WO2020169840A1 - Protéines bispécifiques ayant un échafaudage chimérique - Google Patents

Protéines bispécifiques ayant un échafaudage chimérique Download PDF

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Publication number
WO2020169840A1
WO2020169840A1 PCT/EP2020/054700 EP2020054700W WO2020169840A1 WO 2020169840 A1 WO2020169840 A1 WO 2020169840A1 EP 2020054700 W EP2020054700 W EP 2020054700W WO 2020169840 A1 WO2020169840 A1 WO 2020169840A1
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WIPO (PCT)
Prior art keywords
scaffold
seq
residues
loop
chimeric protein
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PCT/EP2020/054700
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English (en)
Inventor
Laura ITZHAKI
Pam ROWLING
Graham LADDS
Alberto PEREZ RIBA
Christine Martin
Beatriz GOYENECHEA CORZO
Joseph MABBITT
Marco BARDELLI
Simon GILBERT
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Cambridge Enterprise Limited
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Priority claimed from GBGB1902370.4A external-priority patent/GB201902370D0/en
Priority claimed from GBGB1902396.9A external-priority patent/GB201902396D0/en
Priority claimed from GBGB1902398.5A external-priority patent/GB201902398D0/en
Priority claimed from GBGB1902393.6A external-priority patent/GB201902393D0/en
Priority claimed from GBGB1902375.3A external-priority patent/GB201902375D0/en
Priority claimed from GBGB1902384.5A external-priority patent/GB201902384D0/en
Priority claimed from GBGB1902378.7A external-priority patent/GB201902378D0/en
Priority claimed from GBGB1902394.4A external-priority patent/GB201902394D0/en
Priority claimed from GBGB1902397.7A external-priority patent/GB201902397D0/en
Priority claimed from GBGB1902380.3A external-priority patent/GB201902380D0/en
Priority claimed from GBGB1902391.0A external-priority patent/GB201902391D0/en
Priority claimed from GBGB1902401.7A external-priority patent/GB201902401D0/en
Priority claimed from GBGB1902402.5A external-priority patent/GB201902402D0/en
Priority claimed from GBGB1902403.3A external-priority patent/GB201902403D0/en
Application filed by Cambridge Enterprise Limited filed Critical Cambridge Enterprise Limited
Publication of WO2020169840A1 publication Critical patent/WO2020169840A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins

Definitions

  • This invention relates to bispecific proteins and their production and uses.
  • PPIs protein-protein interactions
  • chimeric proteins capable of binding to two or more target molecules can be generated by displaying peptidyl binding motifs, such as short linear motifs (SLiMs), on a peptidyl scaffold.
  • a peptide ligand comprises a peptidyl binding motif.
  • Peptidyl binding motifs are grafted to peptidyl scaffolds by insertion or substitution of amino acid residues into the scaffold.
  • These chimeric proteins may be useful for example, as single- or multi-function protein therapeutics.
  • bispecific or hetero-bifunctional chimeric proteins may be useful in promoting the degradation of target molecules via cellular protein degradation pathways.
  • the peptidyl scaffold may be a (i) CKS scaffold, (ii) coiled-coil scaffold, (iii) Affibody scaffold, (iv) trefoil scaffold, (v) PDZ domain scaffold, (vi) ubiquitin or ubiquitin-like domain scaffold, (vii) GB1 scaffold, (viii) VWV scaffold (ix) Fibritin scaffold (x) aPP scaffold, (xi) fibronectin scaffold (xii) Zn finger scaffold, (xiii) SH3 scaffold or (xiv) Cystine knot (CK) scaffold.
  • a first aspect of the invention is directed to chimeric proteins comprising a CKS (Cycl in dependent kinases regulatory subunit) scaffold.
  • a first set of embodiments of the first aspect of the invention provide a chimeric protein comprising;
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the first aspect of the invention provides a method of producing a chimeric protein comprising;
  • a third set of embodiments of the first aspect provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second and third loops and the helical region of the CKS scaffold of the chimeric protein; and
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fourth set of embodiments of the first aspect provides a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the first aspect provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub libraries comprising;
  • peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second and third loops and the helical region of the CKS scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second and third loops and the helical region of the CKS scaffold.
  • a sixth set of embodiments of the first aspect provides a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the first aspect provides a method of screening a library comprising;
  • a second aspect of the invention is directed to chimeric proteins comprising coiled-coil scaffolds.
  • a first set of embodiments of the second aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at one or more helices or loops of the coiled-coil scaffold.
  • a second set of embodiments of the second aspect of the invention provide a chimeric protein comprising
  • said peptide ligands being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of any one of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the second aspect of the invention provide a method of producing a chimeric protein comprising;
  • a fourth set of embodiments of the second aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said coiled-coil scaffold and said first and second peptide ligands,
  • peptide ligands are located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NO: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold; and expressing the nucleic acid to produce said protein.
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fifth set of embodiments of the second aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein.
  • a seventh set of embodiments of the second aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • each said peptide ligand being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,
  • An eighth set of embodiments of the second aspect of the invention provide a method of screening a library comprising;
  • each said peptide ligand being located at first helix, second helix and first loop (for example at a position between residues 55 to 57 or between residues 35 to 46 or between residues 66 to 77 of SEQ ID NOs: 8 or 10 or 12 or 13 or 14) of the coiled-coil scaffold of the chimeric protein,
  • a third aspect of the invention is directed to chimeric proteins comprising Affibody scaffolds.
  • a first set of embodiments of the third aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at one or more helices and loops of the Affibody scaffold.
  • a second set of embodiments of the third aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the third aspect of the invention provide a method of producing a chimeric protein comprising;
  • a fourth set of embodiments of the third aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fifth set of embodiments of the third aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a sixth set of embodiments of the third aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in two or more of the first, second, and third helices and the first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the of the Affibody scaffold of the chimeric protein, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in two or more of the first, second and third helices and the first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein.
  • a seventh set of embodiments of the third aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • each said peptide ligand being located in two or more of the first, second, and third helices and first and second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 53) of the Affibody scaffold of the chimeric protein,
  • An eighth set of embodiments of the third aspect of the invention provide a method of screening a library comprising;
  • each said peptide ligand being located in two or more of the first, second, third helices and first, second loops (for example, at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of any one of SEQ ID NOs: 16, 18 and 20 to 5) of the Affibody scaffold of the chimeric protein,
  • a fourth aspect of the invention is directed to chimeric proteins comprising trefoil scaffolds.
  • a first set of embodiments of the fourth aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at loops or b strands of the Trefoil scaffold.
  • a second set of embodiments of the fourth aspect of the invention provide a chimeric protein comprising; (i) a Trefoil scaffold, and
  • said peptide ligands being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the fourth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a fourth set of embodiments of the fourth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fifth set of embodiments of the fourth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a sixth set of embodiments of the fourth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues
  • a seventh set of embodiments of the fourth aspect of the invention provide a method of producing a library of chimeric proteins comprising; (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
  • each said peptide ligand being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein,
  • An eighth set of embodiments of the fourth aspect of the invention provide a method of screening a library comprising;
  • each said peptide ligand being located at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of any one of SEQ ID NOs: 54 , 56 and 58 to 78) of the Trefoil scaffold of the chimeric protein,
  • a fifth aspect of the invention is directed to chimeric proteins comprising PDZ domain scaffolds.
  • a first set of embodiments of the fifth aspect of the invention provide a chimeric protein comprising;
  • a second set of embodiments of the fifth aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of SEQ ID NO: 79) of the PDZ scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the fifth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a fourth set of embodiments of the fifth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said PDZ scaffold and said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at position between residues 20 to 24 or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the SEQ ID NO: 79) of the PDZ scaffold; and
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fifth set of embodiments of the fifth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a sixth set of embodiments of the fifth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at wo or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein.
  • a seventh set of embodiments of the fifth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • each said peptide ligand being located at two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein
  • An eighth set of embodiments of the fifth aspect of the invention provide a method of screening a library comprising;
  • each said peptide ligand being located at two or more of the first, second, third, fourth loops and a first helix of the PDZ scaffold of the chimeric protein
  • a sixth aspect of the invention is directed to chimeric proteins comprising ubiquitin or ubiquitin-like domain scaffolds
  • a first set of embodiments of the sixth aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at one or more helices and or loops of the Ubiquitin scaffold.
  • a second set of embodiments of the sixth aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; and between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305 and in structurally similar positions in SEQ ID NO: 293, 295, 297 and 299) of the Ubiquitin scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the sixth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a fourth set of embodiments of the sixth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid encoding an Ubiquitin scaffold
  • a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said Ubiquitin scaffold and said first and second peptide ligands, wherein said peptide ligands are located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold; and
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fifth set of embodiments of the sixth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a sixth set of embodiments of the sixth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • a Ubiquitin scaffold (i) a Ubiquitin scaffold; and (ii) a peptide ligand comprising at least one diverse amino acid residue, wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein.
  • a seventh set of embodiments of the sixth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • each said peptide ligand being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, wherein at least one peptide ligand is diverse in said population, and
  • An eighth set of embodiments of the sixth aspect of the invention provide a method of screening a library comprising;
  • each said peptide ligand being located at first, second, third loops and a first helix (for example at positions between residues 8 to 9; between residues 23 to 33; between residues 53 to 54; between residues 62 to 64 of SEQ ID NOs: 291 , 303 and 305) of the Ubiquitin scaffold of the chimeric protein, wherein at least one amino acid residue in at least one peptide ligand in the Ubiquitin scaffold of the chimeric proteins in said library is diverse,
  • a seventh aspect of the invention is directed to chimeric proteins comprising GB1 scaffolds.
  • a first set of embodiments of the seventh aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at loops or a- helices of the GB1 scaffold
  • a second set of embodiments of the seventh aspect of the invention provide a chimeric protein comprising;
  • said peptide ligands being located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the seventh aspect of the invention provide a method of producing a chimeric protein comprising;
  • a fourth set of embodiments of the seventh aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid encoding a GB1 scaffold
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said GB1 scaffold and said first and second peptide ligands, wherein said peptide ligands are located between residues 22 and 35 (preferably replacing residues 23, 24, 26, 27, 28 and 34-35 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) and between residues 46 and 49 of the GB1 scaffold; and expressing the nucleic acid to produce said protein.
  • a fifth set of embodiments of the seventh aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid encoding a GB1 scaffold
  • a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said GB1 scaffold and said first and second peptide ligands, wherein said peptide ligands are located at two or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold; and
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a sixth set of embodiments of the seventh aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a seventh set of embodiments of the seventh aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • a GB1 scaffold (i) a GB1 scaffold; and (ii) a peptide ligand comprising at least one diverse amino acid residue, wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein.
  • An eighth set of embodiments of the seventh aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348) of the GB1 scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located between at one or more of the first, second, third, fourth loops and a first helix (for example, at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID Nos: 307, 309, 311 and 313 to 348) of the GB1 scaffold of the chimeric protein.
  • a ninth set of embodiments of the seventh aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • each said peptide ligand being located at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348 of the GB1 scaffold of the chimeric protein, wherein at least one peptide ligand is diverse in said population, and (b) expressing said population of nucleic acids to produce the diverse population,
  • a tenth set of embodiments of the seventh aspect of the invention provide a method of screening a library comprising;
  • each said peptide ligand being located at positions between residues 22 to 35 or between residues 9 and 10 or between residues 46 and 49 of any one of SEQ ID NOs: 307, 309, 311 and 313 to 348 of the GB1 scaffold of the chimeric protein,
  • An eighth aspect of the invention is directed to chimeric proteins comprising VWV scaffolds.
  • a first set of embodiments of the eighth aspect of the invention provide a chimeric protein comprising;
  • the first loop may be at a position corresponding to residues 12 to 15 of SEQ ID NO: 349 or SEQ ID NO: 357, residues 18 to 20 of SEQ ID NO: 351 , residues 24 to 26 of SEQ ID NO: 353, residues 14 to 16 of SEQ ID NO: 355, and residues 51 to 53 of SEQ ID NO: 359.
  • the second loop may be at a position corresponding to residues 23 to 25 of SEQ ID NO:
  • the VWV scaffold may further comprise a helical region.
  • Peptide ligands may be located in the helical region of the VWV scaffold.
  • the helical region may be at a position corresponding to residues 14 to 32 of SEQ ID NOs: 412, 414 or 415 or residues
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the eighth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a third set of embodiments of the eighth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in (a) the first and second loops, (b) the first loop and the helical region or (c) the second loop and the helical region of the VWV scaffold of the chimeric protein; and
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • each chimeric protein in the library comprising;
  • a fifth set of embodiments of the eighth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the first loop (b) the second loop or (c) the helical region of the V V scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (a) the first loop (b) the second loop or (c) the helical region of the VWV scaffold.
  • a sixth set of embodiments of the eighth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the eighth aspect of the invention provide a method of screening a library comprising;
  • a ninth aspect of the invention is directed to chimeric proteins comprising Fibritin scaffolds.
  • a first set of embodiments of the ninth aspect of the invention provide a chimeric protein comprising;
  • the coiled-coil subdomain may be at a position corresponding to residues 1 to 38 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 1 to 15 of SEQ ID NO 365; and the disordered region may be at a position corresponding to residues 39 to 50 of SEQ ID NO: 363 or SEQ ID NO: 367 or residues 16 to 27 of SEQ ID NO: 365.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the ninth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a third set of embodiments of the ninth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in the coiled-coil domain and the disordered region of the Fibritin scaffold of the chimeric protein; and
  • one of the first or second target molecules is a member of a cellular degradation pathway, such as an E3 ubiquitin ligase.
  • a fourth set of embodiments of the ninth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the ninth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the disordered region and (b) the coiled-coil subdomain, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in the other of (a) the disordered region and (b) the coiled-coil subdomain of the Fibritin scaffold.
  • a sixth set of embodiments of the ninth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the ninth aspect of the invention provide a method of screening a library comprising;
  • a tenth aspect of the invention is directed to chimeric proteins comprising aPP scaffolds.
  • a first set of embodiments of the tenth aspect of the invention provide a chimeric protein comprising;
  • the loop may at a position in the aPP scaffold corresponding to residues 9 to 13 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415; and the helical region may at a position in the aPP scaffold corresponding to residues 14 to 32 of SEQ ID NO: 412, SEQ ID NO: 414 or SEQ ID NO: 415.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the tenth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a third set of embodiments of the tenth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands,
  • peptide ligands are located in (a) the loop and (b) the helical region of the aPP scaffold of the chimeric protein;
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fourth set of embodiments of the tenth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the tenth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (a) the loop and (b) the helical region of the aPP scaffold of the aPP scaffold.
  • a sixth set of embodiments of the tenth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the tenth aspect of the invention provide a method of screening a library comprising;
  • An eleventh aspect of the invention is directed to chimeric proteins comprising fibronectin scaffolds.
  • a first set of embodiments of the eleventh aspect of the invention provide a chimeric protein comprising;
  • the first loop may be at a position in the FN3 scaffold corresponding to residues 14 to 15 of SEQ ID NO: 418 or SEQ ID NO: 420; the second loop may be at a position in the FN3 scaffold corresponding to residues 25 to 26 of SEQ ID NO: 418 or SEQ ID NO: 420; the third loop may be at a position in the FN3 scaffold corresponding to residues 43 to 44 of SEQ ID NO: 418 or SEQ ID NO: 420; and the fourth loop may be at a position in the FN3 scaffold corresponding to residues 81 to 82 of SEQ ID NO: 418 or SEQ ID NO: 420.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the eleventh aspect of the invention provide a method of producing a chimeric protein comprising;
  • a third set of embodiments of the eleventh aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the fibronectin scaffold of the chimeric protein; and
  • one of the first or second target molecules is a member of a cellular degradation pathway, preferably a E3 ubiquitin ligase.
  • a fourth set of embodiments of the eleventh aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the eleventh aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the fibronectin scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the fibronectin scaffold.
  • a sixth set of embodiments of the eleventh aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the eleventh aspect of the invention provide a method of screening a library comprising;
  • a twelfth aspect of the invention is directed to chimeric proteins comprising Zn finger scaffolds.
  • a first set of embodiments of the twelfth aspect of the invention provide a chimeric protein comprising;
  • the first loop may be at a position in the Zn finger scaffold corresponding to residues 6 to 9 of SEQ ID NO: 423; the second loop may be at a position in the Zn finger scaffold corresponding to residues 11 to 12 of SEQ ID NO: 423 and the helical region may be at a position in the Zn finger scaffold corresponding to 17 to 28 of SEQ ID NO: 423.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the twelfth aspect of the invention provide a method of producing a chimeric protein comprising;
  • a third set of embodiments of the twelfth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein the first peptide ligand is located in one of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein and the second peptide ligand is located in another of the first loop, second loop and helical region of the Zn finger scaffold of the chimeric protein; and expressing the nucleic acid to produce said protein.
  • one of the first or second target molecules is a member of a cellular degradation pathway, such as an E3 ubiquitin ligase.
  • a fourth set of embodiments of the twelfth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the twelfth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first loop, second loop and helical region of the Zn finger scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first loop, second loop and helical region of the Zn finger scaffold.
  • a sixth set of embodiments of the twelfth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the twelfth aspect of the invention provide a method of screening a library comprising;
  • a thirteenth aspect of the invention is directed to chimeric proteins comprising SH3 scaffolds.
  • a first set of embodiments of the thirteenth aspect of the invention provide a chimeric protein comprising;
  • the first loop may be at a position in the SH3 scaffold corresponding to residues 9 to 24 of SEQ ID NO: 653; residues 24 to 39 of SEQ ID NO: 655; or 13 to 21 of SEQ ID NO: 657; the second loop may be at a position in the SH3 scaffold corresponding to residues 31 to 35 of SEQ ID NO: 653; residues 45 to 56 of SEQ ID NO: 655; or residues 32 to 41 of SEQ ID NO: 657; the third loop may be at a position in the SH3 scaffold corresponding to residues 44 to 46 of SEQ ID NO: 653 residues 62 to 63 of SEQ ID NO: 655; or residues 53 to 62 of SEQ ID NO: 657; and the fourth loop may be at a position in the SH3 scaffold corresponding to residues 55 to 56 of SEQ ID NO: 653; residues 69 to 71 of SEQ ID NO: 655; or residues 68 to 70 of SEQ ID NO: 657.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a third set of embodiments of the thirteenth aspect of the invention provide a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third and fourth loops of the SH3 scaffold of the chimeric protein; and
  • one of the first or second target molecules is a member of a cellular degradation pathway, such as an E3 ubiquitin ligase.
  • a fourth set of embodiments of the thirteenth aspect of the invention provide a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the thirteenth aspect of the invention provide a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third and fourth loops of the SH3 scaffold
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third and fourth loops of the SH3 scaffold.
  • a sixth set of embodiments of the thirteenth aspect of the invention provide a method of producing a library of chimeric proteins comprising;
  • a seventh set of embodiments of the thirteenth aspect of the invention provide a method of screening a library comprising;
  • chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, said chimeric proteins comprising different combinations of said first and second peptide ligands,
  • a fourteenth aspect of the invention is directed to chimeric proteins comprising a cystine knot (CK) scaffold (also called a cyclotide scaffold).
  • CK cystine knot
  • a first set of embodiments of the fourteenth aspect of the invention provide a chimeric protein comprising;
  • the first loop may be at a position in the CK scaffold corresponding to residues 2 to 4 of SEQ ID NO: 840 or SEQ ID NO: 842, residues 2 to 7 of SEQ ID NO: 844; residues 3 to 8 of SEQ ID NO: 846 and residues 3 to 4 of SEQ ID NO: 848;
  • the second loop may be at a position in the CK scaffold corresponding to residues 6 to 9 of SEQ ID NO: 840 or SEQ ID NO: 3, residues 9 to 13 of SEQ ID NO: 844; residues 10 to 14 of SEQ ID NO: 846 and residues 6 to 15 of SEQ ID NO: 848;
  • the third loop may be at a position in the CK scaffold corresponding to residues 11 to 14 of SEQ ID NO: 840; residues 11 to 16 of SEQ ID NO: 3, residues 15 to 17 of SEQ ID NO: 844; residues 16 to 18 of SEQ ID NO: 846 and residues 17 to 19 of SEQ ID NO: 848;
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule.
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • a second set of embodiments of the fourteenth aspect of the invention provides a method of producing a chimeric protein comprising;
  • a third set of embodiments of the fourteenth aspect provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising;
  • nucleic acid incorporating into said nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising said first and second peptide ligands, wherein said peptide ligands are located in two of the first, second, third, fourth, fifth and sixth loops of the cystine knot scaffold of the chimeric protein; and
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • a fourth set of embodiments of the fourteenth aspect provides a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • a fifth set of embodiments of the fourteenth aspect provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub libraries comprising;
  • peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of the first, second, third, fourth, fifth and sixth loops of the CKS scaffold, and
  • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of the first, second, third, fourth, fifth and sixth loops of the CKS scaffold.
  • a sixth set of embodiments of the fourteenth aspect provides a method of producing a library of chimeric proteins comprising; (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
  • a seventh set of embodiments of the fourteenth aspect provides a method of screening a library comprising;
  • Figure 1 shows a schematic representation of a library of degradation-inducing chimeric proteins.
  • the library shown is for use in targeting b-catenin for degradation.
  • These proteins comprise a scaffold (grey rectangles) onto which are grafted: (1) a target-binding peptide ligand and (2) a binding peptide for an E3 ubiquitin ligase or a component of another degradation pathway.
  • Each of the target-binding peptides is derived from a different protein that interacts with b-catenin (see Table 1).
  • Each of the degradation pathway-binding peptides (referred to as“degrons”) is derived from a substrate or binding partner of one of many different E3s or from a binding partner for one of a component of another cellular degradation pathway (including chaperone-mediated autophagy, selective autophagy and ESCRT (endosome-lysosome) pathways);‘etc.’ denotes the fact that there are many such proteins that can be harnessed for degradation, as detailed further in Table 2.
  • the schematic illustrates the combinatorial“plug-and-play” nature of these libraries, in terms of the ability to slot in any target-recruiting peptide ligand and degradation-pathway-recruiting peptide ligand.
  • the other factor that can be varied in the library arises from the fact that the two peptides can also be grafted onto different positions in the scaffold so as to present the target in different configurations with respect to the E3 or other degradation machinery.
  • the library Once the library is constructed, it can then be screened in cell-based assays in order to identify the best combination of two peptide ligands and their positions within the scaffold that induces the greatest reduction in target protein levels.
  • the same panel of diverse degradation pathway components can be used for screen for degradation of any target.
  • Figure 2 shows a schematic of the structure of the CKS1 scaffold (2A) and modelled structures of bifunctional CKS1 molecules (2B and C).
  • Figure 2A shows a model of the CKS1 scaffold with sites for peptide grafting and the natural binding site for Skp2 (the substrate-recognition subunit of the E3 ubiquitin ligase SCF Skp2 ) highlighted and annotated.
  • Figure 2B shows a model of a bifunctional CKS1 molecule with a peptide targeting B- catenin-/ KRAS grafted onto a loop (site 1) and the natural binding site for Skp2 indicated.
  • Figure 2C shows a model of a bifunctional CKS1 molecule with a peptide targeting B-catenin / KRAS grafted onto a loop (site 2) and the natural Skp2-binding site indicated. Highlighted in black are the loops or helices targeting B-catenin / KRAS and the Skp2-binding site. The distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 1 DKS as template. Images were generated using PyMol.
  • Figure 3 shows the modelled structure of the complex of Skp2 (the substrate-recognition subunit of the E3 ubiquitin ligase SCF Skp2 ) and the Cks1 scaffold with a grafted loop.
  • Figure 4 shows a helical wheel model of SEQ ID NO: 1 residues 40 to 45 where X represents substitutions to the helix on the solvent accessible face to create a new ligand binding surface.
  • FIG. 5A shows b-catenin degradation using bispecific CKS1 constructs as measured using a HiBiT lytic assay (UNT: Untreated, Scr: Scrambled siRNA, LIPO: Lipofectamine only siRNA: b-catenin-targeted siRNA).
  • Figure 5B shows KRAS degradation using bispecific CKS1 constructs as measured using a HiBiT lytic assay.
  • Figure 5C shows a schematic representation of components used to build the CKS1 constructs.
  • CKS1 CKS1 protein, which acts as a substrate adaptor for the E3 ligase SCF Skp2 . Residues involved in binding to the protein Cdk2 and to the protein p27 have been mutated.
  • HA HA tag
  • Phospho a beta- catenin binding sequence from the protein ARC (Adenomatous polyposis coli); SOX: a beta- catenin binding sequence from the protein SOX; KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605- 61 1).
  • RBP a KRAS-binding sequence identified by phage display (Gareiss P.C.
  • aFT a KRAS-binding sequence from the protein alpha farnesyl transferase
  • FT a KRAS-binding sequence from the protein alpha farnesyl transferase
  • Skp2-binding indicates residues from native CKS1 required for binding to the E3 sCF Skp2
  • Skp2-, cdk2- and p27-binding this construct has the residues from native CKS1 required for binding to the E3 SCF Skp2 and also for binding to cdk2 and p27.
  • the amino acid sequences of the constructs shown in Figure 5C are set out in Table 4.
  • Figure 6 shows the quantification of expression of mono- and bispecific CKS1 constructs in the cell line MIA PaCa-2 24 hours after transfection (Scram: Scrambled siRNA, siRNA: b- catenin targeted siRNA).
  • Figure 7 shows a schematic of the structure of a representative coiled-coil scaffold derived from the PDB structure.
  • Figure 8A shows a schematic of the coiled-coil scaffold, based on PDB 1 cxz, with examples of sites at which binding peptides can be grafted highlighted.
  • Figure 8B and 8C show models of bifunctional coiled-coil molecules with peptides for binding to B-catenin/ KRAS and to an E3 ligase grafted onto alpha-helices.
  • Figure 8D shows a model of a bifunctional coiled-coil molecule with peptides for binding to B-catenin-/ KRAS-binding and to an E3 ligase grafted onto a loop and a helix, respectively, highlighted. B-catenin Distances between the two targeting peptides are shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 1CXZ as template. Images were generated using PyMol.
  • Figure 9 shows a model of coiled-coil scaffold-mediated beta-eaten in ubiquitination through the MDM2 ubiquitin ligase. Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. The geometry of the complex between MDM2, the coiled-coil scaffold and the beta-eaten in is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the p53 degron peptide (sequence FAAYWNLLSAYG) bound to the N- terminal domain of MDM2.
  • Figure 10 shows b-catenin degradation using bispecific coiled-coil constructs as measured using a HiBiT lytic assay ( Figures 10A and 10B).
  • Figure 10C shows a schematic representation of components used to build the coiled-coil (CC) construct.
  • CC is a coiled-coil from the RHOA-binding effector domain of the protein kinase PKN/PRK1 (PDB 1CXZ).
  • lysines all of the lysines, one of which is functionally important, and two other residues required for the native function, have been substituted; HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein APC (Adenomatous polyposis coli): AXIN: an alpha- helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta- catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al.
  • p27 a degron sequence from the protein p27 that binds the E3 SCFSkp2; Puc: a degron sequence from the protein Puc that binds the E3 Cul3-SPOP; NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1 ; PHYL: a degron sequence from the protein PHYL that binds the E3 SI AH; Trib: a degron sequence from the protein Trib that binds the E3 COP1 ; PAM2: a degron sequence from the protein PAM2 that binds the E3 UBR5; CDC25B: a degron sequence from the protein CDC25B that binds the E3 beta- TRCP; p53: an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2.
  • Figure 11 A and 1 1 B show the quantification of expression of bispecific coiled-coil constructs in the cell line MIA PaCa-2 24 hours after transfection.
  • Figure 1 1C shows a western blot of b-catenin using HEK293 transfected with bifunctional constructs or controls. Total protein staining SDS-PAGE of HEK293 cell extract 24hrs (top left panel) and 48hrs (top right panel) after transfection with bifunctional constructs or controls is shown.
  • Figure 12 shows a schematic of the structure of a representative Affibody scaffold with a grafted loop 1 (between H1 and H2 helices) and a grafted H3 helix. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of the loop motif was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.
  • Figure 13 shows a CLUSTAL W alignment of representative sequences of Affibody domains
  • Figure 14A shows a schematic of the modelled structures of a bifunctional Affibody molecule with binding peptides for B-catenin and for an E3 ligase grafted onto a helix and a loop, respectively.
  • Figure 14B shows a model of a bifunctional Affibody molecule, with peptides for binding to B-catenin and to an E3 ligase grafted onto two helices, in complex with B-catenin and the N-terminal domain of MDM2.
  • Figure 15 shows a model of Affibody scaffold-mediated KRAS ubiquitination via the Cul3- Keapl E3 ubiquitin ligase complex.
  • the E3 Cul3-Keap1-E2 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107).
  • the Affibody scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .
  • the geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl . Two different views are shown.
  • Figure 16A shows b-catenin degradation using bispecific Affibody constructs as measured using a HiBiT lytic assay (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 16B shows a schematic representation of components used to build the Affibody constructs.
  • Affibody A protein scaffold based on the Z domain of a Staphylococcus aureus protein A (PMID: 18435759); HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein A PC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha- helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS- binding sequence from the protein SOS1 (Son of sevenless homolog 1); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 16.
  • Figure 17 shows the quantification of expression of mono- and bispecific Affibody constructs in the cell line MIA PaCa-2 24 hours after transfection.
  • Scram Scrambled siRNA
  • siRNA b- catenin targeted siRNA.
  • Figure 18 shows a schematic of the structure of a representative Trefoil scaffold derived from PDB structure code 2AFG.
  • Figure 19 shows the secondary structure of a representative Trefoil scaffold.
  • the beta- strands represented as arrows and italic text, helices as rectangles and bold text.
  • FIG. 20A shows a schematic of the structure of the Trefoil scaffold with examples of sites for peptide grafting highlighted in black and annotated.
  • Figure 20B shows a model of a bifunctional Trefoil molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto loops. Highlighted in black are loops for binding to b-catenin / KRAS and to an E3 ligase. The distance between the two targeting peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 3PG0 as template. Images were generated using PyMol.
  • Figure 21 A shows b-catenin degradation determined using a HiBiT lytic assay for Trefoil constructs.
  • Figure 21 B shows KRAS degradation determined using a HiBiT lytic assay for Trefoil constructs.
  • Figure 21 C show a schematic representation of components used to build the Trefoil constructs.
  • Trefoil The Trefoil scaffold based on a designed 3-fold symmetric non-functional protein (PMID: 22178248, pdb code 3PG0) with lysines removed based on known substitutions at these positions in an NCBI alignment of the protein family, from which the designed sequence is derived; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522); KBL: a KRAS-binding sequence identified by phage display (Sakamoto K. et al. ,
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1.
  • the amino acid sequences of the constructs are shown in Table 20.
  • Figure 22 shows a quantification of the expression of single and bifunctional Trefoil constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA.
  • Scram Scrambled siRNA
  • siRNA b-catenin targeted siRNA.
  • Figure 23 shows a schematic of the structure of a representative PDZ scaffold.
  • Figure 24 shows a schematic of the structure of the PDZ scaffold (24A) and the modelled structure of a bifunctional PDZ protein (24 B).
  • Figure 24A shows a model of the PDZ scaffold with examples of sites for peptide grafting highlighted in black and annotated.
  • Figure 24B shows a model of the bifunctional PDZ molecule with peptides highlighted in black for binding to B-catenin / KRAS and to an E3 ligase grafted onto a helix and a loop respectively. The distance between the two targeting peptides is shown. All models were generated using SWSS-MODEL (expasy Webserver) with pdb 3JXT as template. Images were generated using PyMol.
  • Figure 25 shows a model of PDZ scaffold-mediated KRAS ubiquitination through the Cul3- Keapl E3 ubiquitin ligase complex.
  • the Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER®, and the energy of the resulting proteins was minimized by UCSF Chimera.
  • the geometry of the complex between KRAS, SOS peptide, PDZ scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (from the protein Nrf2) (sequence ETGE) bound to the b-propeller domain of Keapl .
  • Figure 26A shows b-catenin degradation determined using a HiBiT lytic assay for PDZ constructs (UNT: Untreated SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 26B shows KRAS degradation determined using a HiBiT lytic assay for PDZ constructs (UNT: Untreated, SRC: Scrambled siRNA, LIPO:
  • FIG. 26C shows a schematic representation of components used to build the PDZ constructs; PDZ: PSD95 PDZ3 domain was used as a scaffold (PMID: 15820976) with lysine residues substituted. PDZ domains have low affinity for their natural ligands, as they participate in multivalent interactions, and therefore the isolated domain will not be functional; Phospho: a beta-eaten in binding sequence from the protein APC (Adenomatous polyposis coli); AXIN: an alpha-helical beta- catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9.
  • SOS an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1);
  • RBP a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522);
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of the constructs are shown in Table 25.
  • Figure 27 shows a quantification of the expression of single and bifunctional PDZ constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA).
  • Figure 28 shows a schematic of the structure of a representative Ubiquitin scaffold derived from PDB structure code 2KOX.
  • Figure 29 shows a schematic of the modelled structure of a bifunctional ubiquitin molecule with binding peptides for B-catenin and for an E3 ligase grafted onto loops. All models were generated using SWISS-MODEL (expasy Webserver). Images were generated using PyMol.
  • Figure 30 shows a model of Ubiquitin scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex.
  • the Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with
  • Figure 31A shows b-catenin degradation determined using a HiBiT lytic assay for ubiquitin constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 31 B shows a schematic representation of components used to build the Ub (ubiquitin)/ UBL (Ubiquitin-like) constructs; hPLIC2: A ubiquitin-like domain that binds to the Rpn13 subunit of the proteasome; Raf-RBD: RAS-binding domain of the protein C-Raf (also known as Raf-1).
  • Raf-RBD has a Ubiquitin structure
  • HA HA tag
  • Phospho a beta-eaten in binding sequence from the protein APC (Adenomatous polyposis coli)
  • AXIN an alpha-helical beta-eaten in binding sequence from the protein AXIN
  • BCL9 an alpha-helical beta-catenin binding sequence from the protein BCL9
  • SOS an alpha- helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1)
  • RBP a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010,
  • KBL a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611); For PPX107, PPX108, PPX110 and PPX11 , the residues involved in RAS-binding function of Raf-RBD have been replaced with the RAS-binding motif RBP or KBL; p27: a degron sequence from the protein p27 that binds the E3 SCFSkp2; NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1 ; KRAS-binding: indicates that the PPX sequence contains residues of Raf-RBD required for binding to KRAS; Proteasome-binding: indicates that the PPX sequence contains residues of hPLIC2 required for binding to the proteasome.
  • Figure 32 shows a quantification of the expression of single and bifunctional ubiquitin constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA.
  • Scrambled siRNA siRNA: b-catenin targeted siRNA
  • Lipo a quantification of the expression of single and bifunctional ubiquitin constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA.
  • Figure 33 shows a schematic of the structure of representative GB1 domain with examples of sites for peptide grafting annotated.
  • Figure 34 shows a schematic of the modelled structure of a bifunctional GB1 molecule with peptides binding to B-catenin / KRAS and to an E3 ligase grafted onto loops. The distance between the two targeting peptides is shown.
  • AH models were generated using SWISS- MODEL (expasy Webserver) with pdb 1GB4 as template. Images were generated using
  • Figure 35 shows two different views of the modelled structure of the complex of loop-helix- grafted GB1 domain (in grey) in complex with KRAS and the E2-E3 Cullin-Keapl .
  • the figure shows a model of GB1 scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex.
  • the Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al (Free Rad Biol & Med (2015) 88 101-107).
  • the GB1 scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .
  • the geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl Two different views are shown). Grafting of loop motifs was modelled with
  • Figure 36A shows b-catenin degradation determined using a HiBiT lytic assay for GB1 constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 36B shows a schematic representation of components used to build the GB1 constructs.
  • GB1 Stabilised version of the B1 IgG binding domain of protein G from Streptococcus (PMID: 9628485) with the lysine residues substituted.
  • HA HA tag
  • Phospho a beta-eaten in binding sequence from the protein A PC (Adenomatous polyposis coli);
  • AXIN an alpha-helical beta-catenin binding sequence from the protein AXIN;
  • BCL9 an alpha- helical beta-catenin binding sequence from the protein BCL9;
  • SOS an alpha-helical KRAS- binding sequence from the protein SOS1 (Son of sevenless homolog 1);
  • RBP a KRAS- binding sequence identified by phage display (Gareiss P.C.
  • p27 a degron sequence from the protein p27 that binds the E3 SCFSkp2.
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of these constructs are shown in Table 33.
  • Figure 37 shows a quantification of the expression of single and bifunctional GB1 constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo: Lipofectamine only).
  • Figure 38 shows a schematic representation of the structure of the VWV domain of PIN1.
  • Figure 39 shows modelled structures of a VWV scaffold with two grafted loops and with a grafted helix and a grafted loop. Grafting of the loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera.
  • Figure 40 shows a schematic of structure of the WW scaffold and a modelled structure of the V V scaffold with two grafted peptides.
  • Figure 40 shows the WW scaffold with examples of sites for peptide grafting highlighted and annotated.
  • Figure 40 shows a model of the WW scaffold with binding peptides for B-catenin / KRAS and for an E3 ligase grafted onto loops highlighted. B-cateninThe distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 2M8I as template. Images were generated using PyMol.
  • Figure 41 shows a helical wheel of the helixWW scaffold of SEQ ID NO: 11.
  • the Xs represent the isomorphic replacement of amino acids to form a binding interface on the solvent-accessible side of the helix.
  • Figure 42A shows b-catenin degradation determined using a HiBiT lytic assay for WW constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LI PO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 42 B shows a schematic representation of components used to build the WW constructs. WW: a thermodynamically stabilised version of the WW domain from PIN1 (PMID: 23378640) with the lysine residue substituted. Sequences are placed into a loop used to bind proline- rich peptide ligands.
  • WW domains participate in multivalent interactions in nature and have low affinity for their natural ligands, so the isolated domain is not expected be functional;
  • HA HA tag;
  • Phospho a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli);
  • RBP a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517- 522);
  • KBL a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611).
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of these constructs are shown in Table 38.
  • Figure 43 shows a quantification of the expression of single and bifunctional VWV constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, Lipo: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 44 shows the modelled structure of the longer (44A) and shorter (44 B) Fibritin domains with a grafted loop. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of the loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.
  • Figure 45A shows a schematic of the modelled structure of a Fibritin domain with a grafted loop and a grafted helix.
  • Figures 45B-D show schematics of the modelled structures of a single chain of a Fibritin domain (pdb 1aa0) with binding peptides for B-catenin / KRAS and for an E3 ligase grafted onto different helices and loops. Distances between the two binding peptides are shown.
  • Figure 46 shows a model of Fibritin scaffold-mediated KRAS ubiquitination through the Cul3- Keapl E3 ubiquitin ligase complex.
  • the Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107).
  • the Fibritin scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .
  • the geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl . Two different views are shown.
  • Figure 47A shows b-catenin degradation determined using a HiBiT lytic assay for Fibritin constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 47B shows a schematic representation of components used to build the Fibritin constructs.
  • Fibritin The trimerization domain of T4 phage Fibritin with the lysines replaced; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); RBP: a KRAS-binding sequence identified by phage display (Gareiss P.C.
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1.
  • the amino acid sequences of the constructs are shown in Table 41.
  • Figure 48 shows a quantification of the expression of single and bifunctional Fibritin constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA.
  • Figure 49 shows schematics of the structures of aPP scaffold (49A) and PPa scaffold (49C) and the modelled structures of their bifunctional molecules (49 B and 49D).
  • Figure 49A shows the aPP scaffold with examples of sites for peptide grafting highlighted in black and annotated.
  • Figure 49 B shows a model of a bifunctional aPP molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto a loop and a helix, respectively, highlighted in black.
  • Figure 49C shows a model of PPa scaffold with sites for peptide grafting highlighted in black and annotated.
  • Figure 49D shows a model of a bifunctional PPa molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto a helix and a loop, respectively, highlighted in black. The distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 2BF9 (for aPP constructs) and with pdb 5L02 as template. Images were generated using PyMol.
  • Figure 50 shows b-catenin degradation determined using a HiBiT lytic assay for aPP constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 51 shows a schematic representation of components used to build the aPP and PPa constructs.
  • aPP aPP is an avian pancreatic polypeptide domain from a peptide hormone. Grafting of degron and target-binding sequences will abolish its native function. The native sequence contains no lysine residues; HA: HA tag; PPa: PPa is a designed (non-functional) aPP protein in which the sequence optimised for stability (PMID 31251570).
  • Lysine residues in the original sequence have been replaced with arginine residues;
  • Phospho a beta-catenin binding sequence from the protein APC (Adenomatous polyposis coli);
  • AXIN an alpha- helical beta-catenin binding sequence from the protein AXIN;
  • BCL9 an alpha-helical beta- catenin binding sequence from the protein BCL9;
  • RBP a KRAS-binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522);
  • KBL a KRAS- binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun.
  • p27 a degron sequence from the protein p27 that binds the E3 SCFSkp2
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1
  • WNK4 a degron sequence from the protein WNK4 that binds the E3 KLHL2
  • p53 an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2.
  • the amino acid sequences of these constructs are shown in Table 45.
  • Figure 52 shows schematics of the structure of the Fibronectin type III scaffold (52A) and the modelled structure of a bifunctional Fibronectin type III molecule (52 B).
  • Figure 52A shows a Fibronectin type III scaffold with examples of sites for peptide grafting highlighted and annotated.
  • Figure 52 B shows a model of a bifunctional Fibronectin type III molecule with binding peptides for b-catenin / KRAS and for an E3 ligase grafted onto loops, highlighted in black. The distance between the two binding peptides is shown. All models were generated using SWISS-MODEL (expasy Webserver) with pdb 4U3H as template. Images were generated using PyMol.
  • Figure 53A shows b-catenin degradation determined using a HiBiT lytic assay for FN3 constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 53B shows a schematic representation of components used to build the Fibronectin type III (FN3) constructs.
  • FN3 A non-functional stabilised Fibronectin type III consensus sequence based on the alignment of multiple FN3 domains (PMID:25691761) with the single lysine replaced with an arginine; HA: HA tag; Phospho: a beta-eaten in binding sequence from the protein A PC (Adenomatous polyposis coli); NRF2: a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1. The amino acid sequences of these constructs are shown in Table 48.
  • Figure 54 shows a quantification of the expression of single and bifunctional FN3 constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo: Lipofectamine only).
  • Figure 55 shows a modelled structure of a Zinc finger domain with a grafted loop and a grafted helix. Grafting of the helical motif was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of the loop motifs was modelled with
  • Figure 56 shows a model of Zn finger scaffold-mediated KRAS ubiquitination via the Cul3- Keapl E3 ubiquitin ligase complex.
  • the E3 Cul3-Keap1-E2 model was constructed from multiple crystal structures as described in Canning et al (Free Rad Biol & Med (2015) 88 101-107).
  • the Zn finger scaffold is grafted with a helical peptide to bind to KRAS and a loop degron peptide to bind to Keapl .
  • the geometry of the complex is that predicted based on a structural alignment of the modelled loop-grafted degron and the crystal structure of the degron of Nrf2 bound to Keapl
  • Figure 57 A shows b-catenin degradation determined using a HiBiT lytic assay for ZF constructs (UNT: Untreated, SCRAM: Scrambled siRNA, LIPO: Lipofectamine only, siRNA: b-catenin targeted siRNA).
  • Figure 57B shows a schematic representation of components used to build the Zinc-finger (ZF) constructs. ZF: based on the second C2H2 type Zinc-finger domain of ZNF32.
  • Lysines are substituted, which abolishes the natural DNA binding function; HA: HA tag; Phospho: a beta-catenin binding sequence from the protein A PC (Adenomatous polyposis coli); AXIN: an alpha-helical beta-catenin binding sequence from the protein AXIN; BCL9: an alpha-helical beta-catenin binding sequence from the protein BCL9; SOS: an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1); KBL: a KRAS-binding sequence identified by phage display
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1.
  • the amino acid sequences of the constructs are shown in Table 51.
  • Figure 58 shows a quantification of the expression of single and bifunctional ZF constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA (Scram: Scrambled siRNA, siRNA: b-catenin targeted siRNA, Lipo: Lipofectamine only).
  • Figure 59 shows a modelled example of a bifunctional SH3 scaffold created by grafting first and second peptide ligands onto the first and second loops (R-loop and Src loop, respectively). Grafting of the loop peptides was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.
  • Figure 60 shows the SH3 domain from the T cell adapter protein ADAP that has an N- terminal helix (referred to here as hSH3) (pdb 1 R 19) , with examples of sites for peptide grafting highlighted and annotated. Images were generated using PyMol.
  • Figure 61 shows a modelled example of an SH3 scaffold bound to its natural ligand, namely C-Myc, with a degron peptide grafted onto the third loop.
  • Figure 62 shows b-catenin degradation determined using a HiBiT lytic assay for src
  • FIG. 62C shows a schematic representation of components used to build the src Homology-3 (SH3) constructs.
  • Fyn-SH3 the SH3 domain of Fyn (a Src family tyrosine kinase). Degrons have been inserted into n-src loop, thereby abolishing its native function. Lysine residues in the native sequence have been replaced with arginine residues.
  • hSH3 the SH3 domain from the T cell adapter protein ADAP that has an N-terminal helix. All helical binding sequences are grafted onto this helix.
  • grafting process together with the substitution of several lysine residues, knocks out the native lipid-binding activity of this protein.
  • Other lysine residues in the native sequence have been replaced with arginine residues.
  • Phospho a beta-catenin binding sequence from the protein A PC (Adenomatous polyposis coli).
  • AXIN an alpha-helical beta-catenin binding sequence from the protein AXIN.
  • BCL9 an alpha-helical beta-catenin binding sequence from the protein BCL9.
  • SOS an alpha-helical KRAS-binding sequence from the protein SOS1 (Son of sevenless homolog 1).
  • KBL a KRAS-binding sequence identified by phage display (Sakamoto K. et al. , Biochem. Biophys. Res. Commun. 2017 484: 605-611).
  • P27 a degron sequence from the protein p27 that binds the E3 SCFSkp2.
  • Puc a degron sequence from the protein Puc that binds the E3 Cul3-SPOP.
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1.
  • PHYL a degron sequence from the protein PHYL that binds the E3 SIAH.
  • Trib a degron sequence from the protein Trib that binds the E3 COP1.
  • PAM2 a degron sequence from the protein PAM2 that binds the E3 UBR5.
  • CDC25B a degron sequence from the protein CDC25B that binds the E3 beta-TRCP.
  • p53 an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2. The amino acid sequences of the constructs are shown in Table 55.
  • Figure 63 shows Western blot of B-catenin using HEK293 transfected with bifunctional SH3 construct or controls.
  • Figure 64 shows a quantification of the expression of single and bifunctional SH3 constructs in MIA PaCa-2 24 hours after transfection using Cell Profiler analysis of IF Staining for HA.
  • Scram Scrambled sRNA
  • siRNA B-catenin targeted siRNA
  • Lipo Lipofectamine only.
  • Figure 65 shows isothermal titration calorimetry (ITC) data of grafted cystine knot scaffold (PPX259) binding to Keapl in the presence of reducing agent (0.3 mM TCEP)
  • Figure 66 shows ITC data of grafted cystine knot scaffold (PPX259) binding to Keapl in very low concentration of reducing agent (0.015 mM TCEP)
  • Figure 67 shows competition fluorescence polarisation data for grafted cystine knot scaffolds (PPX252 and PPX253) binding to MDM2.
  • FIG. 68 shows a schematic representation of components used to build the cystine knot constructs.
  • Cyclotide is a disulphide-rich peptide sequence based on the cyclotide MCoTI-ll (Momordica cochinchinensis trypsin inhibitor-ll) (Felizmenio-Quimio, M. E. et al., J Biol. Chem. 2001 276: 22875-22882). Under oxidising conditions, disulphide bonds form, which creates loops onto which the target-binding or E3 ligase-engaging peptides can be grafted.
  • MCoTI-ll Momordica cochinchinchinensis trypsin inhibitor-ll
  • PPX250, PPX251 , PPX252, PPX253, PPX254, PPX255 and PPX256 are N-to-C cyclised, thereby creating an additional loop for peptide grafting.
  • Biotin Biotin tag.
  • Phospho a beta-catenin binding sequence from the protein A PC (Adenomatous polyposis coli).
  • AXIN an alpha-helical beta-catenin binding sequence from the protein AXIN.
  • BCL9 an alpha- helical beta-catenin binding sequence from the protein BCL9.
  • SOS an alpha-helical KRAS- binding sequence from the protein SOS1 (Son of sevenless homolog 1).
  • KBL a KRAS- binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611).
  • Abltide A peptide that binds to BCR-ABL.
  • p27 a degron sequence from the protein p27 that binds the E3 SCFSkp2.
  • NRF2 a degron sequence from the protein NRF2 that binds the E3 Cul3-KEAP1.
  • Trib a degron sequence from the protein Trib that binds the E3 COP1.
  • p53 an alpha-helical degron sequence from the protein p53 that binds the E3 MDM2.
  • KBL a KRAS-binding sequence identified by phage display (Sakamoto K. et al., Biochem. Biophys. Res. Commun. 2017 484: 605-611).
  • RBP a KRAS- binding sequence identified by phage display (Gareiss P.C. et al., 2010, ChemBioChem 11 : 517-522).
  • Figure 69 shows a schematic of the modelled structure of a cyclotide with two grafted loops, Loop 2 and Loop 6.
  • This invention relates to chimeric proteins that comprise a monomeric peptide scaffold (i.e. grafted peptide scaffolds).
  • One or more peptide ligands are located in the scaffold of a chimeric protein.
  • the peptide ligands may be to the same or different target molecules and the chimeric proteins of the first to fourteenth aspects may be multi-functional and/or multi valent.
  • Chimeric proteins as described herein may be useful in a range of therapeutic and diagnostic applications.
  • a scaffold is a protein with stable secondary and tertiary structures that tolerates the insertion or grafting of one or more heterologous peptide ligands into the protein sequence i.e. the scaffold retains its structure in the presence of inserted/grafted peptide ligand(s).
  • a scaffold in which one or more heterologous peptide ligands have been inserted may be referred to herein as a grafted scaffold or a chimeric protein.
  • a variant of a reference chimeric protein, construct, scaffold or peptide ligand sequence set out herein may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence.
  • Particular amino acid sequence variants may differ from a reference sequence shown herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3,
  • GAP Garnier et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith- Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147.
  • a scaffold of the first to the fourteenth aspects may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands.
  • aromatic residues in the scaffold may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of scaffold sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions.
  • lysine residues in a scaffold of the first to the fourteenth aspects may be replaced by a different residue, such as arginine to prevent unwanted ubiquitination and subsequent degradation.
  • a different residue such as arginine to prevent unwanted ubiquitination and subsequent degradation.
  • the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC).
  • a first aspect relates to chimeric proteins that comprise CKS scaffolds (i.e. grafted CKS scaffolds).
  • CKS scaffolds i.e. grafted CKS scaffolds.
  • One or more peptide ligands are located in the CKS scaffold of the chimeric protein, for example in the first, second or third loops or the helical region.
  • CKS1 Cyclin-dependent kinases regulatory subunit 1 domain
  • CKS2 Cycl in dependent kinases regulatory subunit 2 domain
  • CKS1 acts as a substrate adaptor for the E3 ubiquitin ligase SCF Sk P 2 by binding both SCF Sk P 2 and a subset of substrates resulting in ubiquitination of those substrates.
  • One such substrate is the cell-cycle inhibitor p27
  • CKS proteins have a binding site for cyclin-dependent kinase 2 (CDK2) that further enhances the efficiency of p27 ubiquitination by SCF Skp2 .
  • CDK2 cyclin-dependent kinase 2
  • the structure of the CKS domain is well known in the art (see for example PFAM 01 11).
  • the invention adopts the well understood sequence-structure relationships of CKS domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.
  • a CKS scaffold 4-stranded b-sheet protein with a short alpha-helix (i.e. a CKS domain structure).
  • a CKS scaffold as used herein, has a length of 60 to 80 amino acids, preferably about 70 amino acids or 69 amino acids.
  • a CKS scaffold may have a MW of about 7600 Da.
  • Suitable CKS scaffolds useful according to the invention include Human CKS protein Cks1 (Uniprot P61024; Gene ID 1163; NP_001817.1) and Human CKS protein Cks2 (Uniprot P33552) or variants of either of these.
  • the sequences of Cks1 and Cks2 are shown in Table 3.
  • PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable CKS scaffolds useful according to the invention without any limitation: 2ASS, 2AST, 4YC3, 1 DKS and 4YC6.
  • Suitable CKS scaffolds may also be identified using the PFAM database (see for example Finn et al Nucleic Acids Research (2016) Database Issue 44: D279-D285).
  • a CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 1 or a variant thereof.
  • the CKS scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 2 or a variant thereof.
  • a CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 3 or a variant thereof.
  • the CKS scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 4 or a variant thereof.
  • Preferred CKS scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination.
  • the lysine residues in a CKS domain may be replaced by either a polar or charged amino acid, preferably Arg or Glu, to generate a CKS scaffold.
  • a suitable lysine-free CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 5 or a variant thereof.
  • the lysine-free CKS scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 6 or a variant thereof.
  • the first loop of the CKS scaffold is located within the position corresponding to residues 25 to 39 of SEQ ID NO: 1 and SEQ ID NO: 665 and residues 31 to 39 of SEQ ID NO: 3.
  • the second loop of the CKS scaffold is located within the position corresponding to residues 46 to 54 of SEQ ID NO: 1 and SEQ ID NO: 665, and residues 46 to 53 of SEQ ID NO: 3.
  • the third loop of the CKS scaffold is located within the position corresponding to residues 58 to 64 of SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 665.
  • the helix of the CKS scaffold is located within the position corresponding to 40 to 45 of SEQ ID NO: 1 , SEQ ID NO: 3 and SEQ ID NO: 665.
  • a CKS scaffold may display binding activity.
  • a CKS scaffold may bind to the E3 ubiquitin ligase SCF Skp2 , in the absence of an inserted peptide ligand. Skp2 binding may be mediated by the helical region corresponding to residues 40 to 45 of SEQ ID NO: 1 and SEQ ID NO: 3.
  • a grafted CKS scaffold that binds to Skp2 may further comprise a peptide ligand in a loop of the CKS scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop).
  • the peptide ligand may be heterologous.
  • a grafted CKS scaffold may contain a peptide ligand within the first loop (a“loop peptide”) i.e. the loop corresponding to residues 25 to 39 of SEQ ID NO: 1 and residues 31 to 39 of SEQ ID NO: 3.
  • a peptide ligand may be located between residues corresponding to 25 and 28 of SEQ ID NO: 1 and 31 to 34 of SEQ ID NO: 3.
  • a peptide ligand may be located in the CKS scaffold immediately after (i.e.
  • the peptide ligand may be inserted into the CKS scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 26, 27, 28, 29, 30, 31 ,
  • the peptide ligand may be added to the first loop or may replace one or more residues of the first loop.
  • the peptide ligand may replace the first loop.
  • a peptide ligand may replace the loop residues corresponding to residues 25 to 39 of SEQ ID NO: 1.
  • a grafted CKS scaffold may contain a peptide ligand within the second loop (a“loop peptide”) i.e. the loop corresponding 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3.
  • a peptide ligand may be located in the CKS scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 45, 46, 47, 48, 49, 50, 51 , 52, or 53 of SEQ ID NO: 1.
  • the peptide ligand may be inserted into the CKS scaffold
  • the peptide ligand may be added to the second loop or may replace one or more residues of the second loop.
  • the peptide ligand may replace the second loop.
  • a peptide ligand may replace the loop residues corresponding 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3.
  • a grafted CKS scaffold may contain a peptide ligand within the third loop (a“loop peptide”) i.e. the loop corresponding to residues 58 to 64 of SEQ ID NO: 1 and SEQ ID NO: 3.
  • a peptide ligand may be located between residues 61 and 64 of SEQ ID NO: 1 and 3.
  • a peptide ligand may be located in the CKS scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 57, 58, 59, 60, 61 , 62, or 63 of SEQ ID NO: 1 or 3.
  • the peptide ligand may be inserted into the CKS scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 59, 60, 61 , 62, 63, 64, or 65 of SEQ ID NO: 1.
  • the peptide ligand may be added to the third loop or may replace one or more residues of the third loop.
  • the peptide ligand may replace the third loop.
  • a peptide ligand may replace the loop residues corresponding 58 to 64 of SEQ ID NO: 1 and SEQ ID NO: 3.
  • a peptide ligand of more than 4 residues may be added to the scaffold sequence without replacing scaffold residues.
  • a peptide ligand of 4 or fewer residues may replace the corresponding number of residues in the CKS scaffold sequence.
  • a grafted CKS scaffold may comprise the amino acid sequence of SEQ ID NO: 6 or a variant thereof.
  • n is 0-30 and Xi, X2...X n are independently any amino acid, for example, independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, pyroglutamatic, serine, threonine, tryptophan, tyrosine and valine, phospho-serine, phospho-threonine and phospho- tyrosine, acetylated amino acids.
  • alanine arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, p
  • the Skp2-binding region of the grafted CKS scaffold may be replaced by a helical peptide ligand. This may alter the binding specificity of the grafted CKS scaffold.
  • the helical peptide ligand may bind to a target molecule other than Skp2.
  • a grafted CKS scaffold may comprise a peptide ligand in the helix portion of the CKS scaffold.
  • a helical peptide ligand may be inserted in a helical portion of the CKS scaffold at the position corresponding to residues 40 to 45 of SEQ ID NO: 1 and SEQ ID NO: 3.
  • A“helical peptide ligand” is a peptide ligand which is positioned in a helical structure of the scaffold.
  • a helical peptide ligand replaces the helix of the CKS scaffold.
  • a peptide ligand may replace residues 40 to 45 of SEQ ID NO: 1 or 3.
  • a peptide ligand may replace one or more of residues S41 , E42 and N45 of SEQ ID NO: 1.
  • a grafted CKS scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 40, 41 or 44 of SEQ ID NO: 1 or 3.
  • a grafted CKS scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 42, 43 or 46 of SEQ ID NO: 1 or 3).
  • Suitable peptide ligands include helical peptide ligands as described herein.
  • a grafted CKS scaffold is created by isomorphic replacement of those residues (for example in SEQ ID NO: 1 one or more of residues S41 , E42 and N45) of the helix portion of the CKS scaffold that are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the helix peptide ligand.
  • residues corresponding to E40, W43, R44 of SEQ ID NO: 1 are not replaced.
  • a helix peptide ligand may comprise the amino acid sequence of SEQ ID NO:
  • SEQ ID NO: 7 a fragment of SEQ ID NO: 7 or a variant of either of these.
  • a grafted CKS scaffold may comprise a CKS scaffold comprising a first peptide ligand inserted within the first loop located at a position corresponding to residues 25 to 39 of SEQ ID NO: 1 and residues 31 to 39 of SEQ ID NO: 3 or in the second loop between residues 46 to 54 of SEQ ID NO: 1 and residues 46 to 53 of SEQ ID NO: 3 and a second peptide ligand located within the helical region located at positions corresponding to residues 40 to 45 of SEQ ID NO: 1 or 3.
  • the CKS scaffold of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the CKS scaffolds.
  • a CKS scaffold of a chimeric protein may also display binding activity i.e. the CKS scaffold may mediate binding of the chimeric protein to a first target molecule, such as Skp2.
  • the peptide ligand may mediate binding of the chimeric protein to a second target molecule.
  • a grafted CKS scaffold may comprise a CKS scaffold with the amino acid sequence of residues 1 to 73 of SEQ ID NO: 665 (PPX69 of Table 4 without the HA Tag).
  • a target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of first loop (loop 1 ; residues 25 to 39 of SEQ ID NO: 665) or third loop (loop 3; residues 58 to 64 of SEQ ID NO: 665) or the helical region (helix 1 ; residues 40 to 45 of SEQ ID NO: 665) of the CKS scaffold.
  • the target-binding peptide ligand is located in the first or the third loop.
  • the E3 ligase-binding peptide ligand is located in helix 1.
  • the E3 ligase-binding peptide ligand is the endogenous SCF Skp2 binding sequence of CKS1 which is present in the helical region (helix 1).
  • the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the helix or the target-binding peptide ligand may be in the third loop and the E3 ligase-binding peptide ligand may be in the helix.
  • Suitable target-binding peptide ligands include bcatenin binding ligands, for example peptides from ARC (Adenomatous polyposis coli), such as SEELEALEALELDE and variants thereof, and peptides from SOX, such as DDIEFDQYL and variants thereof, and KRAS binding ligands, for example peptides from KBL, such as PLYISY and variants thereof, peptides from alpha farnesyl transferase, such as ENPKQYN and variants thereof, peptides from beta farnesyl transferase, such as DAYECLDASRPW or KSRDFYH and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS and variants thereof.
  • a grafted CKS scaffold of the first aspect may comprise an amino acid sequence shown in Table 4 (SEQ ID NOs: 660-682) or a variant of an amino acid sequence shown in Table 4.
  • Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 4 are replaced by a different peptide ligand. Suitable peptide ligands are described below.
  • Variants may also include variants in which the CKS scaffold sequence in a reference amino acid sequence of Table 4 is replaced by a different CKS scaffold sequence. Suitable CKS scaffold sequences are described above.
  • a second aspect relates to chimeric proteins that comprise coiled-coil scaffolds (i.e. grafted coiled-coil scaffolds).
  • coiled-coil scaffolds i.e. grafted coiled-coil scaffolds.
  • One or more peptide ligands are located in the coiled-coil scaffold of the chimeric protein, for example at positions between residues 55 to 57 or between residues 17 to 54 or between residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13 or 14.
  • Coiled-coil domains are well-known and well-characterized example in the prior art. Coiled- coil domains are among the most extensively used model systems in the area of protein folding and design. A coiled-coil domain is a structural fold in proteins in which 2-7 alpha- helices are coiled together like the strands of a rope. Many coiled-coil-type proteins are involved in important biological functions such as the regulation of gene expression, e.g. transcription factors (Chembiochem. 2004 Feb 6;5(2):170-6 PNAS October 17, 2006 103 (42) 15457-15462; Science. 262 (5138): 1401-7). The invention adopts the well understood sequence/structure relationships of coiled-coil domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.
  • a“coiled-coil scaffold” is a peptidyl structure composed of at least two alpha- helices coiled together like the strands of a rope.
  • a coiled-coil may be a dimer or a trimer (of alpha-helices), or may include or consist of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or up to 12 helices.
  • the alpha-helices may be parallel or anti-parallel, and may adopt a left or right handed supercoil. Left-handed supercoils maybe preferred in some embodiments.
  • a coiled-coil scaffold contains a heptad repeat, which is a repeated pattern of hydrophobic (H) or charged (C) amino acid residues, and the pattern may include the heptad HxxHCxC (to specify each position, the heptad is labelled abcdefg, where“a” and“d” are hydrophobic positions). It may be in some embodiments that“a” and“d” are Isoleucine, leucine or valine.
  • a coiled-coil scaffold, as used herein, has a length in the amino acid range of 1-50, 1-20, 1- 1385, and any amino acid length there between.
  • a coiled-coil scaffold is typically 7400 Da in MW, and such scaffolds are in the MW range of 2500 to 158275 Da.
  • a left-handed coiled-coil scaffold comprises
  • a heptad repeat in which the a and d positions are hydrophobic (e.g. leucine, valine, or isoleucine), thus stabilizing helix dimerization through hydrophobic and van der Waals interactions;
  • residues e and g are charged (e.g. glutamate or lysine) in order to form inter helical electrostatic interactions.
  • Such interaction patterns should be of the opposite charge in heterodimers to stabilize their interaction, and of the same charge in homodimers to destabilize them;
  • the length of a coiled-coil scaffold is from 14 amino acids (2 heptad repeats) to 140 amino acids (20 heptad repeats) in length, or may be 21 - 105 amino acids, 28 - 100 amino acids, 35 - 70 amino acids, and any lengths in between the stated length ranges.
  • a coiled-coil scaffold serves as a molecular spacer with respect to the grafted peptide ligands. With respect to the physical size of a coiled-coil, the size is in the range of 40 - 90nm in length, and may be 50nm - 75nm.
  • a coiled-coil scaffold may comprise helices with repeating sequences which exhibit distinct amphipathic character, with both hydrophobic and polar faces.
  • a coiled-coil scaffold may comprise two (or more) helices which associate via their hydrophobic faces, which then drives coiled-coil formation.
  • the helices of a coiled-coil scaffold may arrange such that the hydrophobic strands wrap against each other and are sandwiched between the hydrophilic amino acid residues of the strands, with tight packing and van der Waals contacts between the side-chains of the“a” and“d” residues leading to higher stability of the coiled-coil scaffold.
  • a representative coiled-coil scaffold, as used herein, may be 14-140 amino acids in length.
  • Figure 7 depicts a representative coiled-coil scaffold. [PDB code 1CXZ]
  • a coiled-coil scaffold may be homotypic (same coiled-coils interact) or heterotypic (different coiled-coils interact).
  • a coiled-coil scaffold may be a homo- or hetero-oligomer, and may be formed from separate chains, or from consecutive helices of the same chain.
  • a GCN4 coiled-coil is an example of a coiled-coil scaffold.
  • a GCN4 coiled-coil is a 31- amino-acid (which equates to just over four heptads) parallel, dimeric (i.e. , consisting of two alpha-helices) coiled-coil and has a repeated isoleucine (I) and leucine (L) at the“a” and“d” positions, respectively, and forms a dimeric coiled-coil.
  • a trimeric (three alpha-helices) coiled-coil is formed where“a” and“d” are Leucine and Isoleucine, respectively.
  • a tetrameric (four alpha-helices) coiled-coil is formed where both“a” and“d” are Leucine.
  • a polar residue in particular asparagine, N
  • asparagine is a preferred residue in the opposition“a” positions of a coiled-coil
  • any polar residue which permits self-complementary hydrogen bonding between these residues is contemplated to be within a coiled-coil scaffold.
  • Coiled-coils have underlying sequence repeats that govern their assembly, hence coiled- coils can be reliably predicted from primary sequence (J Struct Biol 155: 140-5).
  • An extensive database of genomic and structural information for coiled-coils J Mol Biol 403: 480-93), and in the periodic table of coiled-coil structures (J Mol Biol 385: 726-32). and the CCp database (Nucleic Acids Res 37: D315-22) of coiled-coil structures are available in art. (http://coiledcoils.chm.bris.ac.uk/ccplus/search/dynamic interface) ( Bioessays 2016 Sep; 38(9): 903-916).
  • Suitable coiled-coil scaffolds may include the coiled-coil domain selected from the following, without limitation: effector domain of the protein kinase Serine/ threonine- protein kinase N1 (HR1 family, UniProtKB - Q16512 (PKN1 JHUMAN) ), General control protein GCN4 (UniProtKB - P03069 (GCN4_YEAST)), Golgin subfamily B member 1 (UniProtKB - Q14789 (GOGB1_HUMAN), Spindle assembly abnormal protein 6 homolog (UniProtKB - Q6UVJ0 (SAS6_HUMAN)), Nuclear mitotic apparatus protein 1 (UniProtKB - Q 14980
  • NDC80_HUMAN Structural maintenance of chromosomes protein 1A (UniProtKB - Q 14683 (SMC1AJHUMAN)), DNA repair protein RAD50 (UniProtKB - Q92878
  • PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable coiled-coil scaffolds and it may include without any limitation: 1CXZ, 2ZTA, 1WT6, 2HY6, 2V71 , 2XU6, 4LTB, 4DZM, 5D3A, 5JXC, 5LXN, 5LXO, 5M48, 1 L8D and 3QH9.
  • the present invention contemplates the use of dimeric and multimeric coiled-coil domains to generate coiled-coil scaffolds that comprise one or more peptide ligands.
  • the coiled-coil scaffolds may comprise amino terminal peptide ligands (N- terminal degrons) and/or carboxy terminal peptide ligands (C- terminal degrons.
  • GCN4 homodimeric parallel coiled-coil (CC) (PDB ID 2ZTA)
  • TAT-p53LZ2 effectively inhibits the cancer cell growth in wild-type but not mutant p53 by arresting cell cycle and inducing apoptosis in vitro
  • the invention contemplates a homo-dimeric parallel coiled-coil scaffold (for example as in CC-Di (4dzm)) and derivatives of it that can hetero-dimerise.
  • the NOXA-B sequence was grafted on to the designed homodimeric parallel coiled-coil CC-Di (4dzm) to produce an inhibitor of the MCL-1/BID complex (PMID: 30393526).
  • the invention also contemplates generation of hetero-dimer coiled-coil scaffolds (for example CC-Di-A/CC-Di-B) that may have only one copy of the binding motif.
  • a coiled-coil scaffold may comprise the amino acid sequence of SEQ ID NO: 8 or a variant thereof.
  • the above coiled-coil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 9 or a variant thereof.
  • the heptad repeat is conserved in coiled-coil domains and is represented as follows
  • lysine residues in a coiled-coil domain may be replaced by E, N, Q, L, T, S or R to generate a coiled-coil scaffold.
  • a suitable lysine-free coiled-coil scaffold may comprise the amino acid sequence of SEQ ID NO: 8 or a variant thereof.
  • Table 7 illustrates a representative example of coiled-coil domains in various proteins with details on sequence conservation, length conservation (Leonard et al Bioessays 2016 Sep; 38(9): 903-916)
  • coiled-coil-scaffold may have 2-3 lysine residues.
  • all lysine residues of the coiled-coil scaffold are replaced with non-lysine residues.
  • residue Lys36 of a coiled-coil scaffold is replaced with Arginine.
  • residue Lys39 of a coiled-coil scaffold is replaced with Arginine.
  • residue Lys41 of a coiled-coil scaffold is replaced with Arginine.
  • any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold.
  • a lysine-attenuated coiled-coil scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids may comprise the amino acid sequence of SEQ ID NO: 10 or a variant thereof.
  • a lysine-free coiled-coil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 11 or a variant thereof.
  • a peptide loop ligand has been inserted into the loop that connects two helices of a coiled-coil scaffold.
  • the peptide ligand is represented by residue X, where n is 0-30 and X is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine,
  • a peptide loop ligand has been inserted into the loop that connects two helices of a lysine-free coiled-coil scaffold.
  • the peptide ligand is represented by residue X, where n is 0-30 and X is each independently selected from a group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline,
  • a coiled-coil scaffold comprising a peptide ligand may be encoded by a nucleic acid sequence of SEQ ID NO: 14 or a variant thereof.
  • n is 0-30 and Y is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 5. For instance, Y is selected from the group of codons“att, ate, and ata” if X of SEQ ID NO: 5 were to be Isoleucine. Similarly Y is selected from the group of codons“ett, etc, eta, ctg, tta, ttg” if X of SEQ ID NO: 5 were to be Leucine.
  • a lysine-free coiled-coil scaffold comprising a peptide ligand may be encoded by a nucleic acid sequence of SEQ ID NO:8 or a variant thereof.
  • n is 0-30 and Y is each independently selected from a group of codons that encode the residue X of SEQ ID NO: 6.
  • Y is selected from the group of codons“cgt, ege, ega, egg, aga, agg” if X of SEQ ID NO: 12 were to be Arginine.
  • Y is selected from the group of codons“aat, aac” if X of SEQ ID NO: 13 were to be Asparagine.
  • Table 8 provides codons for each amino acid that can be used to construct a suitable nucleic acid sequence that encodes variants of any of aforesaid nucleic acid sequences.
  • the a-1 helix of a coiled-coil scaffold is located within the positions corresponding to residues 16 (the CC heptad repeat starts at 26) to 54 of SEQ ID NO: 8 or residues 16 to 54 of SEQ ID NO: 10 or residues 16 to 54 of SEQ ID NO: 12, or residues 16 to 54 of SEQ ID NO: 13.
  • the a-2 helix of a coiled-coil scaffold is located within the positions corresponding to residues 59 to 82 of SEQ ID NO: 8 or 59 to 83 of SEQ ID NO: 10, or residues 59 to 83 of SEQ ID NO: 12 or residues 59 to 83 of SEQ ID NO: 13.
  • the disordered regions or the loop regions of a coiled-coil scaffold that connect the alpha helices are located within the positions corresponding to residues 55 to 58 of SEQ ID NO: 8 or residues 55 to 58 of SEQ ID NO: 10 or residues 55 to 58 of SEQ ID NO: 12, or residues 55 to 58 of SEQ ID NO: 13.
  • a grafted coiled-coil scaffold comprises a peptide ligand within a loop (a“loop peptide”).
  • a peptide ligand may be inserted between residues 10 to 12 of SEQ ID NO: 8, 10, 12 or 13 or residues 11 to 13 of SEQ ID NO: 702 (loop 1) residues 55 to 58 of SEQ ID NO: 8, 10, 12 or 13 or residues 55 to 57 of SEQ ID NO: 702 (loop 2).
  • loop 1 may be located in the N terminal tail of the coiled-coil scaffold.
  • a grafted coiled-coil scaffold comprises a peptide ligand within a helix (a“helix peptide”) (for example, inserted between residues corresponding to residues 16 to 54 or residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13).
  • a peptide ligand may be inserted between residues 25 to 36 of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of SEQ ID NO: 702 (helix 1) or residues 62 to 69 of SEQ ID NO: 8, 10, 12 or 13 or residues 62 to 70 of SEQ ID NO: 702 (helix 2).
  • a grafted coiled-coil scaffold comprises a first peptide ligand between positions 16 and 54 of the coiled-coil scaffold of SEQ ID NO: 8 or 10 or 12 or 13 or between residues 25 to 36 of the coiled-coil scaffold of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of the coiled-coil scaffold of SEQ ID NO: 702.
  • a grafted coiled-coil scaffold comprises a second peptide ligand inserted between the positions 55 and 58 of the coiled-coil scaffold of SEQ ID NO: 8 or 10 or 12 or 13 or residues 55 to 57 of the coiled-coil scaffold of SEQ ID NO: 702 (loop 2).
  • a grafted coiled-coil scaffold comprises a first peptide ligand and a second peptide ligand in relative positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted coiled-coil scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e., a target protein and an E3 ligase.
  • a grafted coiled-coil scaffold comprises a first peptide ligand within a first or second helix described herein (for example, inserted or replaced at position between residues 16 to 54 of SEQ ID NO: 8 or 10 or 12 or 13, or residues 23-37 or 62 to 70 of SEQ ID NO: 72) and a second peptide ligand within a first or second loop (for example, inserted or replaced at position between residues 55 and 58 of SEQ ID NO: 8 or 10 or 12 or 13 or residues 11 to 13 or 55 to 57 of SEQ ID NO: 702).
  • a grafted coiled-coil scaffold comprises a first helical peptide ligand within a first helix (helix 1); for example, inserted or replaced at position between residues 16 to 54 of SEQ ID NO: 1 , 3 or 5-7 or residues 25 to 36 of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of SEQ ID NO: 702 and a second helical peptide ligand within a second helix (helix 2); for example, inserted or replaced at position between residues 66 to 77 or residues 62 to 69 of SEQ I D NO: 8 or 10 or 12 or 13 or residues 62 to 70 of SEQ ID NO:
  • a grafted coiled-coil scaffold is created by grafting a first helical peptide ligand onto a first helix at a position defined for instance by residues 16 to 54 of SEQ ID NO: 8 or 10 or 12 or 13; or residues 25 to 36 of SEQ ID NO: 8, 10, 12 or 13 or residues 23 to 37 of SEQ ID NO: 702.
  • a grafted coiled-coil scaffold is created by grafting a second helical peptide ligand onto a second helix at a position defined for instance by residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13; residues 62 to 69 of SEQ ID NO: 8 or 10 or 12 or 13 or residues 62 to 70 of SEQ ID NO: 702.
  • a grafted coiled-coil scaffold is created by isomorphic replacement of solvent exposed residues of a loop (for example, inserted or replaced at position between residues 55 to 58 of SEQ I D NO: 8 or 10 or 12 or 13, or residues 11 to 13 or 55 to 57 of SEQ ID NO: 702) with solvent facing residues of the loop peptide ligand.
  • a grafted coiled-coil scaffold is created by isomorphic replacement of those residues (for example, at position between residues 55 to 57 of SEQ ID NO: 8 or 10 or 12 or 13) of a loop which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the loop peptide ligand.
  • a grafted coiled-coil scaffold comprises a loop peptide ligand in a loop portion of the coiled- coil scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a coiled-coil domain generally show a high occurrence of insertions across the HMM logo.
  • the loops connecting helices sheets are ideal places for loop grafting of peptide ligands because the loop regions rarely have any conserved residues.
  • a grafted coiled-coil scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region.
  • a loop peptide ligand may be located in the coiled-coil scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to position at residue 54, 55, 56 or 57 of SEQ ID NO: 8 or 10 or 12 or 13, or residues 11 , 12 or 13 or residues 55, 56 or 57 of SEQ ID NO: 702
  • a loop peptide ligand may be inserted into the coiled-coil scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to position at residue 56, 57, 58 and 59 of variants of SEQ ID NO: 8 or 10 or 12 or 13 or residues 11 , 12 or 13 or residues 55, 56 or 57 of SEQ ID NO: 702.
  • a grafted coiled-coil scaffold comprises a helical peptide ligand in a helical portion of the coiled-coil scaffold, for example, isomorphically replaced into a specific location (for example, at position between residues 16 to 54 or at position between residues 59 to 83 of SEQ ID NO: 8 or 10 or 12 or 13 or at position between residues 23 to 37 or at position between residues 62 to 70 of SEQ ID NO: 702) of a helix (thus preserving the existing helical structure).
  • the long helix in a coiled-coil scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.
  • a grafted coiled-coil scaffold also may contain a peptide ligand within a helical region of the scaffold (i.e., a“helical peptide” is a peptide which is positioned in a helical structure of the scaffold.
  • a grafted coiled-coil scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to position at residue 83 of variants of SEQ ID NO: 8 or 10 or 12 or 13.
  • a grafted coiled-coil scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 16 of variants of SEQ ID NO: 8 or 10 or 12 or 13.
  • Figure 9 shows an example of coiled-coil scaffold created by a grafting two helical peptide ligands in two helix regions. The first peptide ligand binds to beta-catenin and the second peptide ligand binds to MDM2-amino terminal domain. Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera.
  • a coiled-coil scaffold may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands.
  • aromatic residues in the coiled-coil scaffold may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of coiled-coil scaffold sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions.
  • lysine residues in the coiled-coil scaffold may be replaced by a different residue, such as arginine to prevent unwanted ubiquitination and subsequent degradation.
  • a different residue such as arginine to prevent unwanted ubiquitination and subsequent degradation.
  • the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC).
  • the coiled-coil scaffolds of a chimeric protein of the second aspect may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the coiled-coil scaffolds.
  • a grafted coiled-coil scaffold may comprise a coiled-coil scaffold with the amino acid sequence of residues 1 to 94 of SEQ ID NO: 702 (PPX59 of Table 12 without the HA Tag).
  • a target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of first helix (helix 1 ; residues 23 to 37 of SEQ ID NO: 702), second helix (helix 2; residues 62 to 70 of SEQ ID NO: 702), first loop (loop 1 ; residues 11 to 13 of SEQ ID NO: 702) and second loop (loop 2; residues 55 to 57 of SEQ ID NO: 702) of the coiled- coil scaffold.
  • the target-binding peptide ligand is located in the first helix or the first loop.
  • the E3 ligase-binding peptide ligand is located in the second loop or second helix.
  • the target-binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the second helix or the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop.
  • Suitable target-binding peptide ligands include b-catenin binding ligands, for example helical beta-catenin binding sequence from the protein AXIN, such as ILxxHV and variants thereof, and peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE or GSEELEALEALELDEA and variants thereof, and KRAS binding ligands, for example alpha-helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as TNxxKxxE and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS or HYPWFKARLYPL and variants thereof;
  • Suitable E3 ligase-binding peptide ligands include SCF Skp2 binding sequences from p27, such as
  • LRPVAMVRPWVR LRPVAMVRPWVR, and variants thereof, COP1 binding sequences from Trib, such as SDQIVPEYQE, and variants thereof, UBR5 binding sequences from PAM2, such as
  • LSVNAPEFYP and variants thereof, beta-TRCP binding sequences from CDC25B, such as TEEDDGFVDI, and variants thereof, and MDM2 binding sequences from p53, such as FSxxWxxL and variants thereof.
  • a grafted coiled-coil scaffold of the second aspect may comprise an amino acid sequence shown in Table 12 (SEQ ID NOs: 683-706) or a variant of an amino acid sequence shown in Table 12.
  • Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 12 are replaced by a different peptide ligand. Suitable peptide ligands are described below.
  • Variants may also include variants in which the coiled-coil scaffold sequence in a reference amino acid sequence of Table 12 is replaced by a different coiled-coil scaffold sequence. Suitable coiled-coil scaffold sequences are described above.
  • a third aspect relates to chimeric proteins that comprise Affibody scaffolds (i.e. grafted Affibody scaffolds).
  • Affibody scaffolds i.e. grafted Affibody scaffolds.
  • One or more peptide ligands are located in the Affibody scaffold of the chimeric protein, for example at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of SEQ ID NO: 16, 18, and 20 to 53.
  • Affibody is a well-known and well-characterized example of a structural fold. Affibodies are among the most extensively used model systems in the area of protein folding and design. Affibody molecules were originally derived from the B-domain in the immunoglobulin-binding region of staphylococcal protein A.
  • the B-domain is a relatively short cysteine- free peptide of 58 amino acids that is folded into a three-helical bundle structure.
  • the engineered Z- domain retained its affinity for the Fc part of the antibody while the weaker affinity for the Fab region was almost completely lost. (FEBS Letters 584 (2010) 2670-2680).
  • the invention adopts the well understood sequence/structure relationships of Affibody domain of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.
  • an“Affibody scaffold” refers to a polypeptide which is composed of one or more three-helix bundles.
  • An Affibody scaffold with Z domain may be preferred over an Affibody scaffold with B domain.
  • An Affibody scaffold is 30-90 amino acids in length, such as 35-85, 40-80, 50-70, and 55-65 amino acids in length, e.g., 58 amino acids in length.
  • An Affibody scaffold has a molecular weight in the range of 4 kDa - 10 kDa, 5 - 9 kDa, 6 - 8 kDa, or about 6.7 kDa.
  • a representative Affibody scaffold may be 58 amino acids in length.
  • Fig. 12 depicts a representative Affibody scaffold. (PDB code -2KZJ).
  • Suitable Affibody scaffolds may include the Affibody domain selected from the sequences (SEQ ID NO: 20 to 53) in Tables 14 and 15 without limitation:
  • PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable Affibody scaffolds, and it may include without any limitation: 2KZI, 2KZJ, 3MZW, 2M5A, 1 H0T, 1 LP1 , 5EFW, 2B87, 2B89 and 20TK, 5U3D 5U5F 5U5M 5U6A 1ZXG 2B88 1 DEE 1 DEE 5U4Y 5U4Y 4HJG 4HKZ 4IOI 5CBO 5H7C 5H79 5H7A 5H7B 4NPF 5H7C 5H7A 5H7A 5H7A 5H7B 5H7B 5H79 1 BDC 1 BDD 1Q2N 1SS1 2JWD 2SPZ 4NPF 5H7C 1 FC2 3MZW 5CBN 5COC 5XBY 5EWX 5H7B 5H77 5H77 5H75 5H76 5H77 5H77
  • Table 15 shows the alignment score with consensus sequence as the query with a representative sequence of the Affibody scaffold as the subject. Similar comparisons may be done with other representative sequences to obtain relative residue positions and alignment scores.
  • Figure 14 shows a representative example of a grafted Affibody scaffold with peptide ligands grafted on to them, indicated by shaded loops.
  • a threading program such as SwissModeller (Nucleic Acids Res. 46 (W1), W296- W303 (2016)) and an ab initio folding program such as l_tasser (Protein structure and function prediction. Nature Methods, 12: 7-8 (2015)) or Robetta (Nucleic Acids Research,
  • Suitable Affibody scaffolds include the Affibody domains shown in Figure 13 and Tables 14- 16 (SEQ ID NOs: 16, 18, 20 to 53 and residues 1 to 66 of SEQ ID NO: 715) or variants thereof.
  • Suitable positions for peptide ligand insertions or replacements for the Affibody scaffolds for variants of sequence listed in Figure 13 and Tables 14 and 15 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID 16, 18, and 20 to 53.
  • one or more peptide ligands may be inserted or replaced at positions between residues 20 to 22; between residues 38 to 39; between residues 5 to 19; between residues 23 to 37; between residues 40 to 56 of SEQ ID Nos: 16, 18, and 20 to 53 of the Affibody scaffold or between residues 5 to 16 (helix 1), 23 to 37 (helix 2), 20 to 22 (loop 1) or 37 to 41 (loop 2) of SEQ ID NO: 715.
  • Figure 15 shows a model of Affibody scaffold-mediated KRAS ubiquitination through the Cul3-Keap1 E3 ubiquitin ligase complex.
  • the Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER®, and the energy of the resulting proteins was minimized by UCSF Chimera.
  • An Affibody scaffold may comprise the amino acid sequence of SEQ ID NO: 16 or a variant thereof
  • the above Affibody scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 17 or a variant thereof.
  • Preferred Affibody scaffolds lack lysine residues, for example, to avoid unwanted
  • the lysine residues in an Affibody domain may be replaced by (Glutamic acid) E, (Asparagine) N, (Aspartic acid) D, (Glutamine) Q, (Leucine) L, (Threonine) T, (Serine) S, Valine (V), or (Arginine) R to generate an Affibody scaffold.
  • a suitable lysine- free Affibody scaffold may comprise the amino acid sequence of SEQ ID NO: 1 or a variant thereof.
  • an Affibody scaffold may have 4-5 lysine residues. In some embodiments, all lysine residues of an Affibody scaffold are replaced with non-lysine residues. In some embodiments, residue Lys4 of an Affibody scaffold is replaced with Aspartic acid or Asparagine. In some embodiments, residue Lys7 of an Affibody scaffold is replaced with glutamic acid or threonine. In some embodiments, residue Lys27 of an Affibody scaffold is replaced with glutamine, glutamic acid or Arginine. In some
  • residue Lys49 forms a salt bridge with residue Glu15 at a1 helix of an
  • residue Lys50 of an Affibody scaffold is replaced with arginine or glutamine.
  • residue Lys58 at the C-terminus of an Affibody scaffold is replaced with a residue selected from a group consisting of glutamine, arginine or aspartic acid.
  • residue Glu15 maybe mutated to Gln15 to enable better hydrophobic packing with residue Val49.
  • any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold.
  • a lysine-attenuated Affibody scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids may comprise the amino acid sequence of SEQ ID NO: 18 or a variant thereof.
  • a lysine-attenuated Affibody scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 19 or a variant thereof.
  • the a-helix 1 (a1) of an Affibody scaffold is located within the positions corresponding to residues 5 to 19 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53 and 5 to 16 of SEQ ID NO: 715 (helix 1).
  • the a-helix 2 (a2) of an Affibody scaffold is located within the positions corresponding to residues 23 to 37 of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NOs: 20 to 53 and SEQ ID NO: 715 (helix 2).
  • the a-helix 3 (a3) of an Affibody scaffold is located within the positions corresponding to residues 40 to 56 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53.
  • the disordered regions or the loop regions of an Affibody scaffold that connect the a-helices are located within the positions corresponding to residues 20 to 22 and 38 to 39 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53.
  • the loop 1 of an Affibody scaffold that connects the a-helices a1 and a2 is located within the positions corresponding to residues 20 to 22 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53 and SEQ ID NO: 715.
  • the loop 2 of an Affibody scaffold that connect the a-alpha a2 and a3 are located within the positions corresponding to residues 38 to 39 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53 and residues 37 to 41 of SEQ ID NO: 715.
  • a grafted Affibody scaffold comprises a peptide ligand within a loop (a“loop peptide”) (for example, inserted into or replaced at a position between residues 20 to 21 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53).
  • a grafted Affibody scaffold comprises a peptide ligand within a helix (a“helix peptide”) (for example, inserted into or replaced at a position between residues 40 to 56 of SEQ ID NO: 16 and SEQ ID NO: 18 and SEQ ID NOs: 20 to 53).
  • a grafted Affibody scaffold comprises a first and a second peptide ligands in two different loops, or at the two different termini of the scaffold, or in a loop and at a terminus of the scaffold.
  • a grafted Affibody scaffold comprises a peptide ligand in the loop 1 (for example, inserted into or replaced at a position between residues 20 to 22 of SEQ ID NO: 16 or 18 or 20 to 53 or 715) that connect the a1 and a2 helices.
  • a grafted Affibody scaffold comprises a peptide ligand in the loop 2 (for example, inserted into or replaced at a position between residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 37 to 41 of SEQ ID NO: 715) that connect the a2 and a3 helices.
  • a grafted Affibody scaffold to comprise a peptide ligand in the loop 1 (residues 20 to 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715) that connect the a1 and a2 helices rather than the loop 2 (residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 37 to 41 of SEQ ID NO: 715) that connects the a2 and a3 helices, as grafting onto loop 1 may have less impact on the thermodynamic stability of the scaffold.
  • a grafted Affibody scaffold comprises a first peptide ligand in the loop 1 (residues 20 and 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715) and a second peptide ligand in the loop 2 (residues 38 and 39 of SEQ ID NO: 16 or 18 or 20 to 53 residues 37 to 41 of SEQ ID NO: 715).
  • a grafted Affibody scaffold comprises a first peptide ligand and a second peptide ligand in respective positions and relative orientations such that the two peptide ligands do not interfere sterically with each other and in such a way that they are displayed such that they face in opposite directions to each other.
  • This type of relative arrangement ensures that the grafted peptides of the scaffold can each interact with their cognate binding partners, i.e. , a target protein and an E3 ligase.
  • a grafted Affibody scaffold comprises a first peptide ligand grafted onto a loop (e.g. residues 20 to 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715) and a second peptide ligand grafted onto a helix (e.g. residues 40 to 46 of SEQ ID NO: 16 or 18 or 20 to 53; or residues 5 to 16 or 23 to 37 of SEQ ID NO: 715).
  • a loop e.g. residues 20 to 21 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 of SEQ ID NO: 715
  • a second peptide ligand grafted onto a helix e.g. residues 40 to 46 of SEQ ID NO: 16 or 18 or 20 to 53; or residues 5 to 16 or 23 to 37 of SEQ ID NO: 715.
  • a grafted Affibody scaffold comprises a first helical peptide ligand grafted onto a first helix (e.g. residues 5 to 19 of SEQ ID NO: 16 or 18 or 20 to 53; residues 5 to 16 of SEQ ID NO: 715) and a second helical peptide ligand grafted onto a second helix (e.g. residues 40 to 56 of SEQ ID NO: 16 or 18 or 20 to 53; or residues 37 to 41 of SEQ ID NO: 715).
  • a first helical peptide ligand grafted onto a first helix e.g. residues 5 to 19 of SEQ ID NO: 16 or 18 or 20 to 53; residues 5 to 16 of SEQ ID NO: 715
  • a second helical peptide ligand grafted onto a second helix e.g. residues 40 to 56 of SEQ ID NO: 16 or 18 or 20 to 53; or residues 37 to 41 of SEQ ID
  • a grafted Affibody scaffold is created by grafting a first helical peptide ligand onto a first helix at a position defined for instance by residues 5 to 19 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 5 to 16 of SEQ ID NO: 715.
  • a grafted Affibody scaffold is created by grafting a second helical peptide ligand onto a second helix at a position defined for instance by residues 40 to 56 or 23 to 37 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 23 to 37 of SEQ ID NO: 715.
  • a grafted Affibody scaffold is created by isomorphic replacement of solvent exposed residues of a loop (e.g residues 20 to 22 or residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715) or in some embodiments a grafted Affibody scaffold is created by insertion of residues within the loop (e.g. between residues 20 to 22 or between residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715) with solvent facing residues of the loop peptide ligand.
  • a grafted Affibody scaffold is created by isomorphic replacement of those residues (e.g. residues 20 to 22 or residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715) of a loop which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the loop peptide ligand.
  • residues e.g. residues 20 to 22 or residues 38 to 39 of SEQ ID NO: 16 or 18 or 20 to 53 or residues 20 to 22 or 37 to 41 of SEQ ID NO: 715
  • a grafted Affibody scaffold comprises a loop peptide ligand in a loop portion of the Affibody scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in an Affibody domain generally show a high occurrence of insertions across the HMM logo.
  • the loops connecting the alpha-helices are ideal places for loop grafting of peptide ligands because the loop regions do not have any conserved residues.
  • a grafted Affibody scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region.
  • a loop peptide ligand may be located in the Affibody scaffold immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 20 of SEQ ID NO: 16 or 18 or 20 to 53 or 715.
  • a loop peptide ligand may be inserted into the Affibody scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 21 of variants of SEQ ID NO: 16 or 18 or 20 to 53 or 715.
  • a grafted Affibody scaffold comprises a helical peptide ligand in a helical portion of the Affibody scaffold, for example, isomorphically replaced into a specific location of a helix (thus preserving the existing helical structure).
  • the long helix in Affibody scaffold is well presented and solvent exposed hence ideal for helical grafting of peptide ligands.
  • a grafted Affibody scaffold also may contain a peptide ligand within a helical region of the scaffold (i.e., a“helical peptide” is a peptide which is positioned in a helical structure of the scaffold.
  • a grafted Affibody scaffold may contain a helical peptide ligand immediately after (i.e. adjacent in a C terminal direction or having a peptidyl linkage to the carboxyl group of) the residue corresponding to 40 of variants of SEQ ID NO: 16 or 18 or 20 to 53.
  • a grafted Affibody scaffold may contain a helical peptide ligand immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to residue 41 of variants of SEQ ID NO: 16 or 18 or 20 to 53.
  • a grafted Affibody scaffold may contain a helical peptide ligand immediately after a proline residue in a helix.
  • a grafted Affibody scaffold may comprise an Affibody scaffold with the amino acid sequence of residues 1 to 66 of SEQ ID NO: 715 (PPX86 of Table 16 without the HA Tag).
  • a target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first helix (helix 1 ; residues 5 to 16 of SEQ ID NO: 715), second helix (helix 2; residues 23 to 37 of SEQ ID NO: 715), first loop (loop 1 ; residues 20 to 22 of SEQ ID NO: 715), and second loop (loop 2; residues 37 to 41 of SEQ ID NO: 715) of the Affibody scaffold.
  • the peptide ligand is located in any of the first or second helix or first loop of the Affibody scaffold.
  • the E3 ligase-binding peptide ligand is located in the second helix or second loop of the Affibody scaffold.
  • the target binding peptide ligand may be in the first helix and the E3 ligase-binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the second helix and the E3 ligase-binding peptide ligand may be in the second loop; the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop or the target-binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second helix.
  • Suitable target-binding peptide ligands include b-catenin binding ligands, for example helical beta-catenin binding sequence from the protein AXIN, such as ILxxHV, AxxILDXHV or ILDxHV and variants thereof, peptides from BCL9, such as TLxxlQxxL, LxTLxxlQ, and SLxxlxxML and variants thereof, and peptides from ARC
  • adenomatous polyposis coli such as SEELEALEALELDE, SEELEALEALELDEAS or GGSEELEALEALELDEASGS and variants thereof
  • KRAS binding ligands for example alpha-helical sequences from the protein SOS1 (Son of sevenless homolog 1), such as TNxxKxxE or IxxTNxxKTXE and variants thereof, and peptides from RBP, such as
  • Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, LDPETGELLS and GLDPETGELLG and variants thereof.
  • a grafted Affibody scaffold of the third aspect may comprise an amino acid sequence shown in Table 16 (SEQ ID NOs: 707-715) or a variant of an amino acid sequence shown in Table 16.
  • Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 16 are replaced by a different peptide ligand. Suitable peptide ligands are described below.
  • Variants may also include variants in which the Affibody scaffold sequence in a reference amino acid sequence of Table 16 is replaced by a different Affibody scaffold sequence. Suitable Affibody scaffold sequences are described above.
  • the Affibody scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Affibody scaffolds.
  • a fourth aspect relates to chimeric proteins that comprise Trefoil scaffolds (i.e. grafted Trefoil scaffolds).
  • One or more peptide ligands are located in the Trefoil scaffold of the chimeric protein, for example at two or more loops (for example, at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of SEQ ID NOs: 54, 56 or 28 to 78) of SEQ ID NOs: 54, 56 or 28 to 78.
  • Trefoil domains are among the most extensively used model systems in the area of protein folding and design. (Proc Natl Acad Sci U S A. 2013 Feb 5;110(6) :2135-9). A detailed analysis of the geometry and architecture of the b-trefoil fold was done by Chothia et al. (J Mol Biol. 1992 Jan 20; 223(2) :531 -43; Protein Sci. 2001 Dec; 10(12): 2587-2599.). A Trefoil domain is composed of six anti parallel beta strands closed off at one end by b-hairpin structures and exhibiting a threefold rotational symmetry at the tertiary structure level.
  • the invention adopts the well understood sequence-structure relationships of Trefoil domains of proteins, and provides a Trefoil domain as a discrete scaffold onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.
  • A“Trefoil scaffold” is a small, soluble, stable protein that exhibits a threefold rotational symmetry.
  • a Trefoil scaffold is composed of six anti-parallel beta-strands closed off at one end by b-hairpin structures (Fig. 18).
  • a Trefoil scaffold has a length in the amino acid range of 47-140, 140-155, 140-233 amino acids.
  • a Trefoil scaffold is typically 15265Da, and is in the MW range of 5120-15265, 15265-18034, 15270-25520 Da.
  • a representative Trefoil scaffold is one which is about 140 amino acids in length.
  • Figure 18 depicts a representative Trefoil scaffold, (PDB code 2AFG) which has b strands tilted at -56° to the barrel axis, a barrel diameter of ⁇ 16A, and a b- barrel shear number (i.e., the stagger of the strands in the barrel) of 12 (J Mol Biol. 1992 Jan 20; 223(2):531-43.).
  • the Trefoil domain of Human acidic fibroblast growth factor (FGF-1) is a non-limiting example of a Trefoil scaffold which exhibits a characteristic pseudo-threefold axis of symmetry when viewed down the b-barrel axis.
  • the monomeric structural unit of this threefold symmetry consists of a pair of anti parallel b-sheets, referred to as a b hairpin.
  • This architecture is composed of three repeating“trefoil” subdomains, each of 40-50 amino acids in length and composed of a pair of anti parallel b-hairpin structures. Within the structure are a total of 12 b-strands (numbered #1-12) and 11 reverse turns. (Protein Sci. 201221(12): 1911-1920.)
  • Suitable Trefoil scaffolds may include the Trefoil domain selected from the following, without limitation: fibroblast growth factors (J Biochem. 1991 110(3):360-3; Science 1991
  • SEQ ID NO: 54 is a designed modular protein made of three identical repetitions.
  • the modular repeat unit is blasted in Uniprot and presented in Table 17 with the top 20 best matches.
  • Sequence numbers refer to the position of the Trefoil repeat unit in the context of the full-length protein.
  • Suitable Trefoil scaffolds include the Trefoil domains shown in Figure 19 and Tables 17, 19 and 20 (SEQ ID NOs: 54, 55 or 28 to 78 and residues 1 to 149 of SEQ ID NO: 722) or variants thereof.
  • Suitable positions for peptide ligand insertions or replacements for the Trefoil scaffolds for variants of sequence listed in Figure 19 and Tables 17, 19 and 20 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID 54, 56 or 28 to 78 or 722.
  • one or more peptide ligands may be inserted or replaced at positions between residues 21 to 22 or between residues 93 to 94 or between residues 13 to 14 or between residues 82 to 83 or between residues 10 to 14 or between residues 35 to 36 or between residues 106 and 107 or between residues 59 to 60 or between residues 129 to 130, or between residues 21 to 28 or between residues 94 to 98 of SEQ ID NO: 54, 56 or 28 to 78.
  • one or more peptide ligands may be inserted or replaced at positions between residues in a trefoil scaffold corresponding to residues 47 to 49 (loop 1) or residues 116 to 118 (loop 2) of SEQ ID NO: 54, 56 or 58 to 78 or residues 49 to 51 (loop 1) or residues 117 to 119 (loop 2) of SEQ ID NO: 722.
  • PDB codes from the protein data bank (https://www.rcsb.org/) identify representative structures of suitable Trefoil scaffolds and it may include without any limitation: 1 PZZ, 1Q03, 1Q04, 2AQZ, 3JUT, 3K1X, 3H6Q, 3H6R, 4I4R, 40W4 and 4XKI.
  • a Trefoil scaffold may comprise the amino acid sequence of SEQ ID NO: 54 or a variant thereof.
  • the secondary structure of a representative example of a Trefoil scaffold is shown in Figure 19.
  • the sequence in figure 19 corresponds to SEQ ID NO: 54.
  • the beta-strands represented as arrows and italic text, helices as rectangles and bold text. Unstructured regions connect the secondary structure elements.
  • the helices are very short and are part of the long beta- turns and are not present in all trefoils.
  • the above Trefoil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 55 or a variant thereof.
  • Preferred Trefoil scaffolds lack lysine residues, for example, to avoid unwanted
  • the lysine residues in a Trefoil domain may be replaced by E, N, Q, L, T, S or R to generate a Trefoil scaffold.
  • a suitable lysine-free Trefoil scaffold may comprise the amino acid sequence of SEQ ID NO: 54 or a variant thereof.
  • each of the three repeating sub-domains of the trefoil scaffold has two lysine residues.
  • all six lysines of the Trefoil scaffold are replaced with non-lysine residues.
  • residue Lys 6 of a first repeating subdomain of a Trefoil scaffold is replaced with Threonine or Arginine.
  • residue Lys 14 of a first repeating subdomain of a Trefoil scaffold is replaced with Leucine or Arginine.
  • residue Lys53 of a second repeating subdomain of a Trefoil scaffold is replaced with Threonine or Arginine.
  • residue Lys 61 of a second repeating subdomain of a Trefoil scaffold is replaced with Leucine or Arginine.
  • residue Lys 100 of a third repeating domain of a Trefoil scaffold is replaced with Threonine or Arginine.
  • residue Lys 108 of a third repeating subdomain of a Trefoil scaffold is replaced with Leucine or Arginine.
  • any replaced Lys residue may be combined with any or all other replaced Lys residues in a single scaffold.
  • a lysine- attenuated Trefoil scaffold, in which all Lys residues or most of the Lys residues have been replaced with other amino acids may comprise the amino acid sequence of SEQ ID NO: 56 or a variant thereof.
  • a lysine-free Trefoil scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 57 or a variant thereof.
  • the b-1 strand of a Trefoil scaffold is located within the positions corresponding to residues 4 to 9 of SEQ ID NO: 54 or 4 to 9 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the b-2 strand of a Trefoil scaffold is located within the positions corresponding to residues 14 to 18 of SEQ ID NO: 54 or 14 to 18 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the b-3 strand of a Trefoil scaffold is located within the positions corresponding to residues 29 to 32 of SEQ ID NO: 54 or 29 to 32 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the b-4 strand of a Trefoil scaffold is located within the positions corresponding to residues 42 to 48 of SEQ ID NO: 54 or 42 to 48 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the b-5 strand of a Trefoil scaffold is located within the positions corresponding to residues 51 to 56 of SEQ ID NO: 54 or 51 to 56 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the b-6 sheet of a Trefoil scaffold is located within the positions corresponding to residues 62 to 65 of SEQ ID NO: 54 or 62 to 65 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the b-7 strand of a Trefoil scaffold is located within the positions corresponding to residues 76 to 79 of SEQ ID NO: 54 or 76 to 79 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the b-8 strand of a Trefoil scaffold is located within the positions corresponding to residues 89 to 93 of SEQ ID NO: 54 or 89 to 93 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the b-9 strand of a Trefoil scaffold is located within the positions corresponding to residues 99 to 103 of SEQ ID NO: 54 or 99 to 103 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the b-10 strand of a Trefoil scaffold is located within the positions corresponding to residues 109 to 1 12 of SEQ ID NO: 54 or 109 to 112 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the b-1 1 strand of a Trefoil scaffold is located within the positions corresponding to residues 123 to 126 of SEQ ID NO: 54 or 123 to 126 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the b-12 strand of a Trefoil scaffold is located within the positions corresponding to residues 136 to 140 of SEQ ID NO: 54 or 136 to 140 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the b-1 1 strand of a Trefoil scaffold is located within the positions corresponding to residues 123 to 126 of SEQ ID NO: 54 or 123 to 126 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78
  • the disordered regions or the loop regions of a Trefoil scaffold are located within the positions corresponding to residues 10 to 14 of SEQ ID NO: 54 or 10 to 14 of SEQ ID NO:
  • SEQ ID NO: 58 to 78 23 to 28 of SEQ ID NO: 54 or 23 to 28 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 33 to 36 of SEQ ID NO: 54 or 33 to 36 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 57 to 61 of SEQ ID NO: 54 or 57 to 61 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 70 to 75 of SEQ ID NO: 56 or 70 to 75 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 80 to 83 of SEQ ID NO: 54 or 80 to 83 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 94 to 97 of SEQ ID NO: 54 or 94 to 97 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78, 104 to 108 of SEQ ID NO: 54 or 104 to 108 of SEQ ID NO: 56 or of SEQ ID
  • the first loop of the Trefoil scaffold is located within the positions corresponding to residues 10 to 14 of SEQ ID NO: 54 or 10 to 14 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the second loop of the Trefoil scaffold is located within the positions corresponding to residues 23 to 28 of SEQ ID NO: 54 or 23 to 28 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the third loop of the Trefoil scaffold is located within the positions corresponding to residues 33 to 36 of SEQ ID NO: 54 or 33 to 36 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the fourth loop of the Trefoil scaffold is located within the positions corresponding to residues 57 to 61 of SEQ ID NO: 54 or 57 to 61 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78 or 49 to 51 of SEQ I D NO: 722.
  • the fifth loop of the Trefoil scaffold is located within the positions corresponding to residues 70 to 75 of SEQ ID NO: 54 or 70 to 75 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the sixth loop of the Trefoil scaffold is located within the positions corresponding to residues 80 to 83 of SEQ ID NO: 54 or 80 to 83 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the seventh loop of the Trefoil scaffold is located within the positions corresponding to residues 94 to 97 of SEQ ID NO: 54 or 94 to 97 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the eighth loop of the Trefoil scaffold is located within the positions corresponding to residues 104 to 108 of SEQ ID NO: 54 or 104 to 108 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78.
  • the ninth loop of the Trefoil scaffold is located within the positions corresponding to residues 117 to 122 of SEQ ID NO: 54 or 117 to 122 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78 or 117 to 119 of SEQ ID NO: 722.
  • the tenth loop of the Trefoil scaffold is located within the positions corresponding to residues 127 to 130 of SEQ ID NO: 54 or 127 to 130 of SEQ ID NO: 56 or of SEQ ID NO: 58 to 78,
  • a grafted Trefoil scaffold comprises a peptide ligand within a loop (a “loop peptide”).
  • a grafted Trefoil scaffold comprises a helical peptide ligand within a loop (a“helix peptide”).
  • a grafted Trefoil scaffold comprises a peptide ligand within a b strand (a“strand peptide”).
  • a grafted Trefoil scaffold comprises a peptide ligand that is less than or equal to 30 amino acids in length.
  • a grafted Trefoil scaffold is created by inserting peptide ligands into one or more of loops or strands of the Trefoil scaffold.
  • a grafted Trefoil scaffold comprises a first peptide ligand and a second peptide ligand, both of which are loop peptide ligands.
  • a grafted Trefoil scaffold comprises a first peptide ligand which is a helix peptide and a second peptide ligand which is a loop peptide ligand.
  • a grafted Trefoil scaffold comprises a first peptide ligand which is a strand peptide and a second peptide ligand which is a loop peptide ligand.
  • a grafted Trefoil scaffold comprises a first peptide ligand which is a strand peptide and a second peptide ligand which is a helix peptide ligand.
  • a grafted Trefoil scaffold comprises more than one loop peptide ligand. In some embodiments, a grafted Trefoil scaffold comprises more than one strand peptide ligand. In some embodiments, a grafted Trefoil scaffold comprises more than one helix peptide ligand.
  • a grafted Trefoil scaffold comprises a first peptide ligand between positions 21 and 22 of SEQ ID NO: 54 or 56 or 58 to 78 in the Trefoil scaffold. In some embodiments, a grafted Trefoil scaffold comprises a second peptide ligand between the positions 93 and 94 of the Trefoil scaffold.
  • a grafted Trefoil scaffold comprises a first peptide ligand between residues corresponding to residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722 in the Trefoil scaffold.
  • a grafted Trefoil scaffold comprises a second peptide ligand between the residues
  • a grafted Trefoil scaffold comprises a first peptide ligand and a second peptide ligand in respective positions and orientations such that the two peptide ligands so that they do not come into contact with each other. It is preferred that a grafted Trefoil scaffold comprises first and second peptide ligands that do not interfere sterically with each other. This ensures that the grafted peptides of the scaffold can each interact with their cognate first and second ligand binding partners, i.e. , a target protein and an E3 ligase.
  • a grafted Trefoil scaffold comprises a first peptide ligand within a first loop (residues 21 to 22 of SEQ ID NO: 54 or 56 or 58 to 78) and a second peptide ligand within a second loop (residues 93 to 94 of SEQ ID NO: 54 or 56 or 58 to 78).
  • a grafted Trefoil scaffold comprises a first loop peptide ligand in the loop region starting at position 21 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold comprises a second loop peptide ligand in the loop region starting at position 98 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold comprises a first peptide ligand within a first loop (e.g. loop 1 ; residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722) and a second peptide ligand within a second loop (e.g. loop 2;
  • a grafted Trefoil scaffold may comprises a first loop peptide ligand between residues of the Trefoil scaffold corresponding to residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722 and a second loop peptide ligand between residues of the Trefoil scaffold corresponding to residues 116 to 118 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 117 to 119 of SEQ ID NO: 722.
  • a grafted Trefoil scaffold may be created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722
  • a grafted Trefoil scaffold may be created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 47 and 49 of SEQ ID NO: 54 or 56 or 58 to 78 or residues 49 and 51 of SEQ ID NO: 722.
  • a grafted Trefoil scaffold comprises a first loop peptide ligand between positions 13 and 14 of SEQ ID NO: 54 or 56 or 58 to 78 of the Trefoil scaffold and a second loop peptide ligand between the positions 82 and 83 of SEQ ID NO: 54 or 56 or 58 to 78 of the Trefoil scaffold.
  • a grafted Trefoil scaffold is created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 10 to 14 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold is created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 80 to 83 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold is created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 35 to 36 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold is created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 106 to 107 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold is created by inserting a first loop peptide ligand in to a first loop at a position defined for instance by residues 59 to 60 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold is created by inserting a second loop peptide ligand in to a second loop at a position defined for instance by residues 129 to 130 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted Trefoil scaffold is created by isomorphic replacement of solvent-exposed residues of a loop (for example, residues 21 to 28 of SEQ ID NO: 54 or 56 or 58 to 78) with solvent-facing residues of the loop peptide ligand.
  • a grafted Trefoil scaffold is created by isomorphic replacement of those residues (for example, residues 94 to 98 of SEQ ID NO: 54, 56 or 58 to 78) of a loop which are solvent exposed and not buried into the hydrophobic core of the scaffold with solvent facing residues of the loop peptide ligand.
  • a grafted Trefoil scaffold comprises a loop peptide ligand in a loop portion of the Trefoil scaffold, for example, inserted into a specific location of a loop (thus providing an extended loop). Loops in a Trefoil domain generally show a high occurrence of insertions across the HMM logo. The loops connecting beta sheets are ideal places for loop grafting of peptide ligands because the loop regions do not have any conserved residues.
  • a grafted Trefoil scaffold comprises a loop peptide ligand which when inserted into the disordered or loop region becomes a part of the loop region. For example, a loop peptide ligand may be located in the Trefoil scaffold immediately after (i.e.
  • a loop peptide ligand may be inserted into the Trefoil scaffold immediately before (i.e. adjacent in an N terminal direction or having a peptidyl linkage to the amino group of) the residue corresponding to 94 of SEQ ID NO: 54 or 56 or 58 to 78.
  • a grafted trefoil scaffold may comprise a trefoil scaffold with the amino acid sequence of residues 1 to 149 of SEQ ID NO: 722 (PPX93 of Table 20 without the HA Tag).
  • a target-binding peptide ligand and the E3 ligase-binding peptide ligand may be grafted into any two of the first, second, third and fourth loops of the trefoil scaffold.
  • the peptide ligand is located in the first or second loop or the helical region.
  • the E3 ligase-binding peptide ligand is located in the second or first loop.
  • the target binding peptide ligand may be in the first loop and the E3 ligase-binding peptide ligand may be in the second loop or the target-binding peptide ligand may be in the second loop and the E3 ligase-binding peptide ligand may be in the first loop.
  • Suitable target-binding peptide ligands include b-catenin binding ligands, for example peptides from A PC (Adenomatous polyposis coli), such as SEELEALEALELDE, SEELEALEALELDEAS and
  • GSEELEALEALELDEASGS and variants thereof peptides from AXIN, such as ILxxHV, AxxILDxHV, ILDVHV, or ILDxHV and variants thereof, peptides from BCL9, such as
  • KRAS binding ligands for example peptides from KBL, such as PLYISY, PLYISYDPV and PLYISYPV and variants thereof, and peptides from RBP, such as SHYPWFKARLYPLS, GHYPWFKARLYPLS, GHYPWFKARLYPL and HYPWFKARLYPL and variants thereof.
  • Suitable E3 ligase-binding peptide ligands include Cul3-KEAP1 binding sequences from NRF2, such as LDPETGEL, and GLDPETGELL and variants thereof.
  • a grafted trefoil scaffold of the fourth aspect may comprise an amino acid sequence shown in Table 20 (SEQ ID NOs: 716-722) or a variant of an amino acid sequence shown in Table 20.
  • Variants of a reference sequence are described above and may include variants in which the one or both of the target-binding peptide ligand and the E3 ligase-binding peptide ligand in a reference amino acid sequence of Table 20 are replaced by a different peptide ligand. Suitable peptide ligands are described below.
  • Variants may also include variants in which the trefoil scaffold sequence in a reference amino acid sequence of Table 20 is replaced by a different trefoil scaffold sequence. Suitable trefoil scaffold sequences are described above.
  • Figure 20 shows an example of Trefoil -scaffold created by a grafting two loop peptide ligands onto two loop regions. Loop insertions were modelled with MODELLER (Current Protocols in Bioinformatics 54, John Wiley & Sons, Inc., 5.6.1-5.6.37, 2016) and the energy of the resulting proteins was minimized by UCSF Chimera (J Comput Chem. 2004
  • the Trefoil scaffolds of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the Trefoil scaffolds (which have no native functions in binding to protein or DNA).
  • a fifth aspect relates to PDZ scaffolds (i.e. grafted PDZ scaffolds).
  • One or more peptide ligands are located in the PDZ scaffold of the chimeric protein, for example between residues 20 to 24, or between residues 73 to 82 or between residues 12 to 18 or between residues 95 to 101 or between residues 34 to 35 of the PDZ scaffold (SEQ ID NOs: 79 to 85, 89 to 189 and 190 to 290) or between residues 28 to 30, or between residues 48 to 51 or between residues 90 to 91 , or between residues 70 to 80 of the PDZ scaffold (residues 1 to 108 of SEQ ID NO: 727).
  • a PDZ domain is a common structural domain of 70-110 amino-acids found in signalling proteins of bacteria, yeast, plants, viruses and animals.
  • PDZ is an initialism combining the first letters of the first three proteins discovered to share the domain— post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1).
  • the invention adopts the well understood sequence/structure relationships of PDZ domains of proteins, and provides them as discrete scaffolds onto which one or more peptide ligands can be presented in order to recruit two or more biological molecules to facilitate destruction of one of the molecules.
  • a PDZ scaffold is a folded protein composed of 4 to 6 b-strands, one short a-helix and one long a-helix.
  • a PDZ scaffold has a length in the range of 70-110 amino acids or any length there between.
  • a PDZ scaffold is globular and has a molecular weight in the range of 7500 Da to 12,300 Da.
  • a PDZ scaffold may have a natural binding site located between one of the b-strands and the long a-helix, forming a binding pocket constituted by several hydrophobic amino acids, the main chain atoms of which form a nest (protein structural motif) binding the C-terminal carboxyl ate of the protein or peptide ligand.
  • Suitable PDZ scaffolds may include the PDZ domains selected from the following, without limitation: Disks large homolog 4 (PSD-95 ;UniProtKB - P78352 (DLG4_HUMAN); Tyrosine- protein phosphatase non-receptor type 13 (PTPN13, PTB-BL, UniProtKB - Q12923
  • Lin-7 homolog A (UniProtKB - 014910 (LIN7AJHUMAN)), LIM domain only protein 7 (UniProtKB - Q8WWI1 (LM07_HUMAN)), E3 ubiquitin-protein ligase LNX1 ( UniProtKB - Q8TBB1 (LNX1_HUMAN) ), Ligand of Numb protein X 2 (UniProtKB - Q8N448 (LNX2_HUMAN)), or variants thereof.
  • Table 24 shows the multiple alignment of representative sequences (SEQ ID NO: 89 to 189) of PDZ domains aligned with SEQ ID NO.79 and the multiple alignment of representative sequences of PDZ domains (SEQ ID NO: 190 to 290) aligned with SEQ ID NO. 80 with the top 100 hits from a BLAST® search. Secondary structural elements are represented by arrows for beta-strands and boxes for alpha helices.
  • Suitable PDZ scaffolds include the PDZ domains shown in Tables 21 , 22 and 24 (SEQ ID NOs: 79-85, 87, 89 to 189 and 190 to 290) or variants thereof.
  • Other suitable PDZ scaffolds include the PDZ domain of residues 1 to 108 of SEQ ID NO: 727 or variants thereof.
  • Suitable positions for peptide ligand insertions or replacements for the PDZ scaffolds for variants of sequence listed in Tables 21 , 22, 24 and 25 can be determined by using the consensus sequence and matching it with the corresponding residue positions of SEQ ID NOs: 79-85, 87, 89 to 290 and 727.
  • one or more peptide ligands may be inserted or replaced at loops or helices marked by positions residues 20 to 24; or residues 18 to 54; or residues 19 to 22; or residues 13-17; or residues 13 to 23; or residues 20 to 24; or residues 10 to 15; or residues 20 to 24; or residues 51 to 56; or residues 59 to 65; or residues 30 to 42; or residues 23-34; or residues 32 to 34; or residues 30 to 38; or residues 21 to 31 ; or residues 51 to 56; or residues 69 to 72 ; or residues 76 to 81 ; or residues 51 to 54; or residues 42 to 44; or residues 52 to 56; or residues 45 to 49; or residues 46 to 52; or residues 69 to 72; or residues 57 to 62; or residues 50 to 55; or residues 64 to 71 ; or residues 52 to 62; or residues 63 to 67; or residues 73 to 82
  • one or more peptide ligands may be inserted or replaced at loops at positions correspond to residues 28 to 30, residues 48 to 51 or residues 90 to 91 of SEQ ID NO: 727 (loops 1 , 2, and 3, respectively) or a helix at a position correspond to residues 70 to 80 of SEQ ID NO: 727 (helix 1).
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 79 or a variant thereof.
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 80 or a variant thereof.
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 81 or a variant thereof.
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 82 or a variant thereof.
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 83 or a variant thereof.
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 84 or a variant thereof.
  • a PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 85 or a variant thereof.
  • Table 21 shows the consensus sequence from the SMART database of the PDZ domains. The positions at which loops can be inserted and the maximum number of amino acids that are found in natural variants at these points are shown. At positions in the consensus sequence where there is a lysine that is not a highly conserved position, it is preferable to substitute with a polar or small amino acid, thereby preserving thermostability of the molecule.
  • the two PDZ domains that show the highest thermodynamic stability is the 3 rd PDZ domain from PSD-95(SEQ ID NO: 79) and the second PDZ domain from protein tyrosine phosphatase non-receptor type 13 (PTP-BL, SEQ ID NO: 80). Extensive folding studies have been performed on these two PDZ domains and this give us key information necessary for successful protein engineering to create multiple interaction sites. An alignment of each one of these PDZ is shown in Table 22.
  • SEQ ID NOs 79 and 80 Multiple alignment of SEQ ID NOs 79 and 80 is shown in Table 24 with the top 100 hits from a BLAST search. Secondary structural elements are represented by arrows for beta-strands and boxes for alpha helices.
  • Beta-strands are boxed with a solid line and alpha helices are boxed with a dashed line.
  • the loop regions suitable for insertion or grafting of molecules are labelled.
  • FIG. 25 shows a model of PDZ1 scaffold-mediated KRAS ubiquitination through the Cul3- Keapl E3 ubiquitin ligase complex.
  • the Cul3-Keap1 E3 model was constructed from multiple crystal structures as described in (Canning et al. 2015). Grafting of helical motifs was performed by structural alignment between two crystal structures with UCSF Chimera. Grafting of loop motifs was modelled with MODELLER, and the energy of the resulting proteins was minimized by UCSF Chimera.
  • the geometry of the complex between KRAS, SOS peptide, PDZ scaffold and the Cul3-Keap1 E3 is predicted by the structural alignment between the modelled loop insertion and the crystal structure of the Keapl degron peptide (sequence ETGE) bound to the b-propeller domain of Keapl .
  • a PDZ scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 86 or a variant thereof.
  • Preferred PDZ scaffolds lack lysine residues, for example, to avoid unwanted ubiquitination.
  • the lysine residues in a PDZ domain may be replaced by E, Q or R to generate a PDZ scaffold.
  • a suitable lysine-free PDZ scaffold may be formed by substituting the 3 lysines with E, Q or R in SEQ ID NO: 79 or a variant thereof.
  • a lysine- free PDZ scaffold may comprise the amino acid sequence of SEQ ID NO: 87 or a variant thereof.
  • a lysine-free PDZ scaffold may be encoded by a nucleic acid sequence of SEQ ID NO: 88 or a variant thereof.
  • a PDZ scaffold or a grafted PDZ SLIM scaffold is 3k82.
  • the PDZ domain of 3k82 has only three Lys residues.
  • the residues Lys46 and Lys71 of the PDZ domain are replaced with glutamine.
  • the residues Lys46 and Lys71 of the PDZ domain are replaced with glutamic acid.
  • residues Lys46 and Lys71 of the PDZ domain are replaced with aspartic acid. . In some embodiments, the residues Lys46 and Lys71 of the PDZ domain are replaced with arginine.
  • the b-1 strand of a PDZ scaffold is located within the positions corresponding to residues 13 to 19 of SEQ ID NO: 79 or 9 to 17 of SEQ ID NO: 80 or 8 to 18 of SEQ ID NO: 81 or 2 to 12 of SEQ ID NO: 82 or 8 to 12 of SEQ ID NO: 83 or 13 to 19 of SEQ ID NO: 84 or 1 to 9 of SEQ ID NO: 85 or 13 to 19 of SEQ ID NO: 87.
  • the b-2 strand of a PDZ scaffold is located within the positions corresponding to residues 25 to 32 of SEQ ID NO: 79 or 66 to 71 of SEQ ID NO: 80 or 23 to 29 of SEQ ID NO: 81 or 18 to 22 of SEQ ID NO: 82 or 24 to 31 of SEQ ID NO: 83 or 25 to 29 of SEQ ID NO: 84 or 16 to 20 of SEQ ID NO: 85 or 25 to 32 of SEQ ID NO: 87.
  • the b-3 strand of a PDZ scaffold is located within the positions corresponding to residues 35 to 43 of SEQ ID NO: 79 or 73 to 75 of SEQ ID NO: 80 or 43 to 50 of SEQ ID NO: 81 or 35 to 41 of SEQ ID NO: 82 or 35 to 43 of SEQ ID NO: 83 or 39 to 44 of SEQ ID NO: 84 or 32 to 37 of SEQ ID NO: 85 or 35 to 43 of SEQ ID NO: 87.
  • the b-4 strand of a PDZ scaffold is located within the positions corresponding to residues 57 to 64 of SEQ ID NO: 79 or 92 to 100 of SEQ ID NO: 80 or 63 to 69 of SEQ ID NO: 81 or 56 to 62 of SEQ ID NO: 82 or 57 to 59 of SEQ ID NO: 83 or 63 to 65 of SEQ ID NO: 84 or 53 to 58 of SEQ ID NO: 85 or 57 to 64 of SEQ ID NO: 87.
  • the b-5 strand of a PDZ scaffold is located within the positions corresponding to residues 66 to 68 of SEQ ID NO: 79 or 71 to 73 of SEQ ID NO: 81 or 64 to 66 of SEQ ID NO: 82 or 61 to 63 of SEQ ID NO: 83 or 86 to 92 of SEQ ID NO: 84 or 60 to 62 of SEQ ID NO: 85 or 66 to 68 of SEQ ID NO: 87.
  • the b-6 strand of a PDZ scaffold is located within the positions corresponding to residues 85 to 94 of SEQ ID NO: 79 or 90 to 100 of SEQ ID NO: 81 or 83 to 93 of SEQ ID NO: 82 or 86 to 91 of SEQ ID NO: 83 or 81 to 89 of SEQ ID NO: 85 or 85 to 94 of SEQ ID NO: 87.
  • the long a helix of a PDZ scaffold is located within the positions corresponding to residues 73 to 82 of SEQ ID NO: 79 or 82 to 89 of SEQ ID NO: 80 or 78 to 86 of SEQ ID NO: 81 or 71 to 79 of SEQ ID NO: 82 or 72 to 76 of SEQ ID NO: 83 or 75 to 83 of SEQ ID NO: 84 or 68 to 76 of SEQ ID NO: 85 or 73 to 82 of SEQ ID NO: 87.
  • the short a helix of a PDZ scaffold is located within the positions corresponding to residues 47 to 50 of SEQ ID NO: 79 or 55 to 58 of SEQ ID NO: 80 or 55 to 56 of SEQ ID NO: 81 or 45 to 49 of SEQ ID NO: 82 or 47 to 51 of SEQ ID NO: 83 or 49 to 51 of SEQ ID NO: 84 or 41 to
  • the third a helix of a PDZ scaffold is located within the positions corresponding to residues 95 to 101 of SEQ ID NO: 79 or 95 to 101 of SEQ ID NO: 87.
  • the disordered regions or the loop regions of a PDZ scaffold are located within the positions corresponding to residues 20 to 24, 51 to 56 and 69 to 72 of SEQ ID NO: 79 or 18 to 54, 59 to 65 and 76 to 81 of SEQ ID NO: 80 or 19 to 22, 30 to 42, 51 to 54, 57 to 62, 74 to 77 and 87 to 89 of SEQ ID NO: 81 or 13 to 17, 23 to 34, 42 to 44, 50 to 55, 67 to 70 and 80 to 82 of SEQ ID NO: 82 or 13 to 23, 32 to 34, 52 to 56, 64 to 71 and 79 to 85 of SEQ ID NO: 83 or 20 to 24, 30 to 38, 45 to 49, 52 to 62 and 66 to 74 of SEQ ID NO: 84 or 10 to 15, 21 to 31 ,
  • the first loop of the PDZ scaffold is at a position corresponding to residues 20 to 24 of SEQ ID NO: 79; residues 18 to 54 of SEQ ID NO: 80; residues 19 to 22 of SEQ ID NO: 81 ;
  • the second loop of the PDZ scaffold is at a position corresponding to residues 51 to 56 of SEQ ID NO: 79; residues 59 to 65 of SEQ ID NO: 80; residues 30 to 42 of SEQ ID NO: 81 ; residues 23-34 of SEQ ID NO: 82; residues 32 to 34 of SEQ ID NO: 83; residues 30 to 38 of SEQ ID NO: 84; residues 21 to 31 of SEQ ID NO: 85; or residues 51 to 56 of SEQ ID NO:
  • the third loop of the PDZ scaffold is at a position corresponding to residues 69 to 72 of SEQ ID NO: 79 ; residues 76 to 81 of SEQ ID NO: 80; residues 51 to 54 of SEQ ID NO: 81 ;
  • the fourth loop of the PDZ scaffold is at a position corresponding to residues 57 to 62 of SEQ ID NO: 81 ; residues 50 to 55 of SEQ ID NO: 82; residues 64 to 71 of SEQ ID NO: 83; residues 52 to 62 of SEQ ID NO: 84; or residues 63 to 67 of SEQ ID NO: 7.
  • a grafted PDZ scaffold comprises a peptide ligand within a loop, (a “loop peptide”) for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 20 to 24, or in SEQ ID NO: 727 residues 28 to 30, 48 to 51 or 90 to 91.
  • loop peptide for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 20 to 24, or in SEQ ID NO: 727 residues 28 to 30, 48 to 51 or 90 to 91.
  • a grafted PDZ scaffold comprises a peptide ligand within a helix (a “helix peptide”), for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 73 to 82 or in SEQ ID NO: 727 residues 70 to 80.
  • a helix peptide for example in SEQ ID NO: 79-85, 9, 20-120, 220-320, residues 73 to 82 or in SEQ ID NO: 727 residues 70 to 80.

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Abstract

La présente invention concerne des protéines chimériques qui sont aptes à se lier à une ou plusieurs molécules cibles. Par exemple, la protéine chimérique peut se lier à une molécule cible et à un composant de la voie de dégradation cellulaire, telle qu'une ligature de protéasome ou E3. Les protéines chimériques comprennent un échafaudage peptidyle monomérique qui peut être, par exemple, un échafaudage CKS, un échafaudage spiralé, un échafaudage d'afficorps, un échafaudage en feuille de trèfle, un échafaudage à domaine PDZ, un échafaudage à domaine d'ubiquitine ou de type ubiquitine, un échafaudage GB1, un échafaudage WW, un échafaudage à fibritine, un échafaudage aPP, un échafaudage à fibronectine, un échafaudage à doigt de Zn, un échafaudage SH3 ou un échafaudage à nœud de cystine (CK). Un ou plusieurs ligands peptidiques hétérologues qui se lient à une molécule cible ou à un composant de la voie de dégradation cellulaire peuvent être greffés par insertion ou substitution de résidus d'acides aminés dans l'échafaudage peptidyle. L'invention concerne également des protéines chimériques ayant diverses configurations et des procédés pour leur production et leur utilisation.
PCT/EP2020/054700 2019-02-21 2020-02-21 Protéines bispécifiques ayant un échafaudage chimérique WO2020169840A1 (fr)

Applications Claiming Priority (28)

Application Number Priority Date Filing Date Title
GBGB1902398.5A GB201902398D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with an ubiquitin or ubiquitin-like scaffold
GB1902402.5 2019-02-21
GB1902380.3 2019-02-21
GB1902397.7 2019-02-21
GB1902370.4 2019-02-21
GB1902398.5 2019-02-21
GBGB1902393.6A GB201902393D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a fibritin scaffold
GB1902396.9 2019-02-21
GBGB1902397.7A GB201902397D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a cystine knot scaffold
GB1902378.7 2019-02-21
GBGB1902384.5A GB201902384D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a fibronectin scaffold
GB1902403.3 2019-02-21
GBGB1902394.4A GB201902394D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a Zn finger scaffold
GB1902393.6 2019-02-21
GBGB1902391.0A GB201902391D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a cks scaffold
GBGB1902370.4A GB201902370D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a coiled-coil scaffold
GBGB1902380.3A GB201902380D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with an aPP scaffold
GB1902394.4 2019-02-21
GB1902401.7 2019-02-21
GB1902384.5 2019-02-21
GBGB1902396.9A GB201902396D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with an SH3 scaffold
GBGB1902401.7A GB201902401D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a PDZ scaffold
GBGB1902402.5A GB201902402D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a trefoil scaffold
GB1902375.3 2019-02-21
GB1902391.0 2019-02-21
GBGB1902403.3A GB201902403D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a GB1 scaffold
GBGB1902378.7A GB201902378D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with a WW scaffold
GBGB1902375.3A GB201902375D0 (en) 2019-02-21 2019-02-21 Bispecific proteins with an affibody scaffold

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023003874A3 (fr) * 2021-07-19 2023-03-02 Aro Biotherapeutics Company Échafaudages protéiques de la fibronectine de type iii humaine
WO2023173094A3 (fr) * 2022-03-10 2023-10-19 Cornell University Variants de chimères « ubiquibodies » sans lysine pour le silençage des protéines intracellulaires à longue durée de vie
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992001047A1 (fr) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Procede de production de chainon de paires a liaison specifique
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5858657A (en) 1992-05-15 1999-01-12 Medical Research Council Methods for producing members of specific binding pairs
US5872215A (en) 1991-12-02 1999-02-16 Medical Research Council Specific binding members, materials and methods
US5871907A (en) 1991-05-15 1999-02-16 Medical Research Council Methods for producing members of specific binding pairs
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5962255A (en) 1992-03-24 1999-10-05 Cambridge Antibody Technology Limited Methods for producing recombinant vectors
US6140471A (en) 1992-03-24 2000-10-31 Cambridge Antibody Technology, Ltd. Methods for producing members of specific binding pairs
US6172197B1 (en) 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US6225447B1 (en) 1991-05-15 2001-05-01 Cambridge Antibody Technology Ltd. Methods for producing members of specific binding pairs
US6492160B1 (en) 1991-05-15 2002-12-10 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
WO2010133893A1 (fr) * 2009-05-21 2010-11-25 Royal Holloway And Bedford New College Procédé

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5969108A (en) 1990-07-10 1999-10-19 Medical Research Council Methods for producing members of specific binding pairs
WO1992001047A1 (fr) 1990-07-10 1992-01-23 Cambridge Antibody Technology Limited Procede de production de chainon de paires a liaison specifique
US6291650B1 (en) 1991-05-15 2001-09-18 Cambridge Antibody Technology, Ltd. Methods for producing members of specific binding pairs
US6492160B1 (en) 1991-05-15 2002-12-10 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5871907A (en) 1991-05-15 1999-02-16 Medical Research Council Methods for producing members of specific binding pairs
US6225447B1 (en) 1991-05-15 2001-05-01 Cambridge Antibody Technology Ltd. Methods for producing members of specific binding pairs
US6172197B1 (en) 1991-07-10 2001-01-09 Medical Research Council Methods for producing members of specific binding pairs
US5565332A (en) 1991-09-23 1996-10-15 Medical Research Council Production of chimeric antibodies - a combinatorial approach
US5885793A (en) 1991-12-02 1999-03-23 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US5872215A (en) 1991-12-02 1999-02-16 Medical Research Council Specific binding members, materials and methods
US6521404B1 (en) 1991-12-02 2003-02-18 Medical Research Council Production of anti-self antibodies from antibody segment repertoires and displayed on phage
US6140471A (en) 1992-03-24 2000-10-31 Cambridge Antibody Technology, Ltd. Methods for producing members of specific binding pairs
US5733743A (en) 1992-03-24 1998-03-31 Cambridge Antibody Technology Limited Methods for producing members of specific binding pairs
US5962255A (en) 1992-03-24 1999-10-05 Cambridge Antibody Technology Limited Methods for producing recombinant vectors
US5858657A (en) 1992-05-15 1999-01-12 Medical Research Council Methods for producing members of specific binding pairs
WO2010133893A1 (fr) * 2009-05-21 2010-11-25 Royal Holloway And Bedford New College Procédé

Non-Patent Citations (135)

* Cited by examiner, † Cited by third party
Title
"Culture of Animal Cells: A Manual of Basic Technique", 1987, LISS, INC.
"Methods in Enzymology", vol. 85, 1990, ACADEMIC PRESS, INC., article "Guide to Protein Purification"
"Protein structure and function prediction", NATURE METHODS, vol. 12, 2015, pages 7 - 8
"Protocols in Molecular Biology", 1992, JOHN WILEY & SONS
"Recombinant Gene Expression Protocols", March 1997, HUMANA PRESS INC
"UniProt", Database accession no. P06241
"Uniprot", Database accession no. P33552
"UniProtKB", Database accession no. AOA3F2YM25
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 405 - 410
ANONYMOUS: "cyclin-dependent kinases regulatory subunit 1 isoform X1 [Ovis aries] - Protein - NCBI", 6 February 2019 (2019-02-06), XP055691346, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/XP_014947767> [retrieved on 20200504] *
ANONYMOUS: "LOW QUALITY PROTEIN: cyclin-dependent kinases regulatory subunit 2-lik - Protein - NCBI", 28 March 2017 (2017-03-28), XP055690568, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/protein/XP_020769355> [retrieved on 20200430] *
APPLEBAUM ET AL., CHEM BIOL., vol. 19, no. 7, 27 July 2012 (2012-07-27), pages 819 - 830
BAYLISS ET AL., MOL. CELL, vol. 12, 2003, pages 851 - 62
BAZAN JF, PNAS USA, vol. 87, 1990, pages 6934 - 6938
BECHARA ET AL., FEBS LETTERS, vol. 587, no. 1, 2013, pages 1693 - 1702
BINZ H ET AL: "Designing Repeat Proteins: Well-expressed, Soluble and Stable Proteins from Combinatorial Libraries of Consensus Ankyrin Repeat Proteins", JOURNAL OF MOLECULAR BIOLOGY, ACADEMIC PRESS, UNITED KINGDOM, vol. 332, no. 2, 12 September 2003 (2003-09-12), pages 489 - 503, XP027101280, ISSN: 0022-2836, [retrieved on 20030912], DOI: 10.1016/S0022-2836(03)00896-9 *
BIOCHEM BIOPHYS RES COMMUN, vol. 257, no. 2, 1999, pages 418 - 24
BIOCHEMISTRY, vol. 13, no. 20, 1974, pages 4212 - 28
BIRTS ET AL., CHEM. SCI., vol. 4, 2013, pages 3046 - 57
BLUNDELL ET AL., PNAS, vol. 78, 1981, pages 4175 - 4179
BOEHM S ET AL., NUCLEIC ACIDS RES, vol. 25, 1997, pages 2464 - 2469
BOERSMA Y L ET AL: "Bispecific designed ankyrin repeat proteins (DARPins) targeting epidermal growth factor receptor inhibit A431 cell proliferation and receptor recycling", JOURNAL OF BIOLOGICAL CHEMISTRY : JBC, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, vol. 286, no. 48, 2 December 2011 (2011-12-02), pages 41273 - 41285, XP002733242, ISSN: 1083-351X, [retrieved on 20111006], DOI: 10.1074/JBC.M111.293266 *
BONDESON ET AL., NAT. CHEM. BIOL., 2015
CAMARERO ET AL., J MOL BIOL, vol. 308, 2001, pages 1045 - 1062
CANNING ET AL., FREE RAD BIOL & MED, vol. 88, 2015, pages 101 - 107
CHEMBIOCHEM., vol. 5, no. 2, 6 February 2004 (2004-02-06), pages 170 - 6
CHEN J ET AL., CELL MOL LIFE SCI., vol. 65, 2008, pages 2431 - 2444
CHIN ET AL., BIOORG MED CHEM LETT, vol. 11, no. 12, 2001, pages 1501 - 1505
CHOTHIA ET AL., J MOL BIOL., vol. 223, no. 2, 20 January 1992 (1992-01-20), pages 531 - 43
CRAIK DJ ET AL., J MOL BIOL, vol. 294, 1999, pages 1327 - 1336
DAVEY ET AL., NUCLEIC ACIDS RES., vol. 39, 1 July 2011 (2011-07-01), pages W56 - W60
DAVEY ET AL., TRENDS BIOCHEM. SCI., vol. 36, 2011, pages 159 - 169
DE VEER ET AL., ACC CHEM RES, vol. 50, 2017, pages 1557 - 1565
DEECHONGKITKELLY, JACS, vol. 124, 2002, pages 4980 - 4986
DICE J.F., TRENDS BIOCHEM. SCI., vol. 15, 1990, pages 305 - 309
DOWLING ET AL., BIOCHEM., vol. 47, 2008, pages 13554 - 63
ERKIZAN ET AL., CELL CYCLE, vol. 10, 2011, pages 3397 - 408
FAN, X. ET AL., NATURE NEUROSCIENCE, vol. 17, 2014, pages 471 - 480
FEBS LETT., vol. 460, no. 1, 1999, pages 61 - 6
FEBS LETTERS, vol. 398, 1996, pages 312 - 316
FEBS LETTERS, vol. 584, 2010, pages 2670 - 2680
FEBS OPEN BIO., vol. 5, 10 July 2015 (2015-07-10), pages 579 - 93
FELIZMENIO-QUIMIO, M. E. ET AL., J BIOL. CHEM., vol. 276, 2001, pages 22875 - 22882
FILIMONOV F ET AL., BIOPHYS CHEM, vol. 77, 1999, pages 195
FINN ET AL., NUCLEIC ACIDS RESEARCH, 2016, pages D279 - D285
GAREISS P.C. ET AL., CHEMBIOCHEM, vol. 11, 2010, pages 517 - 522
GARTON ET AL., PROC NATL ACAD SCI USA, vol. 115, 2018, pages 1505 - 1510
GONDEAU ET AL., J. BIOL. CHEM., vol. 280, 2005, pages 13793 - 800
GUTHE ET AL., MOL BIOL, vol. 337, 2004, pages 905 - 915
H KASPAR BINZ ET AL: "Engineering novel binding proteins from nonimmunoglobulin domains", NATURE BIOTECHNOLOGY, vol. 23, no. 10, 1 October 2005 (2005-10-01), pages 1257 - 1268, XP055023079, ISSN: 1087-0156, DOI: 10.1038/nbt1127 *
HETZ ET AL., MOL. CELL, vol. 63, 2016, pages 686 - 95
HEY T ET AL: "Artificial, non-antibody binding proteins for pharmaceutical and industrial applications", TRENDS IN BIOTECHNOLOGY, ELSEVIER PUBLICATIONS, CAMBRIDGE, GB, vol. 23, no. 10, 1 October 2005 (2005-10-01), pages 514 - 522, XP027778157, ISSN: 0167-7799, [retrieved on 20051001] *
HODGES ET AL., J AM CHEN SOC, vol. 129, 2007, pages 11024 - 11025
HODGES ET AL., J. AM. CHEM. SOC., vol. 129, no. 36, 2007, pages 11024 - 11025
HOSSE RALF J ET AL: "A new generation of protein display scaffolds for molecular recognition", PROTEIN SCIENCE, WILEY, US, vol. 15, no. 1, 1 January 2006 (2006-01-01), pages 14 - 27, XP002541836, ISSN: 0961-8368, DOI: 10.1110/PS.051817606 *
INNIS ET AL.: "PCR Protocols: A Guide to Methods and Applications", 1990, MACK PUBLISHING COMPANY
J BIOCHEM., vol. 110, no. 3, 1991, pages 360 - 3
J BIOL CHEM, vol. 275, no. 12, 2000, pages 8889 - 94
J COMPUT CHEM., vol. 25, no. 13, October 2004 (2004-10-01), pages 1605 - 12
J EXP MED, vol. 191, no. 7, 2000, pages 1105 - 16
J MOL BIOL, vol. 217, no. 1, 5 January 1991 (1991-01-05), pages 153 - 76
J MOL BIOL, vol. 250, no. 3, 14 July 1995 (1995-07-14), pages 354 - 67
J MOL BIOL, vol. 275, no. 2, 1998, pages 347 - 63
J MOL BIOL, vol. 302, no. 5, 2000, pages 1041 - 7
J MOL BIOL., vol. 333, no. 1, 10 October 2003 (2003-10-10), pages 141 - 52
J MOL EVOL, vol. 50, no. 3, 2000, pages 214 - 23
J STRUCT BIOL, vol. 155, pages 140 - 5
J. MOL. BIOL., vol. 194, no. 3, 5 April 1987 (1987-04-05), pages 531 - 44
KIM ET AL., NAT. CHEM. BIOL., vol. 9, 2013, pages 643 - 50
KINTZING ET AL., CURR OPIN CHEM BIOL, vol. 34, 2016, pages 5.6.1 - 5.6.37
KOLASINAC ET AL., INT. J. MOL. SCI., vol. 19, 2018, pages 346
KONTERMANN, RDUBEL, S: "Molecular Cloning: a Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS, pages: 109 - 1 28
KRAMMER ET AL., PLOS ONE, vol. 7, no. 8, 2012, pages e43603
KRITZER ET AL., CHEMBIOCHEM, vol. 7, 2006, pages 29 - 31
KUBE ET AL., LANGMUIR, vol. 33, 2017, pages 1051 - 1059
LEE ET AL., CELL COMMUNICATION AND SIGNALLING, pages 8
LEE ET AL., CHEMBIOCHEM, vol. 14, 2013, pages 445 - 451
LEE ET AL., NATURE COMM, vol. 5, 2014, pages 1
LEONARD ET AL., BIOESSAYS, vol. 38, no. 9, September 2016 (2016-09-01), pages 903 - 916
LESHCHINER ET AL., PNAS, vol. 112, no. 6, 2015, pages 1761 - 1766
LI ET AL., NAT. STRUCT. MOL. BIOL., vol. 17, 2010, pages 105 - 111
LLOUZ ET AL., J. BIOL. CHEM., vol. 281, 2006, pages 30621 - 30630
MACCIONI ET AL., EMBO J., vol. 7, 1988, pages 1957 - 63
MACIAS ET AL., NAT STRUCT MOL BIOL, vol. 7, 2000, pages 375
MACIAS, NATURE, vol. 382, 1996, pages 646 - 649
MALAKAUSKAS SMMAYO SL: "Design, structure and stability of a hyperthermophilic protein variant", NAT. STRUCT. BIOL., vol. 5, 1998, pages 470 - 475, XP008008261, DOI: 10.1038/nsb0698-470
MARGARIT ET AL., CELL, vol. 112, no. 5, 2003, pages 685 - 695
MCGEE ET AL., JBC, vol. 293, no. 9, 2018, pages 3265 - 3280
MEIER ET AL., J MOL BIOL., vol. 344, no. 4, 2004, pages 1051 - 69
MEIER ET AL., J. MOL. BIOL., vol. 344, no. 4, 2004, pages 1051 - 69
MENDOZA ET AL., CANCER RES., vol. 63, 2003, pages 1020 - 4
MEYER ET AL., JOURNAL OF CELL SCIENCE, vol. 114, pages 1253 - 63
MOELLERING ET AL., NATURE, vol. 462, 2009, pages 182 - 8
NAT STRUCT BIOL., vol. 5, no. 10, October 1998 (1998-10-01), pages 898 - 902
NATURE, vol. 359, no. 6398, 1992, pages 855 - 8
NUCL. ACIDS RES., vol. 25, 1997, pages 3389 - 3402
NUCLEIC ACIDS RES., vol. 46, no. W1, 2018, pages W296 - W303
OTTE ET AL., PROTEIN SCIENCE, vol. 12, no. 3, 2003, pages 491 - 500
P.E. BOURNE, NUCLEIC ACIDS RESEARCH, vol. 28, 2000, pages 235 - 242
PAPANIKOLOPOULOU ET AL., JBC 2004, vol. 279, 2004, pages 8991 - 8998
PARK ET AL., BIOCHEMISTRY, vol. 36, 1997, pages 14277 - 14283
PATEL ET AL., J. BIOL. CHEM., vol. 283, 2008, pages 32158 - 61
PATEL ET AL., PROTEIN ENG DES SEL, vol. 26, 2013, pages 307 - 315
PEARSONLIPMAN, PNAS USA, vol. 85, 1988, pages 2444 - 2448
PLOTKIN ET AL., J. PHARMACOL. EXP. THER., vol. 305, 2003, pages 974 - 980
PNAS, vol. 103, no. 42, 17 October 2006 (2006-10-17), pages 15457 - 15462
POTH ET AL., PEPTIDE SCIENCE, vol. 100, no. 5, 2013, pages 480 - 491
PROC NATL ACAD SCI U S A, vol. 110, no. 6, 5 February 2013 (2013-02-05), pages 2135 - 9
PROC NATL ACAD SCI USA, vol. 104, no. 29, 17 July 2007 (2007-07-17), pages 11963 - 8
PROC NATL ACAD SCI USA, vol. 86, no. 24, 1989, pages 9667 - 71
PROTEIN SCI., vol. 10, no. 12, December 2001 (2001-12-01), pages 2587 - 2599
PROTEIN SCI., vol. 21, no. 12, 2012, pages 1911 - 1920
PROTEINS, vol. 10, no. 3, 1991, pages 240 - 50
REMENYI A ET AL: "Docking interactions in protein kinase and phosphatase networks", CURRENT OPINION IN STRUCTURAL BIOLOGY, ELSEVIER LTD, GB, vol. 16, no. 6, 1 December 2006 (2006-12-01), pages 676 - 685, XP024959927, ISSN: 0959-440X, [retrieved on 20061201], DOI: 10.1016/J.SBI.2006.10.008 *
RICHARDS ET AL., PNAS, vol. 113, 2016, pages 13726 - 31
RIVAS ET AL., PNAS, vol. 85, 1988, pages 6092 - 6
ROBETTA, NUCLEIC ACIDS RESEARCH, vol. 32, no. S2, 2004, pages W526 - W531
ROSENFELD ET AL., J. BIOMOL. STRUCT. DYN., vol. 11, 1993, pages 557 - 570
SAKAMOTO ET AL., BBRC, vol. 484, 2017, pages 605 - 11
SAKAMOTO ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 484, 2017, pages 605 - 611
SAKAMOTO K. ET AL., BIOCHEM. BIOPHYS. RES. COMMUN., vol. 484, 2017, pages 605 - 611
SAMBROOK ET AL.: "Molecular Cloning: A laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SCHLATTER D ET AL., MABS, vol. 4, 2012, pages 497
SCHUMAN ET AL., FRONTIERS CHEM, 2015, pages 3
SCIENCE, vol. 251, no. 4989, 1991, pages 1401 - 3
SCWAHER KL ET AL., PROT SCI, vol. 16, 2007, pages 2694
SLOAN DJHELLINGA HW, PROT SCI, vol. 8, 1999, pages 1643 - 1648
SMITHWATERMAN, J. MOL BIOL., vol. 147, 1981, pages 195 - 197
STAUB ET AL., STRUCTURE, vol. 4, no. 5, 1996, pages 495 - 499
STEWART ET AL., NAT. CHEM. BIOL., vol. 6, 2010, pages 595 - 601
STRUCTURE, vol. 6, no. 5, 1998, pages 649 - 59
WATSON ET AL., NAT. COMM., vol. 7, 2016, pages 11262
WINDLEY MJ ET AL., TOXINS (BASEL, vol. 4, 2012, pages 191 - 227
Y. H. LAU ET AL., CHEM. SCI., vol. 5, 2014, pages 1804 - 1809
Z.-X WANG, FEBS LETTERS, vol. 360, 1995, pages 111 - 114

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023003874A3 (fr) * 2021-07-19 2023-03-02 Aro Biotherapeutics Company Échafaudages protéiques de la fibronectine de type iii humaine
WO2023173094A3 (fr) * 2022-03-10 2023-10-19 Cornell University Variants de chimères « ubiquibodies » sans lysine pour le silençage des protéines intracellulaires à longue durée de vie
WO2023225459A2 (fr) 2022-05-14 2023-11-23 Novozymes A/S Compositions et procédés de prévention, de traitement, de suppression et/ou d'élimination d'infestations et d'infections phytopathogènes

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