CA2421447A1 - Collections of repeat proteins comprising repeat modules - Google Patents

Collections of repeat proteins comprising repeat modules Download PDF

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CA2421447A1
CA2421447A1 CA002421447A CA2421447A CA2421447A1 CA 2421447 A1 CA2421447 A1 CA 2421447A1 CA 002421447 A CA002421447 A CA 002421447A CA 2421447 A CA2421447 A CA 2421447A CA 2421447 A1 CA2421447 A1 CA 2421447A1
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collection
repeat
amino acid
nucleic acid
modules
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CA2421447C (en
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Michael Tobias Stumpp
Patrick Forrer
Hans Kaspar Binz
Andreas Pluckthun
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Universitaet Zuerich
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4703—Inhibitors; Suppressors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups

Abstract

The present invention relates to collections of repeat proteins comprising repeat modules which are derived from one or more repeat units of a family o f naturally occurring repeat proteins, to collections of nucleic acid molecule s encoding said repeat proteins, to methods for the construction and applicati on of such collections and to individual members of such collections.

Claims (48)

1. A collection of nucleic acid molecules encoding a collection of repeat proteins, each repeat protein comprising a repeat domain, which comprises a set of consecutive repeat modules, wherein each of said repeat modules is derived from one or more repeat units of one family of naturally occurring repeat proteins, wherein said repeat units comprise framework residues and target interaction residues, wherein said repeat proteins differ in at least one position.
2. The collection of claim 1, wherein each of said repeat modules has an amino acid sequence, wherein at least 70% of the amino acid residues correspond either (i) to consensus amino acid residues deduced from the amino acid residues found at the corresponding positions of at least two naturally occurring repeat units; or (ii) to the amino acid residues found at the corresponding positions in a naturally occurring repeat unit.
3. The collection of claim 1 or 2, wherein said set consists of between two and about 30 repeat modules.
4. The collection of any one of claims 1 to 3, wherein said repeat modules are directly connected.
5. The collection of any one of claims 1 to 3, wherein said repeat modules are connected by a (poly)peptide linker.
6. The collection of any one of claims 1 to 5, wherein said repeat domain further comprises an N- and/or a C-terminal capping module having an amino acid sequence different from any one of said repeat modules.
7. The collection of any one of claims 1 to 6, wherein said repeat units are ankyrin repeats.
8. The collection of claim 7, wherein each of said repeat modules comprises the ankyrin repeat sequence motif . DxxGxTPLHLAaxx~GpxpaVpxLLpxGA~pVNAx, wherein "x" denotes any amino acid, "~" denotes any amino acid or a deletion, "a" denotes an amino acid with an apolar side chain, and "p" denotes a residue with a polar sidechain.
9. The collection of claim 7, wherein each of said repeat modules comprises the ankyrin repeat sequence motif DxxGxTPLHLAxxxGxxxVVxLLLxxGADVNAx, wherein "x" denotes any amino acid.
10. The diverse collection of claim 7, wherein each of said repeat modules comprises the ankyrin repeat sequence motif DxxGxTPLHLAxxxGxxxIVxVLLxxGADVNAx, wherein "x" denotes any amino acid.
11. The collection of any one of claims 8 to 10, wherein one or more of the positions denoted "x" are randomised.
12. The diverse collection of claim 7, wherein each of said repeat modules comprises the ankyrin repeat sequence motif D11G1TPLHLAA11GHLEIVEVLLK2GADVNA1, wherein 1 represents an amino acid residue selected from the group:
A, D, E, F, H, I, K, L, M, N, Q, R, S, T, V, W and Y;
wherein 2 represents an amino acid residue selected from the group:
H, N and Y.
13. The collection of any one of claims 1 to 6, wherein said repeat units are leucine-rich repeats (LRR).
14. The collection of claim 13, wherein each of said modules comprises the LRR
sequence motif xLxxLxLxxN~xaxx~a~a~a~x~, wherein "x" denotes any amino acid, "a" denotes an aliphatic amino acid, and "~" denotes any amino acid or a deletion.
15. The collection of claim 13, wherein at least one of said modules comprises the LRR sequence motif xLExLxLxxCxLTxxxCxxLxxaLxxxx, wherein "x" denotes any amino acid, and "a" denotes an aliphatic amino acid (A-type LRR).
16. The collection of claim 13, wherein at least one of said modules comprises the LRR sequence motif xLxELxLxxNxLGDxGaxxLxxxLxxPxx, wherein "x" denotes any amino acid, and "a" denotes an aliphatic amino acid (B-type LRR).
17. The collection of any one of claims 14 to 16, wherein one or more of the positions denoted "x" and/or "~" are randomised.
18. The collection of claim 15, wherein the cysteine residue at position 10 in the A-type LRR consensus sequence is replaced by a hydrophilic amino acid residue, and wherein the cystein residue at position 17 is replaced by a hydrophobic amino acid residue.
19. The collection of any one of claims 8 to 12 or 14 to 18, wherein one or more of the amino acid residues in said consensus sequences are exchanged by an amino acid residue found at the corresponding position in a corresponding naturally occurring repeat unit.
20. The collection of any one of claims 1 to 19, wherein said set consists of one type of repeat modules.
21. The collection of any one of claims 1 to 19, wherein said set consists of two different types of repeat modules.
22. The collection of claim 20, wherein said set comprises two different types of consecutive repeat modules as pairs in said repeat domain.
23. The collection of claim 21 or 22, wherein said two different types of modules are based on said A-type LRR and B-type LRR.
24. The collection of any one of claims 20 to 23, wherein the amino acid sequences of the repeat modules comprised in said set are identical for each said type except for the randomised residues.
25. The collection of claim 24, wherein the nucleic acid sequences encoding the copies of each said type are identical except for the codons encoding amino acid residues at positions being randomised.
26. The collection of any one of claims 1 to 25, wherein said nucleic acid molecules comprise identical nucleic acid sequences of at feast 9 nucleotides between said repeat modules.
27. The collection of claims 22 or 23, wherein said nucleic acid molecules comprise identical nucleic acid sequences of at least 9 nucleotides between said pairs.
28. The collection of any one of claims 1 to 26, wherein each of the nucleic acid sequences between said modules, or said pairs, comprises a restriction enzyme recognition sequence.
29. The collection of any one of claims 1 to 27, wherein each of the nucleic acid sequences between said modules, or said pairs, comprises a nucleic acid sequence formed from cohesive ends created by two compatible restriction enzymes.
30. The collection of claims 26 or 27, wherein said identical nucleic acid sequences allow a PCR-based assembly of said nucleic acid molecules.
31. The collection of claim 24, wherein said repeat domain comprises one or more pairs of modules based on said A-type LRR and B-type LRR, wherein each of said pairs has the sequence LDPGT, wherein 1 represents an amino acid residue selected from the group:
D, E, N, Q, S, R, K, W and Y;
wherein 2 represents an amino acid residue selected from the group:
N, S and T;
wherein 3 represents an amino acid residue selected from the group:
G, S, D, N, H and T; and wherein 4 represents an amino acid residue selected from the group:
L, V and M.
32. The collection of claim 31, wherein each of said pairs of modules is encoded by the nucleic acid molecule GAT GCA GGC GTG CGG CTG CTC TTG CAG GGG CTG CTG GAC
CCC GGC ACG

wherein 111 represents a codon encoding an amino acid residue selected from the group:
D, E, N, Q, S, R, K, W and Y;
wherein 222 represents a codon encoding an amino acid residue selected from the group:
N, S and T;

wherein 333 represents a codon encoding an amino acid residue selected from the group:
G, S, D, N, H and T; and wherein 444 represents a codon encoding an amino acid residue selected from the group:
L, V and M.
33. The collection of claim 31, wherein one or more of the amino acid residues in at least one of said pair of modules are exchanged by an amino acid residue found at the corresponding position in a naturally occurring LRR.
34. The collection of claim 32, wherein one or more of the amino acid codons in at least one of said pairs of modules are exchanged by a codon encoding an amino acid residue found at the corresponding position in a naturally occurring LRR.
35. A collection of recombinant nucleic acid molecules comprising a collection of nucleic acid molecules according to any one of claims 1 to claim 34.
36. A collection of vectors comprising a collection of nucleic acid molecules according to any one of claims 1 to claim 34, or a collection of recombinant nucleic acid molecules according to claim 35.
37. A collection of host cells comprising a collection of nucleic acid molecules according to any one of claims 1 to 34, a collection of recombinant nucleic acid molecules according to claim 35, or a collection of vectors according to claim 36.
38. A collection of repeat proteins encoded by a collection of nucleic acid molecules according to any one of claims 1 to 34, by a collection of recombinant nucleic acid molecules according to claim 35, by a collection of vectors according to claim 36, or produced by a collection of host cells according to claim 37.
39. A method for the construction of a collection of nucleic acid molecules according to any one of claims 1 to 34, comprising the steps of (a) identifying a repeat unit from a repeat protein family;
(b) identifying framework residues and target interaction residues in said repeat unit;
(c) deducing at least one type of repeat module comprising framework residues and randomised target interaction residues from at least one member of said repeat protein family; and (d) constructing nucleic acid molecules each encoding a repeat protein comprising two or more copies of said at least one type of repeat module deduced in step (c).
40. The method of claim 38, wherein said at least one repeat module deduced in step (c) has an amino acid sequence, wherein at least 70% of the amino acid residues correspond either (i) to consensus amino acid residues deduced from the amino acid residues found at the corresponding positions of at least two naturally occurring repeat units; or (ii) to the amino acid residues found at the corresponding positions in a naturally occurring repeat unit.
41. A method for the production of a collection of repeat proteins according to claim 38, comprising the steps of (a) providing a collection of host cells according to claim 37; and (b) expressing the collection of nucleic acid molecules comprised in said host cells.
42. A method for obtaining a repeat protein having a predetermined property, comprising the steps of (a) providing a collection of repeat proteins according to claim 38 or 39 or produced according to claim 41; and (b) screening said collection and/or selecting from said collection to obtain at least one repeat protein having said predetermined property.
43. The method of claim 42, wherein said predetermined property is binding to a target.
44. A repeat protein from a collection according to any one of claims 24 to 34.
45. A nucleic acid molecule encoding the repeat protein of claim 44.
46. A vector containing the nucleic acid molecule of claim 45.
47. A pharmaceutical composition comprising the repeat protein of claim 44 or the nucleic acid molecule of claim 45, and optionally a pharmaceutically acceptable carrier and/or diluent.
48. A nucleic acid molecule encoding a pair of repeat modules for the construction of a collection according to claims 31 or 32, wherein said nucleic acid molecule is:

GAT GCA GGC GTG CGG CTG CTC TTG CAG GGG CTG CTG GAC
CCC GGC ACG, wherein 111 represents a colon encoding an amino acid residue selected from the group:
D, E, N, Q, S, R, K, W and Y;
wherein 222 represents a colon encoding an amino acid residue selected from the group:
N, S and T;
wherein 333 represents a colon encoding an amino acid residue selected from the group:
G, S, D, N, H and T; and wherein 444 represents a colon encoding an amino acid residue selected from the group:
L, V and M.
CA2421447A 2000-09-08 2001-09-10 Collections of repeat proteins comprising repeat modules Expired - Lifetime CA2421447C (en)

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EP00119670 2000-09-08
EP00119670.8 2000-09-08
PCT/EP2001/010454 WO2002020565A2 (en) 2000-09-08 2001-09-10 Collections of repeat proteins comprising repeat modules

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EP (2) EP1332209B1 (en)
JP (3) JP5291279B2 (en)
AT (1) ATE448301T1 (en)
AU (1) AU2002218166A1 (en)
CA (1) CA2421447C (en)
DE (1) DE60140474D1 (en)
DK (1) DK1332209T3 (en)
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