Carrier for targeting nerve cells
The present invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by Clostridium botulinum wherein (i) the protein binds specifically to nerve cells with a higher or lower affinity as the native neurotoxin; (ii) the protein has an increased or reduced neurotoxicity compared to the native neurotoxin, the neurotoxicity being preferably determined in the hemidiaphragm assay; and/or (iii) the protein comprises a lower affinity against neutralizing antibodies compared to the native neurotoxin. The invention also relates to methods for producing the same and the use thereof in cosmetic and pharmaceutical compositions.
1. A transport protein comprising a modified Clostridium botulinum serotype B neurotoxin Hcc domain that binds to a nerve cell with a higher or lower affinity than the native Clostridium botulinum serotype B neurotoxin from which said Hcc domain is derived, said transport protein comprises substitution by a non-polar amino acid of at least one amino acid in the amino acid positions selected from the group consisting of: amino acid positions corresponding to aspartate 1114, serine 1116, proline 1117, valine 1118, tyrosine 1183, phenylalanine 1186, lysine 1188, glutamate 1191, lysine 1192, leucine 1193, phenylalanine 1194, phenylalanine 1204, phenylalanine 1243, glutamate 1245, lysine 1254, aspartate 1255 and tyrosine 1256 of Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11, wherein the transport protein binds to synaptotagmin I or II, and wherein the transport protein is covalently linked to a Clostridium botulinum serotype B protease.
2. The transport protein according to claim 1 , wherein the protein exhibits an affinity which is at least 15% higher or at least 15% lower than the native Clostridium botulinum serotype B neurotoxin from which said H CC domain is derived.
3. The transport protein according to claim 1 , wherein at least one amino acid in the positions corresponding to valine 1118, tyrosine 1183, glutamate 1191, lysine 1192, glutamate 1245 and/or tyrosine 1256 of the Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11 is substituted by the non-polar amino acid.
4. The transport protein according to claim 3 , wherein the amino acid tyrosine is substituted in the position corresponding to position 1183 by leucine, or wherein the amino acid glutamate is substituted in the position corresponding to position 1191 by leucine.
5. The transport protein according to claim 1 , which binds to said nerve cell with a higher affinity than native Clostridium botulinum serotype B neurotoxin.
6. The transport protein according to claim 1 , wherein said non-polar amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan.
7. The transport protein according to claim 1 covalently linked to a clostridial translocation domain.
8. The transport protein according to claim 7 , including an amino acid sequence that is recognized and cleaved by the Clostridium botulinum serotype B protease, thereby providing a di-chain polypeptide in which the clostridial translocation domain and the Clostridium botulinum serotype B protease are linked together by a disulfide-bridge.
9. The transport protein according to claim 8 , wherein said amino acid sequence is a native clostridial amino acid sequence or a non-clostridial (exogenous) amino acid sequence.
10. A method for treating an individual suffering from a disease or disorder selected from: hemi-facial spasm, spasmodic torticollis, blepharospasm, spasticities, dystonias, migraine, pain, disorders of the neck and lumber vertebral column, strabism, hypersalivation, snoring, wound healing, and depressive disorders, said method comprising administering a therapeutically effective amount of a transport protein according to claim 8 to said patient.
11. A method for treating an individual suffering from a cosmetic indication selected from hyperhidrosis or facial wrinkles, said method comprising administering a therapeutically effective amount of a transport protein according to claim 8 to said patient.
12. A transport protein comprising a modified clostridial neurotoxin Hcc domain that binds to a nerve cell with a higher affinity than the native Clostridium botulinum serotype B neurotoxin from which said Hcc domain is derived, comprising substitution by a non-polar amino acid of at least one amino acid in the amino acid positions selected from the group consisting of: amino acid positions corresponding to aspartate 1114, serine 1116, proline 1117, valine 1118, tyrosine 1183, phenylalanine 1186, lysine 1188, glutamate 1191, lysine 1192, leucine 1193, phenylalanine 1194, phenylalanine 1204, phenylalanine 1243, glutamate 1245, lysine 1254, aspartate 1255 and tyrosine 1256 of Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11; wherein the transport protein binds to synaptotagmin I or II.
13. The transport protein according to claim 12 , which is covalently linked to a Clostridium botulinum serotype B protease.
14. The transport protein according to claim 12 , wherein said non-polar amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan.
15. The transport protein according to claim 12 , wherein at least one amino acid in the positions corresponding to aspartate 1114, proline 1117, phenylalanine 1186, lysine 1188, glutamate 1191, lysine 1192, glutamate 1245, lysine 1254, aspartate 1255 and tyrosine 1256 of the Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11 is substituted by the non-polar amino acid.
16. A transport protein comprising a modified clostridial neurotoxin Hcc domain that binds to a nerve cell with a higher or lower affinity than the native Clostridium botulinum serotype B neurotoxin from which said Hcc domain is derived, comprising a substitution by a non-polar amino acid in the amino acid position corresponding to glutamate 1191 of Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11;
wherein the transport protein binds to synaptotagmin I or II, and wherein the transport protein is covalently linked to a Clostridium botulinum serotype B protease.
17. The transport protein according to claim 16 , which binds to said nerve cell with a higher affinity than native Clostridium botulinum serotype B neurotoxin.
18. The transport protein according to claim 16 , wherein said non-polar amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan.
19. A transport protein comprising a modified clostridial neurotoxin Hcc domain that binds to a nerve cell with a higher affinity than the native Clostridium botulinum serotype B neurotoxin from which said Hcc domain is derived, comprising a substitution by a non-polar amino acid in the amino acid position corresponding to glutamate 1191 of Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11;
wherein the transport protein binds to synaptotagmin I or II.
20. The transport protein according to claim 19 , which is covalently linked to a Clostridium botulinum serotype B protease.
21. The transport protein according to claim 19 , wherein said non-polar amino acid is selected from the group consisting of glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, and tryptophan.
22. The transport protein according to claim 1 , wherein at least one amino acid in the positions corresponding to aspartate 1114, proline 1117, phenylalanine 1186, lysine 1188, glutamate 1191, lysine 1192, glutamate 1245, lysine 1254, aspartate 1255 and tyrosine 1256 of the Clostridium botulinum serotype B neurotoxin of SEQ ID NO.: 6, 7, 8, 9, 10 or 11 is substituted by the non-polar amino acid.