EP2038299A2 - Modifizierte clostridientoxine mit verbesserten translokationsfähigkeiten und geänderter targeting-aktivität für nichtclostridientoxin-targetzellen - Google Patents

Modifizierte clostridientoxine mit verbesserten translokationsfähigkeiten und geänderter targeting-aktivität für nichtclostridientoxin-targetzellen

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Publication number
EP2038299A2
EP2038299A2 EP07812774A EP07812774A EP2038299A2 EP 2038299 A2 EP2038299 A2 EP 2038299A2 EP 07812774 A EP07812774 A EP 07812774A EP 07812774 A EP07812774 A EP 07812774A EP 2038299 A2 EP2038299 A2 EP 2038299A2
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EP
European Patent Office
Prior art keywords
amino acids
bont
seq
clostridial toxin
amino acid
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.)
Withdrawn
Application number
EP07812774A
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English (en)
French (fr)
Inventor
Lance E. Steward
Joseph Francis
Ester Fernandez-Salas
Marcella A. Gilmore
Shengwen Li
Kei Roger Aoki
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Allergan Inc
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Allergan Inc
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Publication of EP2038299A2 publication Critical patent/EP2038299A2/de
Withdrawn legal-status Critical Current

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    • 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin

Definitions

  • FIG. 5 shows modified Clostridial toxins with an enhanced translocation capability and an altered targeting activity located between two other domains.
  • FIG. 5A depicts the single polypeptide form of a modified Clostridial toxin with an amino to carboxyl linear organization comprising an enzymatic domain, an altered targeting domain, a translocation domain and a translocation facilitating domain, with the di- chain loop region depicted by the double SS bracket.
  • a proteolytic cleavage site (P) within a di-chain loop region is located between the enzymatic and targeting domains. Upon proteolytic cleavage with a P protease, the single chain form of the toxin is converted to the di-chain form.
  • P proteolytic cleavage site
  • the H N domain also forms a long unstructured loop called the 'translocation belt,' which wraps around a large negatively charged cleft of the light chain that blocks access of the zinc atom to the catalytic-binding pocket of active site.
  • the H c domain comprises two distinct structural features of roughly equal size that indicate function. The first, designated the H CN domain, is located in the amino half of the H c domain. The H CN domain forms a ⁇ -barrel, jelly-roll fold. The H C c domain is the second domain that comprises the H c domain.
  • a conservative Clostridial toxin light chain variant can function in substantially the same manner as the reference Clostridial toxin light chain on which the conservative Clostridial toxin light chain variant is based, and can be substituted for the reference Clostridial toxin light chain in any aspect of the present invention.
  • a BoNT/G light chain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 1 -446 of SEQ ID NO: 7.
  • a BoNT/G light chain comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 non-contiguous amino acid deletions relative to amino acids 1 -446 of SEQ ID NO: 7.
  • a TeNT light chain comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 1 -457 of SEQ ID NO: 8, at least 75% amino acid identity with amino acids 1 -457 of SEQ ID NO: 8, at least 80% amino acid identity with amino acids 1 -457 of SEQ ID NO: 8, at least 85% amino acid identity with amino acids 1 -457 of SEQ ID NO: 8, at least 90% amino acid identity with amino acids 1 -457 of SEQ ID NO: 8 or at least 95% amino acid identity with amino acids 1 -457 of SEQ ID NO: 8.
  • the term "conservative Clostridial toxin H N region variant” means a Clostridial toxin H N region that has at least one amino acid substituted by another amino acid or an amino acid analog that has at least one property similar to that of the original amino acid from the reference Clostridial toxin H N region sequence (Table 1 ).
  • properties include, without limitation, similar size, topography, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent-bonding capacity, hydrogen-bonding capacity, a physicochemical property, of the like, or any combination thereof.
  • a Clostridial toxin translocation domain comprises a non-naturally occurring Clostridial toxin H N region variant, such as, e.g., a conservative Clostridial toxin H N region variant, a non-conservative Clostridial toxin H N region variant, a Clostridial toxin chimeric H N region, an active Clostridial toxin H N region fragment, or any combination thereof.
  • a Clostridial toxin translocation domain comprises a BoNT/A H N region.
  • a BoNT/A H N region comprises amino acids 449-873 of SEQ ID NO: 1 .
  • a BoNT/A H N region comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 449-873 of SEQ ID NO: 1 .
  • a BoNT/A H N region comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid substitutions relative to amino acids 449-873 of SEQ ID NO: 1.
  • a BoNT/D H N region comprises a polypeptide having, e.g., at least 70% amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at least 75% amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at least 80% amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at least 85% amino acid identity with amino acids 446-864 of SEQ ID NO: 4, at least 90% amino acid identity with amino acids 446-864 of SEQ ID NO: 4 or at least 95% amino acid identity with amino acids 446-864 of SEQ ID NO: 4.
  • a TeNT H N region comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino acids 458-881 of SEQ ID NO: 8.
  • a TeNT H N region comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to amino acids 458-881 of SEQ ID NO: 8.
  • a non-conservative Clostridial toxin H CN region variant can function in substantially the same manner as the reference Clostridial toxin H CN region on which the non-conservative Clostridial toxin H CN region variant is based, and can be substituted for the reference Clostridial toxin H CN region in any aspect of the present invention.
  • a non-conservative Clostridial toxin H CN region variant can delete one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, and ten or more amino acids from the reference Clostridial toxin H CN region on which the non-conservative Clostridial toxin H CN region variant is based.
  • a BoNT/D H CN region comprises amino acids 865-1098 of a non-naturally occurring BoNT/D H CN region variant of SEQ ID NO: 4, such as, e.g., amino acids 865-1098 of a conservative BoNT/D H CN region variant of SEQ ID NO: 4, amino acids 865-1098 of a non-conservative BoNT/D H CN region variant of SEQ ID NO: 4, amino acids 865-1098 of an active BoNT/D H CN region fragment of SEQ ID NO: 4, or any combination thereof.
  • Non-limiting examples of a conservative enveloped virus fusogenic peptide domain variant include, e.g., conservative influenzavirus fusogenic peptide domain variants, conservative alphavirus fusogenic peptide domain variants, conservative vesiculovirus fusogenic peptide domain variants, conservative respirovirus fusogenic peptide domain variants, conservative morbillivirus fusogenic peptide domain variants, conservative avulavirus fusogenic peptide domain variants, conservative henipavirus fusogenic peptide domain variants, conservative metapneumovirus fusogenic peptide domain variants and conservative foamy virus fusogenic peptide domain variants.
  • an influenzavirus fusogenic peptide domain comprises a naturally occurring influenzavirus fusogenic peptide domain variant of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198, such as, e.g., an influenzavirus fusogenic peptide domain isoform of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198 or an influenzavirus fusogenic peptide domain subtype of SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198.
  • an influenzavirus fusogenic peptide domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid substitutions relative to SEQ ID NO: 194, SEQ ID NO: 195, SEQ ID NO: 196, SEQ ID NO: 197 or SEQ ID NO: 198.
  • SEQ ID NO: 232 SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, at most 85% amino acid identity with SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO: 228, SEQ ID NO: 229, SEQ ID NO: 230, SEQ ID NO: 231 , SEQ ID NO: 232, SEQ ID NO: 233, SEQ ID NO: 234, SEQ ID NO: 235 or SEQ ID NO: 236, at most 90% amino acid identity with SEQ ID NO: 226, SEQ ID NO: 227, SEQ ID NO:
  • a Clostridial toxin translocation facilitating domain comprises a morbillivirus fusogenic peptide domain.
  • a morbillivirus fusogenic peptide domain comprises a naturally occurring morbillivirus fusogenic peptide domain variant, such as, e.g., a morbillivirus fusogenic peptide domain isoform or a morbillivirus fusogenic peptide domain subtype.
  • a morbillivirus fusogenic peptide domain comprises a naturally occurring morbillivirus fusogenic peptide domain variant of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251 , SEQ ID NO: 252 or SEQ ID NO: 253, such as, e.g., a morbillivirus fusogenic peptide domain isoform of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251 , SEQ ID NO: 252 or SEQ ID NO: 253 or a morbillivirus fusogenic peptide domain subtype of SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 245, SEQ ID NO:
  • a morbillivirus fusogenic peptide domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid deletions relative to SEQ ID NO: 244, SEQ ID NO: 245, SEQ ID NO: 246, SEQ ID NO: 247, SEQ ID NO: 248, SEQ ID NO: 249, SEQ ID NO: 250, SEQ ID NO: 251 , SEQ ID NO: 252 or SEQ ID NO: 253.
  • an avulavirus fusogenic peptide domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 non-contiguous amino acid additions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261 , SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266.
  • an avulavirus fusogenic peptide domain comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine or 10 contiguous amino acid deletions relative to SEQ ID NO: 254, SEQ ID NO: 255, SEQ ID NO: 256, SEQ ID NO: 257, SEQ ID NO: 258, SEQ ID NO: 259, SEQ ID NO: 260, SEQ ID NO: 261 , SEQ ID NO: 262, SEQ ID NO: 263, SEQ ID NO: 264, SEQ ID NO: 265 or SEQ ID NO: 266.
  • a metapneumovirus fusogenic peptide domain comprises amino acids a non-naturally occurring metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, such as, e.g., a conservative metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, a non-conservative metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, an active metapneumovirus fusogenic peptide domain fragment of SEQ ID NO: 269, or any combination thereof.
  • a conservative metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269 such as, e.g., a conservative metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, a non-conservative metapneumovirus fusogenic peptide domain variant of SEQ ID NO: 269, an active metapneumovirus fusogenic peptide domain fragment of SEQ
  • an endorphin- ⁇ variant derived from SEQ ID NO: 17 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to SEQ ID NO: 17;
  • a Dymorphin A variant derived from SEQ ID NO: 21 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to SEQ ID NO: 21 ;
  • a nociceptin variant derived from SEQ ID NO: 52 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to SEQ ID NO: 52;
  • a galanin variant derived from SEQ ID NO: 72 will have at least one amino acid difference, such as, e.g., an amino acid substitution, deletion or addition, as compared to SEQ ID NO: 72;
  • an adrenomedullary peptide variant derived from SEQ ID NO: 83 will have at least one amino acid
  • a naturally occurring altered targeting domain variant may substitute one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, ten or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, 50 or more amino acids or 100 or more amino acids from the reference altered targeting domain on which the naturally occurring altered targeting domain variant is based.
  • a peptide comprising an altered targeting domain has, e.g., at most one, two, three, four or five non-contiguous amino acid deletions relative to amino acids 1 -12, amino acids 6-22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 172; amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 173; amino acids 1 - 12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 174; amino acids 1 -12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino acids 1 -12, amino acids 6- 22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 176; or amino acids 1-12, amino acids 6-22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 177.
  • a peptide comprising an altered targeting domain has, e.g., at least one, two, three, four or five contiguous amino acid substitutions relative to amino acids 1 -12, amino acids 6-22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 172; amino acids 1 -12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 173; amino acids 1 -12, amino acids 6- 22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 174; amino acids 1 -12, amino acids 6-22, amino acids 8-22 or amino acids 1-22 of SEQ ID NO: 175; amino acids 1-12, amino acids 6-22, amino acids 8- 22 or amino acids 1 -22 of SEQ ID NO: 176; or amino acids 1 -12, amino acids 6-22, amino acids 8-22 or amino acids 1 -22 of SEQ ID NO: 177.
  • a dynorphin comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 21 , SEQ ID NO: 30 or SEQ ID NO: 46.
  • a dynorphin comprising an altered targeting domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten non-contiguous amino acid substitutions relative to SEQ ID NO: 21 , SEQ ID NO: 30 or SEQ ID NO: 46.
  • a dynorphin comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 21 , SEQ ID NO: 30 or SEQ ID NO: 46.
  • a dynorphin comprising an altered targeting domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 21 , SEQ ID NO: 30 or SEQ ID NO: 46.
  • a nociceptin comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61 .
  • a nociceptin comprising an altered targeting domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 52, SEQ ID NO: 59, SEQ ID NO: 60 or SEQ ID NO: 61 .
  • an adrenocorticotropin comprising an altered targeting domain has, e.g., at least one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 65 or SEQ ID NO: 66. In yet other aspects of this embodiment, an adrenocorticotropin comprising an altered targeting domain has, e.g., at most one, two, three, four or five contiguous amino acid deletions relative to SEQ ID NO: 65 or SEQ ID NO: 66.
  • a melanocortin peptide comprising an altered targeting domain is a lipotropin.
  • a lipotropin comprising an altered targeting domain is derived from a ⁇ -lipotropin ( ⁇ -LPH) or a ⁇ -lipotropin ( ⁇ -LPH).
  • a lipotropin comprising an altered targeting domain is SEQ ID NO: 67 or SEQ ID NO: 68.
  • a chromogranin A peptide comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80 or SEQ ID NO: 81 .
  • a chromogranin B peptide comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • a chromogranin B peptide comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid additions relative to SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85 or SEQ ID NO: 86.
  • a chromogranin C peptide comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 87.
  • a chromogranin C peptide comprising an altered targeting domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 87.
  • an altered targeting domain disclosed in the present specification is, e.g., a tachykinin peptide, such as, e.g., a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin.
  • a tachykinin peptide such as, e.g., a Substance P, a neuropeptide K (NPK), a neuropeptide gamma (NP gamma), a neurokinin A (NKA; Substance K, neurokinin alpha, neuromedin L), a neurokinin B (NKB), a hemokinin or a endokinin.
  • a tachykinin peptide such as, e.g., a Substance P, a neuropeptide K
  • a cholecystokinin peptide comprising an altered targeting domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ IDNO: 114 or SEQ ID NO: 115, at least 75% amino acid identity with SEQ IDNO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109
  • SEQ ID NO: 106 SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 1 10, SEQ ID NO: 1 1 1 , SEQ ID NO: 1 12, SEQ ID NO: 113, SEQ ID NO: 1 14 or SEQ ID NO: 115.
  • an altered targeting domain is, e.g., a Neuropeptide Y related peptide, such as, e.g., a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) or a Pancreatic icosapeptide (PIP).
  • a Neuropeptide Y related peptide such as, e.g., a Neuropeptide Y (NPY), a Peptide YY (PYY), Pancreatic peptide (PP) or a Pancreatic icosapeptide (PIP).
  • NPY Neuropeptide Y
  • PYYY Peptide YY
  • PP Pancreatic peptide
  • PIP Pancreatic icosapeptide
  • a Neuropeptide Y related peptide comprising an altered targeting domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid deletions relative to SEQ ID NO: 116, SEQ ID NO: 1 17, SEQ ID NO: 1 18, SEQ ID NO: 119 or SEQ ID NO: 120.
  • a kinin peptide comprising an altered targeting domain has, e.g., at least 70% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 , at least 75% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 , at least 80% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 , at least 85% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 , at least 90% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 or at least 95% amino acid identity with SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO
  • a kinin peptide comprising an altered targeting domain has, e.g., at least one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 .
  • a kinin peptide comprising an altered targeting domain has, e.g., at most one, two, three, four, five, six, seven, eight, nine or ten contiguous amino acid substitutions relative to SEQ ID NO: 178, SEQ ID NO: 179, SEQ ID NO: 180 or SEQ ID NO: 181 .
  • a PAR peptide comprising an altered targeting domain has, e.g., at most 70% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1 -64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1 -60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61 , amino acids 35-70 or amino acids 1 -70 of SEQ ID NO: 185, at most 75% amino acid identity with amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1 -64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1 -59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or
  • a PAR peptide comprising an altered targeting domain has, e.g., at most one, two, three, four or five contiguous amino acid substitutions relative to amino acids 42-47, amino acids 42-55, amino acids 29-64 or amino acids 1 -64 of SEQ ID NO: 182; amino acids 35-40, amino acids 35-48, amino acids 24-59 or amino acids 1-59 of SEQ ID NO: 183; amino acids 39-44, amino acids 39-52, amino acids 26-60 or amino acids 1 -60 of SEQ ID NO: 184; amino acids 48-53, amino acids 48-61 , amino acids 35-70 or amino acids 1 -70 of SEQ ID NO: 185.
  • a ⁇ - trefoil domain comprises four ⁇ -sheets of the first ⁇ -trefoil fold, a ⁇ -hairpin turn, four ⁇ -sheets of the second ⁇ -trefoil fold, a second ⁇ -hairpin turn four ⁇ -sheets of the third ⁇ -trefoil fold. Because the first hairpin turn is located between the fourth and fifth ⁇ -sheets of the ⁇ -trefoil domain, it is designated the ⁇ 4/ ⁇ 5 ⁇ -hairpin turn. Likewise, since the second hairpin turn is located between the eight and ninth ⁇ - sheets of the ⁇ -trefoil domain, it is designated the ⁇ 8/ ⁇ 9 ⁇ -hairpin turn.
  • an altered Clostridial toxin H C c targeting region comprises a polypeptide having, e.g., at most one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to its reference sequence.
  • an altered Clostridial toxin H C c targeting region comprises a polypeptide having, e.g., at least one, two, three, four, five, six, seven, eight, nine, 10, 20, 30, 40 , 50, 100 or 200 contiguous amino acid additions relative to its reference sequence.
  • a modified Clostridial toxin with an altered targeting activity comprises, in part, a protease cleavage site is located within a di-chain loop region.
  • the term "di-chain loop region" means the amino acid sequence of a Clostridial toxin containing a protease cleavage site used to convert the single-chain form of a Clostridial toxin into the di-chain form.
  • a modified Clostridial toxin disclosed in the present specification can comprise a flexible spacer in any and all locations with the proviso that modified Clostridial toxin is capable of performing the intoxication process.
  • a flexible spacer is positioned between, e.g., an enzymatic domain and a translocation domain, an enzymatic domain and an altered targeting domain, an enzymatic domain and a protease cleavage site.

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EP07812774A 2006-07-11 2007-07-11 Modifizierte clostridientoxine mit verbesserten translokationsfähigkeiten und geänderter targeting-aktivität für nichtclostridientoxin-targetzellen Withdrawn EP2038299A2 (de)

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