Claims
- 1. A pharmaceutical composition comprising a chimeric, folded protein domain and a pharmaceutically acceptable carrier, wherein said chimeric folded protein domain is selected from a repertoire of chimeric protein domains, said domain comprising two or more sequence segments from parent amino acid sequences that are non-homologous, wherein said two or more sequence segments do not form stable folds in isolation from said parent amino acid sequences.
- 2. The pharmaceutical composition of claim 1, wherein said sequence segments do not consist of complete protein domains, and wherein said sequence segments do not consist solely of single and complete protein structural elements.
- 3. The pharmaceutical composition of claim 1, wherein said two or more sequence segments are combined non-covalently.
- 4. The pharmaceutical composition of claim 1, wherein at least one of the parent amino acid sequences is from a protein.
- 5. The pharmaceutical composition of claim 3, wherein at least one of the parent amino acid sequences is from a protein selected from the group consisting of a naturally occurring protein, an engineered protein, a protein with a known binding activity, a protein with a known binding activity for an organic compound, a protein with a known binding activity for a peptide or polypeptide, a protein with a known binding activity for a carbohydrate, a protein with a known binding activity for a nucleic acid, a known binding activity for a hapten, a protein with a known binding activity for a steroid, a protein with a known binding activity for an inorganic compound, and a protein with an enzymatic activity.
- 6. The pharmaceutical composition of claim 1, wherein the parent amino acid sequences are from the open reading frames of a single genome, and:
(a) said reading frames are those used in nature; or (b) said reading frames are not those used in nature.
- 7. The pharmaceutical composition of claim 1, wherein the parent amino acid sequences are derived from the open reading frames of two or more genomes, and
(a) said reading frames are those used in nature; or (b) said reading frames are not those used in nature.
- 8. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain is resistant to in vivo or in vitro proteolysis by protease enzymes.
- 9. The pharmaceutical composition of claim 1, wherein the sequence segments are from parent domains with the same polypeptide fold in their structures.
- 10. The pharmaceutical composition of claim 1, wherein the sequence segments are from parent domains with different polypeptide folds in their structures.
- 11. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain has a free energy of folding greater than 1.6 kcal/mol.
- 12. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain has a free energy of folding greater than 3 kcal/mol.
- 13. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain has a free energy of folding greater than 5 kcal/mol.
- 14. The pharmaceutical composition of claim 1, wherein one or more of said sequence segments is fused to one or more additional and complete protein domains.
- 15. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain is fused to the coat protein of a filamentous bacteriophage, said bacteriophage encapsidating a nucleic acid encoding said protein domain.
- 16. The pharmaceutical composition of claim 1, wherein a single sequence segment is from a human protein.
- 17. The pharmaceutical composition of claim 1, wherein two or more sequence segments are from human proteins.
- 18. The pharmaceutical composition of claim 16 or 17, wherein at least one of said sequence segments is from a source other than a human protein.
- 19. The pharmaceutical composition of claim 16 or 17, wherein all sequence segments are from human proteins.
- 20. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain comprises a B cell epitope of at least one of the parent amino acid sequences.
- 21. The pharmaceutical composition of claim 20, wherein said chimeric, folded protein domain comprises a conformational B cell epitope of at least one of said parent amino acid sequences.
- 22. The pharmaceutical composition of claim 20, wherein said chimeric, folded protein domain comprises a conformational B cell epitope of at least one of said parent amino acid sequences and at least one T cell epitope.
- 23. The pharmaceutical composition of claim 22, wherein said conformational B cell epitope and at least one T cell epitope are derived from the same amino acid sequence.
- 24. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain cross reacts with antibodies raised against a parent amino acid sequence.
- 25. The pharmaceutical composition of claim 24, wherein said chimeric, folded protein domain cross reacts with antibodies raised against the folded parent protein of one of said sequence segments.
- 26. The pharmaceutical composition of claim 24, wherein said chimeric, folded protein domain cross reacts with antibodies specific for the folded parent protein of one of said sequence segments, but not with antibodies specific for the unfolded parent protein or fragments thereof.
- 27. The pharmaceutical composition of claim 1 wherein the amino acid sequences of said chimeric, folded protein domain are altered to increase stability or function of the chimeric protein.
- 28. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain comprises at least one reaction group for covalent linkage.
- 29. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain comprises at least one reaction group for non-covalent linkage.
- 30. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain comprises at least one D-amino acid.
- 31. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain comprises at lease one non-naturally occurring amino acid.
- 32. The pharmaceutical composition of claim 1, wherein said chimeric, folded protein domain comprises at least one amino acid having a label or a tag.
- 33. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a chimeric, folded protein domain selected from a repertoire of chimeric folded proteins, said protein domain comprising two or more sequence segments from parent amino acid sequences wherein each of said segments in said chimeric protein domain comprises a region of common sequence, and in which said region of common sequence does not consist solely of one or more complete structural elements.
- 34. The pharmaceutical composition of claim 33, wherein said region of common sequence is at least 10 identical contiguous amino acid residues in length.
- 35. The pharmaceutical composition of claim 34, wherein said region of common sequence is at least 20 identical contiguous amino acid residues in length.
- 36. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a chimeric, folded protein domain selected from a repertoire of chimeric folded proteins comprising two or more sequence segments, wherein each of said segments:
(a) is from parent proteins with a common fold; and (b) comprises a common region of the common fold and in which said common region of the common fold does not consist of one or more complete structural elements.
- 37. The chimeric, folded protein domain of claim 36 wherein each of said segments is from different proteins which are homologous in sequence.
- 38. The pharmaceutical composition of claim 36 wherein each of said segments is from the same protein.
- 39. The pharmaceutical composition of claim 36 in which the common region of the common fold is at least 10 contiguous amino acid residues in length.
- 40. The pharmaceutical composition of claim 36 in which the common region of the common fold is at least 20 contiguous amino acid residues in length.
- 41. The pharmaceutical composition of claim 36, in which the amino acid sequences of said parent proteins are from the open reading frames of a genome, wherein said reading frames are the natural reading frame of the genes encoded by said genome.
- 42. The pharmaceutical composition of claim 33 wherein said chimeric, folded protein domain is resistant to in vivo or in vitro proteolysis by protease enzymes.
- 43. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain has a free energy of folding greater than 1.6 kcal/mol.
- 44. The pharmaceutical composition of claim 33, wherein one or more of the sequence segments of said chimeric, folded protein is fused to one or more additional and complete protein domains.
- 45. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain fused to the coat protein on a filamentous bacteriophage, said bacteriophage encapsidating a nucleic acid encoding said protein domain.
- 46. The pharmaceutical composition of claim 33, wherein a single sequence segment of said chimeric, folded protein domain is from a human protein.
- 47. The pharmaceutical composition of claim 33, wherein two or more of the sequence segments of said chimeric, folded protein domain are from a human protein.
- 48. The pharmaceutical composition of claim 33, wherein at least one of the segments of said chimeric, folded protein domain is not from a human protein.
- 49. The pharmaceutical composition of claim 33, wherein all segments of said chimeric, folded protein domain are from human proteins.
- 50. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain comprises a B cell epitope of at least one of the parent amino acid sequences.
- 51. The pharmaceutical composition of claim 50, wherein said B cell epitope is a confirmational epitope of at least one of the parent amino acid sequences.
- 52. The pharmaceutical composition of claim 50, wherein said chimeric, folded protein domain comprises a conformational B cell epitope of at least one of the parent amino acid sequences and at least one T cell epitope.
- 53. The pharmaceutical composition of claim 50 wherein said chimeric, folded protein domain comprises a conformational B cell epitope of at least one of the parent amino acid sequence and at least one T cell epitope, and wherein said epitopes are derived from the same parent amino acid sequence.
- 54. The pharmaceutical composition of claim 33 wherein said chimeric, folded protein domain cross reacts with antibodies raised against a parent amino acid sequence.
- 55. The pharmaceutical composition of claim 33 wherein said chimeric, folded protein domain cross reacts with antibodies raised against the folded parent protein.
- 56. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain cross reacts with antibodies raised against the folded parent protein, but not with antibodies specific for the unfolded parent protein or fragments thereof.
- 57. The pharmaceutical composition of claim 33, wherein the amino acid sequences of said chimeric, folded protein domain are altered to increase stability or function of the chimeric protein.
- 58. A pharmaceutical composition of claim 32, wherein said chimeric, folded protein domain comprises at least one reaction group for covalent linkage.
- 59. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain comprises at least one reaction group for non-covalent linkage.
- 60. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain comprises at least one D-amino acid.
- 61. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain comprises at least one non-naturally-occurring amino acid.
- 62. The pharmaceutical composition of claim 33, wherein said chimeric, folded protein domain comprises at least one amino acid having a label or a tag.
- 63. A method for preparing a pharmaceutical composition of claim 1, comprising the steps of:
(a) providing a first library of nucleic acids, said library comprising coding sequences encoding sequence segments from one or more amino acid sequences; (b) providing a second library of nucleic acids, said library comprising coding sequences encoding sequence segments derived from one or more amino acid sequences; (c) combining the coding sequences to form a combinatorial library of nucleic acids, said nucleic acids comprising contiguous coding sequences encoding sequence fragments derived from the first and second libraries; (d) transcribing and/or translating the contiguous coding sequences to produce the encoded protein domains; (e) selecting a chimeric protein domain which adopts a folded structure; and (f) combining said chimeric, folded protein domain with a pharmaceutically acceptable carrier.
- 64. The method of claim 63, further comprising the steps of:
(i) analysing the sequence of the selected chimeric protein domains to identify the parent amino acid sequences of said sequence segments; and (ii) comparing the sequences of each of said parent amino acid sequences to determine whether said parent amino acid sequences are non-homologous.
- 65. A method for preparing a pharmaceutical composition of claim 1 wherein said chimeric, folded protein domain comprises two or more sequence segments derived from parent amino acid sequences that are non-homologous, wherein said two or more sequence segments do not form stable folds in isolation from said parent amino acid sequences, and wherein the sequence segments are from parent domains with different polypeptide folds in the structure, said method comprising the steps of claim 63 or 64 and the additional step of comparing the structures of each of said parent amino acid sequences to identify whether they have the same polypeptide folds.
- 66. The method of claim 63, wherein steps (b) and (c) are modified as follows:
(b) providing a partner coding sequence encoding a sequence segment derived from one protein; (c) combining the library of step (a) and the partner coding sequence of step (b) to form a combinatorial library of nucleic acids, said nucleic acids comprising contiguous coding sequences encoding sequence fragments from the first library, joined to said partner coding sequence.
- 67. The method of claim 63, wherein the domains which adopt a folded structure are selected by one or more methods selected from the group consisting of in vivo proteolysis, in vitro proteolysis, binding ability, functional activity and expression.
- 68. The method of claim 67, wherein said binding ability is to an antibody raised against a parent protein.
- 69. A method for preparing a pharmaceutical composition of claim 27, wherein said sequence segments of said parent amino acid sequences are altered subsequent to their juxtaposition in said chimeric protein domain, comprising a step selected from the group consisting of:
(a) preselecting and introducing specific or random mutations at predefined positions within the gene of the chimeric protein; (b) deleting nucleotides within the gene of the chimeric protein so as to delete amino acid residues; (c) inserting nucleotides within the gene of the chimeric protein so as to insert amino acid residues; (d) appending nucleotides to the gene of the chimeric protein so as to append amino acid residues; (e) randomly introducing mutations in all or part of the gene encoding the chimeric protein through recombinant DNA technology; (f) randomly introducing mutations in the gene of the chimeric protein through propagation in mutator cells; (g) introducing derivatives of natural amino acid during chemical synthesis; (h) chemically derivatizing amino acid groups after synthesis; (i) multimerizing the chimeric proteins through concatenation of two or more copies of the gene in a single open reading frame; (j) multimerizing the chimeric proteins through covalent linkage of two or more copies of the chimeric protein domain after translation; and (k) multimerizing the chimeric proteins through fusion to a multimeric partner.
- 70. A method for preparing a pharmaceutical composition of claim 33, comprising the steps of:
(a) providing a first library of nucleic acids, said library comprising coding sequences encoding sequence segments from one or more amino acid sequences; (b) providing a second library of nucleic acids, said library comprising coding sequences encoding sequence segments from one or more amino acid sequences; (c) combining the coding sequences to form a combinatorial library of nucleic acids, said nucleic acids comprising contiguous coding sequences encoding sequence fragments derived from the first and second libraries; (d) transcribing and/or translating the contiguous coding sequences to produce the encoded protein domains; and (e) selecting the chimeric protein domains which adopt a folded structure; (f) combining said chimeric, folded protein domain with a pharmaceutically acceptable carrier.
- 71. The method of claim 70, further comprising the steps of:
(i) analysing the sequence of the selected chimeric protein domains to identify the parent amino acid sequences of said sequence segments; and (ii) comparing said parent amino acid sequences to determine whether they comprise common sequences that do not consist solely of one or more complete structural elements.
- 72. The method for preparing a chimeric, folded protein domain of claim 36, comprising the steps of:
(a) providing a first library of nucleic acids, said library comprising coding sequences encoding sequence segments from one or more amino acid sequences; (b) providing a second library of nucleic acids, said library comprising coding sequences encoding sequence segments from one or more amino acid sequences; (c) combining the coding sequences to form a combinatorial library of nucleic acids, said nucleic acids comprising contiguous coding sequences encoding sequence fragments from the first and second libraries; (d) transcribing and/or translating the contiguous coding sequences to produce the encoded protein domains; (e) selecting the chimeric protein domains, which adopt a folded structure; (f) analysing the sequence of the selected chimeric protein domains to identify the parent amino acid sequences of said sequence segments; (g) comparing the structures of the parent amino acid sequences to determine whether the parent amino acid sequences have a common fold; and (h) identifying a selected chimeric protein domain wherein the segments comprise a common region of the common fold; (i) combining chimeric, folded protein domain of step (h) with a pharmaceutically acceptable carrier.
- 73. The method of claim 70, wherein step (b) and (c) are modified such that:
(b) providing a partner coding sequence encoding a sequence segment from one protein; (c) combining the library and partner coding sequence to form a combinatorial library of nucleic acids, said nucleic acids comprising contiguous coding sequences encoding sequence fragments from the first library and the partner coding sequence.
- 74. The method of claim 70 wherein the domains which adopt a folded structure are selected by one or more methods selected from the group consisting of in vivo proteolysis, in vitro proteolysis, binding ability, functional activity and expression.
- 75. The method of claim 74, wherein said binding ability is to an antibody raised against a parent protein
- 76. A method for preparing a pharmaceutical composition of claim 33, wherein the sequence segments of the parent amino acid sequences are altered subsequent to their juxtaposition using a step selected from the group consisting of:
(a) preselecting and introducing specific or random mutations at predefined positions within the gene of the chimeric protein; (b) deleting nucleotides within the gene of the chimeric protein so as to delete amino acid residues; (c) inserting nucleotides within the gene of the chimeric protein so as to insert amino acid residues; (d) appending nucleotides to the gene of the chimeric protein so as to append amino acid residues; (e) randomly introducing mutations in all or part of the gene encoding the chimeric protein through recombinant DNA technology; (f) randomly introducing mutations in the gene of the chimeric protein through propagation in mutator cells; (g) introducing derivatives of natural amino acid during chemical synthesis; (h) chemically derivatising amino acid groups after synthesis; (i) multimerising the chimeric proteins through concatenation of two or more copies of the gene in a single open reading frame; (j) multimerising the chimeric proteins through covalent linkage of two or more copies of the chimeric protein domain after translation; (k) multimerising the chimeric proteins through fusion to a multimeric partner.
- 77. A method of raising, in an individual, an immune response against one or more of the parent amino acid sequences from which the sequence segments of a chimeric, folded protein domain of a pharmaceutical composition of claim 1 are taken, the method comprising administering an effective amount of a pharmaceutical composition of claim 1 to said individual.
- 78. A method of raising, in an individual, an immune response against one or more of the parent amino acid sequences from which the sequence segments of a chimeric, folded protein domain of a pharmaceutical composition of claim 33 are taken, the method comprising administering an effective amount of a pharmaceutical composition of claim 33 to said individual.
- 79. A method of raising, in an individual, an immune response against one or more of the parent amino acid sequences from which the sequence segments of a chimeric, folded protein domain of a pharmaceutical composition of claim 36 are taken, the method comprising administering an effective amount of a pharmaceutical composition of claim 36 to said individual.
Priority Claims (3)
Number |
Date |
Country |
Kind |
0002492.7 |
Feb 2000 |
GB |
|
0019362.3 |
Aug 2000 |
GB |
|
0016346.9 |
Jul 2000 |
GB |
|
Parent Case Info
[0001] This application is a continuation-in-part of PCT/GB01/00445, filed Feb. 2, 2001, which claims the priority of U.K. Patent Application 0002492.7, filed Feb. 3, 2000, U.S. Provisional Application No. 60/180,326, filed Feb. 4, 2000, U.K. Patent Application 0016346.9, filed Jul. 3, 2000, and U.K Patent Application 0019362.3, filed Aug. 7, 2000. This application also claims the priority of U.S. Provisional Application No. 60/228,078, filed Aug. 25, 2000, and U.S. Utility application Ser. No. 09/938,945, filed Aug. 24, 2001.
Provisional Applications (1)
Continuation in Parts (2)
|
Number |
Date |
Country |
Parent |
09938945 |
Aug 2001 |
US |
Child |
10119556 |
Apr 2002 |
US |
Parent |
PCT/GB01/00445 |
Feb 2001 |
US |
Child |
10119556 |
Apr 2002 |
US |