoxin serotype A according to the invention comprises at least one amino acid modification which is located at the alpha-exosite at at least one position selected from D102, T109, K340, I348, N353, K356.
- the recombinant botulinum neurotoxin serotype A according to the invention comprises at least one amino acid modification which is located at the beta-exosite at at least one position selected from G169, T220, P239, S254.
- the recombinant botulinum neurotoxin serotype A comprises the following six modifications at the alpha-exosite of the light chain:
- the recombinant botulinum neurotoxin serotype A comprises the following four modifications at the beta-exosite of the light chain:
- the recombinant botulinum neurotoxin serotype A comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues, particularly 100 amino acid residues, 150 amino acid residues, or 200 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one serine residues.
- the recombinant botulinum neurotoxin serotype A comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 150 amino acid residues or between 80 and 120 amino acid residues or between 90 and 110 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one serine residues.
- the term “functional variant of a botulinum neurotoxin” refers to a botulinum neurotoxin serotype A that differs in the amino acid sequence and/or the nucleic acid sequence encoding the amino acid sequence from a botulinum neurotoxin serotype A, but is still functionally active.
- the term “functionally active” refers to the property of a recombinant botulinum neurotoxin serotype A to exhibit a biological activity of at least about 20%, particularly to at least about 40%, at least about 70%, at least about 80%, and most particularly at least about 90% of the biological activity of a naturally occurring parental botulinum neurotoxin, i.e.
- a parental botulinum neurotoxin serotype A without modifications at the C-terminus of the light chain where the biological functions include, but are not limited to, binding to the neurotoxin receptor, entry of the neurotoxin into a neuronal cell, release of the light chain from the two-chain neurotoxin, and endopeptidase activity of the light chain, and thus inhibition of neurotransmitter release from the affected nerve cell.
- In vivo assays for assessing biological activity include the mouse LD50 assay and the ex vivo mouse hemidiaphragm assay as described by Pearce et al. (Pearce 1994, Toxicol. Appl. Pharmacol. 128: 69-77) and Dressler et al.
- MU Mouse Units
- 1 MU is the amount of neurotoxic component, which kills 50% of a specified mouse population after intraperitoneal injection, i.e. the mouse i.p. LD50.
- a functional variant will maintain key features of the corresponding botulinum neurotoxin serotype A, such as key residues for the endopeptidase activity in the light chain, or key residues for the attachment to the neurotoxin receptors or for translocation through the endosomal membrane in the heavy chain, but may contain modifications comprising a substitution of one or more amino acids of the corresponding botulinum neurotoxin.
- the functional variant of a botulinum neurotoxin serotype A additionally comprises a signal peptide.
- said signal peptide will be located at the N-terminus of the neurotoxin.
- Many such signal peptides are known in the art and are comprised by the present invention.
- the signal peptide results in transport of the neurotoxin across a biological membrane, such as the membrane of the endoplasmic reticulum, the Golgi membrane or the plasma membrane of a eukaryotic or prokaryotic cell. It has been found that signal peptides, when attached to the neurotoxin, will mediate secretion of the neurotoxin into the supernatant of the cells.
- the signal peptide will be cleaved off in the course of, or subsequent to, secretion, so that the secreted protein lacks the N-terminal signal peptide, is composed of separate light and heavy chains, which are covalently linked by disulfide bridges, and is proteolytically active.
- identity refers to sequence identity characterized by determining the number of identical amino acids between two nucleic acid sequences or two amino acid sequences wherein the sequences are aligned so that the highest order match is obtained. It can be calculated using published techniques or methods codified in computer programs such as, for example, BLASTP, BLASTN or FASTA (Altschul 1990, J Mol Biol 215, 403). The percent identity values are, in one aspect, calculated over the entire amino acid sequence. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results.
- the program PileUp Higgins 1989, CABIOS 5, 151
- the programs Gap and BestFit Gap and BestFit (Needleman 1970, J Mol Biol 48; 443; Smith 1981, Adv Appl Math 2, 482), which are part of the GCG software packet (Genetics Computer Group 1991, 575 Science Drive, Madison, Wis., USA 53711)
- the sequence identity values recited above in percent (%) are to be determined, in another aspect of the invention, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments.
- the nucleic acid sequences encoding the functional homologue and the parental botulinum neurotoxin may differ to a larger extent due to the degeneracy of the genetic code. It is known that the usage of codons is different between prokaryotic and eukaryotic organisms. Thus, when expressing a prokaryotic protein such as a botulinum neurotoxin, in a eukaryotic expression system, it may be necessary, or at least helpful, to adapt the nucleic acid sequence to the codon usage of the expression host cell, meaning that sequence identity or homology may be rather low on the nucleic acid level.
- the term “variant” refers to a botulinum neurotoxin serotype A that is a chemically, enzymatically, or genetically modified derivative of a corresponding neurotoxin of C. botulinum neurotoxin serotype A.
- a chemically modified derivative may be one that is modified by pyruvation, phosphorylation, sulfatation, lipidation, pegylation, glycosylation and/or the chemical addition of an amino acid or a polypeptide comprising between 2 and about 100 amino acids, including modification occurring in the eukaryotic host cell used for expressing the derivative.
- An enzymatically modified derivative is one that is modified by the activity of enzymes, such as endo- or exoproteolytic enzymes, including modification by enzymes of the eukaryotic host cell used for expressing the derivative.
- a genetically modified derivative is one that has been modified by deletion or substitution of one or more amino acids contained in, or by addition of one or more amino acids (including polypeptides comprising between 2 and about 100 amino acids) to, the amino acid sequence of said botulinum neurotoxin.
- the recombinant botulinum neurotoxin serotype A is used in the treatment of a disease requiring improved chemodenervation, wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- the term “increased duration of effect” or “increased duration of action” refers to a longer lasting denervation mediated by a botulinum neurotoxin serotype A of the present invention.
- administration of a disulfide-linked di-chain botulinum neurotoxin serotype A comprising two domains according to the invention results in localized paralysis for a longer period of time relative to administration of an identical disulfide-linked di-chain botulinum neurotoxin serotype A without the at least two domains according to the present invention.
- the term “increased duration of effect/action” is defined as a more than about 20%, particularly more than about 50%, more particularly more than about 90% increased duration of effect of the recombinant neurotoxin of the present invention relative to the identical neurotoxin without the two domains according to the invention.
- an “increased duration of effect” can be determined using the “Mouse Running Assay”.
- the “Mouse Running Assay” is well-known to the person skilled in the art and measures the daily running distance of a mouse in a treadmill after a botulinum neurotoxin was injected into the M. gastrocnemius (see Keller J E. Recovery from botulinum neurotoxin poisoning in vivo. Neuroscience.
- the distance which a mouse is able to run in the treadmill the day before the botulinum neurotoxin is injected is used as comparison and is set as 100%.
- a daily running distance of no more than 80% of the initial running distance is regarded as paralysis of the muscle.
- the duration of effect is determined by the time period between the time point attaining a half-maximal paralysis, i.e. about 40% of the initial running distance and the time point when paralysis reaches recovery, i.e. 40% of the initial running distance.
- the botulinum neurotoxin is considered to exhibit an “increased duration of effect/action” provided that the mutated BoNT exhibits a similar potency i.e shows a similar maximal paralysis (reduction of the running distance) of about 80-90%.
- the “specific endoprotease activity” is measured in an assay which is described in Jones et al. (J Immunol Methods. 2008 Jan. 1; 329(1-2):92-101) and Hallis et al. (J Clin Microbiol. 1996 (8):1934-8; 1996) and is described further below in example 2.
- the term “decreased specific endoprotease activity” is defined as an at least 20% lower amount of cleavage product produced by a BoNT/A mutant in the endoprotease assay compared to the BoNT/A wildtype applying the same amount of protein.
- the term “specific biological activity” relates to the “specific endoprotease activity” mentioned above and can be derived also from the maximal paralysis shown in the “Mouse Running Assay” described above.
- denervation refers to denervation resulting from administration of a chemodenervating agent, for example a neurotoxin.
- localized denervation or “localized paralysis” refers to denervation of a particular anatomical region, usually a muscle or a group of anatomically and/or physiologically related muscles, which results from administration of a chemodenervating agent, for example a neurotoxin, to the particular anatomical region.
- a chemodenervating agent for example a neurotoxin
- the recombinant botulinum neurotoxins of the present invention might show increased biological half-life, reduced degradation rates, decreased diffusion rates, increased uptake by neuronal cells, and/or modified intracellular translocation rates, in each case relative to an identical parental clostridial neurotoxin without the at least two domains according to the invention.
- biological half-life specifies the lifespan of a protein, for example of a botulinum neurotoxin serotype A, in vivo.
- biological half-life refers to the period of time, by which half of a protein pool is degraded in vivo. For example it refers to the period of time, by which half of the amount of botulinum neurotoxin of one administered dosage is degraded.
- the present invention relates to a composition, in particular a pharmaceutical or cosmetic composition comprising the recombinant botulinum neurotoxin of the present invention.
- a composition in particular a pharmaceutical or cosmetic composition
- the toxin can be formulated by various techniques dependent on the desired application purposes which are known in the art.
- the (biologically active) botulinum neurotoxin polypeptide can be used in combination with one or more pharmaceutically acceptable carriers as a pharmaceutical composition.
- the pharmaceutically acceptable carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof.
- the pharmaceutical carrier employed may include a solid, a gel, or a liquid.
- Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and the like.
- Exemplary of liquid carriers are glycerol, phosphate buffered saline solution, water, emulsions, various types of wetting agents, and the like. Suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.
- the pharmaceutical composition can be dissolved in a diluent, prior to administration.
- the diluent is also selected so as not to affect the biological activity of the Neurotoxin product.
- the pharmaceutical composition or formulation may also include other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like.
- the formulated Neurotoxin product can be present, in an aspect, in liquid or lyophilized form. In an aspect, it can be present together with glycerol, protein stabilizers (HSA) or non-protein stabilizers such as polyvinyl pyrrolidone (PVP), hyaluronic acid or free amino acids.
- suitable non-proteinaceous stabilizers are disclosed in WO 2005/007185 or WO 2006/020208.
- the formulated Neurotoxin product may be used for human or animal therapy of various diseases or disorders in a therapeutically effective dose or for cosmetic purposes.
- the recombinant botulinum neurotoxin of the present invention or the pharmaceutical composition of the present invention is for use in the treatment of a disease or condition taken from the list of: cervical dystonia (spasmodic torticollis), blepharospasm, severe primary axillary hyperhidrosis, achalasia, lower back pain, benign prostate hypertrophy, chronic focal painful neuropathies, migraine and other headache disorders.
- Additional indications where treatment with botulinum neurotoxins is currently under investigation and where the pharmaceutical composition of the present invention may be used include pediatric incontinence, incontinence due to overactive bladder, and incontinence due to neurogenic bladder, anal fissure, spastic disorders associated with injury or disease of the central nervous system including trauma, stroke, multiple sclerosis, Parkinson's disease, or cerebral palsy, focal dystonias affecting the limbs, face, jaw or vocal cords, temporomandibular joint (TMJ) pain disorders, diabetic neuropathy, wound healing, excessive salivation, vocal cord dysfunction, reduction of the Masseter muscle for decreasing the size of the lower jaw, treatment and prevention of chronic headache and chronic musculoskeletal pain, treatment of snoring noise, assistance in weight loss by increasing the gastric emptying time.
- pediatric incontinence incontinence due to overactive bladder
- incontinence due to neurogenic bladder anal fissure
- spastic disorders associated with injury or disease of the central nervous system including trauma, stroke,
- clostridial neurotoxins have been evaluated for the treatment of other new indications, for example painful keloid, diabetic neuropathic pain, refractory knee pain, trigeminal neuralgia trigger-zone application to control pain, scarring after cleft-lip surgery, cancer and depression.
- the present invention relates to the use of the composition of the present invention for cosmetic treatment.
- the present invention relates to a method of cosmetically treating a patient, comprising the step of administering a composition comprising a recombinant clostridial neurotoxin according to the present invention to a patient desiring such cosmetic treatment
- cosmetic treatment relates to uses in cosmetic or aesthetic applications, such as the treatment of wrinkles, crow's feet, glabella frown lines, reduction of the masseter muscle, reduction of the calves, removing of facial asymmetries etc.
- the present invention relates to a method for the generation of the recombinant botulinum neurotoxin of the present invention, comprising the step of obtaining a recombinant nucleic acid sequence encoding a recombinant single-chain precursor botulinum neurotoxin by the insertion of a nucleic acid sequence encoding at least two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues into a nucleic acid sequence encoding a parental clostridial neurotoxin and by modifying the nucleic acid sequence encoding a parental botulinum neurotoxin at the alpha-exosite and/or at the beta-exosite of the light chain.
- the term “recombinant nucleic acid sequence” refers to a nucleic acid, which has been generated by joining genetic material from two different sources.
- single-chain precursor botulinum neurotoxin refers to a single-chain precursor for a disulfide-linked di-chain botulinum neurotoxin, comprising a functionally active botulinum neurotoxin light chain, a functionally active neurotoxin heavy chain, and a loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain.
- the term “recombinant single-chain precursor botulinum neurotoxin” refers to a single-chain precursor botulinum neurotoxin comprising at least two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues, and a modified alpha-exosite and/or beta-exosite of the light chain of the neurotoxin.
- the recombinant single-chain precursor botulinum neurotoxin comprises a protease cleavage site in said loop region.
- Single-chain precursor botulinum neurotoxins have to be proteolytically cleaved to obtain the final biologically active botulinum neurotoxins.
- Proteolytic cleavage may either occur during heterologous expression by host cell enzymes, or by adding proteolytic enzymes to the raw protein material isolated after heterologous expression.
- Naturally occurring botulinum neurotoxins usually contain one or more cleavage signals for proteases which post-translationally cleave the single-chain precursor molecule, so that the final di- or multimeric complex can form.
- botulinum neurotoxins are still predominantly produced by fermentation processes using appropriate Clostridium strains.
- the single-chain precursors are proteolytically cleaved by an unknown clostridial protease to obtain the biologically active di-chain clostridial neurotoxin.
- the single-chain precursor molecule is the precursor of a protease
- autocatalytic cleavage may occur.
- the protease can be a separate non-clostridial enzyme expressed in the same cell.
- WO 2006/076902 describes the proteolytic cleavage of a recombinant clostridial neurotoxin single-chain precursor at a heterologous recognition and cleavage site by incubation of the E. coli host cell lysate.
- proteolytic cleavage is carried out by an unknown E. coli protease.
- modified protease cleavage sites have been introduced recombinantly into the interchain region between the light and heavy chain of clostridial toxins, e.g. protease cleavage sites for human thrombin or non-human proteases (see WO 01/14570).
- the protease cleavage site is a site that is cleaved by a protease selected from the list of: thrombin, trypsin, enterokinase, factor Xa, plant papain, insect papain, crustacean papain, enterokinase, human rhinovirus 3C protease, human enterovirus 3C protease, tobacco etch virus protease, Tobacco Vein Mottling Virus, subtilisin and caspase 3.
- a protease selected from the list of: thrombin, trypsin, enterokinase, factor Xa, plant papain, insect papain, crustacean papain, enterokinase, human rhinovirus 3C protease, human enterovirus 3C protease, tobacco etch virus protease, Tobacco Vein Mottling Virus, subtilisin and caspase 3.
- the recombinant single-chain precursor botulinum neurotoxin serotype A further comprises a binding tag, particularly selected from the group comprising: glutathione-S-transferase (GST), maltose binding protein (MBP), a His-tag, a StrepTag, or a FLAG-tag.
- GST glutathione-S-transferase
- MBP maltose binding protein
- His-tag a StrepTag
- FLAG-tag FLAG-tag
- parental botulinum neurotoxin refers to an initial botulinum neurotoxin without modifications selected from a natural botulinum neurotoxin, a functional variant of a natural botulinum neurotoxin or a chimeric botulinum neurotoxin.
- the method for the generation of the recombinant botulinum neurotoxin of the present invention further comprises the step of heterologously expressing said recombinant nucleic acid sequence in a host cell, particularly in a bacterial host cell, more particularly in an E. coli host cell.
- the E. coli cells are selected from E. coli XL1-Blue, Nova Blue, TOP10, XL10-Gold, BL21, and K12.
- the method for the generation of the recombinant botulinum neurotoxin of the present invention additionally comprises at least one of the steps of (i) generating a disulfide-linked di-chain recombinant botulinum neurotoxin according to the invention by causing or allowing contacting of said recombinant single-chain precursor botulinum neurotoxin with an endoprotease and (ii) purification of said recombinant single-chain precursor botulinum neurotoxin or said disulfide-linked di-chain recombinant botulinum neurotoxin by chromatography.
- the recombinant single-chain precursor botulinum neurotoxin of the present invention, or the recombinant disulfide-linked di-chain botulinum neurotoxin of the present invention is purified after expression, or in the case of the recombinant disulfide-linked di-chain botulinum neurotoxin, after the cleavage reaction.
- the protein is purified by chromatography, particularly by immunoaffinity chromatography, or by chromatography on an ion exchange matrix, a hydrophobic interaction matrix, or a multimodal chromatography matrix, particularly a strong ion exchange matrix, more particularly a strong cation exchange matrix.
- the term “causing . . . contacting of said recombinant single-chain precursor botulinum neurotoxin . . . with an endoprotease” refers to an active and/or direct step of bringing said neurotoxin and said endoprotease in contact
- the term “allowing contacting of a recombinant single-chain precursor botulinum neurotoxin . . . with an endoprotease” refers to an indirect step of establishing conditions in such a way that said neurotoxin and said endoprotease are getting in contact to each other.
- endoprotease refers to a protease that breaks peptide bonds of non-terminal amino acids (i.e. within the polypeptide chain). As they do not attack terminal amino acids, endoproteases cannot break down peptides into monomers.
- cleavage of the recombinant single-chain precursor botulinum neurotoxin is near-complete.
- the term “near-complete” is defined as more than about 95% cleavage, particularly more than about 97.5%, more particularly more than about 99% as determined by SDS-PAGE and subsequent Western Blot or reversed phase chromatography.
- cleavage of the recombinant single-chain precursor botulinum neurotoxin of the present invention occurs at a heterologous cleavage signal located in the loop region of the recombinant precursor botulinum neurotoxin.
- the cleavage reaction is performed with crude host cell lysates containing said single-chain precursor protein.
- the single-chain precursor protein is purified or partially purified, particularly by a first chromatographic enrichment step, prior to the cleavage reaction.
- the term “purified” relates to more than about 90% purity. In the context of the present invention, the term “partially purified” relates to purity of less than about 90% and an enrichment of more than about two fold.
- the present invention relates to a recombinant single-chain botulinum neurotoxin serotype A, which is a precursor for the recombinant botulinum neurotoxin of the present invention.
- the present invention relates to a recombinant single-chain precursor botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues, and a modified alpha-exosite and/or beta-exosite of the light chain of the neurotoxin.
- the present invention relates to a method for obtaining the nucleic acid sequence of the present invention, comprising the step of modifying a nucleic acid sequence encoding a parental botulinum neurotoxin serotype A.
- the present invention relates to a vector comprising the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention.
- the present invention relates to a recombinant host cell comprising the nucleic acid sequence of the present invention, the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention.
- the present invention relates to a method for producing the recombinant single-chain precursor botulinum neurotoxin of the present invention, comprising the step of expressing the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention in a recombinant host cell, or cultivating the recombinant host cell of the present invention under conditions that result in the expression of said nucleic acid sequence.
- Example 1 Generation and Purification of a PASylated Botulinum Toxin Type A (PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100)
- PAS100-BoNT/A D102F T109R K340M I348L N353M K356R
- the nucleic acid construct encoding a “PAS” module comprising 100 amino acid residues built from the amino acids proline (P), alanine (A) and serine (S) was synthetically produced, wherein the following motive was used (ASPAAPAPASPAAPAPSAPA) 5 .
- ASPAAPAPASPAAPAPSAPA restriction enzymes NdeI and SwaI
- the corresponding gene module was first inserted at the N-terminus of recombinant BoNT/A (rBoNT/A).
- the PAS module was inserted at the C-terminus of the heavy chain by using restriction enzymes BglII and AatII. The correct cloning was verified by sequencing.
- BoNT/A in E.
- coli gene constructs were cloned into pET29c.
- the variants contain fused His6- and Strep-affinity tags which can be cleaved after protein purification via thrombin.
- a synthetic gene with the corresponding mutations of D102F T109R K340M I348L N353M K356R in the BoNT/A1 wild type sequence and restriction sites SwaI and ScaI was used.
- rBoNT/A variants were performed in Riesenberg media with 50 ⁇ g/mL Kanamycin ⁇ Riesenberg, 1991 #1 ⁇ . Cells were grown in shake flasks (37° C., 175 r.p.m) until an OD600 of 1.5-2 was reached. For induction of protein expression 1 mM IPTG (Fermentas) was added to the E. coli culture. Protein synthesis was performed for 24 h (15° C., 175 r.p.m.).
- the resulting crude extracts were centrifuged (20,000 r.p.m., 30 min, 4° C.), and the supernatants with the soluble proteins were recovered.
- Protein purification was carried out by fast protein liquid chromatography (GE Healthcare) using a three step purification protocols. The first capture step was performed by IMAC using a HisTrap HP 1 mL column (GE Healthcare). The column was washed (1 ml min-1 working flow) using a two-step protocol with His elution buffer (50 mM Tris, 150 mM NaCl, 400 mM Imidazol pH 8.0). The elution of the toxin proteins occurred at 400 mM Imidazol.
- a Strep-Tactin affinity chromatography was performed as previously described (IBA GmbH).
- a cation exchange chromatography with a HiTrap SP HP 1 mL column (GE Healthcare, Freiburg, Germany) was used.
- the corresponding samples were diluted with SP binding buffer (50 mM Tris, pH 8) and eluted with SP elution buffer (50 mM Tris, 1 M NaCl, pH 8).
- SP binding buffer 50 mM Tris, pH 8
- SP elution buffer 50 mM Tris, 1 M NaCl, pH 8
- the SEC running buffer (20 mM Tris, 150 mM NaCl, 2.5 mM CaCl 2 ) pH 7.7) was also used to store the purified protein solutions in aliquots at ⁇ 20° C. Each protein was analyzed by applying 0.5-1 ⁇ g on 4-12% gradient SDS-PAGE (Novex Life Technologies) and stained with Coomassie G-250 based SimplyBlue safe stain (Pierce). Each protein was judged >98% pure before applying in vitro or in vivo experiments.
- BoNT/A preparations were activated with Thrombin (Merck Millipore; 8 U/1 mg BoNT) for 24 h at 20° C. yielding >99% of di-chain toxin. Afterwards the cleavage protease was eliminated with the previously described Strep-Tactin Kit (IBA GmbH).
- FIG. 2 summarizes the results of purification and activation.
- the specific endoproteinase activity of wildtype BoNT/A and modified BoNT/A variants was determined by an assay which is described in Jones R G, Ochiai M, Liu Y, Ekong T, Sesardic D. Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins. J Immunol Methods. 2008 Jan. 1; 329(1-2):92-101 and Hallis B, James B A, Shone C C Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. J Clin Microbiol. 1996 (8):1934-8. The “1-step TMB Ultra” solution from Perbio Science was used for staining and quantification.
- a C-terminal fragment of 70 amino acids of SNAP25 from IBA GmBH was used in the reaction as a substrate.
- the primary mouse antibody from R&D Systems specifically recognizes SNAP25 fragments cleaved only by BoNT/A.
- the secondary antibody was an anti-mouse-IgG-HRP-antibody conjugate from Dianova.
- the assay was performed on a 96 microtiter plate and the amount of cleaved material was determined after 120 minutes by measuring the absorption at 450 nm (Molecular Devices, FilterMax F5). For the assay equal amounts of proteins were used.
- Table 1 shows that the specific endoproteinase activity of PASylated botulinum toxin A was increased by introduced mutations.
- Example 3 Generation and Purification of a PASylated Botulinum Toxin Type A (PAS100-BoNT/A G169I, T220R, P239M, S254-PAS100)
- the nucleic acid construct encoding a “PAS” module comprising 100 amino acid residues built from the amino acids proline (P), alanine (A) and serine (S) was synthetically produced, wherein the following motive was used (ASPAAPAPASPAAPAPSAPA) 5 .
- ASPAAPAPASPAAPAPSAPA restriction enzymes NdeI and SwaI
- the corresponding gene module was first inserted at the N-terminus of recombinant BoNT/A (rBoNT/A).
- the PAS module was inserted at the C-terminus of the heavy chain by using restriction enzymes BglII and AatII. The correct cloning was verified by sequencing.
- BoNT/A in E.
- coli gene constructs were cloned into pET29c.
- the variants contain fused His6- and Strep-affinity tags which can be cleaved after protein purification via thrombin.
- a synthetic gene with the corresponding mutations of G169I, T220R, P239M, S254 in the BoNT/A1 wild type sequence and restriction sites SwaI and Scat was used.
- Protein expression was performed as described above (see Example 1) in expression strain E. coli BL21. Purification was done using a combination of his affinity, ion exchange and size exclusion chromatography, followed by activation using thrombin. FIG. 3 summarizes the results of purification and activation.
- SEQ ID NO 1 recombinant BoNT A including His.tag
- SEQ ID NO 2 recombinant PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100 including His.tag
- SEQ ID NO 3 recombinant PAS100-BoNT/A (G169I, T220R, P239M, S254)-PAS100 including His.tag)
- SEQ ID NO 4 recombinant PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100 including His.tag (nucleic acid sequence)
- SEQ ID NO 5 recombinant PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS100 including His.tag) (nucleic acid sequence)
Abstract
This invention relates to novel recombinant botulinum neurotoxins serotype A exhibiting both (i) an increased duration of effect and (ii) a high specific biological activity. These novel recombinant botulinum neurotoxins comprise at least two additional domains consisting of proline, alanine and an additional amino acid residue and at least one amino acid modification which is located at the alpha-exosite or at the beta-exosite of the light chain of the neurotoxin. The invention further relates to novel recombinant single-chain precursor botulinum neurotoxins and compositions comprising the recombinant botulinum neurotoxin with an increased duration of effect and a high specific biological activity.
Description
- This application is a divisional of U.S. application Ser. No. 16/498,257, filed Sep. 26, 2019, which is a U.S. National Stage Application filed under 35 U.S.C. § 371 of International Application No. PCT/EP2017/066891, filed Jul. 6, 2017, both of which are hereby incorporated by reference in their entirety.
- This invention relates to novel recombinant botulinum neurotoxins serotype A exhibiting both (i) an increased duration of effect and (ii) a high specific biological activity. The invention also relates to methods for the manufacture of such recombinant botulinum neurotoxins. These novel recombinant botulinum neurotoxins comprise at least two additional domains and least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin. The invention further relates to pharmaceutical compositions comprising said recombinant neurotoxins. The invention further relates to novel recombinant single-chain precursor botulinum neurotoxins and nucleic acid sequences encoding such recombinant single-chain precursor botulinum neurotoxins.
- Clostridium is a genus of anaerobe gram-positive bacteria, belonging to the Firmicutes. Clostridium consists of around 100 species that include common free-living bacteria as well as important pathogens, such as Clostridium botulinum and Clostridium tetani. Both species produce neurotoxins, botulinum toxin and tetanus toxin, respectively. These neurotoxins are potent inhibitors of calcium-dependent neurotransmitter secretion of neuronal cells and are among the strongest toxins known to man. The lethal dose in humans lies between 0.1 ng and 1 ng per kilogram of body weight.
- Oral ingestion of botulinum toxin via contaminated food or generation of botulinum toxin in wounds can cause botulism, which is characterised by paralysis of various muscles. Paralysis of the breathing muscles can cause death of the affected individual.
- Although both botulinum neurotoxin (BoNT) and tetanus neurotoxin (TxNT) function via a similar initial physiological mechanism of action, inhibiting neurotransmitter release from the axon of the affected neuron into the synapse, they differ in their clinical response. While the botulinum toxin acts at the neuromuscular junction and other cholinergic synapses in the peripheral nervous system, inhibiting the release of the neurotransmitter acetylcholine and thereby causing flaccid paralysis, the tetanus toxin acts mainly in the central nervous system, preventing the release of the inhibitory neurotransmitters GABA (gamma-aminobutyric acid) and glycine by degrading the protein synaptobrevin. The consequent overactivity in the muscles results in generalized contractions of the agonist and antagonist musculature, termed a tetanic spasm (rigid paralysis).
- While the tetanus neurotoxin exists in one immunologically distinct type, the botulinum neurotoxins are known to occur in seven different immunogenic types, termed BoNT/A through BoNT/G. Most Clostridium botulinum strains produce one type of neurotoxin, but strains producing multiple toxins have also been described.
- Botulinum and tetanus neurotoxins have highly homologous amino acid sequences and show a similar domain structure. Their biologically active form comprises two peptide chains, a light chain of about 50 kDa and a heavy chain of about 100 kDa, linked by a disulfide bond. A linker or loop region, whose length varies among different clostridial toxins, is located between the two cysteine residues forming the disulfide bond. This loop region is proteolytically cleaved by an unknown clostridial endoprotease to obtain the biologically active toxin.
- The molecular mechanism of intoxication by TeNT and BoNT appears to be similar as well: entry into the target neuron is mediated by binding of the C-terminal part of the heavy chain to a specific cell surface receptor; the toxin is then taken up by receptor-mediated endocytosis. The low pH in the so formed endosome then triggers a conformational change in the clostridial toxin which allows it to embed itself in the endosomal membrane and to translocate through the endosomal membrane into the cytoplasm, where the disulfide bond joining the heavy and the light chain is reduced. The light chain can then selectively cleave so called SNARE-proteins, which are essential for different steps of neurotransmitter release into the synaptic cleft, e.g. recognition, docking and fusion of neurotransmitter-containing vesicles with the plasma membrane. TeNT, BoNT/B, BoNT/D, BoNT/F, and BoNT/G cause proteolytic cleavage of synaptobrevin or VAMP (vesicle-associated membrane protein), BoNT/A and BoNT/E cleave the plasma membrane-associated protein SNAP-25, and BoNT/C cleaves the integral plasma membrane protein syntaxin and SNAP-25.
- Clostridial neurotoxins display variable durations of action that are serotype specific. The clinical therapeutic effect of BoNT/A lasts approximately 3 months for neuromuscular disorders and 6 to 12 months for hyperhidrosis. The effect of BoNT/E, on the other hand, lasts less than 4 weeks. The longer lasting therapeutic effect of BoNT/A makes it preferable for certain clinical use compared to the other serotypes, for example serotypes B, C1, D, E, F, G. One possible explanation for the divergent durations of action might be the distinct subcellular localizations of BoNT serotypes. The protease domain of BoNT/A light chain localizes in a punctate manner to the plasma membrane of neuronal cells, co-localizing with its substrate SNAP-25. In contrast, the short-duration BoNT/E serotype is cytoplasmic. Membrane association might protect BoNT/A from cytosolic degradation mechanisms allowing for prolonged persistence of BoNT/A in the neuronal cell.
- In Clostridium botulinum, the botulinum toxin is formed as a protein complex comprising the neurotoxic component and non-toxic proteins. The accessory proteins embed the neurotoxic component thereby protecting it from degradation by digestive enzymes in the gastrointestinal tract. Thus, botulinum neurotoxins of most serotypes are orally toxic. Complexes with, for example, 450 kDa or with 900 kDa are obtainable from cultures of Clostridium botulinum.
- In recent years, botulinum neurotoxins have been used as therapeutic agents in the treatment of dystonias and spasms. Preparations comprising botulinum toxin complexes are commercially available, e.g. from Ipsen Ltd (Dysport®) or Allergan Inc. (Botox®). A high purity neurotoxic component, free of any complexing proteins, is for example available from Merz Pharmaceuticals GmbH, Frankfurt (Xeomin®).
- Clostridial neurotoxins are usually injected into the affected muscle tissue, bringing the agent close to the neuromuscular end plate, i.e. close to the cellular receptor mediating its uptake into the nerve cell controlling said affected muscle. Various degrees of neurotoxin spread have been observed. The neurotoxin spread is thought to depend on the injected amount and the particular neurotoxin preparation. It can result in adverse side effects such as paralysis in nearby muscle tissue, which can largely be avoided by reducing the injected doses to the therapeutically relevant level. Overdosing can also trigger the immune system to generate neutralizing antibodies that inactivate the neurotoxin preventing it from relieving the involuntary muscle activity. Immunologic tolerance to botulinum toxin has been shown to correlate with cumulative doses.
- At present, clostridial neurotoxins are still predominantly produced by fermentation processes using appropriate Clostridium strains. However, industrial production of clostridial neurotoxin from anaerobic Clostridium culture is a cumbersome and time-consuming process. Due to the high toxicity of the final product, the procedure must be performed under strict containment. During the fermentation process, the single-chain precursors are proteolytically cleaved by an unknown clostridial protease to obtain the biologically active di-chain clostridial neurotoxin. The degree of neurotoxin activation by proteolytic cleavage varies between different strains and neurotoxin serotypes, which is a major consideration for the manufacture due to the requirement of neurotoxin preparations with a well-defined biological activity. Furthermore, during fermentation processes using Clostridium strains the clostridial neurotoxins are produced as protein complexes, in which the neurotoxic component is embedded by accessory proteins. These accessory proteins have no beneficial effect on biological activity or duration of effect. They can however trigger an immune reaction in the patient, resulting in immunity against the clostridial neurotoxin. Manufacture of recombinant clostridial neurotoxins, which are not embedded by auxiliary proteins, might therefore be advantageous.
- Methods for the recombinant expression of clostridial neurotoxins in E. coli are well known in the art (see, for example, WO 00/12728, WO 01/14570, or WO 2006/076902). Furthermore, clostridial neurotoxins have been expressed in eukaryotic expression systems, such as in Pichia pastoris, Pichia methanolica, Saccharomyces cerevisiae, insect cells and mammalian cells (see WO 2006/017749).
- Recombinant botulinum neurotoxins may be expressed as single-chain precursors, which subsequently have to be proteolytically cleaved to obtain the final biologically active botulinum neurotoxin. Thus, botulinum neurotoxins may be expressed in high yield in rapidly-growing bacteria as relatively non-toxic single-chain polypeptides.
- Furthermore, it might be advantageous to modify botulinum neurotoxin characteristics regarding biological activity, cell specificity, antigenic potential and duration of effect by genetic engineering to obtain recombinant neurotoxins with new therapeutic properties in specific clinical areas. Genetic modification of botulinum neurotoxins might allow altering the mode of action or expanding the range of therapeutic targets.
- Botulinum toxin variants exhibiting an increased duration of effect and a high specific biological activity in neuromuscular tissue than naturally occurring botulinum toxins would be very advantageous in order to reduce administration frequency and the incidence of neutralizing antibody generation since immunologic tolerance to botulinum toxin is correlated with cumulative doses.
- US 2002/0127247 describes clostridial neurotoxins comprising modifications in secondary modification sites and exhibiting altered biological persistence.
- There is a strong demand to produce new botulinum neurotoxins serotype A with an increased duration of effect and with improved properties, in order to allow for exploitation of the therapeutic potential of BoNT serotype A, which have so far been considered impractical for certain clinical application. Ideally, the increased duration of effect of a particular botulinum neurotoxin serotype A could be adjusted in a tailor-made fashion in order to address any particular features and demands of a given indication, such as the amount of neurotoxin being administered, frequency of administration etc. In addition, it would be desirable to produce botulinum neurotoxins serotype A which exhibit an increased duration of effect than naturally occurring botulinum toxins serotype A with a high specific biological activity. To date, such aspects have not been solved satisfactorily.
- It was an object of the invention to overcome the above illustrated drawbacks. In particular, it was an object of the invention to provide recombinant botulinum neurotoxins serotype A exhibiting an increased duration of effect than naturally occurring botulinum toxins with a high specific biological activity in neuromuscular tissue and to establish a reliable and accurate method for manufacturing and obtaining such recombinant botulinum neurotoxins. Such a method and novel precursor botulinum neurotoxins used in such methods would serve to satisfy the great need for recombinant botulinum neurotoxins exhibiting an increased duration of effect with a high specific biological activity.
- The naturally occurring botulinum toxin serotypes display highly divergent durations of effect, probably due to their distinct subcellular localization. BoNT/A exhibits the longest persistence and was shown to localize in the vicinity of the plasma membrane of neuronal cells. However, additional factors such as degradation, spread or diffusion, and/or translocation rates might have a decisive impact on the differences in the duration for the individual botulinum toxin serotypes.
- So far, except for the approach described and claimed in WO 2015/132004, no generally applicable method for modifying clostridial neurotoxins to increase their duration of effect is available. According to WO 2015/132004, a recombinant botulinum neurotoxin comprising a domain consisting of proline (P), alanine (A) and serine (S) residues (hereafter referred to “PASylated” botulinum neurotoxins) exhibits an increased duration of effect compared to a corresponding wildtype botulinum neurotoxin, but it was shown that such a “PASylated” botulinum neurotoxin also exhibits a decreased specific potency, i.e. a decreased specific biological activity compared to a corresponding wildtype botulinum neurotoxin. This means for the clinical setting, that a higher amount of such a modified botulinum neurotoxin has to be injected into a subject/patient to reach the same paralytic effect in comparison to a wildtype botulinum neurotoxin which in turn increases the risk of generating neutralizing antibodies that inactivate the neurotoxin.
- Surprisingly, it has been found that recombinant botulinum neurotoxins serotype A having an increased duration of effect compared to a corresponding wildtype botulinum neurotoxin and a high specific potency, i.e. a high specific biological activity can be obtained by a two-fold modification. On the one hand these neurotoxins comprise at least two additional domains consisting of proline, alanine and an additional amino acid residue. Secondly, these neurotoxins according to the invention comprise at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- Thus, in one aspect, the present invention relates to a recombinant botulinum neurotoxin serotype A comprising at least two domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- In another aspect, the present invention relates to a composition, in particular to a pharmaceutical composition comprising the recombinant botulinum neurotoxin of the present invention.
- In yet another aspect, the present invention relates to the use of the composition of the present invention for cosmetic treatment.
- In another aspect, the present invention relates to a method for the generation of the recombinant botulinum neurotoxin of the present invention, comprising the step of obtaining a recombinant nucleic acid sequence encoding a recombinant single-chain precursor botulinum neurotoxin serotype A by modifying the nucleic acid sequence of the botulinum neurotoxin serotype A accordingly.
- In another aspect, the present invention relates to a recombinant single-chain precursor botulinum neurotoxin serotype A comprising at least two additional domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, and a modified alpha-exosite and/or beta-exosite of the light chain.
- In another aspect, the present invention relates to a nucleic acid sequence encoding the recombinant single-chain precursor botulinum neurotoxin serotype A of the present invention.
- In another aspect, the present invention relates to a method for obtaining the nucleic acid sequence of the present invention, comprising the step of adding nucleic acid sequences to the heavy and the light chain and in addition modifying a nucleic acid sequence encoding the alpha-exosite and/or at the beta-exosite of the light chain of the parental botulinum neurotoxin serotype A.
- In another aspect, the present invention relates to a vector comprising the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention.
- In another aspect, the present invention relates to a recombinant host cell comprising the nucleic acid sequence of the present invention, the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention.
- In another aspect, the present invention relates to a method for producing the recombinant single-chain precursor botulinum neurotoxin of the present invention, comprising the step of expressing the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention in a recombinant host cell, or cultivating the recombinant host cell of the present invention under conditions that result in the expression of said nucleic acid sequence.
- A sequence listing containing SEQ ID NOs: 1-5, created Nov. 8, 2019, 48 KB is provided herewith in a computer-readable nucleotide/amino acid .txt file and is specifically incorporated by reference.
-
FIG. 1 : Schematic Presentation of a modified botulinum toxin A (MaJ007=PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100=PAS100-BoNT/A-ISA2-PAS100), wherein both the light chain (LC) and the heavy chain (HC) each comprise an additional amino acid sequence consisting of 100 amino acid residues, wherein said amino acid sequence consists of proline (P), alanine (A) and serine (S) residues (PAS) and wherein the light chain (LC) comprises six amino acid modifications which are located at the alpha-exosite at positions D102, T109, K340, I348, N353, K356, wherein these amino acids are substituted as follows D102F, T109R, K340M, I348L, N353M, K356R. -
FIG. 2 : SDS⋅PAGE of purified BoNT/A MaJ007 (PAS100-BoNT/A-ISA2-PAS100). Lane 1: Molecular weight marker. Prior to applying the samples to the gel, R-mercaptoethanol was added. Lane “v.A.” (before activation): purified, non-activated single-chain PAS100-BoNT/A-ISA2-PAS100. Lanes “n.A.” (after activation) and “n.R.” (after purification) show light chain (LC) and heavy chain (HC) obtained after activation by thrombin under reducing conditions. -
FIG. 3 : Schematic Presentation of a modified botulinum toxin PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS(100)=PAS100-BoNT/A-ISA5-PAS100), wherein both the light chain (LC) and the heavy chain (HC) each comprise an additional amino acid sequence consisting of 100 amino acid residues, wherein said amino acid sequence consists of proline, alanine and serine residues (PAS) and wherein the light chain (LC) comprises four amino acid modifications are located at the beta-exosite at positions G169, T220, P239, S254, wherein these amino acids are substituted as follows G169I, T220R, P239M, S254T. -
FIG. 4 : SDS⋅PAGE of purified BoNT/A MaJ024 (PAS100-BoNT/A-ISA5-PAS(100). Lane 1: Molecular weight marker. Prior to applying the samples to the gel, R-mercaptoethanol was added. Lane “v.A.” (before activation): purified, non-activated single-chain PAS100-BoNT/A-ISA5-PAS(100. Lanes “n.A.” (after activation) and “n.R.” (after purification) show light chain (LC) and heavy chain (HC) obtained after activation by thrombin under reducing conditions. -
FIG. 5 : Mouse running assay with BoNT/A PAS100-BoNT/A-ISA5-PAS100 (MaJ024) - The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.
- In one aspect, the present invention relates to a recombinant botulinum neurotoxin serotype A comprising at least two domains wherein each domain comprises an amino acid sequence consisting of at least 50 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- In the context of the present invention, the term “botulinum neurotoxin serotype A” refers to a natural neurotoxin serotype A obtainable from bacteria Clostridium botulinum, or to a neurotoxin obtainable from alternative sources, including from recombinant technologies or from genetic or chemical modification. Particularly, the botulinum neurotoxins have a specific biological activity, i.e. endopeptidase activity.
- Botulinum neurotoxins are produced as single-chain precursors that are proteolytically cleaved by an unknown clostridial endoprotease within the loop region to obtain the biologically active disulfide-linked di-chain form of the neurotoxin, which comprises two chain elements, a functionally active light chain and a functionally active heavy chain, where one end of the light chain is linked to one end of the heavy chain not via a peptide bond, but via a disulfide bond.
- In the context of the present invention, the term “botulinum neurotoxin light chain” refers to that part of a botulinum neurotoxin serotype A that comprises an endopeptidase activity responsible for cleaving one or more proteins that is/are part of the so-called SNARE-complex involved in the process resulting in the release of neurotransmitter into the synaptic cleft: In naturally occurring botulinum neurotoxins, the light chain has a molecular weight of approx. 50 kDa.
- In the context of the present invention, the term “alpha-exosite of the light chain” refers to four LC alpha-helices (102-113, 310-321, 335-348, and 351-358) that interface a helical motif of SNAP-25 that is approximately 30-50 amino acids away from the cleavage site of SNAP-25 which interacts with the substrate SNAP25 (see Xue S, Javor S, Hixon M S, Janda K D, Probing BoNT/A protease exosites: implications for inhibitor design and light chain longevity. Biochemistry. 2014; 53(43):6820-4).
- In the context of the present invention, the term “beta-exosite of the light chain” refers to a beta-sheet region located in a beta-sheet close to the active site which interacts with the substrate SNAP25 comprising AS 162-254 (see Breidenbach MA1, Brunger A T. Substrate recognition strategy for botulinum neurotoxin serotype A, Nature. 2004, 432(7019):925-9).
- In the context of the present invention, the term “botulinum neurotoxin heavy chain” refers to that part of a botulinum neurotoxin serotype A that is responsible for entry of the neurotoxin into the neuronal cell: In naturally occurring botulinum neurotoxins, the heavy chain has a molecular weight of approx. 100 kDa.
- In the context of the present invention, the term “functionally active botulinum neurotoxin chain” refers to a recombinant botulinum neurotoxin serotype A chain able to perform the biological functions of a naturally occurring botulinum neurotoxin serotype A chain to at least about 50%, particularly to at least about 60%, to at least about 70%, to at least about 80%, and most particularly to at least about 90%, where the biological functions of botulinum neurotoxin chains include, but are not limited to, binding of the heavy chain to the neuronal cell, entry of the neurotoxin into a neuronal cell, release of the light chain from the di-chain neurotoxin, and endopeptidase activity of the light chain. Methods for determining a neurotoxic activity can be found, for example, in WO 95/32738, which describes the reconstitution of separately obtained light and heavy chains of tetanus toxin and botulinum toxin. Also cell-based assay methods as described for example in WO2009/114748, WO 2013/049508 and WO2014/207109.
- In the context of the present invention, the term “about” or “approximately” means within 20%, alternatively within 10%, including within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e. an order of magnitude), including within a factor of two of a given value.
- In the context of the present invention, the term “recombinant botulinum neurotoxin” refers to a composition comprising a botulinum neurotoxin serotype A that is obtained by expression of the neurotoxin in a heterologous cell such as E. coli, and including, but not limited to, the raw material obtained from a fermentation process (supernatant, composition after cell lysis), a fraction comprising a botulinum neurotoxin serotype A obtained from separating the ingredients of such a raw material in a purification process, an isolated and essentially pure protein, and a formulation for pharmaceutical and/or aesthetic use comprising a botulinum neurotoxin serotype A and additionally pharmaceutically acceptable solvents and/or excipients.
- In the context of the present invention, the term “comprises” or “comprising” means “including, but not limited to”. The term is intended to be open-ended, to specify the presence of any stated features, elements, integers, steps or components, but not to preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. The term “comprising” thus includes the more restrictive terms “consisting of” and “consisting essentially of”.
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention comprises at least one amino acid modification which is located at the alpha-exosite at at least one position selected from D102, T109, K340, I348, N353, K356.
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention comprises at least one amino acid modification which is located at the beta-exosite at at least one position selected from G169, T220, P239, S254.
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention comprises the following six modifications at the alpha-exosite of the light chain:
-
- (i) D102F modification (aspartic acid at position 102 of the light chain is replaced by a phenylalanine),
- (ii) T109R modification (threonine at position 109 is replaced by an arginine),
- (iii) K340M modification (lysine at position 340 is replaced by a methionine),
- (iv) I348L modification (isoleucine at position 348 of the light chain is replaced by a leucine),
- (v) N353M modification (asparagine at position 353 of the light chain is replaced by a methionine),
- (vi) K356R modification (lysine at position 356 of the light chain is replaced by an arginine).
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention comprises the following four modifications at the beta-exosite of the light chain:
-
- (i) G169I modification (glycine at position 169 of the light chain is replaced by a isoleucine),
- (ii) T220R modification (threonine at position 220 is replaced by an arginine),
- (iii) P239M modification (proline at position 239 is replaced by a methionine),
- (iv) S254T modification (serine at position 254 of the light chain is replaced by a threonine).
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues, particularly 100 amino acid residues, 150 amino acid residues, or 200 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one serine residues.
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 150 amino acid residues or between 80 and 120 amino acid residues or between 90 and 110 amino acid residues, wherein said amino acid sequence consists of at least one proline, at least one alanine and at least one serine residues.
- In the context of the present invention, the term “functional variant of a botulinum neurotoxin” refers to a botulinum neurotoxin serotype A that differs in the amino acid sequence and/or the nucleic acid sequence encoding the amino acid sequence from a botulinum neurotoxin serotype A, but is still functionally active. In the context of the present invention, the term “functionally active” refers to the property of a recombinant botulinum neurotoxin serotype A to exhibit a biological activity of at least about 20%, particularly to at least about 40%, at least about 70%, at least about 80%, and most particularly at least about 90% of the biological activity of a naturally occurring parental botulinum neurotoxin, i.e. a parental botulinum neurotoxin serotype A without modifications at the C-terminus of the light chain, where the biological functions include, but are not limited to, binding to the neurotoxin receptor, entry of the neurotoxin into a neuronal cell, release of the light chain from the two-chain neurotoxin, and endopeptidase activity of the light chain, and thus inhibition of neurotransmitter release from the affected nerve cell. In vivo assays for assessing biological activity include the mouse LD50 assay and the ex vivo mouse hemidiaphragm assay as described by Pearce et al. (Pearce 1994, Toxicol. Appl. Pharmacol. 128: 69-77) and Dressler et al. (Dressler 2005, Mov. Disord. 20:1617-1619, Keller 2006, Neuroscience 139: 629-637) or a cell-based assay as described in WO2009/114748, WO2014/207109 or WO 2013/049508. The biological activity is commonly expressed in Mouse Units (MU). As used herein, 1 MU is the amount of neurotoxic component, which kills 50% of a specified mouse population after intraperitoneal injection, i.e. the mouse i.p. LD50.
- On the protein level, a functional variant will maintain key features of the corresponding botulinum neurotoxin serotype A, such as key residues for the endopeptidase activity in the light chain, or key residues for the attachment to the neurotoxin receptors or for translocation through the endosomal membrane in the heavy chain, but may contain modifications comprising a substitution of one or more amino acids of the corresponding botulinum neurotoxin.
- In another embodiment, the functional variant of a botulinum neurotoxin serotype A additionally comprises a signal peptide. Usually, said signal peptide will be located at the N-terminus of the neurotoxin. Many such signal peptides are known in the art and are comprised by the present invention. In particular, the signal peptide results in transport of the neurotoxin across a biological membrane, such as the membrane of the endoplasmic reticulum, the Golgi membrane or the plasma membrane of a eukaryotic or prokaryotic cell. It has been found that signal peptides, when attached to the neurotoxin, will mediate secretion of the neurotoxin into the supernatant of the cells. In certain embodiments, the signal peptide will be cleaved off in the course of, or subsequent to, secretion, so that the secreted protein lacks the N-terminal signal peptide, is composed of separate light and heavy chains, which are covalently linked by disulfide bridges, and is proteolytically active.
- In particular embodiments, the functional variant has a sequence identity of at least about 40%, at least about 50%, at least about 60%, at least about 70% or most particularly at least about 80%, and a sequence homology of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or most particularly at least about 95% to the corresponding part in the parental botulinum neurotoxin serotype A. Methods and algorithms for determining sequence identity and/or homology, including the comparison of variants having deletions, additions, and/or substitutions relative to a parental sequence, are well known to the practitioner of ordinary skill in the art. The term “identity” as used herein refers to sequence identity characterized by determining the number of identical amino acids between two nucleic acid sequences or two amino acid sequences wherein the sequences are aligned so that the highest order match is obtained. It can be calculated using published techniques or methods codified in computer programs such as, for example, BLASTP, BLASTN or FASTA (Altschul 1990, J Mol Biol 215, 403). The percent identity values are, in one aspect, calculated over the entire amino acid sequence. A series of programs based on a variety of algorithms is available to the skilled worker for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To carry out the sequence alignments, the program PileUp (Higgins 1989,
CABIOS 5, 151) or the programs Gap and BestFit (Needleman 1970, J Mol Biol 48; 443; Smith 1981, Adv Appl Math 2, 482), which are part of the GCG software packet (Genetics Computer Group 1991, 575 Science Drive, Madison, Wis., USA 53711), may be used. The sequence identity values recited above in percent (%) are to be determined, in another aspect of the invention, using the program GAP over the entire sequence region with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10.000 and Average Mismatch: 0.000, which, unless otherwise specified, shall always be used as standard settings for sequence alignments. On the DNA level, the nucleic acid sequences encoding the functional homologue and the parental botulinum neurotoxin may differ to a larger extent due to the degeneracy of the genetic code. It is known that the usage of codons is different between prokaryotic and eukaryotic organisms. Thus, when expressing a prokaryotic protein such as a botulinum neurotoxin, in a eukaryotic expression system, it may be necessary, or at least helpful, to adapt the nucleic acid sequence to the codon usage of the expression host cell, meaning that sequence identity or homology may be rather low on the nucleic acid level. - In the context of the present invention, the term “variant” refers to a botulinum neurotoxin serotype A that is a chemically, enzymatically, or genetically modified derivative of a corresponding neurotoxin of C. botulinum neurotoxin serotype A. A chemically modified derivative may be one that is modified by pyruvation, phosphorylation, sulfatation, lipidation, pegylation, glycosylation and/or the chemical addition of an amino acid or a polypeptide comprising between 2 and about 100 amino acids, including modification occurring in the eukaryotic host cell used for expressing the derivative. An enzymatically modified derivative is one that is modified by the activity of enzymes, such as endo- or exoproteolytic enzymes, including modification by enzymes of the eukaryotic host cell used for expressing the derivative. As pointed out above, a genetically modified derivative is one that has been modified by deletion or substitution of one or more amino acids contained in, or by addition of one or more amino acids (including polypeptides comprising between 2 and about 100 amino acids) to, the amino acid sequence of said botulinum neurotoxin. Methods for designing and constructing such chemically or genetically modified derivatives and for testing of such variants for functionality are well known to anyone of ordinary skill in the art.
- In particular embodiments, the recombinant botulinum neurotoxin serotype A according to the invention is used in the treatment of a disease requiring improved chemodenervation, wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
- In the context of the present invention, the term “increased duration of effect” or “increased duration of action” refers to a longer lasting denervation mediated by a botulinum neurotoxin serotype A of the present invention. For example, as disclosed herein, administration of a disulfide-linked di-chain botulinum neurotoxin serotype A comprising two domains according to the invention results in localized paralysis for a longer period of time relative to administration of an identical disulfide-linked di-chain botulinum neurotoxin serotype A without the at least two domains according to the present invention.
- In the context of the present invention, the term “increased duration of effect/action” is defined as a more than about 20%, particularly more than about 50%, more particularly more than about 90% increased duration of effect of the recombinant neurotoxin of the present invention relative to the identical neurotoxin without the two domains according to the invention. For example, an “increased duration of effect” can be determined using the “Mouse Running Assay”. The “Mouse Running Assay” is well-known to the person skilled in the art and measures the daily running distance of a mouse in a treadmill after a botulinum neurotoxin was injected into the M. gastrocnemius (see Keller J E. Recovery from botulinum neurotoxin poisoning in vivo. Neuroscience. 2006 May 12; 139(2):629-37). The distance which a mouse is able to run in the treadmill the day before the botulinum neurotoxin is injected is used as comparison and is set as 100%. A daily running distance of no more than 80% of the initial running distance is regarded as paralysis of the muscle. The duration of effect is determined by the time period between the time point attaining a half-maximal paralysis, i.e. about 40% of the initial running distance and the time point when paralysis reaches recovery, i.e. 40% of the initial running distance. If this time period is longer than 2 days compared with the standard (wildtype BoNT), the botulinum neurotoxin is considered to exhibit an “increased duration of effect/action” provided that the mutated BoNT exhibits a similar potency i.e shows a similar maximal paralysis (reduction of the running distance) of about 80-90%.
- In the context of the present invention, the “specific endoprotease activity” is measured in an assay which is described in Jones et al. (J Immunol Methods. 2008 Jan. 1; 329(1-2):92-101) and Hallis et al. (J Clin Microbiol. 1996 (8):1934-8; 1996) and is described further below in example 2.
- In the context of the present invention, the term “decreased specific endoprotease activity” is defined as an at least 20% lower amount of cleavage product produced by a BoNT/A mutant in the endoprotease assay compared to the BoNT/A wildtype applying the same amount of protein.
- In the context of the present invention, the term “specific biological activity” relates to the “specific endoprotease activity” mentioned above and can be derived also from the maximal paralysis shown in the “Mouse Running Assay” described above.
- In the context of the present invention the term “denervation” refers to denervation resulting from administration of a chemodenervating agent, for example a neurotoxin.
- In the context of the present invention, the term “localized denervation” or “localized paralysis” refers to denervation of a particular anatomical region, usually a muscle or a group of anatomically and/or physiologically related muscles, which results from administration of a chemodenervating agent, for example a neurotoxin, to the particular anatomical region.
- Without wishing to be bound by theory, the recombinant botulinum neurotoxins of the present invention might show increased biological half-life, reduced degradation rates, decreased diffusion rates, increased uptake by neuronal cells, and/or modified intracellular translocation rates, in each case relative to an identical parental clostridial neurotoxin without the at least two domains according to the invention.
- In the context of the present invention, the term “biological half-life” specifies the lifespan of a protein, for example of a botulinum neurotoxin serotype A, in vivo. In the context of the present invention, the term “biological half-life” refers to the period of time, by which half of a protein pool is degraded in vivo. For example it refers to the period of time, by which half of the amount of botulinum neurotoxin of one administered dosage is degraded.
- In another aspect, the present invention relates to a composition, in particular a pharmaceutical or cosmetic composition comprising the recombinant botulinum neurotoxin of the present invention. For preparing a preparation comprising a botulinum neurotoxin serotype A the toxin can be formulated by various techniques dependent on the desired application purposes which are known in the art. For example, the (biologically active) botulinum neurotoxin polypeptide can be used in combination with one or more pharmaceutically acceptable carriers as a pharmaceutical composition. The pharmaceutically acceptable carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may include a solid, a gel, or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are glycerol, phosphate buffered saline solution, water, emulsions, various types of wetting agents, and the like. Suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. In an aspect, the pharmaceutical composition can be dissolved in a diluent, prior to administration. The diluent is also selected so as not to affect the biological activity of the Neurotoxin product. Examples of such diluents are distilled water or physiological saline. In addition, the pharmaceutical composition or formulation may also include other carriers or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. Thus, the formulated Neurotoxin product can be present, in an aspect, in liquid or lyophilized form. In an aspect, it can be present together with glycerol, protein stabilizers (HSA) or non-protein stabilizers such as polyvinyl pyrrolidone (PVP), hyaluronic acid or free amino acids. In an aspect, suitable non-proteinaceous stabilizers are disclosed in WO 2005/007185 or WO 2006/020208. The formulated Neurotoxin product may be used for human or animal therapy of various diseases or disorders in a therapeutically effective dose or for cosmetic purposes.
- In particular embodiments, the recombinant botulinum neurotoxin of the present invention or the pharmaceutical composition of the present invention is for use in the treatment of a disease or condition taken from the list of: cervical dystonia (spasmodic torticollis), blepharospasm, severe primary axillary hyperhidrosis, achalasia, lower back pain, benign prostate hypertrophy, chronic focal painful neuropathies, migraine and other headache disorders.
- Additional indications where treatment with botulinum neurotoxins is currently under investigation and where the pharmaceutical composition of the present invention may be used, include pediatric incontinence, incontinence due to overactive bladder, and incontinence due to neurogenic bladder, anal fissure, spastic disorders associated with injury or disease of the central nervous system including trauma, stroke, multiple sclerosis, Parkinson's disease, or cerebral palsy, focal dystonias affecting the limbs, face, jaw or vocal cords, temporomandibular joint (TMJ) pain disorders, diabetic neuropathy, wound healing, excessive salivation, vocal cord dysfunction, reduction of the Masseter muscle for decreasing the size of the lower jaw, treatment and prevention of chronic headache and chronic musculoskeletal pain, treatment of snoring noise, assistance in weight loss by increasing the gastric emptying time.
- Most recently, clostridial neurotoxins have been evaluated for the treatment of other new indications, for example painful keloid, diabetic neuropathic pain, refractory knee pain, trigeminal neuralgia trigger-zone application to control pain, scarring after cleft-lip surgery, cancer and depression.
- In yet another aspect, the present invention relates to the use of the composition of the present invention for cosmetic treatment.
- Thus, in another aspect, the present invention relates to a method of cosmetically treating a patient, comprising the step of administering a composition comprising a recombinant clostridial neurotoxin according to the present invention to a patient desiring such cosmetic treatment
- In the context of the present invention, the term “cosmetic treatment” relates to uses in cosmetic or aesthetic applications, such as the treatment of wrinkles, crow's feet, glabella frown lines, reduction of the masseter muscle, reduction of the calves, removing of facial asymmetries etc.
- In another aspect, the present invention relates to a method for the generation of the recombinant botulinum neurotoxin of the present invention, comprising the step of obtaining a recombinant nucleic acid sequence encoding a recombinant single-chain precursor botulinum neurotoxin by the insertion of a nucleic acid sequence encoding at least two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues into a nucleic acid sequence encoding a parental clostridial neurotoxin and by modifying the nucleic acid sequence encoding a parental botulinum neurotoxin at the alpha-exosite and/or at the beta-exosite of the light chain.
- In the context of the present invention, the term “recombinant nucleic acid sequence” refers to a nucleic acid, which has been generated by joining genetic material from two different sources.
- In the context of the present invention, the term “single-chain precursor botulinum neurotoxin” refers to a single-chain precursor for a disulfide-linked di-chain botulinum neurotoxin, comprising a functionally active botulinum neurotoxin light chain, a functionally active neurotoxin heavy chain, and a loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain.
- In the context of the present invention, the term “recombinant single-chain precursor botulinum neurotoxin” refers to a single-chain precursor botulinum neurotoxin comprising at least two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues, and a modified alpha-exosite and/or beta-exosite of the light chain of the neurotoxin.
- In particular embodiments, the recombinant single-chain precursor botulinum neurotoxin comprises a protease cleavage site in said loop region.
- Single-chain precursor botulinum neurotoxins have to be proteolytically cleaved to obtain the final biologically active botulinum neurotoxins. Proteolytic cleavage may either occur during heterologous expression by host cell enzymes, or by adding proteolytic enzymes to the raw protein material isolated after heterologous expression. Naturally occurring botulinum neurotoxins usually contain one or more cleavage signals for proteases which post-translationally cleave the single-chain precursor molecule, so that the final di- or multimeric complex can form. At present, botulinum neurotoxins are still predominantly produced by fermentation processes using appropriate Clostridium strains. During the fermentation process, the single-chain precursors are proteolytically cleaved by an unknown clostridial protease to obtain the biologically active di-chain clostridial neurotoxin. In cases, where the single-chain precursor molecule is the precursor of a protease, autocatalytic cleavage may occur. Alternatively, the protease can be a separate non-clostridial enzyme expressed in the same cell. WO 2006/076902 describes the proteolytic cleavage of a recombinant clostridial neurotoxin single-chain precursor at a heterologous recognition and cleavage site by incubation of the E. coli host cell lysate. The proteolytic cleavage is carried out by an unknown E. coli protease. In certain applications of recombinant expression, modified protease cleavage sites have been introduced recombinantly into the interchain region between the light and heavy chain of clostridial toxins, e.g. protease cleavage sites for human thrombin or non-human proteases (see WO 01/14570).
- In particular embodiments, the protease cleavage site is a site that is cleaved by a protease selected from the list of: thrombin, trypsin, enterokinase, factor Xa, plant papain, insect papain, crustacean papain, enterokinase, human rhinovirus 3C protease, human enterovirus 3C protease, tobacco etch virus protease, Tobacco Vein Mottling Virus, subtilisin and
caspase 3. - In a particular embodiment, the recombinant single-chain precursor botulinum neurotoxin serotype A further comprises a binding tag, particularly selected from the group comprising: glutathione-S-transferase (GST), maltose binding protein (MBP), a His-tag, a StrepTag, or a FLAG-tag.
- In the context of the present invention, the term “parental botulinum neurotoxin” refers to an initial botulinum neurotoxin without modifications selected from a natural botulinum neurotoxin, a functional variant of a natural botulinum neurotoxin or a chimeric botulinum neurotoxin.
- In particular embodiments, the method for the generation of the recombinant botulinum neurotoxin of the present invention further comprises the step of heterologously expressing said recombinant nucleic acid sequence in a host cell, particularly in a bacterial host cell, more particularly in an E. coli host cell.
- In certain embodiments, the E. coli cells are selected from E. coli XL1-Blue, Nova Blue, TOP10, XL10-Gold, BL21, and K12.
- In particular embodiments, the method for the generation of the recombinant botulinum neurotoxin of the present invention additionally comprises at least one of the steps of (i) generating a disulfide-linked di-chain recombinant botulinum neurotoxin according to the invention by causing or allowing contacting of said recombinant single-chain precursor botulinum neurotoxin with an endoprotease and (ii) purification of said recombinant single-chain precursor botulinum neurotoxin or said disulfide-linked di-chain recombinant botulinum neurotoxin by chromatography.
- In particular embodiments, the recombinant single-chain precursor botulinum neurotoxin of the present invention, or the recombinant disulfide-linked di-chain botulinum neurotoxin of the present invention, is purified after expression, or in the case of the recombinant disulfide-linked di-chain botulinum neurotoxin, after the cleavage reaction. In particular such embodiments, the protein is purified by chromatography, particularly by immunoaffinity chromatography, or by chromatography on an ion exchange matrix, a hydrophobic interaction matrix, or a multimodal chromatography matrix, particularly a strong ion exchange matrix, more particularly a strong cation exchange matrix.
- In the context of the present invention, the term “causing . . . contacting of said recombinant single-chain precursor botulinum neurotoxin . . . with an endoprotease” refers to an active and/or direct step of bringing said neurotoxin and said endoprotease in contact, whereas the term “allowing contacting of a recombinant single-chain precursor botulinum neurotoxin . . . with an endoprotease” refers to an indirect step of establishing conditions in such a way that said neurotoxin and said endoprotease are getting in contact to each other.
- In the context of the present invention, the term “endoprotease” refers to a protease that breaks peptide bonds of non-terminal amino acids (i.e. within the polypeptide chain). As they do not attack terminal amino acids, endoproteases cannot break down peptides into monomers.
- In particular embodiments, cleavage of the recombinant single-chain precursor botulinum neurotoxin is near-complete.
- In the context of the present invention, the term “near-complete” is defined as more than about 95% cleavage, particularly more than about 97.5%, more particularly more than about 99% as determined by SDS-PAGE and subsequent Western Blot or reversed phase chromatography.
- In particular embodiments, cleavage of the recombinant single-chain precursor botulinum neurotoxin of the present invention occurs at a heterologous cleavage signal located in the loop region of the recombinant precursor botulinum neurotoxin.
- In particular embodiments, the cleavage reaction is performed with crude host cell lysates containing said single-chain precursor protein.
- In other particular embodiments, the single-chain precursor protein is purified or partially purified, particularly by a first chromatographic enrichment step, prior to the cleavage reaction.
- In the context of the present invention, the term “purified” relates to more than about 90% purity. In the context of the present invention, the term “partially purified” relates to purity of less than about 90% and an enrichment of more than about two fold.
- In another aspect, the present invention relates to a recombinant single-chain botulinum neurotoxin serotype A, which is a precursor for the recombinant botulinum neurotoxin of the present invention. Thus, in such aspect, the present invention relates to a recombinant single-chain precursor botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and additional amino acid residues, selected from the group consisting of serine, threonine, tyrosine and glutamine residues, and a modified alpha-exosite and/or beta-exosite of the light chain of the neurotoxin.
- In another aspect, the present invention relates to a method for obtaining the nucleic acid sequence of the present invention, comprising the step of modifying a nucleic acid sequence encoding a parental botulinum neurotoxin serotype A.
- In another aspect, the present invention relates to a vector comprising the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention.
- In another aspect, the present invention relates to a recombinant host cell comprising the nucleic acid sequence of the present invention, the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention.
- In another aspect, the present invention relates to a method for producing the recombinant single-chain precursor botulinum neurotoxin of the present invention, comprising the step of expressing the nucleic acid sequence of the present invention, or the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention in a recombinant host cell, or cultivating the recombinant host cell of the present invention under conditions that result in the expression of said nucleic acid sequence.
- The nucleic acid construct encoding a “PAS” module comprising 100 amino acid residues built from the amino acids proline (P), alanine (A) and serine (S) was synthetically produced, wherein the following motive was used (ASPAAPAPASPAAPAPSAPA)5. By using restriction enzymes NdeI and SwaI, the corresponding gene module was first inserted at the N-terminus of recombinant BoNT/A (rBoNT/A). In a second step, the PAS module was inserted at the C-terminus of the heavy chain by using restriction enzymes BglII and AatII. The correct cloning was verified by sequencing. For expression of BoNT/A in E. coli gene constructs were cloned into pET29c. The variants contain fused His6- and Strep-affinity tags which can be cleaved after protein purification via thrombin. In order to construct a plasmid for the expression of BoNT/A a synthetic gene with the corresponding mutations of D102F T109R K340M I348L N353M K356R in the BoNT/A1 wild type sequence and restriction sites SwaI and ScaI was used.
- Expression of rBoNT/A variants was performed in Riesenberg media with 50 μg/mL Kanamycin{Riesenberg, 1991 #1}. Cells were grown in shake flasks (37° C., 175 r.p.m) until an OD600 of 1.5-2 was reached. For induction of protein expression 1 mM IPTG (Fermentas) was added to the E. coli culture. Protein synthesis was performed for 24 h (15° C., 175 r.p.m.). Cells were collected by centrifugation (5,000 r.p.m., 20 min, 4° C.) and resuspended in His binding buffer pH 8.0 (50 mM Tris, 150 mM NaCl, 5 mM Imidazol) containing EDTA-free protease inhibitor complete (Roche Diagnostics). For the determination of endopeptidase activity and in vivo characterization, the different toxin variants were extracted and purified. Resuspended pellets were disrupted in 2-3 cycles by a French Press Cell Disrupter (Thermo Electron Corporation) at 4° C. The resulting crude extracts were centrifuged (20,000 r.p.m., 30 min, 4° C.), and the supernatants with the soluble proteins were recovered. Protein purification was carried out by fast protein liquid chromatography (GE Healthcare) using a three step purification protocols. The first capture step was performed by IMAC using a HisTrap HP 1 mL column (GE Healthcare). The column was washed (1 ml min-1 working flow) using a two-step protocol with His elution buffer (50 mM Tris, 150 mM NaCl, 400 mM Imidazol pH 8.0). The elution of the toxin proteins occurred at 400 mM Imidazol. In a further step a Strep-Tactin affinity chromatography was performed as previously described (IBA GmbH). As an alternative instead of a second affinity chromatography a cation exchange chromatography with a HiTrap SP HP 1 mL column (GE Healthcare, Freiburg, Germany) was used. The corresponding samples were diluted with SP binding buffer (50 mM Tris, pH 8) and eluted with SP elution buffer (50 mM Tris, 1 M NaCl, pH 8). This procedure was followed by a SEC using a Superdex 200 10/300 column (GE Healthcare). The SEC running buffer (20 mM Tris, 150 mM NaCl, 2.5 mM CaCl2) pH 7.7) was also used to store the purified protein solutions in aliquots at −20° C. Each protein was analyzed by applying 0.5-1 μg on 4-12% gradient SDS-PAGE (Novex Life Technologies) and stained with Coomassie G-250 based SimplyBlue safe stain (Pierce). Each protein was judged >98% pure before applying in vitro or in vivo experiments.
- BoNT/A preparations were activated with Thrombin (Merck Millipore; 8 U/1 mg BoNT) for 24 h at 20° C. yielding >99% of di-chain toxin. Afterwards the cleavage protease was eliminated with the previously described Strep-Tactin Kit (IBA GmbH).
- Expression was performed in expression strain E. coli B121. Purification was done using a combination of his affinity, ion exchange and size exclusion chromatography, followed by activation using thrombin.
FIG. 2 summarizes the results of purification and activation. - The specific endoproteinase activity of wildtype BoNT/A and modified BoNT/A variants was determined by an assay which is described in Jones R G, Ochiai M, Liu Y, Ekong T, Sesardic D. Development of improved SNAP25 endopeptidase immuno-assays for botulinum type A and E toxins. J Immunol Methods. 2008 Jan. 1; 329(1-2):92-101 and Hallis B, James B A, Shone C C Development of novel assays for botulinum type A and B neurotoxins based on their endopeptidase activities. J Clin Microbiol. 1996 (8):1934-8. The “1-step TMB Ultra” solution from Perbio Science was used for staining and quantification. A C-terminal fragment of 70 amino acids of SNAP25 from IBA GmBH was used in the reaction as a substrate. The primary mouse antibody from R&D Systems specifically recognizes SNAP25 fragments cleaved only by BoNT/A. The secondary antibody was an anti-mouse-IgG-HRP-antibody conjugate from Dianova. The assay was performed on a 96 microtiter plate and the amount of cleaved material was determined after 120 minutes by measuring the absorption at 450 nm (Molecular Devices, FilterMax F5). For the assay equal amounts of proteins were used.
- The results of the measured endopeptidase activities are shown in the following table 1:
-
TABLE 1 Endoproteinase activities of mutants Batch Construct Endopeptidase assay Xeomin Wildtype BoNT/ A #100% DaSch021 PAS100-BoNT/A-PAS100 30-40% MaJ007 PAS(100)-BoNT/A-ISA2-PAS(100) 85-95% ISA2 = (D102F T109R K340M I348L N353M K356R); # Standard - Table 1 shows that the specific endoproteinase activity of PASylated botulinum toxin A was increased by introduced mutations.
- The nucleic acid construct encoding a “PAS” module comprising 100 amino acid residues built from the amino acids proline (P), alanine (A) and serine (S) was synthetically produced, wherein the following motive was used (ASPAAPAPASPAAPAPSAPA)5. By using restriction enzymes NdeI and SwaI the corresponding gene module was first inserted at the N-terminus of recombinant BoNT/A (rBoNT/A). In a second step, the PAS module was inserted at the C-terminus of the heavy chain by using restriction enzymes BglII and AatII. The correct cloning was verified by sequencing. For expression of BoNT/A in E. coli gene constructs were cloned into pET29c. The variants contain fused His6- and Strep-affinity tags which can be cleaved after protein purification via thrombin. In order to construct a plasmid for the expression of BoNT/A a synthetic gene with the corresponding mutations of G169I, T220R, P239M, S254 in the BoNT/A1 wild type sequence and restriction sites SwaI and Scat was used.
- Protein expression was performed as described above (see Example 1) in expression strain E. coli BL21. Purification was done using a combination of his affinity, ion exchange and size exclusion chromatography, followed by activation using thrombin.
FIG. 3 summarizes the results of purification and activation. - Two different dosages of PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS100 (=MaJ024), i.e. 6.0 pg and 9.0 pg were injected into the M. gastrocnemius of each mice in comparison to standard Xeomin® (3 pg; 0.6 U) and PASylated Botulinum Toxin Type A without the introduced mutations (=Dasch021). The mice had been trained in a treadmill. The daily running distance in the treadmill was measured over 21 days. The paralysis caused by the toxins was plotted as percentage of the running distance on the day before the injection, which was set as 100%, against the time (see
FIG. 5 ). - As shown in
FIG. 5 , only 6 pg of PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS(100) (=MaJ024) was required to achieve the same paralysis in comparison to 9 pg of Dasch021 (PASylated BoNT/A without introduced mutations) which indicates that the specific potency of MaJ024 was clearly increased in comparison to Dasch021. - SEQ ID NO 1: recombinant BoNT A including His.tag
SEQ ID NO 2: recombinant PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100 including His.tag
SEQ ID NO 3: recombinant PAS100-BoNT/A (G169I, T220R, P239M, S254)-PAS100 including His.tag)
SEQ ID NO 4: recombinant PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100 including His.tag (nucleic acid sequence)
SEQ ID NO 5: recombinant PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS100 including His.tag) (nucleic acid sequence) -
SEQ ID NO 1: MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKAGAGKSLVPRGSAGAGALNDLCIKVNNWDLFFSPSEDNFTND LNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNI ERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKV NKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVG ALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIV TNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLN ESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQV DRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRY ASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFN SISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRW IFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNL FDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYM YLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLA TNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQ FNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLGDLVPRGSANSSSVDKLW SHPQFEKLEHHHHHH
SEQ ID NO 2: recombinant PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100 including His.tag -
MGSSHHHHHHGSLVPRSSSASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNP PPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTFLGRMLLRSIVRGIPFWGGS TIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNR VFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAK SIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDMLYKMLTELYTEDMFVRFFKVL NRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTG LFEFYKLLCVRGIITSKAGAGKSLVPRGSAGAGALNDLCIKVNNWDLFFSPSEDNFTNDL NKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIE RFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVN KATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGA LIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVT NWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNE SINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVD RLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYA SKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWI FVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLF DKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMY LKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLAT NASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQF NNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAGDLVPRGSANSSSVDKLWSHPQFEK
SEQ ID NO 3: recombinant PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS100 including His.tag) -
MGSSHHHHHHGSLVPRSSSASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA SPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAA PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNP PPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGS TIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFIHEVLNLTRNGYG STQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVRLAHELIHAGHRLYGIAINMNR VFKVNTNAYYEMTGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAK SIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVL NRKTYLNFDKAVFKINIVPKVNYTIYDGFNLSNTNLAANFNGQNTEINNMNFTKLKNFTG LFEFYKLLCVRGIITSKAGAGKSLVPRGSAGAGALNDLCIKVNNWDLFFSPSEDNFTNDL NKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIE RFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVN KATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGA LIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVT NWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNE SINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVD RLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYA SKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNS ISLNNEYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWI FVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLF DKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMY LKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLAT NASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQF NNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPLASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAPAASPAAPAPASPAAPAPSAP AASPAAPAPASPAAPAPSAPAGDLVPRGSANSSSVDKLWSHPQFEK
SEQ ID NO 4: recombinant PAS100-BoNT/A (D102F T109R K340M I348L N353M K356R)-PAS100 including His.tag (nucleic acid sequence) -
ATGGGTAGCAGCCATCATCATCACCATCATGGTAGCCTGGTTCCGCGTAGCTCTTCTGCA AGTCCGGCAGCACCGGCACCGGCTTCACCAGCTGCACCAGCACCTAGCGCACCGGCAGCA TCTCCAGCAGCCCCTGCACCGGCAAGCCCTGCAGCTCCAGCACCGTCAGCACCAGCAGCA AGCCCAGCTGCTCCTGCTCCAGCGAGCCCAGCAGCGCCAGCTCCTAGTGCCCCTGCTGCC TCTCCTGCTGCTCCGGCACCAGCAAGTCCTGCTGCGCCTGCACCGAGTGCTCCGGCTGCT AGTCCTGCCGCACCAGCTCCGGCTAGTCCAGCTGCTCCAGCCCCTTCAGCCCCTGCAGCA CCATTTGTGAACAAGCAGTTTAACTATAAGGACCCGGTGAACGGTGTGGATATCGCGTAT ATCAAAATCCCGAATGCGGGCCAGATGCAACCAGTCAAGGCGTTCAAGATTCATAACAAG ATTTGGGTTATTCCGGAACGTGATACCTTCACCAATCCGGAAGAAGGCGATTTAAATCCG CCGCCAGAAGCCAAACAAGTGCCGGTGAGCTACTATGATAGCACGTATCTTAGCACCGAT AATGAAAAAGACAATTACCTGAAGGGCGTGACCAAGTTGTTCGAGCGCATCTACAGTACC TTCTTAGGCCGCATGTTGTTGCGTAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGCTCG ACCATTGATACCGAGTTGAAAGTCATTGACACGAACTGTATCAATGTTATCCAACCGGAC GGCAGTTATCGCAGCGAGGAGTTAAATTTGGTCATCATCGGTCCAAGCGCAGATATTATT CAGTTCGAATGCAAGAGCTTCGGCCATGAGGTCTTGAATTTGACGCGCAACGGTTACGGC AGCACCCAATACATCCGCTTTAGCCCGGATTTCACCTTTGGCTTCGAGGAGAGCTTGGAG GTGGACACCAACCCGCTGTTAGGTGCCGGCAAATTCGCAACCGACCCGGCAGTGACGTTG GCGCACGAATTGATTCATGCGGGTCACCGCTTATACGGTATCGCGATCAATCCGAATCGC GTCTTTAAAGTCAATACCAACGCGTACTACGAAATGAGCGGCTTAGAGGTTAGCTTTGAA GAATTACGCACCTTCGGTGGCCACGACGCCAAGTTCATCGACAGCCTGCAGGAAAATGAG TTCCGCTTGTACTATTACAATAAATTCAAGGACATCGCGAGCACCTTAAATAAAGCAAAG AGCATTGTGGGCACCACCGCAAGCTTGCAGTACATGAAGAACGTATTTAAGGAAAAATAT TTGTTGTCGGAGGATACCAGCGGGAAATTCAGCGTCGATAAGCTGAAATTCGACATGTTG TATAAAATGCTGACCGAGCTGTACACCGAGGATATGTTCGTCCGTTTTTTTAAGGTGTTA AATCGTAAGACCTATTTAAACTTTGATAAAGCGGTGTTTAAAATTAATATCGTGCCGAAG GTGAATTACACCATCTACGATGGTTTCAATTTACGCAACACGAATCTGGCGGCGAATTTT AATGGCCAAAACACCGAAATTAACAACATGAACTTTACGAAGTTAAAGAATTTCACGGGC TTATTCGAATTCTACAAGTTATTATGCGTGCGCGGCATCATTACCAGCAAGGCAGGTGCG GGCAAGTCCTTGGTTCCGCGTGGCAGCGCCGGCGCCGGCGCGCTCAATGATCTGTGTATT AAAGTCAATAACTGGGACCTGTTCTTCAGCCCGAGCGAGGATAACTTTACCAACGACTTA AACAAAGGCGAGGAGATCACGAGCGATACGAACATCGAGGCGGCGGAGGAAAATATTAGC CTGGACCTCATTCAGCAGTACTATCTGACGTTCAATTTTGACAATGAGCCGGAGAACATC AGCATTGAAAATCTCAGCAGCGACATCATCGGTCAGTTGGAACTGATGCCGAACATTGAA CGCTTTCCGAACGGCAAAAAATATGAACTGGACAAGTATACCATGTTCCATTACTTACGC GCACAGGAATTTGAGCACGGCAAGAGCCGCATTGCGCTGACCAATAGCGTTAACGAGGCC TTGTTAAATCCGAGCCGTGTCTACACGTTCTTCAGCAGCGATTATGTCAAAAAAGTGAAC AAGGCGACCGAAGCCGCGATGTTTTTGGGCTGGGTCGAGCAATTGGTTTACGATTTTACC GACGAAACCAGCGAGGTGAGCACGACCGACAAAATTGCAGATATCACCATCATCATTCCG TACATCGGTCCGGCGCTCAATATCGGCAATATGTTATACAAGGACGACTTTGTGGGCGCG CTGATCTTTAGCGGCGCGGTTATCTTATTAGAATTCATCCCGGAGATCGCAATCCCGGTC TTGGGCACCTTTGCGTTGGTGAGCTATATCGCGAATAAAGTGCTCACGGTCCAAACCATC GATAACGCGCTCAGCAAGCGTAATGAGAAATGGGACGAGGTTTATAAGTATATCGTGACC AACTGGTTAGCAAAAGTCAATACGCAGATCGATCTCATCCGCAAAAAAATGAAAGAAGCC TTGGAAAATCAAGCGGAGGCAACCAAAGCCATCATTAATTACCAGTATAACCAATATACC GAAGAAGAAAAAAACAATATCAACTTCAATATCGATGATTTGAGCAGCAAACTGAACGAG AGCATTAACAAAGCGATGATTAACATCAACAAGTTCTTGAATCAATGCAGCGTGAGCTAT CTCATGAACAGCATGATCCCGTATGGCGTCAAACGCTTGGAAGATTTTGACGCCAGCCTG AAAGATGCGCTCCTCAAGTATATTTATGACAACCGCGGCACCCTCATTGGCCAGGTGGAC CGCTTGAAGGATAAAGTGAACAATACGCTCAGCACGGATATCCCGTTCCAGCTGAGCAAG TACGTCGACAACCAGCGCTTACTGAGCACCTTTACCGAGTATATCAAGAACATCATTAAT ACCAGCATCCTCAACTTGCGCTATGAGAGCAATCACCTGATCGACCTCAGCCGCTACGCC AGCAAGATCAACATCGGCAGCAAGGTCAATTTCGACCCGATCGATAAGAATCAGATCCAA TTGTTTAACCTGGAAAGCAGCAAGATCGAGGTTATCTTGAAGAACGCGATTGTGTACAAC AGCATGTACGAGAACTTTAGCACGAGCTTCTGGATTCGTATCCCGAAGTATTTCAATAGC ATTAGCCTGAATAACGAATATACCATTATCAACTGCATGGAAAATAATAGCGGCTGGAAG GTGAGCTTAAATTACGGCGAGATCATTTGGACCTTACAGGATACCCAAGAAATCAAACAG CGCGTCGTCTTTAAGTATAGCCAGATGATCAACATCAGCGATTACATCAACCGCTGGATC TTCGTGACCATCACCAATAATCGCTTGAATAATAGCAAGATTTACATCAATGGTCGCTTG ATTGATCAAAAACCGATCAGCAATCTCGGTAATATCCATGCCAGCAATAACATCATGTTT AAGTTAGACGGTTGCCGCGATACCCACCGCTATATCTGGATCAAGTATTTTAACTTATTT GATAAGGAACTCAACGAAAAGGAAATTAAAGACTTATATGACAATCAGAGCAATAGCGGC ATCCTGAAGGATTTCTGGGGCGACTACCTGCAGTACGATAAGCCGTACTATATGTTGAAC TTGTATGACCCGAACAAATATGTCGATGTGAACAATGTGGGTATTCGTGGCTATATGTAC TTAAAGGGCCCGCGTGGTAGCGTGATGACCACGAATATTTACTTAAACAGCAGCTTATAC CGCGGCACGAAGTTTATTATCAAGAAGTATGCCAGCGGCAACAAGGACAATATCGTCCGC AACAACGACCGTGTGTATATTAACGTGGTGGTGAAGAATAAAGAGTACCGCTTGGCCACG AATGCGAGCCAGGCGGGCGTGGAAAAAATCTTGAGCGCGTTGGAGATCCCGGACGTCGGC AACCTCAGCCAGGTTGTGGTGATGAAGTCTAAAAACGACCAGGGCATCACGAACAAGTGC AAAATGAATTTGCAAGATAACAACGGCAACGACATCGGCTTTATTGGTTTTCACCAGTTC AATAACATCGCCAAACTCGTGGCCAGCAATTGGTATAACCGCCAAATTGAACGCAGCAGC CGCACGCTCGGCTGTAGCTGGGAGTTCATCCCGGTGGACGATGGCTGGGGCGAGCGCCCG CTCGCAAGTCCGGCAGCACCGGCACCGGCTTCACCAGCTGCACCAGCACCTAGCGCACCG GCAGCATCTCCAGCAGCCCCTGCACCGGCAAGCCCTGCAGCTCCAGCACCGTCAGCACCA GCAGCAAGCCCAGCTGCTCCTGCTCCAGCGAGCCCAGCAGCGCCAGCTCCTAGTGCCCCT GCTGCCTCTCCTGCTGCTCCGGCACCAGCAAGTCCTGCTGCGCCTGCACCGAGTGCTCCG GCTGCTAGTCCTGCCGCACCAGCTCCGGCTAGTCCAGCTGCTCCAGCCCCTTCAGCCCCT GCAGGAGATCTGGTGCCACGCGGTTCCGCGAATTCGAGCTCCGTCGACAAGCTTTGGAGC CACCCGCAGTTCGAAAAATAA
SEQ ID NO 5: recombinant PAS100-BoNT/A (G169I, T220R, P239M, S254T)-PAS100 including His.tag) (nucleic acid sequence) -
ATGGGTAGCAGCCATCATCATCACCATCATGGTAGCCTGGTTCCGCGTAGCTCTTCTGCA AGTCCGGCAGCACCGGCACCGGCTTCACCAGCTGCACCAGCACCTAGCGCACCGGCAGCA TCTCCAGCAGCCCCTGCACCGGCAAGCCCTGCAGCTCCAGCACCGTCAGCACCAGCAGCA AGCCCAGCTGCTCCTGCTCCAGCGAGCCCAGCAGCGCCAGCTCCTAGTGCCCCTGCTGCC TCTCCTGCTGCTCCGGCACCAGCAAGTCCTGCTGCGCCTGCACCGAGTGCTCCGGCTGCT AGTCCTGCCGCACCAGCTCCGGCTAGTCCAGCTGCTCCAGCCCCTTCAGCCCCTGCAGCA CCATTTGTGAACAAGCAGTTTAACTATAAGGACCCGGTGAACGGTGTGGATATCGCGTAT ATCAAAATCCCGAATGCGGGCCAGATGCAACCAGTCAAGGCGTTCAAGATTCATAACAAG ATTTGGGTTATTCCGGAACGTGATACCTTCACCAATCCGGAAGAAGGCGATTTAAATCCG CCGCCAGAAGCCAAACAAGTGCCGGTGAGCTACTATGATAGCACGTATCTTAGCACCGAT AATGAAAAAGACAATTACCTGAAGGGCGTGACCAAGTTGTTCGAGCGCATCTACAGTACC GACTTAGGCCGCATGTTGTTGACGAGCATCGTTCGCGGTATCCCGTTCTGGGGCGGCTCG ACCATTGATACCGAGTTGAAAGTCATTGACACGAACTGTATCAATGTTATCCAACCGGAC GGCAGTTATCGCAGCGAGGAGTTAAATTTGGTCATCATCGGTCCAAGCGCAGATATTATT CAGTTCGAATGCAAGAGCTTCATCCATGAGGTCTTGAATTTGACGCGCAACGGTTACGGC AGCACCCAATACATCCGCTTTAGCCCGGATTTCACCTTTGGCTTCGAGGAGAGCTTGGAG GTGGACACCAACCCGCTGTTAGGTGCCGGCAAATTCGCAACCGACCCGGCAGTGCGCTTG GCGCACGAATTGATTCATGCGGGTCACCGCTTATACGGTATCGCGATCAATATGAATCGC GTCTTTAAAGTCAATACCAACGCGTACTACGAAATGACCGGCTTAGAGGTTAGCTTTGAA GAATTACGCACCTTCGGTGGCCACGACGCCAAGTTCATCGACAGCCTGCAGGAAAATGAG TTCCGCTTGTACTATTACAATAAATTCAAGGACATCGCGAGCACCTTAAATAAAGCAAAG AGCATTGTGGGCACCACCGCAAGCTTGCAGTACATGAAGAACGTATTTAAGGAAAAATAT TTGTTGTCGGAGGATACCAGCGGGAAATTCAGCGTCGATAAGCTGAAATTCGACAAATTG TATAAAATGCTGACCGAGATTTACACCGAGGATAACTTCGTCAAGTTTTTTAAGGTGTTA AATCGTAAGACCTATTTAAACTTTGATAAAGCGGTGTTTAAAATTAATATCGTGCCGAAG GTGAATTACACCATCTACGATGGTTTCAACTTAAGCAACACGAATCTGGCGGCGAATTTT AATGGCCAAAACACCGAAATTAACAACATGAACTTTACGAAGTTAAAGAATTTCACGGGC TTATTCGAATTCTACAAGTTATTATGCGTGCGCGGCATCATTACCAGCAAGGCAGGTGCG GGCAAGTCCTTGGTTCCGCGTGGCAGCGCCGGCGCCGGCGCGCTCAATGATCTGTGTATT AAAGTCAATAACTGGGACCTGTTCTTCAGCCCGAGCGAGGATAACTTTACCAACGACTTA AACAAAGGCGAGGAGATCACGAGCGATACGAACATCGAGGCGGCGGAGGAAAATATTAGC CTGGACCTCATTCAGCAGTACTATCTGACGTTCAATTTTGACAATGAGCCGGAGAACATC AGCATTGAAAATCTCAGCAGCGACATCATCGGTCAGTTGGAACTGATGCCGAACATTGAA CGCTTTCCGAACGGCAAAAAATATGAACTGGACAAGTATACCATGTTCCATTACTTACGC GCACAGGAATTTGAGCACGGCAAGAGCCGCATTGCGCTGACCAATAGCGTTAACGAGGCC TTGTTAAATCCGAGCCGTGTCTACACGTTCTTCAGCAGCGATTATGTCAAAAAAGTGAAC AAGGCGACCGAAGCCGCGATGTTTTTGGGCTGGGTCGAGCAATTGGTTTACGATTTTACC GACGAAACCAGCGAGGTGAGCACGACCGACAAAATTGCAGATATCACCATCATCATTCCG TACATCGGTCCGGCGCTCAATATCGGCAATATGTTATACAAGGACGACTTTGTGGGCGCG CTGATCTTTAGCGGCGCGGTTATCTTATTAGAATTCATCCCGGAGATCGCAATCCCGGTC TTGGGCACCTTTGCGTTGGTGAGCTATATCGCGAATAAAGTGCTCACGGTCCAAACCATC GATAACGCGCTCAGCAAGCGTAATGAGAAATGGGACGAGGTTTATAAGTATATCGTGACC AACTGGTTAGCAAAAGTCAATACGCAGATCGATCTCATCCGCAAAAAAATGAAAGAAGCC TTGGAAAATCAAGCGGAGGCAACCAAAGCCATCATTAATTACCAGTATAACCAATATACC GAAGAAGAAAAAAACAATATCAACTTCAATATCGATGATTTGAGCAGCAAACTGAACGAG AGCATTAACAAAGCGATGATTAACATCAACAAGTTCTTGAATCAATGCAGCGTGAGCTAT CTCATGAACAGCATGATCCCGTATGGCGTCAAACGCTTGGAAGATTTTGACGCCAGCCTG AAAGATGCGCTCCTCAAGTATATTTATGACAACCGCGGCACCCTCATTGGCCAGGTGGAC CGCTTGAAGGATAAAGTGAACAATACGCTCAGCACGGATATCCCGTTCCAGCTGAGCAAG TACGTCGACAACCAGCGCTTACTGAGCACCTTTACCGAGTATATCAAGAACATCATTAAT ACCAGCATCCTCAACTTGCGCTATGAGAGCAATCACCTGATCGACCTCAGCCGCTACGCC AGCAAGATCAACATCGGCAGCAAGGTCAATTTCGACCCGATCGATAAGAATCAGATCCAA TTGTTTAACCTGGAAAGCAGCAAGATCGAGGTTATCTTGAAGAACGCGATTGTGTACAAC AGCATGTACGAGAACTTTAGCACGAGCTTCTGGATTCGTATCCCGAAGTATTTCAATAGC ATTAGCCTGAATAACGAATATACCATTATCAACTGCATGGAAAATAATAGCGGCTGGAAG GTGAGCTTAAATTACGGCGAGATCATTTGGACCTTACAGGATACCCAAGAAATCAAACAG CGCGTCGTCTTTAAGTATAGCCAGATGATCAACATCAGCGATTACATCAACCGCTGGATC TTCGTGACCATCACCAATAATCGCTTGAATAATAGCAAGATTTACATCAATGGTCGCTTG ATTGATCAAAAACCGATCAGCAATCTCGGTAATATCCATGCCAGCAATAACATCATGTTT AAGTTAGACGGTTGCCGCGATACCCACCGCTATATCTGGATCAAGTATTTTAACTTATTT GATAAGGAACTCAACGAAAAGGAAATTAAAGACTTATATGACAATCAGAGCAATAGCGGC ATCCTGAAGGATTTCTGGGGCGACTACCTGCAGTACGATAAGCCGTACTATATGTTGAAC TTGTATGACCCGAACAAATATGTCGATGTGAACAATGTGGGTATTCGTGGCTATATGTAC TTAAAGGGCCCGCGTGGTAGCGTGATGACCACGAATATTTACTTAAACAGCAGCTTATAC CGCGGCACGAAGTTTATTATCAAGAAGTATGCCAGCGGCAACAAGGACAATATCGTCCGC AACAACGACCGTGTGTATATTAACGTGGTGGTGAAGAATAAAGAGTACCGCTTGGCCACG AATGCGAGCCAGGCGGGCGTGGAAAAAATCTTGAGCGCGTTGGAGATCCCGGACGTCGGC AACCTCAGCCAGGTTGTGGTGATGAAGTCTAAAAACGACCAGGGCATCACGAACAAGTGC AAAATGAATTTGCAAGATAACAACGGCAACGACATCGGCTTTATTGGTTTTCACCAGTTC AATAACATCGCCAAACTCGTGGCCAGCAATTGGTATAACCGCCAAATTGAACGCAGCAGC CGCACGCTCGGCTGTAGCTGGGAGTTCATCCCGGTGGACGATGGCTGGGGCGAGCGCCCG CTCGCAAGTCCGGCAGCACCGGCACCGGCTTCACCAGCTGCACCAGCACCTAGCGCACCG GCAGCATCTCCAGCAGCCCCTGCACCGGCAAGCCCTGCAGCTCCAGCACCGTCAGCACCA GCAGCAAGCCCAGCTGCTCCTGCTCCAGCGAGCCCAGCAGCGCCAGCTCCTAGTGCCCCT GCTGCCTCTCCTGCTGCTCCGGCACCAGCAAGTCCTGCTGCGCCTGCACCGAGTGCTCCG GCTGCTAGTCCTGCCGCACCAGCTCCGGCTAGTCCAGCTGCTCCAGCCCCTTCAGCCCCT GCAGGAGATCTGGTGCCACGCGGTTCCGCGAATTCGAGCTCCGTCGACAAGCTTTGGAGC CACCCGCAGTTCGAAAAATAA
Claims (20)
1.-15. (canceled)
16. A method of treating a disease requiring improved chemodenervation, said method comprising the step of administering a recombinant neurotoxin to a patient in need thereof, said recombinant neurotoxin comprising:
at least two domains wherein each domain comprises an amino acid sequence comprising at least 50 amino acid residues, wherein said amino acid sequence comprises at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin,
wherein the said at least one amino acid modification is located at at least one position selected from D102, T109, K340, I348, N353, and K356 of the alpha-exosite, or at at least one position selected from G169, T220, P239, and S254 of the beta-exosite, and
wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
17. The method of claim 16 , wherein six amino acid modifications are located at the alpha-exosite at positions D102, T109, K340, I348, N353, K356.
18. The method of claim 17 , wherein the six amino acids are substituted as follows: D102F, T109R, K340M, I348L, N353M, K356R.
19. The method of claim 16 , wherein four amino acid modifications are located at the beta-exosite at positions G169, T220, P239, S254
20. The method of claim 19 , wherein the four amino acids are substituted as follows: G169I, T220R, P239M, S254T.
21. The method of claim 16 , wherein the recombinant neurotoxin comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues.
22. The method of claim 16 , wherein the recombinant neurotoxin is administered with a solvent or excipient.
23. The method of claim 16 , wherein the recombinant neurotoxin is administered with one or more pharmaceutically acceptable carriers.
24. The method of claim 16 , wherein the recombinant neurotoxin is administered through an injection.
25. The method of claim 16 , wherein the disease is selected from the group consisting of: cervical dystonia (spasmodic torticollis), blepharospasm, severe primary axillary hyperhidrosis, achalasia, lower back pain, benign prostate hypertrophy, chronic focal painful neuropathies, migraines and other headache disorders.
26. A method of using a recombinant neurotoxin for cosmetic treatment, said method comprising the step of administering the recombinant neurotoxin to a subject, said recombinant neurotoxin comprising:
at least two domains wherein each domain comprises an amino acid sequence comprising at least 50 amino acid residues, wherein said amino acid sequence comprises at least one proline, at least one alanine and at least one additional amino acid residue, selected from the group consisting of serine, threonine, tyrosine and glutamine, wherein the neurotoxin further comprises at least one amino acid modification which is located at the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin,
wherein the said at least one amino acid modification is located at at least one position selected from D102, T109, K340, I348, N353, and K356 of the alpha-exosite, or at at least one position selected from G169, T220, P239, and S254 of the beta-exosite, and
wherein the recombinant neurotoxin causes both (i) an increased duration of effect relative to a wildtype botulinum neurotoxin serotype A and (ii) an increased specific biological activity relative to a botulinum neurotoxin serotype A comprising two domains consisting of proline, alanine and serine residues without said modifications in the alpha-exosite and/or at the beta-exosite of the light chain of the neurotoxin.
27. The method of claim 26 , wherein six amino acid modifications are located at the alpha-exosite at positions D102, T109, K340, I348, N353, K356.
28. The method of claim 27 , wherein the six amino acids are substituted as follows: D102F, T109R, K340M, I348L, N353M, K356R.
29. The method of claim 26 , wherein four amino acid modifications are located at the beta-exosite at positions G169, T220, P239, S254
30. The method of claim 29 , wherein the four amino acids are substituted as follows: G169I, T220R, P239M, S254T.
31. The method of claim 26 , wherein the recombinant neurotoxin comprises two domains wherein each domain comprises an amino acid sequence consisting of between 70 and 260 amino acid residues.
32. The method of claim 26 , wherein the recombinant neurotoxin is administered with a solvent or excipient.
33. The method of claim 26 , wherein the recombinant neurotoxin is administered with one or more pharmaceutically acceptable carriers.
34. The method of claim 26 , wherein cosmetic treatment is selected from the group consisting of: treatment of wrinkles, treatment of crow's feet, treatment of glabella frown lines, reduction of masseter muscles, reduction of calves, and removing of facial asymmetries.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US17/482,953 US20220010294A1 (en) | 2017-07-06 | 2021-09-23 | Novel recombinant botulinum neurotoxins with increased duration of effect |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/EP2017/066891 WO2019007509A1 (en) | 2017-07-06 | 2017-07-06 | Novel recombinant botulinum neurotoxins with increased duration of effect |
| US201916498257A | 2019-09-26 | 2019-09-26 | |
| US17/482,953 US20220010294A1 (en) | 2017-07-06 | 2021-09-23 | Novel recombinant botulinum neurotoxins with increased duration of effect |
Related Parent Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US16/498,257 Division US11155802B2 (en) | 2017-07-06 | 2017-07-06 | Recombinant botulinum neurotoxins with increased duration of effect |
| PCT/EP2017/066891 Division WO2019007509A1 (en) | 2017-07-06 | 2017-07-06 | Novel recombinant botulinum neurotoxins with increased duration of effect |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| US20220010294A1 true US20220010294A1 (en) | 2022-01-13 |
Family
ID=59384133
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US16/498,257 Active US11155802B2 (en) | 2017-07-06 | 2017-07-06 | Recombinant botulinum neurotoxins with increased duration of effect |
| US17/482,953 Pending US20220010294A1 (en) | 2017-07-06 | 2021-09-23 | Novel recombinant botulinum neurotoxins with increased duration of effect |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| US16/498,257 Active US11155802B2 (en) | 2017-07-06 | 2017-07-06 | Recombinant botulinum neurotoxins with increased duration of effect |
Country Status (4)
| Country | Link |
|---|---|
| US (2) | US11155802B2 (en) |
| EP (1) | EP3649143B1 (en) |
| ES (1) | ES2930237T3 (en) |
| WO (1) | WO2019007509A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10441640B2 (en) * | 2014-08-12 | 2019-10-15 | Biomadison, Inc. | Botulinum neurotoxins with modified light chain specificity and methods for producing same |
| TW201718627A (en) * | 2015-06-11 | 2017-06-01 | 梅茲製藥有限兩合公司 | Recombinant clostridial neurotoxin, a use thereof, and a method for generating the same, a pharmaceutical composition comprising the same and a precursor corresponding to the same, a nucleic acid sequence encoding the precursor and a method for obtaining |
| WO2018233813A1 (en) | 2017-06-20 | 2018-12-27 | Merz Pharma Gmbh & Co. Kgaa | Novel recombinant botulinum toxin with increased duration of effect |
| US11155802B2 (en) * | 2017-07-06 | 2021-10-26 | Merz Pharma Gmbh & Co. Kgaa | Recombinant botulinum neurotoxins with increased duration of effect |
Family Cites Families (51)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6203794B1 (en) | 1994-05-31 | 2001-03-20 | Allergan Sales, Inc. | Modification of clostridial toxins for use as transport proteins |
| AU4746096A (en) | 1995-06-06 | 1996-12-24 | Wisconsin Alumni Research Foundation | Analogs of botulinum toxin and pharmaceutical compositions o f botulinum toxin |
| US6214602B1 (en) | 1998-08-28 | 2001-04-10 | Promega Corporation | Host cells for expression of clostridial toxins and proteins |
| KR100876060B1 (en) | 1999-08-25 | 2008-12-26 | 알러간, 인코포레이티드 | Activatable Recombinant Neurotoxins |
| US6903187B1 (en) | 2000-07-21 | 2005-06-07 | Allergan, Inc. | Leucine-based motif and clostridial neurotoxins |
| US7491799B2 (en) | 2000-07-21 | 2009-02-17 | Allergan, Inc. | Modified botulinum neurotoxins |
| US20020127247A1 (en) | 2000-11-17 | 2002-09-12 | Allergen Sales, Inc. | Modified clostridial neurotoxins with altered biological persistence |
| DE10333317A1 (en) | 2003-07-22 | 2005-02-17 | Biotecon Therapeutics Gmbh | Formulation for protein medicines without the addition of human serum albumin (HSA) |
| DK1755661T3 (en) | 2004-05-12 | 2014-06-16 | Brigham & Womens Hospital | GELSOLIN FOR USE FOR TREATMENT OF INFECTIONS |
| KR100852822B1 (en) | 2004-07-26 | 2008-08-18 | 메르츠 파마 게엠베하 운트 코. 카가아 | Therapeutic composition with a botulinum neurotoxin |
| AU2005271372B2 (en) | 2004-08-04 | 2012-05-03 | Allergan, Inc. | Optimizing expression of active botulinum toxin type A |
| DE102005002978B4 (en) | 2005-01-21 | 2013-04-25 | Merz Pharma Gmbh & Co. Kgaa | Recombinant expression of proteins in a disulfide-bonded, two-chain form |
| EP2131857B1 (en) | 2007-03-22 | 2015-07-29 | The Regents of the University of Colorado, a body corporate | Method of preparing an immunologically-active adjuvant-bound dried vaccine composition |
| ATE502114T1 (en) | 2007-06-21 | 2011-04-15 | Univ Muenchen Tech | BIOLOGICALLY ACTIVE PROTEINS WITH INCREASED IN-VIVO AND/OR IN-VITRO STABILITY |
| US9044477B2 (en) | 2007-12-12 | 2015-06-02 | Allergan, Inc. | Botulinum toxin formulation |
| US9161970B2 (en) | 2007-12-12 | 2015-10-20 | Allergan, Inc. | Dermal filler |
| BR122012027456A2 (en) | 2008-03-14 | 2015-07-14 | Allergan Inc | Pharmaceutical composition comprising botulinum neurotoxin serotype a |
| BRPI0916964A2 (en) | 2008-08-29 | 2015-11-24 | Merz Pharma Gmbh & Co Kgaa | polypeptide, antibody, nucleic acid, vector, host cell, method for making a polypeptide, composition and use of the polypeptide |
| AU2009288118B2 (en) | 2008-09-02 | 2014-12-11 | Allergan, Inc. | Threads of hyaluronic acid and/or derivatives thereof, methods of making thereof and uses thereof |
| AU2010204445B2 (en) | 2009-01-07 | 2015-09-03 | 2405871 Ontario Inc. | Treatment of soft tissue injury using hyaluronic acid and botulinum toxin |
| CN116925238A (en) | 2009-02-03 | 2023-10-24 | 阿穆尼克斯制药公司 | Extended recombinant polypeptides and compositions comprising the same |
| WO2010136585A2 (en) | 2009-05-29 | 2010-12-02 | Galderma Research & Development | Combination of adrenergic receptor agonist -1 or -2, preferably brimonidine with fillers, preferablyhyaluronic acid |
| IN2012DN00733A (en) | 2009-07-02 | 2015-06-19 | Merz Pharma Gmbh & Co Kgaa | |
| WO2011109130A1 (en) | 2010-03-01 | 2011-09-09 | Tautona Group Lp | Threads of hyaluronic acid and methods of use thereof |
| US8557961B2 (en) | 2010-04-02 | 2013-10-15 | Amunix Operating Inc. | Alpha 1-antitrypsin compositions and methods of making and using same |
| WO2011123813A2 (en) | 2010-04-02 | 2011-10-06 | Amunix Operating Inc. | Binding fusion proteins, binding fusion protein-drug conjugates, xten-drug conjugates and methods of making and using same |
| EA024755B1 (en) | 2010-05-21 | 2016-10-31 | ИксЭль-ПРОТЕИН ГМБХ | Biosynthetic proline/alanine random coil polypeptides and their uses |
| FR2966348B1 (en) | 2010-10-22 | 2012-11-09 | Galderma Res & Dev | COMPOSITIONS COMPRISING A WRINKLE FILLING MEDIUM AND A CHEMICALLY MODIFIED TETRACYCLINE |
| KR101640694B1 (en) | 2011-09-29 | 2016-07-18 | 셀스냅, 엘엘씨 | Compositions and methods for toxigenicity testing |
| RU2646110C2 (en) * | 2011-11-09 | 2018-03-01 | Мерц Фарма Гмбх Унд Ко. Кгаа | Neurotoxins showing shortened biological activity |
| AU2012334076A1 (en) | 2011-11-09 | 2014-05-15 | Merz Pharma Gmbh & Co. Kgaa | Modified neurotoxins with poly-Glycine and uses thereof |
| JP2014534265A (en) | 2011-11-28 | 2014-12-18 | フェーズバイオ ファーマシューティカルズ，インコーポレイテッド | Therapeutic drugs containing insulin amino acid sequence |
| AR089797A1 (en) | 2012-01-27 | 2014-09-17 | Merck Sharp & Dohme | VACCINES AGAINST CLOSTRIDUM DIFFICILE THAT INCLUDE RECOMBINANT TOXINS |
| GB201219602D0 (en) | 2012-10-31 | 2012-12-12 | Syntaxin Ltd | Recombinant clostridium botulinum neurotoxins |
| US20150322118A1 (en) | 2012-12-05 | 2015-11-12 | Merz Pharma Gmbh & Co. Kgaa | Recombinant clostridial neurotoxins with enhanced membrane localization |
| CA2899961C (en) | 2013-02-14 | 2022-08-09 | Cornell University | Methods to protect against and treat multiple sclerosis |
| WO2014207109A1 (en) | 2013-06-28 | 2014-12-31 | Merz Pharma Gmbh & Co. Kgaa | Means and methods for the determination of the biological activity of neurotoxin polypeptides in cells |
| EP3113792B1 (en) | 2014-03-05 | 2018-12-05 | Merz Pharma GmbH & Co. KGaA | Novel recombinant clostridial neurotoxins with increased duration of effect |
| EP3156412B1 (en) | 2014-05-29 | 2020-01-08 | Procell Therapeutics Inc. | Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
| US10441640B2 (en) | 2014-08-12 | 2019-10-15 | Biomadison, Inc. | Botulinum neurotoxins with modified light chain specificity and methods for producing same |
| WO2016073562A1 (en) | 2014-11-04 | 2016-05-12 | Synthetic Biologics, Inc. | Methods and compositions for inhibiting clostridium difficile |
| PL3242884T3 (en) | 2015-01-09 | 2021-08-16 | Ipsen Bioinnovation Limited | Cationic neurotoxins |
| WO2016180533A1 (en) * | 2015-05-12 | 2016-11-17 | Merz Pharma Gmbh & Co. Kgaa | Novel recombinant clostridial neurotoxins with increased duration of effect |
| TW201718627A (en) | 2015-06-11 | 2017-06-01 | 梅茲製藥有限兩合公司 | Recombinant clostridial neurotoxin, a use thereof, and a method for generating the same, a pharmaceutical composition comprising the same and a precursor corresponding to the same, a nucleic acid sequence encoding the precursor and a method for obtaining |
| ES2929347T3 (en) * | 2016-01-20 | 2022-11-28 | Merz Pharma Gmbh & Co Kgaa | Novel recombinant clostridial neurotoxins with increased duration of effect |
| EP3335719A1 (en) * | 2016-12-14 | 2018-06-20 | Merz Pharma GmbH & Co. KGaA | Novel recombinant botulinum neurotoxins with a stabilized light chain |
| WO2018233813A1 (en) | 2017-06-20 | 2018-12-27 | Merz Pharma Gmbh & Co. Kgaa | Novel recombinant botulinum toxin with increased duration of effect |
| US11155802B2 (en) * | 2017-07-06 | 2021-10-26 | Merz Pharma Gmbh & Co. Kgaa | Recombinant botulinum neurotoxins with increased duration of effect |
| EP3700919A1 (en) | 2017-10-26 | 2020-09-02 | Merz Pharma GmbH & Co. KGaA | Novel recombinant botulinum neurotoxins with increased duration of effect |
| WO2019101308A1 (en) * | 2017-11-22 | 2019-05-31 | Merz Pharma Gmbh & Co. Kgaa | Novel recombinant botulinum toxin with increased duration of effect |
| CA3118412A1 (en) * | 2018-11-02 | 2020-05-07 | Institute Of Basic Medical Sciences Chinese Academy Of Medical Sciences | Cell-penetrating peptide based on influenza virus m2 protein |
-
2017
- 2017-07-06 US US16/498,257 patent/US11155802B2/en active Active
- 2017-07-06 EP EP17742665.7A patent/EP3649143B1/en active Active
- 2017-07-06 ES ES17742665T patent/ES2930237T3/en active Active
- 2017-07-06 WO PCT/EP2017/066891 patent/WO2019007509A1/en unknown
-
2021
- 2021-09-23 US US17/482,953 patent/US20220010294A1/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP3649143A1 (en) | 2020-05-13 |
| US11155802B2 (en) | 2021-10-26 |
| ES2930237T3 (en) | 2022-12-09 |
| US20200131494A1 (en) | 2020-04-30 |
| WO2019007509A1 (en) | 2019-01-10 |
| EP3649143B1 (en) | 2022-08-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220010294A1 (en) | Novel recombinant botulinum neurotoxins with increased duration of effect | |
| EP3113792B1 (en) | Novel recombinant clostridial neurotoxins with increased duration of effect | |
| US11357821B2 (en) | Recombinant clostridial neurotoxins with increased duration of effect | |
| US20090018081A1 (en) | Activatable clostridial toxins | |
| EP3335719A1 (en) | Novel recombinant botulinum neurotoxins with a stabilized light chain | |
| US20150322118A1 (en) | Recombinant clostridial neurotoxins with enhanced membrane localization | |
| US11952601B2 (en) | Recombinant botulinum toxin with increased duration of effect | |
| US11078472B2 (en) | Recombinant clostridial neurotoxins with increased duration of effect | |
| US20210008156A1 (en) | Novel recombinant botulinum neurotoxins with increased duration of effect | |
| US20200354706A1 (en) | Novel recombinant botulinum toxin with increased duration of effect | |
| WO2016180533A1 (en) | Novel recombinant clostridial neurotoxins with increased duration of effect | |
| EP3312193A1 (en) | Novel recombinant botulinum neurotoxins with accelerated onset of effect | |
| EP3333179A1 (en) | Novel recombinant botulinum toxin with accelarated onset of effect | |
| EP3290437A1 (en) | Novel recombinant clostridial neurotoxins with decreased duration of effect | |
| US20150232828A1 (en) | Method for the manufacturing of recombinant proteins harbouring an n-terminal lysine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| AS | Assignment |
Owner name: MERZ PHARMA GMBH & CO. KGAA, GERMANY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:FREVERT, JUERGEN;HOFMANN, FRED;JURK, MARCEL;AND OTHERS;SIGNING DATES FROM 20190804 TO 20190823;REEL/FRAME:057597/0420 |
|
| STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
| STPP | Information on status: patent application and granting procedure in general |
Free format text: NOTICE OF ALLOWANCE MAILED -- APPLICATION RECEIVED IN OFFICE OF PUBLICATIONS |
|
| ZAAB | Notice of allowance mailed |
Free format text: ORIGINAL CODE: MN/=. |