Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4702—Regulators; Modulating activity
  • C07K14/4703—Inhibitors; Suppressors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/82—Translation products from oncogenes
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/93—Ligases (6)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
  • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
  • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)

Definitions

• the present disclosure generally relates to binding proteins and protein binding partners for display of peptides and various uses thereof.
• Peptides are attractive diagnostic and therapeutic targets due to their varied physiological roles and potentially high potency and target specificity. However, the identification and development of such peptides remains challenging.
• Peptides sometimes bind to their targets with modest affinity due to entropic thermodynamic reasons because the exhibit insufficient conformational constraint. While constraint may be conferred through cyclisation of the peptides, but this can involve complex chemistries which may not be feasible for the construction of large libraries.
• peptides when expressed in a host cell are susceptible to degradation by proteases and peptidases. This is normally because these peptides are unable to form stable tertiary structures. While one or more disulfide bonds may be added to increase the stability of certain peptides, disulfide bonds are susceptible to reduction in certain extracellular environments such as in blood and within cells.
• peptides that are stably expressed accumulate in insoluble aggregates.
• Peptides that are present in inclusion bodies may be misfolded, inactive and/or denatured.
• the process of obtaining bioactive peptides from inclusion bodies requires extensive processing comprising isolation, solubilization, refolding and purification.
• Peptides can sometimes be used as receptor binding domains (RBD) for targeting specific cell types and/or concentrating drugs to diseased tissues such as tumours.
• RBD receptor binding domains
• Peptides can sometimes be used as receptor binding domains (RBD) for targeting specific cell types and/or concentrating drugs to diseased tissues such as tumours.
• RBDs receptor binding domains
• peptide-derived RBDs are often unstable and can be challenging to conjugate to their payloads.
• the present disclosure provides reagents and methods for displaying peptides, e.g., in a constrained form. Such reagents and methods can provide for additional stability of a peptide.
• the reagents and methods of the disclosure additionally permit modular production of proteins and complexes displaying a plurality of peptides.
• a protein or complex of the disclosure may display a peptide capable of inhibiting an intracellular protein interaction and a cell penetrating peptide to allow for intracellular delivery.
• a protein or complex of the disclosure may display a therapeutic agent or peptide and a peptide that is a receptor binding domain to allow for cell targeting and concentration on a disease tissue or cell of interest.
• a protein or complex of the disclosure could be used for diagnostic or theranostic purposes.
• the present disclosure also provides reagents and methods to facilitate screening for peptides having various biological activities.
• the present disclosure provides a complex comprising:
• a binding protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; b) a peptide;
• binding protein is linked to the protein binding partner via a covalent isopeptide bond
• the binding protein is linked a first terminus of the peptide and the peptide tag is linked to a second terminus of the peptide.
• the complex comprises:
• a binding protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; b) a peptide;
• binding protein is linked to the protein binding partner via a covalent isopeptide bond
• the binding protein is linked the N' terminus of the peptide and the peptide tag is linked to the C terminus of the peptide.
• the complex comprises:
• a binding protein comprising an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; b) a peptide;
• binding protein is linked to the protein binding partner via a covalent isopeptide bond
• the binding protein is linked the C terminus of the peptide and the peptide tag is linked to the N' terminus of the peptide.
• the binding protein disclosed herein comprises an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 2.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 3.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 4.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 5.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 6.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 7.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 8.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 9.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 10.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 11.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 12.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 13.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 14.
• the binding protein disclosed herein comprises the amino acid sequence set forth in SEQ ID NO: 1.
• the protein binding partner described herein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.
• the protein binding partner described herein comprises an amino acid set forth in SEQ ID NO: 15.
• the protein binding partner described comprises an amino acid set forth in SEQ ID NO: 16.
• the protein binding partner comprises an amino acid set forth in SEQ ID NO: 17.
• the protein binding partner comprises an amino acid set forth in SEQ ID NO: 18.
• the intermolecular isopeptide bond formed between any binding protein and protein binding partner described herein is an autocatalytic reaction.
• the intermolecular isopeptide bond formed between any binding protein and protein binding partner described herein occurs within at least 24 hours of contacting a binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 12 hours of contacting a binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 6 hours of contacting a binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 3 hours of contacting a binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 2 hours of contacting a binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 1 hour of contacting a binding protein with a protein binding partner.
• the intermolecular isopeptide bond formed between any binding protein and protein binding partner described herein is stable under conditions that may dissociate non-covalently linked proteins.
• the intermolecular isopeptide bond is stable at 80°C.
• the intermolecular isopeptide bond is stable at 85°C.
• the intermolecular isopeptide bond is stable at 90°C.
• the intermolecular isopeptide bond is stable at 95 °C.
• the intermolecular isopeptide bond is stable at 100°C.
• any binding protein or protein binding partner described herein is linked to a detectable label.
• the detectable label is a fluorescent tag.
• the detectable label is a chemical tag.
• the detectable label is an epitope tag.
• the detectable label is a fluorescent tag.
• the detectable label is an isobaric tag.
• the detectable label is a radiochemical tag.
• the detectable label is a fluorescent tag.
• the detectable label is a biotin.
• the detectable label is a fluorescent tag.
• the detectable label is an acyl carrier protein tag.
• the detectable label is a fluorescent tag.
• the detectable label is streptavidin.
• any binding protein or protein binding partner described herein is linked to a therapeutic agent.
• any binding protein or protein binding partner described herein is linked to a toxin.
• any binding protein or protein binding partner described herein is linked to a cell penetrating peptide. In one example, any binding protein or protein binding partner described herein is linked to a RNAi agent. In one example, the RNAi agent is a small interfering RNA. In one example, the RNAi agent is a short hairpin RNA. In one example, the RNAi agent is a microRNA. In one example, any binding protein or protein binding partner described herein is linked to a nucleic acid therapeutic.
• any binding protein or protein binding partner described herein is linked to a short activating RNA.
• any binding protein or protein binding partner described herein is linked to an antigen.
• any complex described herein is displayed on a bacteriophage.
• the bacteriophage is a T phage. In another example, the bacteriophage is a filamentous phage. In another example, the bacteriophage is a lysogenic bacteriophage. In another example, the bacteriophage is a lambda phage.
• any complex described herein is displayed on a nanoparticle or a micro particle.
• any complex described herein is displayed within in a cell.
• the cell is a bacterial cell.
• the cell is a yeast cell.
• the cell is a mammalian cell.
• the present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising:
• the present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising:
• step ii) incubating the displayed complex of provided in step i) with a molecule; and iii) detecting the presence of an interaction between the molecule and the peptide (i.e. the peptide in the complex which is linked at a first end to a binding protein and at a second end to a protein binding partner).
• the peptide i.e. the peptide in the complex which is linked at a first end to a binding protein and at a second end to a protein binding partner.
• the method additionally comprises a washing step to remove any unbound peptides.
• the method additionally comprises a filtration step to remove any unbound peptides.
• the method additionally comprises incubating the displayed complex with a non-target molecule prior to step ii).
• the non-target molecule is a cell that does not express the molecule.
• the method additionally comprises isolating the peptide that interacts with the molecule.
• detecting the presence of an interaction between the molecule and the peptide comprises detecting a detectable label linked to a binding protein or a protein binding partner.
• the detectable label is a fluorescent tag.
• detecting the presence of an interaction between the molecule and the peptide comprises measuring a refractive index.
• detecting the presence of an interaction between the molecule and the peptide comprises measuring an acoustic or optical resonance.
• detecting the presence of an interaction between the molecule and the peptide additionally comprises detecting a peptide that specifically binds to a molecule.
• detecting the presence of an interaction between the molecule and the peptide comprises a peptide that binds to a molecule with an equilibrium constant (KD) of 100 nM or less.
• the molecule is a cell surface receptor.
• the cell surface receptor is epithelial growth factor receptor (EGFR).
• the cell surface receptor is C-X-C chemokine receptor type 4 (CXCR4).
• the cell surface receptor is folate receptor alpha protein.
• the cell surface receptor is a folate receptor beta protein.
• the cell surface receptor is CD19, CD20 or CD22 receptors expressed on lymphoid cells.
• the molecule is an intracellular receptor.
• the molecule is a transcription factor.
• the transcription factor is a STAT family protein.
• the transcription factor surface receptor is c-Myc.
• the molecule is a RNA binding protein.
• the RNA binding protein is YB-1.
• a molecule described herein is immobilised on a solid support.
• the present disclosure also provides a method for identifying a peptide capable of translocating a membrane of a cell comprising:
• the method additionally comprises incubating the cell and the complex for a time and under conditions sufficient for the complex to enter a cell.
• the method additionally comprises washing the cell to remove any unbound modified binding proteins.
• the method additionally comprises isolating the peptide.
• detecting the peptide in the cell comprises detecting a detectable label linked to the modified binding protein.
• the detectable label is a fluorescent tag.
• the present disclosure also provides a modified binding protein. Any binding protein described herein may be modified.
• a modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises one or more peptides inserted into one or more regions of the binding protein corresponding to amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.
• a modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 or 48-50 of SEQ ID NO: 1.
• a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21, 27-31, 38-42, 48-50 or 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 of SEQ ID NO: 1.
• a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 of SEQ ID NO: 1.
• a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.
• a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• a modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a (e.g.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 12-21 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27-31 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 38-42 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1, wherein the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48-50 and another peptide inserted into a region of the binding protein corresponding to amino acid residues 59-68 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 27 and 29 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 27 and 30 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 28 and 29 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 28 and 30 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 28 and 31 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 29 and 30 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 29 and 31 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 27 and 31 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 30 and 31 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48 and 50 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 48 and 49 of SEQ ID NO: 1.
• the modified binding protein comprises the amino acid sequence set forth in SEQ ID NO: 1 and the modification comprises a peptide inserted into a region of the binding protein corresponding to amino acid residues 48 and 50 of SEQ ID NO: 1, the peptide is inserted into between amino acid residues 49 and 50 of SEQ ID NO: 1.
• a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.
• a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 15.
• a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 16.
• a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 17.
• a modified binding protein described herein is capable of forming an intermolecular isopeptide bond with a protein binding partner comprising an amino acid set forth in SEQ ID NO: 18.
• the intermolecular isopeptide bond formed between a modified binding protein described herein and a protein binding partner described herein is an autocatalytic reaction.
• the intermolecular isopeptide bond formed between a modified binding protein described herein and protein binding partner described herein occurs within at least 24 hours of contacting the modified binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 12 hours of contacting the modified binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 6 hours of contacting the modified binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 3 hours of contacting the modified binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 2 hours of contacting the modified binding protein with a protein binding partner.
• the intermolecular isopeptide bond may form within 1 hour of contacting the modified binding protein with a protein binding partner.
• the intermolecular isopeptide bond formed between a modified binding protein described herein and protein binding partner described herein is stable under conditions that may dissociate non-covalently linked proteins.
• the intermolecular isopeptide bond is stable at 80°C.
• the intermolecular isopeptide bond is stable at 85°C.
• the intermolecular isopeptide bond is stable at 90°C.
• the intermolecular isopeptide bond is stable at 95 °C.
• the intermolecular isopeptide bond is stable at 100°C.
• a modified binding protein described herein is linked to a detectable label.
• a protein binding partner described herein is linked to a detectable label.
• the detectable label is a fluorescent tag.
• the detectable label is a chemical tag.
• the detectable label is an epitope tag.
• the detectable label is an isobaric tag.
• the detectable label is a radiochemical tag.
• the detectable label is a biotin.
• the detectable label is an acyl carrier protein tag.
• the detectable label is streptavidin.
• a modified binding protein described herein is linked to a therapeutic agent.
• a protein binding partner described herein is linked to a therapeutic agent.
• a modified binding protein described herein is linked to a toxin.
• a protein binding partner described herein is linked to a toxin.
• a modified binding protein described herein is linked to a small molecule.
• a protein binding partner described herein is linked to a small molecule.
• a modified binding protein described herein is linked to a cell penetrating peptide.
• a modified binding protein described herein is linked to a cell penetrating peptide.
• a modified binding protein described herein is linked to a RNAi agent.
• a protein binding partner described herein is linked to a RNAi agent.
• the RNAi agent is a small interfering RNA.
• the RNAi agent is a short hairpin RNA.
• the RNAi agent is a microRNA.
• any modified binding protein or protein binding partner described herein is linked to nucleic acid therapeutic.
• a modified binding protein described herein is linked to an antigen.
• a protein binding partner described herein is linked to an antigen.
• a modified binding protein described herein is linked to a short activating RNA.
• a protein binding partner described herein is linked to a short activating RNA.
• a modified binding protein described herein is displayed on a particle.
• a modified binding protein described herein is displayed on a bacteriophage.
• the bacteriophage is a T phage.
• the bacteriophage is a filamentous phage.
• the bacteriophage is a lysogenic bacteriophage.
• the bacteriophage is a lambda phage, any modified binding protein described herein is displayed on a nanoparticle or a microparticle.
• a modified binding protein described herein is displayed on the surface of a cell.
• a modified binding protein described herein is displayed on the surface of a bacterial cell.
• a modified binding protein described herein is displayed on the surface of a yeast cell.
• a modified binding protein described herein is displayed within in a cell.
• a modified binding protein described herein is displayed within in a bacterial cell.
• a modified binding protein described herein is displayed within in a yeast cell.
• a modified binding protein described herein is displayed within in a mammalian cell.
• a modified binding protein is displayed within in a CHO-K1 cell.
• a modified binding protein is displayed within in a HEK293 cell.
• the present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising: i) incubating a modified binding protein disclosed herein with a molecule; and ii) detecting the presence of an interaction between the molecule and the peptide (i.e. the peptide inserted into a region of the binding protein).
• the present disclosure also provides a method for identifying a molecule that interacts with a peptide, the method comprising:
• step ii) incubating the modified binding protein of provided in step i) with a molecule
• the method additionally comprises a washing step to remove any unbound modified binding proteins.
• the method additionally comprises a filtration step to remove any unbound modified binding proteins.
• the method additionally comprises incubating the modified binding protein with a non-target molecule prior to step ii).
• the non-target molecule is a cell that does not express the molecule.
• the method additionally comprises isolating the peptide (i.e. the peptide inserted into a region of the binding protein) that interacts with the molecule.
• detecting the presence of an interaction between the molecule and the peptide comprises detecting a detectable label linked to a binding protein or a protein binding partner.
• the detectable label is a fluorescent tag.
• detecting the presence of an interaction between the molecule and the peptide comprises measuring a refractive index.
• detecting the presence of an interaction between the molecule and the peptide comprises measuring an acoustic or optical resonance.
• detecting the presence of an interaction between the molecule and the peptide additionally comprises detecting a peptide that specifically binds to a molecule.
• detecting the presence of an interaction between the molecule and the peptide comprises a peptide that binds to a molecule with an equilibrium constant (KD) of 100 nM or less.
• the molecule is a cell surface receptor.
• the cell surface receptor is epithelial growth factor receptor (EGFR).
• the cell surface receptor is C-X-C chemokine receptor type 4 (CXCR4).
• the cell surface receptor is folate receptor alpha protein.
• the cell surface receptor is a folate receptor beta protein.
• the molecule is an intracellular receptor.
• the molecule is a transcription factor is a transcription factor, a transcriptional co-activator or a co-repressor.
• the transcription factor is a STAT family protein.
• the transcription factor is Myc.
• the transcriptional co-activator is beta catenin.
• the transcriptional co-activator is Master Mind Like Protein (MAML).
• the intracellular receptor is an RNA binding protein.
• the RNA binding protein is YB-1.
• a molecule described herein is immobilised on a solid support.
• the present disclosure also provides a method for directing a molecule to a target comprising:
• the cell expresses a target linked to a protein binding partner described herein, and wherein the modified binding protein forms a covalent isopeptide bond with the protein binding partner such that the molecule is directed to the target.
• contacting at step i) is for a time and under conditions sufficient for the modified binding protein linked to a molecule to enter a cell.
• the method additionally comprises determining or identifying that the modified binding protein linked to a molecule has been directed to a target.
• the method additionally comprises producing cells that expresses a target linked to the protein binding partner.
• the molecule is a therapeutic agent.
• the molecule is a toxin.
• the molecule is a transcription factor.
• the molecule is an enzyme.
• the molecule is a RNAi agent.
• the RNAi agent is a small interfering RNA.
• the RNAi agent is a short hairpin RNA.
• the RNAi agent is a microRNA.
• the molecule is an antigen.
• the molecule is a nucleic acid therapeutic.
• the molecule is a short activating RNA.
• the target is expressed in a subcellular compartment.
• the target is a cell surface receptor.
• the target is an intracellular receptor.
• the target is a transcription factor
• the target is component of a membrane receptor. In one example, the target is component of a signalling adaptor protein.
• the present disclosure also provides a method for identifying a peptide capable of translocating a membrane of a cell comprising:
• the method additionally comprises incubating the cell and the modified binding protein for a time and under conditions sufficient for the modified binding protein linked to enter a cell.
• the method additionally comprises washing the cell to remove any unbound modified binding proteins.
• the method additionally comprises isolating the peptide.
• detecting the peptide in the cell comprises detecting a detectable label linked to the modified binding protein.
• the detectable label is a fluorescent tag.
• the present disclosure also provides a method for identifying a receptor binding domain, the method comprising:
• a protein binding partner disclosed herein linked to a cell penetrating peptide wherein the modified binding protein is linked with the protein binding partner by a covalent isopeptide bond, and
• the detection of the complex within the cell is indicative that the peptide (i.e. the peptide inserted into a region of the binding protein) is a receptor binding domain.
• the protein binding partner is additionally linked to a molecule.
• a molecule is a detectable label.
• a molecule is a therapeutic agent.
• the molecule is a toxin.
• the present disclosure also provides a method for identifying a receptor binding domain, the method comprising:
• the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a receptor binding domain.
• the protein binding partner is additionally linked to a molecule.
• a molecule is a detectable label.
• a molecule is a therapeutic agent.
• the molecule is a toxin.
• the molecule is a diagnostic or imaging agent.
• the present disclosure also provides a method for identifying a cell penetrating, the method comprising:
• modified binding protein is linked with the protein binding partner by a covalent isopeptide bond
• the detection of a complex within a cell is indicative that the peptide (i.e. the peptide inserted into a region of the binding protein) is a cell penetrating peptide.
• the present disclosure also provides a method for identifying a cell penetrating, the method comprising:
• modified binding protein forms a covalent isopeptide bond with the protein binding partner
• the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a cell penetrating peptide.
• the protein binding partner is additionally linked to a molecule.
• a molecule is a detectable label.
• a molecule is a therapeutic agent.
• the molecule is a toxin.
• the present disclosure also provides a method for identifying a cell penetrating, the method comprising: i) contacting a cell with a complex comprising a modified binding protein disclosed herein linked to a receptor binding domain; and
• the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a cell penetrating peptide.
• the protein binding partner is additionally linked to a molecule.
• a molecule is a detectable label.
• a molecule is a therapeutic agent.
• the molecule is a toxin.
• the present disclosure also provides a method for identifying a cell penetrating, the method comprising:
• the detection of a complex within a cell is indicative that the peptide (i.e. the the peptide inserted into a region of the binding protein) is a cell penetrating peptide.
• the protein binding partner is additionally linked to a molecule.
• a molecule is a detectable label.
• a molecule is a therapeutic agent.
• the molecule is a toxin.
• SEQ ID NO: 1 Amino acid sequence of a binding ; protein
• SEQ ID NO: 2 Amino acid sequence of a binding ; protein
• SEQ ID NO: 3 Amino acid sequence of a binding ; protein
• SEQ ID NO: 4 Amino acid sequence of a binding ; protein
• SEQ ID NO: 5 Amino acid sequence of a binding ; protein
• SEQ ID NO: 6 Amino acid sequence of a binding ; protein
• SEQ ID NO: 7 Amino acid sequence of a binding ; protein
• SEQ ID NO: 8 Amino acid sequence of a binding ; protein
• SEQ ID NO: 9 Amino acid sequence of a binding ; protein
• SEQ ID NO: 10 Amino acid sequence of a binding ; protein
• SEQ ID NO: 11 Amino acid sequence of a binding ; protein
• SEQ ID NO: 12 Amino acid sequence of a binding ; protein
• SEQ ID NO: 13 Amino acid sequence of a binding ; protein
• SEQ ID NO: 14 Amino acid sequence of a binding ; protein
• SEQ ID NO: 15 Amino acid sequence of a protein binding partner
• SEQ ID NO: 16 Amino acid sequence of a protein binding partner
• SEQ ID NO: 17 Amino acid sequence of a protein binding partner
• SEQ ID NO: 18 Amino acid sequence of a protein binding partner
• SEQ ID NO: 19 Amino acid sequence of a mutant protein binding partner
• SEQ ID NO: 23 Amino acid sequence of a bovine pancreatic trypsin inhibitor
• SEQ ID NO: 24 Amino acid sequence of a mutant bovine pancreatic trypsin inhibitor
• SEQ ID NO: 25 Amino acid sequence of a protein binding partner-bovine pancreatic trypsin inhibitor-binding protein cassette
• SEQ ID NO: 26 Amino acid sequence of a binding protein-bovine pancreatic trypsin inhibitor-protein binding partner cassette
• SEQ ID NO: 27 Amino acid sequence of a binding protein-mutant bovine pancreatic trypsin inhibitor-partner cassette
• SEQ ID NO: 28 Amino acid sequence of a binding protein-bovine pancreatic trypsin inhibitor-mutant protein binding partner
• SEQ ID NO: 29 Amino acid sequence of a binding protein-bovine pancreatic trypsin inhibitor -mutant protein binding partner
• SEQ ID NO: 30 Amino acid sequence of a modified binding protein-mutant bovine pancreatic trypsin inhibitor cassette
• SEQ ID NO: 31 Amino acid sequence of a SRC Homology 3 domain
• SEQ ID NO: 32 Amino acid sequence of a protein binding partner-SRC Homology 3 domain-Binding protein cassette
• SEQ ID NO: 33 Amino acid sequence of a SRC Homology 3 domain-binding protein cassette
• SEQ ID NO: 34 Amino acid sequence of a binding protein-SRC Homology 3 domain protein binding partner cassette
• SEQ ID NO: 35 Amino acid sequence of a binding protein-SRC Homology 3 domain cassette
• SEQ ID NO: 36 Amino acid sequence of a binding protein-SRC Homology 3 domain mutant protein binding partner
• SEQ ID NO: 37 Amino acid sequence of a modified binding protein-SRC Homology :
• SEQ ID NO: 38 Amino acid sequence of a peptide mimetic p53 17 28
• SEQ ID NO: 42 Amino acid sequence of a modified binding protein-peptide mimetic p53 17 28 cassette
• SEQ ID NO: 43 Amino acid sequence of a modified binding protein-peptide mimetic
• SEQ ID NO: 44 Amino acid sequence of a modified binding protein-peptide mimetic
• SEQ ID NO: 45 Amino acid sequence of a modified binding protein-peptide mimetic
• SEQ ID NO: 46 Amino acid sequence of a peptide mimotope of the CD20 epitope
• SEQ ID NO: 47 Amino acid sequence of a mutant peptide mimotope of the CD20 epitope
• SEQ ID NO: 48 Amino acid sequence of a binding protein-peptide mimotope of the
• SEQ ID NO: 49 Amino acid sequence of a binding protein-mutant peptide mimotope (the CD20 epitope -protein binding partner cassette
• SEQ ID NO: 50 Amino acid sequence of a binding protein-peptide mimotope of the
• SEQ ID NO: 51 Amino acid sequence of a binding protein- mutant peptide mimotope the CD20 epitope - mutant protein binding partner cassette
• SEQ ID NO: 52 Amino acid sequence of a modified binding protein-peptide mimotopi of the CD20 epitope cassette
• SEQ ID NO: 53 Amino acid sequence of a modified binding protein-mutant peptide mimotope of the CD20 epitope cassette
• SEQ ID NO: 54 Amino acid sequence of a peptide aptamer
• SEQ ID NO: 55 Amino acid sequence of a cell penetrating peptide
• SEQ ID NO: 56 Amino acid sequence of a modified binding protein-peptide aptamer cassette
• SEQ ID NO: 57 Amino acid sequence of a binding protein - peptide aptamer - protein binding partner - cell penetrating peptide cassette
• SEQ ID NO: 58 Amino acid sequence of a EGFR-Affybody
• SEQ ID NO: 62 Amino acid sequence of a C-X-C chemokine receptor type 4
• SEQ ID NO: 63 Amino acid sequence of a peptide linker
• SEQ ID NO: 64 Amino acid sequence of a FITC-V5 -protein binding partner
• SEQ ID NO: 65 Amino acid sequence of a Biotin-V5-protein binding partner
• SEQ ID NO: 66 Amino acid sequence of a binding protein-peptide aptamer- protein binding partner cassette
• SEQ ID NO: 67 Amino acid sequence of a binding protein - peptide aptamer - protein binding partner - cell penetrating peptide cassette
• SEQ ID NO: 68 Amino acid sequence of a Epidermal growth factor receptor (EGFR)
• SEQ ID NO: 70 Amino acid sequence of a Bid BHS sequence
• SEQ ID NO: 72 Amino acid sequence of a modified binding protein-BimBH3-cassette
• SEQ ID NO: 74 Amino acid sequence of a modified binding protein-PumaBH3 -cassette
• SEQ ID NO: 75 Amino acid sequence of a binding protein-BimBH3-cassette
• SEQ ID NO: 76 Amino acid sequence of a binding protein-BidBH3-cassette
• SEQ ID NO: 80 Amino acid sequence of a binding protein-Hisl -binding partner protein cassette
• SEQ ID NO: 82 Amino acid sequence of a folate receptor beta protein
• SEQ ID NO: 83 Amino acid sequence of a mutant FITC -protein binding partner
• SEQ ID NO: 84 Amino acid sequence of a cell penetrating peptide -partner-peptide aptamer-binding protein cassette
• SEQ ID NO: 85 Amino acid sequence of a cell penetrating peptide- mutant protein binding partner-peptide aptamer-binding protein cassette
• SEQ ID NO: 86 Amino acid sequence of a partner -peptide aptamer-binding protein-cell penetrating peptide cassette
• SEQ ID NO: 88 Amino acid sequence of a protein binding partner-peptide aptamer- binding protein cassette
• aptamer-binding protein cassette SEQ ID NO: 90 Nucleic acid sequence of BP library cassette
• SEQ ID NO: 108 Amino acid sequence of MSA20.
• Figure 1 is a graphical representation of showing the inhibition of the proteolytic activity of trypsin upon addition of PBB or BBP.
• Figure 2 is a graphical representation of showing the inhibition of the proteolytic activity of trypsin upon addition of MB.
• Figure 3 is a schematic overview of the modified binding protein and binding protein p53-MDM2/ MDMX/ COPl peptide inhibitor fusion protein constructs and the various p53-MDM2/ MDMX/ COPl peptide inhibitor sequences.
• Figure 4 shows an image of an SDS-PAGE gel analysis of modified binding protein-p53-MDM2/ MDMX/ COPl peptide inhibitor fusion protein ligation.
• the various fusion protein constructs retain the ability to ligate to a protein binding partner in trans.
• Figure 5 shows an image of an SDS-PAGE gel analysis of binding protein p53- MDM2/ MDMX/ COPl peptide inhibitor fusion protein ligation.
• the fusion protein retains the ability to ligate to a fused protein binding partner in cis.
• Figure 6 is a graphical representation of showing the 48-hour Presto Blue
• Figure 7 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D cells upon the addition of MPMI N8A , MPMI N8A -CPP- partner conjugate, MPDI and MPDI-CPP-conjugate.
• Figure 8 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D and CHO-Kl cells upon the addition of Mp28 and Mp28-CPP-conjugate.
• Figure 9 is a graphical representation of showing the 48-hour Presto Blue
• Figure 10 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D cells upon the addition of BPMI N8A and BPMI N8A - CPP-partner conjugate.
• Figure 11 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D and CHO-Kl cells upon the addition of Bp28 and Bp28-CPP-partner conjugate.
• Figure 12 is a graphical representation of showing the 48-hour Presto Blue measured cell viabilities of T47D and CHO-Kl cells upon the addition of CPP-partner (negative control) and PBS Buffer.
• Figure 13 shows results of an assay to determine the ability of MABDcon, MABDcon5, MABDcon9, and MSA20 to retain the specific ABD interaction with the protein binding partner, by test with FITC-partner.
• MABDcon, MABDcon5, MABDcon9, and MSA20 retain the ability to ligate a FITC-partner.
• Figure 14 shows results of an interferometry assay to determine the affinity of biotinylated human serum albumin (HSA) for each of MABDcon, MABDcon5, MABDcon9, and MSA20.
• HSA biotinylated human serum albumin
• Figure 15 shows results of an interferometry assay to determine the affinity of biotinylated human serum albumin (HSA) for each of BP+PBP+ABDcon5; BP+PBP+ABDcon9; and BP alone (negative control).
• HSA biotinylated human serum albumin
• Figure 16 is a schematic overview of possible configurations of Phylomers in a phylomer phage display library constrained by an interaction between binding protein and protein binding partner.
• Phylomer cyclisation is achieved through conjugation of a fused protein binding partner in cis.
• Figure 17 is a schematic overview of possible configurations of Phylomers in a phylomer phage display library constrained by modified binding protein loop display. Phylomers are inserted for cyclisation within Loop2 of the modified binding protein, leaving the modified binding protein available for conjugation with a protein binding partner in trans.
• Fig. 18 is a schematic overview of a cyclised phylomer library enrichment strategy. Addition of magnetic bead-bound protein binding partner results in conjugation to phage displaying properly folded modified binding-protein-phylomer fusion proteins, which can then be magnetically separated from phage displaying partial or improperly folded modified binding protein.
• Fig. 19 is a bar graph summarising the results of the enrichment scheme depicted in Fig. 18. Following reaction of the modified binding protein Phylomer library with protein binding partner magnetic beads, the library is partially enriched for phage having read through in frame modified binding protein-Phylomer fusion proteins.
• protein shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex).
• the series of polypeptide chains can be covalently linked using a suitable chemical or a disulfide bond.
• non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
• the binding protein and protein binding partner disclosed herein are capable of forming an isopeptide bond with each other.
• isopeptide bond refers to a covalent amide bond formed between a first reactive group and a second reactive group.
• the first reactive group is a carboxyl group and the second reactive group is an amino group.
• the first reactive group is a lysine residue and the second reactive amino acid is selected from the group consisting of an asparagine, an aspartic acid, a glutamine, or a glutamic acid residue. Accordingly, in one example, an isopeptide bond is formed between a lysine residue and an asparagine residue.
• an isopeptide bond is formed between a lysine residue and an aspartic acid residue. In another example, an isopeptide bond is formed between a lysine residue and a glutamine residue. In yet another example, an isopeptide bond is formed between a lysine residue and a glutamic acid residue. Two proteins linked via an intermolecular isopeptide bond are defined as complex.
• Isopeptide bond formation may be an enzyme-dependent processes.
• the formation of an isopeptide bond may require an enzyme.
• Any enzyme known in the art to that catalyzes the formation of an isopeptide bond may be used.
• the enzyme may be a transglutaminase.
• Isopeptide bond formation may be an autocatalytic reaction.
• the isopeptide bond may therefore form autocatalytically e.g. without the presence of an enzyme catalyst or other agent.
• the presence of an isopeptide bond may be detected by suitable methods known in the art. For example, mass spectrometry is routinely used to detect an isopeptide bond since the formation of the isopeptide bond results in a loss of a water molecule.
• mass spectrometry is routinely used to detect an isopeptide bond since the formation of the isopeptide bond results in a loss of a water molecule.
• an 18 kDa shift in the molecular mass of a complex may be detected as compared to the combined molecular mass of the binding protein and protein binding partner as determined separately.
• the binding protein is capable of forming an intermolecular isopeptide bond with any protein binding partner described herein.
• the binding protein comprises the first reactive group and the protein binding partner comprises the second reactive group.
• the binding protein comprises a lysine residue ⁇ i.e. the first reactive group) and the protein binding partner comprises an amino acid residue selected from the group consisting of an asparagine, an aspartic acid, a glutamine, or a glutamic acid residue ⁇ i.e. the second reactive group).
• an isopeptide bond is formed between a lysine residue of the binding protein and an asparagine residue of the protein binding partner.
• an isopeptide bond is formed between a lysine residue of the binding protein and an aspartic acid residue of the protein binding partner. In another example, an isopeptide bond is formed between a lysine residue of the binding protein and a glutamine residue of the protein binding partner. In yet another example, an isopeptide bond is formed between a lysine residue of the binding protein and a glutamic acid residue of the protein binding partner.
• the protein binding partner comprises the first reactive group and the binding protein comprises the second reactive group.
• Isopeptide bond formation may occur almost immediately after contact between the binding protein and the protein binding partner.
• an isopeptide bond may form within at least 1 minute or at least 2 minutes or at least 3 minutes or at least 4 minutes or at least 5 minutes or at least 10 minutes or at least 15 minutes or at least 20 minutes or at least 25 minutes or at least 30 minutes of contacting the binding protein with the protein binding partner.
• an isopeptide bond may form within at least 1 hour or at least 2 hours or at least 4 hours or at least 6 hours or at least 8 hours or at least 12 hours or at least 16 hours or at least 20 hours or at least 24 hours of contacting a binding protein with a protein binding partner.
• Isopeptide bond formation between a binding protein and a protein binding partner may occur under any conditions.
• an isopeptide bond may form in phosphate-buffered saline (PBS) at pH 7.0 and at 25°C.
• an isopeptide bond may form in phosphate-buffered saline (PBS) at pH 7.0 and at 4°C.
• a complex comprising a binding protein and a protein binding partner linked via an isopeptide bond may stable under conditions that may dissociate non-covalently linked proteins.
• a complex may be stable at a high temperature.
• a complex may be stable at 80°C or at 85°C or at 90°C or at 95°C or at 100°C.
• a complex may be stable when subjected to chemical treatment.
• complex may be stable at pH 2 or at pH 3.
• a binding protein may be of any length.
• a binding protein may comprise or consist of at least 80, 90, 100, 110, 120, 130, 140, 150, 200, 250, 300 or 350 or more amino acids.
• a binding protein may therefore comprise or consist of from 80 to 150 amino acids, such as from 90 to 140 amino acids, or such as from 100 to 130 amino acids, including any length within said range(s).
• a binding protein may have an amino acid sequence that is substantially similar to the amino acid sequence set forth in SEQ ID NO: 1.
• substantially similar it is meant that the binding protein may comprise or consist of an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% or 100% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 1.
• a binding protein may comprise or consist of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
• a binding protein may comprise or consist of an amino acid sequence having at least 90% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
• a binding protein may comprise or consist of an amino acid sequence having at least 95% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In another example, a binding protein may comprise or consist of an amino acid sequence having at least 98% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. In this example, the binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO 2.
• a binding protein may comprise or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 3 to 14.
• a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 4.
• a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 5. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 6. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 7. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 8. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 9. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 10. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 11.
• a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 12. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 13. In another example, a binding protein may comprise or consist of an amino acid sequence set forth in SEQ ID NO: 14.
• Percentage amino acid sequence identity with respect to a given amino acid sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.
• Amino acid sequence identity may be determined using the EMBOSS Pairwise Alignment Algorithms tool available from The European Bioinformatics Institute (EMBL-EBI), which is part of the European Molecular Biology Laboratory. This tool is accessible at the website located at www.ebi.ac.uk/Tools/emboss/align/. This tool utilizes the Needleman-Wunsch global alignment algorithm (Needleman and Wunsch, 1970). Default settings are utilized which include Gap Open: 10.0 and Gap Extend 0.5. The default matrix "Blosum62" is utilized for amino acid sequences and the default matrix.
• a binding protein may be a fragment of an isopeptide protein e.g. a protein which comprises an intramolecular isopeptide bond.
• isopeptide proteins comprising an intramolecular isopeptide bond are known in the art and are described, for example in Kang et al. 2007.
• a binding protein is linked to a target.
• a binding protein is linked to a molecule.
• a binding protein is linked to a cell penetrating peptide.
• the protein binding partner is capable of forming an intermolecular isopeptide bond with any binding protein described herein.
• the protein binding partner comprises the first reactive group and the binding protein comprises the second reactive group of an intermolecular isopeptide bond.
• the binding protein comprises the first reactive group and the protein binding partner comprises the second reactive group of an intermolecular isopeptide bond.
• the protein binding partner may be a fragment of an isopeptide protein.
• the protein binding partner may be of any length.
• the protein binding partner may comprise or consist of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40 or more amino acids.
• the protein binding partner may therefore comprise or consist of from 8 to 30 amino acids, such as from 8 to 20 amino acids, or such as from 8 to 15 amino acids, including any length within said range(s).
• the protein binding partner may consist of or may comprise from 5 to 10 amino acids.
• the protein binding partner may comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.
• the first sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.
• a protein binding partner may comprise or consist of an amino acid sequence having at least 80% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.
• a protein binding partner may comprise or consist of an amino acid sequence having at least 85% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 15.
• the protein binding partner may comprise or consist of an amino acid sequence set forth in any one of SEQ ID NOs: 16-18.
• the protein binding partner may comprise the amino acid sequence set forth in SEQ ID NO: 16.
• the protein binding partner may comprise the amino acid sequence set forth in SEQ ID NO: 17.
• the protein binding partner may comprise the amino acid sequence set forth in SEQ ID NO: 18.
• a protein binding partner is linked to a target
• a protein binding partner is linked to a molecule.
• a protein binding partner is linked to a cell penetrating peptide.
• peptide is intended to include compounds composed of amino acid residues linked by amide bonds.
• a peptide may be natural or unnatural, ribosome encoded or synthetically derived.
• a peptide will consist of between 2 and 200 amino acids.
• the peptide may have a length in the range of 10 to 20 amino acids or 10 to 30 amino acids or 10 to 40 amino acids or 10 to 50 amino acids or 10 to 60 amino acids or 10 to 70 amino acids or 10 to 80 amino acids or 10 to 90 amino acids or 10 to 100 amino acids, including any length within said range(s).
• the peptide may comprise or consist of fewer than about 150 amino acids or fewer than about 125 amino acids or fewer than about 100 amino acids or fewer than about 90 amino acids or fewer than about 80 amino acids or fewer than about 70 amino acids or fewer than about 60 amino acids or fewer than about 50 amino acids.
• Peptides include peptide analogues, peptide derivatives and peptidomimetic.
• peptide analogues include peptides comprising one or more non-natural amino acids.
• peptide derivatives include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxy-terminus has been derivatized.
• a peptidomimetic s include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules, "inverso" peptides in which all L-amino acids are substituted with the corresponding D- amino acids, “retro-inverso” peptides in which the sequence of amino acids is reversed and all L-amino acidsare replaced with D-amino acids and other isosteres, such as peptide back-bone (i.e., amide bond) mimetics, including modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.
• peptide back-bone i.e., amide bond
• peptide backbone modifications including ⁇
• /[COCH 2 ], and ⁇ [( ⁇ ) or (Z) CH CH].
• ⁇ indicates the absence of an amide bond.
• the structure that replaces the amide group is specified within the brackets.
• N-alkyl (or aryl) substitution ⁇ [CO R] backbone crosslinking to construct lactams and other cyclic structures
• other derivatives including C-terminal hydroxymethyl derivatives, O-modified derivatives and N-terminally modified derivatives including substituted amides such as alkylamides and hydrazides.
• Peptides may be encoded by nucleic acid fragments of genomic DNA or cDNA obtained from an evolutionary diverse range of organisms from Viruses, Bacteria, Archaea, and Eukarya.
• nucleic acid fragments may be obtained from Aeropyrum pernix, Aquifex aeolicus, Archaeoglobus fulgidis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Chlamydia trachomatis, Escherichia coli, Haemophilus influenzae, Helicobacter pylori, Methanobacterium thermoautotrophicum, Methanococcus jannaschii, Mycoplasma pneumoniae, Neisseria meningitidis, Pseudomonas aeruginosa, Pyrococcus horikoshii, Synechocystis PCC 6803, Thermoplasma volcanium and Thermotoga maritima.
• Nucleic acid fragments may be generated using one or more of a variety of methods known to those skilled in the art. Suitable methods include, as well as those described in the examples below, for example, mechanical shearing (e.g. by sonication or passing the nucleic acid through a fine gauge needle), digestion with a nuclease (e.g. Dnase 1), partial or complete digestion with one or more restriction enzymes, preferably frequent cutting enzymes that recognize 4-base restriction enzyme sites and treating the DNA samples with radiation (e.g. gamma radiation or ultra-violet radiation).
• a nuclease e.g. Dnase 1
• restriction enzymes e.g. Dnase 1
• restriction enzymes e.g. gamma radiation or ultra-violet radiation
• a peptide may be linked to one or more peptide linkers to facilitate insertion of the peptide into a region of a binding protein.
• a peptide may linked at a first end to a peptide linker and at a second end to a peptide linker.
• a peptide may be a cell penetrating peptide.
• a complex comprises any binding protein described herein, any peptide described herein and any protein binding partner described herein, wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond, the binding protein is linked a terminus of the peptide and the peptide tag is linked to another terminus of the peptide.
• the binding protein is linked the N' terminus of the peptide and the peptide tag is linked to the C terminus of the peptide.
• the binding protein is linked to the C terminus of the peptide and the peptide tag is linked to the N' terminus of the peptide.
• a complex comprises any modified binding protein described herein and any protein binding partner described herein, wherein the binding protein is linked to the protein binding partner via a covalent isopeptide bond.
• any complex described herein is displayed on a bacteriophage.
• the bacteriophage is a T phage.
• the bacteriophage is a filamentous phage.
• the bacteriophage is a lysogenic bacteriophage.
• the bacteriophage is a lambda phage.
• any complex described herein is displayed on a nanoparticle or a microparticle.
• any complex described herein is displayed within in a cell.
• the cell is a bacterial cell.
• the cell is a yeast cell.
• the cell is a mammalian cell. Modified binding protein
• modified binding protein refers to a binding protein wherein one or more peptides are inserted into one or more regions of the binding protein.
• region refers to one or more subsequences within a protein.
• a protein contains multiple distinct regions. Each region may contain sites for inserting a peptide with respect to a reference sequence.
• the region may comprise or consist of amino acid residues 12-22 of SEQ ID NO: 1.
• the region may comprise or consist of amino acid residues 12-22 of SEQ ID NO: 1.
• the region may comprise or consist of amino acid residues 12-16 or 17- 21 or 13-20, or 14-19 or 15-18 or 12-13 or 13-14 or 14-15 or 15-16 or 16-17 or 17-18 or 18-19 or 19-20 or 20-21.
• the region may comprise or consist of amino acid residues 27-31 of SEQ ID NO: 1.
• a region may therefore comprise or consist of amino acid residues 27-28 or 28-29 or 29- 30 or 30-31.
• the region may comprise or consist of amino acid residues 38-43 of SEQ ID NO: 1.
• a region may therefore comprise or consist of amino acid residues 39-41 or 38-39 or 39-40 or 40-41 or 41-42 or 42-43.
• the region may comprise or consist of amino acid residues 48-50 of SEQ ID NO: 1.
• a region may therefore comprise or consist of amino acid residues 48-49 or 49-50.
• the region may comprise or consist of amino acid residues 59-68 of SEQ ID NO: 1.
• a region may therefore comprise or consist of amino acid residues 60- 67 or 61-66 or 62-65 or 59-60 or 60-61 or 61-62 or 62-63 or 63-64 or 64-65 or 65-66 or 66-67 or 67-68.
• a modified binding protein may be displayed on a particle.
• Various in vitro methods for displaying proteins on particles are known in the art and described, for example, in Ullman et al. 2011.
• the in vitro display method used may be a ribosome display, a covalent display or a mRNA display.
• a modified binding protein is displayed on a ribosome.
• a modified binding protein is displayed on a RepA protein
• a modified binding protein is displayed on a DNA puromycin linker.
• a modified binding protein is displayed on a RNA puromycin linker.
• the particle may be a bacteriophage.
• the bacteriophage may be labelled with fluorescent tag or a fluorophore.
• the bacteriophage may be a filamentous phage.
• the filamentous phage may be a M13 phage or a fl phage or a fd phage or a IKe phage or a Ifl or a If2 phage.
• the filamentous phage may be M13.
• the bacteriophage may be a lysogenic bacteriophage.
• the bacteriophage may be a lambda phage.
• the bacteriophage may be a T phage.
• the T phage may a T3 phage or a T4 phage or a T7 phage.
• the T phage may be a T7 phage.
• a protein to be displayed on a bacteriophage is linked to a coat protein of the bacteriophage phage.
• the coat protein may be a pill coat protein or a pVI coat protein or a pVII coat protein or a pVIII coat protein or a pIX coat protein.
• the particle may be a magnetic particle, a nanoparticle or a microparticle.
• Various methods of displaying a protein on a magnetic particle, a nanoparticle or a microparticle have been used including, for example, electrostatic assembly as described, for example, in Goldman et al. 2002, covalent cross-linking as described, for example, in Gao et al. 2004, avidin-biotin technology as described, for example, in Gref et al. 2003, or membrane integration as described, for example, in Mirzabekov et al. 2000.
• the person skilled in the art will understand that the amount and stability of a displayed protein is dependent on the method used for coupling.
• a modified binding protein may be displayed within a cell.
• a modified binding protein may be displayed within a bacterial cell.
• modified binding protein may be displayed within a yeast.
• modified binding protein may be displayed within a mammalian cell.
• a modified binding protein may be displayed in a cell free system.
• a modified binding protein may be displayed in an in vitro display system such as ribosome display, mRNA display, covalent display or CIS display.
• a peptide inserted into a region of a modified binding protein may be constrained.
• a peptide linked at a first end to a binding protein and at a second end to a protein binding partner may be constrained.
• constrained it is meant that the peptide when inserted into a region of a modified binding protein or when a binding protein is linked to the protein binding partner via a covalent isopeptide bond wherein a peptide when linked at a first end to a binding protein and at a second end to a protein binding partner, has a reduced degree of freedom as compared to the corresponding linear peptide.
• the degree of freedom may be a rotational degree of freedom.
• the degree of freedom may be a translation degree of freedom.
• the degree of freedom may be a vibrational degree of freedom.
• the degree of freedom of a peptide can be measured with any method known in the art as described, for example in Minary and Levitt, 2010.
• CD circular dichroism
• nuclear magnetic resonance (NMR) spectroscopy is used to measure the degree of freedom of a peptide.
• NMR spectroscopy produces information on the relative positions of all atoms including hydrogen for a given protein.
• NMR spectroscopy is an excellent tool to determine macromolecular structure at atomic resolution.
• NMR spectroscopy can follow protein folding as it happens, either by fast data acquisition of heteronuclear single quantum coherence or correlation experiments or by H/D exchange as described, for example, in Bieri et al., 2011.
• the degree of freedom of a peptide is determined by crystallography.
• X-ray crystallography produces information on the relative positions of all non-hydrogen atoms for a given protein as described, for example, in Wlodawer et al. 2013.
• a constrained peptide is capable of forming a stable secondary structure and/or conformation sufficient for binding and/or internalization and/or localization to a subcellular compartment e.g., without a need for intramolecular disulphide bridge formation to produce a loop.
• a molecule refers to any compound.
• a molecule may be a ligand, a carbohydrate, a polymer, an enzyme, a cellular receptor, an antigen mimic of a cellular receptor, a cell or a cell surface protein.
• a molecule is a detectable label.
• a molecule is a therapeutic agent.
• the molecule is a toxin.
• the molecule is a cell surface receptor.
• the molecule is an intracellular receptor.
• the molecule is a transcription factor.
• the molecule is an enzyme.
• the molecule is a ligand.
• the ligand may be a surface receptor ligand, an intracellular receptor ligand or a target ligand.
• the molecule is an antigen.
• the molecule is a nucleic acid therapeutic.
• the molecule is short activating RNA.
• Any molecule described herein that has a molecule weight of less than 1000 Da is described herein as a "small molecule”.
• detectable label refers to any type of molecule which can be detected by optical, fluorescent, isotopic imaging or by mass spectroscopic techniques, or by performing simple enzymatic assays. Any detectable label known in the art may be used.
• the detectable label may be toxic to cells or cytotoxic. Accordingly, the detectable label may also be a therapeutic agent or a cytotoxic agent.
• the detectable label may be selected form the group consisting of a fluorescent tag, a chemical tag, an epitope tag, a isobaric tagor a radiochemical tag.
• a fluorescent tag may be a fluorophore.
• a fluorophore may be fluorescein isothiocyante, fluorescein thiosemicarbazide, rhodamine, Texas Red, a CyDye such as Cy3, Cy5 and Cy5.5, a Alexa Fluor such as Alexa488, Alexa555, Alexa594 and Alexa647) or a near infrared fluorescent dye.
• a fluorescent tag may be a fluorescent protein.
• a fluorescent protein may be green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), AcGFP or TurboGFP, Emerald, Azami Green, ZsGreen, EBFP, Sapphire, T-Sapphire, ECFP, mCFP, Cerulean, CyPet, AmCyanl, Midori-Ishi Cyan, mTFPl (Teal), enhanced yellow fluorescent protein (EYFP), Topaz, Venus, mCitrine, YPet, PhiYFP, ZsYellowl, mBanana, Kusabira,ange, mOrange, dTomato, dTomato-Tandem, AsRed2, mRFPl, Jred, mCherry, HcRedl, mRaspberry, HcRedl, HcRed-Tandem, mPlum, AQ 143.
• GFP green fluorescent protein
• EGFP enhanced green fluorescent protein
• AcGFP or TurboGFP Emerald
• a fluorescent tag may be a quantum dot. Fluorescent tags may be detected using fluorescent microscopes such as epifluorescence or confocal microscopes, fluorescence scanners such as microarray readers, spectrofluorometers, microplate readers and/or flow cytometers.
• fluorescent microscopes such as epifluorescence or confocal microscopes
• fluorescence scanners such as microarray readers, spectrofluorometers, microplate readers and/or flow cytometers.
• a chemical tag may be SNAP tag, a CLIP tag, a HaloTag or a TMP-tag.
• the chemical tag is a SNAP-tag or a CLIP-tag.
• SNAP and CLIP fusion proteins enable the specific, covalent attachment of virtually any molecule to a protein of interest as described, for example, in Correa 2015.
• the chemical tag is a HaloTag.
• HaloTag involves a modular protein tagging system that allows different molecules to be linked onto a single genetic fusion, either in solution, in living cells, or in chemically fixed cells as described, for example, in Los et al. 2008.
• the chemical tag is a TMP-tag. TMP-tags are able to label intracellular, as opposed to cell-surface, proteins with high selectivity as described, for example, in Chen et al. 2012.
• An epitope tag may be a poly-histidine tag such as a hexahistidine tag or a dodecahistidine, a FLAG tag, a Myc tag, a HA tag, a GST tag or a V5 tag.
• Epitope tags are routinely detected with commercially available antibodies. A person skilled in the art will be aware that an epitope tag may facilitate purification and/or detection.
• a conjugate containing a hexahistidine tag may be purified using methods known in the art, such as, by contacting a sample comprising the protein with nickel- nitrilotriacetic acid (Ni-NTA) that specifically binds a hexahistidine tag immobilized on a solid or semi-solid support, washing the sample to remove unbound protein, and subsequently eluting the bound protein.
• Ni-NTA nickel- nitrilotriacetic acid
• a ligand or antibody that binds to an epitope tag may be used in an affinity purification method.
• An isobaric tag may be a mass tag or an isobaric tag for relative absolute quantification (iTRAQ).
• a mass tag is a chemical label used for mass spectrometry based quantification of proteins and peptides. In such methods mass spectrometers recognise the mass difference between the labeled and unlabeled forms of a protein or peptide, and quantification is achieved by comparing their respective signal intensities as described, for example, in Bantscheff et al. 2007. Examples of mass tags include TMTzero, TMTduplex, TMTsixplex and TMT 10-plex.
• An isobacric tag for relative absolute quantification (iTRAQ) is a chemical tag used in quantitative proteomics by tandem mass spectrometry to determine the amount of proteins from different sources in a single experiment as described, for example, in Wiese et al. 2007.
• a radiochemical tag may be a ⁇ -emitting radionuclide, Auger-emitting radionuclide, ⁇ -emitting radionuclide, an a-emitting radionuclide, or a positron- emitting radionuclide.
• the radionuclides may be 3H, 14C, 32P, 33P, 35S, 1251 or 51Cr.
• the detectable labels may be selected from the group consisting of biotin, a acyl carrier protein tag, or streptavidin.
• Radiochemical tags may be detected with an ionization chamber or an autoradiograph.
• detectable labels include biotin, an acyl carrier protein tag, or streptavidin.
• therapeutic agent refers to any type of molecule capable of having a biological effect.
• a therapeutic agent may be selected form the group consisting of a therapeutic compound, a chemotherapeutic agent or a cytotoxic agent.
• a therapeutic compound may induce an immune response.
• the therapeutic compound may be, a chemokine (e.g., BCA-1, BRAK, CTACK, CXCL17, ENA 78, Eotaxin, Interleukine-8, MCP, Platelet Factor-4 and Rantes), a cytokine (e.g., tumor necrosis factor alpha, Interferon, Beta Defensin, Betacellulin, Leukemia Inhibitory Factor, Hedgehog Protein, Follistatin, Flt3 Ligand, Granulocyte-Macrophage Colony-Stimulating Factor), a growth factor (e.g., Growth Hormone, Colony Stimulating Factor, Epidermal Growth Factor, Erythropoietin, Myostatin, RANK Ligand, Osteoprotegerin, Noggin, and VEGF ), a hormone (e.g., Endothelin, Exendin, FSH, Stanniocalcin, Thymosin), a human leukocyte
• a chemotherapeutic agent may be an alkylating agent, a kinase inhibitor, a vinca alkaloid, anthracycline, an anti-metabolite, an aromatase inhibitor or a topoisomerase inhibitor.
• a cytotoxic agent may affect cell division.
• the cytotoxic agent may affect the S phase of the cell cycle.
• examples of cytotoxic agents that affect the S phase of the cell cycle include Ergot alkaloids and Methotrexate.
• the cytotoxic agent may affect the G2 phase of the cell cycle.
• examples of cytotoxic agents that affect the G2 phase of the cell cycle include Etoposide and Bleomycin.
• the cytotoxic agent may affect the M phase of the cell cycle.
• Examples of cytotoxic agents that affect the M phase of the cell cycle include Gresiofulvin, Paclitaxel, Vincristine and Vinblastine.
• a cytotoxic agent may induce cell death.
• determining viability of the cell may comprise determining the doubling rate of the cell e.g., the period of time required for the cell to divide e.g., nucleic acid content or cell counting such as by FACS.
• toxin refers to any molecule capable of causing cell death or impaired cell survival when internalised in a cell.
• a toxin may induce cell death in more than 50% or more 60% or more than 70% or more than 80% or more than 90% or more than 95% or more than 97% or more than 98% or more than 99% of cells in which it is internalized.
• the toxin comprises one or more domains from plant, bacterial or fungal protein toxins.
• the toxin may classified according to their mechanism of action and/or structural organization, such as, for example, ADP- ribosylating toxins; N-glycosidase containing ribosome inactivating toxins; and binary bacterial toxins that comprise separate cell binding and catalytic domains, including, for example, anthrax toxin, pertussis toxins, cholera toxin, E.
• cell viability or cytotoxicity are known in the art such as, for example, plate viability assays, colony regression assays, plating assays, and fluorometric/colorimetric growth indicator assays based on detection of metabolic activity.
• cell viability is determined based on the ability of the membrane of viable test cells to exclude dyes, such as, for example, trypan blue or propidium iodide. Living test cells exclude such dyes and do not become stained. In contrast, dead or dying test cells that have lost membrane integrity allow these dyes to enter the cytoplasm and stain various compounds or organelles within the test cell.
• a number of cell viability assays and cytotoxicity assays are also commercially available. Diagnostic/Theranostic
• diagnosis refers to one or more components, complexes, and/or biomarkers the detection or level of which is indicative of a specific health condition ⁇ e.g., the presence or absence of a cancer).
• theranostic refers to one or more agents that combine a therapeutic modality with a diagnostic modality to facilitate the assessment of disease status following administration of the theranostic agent.
• target refers to any substance expressed by a cell, on a cell or present within a cell, to which a peptide may bind.
• a target may be a ligand, a carbohydrate, a polymer, an enzyme, a cellular receptor or a cell surface protein.
• a target may be expressed by specific cells.
• the target may be expressed by tumor cells, immune cells, bacterial cells, fungal cells, parasites, or virus infected cells.
• the target is expressed by tumor cells. In another example, the target is expressed by bacterial cells. In another example, the target is expressed by fungal cells. In another example, the target is expressed by parasites. In another example, the target is expressed by virus infected cells.
• a target may be expressed in a specific region of a cell.
• the target is expressed in a subcellular compartment.
• the target is a cell surface receptor.
• the target is an intracellular receptor.
• a target may be a transcription factor.
• a target may be a signalling adaptor protein or a component thereof.
• signalling adaptor proteins include, for example, GRAP, GRAP2, LDLRAP1, NCK1, NCK2, NOS 1AP, PIK3AP1, SH2B 1, SH2B2, SH2B3, SH2D3A, SH2D3C, SHB, SLC4A1AP, GAB 2.
• subcellular compartment refers to any compartment in a cell including, but not limited to cytosol, endosome, nucleus, endoplasmic reticulum, golgi, vacuole, mitochondrion, plastid such as chloroplast or amyloplast or chromoplast or nucleus, cytoskeleton, centriole, microtubule-organizing center (MTOC), acrosome, glyoxysome, melanosome, myofibril, nucleolus, peroxisome, nucleosome or microtubule or the cytoplasmic surface such the cytoplasmic membrane or the nuclear membrane.
• cytosol endosome
• nucleus endoplasmic reticulum
• golgi vacuole
• mitochondrion plastid
• plastid such as chloroplast or amyloplast or chromoplast or nucleus
• centriole microtubule-organizing center
• MTOC microtubule-organizing center
• cell surface receptor refers to any protein that is present on the surface of a cell and is capable of transmitting or transducing a signal in the cell. Any cell surface receptor known in the art may be used such as those described, for example, in Uings and Farrow, 2000.
• the cell surface receptor may be a ligand-gated ion channel receptor, an enzyme-coupled receptor or a G-protein-coupled receptor.
• the cell surface receptor is a capable of binding and internalising any molecule via receptor mediated endocytosis.
• the cell surface receptor the epithelial growth factor receptor (EGFR).
• an epithelial growth factor receptor may be linked to an affibody and comprise the amino acid sequence set forth in SEQ ID NO: 58.
• the cell surface receptor is an Ephrin receptor.
• the Ephrin receptor is Ephrin receptor EphA2
• the cell surface receptor is C-X- C chemokine receptor type 4 (CXCR4).
• the cell surface receptor is folate receptor alpha protein.
• the folate receptor alpha protein may comprise the amino acid sequence set forth in SEQ ID NO: 59.
• the cell surface receptor is a folate receptor beta protein.
• the folate receptor beta protein may comprise the amino acid sequence set forth in SEQ ID NO: 82.
• a synthetic cell surface receptor may be used. Examples of synthetic cell surface receptors are described for example in Hymel and Peterson 2010. Intracellular receptor
• intracellular receptor refers to any protein located in a subcellular compartment that is capable of being activated by a ligand.
• the intracellular receptor is a nuclear receptor.
• the intracellular receptor is a constitutive androstane receptor.
• the intracellular receptor is a famesoid X receptor.
• the intracellular receptor is a IP3 receptor.
• the intracellular receptor is a liver X receptor.
• the intracellular receptor is a peroxisome proliferator-activated receptors.
• the intracellular receptor is a pregnane X receptor.
• the intracellular receptor is a retinoic acid receptor.
• the intracellular receptor is a retinoid X receptor.
• transcription factor refers to any protein involved in the process of converting, or transcribing, DNA into RNA.
• a transcription factor may bind DNA alone or a transcription factor may form complexes with other transcription factors and thus may bind DNA directly or indirectly.
• the transcription factor is an activator. In one example, the transcription factor is a transcriptional co-activator. In one example, the transcriptional co-activator may be beta catenin. In one example, the transcriptional co-activator may be Master Mind Like Protein (MAML). In another example, the transcription factor is a repressor. In another example, the transcription factor is a co-repressor. In another example, the transcription factor is an initiator of transcription.
• the transcription factor is an activator. In one example, the transcription factor is a transcriptional co-activator. In one example, the transcriptional co-activator may be beta catenin. In one example, the transcriptional co-activator may be Master Mind Like Protein (MAML). In another example, the transcription factor is a repressor. In another example, the transcription factor is a co-repressor. In another example, the transcription factor is an initiator of transcription.
• MAML Master Mind Like Protein
• the transcription factor is STAT (signal transducers and activators of transcription) family protein.
• STAT family proteins are well known in the art and are described, for example, in Akira 1999.
• the STAT family protein is selected from the group consisting of STAT3 and STAT5.
• the transcription factor is c-Myc.
• c-Myc is known to play a pivotal role in growth control, differentiation and apoptosis, and its abnormal expression is associated with many tumors as described, for example, in Hoffman and Liebermann 2008.
• RNA binding protein refers to any protein involved in binding specifically to RNA.
• An RNA binding protein may bind to RNA alone or a may form complexes with other proteins or nucleic acids and thus may bind RNA directly or indirectly.
• the RNA binding protein is YB-1.
• YB-1 is involved in a wide variety of DNA/RNA-dependent events including cell proliferation and differentiation, stress response, and malignant cell transformation as described, for example, in Bobkova et al. 2005.
• conjugate refers a molecule comprising two or more elements or components that are linked by whatever means including chemical conjugation or recombinant means.
• a conjugate may comprise a binding protein linked to a molecule.
• a peptide linked to a molecule Methods of conjugation are known in the art.
• Two elements or components may be linked together directly. The person skilled in the art will understand that the co-linear, covalent linkage of two or more proteins via their individual peptide backbones and expressed as a single molecule encoding those proteins forms a specific type of conjugate described herein as a "fusion protein". Two elements or components may be linked to each other via one or more peptide linkers.
• cell penetrating peptide refers to a peptide that is capable of crossing a cellular membrane.
• a cell penetrating peptide is capable of translocating across a mammalian cell membrane and entering into a cell.
• a cell penetrating peptide may direct a conjugate to a desired subcellular compartment.
• a cell penetrating peptide may direct or facilitate penetration of a molecule of interest across a phospholipid, mitochondrial, endosomal or nuclear membrane.
• a cell penetrating peptide may direct a molecule of interest from outside a cell through the plasma membrane, and into the cytoplasm or a desired subcellular compartment.
• a cell penetrating peptide may direct a molecule of interest across the blood-brain, trans-mucosal, hematoretinal, skin, gastrointestinal and/or pulmonary barriers.
• RNAi agent refers to an RNA molecule having a structure characteristic of molecules that can inhibit transcription and/or translation a gene.
• RNAi agents include, for example, small interfering RNAs (siRNA), double stranded RNAs (dsRNAs), inverted repeats, short hairpin RNAs (shRNAs), small temporally regulated RNAs (stRNAs), clustered inhibitory RNAs (cRNAs), including radial clustered inhibitory RNA, asymmetric clustered inhibitory RNA, linear clustered inhibitory RNA, and complex or compound clustered inhibitory RNA, dicer substrates, DNA-directed RNAi (ddRNAi), microRNA (miRNA), miRNA antagonists, microRNA mimics, microRNA agonists, blockmirs, microRNA mimetics, microRNA addbacks, and supermiRs.
• siRNA small interfering RNAs
• dsRNAs double stranded RNAs
• stRNAs small
• a peptide linker refers to amino acid sequences that connect, join or link two protein sequences.
• a peptide linker may be of any length.
• the peptide linker may comprise or consist of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.
• the peptide linker may therefore comprise or consist of from 2 to 10 amino acids, such as from 2 to 8 amino acids, or such as from 4 to 6 amino acids.
• the peptide linker may comprise or consist of the amino acid sequence set forth in SEQ ID NOs: 20 to 23 or 63.
• the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 20.
• the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 21.
• the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 22. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 23. In one example, the peptide linker comprises the amino acid sequence set forth in SEQ ID NO: 63.
• vector refers to a nucleic acid capable of transporting another nucleic acid to which it has been linked.
• the vector may a bacterium, a plasmid, a bacteriophage, a cosmid, an episome or a virus.
• expression vector selection of appropriate vectors is within the knowledge of those having skill in the art.
• signal peptide refers to a peptide sequence that directs the transport of a protein. Any signal peptide known in the art may be used. For example, a signal peptide may direct a protein through a secretory pathway of a cell. Alternatively, a signal peptide may bind to an extracellular domain of a receptor on an exterior surface of a cell. Upon internalization of the receptor, the signal peptide may facilitate the localization of a protein to a subcellular compartment. In another example, a signal peptide may be capable of directing a protein to a desired subcellular compartment of a cell. For example, a peptide signal may be a nuclear localisation signal.
• peptide signal may be a golgi localisation sequence.
• peptide signal may be a mitochondria localisation sequence.
• a signal peptide may be a prokaryotic signal peptide.
• the signal peptide may be a DsbA signal peptide, a TorT signal peptide, a TolB signal peptide or a Sfm signal peptide, a Lam signal peptide, a MalE signal peptide, a MglB signal peptide, a OmpA signal peptide, or a PelB signal peptide.
• a signal peptide may be a yeast signal peptide.
• the yeast signal peptide may be a SUC2 signal peptide or a PH05 signal peptide.
• a signal peptide may be a mammalian signal peptide.
• the mammalian signal peptide may a BM-40 signal peptide, a VSVG signal peptide, a chymotrypsinogen signal peptide, an interleukin-2 (IL-2) signal peptide, a Gaussia lucif erase signal peptide, a human serum albumin signal peptide, an influenza haemagglutinin signal peptide, or an insulin signal peptide.
• immobilization refers to various methods and techniques to link proteins to supports. For example, immobilization can serve to stabilize the proteins so that its activity is not reduced or adversely modified by biological, chemical or physical exposure, especially during storage or in single-batch use.
• solid support refers to any solid (flexible or rigid) substrate onto which one or more compounds may be applied.
• the solid support may be in the form of a bead, column, membrane, microwell or centrifuge tube.
• the solid support may be a bead and wherein the bead is a glass bead, or microbead, magnetic bead, or paramagnetic bead.
• cell refers to any prokaryotic or eukaryotic cell.
• suitable prokaryotic cells include, for example, strains of E. coli (e.g., BL21, DH5a, XL-l-Blue, JM105, JM110, and Rosetta), Bacillus subtilis, Salmonella sp., and Agrobacterium tumefaciens.
• the cell may be a plant cell, a yeast, an insect or a mammalian cell.
• suitable mammalian cells include cell lines, such as, for example, human GM12878, K562, HI human embryonic, Hela, HUVEC, HEPG2, HEK-293, H9, MCF7, and Jurkat cells, mouse NIH-3T3, C127, and L cells, simian COSl and COS7 cells, quail QCl-3 cells, and Chinese hamster ovary (CHO) cells.
• cell lines such as, for example, human GM12878, K562, HI human embryonic, Hela, HUVEC, HEPG2, HEK-293, H9, MCF7, and Jurkat cells, mouse NIH-3T3, C127, and L cells, simian COSl and COS7 cells, quail QCl-3 cells, and Chinese hamster ovary (CHO) cells.
• the mammalian cells may be hematopoietic cells, neural cells, mesenchymal cells, cutaneous cells, mucosal cells, stromal cells, muscle spleen cells, reticuloendothelial cells, epithelial cells, endothelial cells, hepatic cells, kidney cells, gastrointestinal cells, pulmonary cells, T- cells.
• a cell may be a cultured cell, e.g. in vitro or ex vivo.
• cells cultured in vitro in a culture medium and environmental stimuli may be added to the culture medium.
• cells may be previously obtained from a subject. Cells can be obtained by biopsy or other surgical means known to those skilled in the art.
• Any protein of the present disclosure may be synthesized using a chemical method known to the skilled artisan.
• synthetic protein are prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof, and can include natural and/or unnatural amino acids.
• any protein of the present disclosure may be expressed by recombinant means.
• the nucleic acid encoding the binding protein may be placed in operable connection with a promoter or other regulatory sequence capable of regulating expression in cellular system or organism.
• Typical promoters suitable for expression in bacterial cells include, for example, the lacz promoter, the Ipp promoter, temperature-sensitive L or R promoters, T7 promoter, T3 promoter, SP6 promoter or semi- artificial promoters such as the IPTG- inducible tac promoter or lacUV5 promoter.
• a number of other gene construct systems for expressing the nucleic acid fragment of the invention in bacterial cells are well- known in the art and are described, for example, in Ausubel et al. (1988), and Sambrook et al. (2001).
• Numerous expression vectors for expression of recombinant polypeptides in bacterial cells have been described, and include, for example, PKC3, pKK173-3, pET28, the pCR vector suite (Invitrogen), pGEM-T Easy vectors (Promega), the pL expression vector suite (Invitrogen) or pBAD/thio— TOPO series of vectors containing an arabinose-inducible promoter (Invitrogen), amongst others.
• Typical promoters suitable for expression in yeast cells such as, for example, a yeast cell selected from the group comprising Pichia pastoris, S. cerevisiae and S.
• pombe include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PH05 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.
• Expression vectors for expression in yeast cells include, for example, the pACT vector (Clontech), the pDBleu-X vector, the pPIC vector suite (Invitrogen), the pGAPZ vector suite (Invitrogen), the pHYB vector (Invitrogen), the pYD 1 vector (Invitrogen), and the pNMT 1, pNMT41, pNMT81 TOPO vectors (Invitrogen), the pPC86-Y vector (Invitrogen), the pRH series of vectors (Invitrogen), pYESTrp series of vectors (Invitrogen).
• Preferred vectors for expression in mammalian cells include, for example, the pcDNA vector suite (Invitrogen), the pTARGET series of vectors (Promega), and the pSV vector suite (Promega).
• nucleic acid may be introduced into prokaryotic cells using for example, electroporation or calcium- chloride mediated transformation.
• nucleic acid may be introduced into mammalian cells using, for example, microinjection, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, transfection mediated by liposomes such as by using Lipofectamine (Invitrogen) and/or cellfectin (Invitrogen), PEG mediated DNA uptake, electroporation, transduction by Adenoviuses, Herpesviruses, Togaviruses or Retroviruses and microparticle bombardment such as by using DNA-coated tungsten or gold particles.
• nucleic acid may be introduced into yeast cells using conventional techniques such as, for example, electroporation, and PEG mediated transformation.
• any protein of the present disclosure can be purified using a method known in the art.
• affinity purification may be used to purify any protein of the present disclosure
• Methods for isolating a protein using affinity chromatography are known in the art and described, for example, in Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).
• any protein of the present disclosure may be determined by various methods, including identification of a major large peak on HPLC OR UPLC. Methods for identifying a molecule that specifically binds with a peptide
• the present disclosure provides for methods for identifying a molecule that specifically binds with a peptide e.g. a peptide contained within any modified binding protein described herein or a peptide contained within any complex described herein.
• a peptide e.g. a peptide contained within any modified binding protein described herein or a peptide contained within any complex described herein.
• the term “specifically binds” or “binds specifically” means that a peptide of the disclosure reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a molecule or cell expressing the same than it does with alternative molecules or cells.
• a peptide that specifically binds to a molecule binds to that molecule with greater affinity, avidity, more readily, and/or with greater duration than it binds to other molecules.
• a peptide that specifically binds to a first molecule may or may not specifically bind to a second molecule.
• binding does not necessarily require exclusive binding or non-detectable binding of another molecule, this is meant by the term “selective binding”.
• binding of a peptide of the disclosure to a molecule means that the peptide binds to the molecule with an equilibrium constant (KD) of 100 nM or less, such as 50 nM or less, for example, 20 nM or less, such as, 15 nM or less or 10 nM or less or 5 nM or less or 1 nM or less or 500 pM or less or 400 pM or less or 300 pM or less or 200 pM or less or 100 pM or less.
• KD equilibrium constant
• An interaction between a molecule and a peptide may be identified using affinity purification.
• Affinity purification techniques are known in the art and are described in, for example, Scopes (In: Protein purification: principles and practice, Third Edition, Springer Verlag, 1994).
• Methods of affinity purification typically involve contacting a peptide with a specific target molecule, e.g., a target protein and, following washing to remove unbound or non- specifically bound peptides, eluting those peptides that remain bound to the target protein. By performing increasingly stringent washes, peptides having higher affinity for the target molecule are identified.
• An interaction between a molecule and a peptide may be identified using a protein chip or series of pins having immobilized thereon a target may be contacted with a peptide.
• the peptide is labelled with a detectable marker, e.g., a fluorescent marker.
• a detectable marker e.g., a fluorescent marker.
• the location of bound label is detected.
• the location of bound label is indicative of a peptide capable of binding to the target molecule.
• the identity of the peptide may then be conformed, e.g., using a method described herein, e.g., MALD-TOF.
• An interaction between a molecule and a peptide may be identified using a surface-plasmon resonance assay, such as, for example, Biacore sensor chip technology (Biacore AB, UK).
• the Biacore sensor chip is a glass surface coated with a thin layer of gold modified with carboxymethylated dextran, to which a target molecule is covalently attached. A peptide is then brought into contact with the target molecule.
• a surface plasmon resonance assay detects changes in the mass of the aqueous layer close to the chip surface, through measuring changes in the refractive index. Accordingly, when a peptide binds to the target protein or nucleic acid the refractive index increases.
• An interaction between a molecule and a peptide may be identified with a biosensor based on the detection of diffractive optics technology (light-scattering).
• biosensors are available commercially, e.g., from Axela Biosensors Inc., Toronto, Canada.
• a biosensor may be used which is based on acoustic resonance, such as that produced by Akubio, Cambridge UK.
• An interaction between a molecule and a peptide may be identified using a commercially available chip.
• a GPCR chip may be used to identify an interaction between a peptide and a G-protein coupled receptor as described in Fang et al., 2002.
• An interaction between a molecule and a peptide may be identified using a screen, such as, for example, a radioimmunoassay (RIA), an enzyme immunoassay, fluorescence resonance energy transfer (FRET), matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), electrospray ionization (ESI), mass spectrometry (including tandem mass spectrometry, eg LC MS/MS), biosensor technology, evanescent fiber-optics technology or protein chip technology.
• a radioimmunoassay RIA
• an enzyme immunoassay fluorescence resonance energy transfer
• MALDI-TOF matrix-assisted laser desorption/ionization time of flight
• ESI electrospray ionization
• mass spectrometry including tandem mass spectrometry, eg LC MS/MS
• biosensor technology evanescent fiber-optics technology or protein chip technology.
• An interaction between a molecule and a peptide may be identified using the two-hybrid assay.
• Modified two-hybird assays may be used including, for example, a PolIII two hybrid system, the Tribrid system, a ubiquitin based split protein sensor system, a Sos recruitment system as described in Vidal and Legrain 1999 or the three hybrid assay.
• biopanning may involve a negative section step wherein peptides are incubated with a non-target population of cells in medium for a period of time and under conditions sufficient to allow peptides to adhere to the cells. Subsequent removal of the non-target population of cells from the medium will result in a proportion of peptides which have adhered to the cells being removed e.g., this negative selection may remove peptides with an affinity for adhering to a broad range of cells.
• biopanning may involve a positive section step wherein peptides from the one or more negative selection steps are incubated with a desirable target population of cells. Incubation of those peptides and cells for a period of time and under conditions sufficient to allow peptides to adhere to the target population of cells thereby isolating cell- specific or cell selective peptides.
• the present disclosure provides a method for directing a molecule to a target comprising:
• a cell i) contacting a cell with a modified binding protein linked to a molecule ⁇ i.e. a conjugate or fusion protein), wherein the cell expresses a target linked to a protein binding partner, and wherein the binding protein forms a covalent isopeptide bond with the protein binding partner such that the molecule is directed to the target.
• a modified binding protein linked to a molecule i.e. a conjugate or fusion protein
• the contacting at step i) may be for a time and under conditions sufficient for the conjugate to enter a cell.
• the peptide inserted into a region of the binding protein may be a cell penetrating peptide.
• the method may additionally comprise determining or identifying that a conjugate has been directed to a target.
• the method may also comprise producing cells that are stably or transiently transformed with a vector encoding the target linked to the protein binding partner.
• the present disclosure provides a method of identifying a peptide capable of translocating a membrane of a cell comprising:
• a modified binding protein ⁇ i.e. a binding protein wherein a peptide is inserted into a region of the binding protein
• a modified binding protein may be provided as a conjugate.
• the conjugate may comprise a modified binding protein and a detectable label.
• a modified binding protein may be displayed on a particle.
• the contacting at step i) may be for a time and under conditions sufficient for the modified binding protein to enter the endosome of the cell.
• the method may comprise an incubating at step after step ii) may be for a time and under conditions such that the modified binding protein is translocated out of the endosome.
• the method may additionally comprise treating the cell at step i) to remove modified binding protein that are associated with the membrane of the cells without disrupting the cell membranes.
• associated with the membrane it is meant that the peptide is in physical relation with the cell other than by means of a mechanism that is capable of transporting the peptide through the membrane of that particular cell or internalizing the peptide in that particular cell.
• treating the cells may comprise incubating the cells with a protease for a time and under conditions sufficient to remove and/or inactivate extrinsic modified binding proteins to the cells without disrupting the cell membrane.
• a FITC labelled binding partner (FITC-Partner) and a FITC labelled binding partner comprising an aspartic acid to alanine mutation (FITC-Partner DA ) are synthesised using the mild Fmoc chemistry method.
• the amino acid sequence for FITC-Partner is set forth in SEQ ID NO: 64.
• the amino acid sequence for FITC- Partner ⁇ is set forth in SEQ ID NO: 83.
• a biotin labelled binding partner (Bio-Partner) is synthesised using the mild conditions:
• Recombinant proteins are expressed in E. coli BL21 (DE3).
• LB media supplemented with 0.5 mg/mL kanamycin was inoculated from a glycerol stock. Overnight cultures are diluted 1000-fold, grown in auto-induction media (Sigma) with 0.5 mg/mL kanamycin and then cultured at 37 °C overnight.
• Ni-NTA Metal-affinity chromatography
• Qiagen Metal-affinity chromatography
• All samples are desalted using PD-10 desalting columns (GE Life Sciences) as per manufacturer's instructions. Protein concentration and purity is determined using the Pierce BCA analysis (Thermo Fisher Scientific) and SDS-PAGE analysis as previously described (Sambrook et al, 1989).
• SDS-PAGE is performed on 10 to 20% gradient gels. Dithiothreitol (DTT, Sigma) is added to a final concentration of 100 mM. Samples are then mixed with 6x SDS-PAGE loading buffer (0.23 M Tris-HCl, 0.24 % glycerol, 6.7 % SDS and 12 mM bromophenol blue). Samples are heated at 95 °C for 7 min before loading onto the gel. Gels were stained with Coomassie Blue, imaged using a Bio-Rad ChemiDoc XRS+, and analyzed using Image Lab 3.0 software (Bio-Rad).
• DTT Dithiothreitol
• T7-PreScission-Avi For T phage, vector construct designated, T7-PreScission-Avi, was generated for mid-copy number display of proteins using T7 Select 10 vector (Novagen) as a template.
• the T7-PreScission-Avi vector encodes a Rhinovirus 3C protease recognition site and an Avi-Tag and.
• the vector T7-PreScission-Avi comprises an EcoRI and Sail site positioned 3' of the nucleic acid encoding a 10B capsid protein to provide for insertion of nucleic acid encoding the desired cassette.
• Cassettes from pET28a+ vectors are amplified using specific primers that bind to the Mfel and Sail restriction sites.
• Amplified fragments are column purified, digested with Mfel/Sall and inserted in the T7-PreScission-Avi vector cut with EcoRI/Sall. Specific methods for cloning, propagation and maintenance are used as specified in the manual supplied with the T7Select Packaging Kit (Merck Millipore).
• pNp3 is an M13 vector comprising nucleic acid encoding a PelB leader, a hexahistidine tag, hemagglutinin tag, and a M13 pill coat protein.
• the vector pNp3 comprises an EcoRI and Sail site positioned 3' of the nucleic acid encoding the M13 pill coat protein to provide for insertion of nucleic acid encoding the desired cassette.
• pNp3 also contains a frameshift base between the EcoRI and Sail site that shifts the reading frame to ablate correct translation of pill when no DNA is inserted into the multiple cloning site. This avoids expression of wild-type phage that do not display a protein.
• Cassettes from pET28a+ vectors are amplified using specific primers that bind to the Mfel and Sail restriction sites. Amplified fragments are column purified, digested with Mfel/Sall and inserted in the pNp3 cut with EcoRI/ Sail.
• binding protein (BP) cassette and modified binding protein (MP) cassettes for use in bacteriophage display
• a BP library cassette was generated using pET28+ as a template.
• the BP library cassette comprises an EcoRI restriction site between nucleic acid encoding the binding protein and nucleic acid encoding binding protein that includes a frameshift base that shifts the reading frame to ablate correct translation of the binding protein partner when no DNA is inserted into the EcoRI restriction site.
• the nucleic acid sequence of the BP library cassette is set forth in SEQ ID NO: 90.
• MB library cassettes are generated using pET28+ as a template.
• the MP library cassette with an EcoRI restriction site in the designated loop region that includes a frameshift base that shifts the reading frame to ablate correct translation of modified binding protein 3' to the EcoRI cloning site when no DNA is inserted is set forth in SEQ ID NO: 91.
• T7 display vector To create a T7 display vector, the BP library cassette and MB library cassettes are cloned into T7-Prescission-Avi cut with EcoRI/ Sail. The resulting vectors are designated, T7-BP-Entry and T7-MP-Entry.
• M13-BP-Entry M13-MP-Entry
• nucleic acid fragments are generated using multiple consecutive rounds of PCR using tagged random oligonucleotides and mixture of nucleic acid fragments produced from viral, prokaryotic and eukaryotic genomes were digested with the restriction endonuclease Mfel, purified e.g., using a QIAquick PCR purification column (QIAGEN) as per manufacturer's instructions, and retained for ligation into a compatible EcoRI site of T7 vector or a M13 vector for subsequent display.
• QIAquick PCR purification column QIAquick PCR purification column
• Nucleic acid fragment libraries for T7 bacteriophage are generated by inserting Mfel digested nucleic acid fragments in T7-BP-Entry cut with EcoRI or T7-MP-Entry cut with EcoRI.
• T7 bacteriophage are prepared as specified in the manual supplied with the T7 Select Packaging Kit (Merck Millipore).
• Nucleic acid fragment libraries for M13 bacteriophage are generated by inserting Mfel digested nucleic acid fragments in M13-BP-Entry cut with EcoRI or cut with EcoRI. M13 bacteriophage are prepared using standard methods.
• PEG precipitated T7 and M13 phage are labelled with either AlexaFluor 488 (AlexaFluor® 488 carboxylic acid 2,3,5, 6-tetrafluorophenyl ester 5-isomer) or Oregon Green 488 (Oregon Green® 488 carboxylic acid, succinimidyl ester 5- isomer).
• AlexaFluor 488 AlexaFluor® 488 carboxylic acid 2,3,5, 6-tetrafluorophenyl ester 5-isomer
• Oregon Green 488 Opgon Green® 488 carboxylic acid, succinimidyl ester 5- isomer.
• SEC size exclusion chromatography
• Bovine pancreatic trypsin inhibitor is a well-characterised single-chain protein which contains three disulfide bonds, an alpha helix, a short 3-helix and a 3- stranded beta sheet.
• BPTI contains 58 amino acid residues and inhibits the proteolytic activity of trypsin.
• the amino acid sequence of BPTI is set forth in SEQ ID NO: 23.
• pET28a+ partner-BPTI-binding protein PBB
• pET28a+ binding protein-BPTI- partner BBP
• pET28a+ binding protein-BPTI m - partner BB m P
• pET28a binding protein-BPTI-partner DA BP DA
• pET28a+ binding protein-BPTI m -partner DA BB m P DA
• MB pET28a+ modified binding protein-BPTI
• MB was mixed with FITC-Partner at a 1: 1 to 1.10 stoichiometric ratio.
• FITC-Partner was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37 °C for 3 h or at 4 °C overnight. The reaction was then stopped.
• the reaction mixture was analysed by SDS PAGE under low light conditions.
• the gels were also then read under a FITC channel. Gels are also stained with Coomassie blue stain. The efficiency of the interactions between the MB and FITC- Partner was determined by comparing the molecular mass of the MB alone or FITC- Partner alone with the molecular mass of a sample of the reaction mixture comprising MB and FITC-Partner. In addition, densitometry was used to evaluate conjugation reaction efficiency.
• BPTI is function when expressed as PBB, BBP, BB m P, BBP DA , BB m P D and MB
• the ability of the recombinant proteins to inhibit the proteolytic activity of trypsin is performed using a colour-metric QuantiCleave protease assay kit (Pierce) as per manufacturer's instructions.
• Protease activity is measured as A450 using a fluorescence microplate reader 20 min after the addition of chromogenic reagent 2,4,6- trinitrobenzene sulfonic acid, which reacts with the primary amine of digested peptide and produces the colour reaction that can be quantified by the absorbance reader.
• BBP When incubated with FITC-partner, BBP did not form a complex as evidenced by the presence of a single band corresponding to the unconjugated mass of BBP at in the Coomassie Blue stained SDS PAGE gel ( ⁇ 21kDa), and the absence of a fluorescent band at the expected mass of BBP-FITC-partner complex in the FITC channel ( ⁇ 25kDa). Together these data indicate that the binding protein and protein binding partner, separated by BPTI, formed a stable, irreversible conjugate for BBP and that BPTI is thus cyclised.
• PBB did bind a small amount of FITC-partner as evidenced by a faint fluorescent band at the expected molecular weight for a PBB-FITC-partner conjugate ( ⁇ 27kDa) and a large band at ⁇ 3.5kDa corresponding to unreacted FITC-partner in the FITC channel analysed SDS PAGE gel.
• the faint band corresponded to a conjugation efficiency of less than 2% by densitometry.
• the Coomassie Blue stain SDS PAGE gel also displayed a faint band at ⁇ 27kDa, but this was less than 1% of the large band at ⁇ 24kDa corresponding to the unconjugated PBB.
• BBP DA was able to form a conjugate with FITC-partner as evidenced by a fluorescent band at ⁇ 25kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the BBP DA -FITC-partner conjugate. This indicates that the BBP DA does not form an internal isopeptide bond between its binding protein and DA mutant binding partner and that BPTI is thus not cyclised.
• MB formed a conjugate with FITC-partner as evidenced by a band at the correct molecular weight for the MB-FITC-partner conjugate in both FITC channel and Coomassie stained SDS gels ( ⁇ 24kDa).
• the efficiency of conjugation was determined by densitometry to be -70%, which was similar to that expected of the binding protein.
• T7 bacteriophage displayed BBP did not form a conjugate when incubated with FITC-partner, as evidenced by the absence of any fluorescent bands in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BBP was unable to conjugate with FITC-partner and is thus cyclised.
• T7 bacteriophage displayed BBPDA when incubated with FITC-partner formed a conjugate as evidenced by the presence of a fluorescent band at the correct molecular weight for the T7-capsid-displayed-BBPDA-FITC-partner conjugate ( ⁇ 63kDa) in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BBPDA does not form an internal isopeptide bond between its binding protein and DA mutant binding partner.
• T7 bacteriophage displayed MB formed a conjugate with FITC-partner as evidenced by a fluorescent band at the correct molecular weight ( ⁇ 62kDa) for the T7- capsid-displayed-MB-FITC-partner conjugate in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed modified binding protein folds correctly when BPTI is included in the modified site, and is able to efficiently conjugate with FITC-partner, and that the BPTI is cyclised.
• Partner-BPTI-binding This cassette comprises a protein binding SEQ ID NO: 25 protein (PBB) partner, a bovine pancreatic trypsin
• Binding protein-BPTI- This cassette comprises a binding protein, a SEQ ID NO: 26 partner (BBP) bovine pancreatic trypsin inhibitor, and a
• Binding protein- This cassette comprises a binding protein a SEQ ID NO: 27 ⁇ -partner (BB m P) bovine pancreatic trypsin inhibitor wherein
• Binding protein-BPTI- This cassette comprises a binding protein, a SEQ ID NO: 28 partner m (BBP DA ) bovine pancreatic trypsin inhibitor and a
• Binding protein- This cassette comprises a binding protein, a SEQ ID NO: 29 BPTI m -partner m bovine pancreatic trypsin inhibitor wherein
• This cassette comprises a modified binding SEQ ID NO: 30 protein-BPTI (MB) protein wherein a bovine pancreatic trypsin
• Example 3 Display of a non-catalytic domain
• SH3 SRC Homology 3 domain
• the basic fold of SH3 domains contain five anti-parallel beta-strands packed to form two perpendicular beta-sheets.
• SH3 contains 58 amino acid residues.
• SH3 does not contain any cysteine residues.
• the amino acid sequence of SH3 is set forth in SEQ ID NO: 31.
• pET28a+ partner-SH3-binding protein PSB
• pET28a+ SH3-binding protein SB
• BSP pET28a+ binding protein- SH3 -partner
• BS pET28a+ binding protein-SH3-partner DA
• MS pET28a+ modified binding protein-SH3
• the synthesised cassettes are cloned into the NcoIZ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 2.
• PSB, SB, BSP, BS, BSP DA and MS are expressed and purified using the method described in Example 1.
• MS was mixed with FITC-Partner at a 1: 1 to 1.10 stoichiometric ratio.
• MS was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37 °C for 3 h or at 4 °C overnight. The reaction is then stopped.
• the reaction mixture is analysed by SDS PAGE under low light conditions.
• the gels are then read under a FITC channel. Gels are also stained with Coomassie blue stain.
• the efficiency of the interactions between the MS and FITC-Partner was determined by comparing the molecular mass of the MS alone or FITC-Partner alone with the molecular mass of a sample of the reaction mixture comprising MB and FITC- Partner. In addition, densitometry was used to evaluate reaction efficiency.
• Partner-SH3- This cassette comprises a protein binding partner, a SEQ ID NO: 32 Binding protein SRC Homology 3 Domain and binding protein.
• This cassette comprises a SRC Homology 3 Domain SEQ ID NO: 33 protein (SB) and a binding protein.
• Binding protein- This cassette comprises a binding protein, a SRC SEQ ID NO: 34 SH3-Partner Homology 3 Domain and a protein binding partner.
• Binding protein- This cassette comprises a binding protein and a SRC SEQ ID NO: 35 SH3 (BS) Homology 3 Domain.
• Binding protein- This cassette comprises a binding protein, a SRC SEQ ID NO: 36
• This cassette comprises a modified isopeptide protein SEQ ID NO: 37 protein-SH3 (MS) wherein a SRC Homology 3 Domain is inserted into
• BSP did not form a conjugate as evidenced by the presence of a single band corresponding to the unconjugated mass of BSP at in the Coomassie Blue stained SDS PAGE gel ( ⁇ 21kDa), and the absence of a fluorescent band at the expected mass of BSP-FITC-partner conjugate in the FITC channel analysed SDS PAGE gel ( ⁇ 25kDa).
• PSB did bind a small amount of FITC-partner upon incubation, as evidenced by a faint fluorescent band at the expected molecular weight for a PBB-FITC-partner conjugate ( ⁇ 28kDa) in the FITC channel analysed SDS PAGE gel, however this corresponded to a conjugation efficiency of less than 1% by densitometry.
• the Coomassie Blue stain SDS PAGE gel also displayed a faint band at ⁇ 28kDa, but again this was less than 1% of the large band at ⁇ 24kDa corresponding to the unconjugated PSB.
• BSP DA was able to form a conjugate with FITC-partner as evidenced by a fluorescent band at ⁇ 25kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the BSP DA -FITC-partner conjugate.
• MS formed a conjugate with FITC-partner as evidenced by a band at the correct molecular weight for the MB -FITC-partner conjugate in both FITC channel analysed and Coomassie stained SDS PAGE gels (-24 kDa).
• the efficiency of conjugation was determined by densitometry to be 60%, and similar to that expected of the binding protein.
• T7 bacteriophage displayed BSP did not form a conjugate when incubated with FITC-partner, as evidenced by the absence of any fluorescent bands in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BSP was unable to conjugate with FITC-partner and that the SH3 domain is thus cyclised.
• T7 bacteriophage displayed BSP DA when incubated with FITC-partner, formed a conjugate with the presence of a fluorescent band at the correct molecular weight for the T7-capsid-displayed-BSP DA -FITC-partner conjugate ( ⁇ 63kDa) in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed BSP DA does not form an internal isopeptide bond between its binding protein and DA mutant binding partner.
• T7 bacteriophage displayed MS formed a conjugate with FITC-partner as evidenced by a fluorescent band at the correct molecular weight (-62 kDa) for the T7- capsid-displayed-MS-FITC-partner conjugate in the FITC channel analysis of an SDS PAGE gel. This indicates that the T7 bacteriophage displayed modified binding protein folds correctly when SH3 is included in the modified site, and is able to efficiently conjugate with FITC-partner, and that the SH3 is cyclised.
• Example 4 Display of a peptide mimetic
• p53 is best known as a tumor suppressor that transcriptionally regulates, in response to cellular stresses such as DNA damage or oncogene activation, the expression of various target genes that mediate cell-cycle arrest, DNA repair, senescence or apoptosis.
• the oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53.
• Several peptide inhibitors of the p53-MDM2/MDMX interactions have been identified including p53 17"28 , PMI, PMI N8A and PDI (see for example Hu et al. 2007, Li et al. 2010 and Ji et al. 2013).
• the amino acid sequence of p53 17 " 28 is set forth in SEQ ID NO: 38.
• the amino acid sequence of PMI is set forth in SEQ ID NO: 39.
• the amino acid sequence of PMI N8A is set forth in SEQ ID NO: 40.
• the amino acid sequence of PDI is set forth in SEQ ID NO
• the constitutive morphogenic protein 1 is an E3 ubiquitin ligase that also regulates the activity of the tumour suppressor protein p53 by binding to its DNA- binding domain.
• a peptide inhibitor of the p53-COPl interaction has been identified (see for example Yamada et al. 2013 and, Yamada, Das Gupta and Beattie 2013).
• the amino acid of p28 is set forth in SEQ ID NO: 100.
• the p53 transcription factor is located in the cell nucleus; to evaluate the biological action of the binding protein and modified binding protein p53 mimetics, they will need to be conjugated to a cell-penetrating peptide protein binding partner (CPP-partner).
• CPP-partner cell-penetrating peptide protein binding partner
• the CPP-component will facilitate the translocation of the binding protein and modified binding protein p53 mimetics from the extracellular space into the cytoplasm from where they can interact with either MDM2/ MDMX or COP1.
• pET28a+ modified binding protein-p53 17-28 (Mp53 17"28 ), pET28a+ modified binding protein-PMI (MPMI), pET28a+ modified binding protein-PMI N8A (MPMI N8A ), pET28a+ modified binding protein-PDI (MPDI) and pET28a+ modified binding protein-p28 (Mp28) are synthesized (DNA 2.0, Menlo Park, CA, USA).
• pET28a+ binding protein-p53 17-28 (Bp53 17"28 ), pET28a+ binding protein-PMI N8A (BPMI N8A ) and pET28a+ binding protein-p28 (Bp28) are synthesized (DNA 2.0, Menlo Park, CA, USA).
• the synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification.
• a description of each cassette (including the amino acid sequence) is provided in Table 3.
• Cassettes from pET28a+ vectors are amplified, column purified and inserted in a mammalian expression vector.
• Bp53 17"28 , BPMI N8A , Bp28, Mp53 17"28 , MPMI, MPMI N8A , MPDI and Mp28 are expressed in bacteria and purified using the method described in Example 1.
• each protein was mixed with CPP-Partner at a 1: 1 to 1.10 stoichiometric ratio.
• the protein was mixed with CPP-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37 °C for 3 h or at 4 °C overnight. The reaction is then stopped.
• the reaction mixture is analysed by Coomassie blue stained SDS PAGE gel.
• the efficiency of the interactions between the Mp53 17"18 , MPMI, MPMI N8A , MPDI and Mp28 with CPP-Partner was determined by comparing the molecular mass of the Mp53 17"18 , MPMI, MPMI N8A , MPDI or Mp28 alone or CPP-Partner alone with the molecular mass of a sample of the reaction mixture comprising Mp53 17 " 18 , MPMI, MPMI N8A , MPDI and Mp28, and CPP-Partner. In addition, densitometry was used to evaluate reaction efficiency.
• T47D breast cancer cell lines are obtained.
• T47D breast cancer cells express a mutated type of p53 which is localized in the cytoplasm and is known to be sensitive to MDM2 inhibition.
• HCT116 human colon cancer cell lines are obtained.
• HCT116's p53 deficient subline HCT116 p537- are obtained.
• U87 glioma cell lines are obtained.
• U87 glioma cell lines express a mutated type of p53.
• As a control U251 glioma cell lines are also obtained which express a wild type p53.
• CHO-K1 cell lines are obtained. CHO-K1 cell lines express wild type p53. Cell viability assay
• the activity of the Bp53 17"28 , BPMI N8A , Bp28, Mp53 17"28 , MPMI, MPMI N8A , MPDI, Mp28, Bp53 17"28 -CPP-partner conjugate, BPMI N8A -CPP-partner conjugate, Bp28-CPP-partner conjugate, Mp53 17"28 -CPP-partner conjugate, MPMI-CPP-partner conjugate, MPMI N8A -CPP-partner conjugate, MPDI-CPP-partner conjugate and Mp28- CPP-partner conjugate is evaluated in selected cells.
• Bp53 17"28 , BPMI N8A , Bp28, Mp53 17"28 , MPMI, MPMI N8A , MPDI, Mp28, Bp53 17"28 - CPP-partner conjugate, BPMI N8A -CPP-partner conjugate, Bp28-CPP-partner conjugate, Mp53 17"28 -CPP-partner conjugate, MPMI-CPP-partner conjugate , MPMI N8A -CPP- partner conjugate, MPDI-CPP-partner conjugate and Mp28-CPP-partner conjugate are added to cells at a range of concentration in complete medium.
• PrestoBlue® reagent is added to the media and the cells are incubated for a further 30 min. Fluorescent signal indicating cell metabolic activity are then be measured as per manufacturer's instructions using an EnSpire® multimode plate reader (Perkin Elmer).
• Increased cell death also indicates restoration of the normal programmed cell death pathways mediated by the P53 tumour suppressor.
• increased cell death indicates inhibition of the p53-COPl interaction, increased levels of p53 and restoration of the normal programmed cell death pathways mediated by the P53 tumour suppressor pathway.
• Bp53 17 " 28 forms a conjugate with CPP-partner when incubated together, as evidenced by an intense band on a Coomassie Blue stained SDS PAGE gel corresponding to the molecular weight of the Bp53 17 " 28 -CPP-partner conjugate
• BPMI N8A forms a conjugate with CPP-partner when incubated together, as evidenced by a band at ⁇ 19.5kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the BPMI N8A -CPP-partner conjugate in comparison to the unconjugated BPMI N8A molecular weight of ⁇ 14kDa. Densitometry measurements indicted that the conjugation efficiency for the reactions of BPMI N8A with CPP-partner was 60%. ( Figure 5).
• Bp28 forms a conjugate with CPP-partner when incubated together, as evidenced by a clear band at ⁇ 21kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the Bp28 -CPP-partner conjugate in comparison to the unconjugated Bp28 molecular weight of ⁇ 15.5kDa. Densitometry measurements indicated that the conjugation efficiency for the reaction of Bp28 with CPP-partner was 43% ( Figure 5).
• Mp53 17 " 28 forms a conjugate with CPP-partner when incubated together, as evidenced by an intense band at ⁇ 20.5kDa in a Coomassie Blue stained SDS PAGE gel. This molecular weight corresponds to the expected molecular weight for the
• Mp53 17 " 28 -CPP-partner conjugate The unconjugated Mp5317 " 28 molecular weight is ⁇ 15kDa. Densitometry measurements indicated that conjugation efficiency for the reaction of Mp53 17 " 28 with CPP-partner was 70% ( Figure 4). This data indicates that the modified binding protein folds correctly when p53 17 28 is included in the modified site, and retains its ability to conjugate CPP-partner. MPMI forms a conjugate with CPP-partner when incubated together, as evidenced by a clear band at ⁇ 20kDa in a Coomassie Blue stained SDS PAGE gel.
• This band corresponds to the expected molecular weight for the MPMI N8A -CPP-partner conjugate in contrast to the unconjugated MPMI N8A molecular weight of ⁇ 15kDa.
• the conjugation efficiency of the reaction between MPMI N8A and CPP-partner was determined to be 67% by densitometry ( Figure 4).
• MPDI forms a conjugate with CPP-partner when incubated together, as evidenced by an intense band at ⁇ 20.5kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the MPDI-CPP- partner conjugate, in comparison to the unconjugated MPDI molecular weight of ⁇ 15kDa.
• the conjugation efficiency of the reaction between MPDI and CPP-partner was determined to be 34% by densitometry (Figure 4).
• Mp28 forms a conjugate with CPP-partner when incubated together, as evidenced by a clear band at ⁇ 22kDa in a Coomassie Blue stained SDS PAGE gel. This band corresponds to the expected molecular weight for the Mp28 -CPP-partner conjugate, in comparison to the unconjugated Mp28 molecular weight of ⁇ 16.5kDa.
• the conjugation efficiency of the reaction between Mp28 and CPP-partner was determined to be 68% by densitometry (Figure 4). This data indicates that the modified binding protein folds correctly when p28 is included in the modified site, and retains its ability to conjugate CPP-partner.
• CHO-K1 cells, with wild type P53 were evaluated in parallel as a negative control.
• Mp53 " does not inhibit cell viability in either T47D (A) or CHO-Kl (C) cell lines.
• the Mp53 17"28 -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner (B) but does not impact CHO-Kl cell viability (D).
• both MPMI N8A and MPDI do not inhibit cell viability in T47D cells.
• the MPMI N8A -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner.
• the MPDI-CPP-partner conjugate also inhibits T47D cell viability in a dose dependent manner.
• the Mp28-CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner, but does not impact on the cell viability of CHO-Kl cells.
• Bp53 17 " 28 does not inhibit cell viability in either T47D (A) or CHO-Kl (C) cell lines ( Figure 9).
• the Bp53 17"28 -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner (B) but does not impact CHO- Kl cell viability (D) ( Figure 9).
• BPMI N8A does not inhibit cell viability in T47D cells ( Figure 10).
• the BPMI N8A -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner ( Figure 10).
• Bp28 does not inhibit cell viability in either T47D or CHO-Kl cells ( Figure 11).
• the Bp28 -CPP-partner conjugate inhibits T47D cell viability in a dose dependent manner, but does not impact on the cell viability of CHO-Kl cells ( Figure 11).
• Modified binding This cassette comprises a modified binding protein SEQ ID NO: 42 protein-p53 17 28 wherein a p53 mimetic, p53 17 28 , is inserted into a
• Modified binding This cassette comprises a modified binding protein SEQ ID NO: 43 protein-PMI (MP) wherein a p53 mimetic, PMI, is inserted into a
• Modified binding This cassette comprises a modified binding protein SEQ ID NO: 44 protein-PMI N8A wherein a p53 mimetic, PMI N8A , is inserted into a
• Modified binding This cassette comprises a modified binding protein SEQ ID NO: 45 protein-PDI wherein a p53 mimetic, PDI, is inserted into a
• Modified binding This cassette comprises a modified binding protein SEQ ID NO: protein-p28 (Mp28) wherein a p53 mimetic, p28, is inserted into a 126
• Binding protein- This cassette comprises a binding protein wherein a SEQ ID NO: p53 17"28 (Bp53 17 ⁇ 28 ) p53 mimetic, p53 17 28 , is appended on the C- 127
• Binding protein- This cassette comprises a binding protein wherein a SEQ ID NO: PMI N8A (BPMI N8A ) p53 mimetic, PMI N8A , is appended on the C- 128
• Binding protein- This cassette comprises a binding protein wherein a SEQ ID NO: p28 (Bp28) p53 mimetic, p28, is appended on the C-terminus of 129
• Rituximab is a widely used monoclonal antibody drug for treating certain lymphomas and autoimmune diseases.
• Rituximab is known to interact with a 15 amino acid loop of CD20.
• a peptide mimotope of the CD20 epitope (PMCD20) has been shown to be recognized by Rituximab (Du et al. 2007).
• the amino acid sequence of PMCD20 is set forth in SEQ ID NO: 46.
• Rituximab does not bind to a mutated version of PMCD20 where the cysteine residues are substituted for serine residues (PMCD20 m ).
• the amino acid sequence of PMCD20 m is set forth in SEQ ID NO: 47.
• BPP, BP m P, BPP DA , BP m P DA , MP and MP m are expressed and purified using the method described in Example 1. Display of peptide mimotopes on bacteriophage
• BPP, BP m P, BPP DA , BP m P DA , MP and MP m are displayed on T phage using the method described in Example 1.
• BPP, BP m P, BPP DA , BP m P DA , MP or MP m The ability of BPP, BP m P, BPP DA , BP m P DA , MP or MP m to bind Rituximab is assessed using an ELISA assay. Briefly, Rituximab is coated onto 96-well polystyrene plates at approximately 2 ug/ml overnight at 4 °C. The plates are blocked with DELFIA assay buffer with PBS 1% BSA (v/v) at room temperature for 1 hour. Following washing of the blocking buffer (3x, PBS/ TBS 0.05% Tween-20), BPP, BP m P, BPP DA , BP m P DA , MP or MP m are added and incubated for 1 hour at room temperature with shaking.
• bacteriophage displayed BPP, BP m P, BPP DA , BP m P DA , MP or MP m is assessed using an ELISA assay. Briefly, Rituximab is coated onto 96- well polystyrene plates at approximately 2 ug/ml overnight at 4 °C. The plates are blocked with DELFIA assay buffer with 1% BSA (v/v) at room temperature for 1 hour.
• bacteriophage displaying BPP, BP m P, BPP DA , BP m P DA , MP or MP m are added and incubated for 1 hour at room temperature with shaking.
• Binding protein-PMCD20- This cassette comprises a binding protein, SEQ ID NO: 48 partner (BPP) PMCD20 and a protein binding partner.
• Binding protein- This cassette comprises a binding protein, SEQ ID NO: 49 PMCD20 m -partner (BP m P) PMCD20 m and a protein binding partner.
• Binding protein-PMCD20- This cassette comprises a binding protein, SEQ ID NO: 50 partner DA (BPP DA ) PMCD20 and a protein binding partner
• Binding protein- This cassette comprises a binding protein, SEQ ID NO: 51
• Modified binding protein- This cassette comprises a modified SEQ ID NO: 52 PMCD20 (MP) binding protein wherein PMCD20 is
• Modified binding protein- This cassette comprises a modified SEQ ID NO: 53 PMCD20 m (MP m ) binding protein wherein PMCD20 m is
• Example 6 Display of a peptide mimotope
• MOMP mitochondrial outer membrane permeabilization
• the amino acid sequence of PUMA BH3 is set forth in SEQ ID NO: 71.
• BAX and Bak proteins are thought to form an oligomeric Mitochondrial Apoptosis-Induced Channel (MAC) in the outer mitochondrial membrane, which in turn results in the release of pro-apoptotic molecules such as cytochrome-c.
• MAC Mitochondrial Apoptosis-Induced Channel
• the importance of the BH3 domains in driving the apoptotic pathways has been recognised with the development of BH3-mimetics targeting some Bcl-2 pro-survival proteins, with molecules showing promise in clinical trials for the treatment of cancers such as leukemia and lymphoma.
• pET28a+ modified binding protein-BimBH3 (MBimBH3), pET28a+ modified binding protein-BidBH3 (MBidBH3), pET28a+ modified binding protein-PumaBH3 (MPumaBH3), pET28a+ binding protein-BimBH3 -protein binding partner (BBimBH3), pET28a+ binding protein-BidBH3- protein binding partner (BBidBH3) and pET28a+ binding protein-BPumaBH3- protein binding partner (BPumaBH3) are synthesized (DNA 2.0, Menlo Park, CA, USA).
• the synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen). All cassettes include a hexahistidine tag to aid purification. A description of each cassette (including the amino acid sequence) is provided in Table 5.
• Cassettes from pET28a+ vectors are amplified, column purified and inserted in a mammalian expression vector.
• cytochrome c release assay The ability of MBimBH3, MBidBH3, MPumaBH3, BBimBH3, BBidBH3 or BPumaBH3 to induce cytochrome c release in the absence of the known direct activators, Bim and Bid is using a cytochrome c release assay as described in Chipuk et al. 2008 and Checco et al. 2015. Briefly, digitonin-permeabilized cell pellets or MEFs are grown in DMEM, harvested and washed with PBS supplemented with 1 mM CaC12 and 1 mM MgC12.
• Cells are permeabilized by resuspension into a buffer containing 20 mM HEPES, pH 7.4, 250 mM sucrose, 100 mM KC1, 5 mM MgC12, 1 mM EDTA, 1 mM EGTA, and 100 ⁇ g/ml digitonin at 3.5 x 107 cells/ml. After incubation on ice for 5 min, 1% BSA is added to remove digitonin, and the cells are pelleted and resuspended in the same volume of buffer without digitonin.
• This suspension of permeabilized cells is incubated with MBimBH3, MBidBH3, MPumaBH3, BBimBH3, BBidBH3, BPumaBH3 at 30 °C for 1 h and is spun down at 1000 g for 5 min to separate the supernatant and the pellet. Equivalent amounts of the supernatant and the pellet are loaded onto 15% SDS-PAGE gels for cytochrome c immunoblotting, using an anti- cytochrome c antibody (BD Pharmingen).
• BBimBH3, BBidBH3 or BPumaBH3 is evaluated using the PrestoBlue® Assay (LifeTech). Briefly, cells are seeded into 96 well plates and allowed to settle overnight. MBimBH3, MBidBH3, MPumaBH3, BBimBH3, BBidBH3 or BPumaBH3 are added to cells at a range of concentration in complete medium. At 24 and 48 h time points a 1/lOth volume of the PrestoBlue® reagent is be added to the media and the cells are incubated for a further 30 min. Fluorescent signal indicating cell metabolic activity are then be measured as per manufacturer's instructions using an EnSpire® multimode plate reader (Perkin Elmer).
• Binding protein- BimBH3- This cassette comprises a binding protein, SEQ ID NO: 75 partner (BBimBH3) BimBH3 and a protein binding partner.
• Binding protein- BidBH3- This cassette comprises a binding protein, SEQ ID NO: 76 partner (BBidBH3) BidBH3 and a protein binding partner.
• Binding protein- This cassette comprises a binding protein, SEQ ID NO: 77 PumaB H3 -partner BPumaBH3 and a protein binding partner.
• Modified binding protein- This cassette comprises a modified binding SEQ ID NO: 72 BimBH3 (MBimBH3) protein wherein BimBH3 is inserted into a
• Modified binding protein- This cassette comprises a modified binding SEQ ID NO: 73 BidBH3 (MBidBH3) protein wherein BidBH3 is inserted into a
• Modified binding protein- This cassette comprises a modified binding SEQ ID NO: 74 PumaBH3 (MPumaBH3) protein wherein PumaBH3 is inserted into a
• Example 7 Display of a peptide aptamer
• the signal transducer and activator of transcription, Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors.
• a peptide aptamer (PA) has been shown to directly interact with the DNA -binding domain of Stat5.
• the amino acid sequence of PA is set forth in SEQ ID NO: 54.
• CPPAB pET28a+ cell penetrating peptide- partner-PA-binding protein
• CP DA PAB pET28a+ cell penetrating peptide- partner DA -PA-binding protein
• PABC pET28a+ partner -PA-binding protein -cell penetrating peptide
• P DA PABC pET28a+ partner-PA-binding protein
• PPAB pET28a+ partner DA -PA-binding protein
• P DA PAB pET28a+ partner DA -PA-binding protein
• PPAB pET28a+ partner - PA-binding protein
• PPAB pET28a+ partner DA -PA-binding protein
• P DA PAB pET28a+ partner - PA-binding protein
• PPAB pET28a+ partner DA -PA-binding protein
• CMPA pET28a+ modified binding protein-PA
• MPA
• the synthesised cassettes are cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen).
• the cassettes include a hexahistidine tag to aid purification.
• a description of the cassettes (including the amino acid sequence) is provided in Table 6.
• Cassettes from pET28a+ vectors are amplified, column purified and inserted in a mammalian expression vector.
• Transient transfection is performed as previously described by Weber et al.
• Sequence pET28a+ cell penetrating This cassette comprises a cell penetrating SEQ ID NO: 84 peptide- partner-PA- peptide, a protein binding partner, a peptide
• CPPAB binding protein
• This cassette comprises a cell penetrating SEQ ID NO: 85 peptide- partnerDA-PA- peptide, a protein binding partner wherein
• binding protein the aspartic acid residue capable of forming
• This cassette comprises a protein binding SEQ ID NO: 86 binding protein-cell partner, a peptide aptamer, a binding
• pET28a+ partner DA -PA- This cassette comprises a protein binding SEQ ID NO: 87 binding protein -cell partner wherein the aspartic acid residue
• pET28a+ partner-PA- This cassette comprises a protein binding SEQ ID NO: 88 binding protein (PPAB) partner, a peptide aptamer and a binding
• pET28a+ partner DA -PA- This cassette comprises a protein binding SEQ ID NO: 89 binding protein (P DA PAB) partner wherein the aspartic acid residue
• This cassette comprises a modified binding SEQ ID NO: 56 binding protein-PA protein wherein a protein aptamer is
• Sequence pET28a+ cell penetrating This cassette comprises a cell penetrating SEQ ID NO: 57 peptide-modified binding peptide and a modified binding protein
• CMP A protein-PA
• Hstl human salivary peptide histatin 1
• EGF extracellular signal-regulated kinases 1/2
• pET28a+ binding protein-Hstl -protein binding partner BHP
• pET28a+ binding protein-Hstl BH
• pET28a+ modified binding protein-Hstl MH
• Titred BHP, BH and MH (1 nM to 20 uM) are added to the wells to determine whether the cyclized Histatin is more potent than the linear displayed peptide. Relative closure will be calculated by dividing the closure in the presence of peptide by that in the absence of peptide.
• TR146 cells are grown in 12-, 24-, or 48-well plates until confluence, and serum deprived for 24 h in keratinocyte serum-free medium (SFM; Invitrogen).
• SFM keratinocyte serum-free medium
• a scratch is made using a sterile tip, and cellular debris is removed by washing with SFM.
• the width of the scratch is determined microscopically immediately after creation and 16 h later.
• the effects of the following conditions on wound closure is analyzed:
• Sequence pET28a+ binding protein This cassette comprises a binding protein, SEQ ID NO: 80 -Histl- partner (BHP) Histl and a protein binding partner.
• pET28a+ binding protein- This cassette comprises a binding protein, SEQ ID NO: 81 Histl (BHP) Histl.
• This cassette comprises a modified binding SEQ ID NO: 79 binding protein-Histl protein wherein Histl is inserted into a
• Example 9 Identification of peptides that interact with a molecule
• a bacteriophage display library is generated and labelled using the method described in Example 1.
• Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.
• a bacteriophage display library is generated and labelled using the method described in Example 1.
• a Chinese hamster ovarian cell line CHO-Kl
• CHO-Kl cells do not express EGFR.
• a CHO-Kl cell line expressing EGFR is used for positive selection as described, for example, in Zhao et al. 2007.
• a human embryonic kidney (HEK)293 cell line is used for negative selection.
• HEK293 cells do not express EGFR.
• a HEK293 cell line expressing EGFR EGFR is used for positive selection as described, for example, in Schmidt et al. 2003 is used.
• a bacteriophage display library is generated and labelled using the method described in Example 1.
• a Chinese hamster ovarian cell line CHO-Kl
• a CHO-Kl cell line over-expressing the Ephrin receptor EphA2 is used for positive selection.
• Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.
• Example 12 Identification of peptides that interact with the CXCR4 receptor
• a bacteriophage display library is generated and labelled using the method described in Example 1.
• a Chinese hamster ovarian cell line CHO-K1
• CHO-K1 cells do not express the CXCR4 receptor.
• a CHO-K1 cell line over-expressing the CXCR4 receptor is used for positive selection.
• Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.
• Example 13 Identification of peptides capable of translocating a membrane of a cell
• a bacteriophage display library is generated using the method described in Example 1.
• phage display libraries are incubated with HEK-293, CHO-K1, NIH- 3T3, HeLa or COS-7 cells. After treatment to remove surface-bound phage, cells are harvested, either by trypsinization or cell scraping, and then lyzed to recover internalized phage. Between 1 to 5 iterative rounds of biopanning were performed for each screen.
• Peptides are identified by recovering the bacteriophage displaying the modified binding proteins and determining the nucleic acid sequence of the modified binding proteins. The deduced amino acid sequences of the peptides within the modified binding protein are then analyzed by:
• Bioinformatics employed PSIPRED algorithm employed PSIPRED algorithm.
• Database queries are performed using a database of known CPPs available at the database "CellPPD: Designing of Cell Penetrating Peptides", which provides in silico prediction of cell penetration efficiency based on a dataset of 708 experimentally- validated CPPs.
• CellPPD permits prediction of peptides having CPP-like properties in each pool of isolated or identified peptides based on their sequences, including the identification of CPP-like motifs in peptides.
• Example 14 Identification of receptor binding domain that interact with Epidermal growth factor receptor (EGFR) using a complex comprising a modified binding protein and a binding protein partner linked to a cell-penetrating peptide
• EGFR Epidermal growth factor receptor
• a bacteriophage display library is generated using the method described in
• Example 1 and labelled with either SEQ ID 92 or SEQ ID 93.
• a Chinese hamster ovarian cell line CHO-K1
• CHO-K1 cells do not express EGFR.
• a CHO-K1 cell line expressing EGFR is used for positive selection as described, for example, in Zhao et al. 2007.
• a human embryonic kidney (HEK)293 cell line is used for negative selection.
• HEK293 cells do not express EGFR.
• a HEK293 cell line expressing EGFR EGFR is used for positive selection as described, for example, in Schmidt et al. 2003 is used.
• Subtractive biopanning is performed as described, for example in Eisenhardt et al. 2007.
• Example 15 Identification of a peptide capable of translocating a membrane of a cell using a complex comprising a modified binding protein linked to an EGRF binding Affibody
• RMP library and MPR library cassettes For the display of a nucleic acid fragment library in any of the designated regions of the modified binding protein, several MP library cassettes are generated using pET28+ as a template, where the EGFR Affibody receptor-binding-domain is either N- or C-terminal to the modified binding protein, named RMP library and MPR library cassettes respectively.
• the RMP library and MPR library cassettes contain an EcoRI restriction site in the designated loop region that includes a frameshift base that shifts the reading frame to ablate correct translation of modified binding protein 3' to the EcoRI cloning site when no DNA is inserted.
• the nucleic acid sequences of the RMP library and MPR library cassettes are set forth in SEQ ID NO: 94 and 95 respectively.
• T7 display vector To create a T7 display vector, the RMP library and MPR library cassettes are cloned into T7-Prescission-Avi cut with EcoRI/ Sail. The resulting vectors are designated, T7-RMP-Entry and T7-MPR-Entry.
• a bacteriophage display library is generated and labelled using the method described in Example 1.
• a Chinese hamster ovarian cell line CHO-K1
• CHO-K1 cells do not express EGFR.
• a CHO-K1 cell line expressing EGFR is used for positive selection as described, for example, in Zhao et al. 2007.
• a human embryonic kidney (HEK)293 cell line is used for negative selection.
• HEK293 cells do not express EGFR.
• a HEK293 cell line expressing EGFR is used for positive selection as described, for example, in Schmidt et al. 2003 is used.
• phage display libraries are incubated with HEK-293-EGFR or CHO- Kl-EGFR cells. After treatment to remove surface-bound phage, cells are harvested, either by trypsinization or cell scraping, and then lyzed to recover internalized phage. Between 1 to 5 iterative rounds of biopanning were performed for each screen.
• Peptides are identified by recovering the bacteriophage displaying the modified binding proteins and determining the nucleic acid sequence of the modified binding proteins. The deduced amino acid sequences of the peptides within the modified binding protein are then analyzed by:
• Bioinformatics employed PSIPRED algorithm employed PSIPRED algorithm.
• Database queries are performed using a database of known CPPs available at the database "CellPPD: Designing of Cell Penetrating Peptides", which provides in silico prediction of cell penetration efficiency based on a dataset of 708 experimentally- validated CPPs.
• CellPPD permits prediction of peptides having CPP-like properties in each pool of isolated or identified peptides based on their sequences, including the identification of CPP-like motifs in peptides.
• Example 16 Display of a protein albumin binding domain (ABD)
• HSA Human serum albumin
• HSA human serum albumin
• IgG immunoglobulin G
• Jacobs et al. 2015 designed an albumin-binding domain based on the consensus sequence of 20 homologs of the GA module, and named it ABDcon. Jacobs et al. 2015, also mutated key residues in the ADBcon consensus sequence to tune the binding to Human Serum Albumin (HSA), Murine Serum Albumin (MSA) and Rat Serum Albumin (RSA).
• HSA Human Serum Albumin
• MSA Murine Serum Albumin
• RSA Rat Serum Albumin
• N-terminal extension of the consensus sequence with TIDEWL improves thermal stability through N-terminal helix extension and hydrogen- bond formation (Jacobs et al 2015, Lejon et al 2004 and Johansson et al 2002).
• the 1TF0 crystal structure also indicates that C-terminal extension with two residues (HA) extends the C-terminal helix of the ABD and appears to stabilise the interaction of HSA and the ABD.
• the albumin binding domains selected for display in the L2 Loop of the modified binding protein were: ABDcon (SEQ ID NO:98), ABDcon5 (SEQ ID NO:99), ABDcon9 (SEQ ID NO: 100) and SA20 (SEQ ID NO: 104).
• the albumin binding ability of the albumin binding domain displaying modified binding proteins was evaluated by an HSA binding affinity assay.
• HSA binding affinity assay To determine the impact on the albumin binding affinity of the above sequences being displayed in the loop of the modified binding protein, linear controls coupled to the protein binding partner were synthesized as peptides and ligated to the binding protein. These complexes were then evaluated in the HSA binding affinity assay in parallel to the modified binding proteins
• pET28a+ modified binding protein- ABDcon MABDcon
• pET28a+ modified binding protein-ABDcon5 MABDcon5
• pET28a+ modified binding protein- ABDcon9 MABDcon9
• pET28a+ modified binding protein-SA20 MSA20
• pET28a+ binding protein (B) were codon optimized for E.coli expression and synthesized (DNA 2.0, Menlo Park, CA, USA).
• the synthesized cassettes were cloned into the Ncol/ Xhol of the pET28a+ expression vector (Novagen).
• the cassettes include a hexahistidine tag and prescission protease cleavage site to aid purification. A description of the peptides and a list of their corresponding SEQ ID Nos is provided in Table 9.
• Modified binding protein- This cassette comprises a modified SEQ ID NO: 105 pep tide ABDcon binding protein wherein ABDcon is
• Modified binding protein- This cassette comprises a modified SEQ ID No: 106 pep tide ABDcon5 binding protein wherein ABDcon5 is
• Modified binding protein- This cassette comprises a modified SEQ ID NO: 107 pep tide ABDcon9 binding protein wherein ABDcon9 is
• Modified binding protein- This cassette comprises a modified SEQ ID NO: 108 peptide SA20 (MSA20) binding protein wherein ABDcon9 is
• Binding protein This cassette comprises a binding SEQ ID NO: 14 protein
• MABDcon, MABDcon5, MABDcon9 and MSA20 were expressed and purified using the method described in Example 1.
• MABDcon To test the ability of MABDcon, MABCon5, MABDcon9 and MSA20 to interact with a protein binding partner, was mixed with FITC-Partner at a 1: 1 to 1.10 stoichiometric ratio.
• MABDcon was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.
• MABDcon5 was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.
• MABDcon9 was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.
• MSA20 was mixed with FITC-Partner at a 1: 1.1 stoichiometric ratio. Reactions were incubated at 37°C for 3 h or at 4°C overnight. The reaction was then stopped.
• the reaction mixture was analysed by SDS PAGE under low light conditions. The gels are also then read under a FITC channel. Gels were also stained with Coomassie blue stain.
• the efficiency of the interactions between MABDcon and FITC- Partner, MABDcon9 and FITC-Partner, MABDcon9 and FITC-Partner, and MSA20 and FITC-Partner was determined by comparing the molecular mass of MABDcon, MABDcon5, MABDcon9 or MSA20 alone or FITC-Partner alone with the molecular mass of a sample of the reaction mixture comprising MABDcon and FITC-Partner, MABDcon9 and FITC-Partner, MABDcon9 and FITC-Partner, and MSA20 and FITC- Partner, respectively.
• densitometry was used to evaluate conjugation reaction efficiency.
• partner-ABDcon5, partner-ABDcon9 and partner-SA20 synthetic peptides were conjugated to binding protein by mixing at a peptide: protein stoichiometric ratio of 1.1: 1, to generate B- partner-ABDcon5, B-partner-ABDcon9 and B-partner-SA20 conjugates respectively. Reactions were incubated at 37°C for 3 h or at 4 °C overnight. The reaction was then stopped.
• biolayer interferometry was used to measure the capture of biotinylated
• HSA bio-HSA
• streptavidin biosensors step 1
• the biosensors were then incubated with solutions containing various concentrations of the albumin binding domain containing modified binding proteins, and binding protein - protein binding partner conjugates, respectively, to measure the rates of association of the proteins to bio-HSA (Step 2).
• the biosensors were then moved from the protein solutions into assay buffer to measure the rates of dissociation (Step 3).
• the rates of association in step 2 and rates of dissociation in step 3 were used to determine the binding affinities of the various proteins, respectively, according the manufacturer's instructions (Pall ForteBio).
• Binding affinity was measured in molar terms, with increased affinity indicated by decreasing concentration.
• Modified binding protein, binding protein and protein binding partner conjugations MABDcon formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at -24 kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MABDcon-FITC- partner conjugate, in comparison to the unconjugated MABDcon molecular weight of ⁇ 20.5kDa. This confirmed that the modified binding protein was able to fold correctly when ABDcon is displayed in the modified site, and retained its ability to conjugate to FITC-partner (Figure 13).
• MABDcon5 formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at -24 kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MABDcon5- FITC-partner conjugate, in comparison to the unconjugated MABDcon5 molecular weight of ⁇ 20.5kDa. This confirmed that the modified binding protein was able to fold correctly when ABDcon5 is displayed in the modified site, and retained its ability to conjugate to FITC-partner (Figure 13).
• MABDcon9 formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at ⁇ 24kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MABDcon9- FITC-partner conjugate, in comparison to the unconjugated MABDcon9 molecular weight of ⁇ 20.5kDa. This confirmed that the modified binding protein was able to fold correctly when ABDcon9 is displayed in the modified site, and retained its ability to conjugate to FITC-partner (Figure 13).
• MSA20 formed a conjugate with FITC-partner when incubated together, as evidenced by the presence of a fluorescent band at -20 kDa in the FITC channel analysed SDS PAGE gel, corresponding to the expected mass for the MSA20-FITC- partner conjugate, in comparison to the unconjugated MSA20 molecular weight of ⁇ 16kDa.
• MABDcon5 when incubated with various concentrations of bio-HSA was determined to have a binding affinity of 58.7nM by biolayer interferometry ( Figure 14).
• B-partner-ABDcon5 conjugate when incubated with various concentration of bio-HSA was determined to have a binding affinity of 20nM by biolayer interferometry
• MABDcon9 when incubated with various concentrations of bio-HS A was determined to have a binding affinity of 75.9 nM by biolayer interferometry
• MSA20 when incubated with various concentrations of bio-HSA was determined to have a binding affinity of 79.7 nM by biolayer interferometry
• Binding protein control, B does not bind to bio-HSA as determined by biolayer interferometry ( Figure 15).
• Example 17 Bacteriophage display of a Phylomer peptide library constrained by interaction between a binding protein and a binding protein partner
• cyclic peptides Compared to linear peptides, cyclic peptides have been shown to be superior protein interaction partners due to i) more biologically relevant conformations, ii) increased structural rigidity and iii) higher stability due to reduced proteolytic attack.
• a subset of the Phylomer peptides will be presented as cyclic peptides when the binding protein partner forms an intramolecular, covalent bond with the preceding binding protein.
• a Phylomer library containing such cyclic peptides is expected to provide superior interaction partners for targets, which require a structured interaction partner.
• One configuration of such a library is schematically illustrated in Figure 16. Materials and Methods
• binding protein-binding protein partner library cassette (BP_BPP_lib) or use in bacteriophage display
• a BP_BPP_lib cassette was generated in pET28a+.
• BP_BPP_lib cassette comprises an EcoRI restriction site between nucleic acids encoding the binding protein and nucleic acids encoding the binding protein partner.
• the EcoRI site is followed by two extra bases that shift the reading frame to ablate correct translation of the binding protein partner when no DNA is inserted into the EcoRI restriction site.
• the nucleic acid sequence and translated amino acid sequence of the BP_BPP_lib cassette are set forth in SEQ ID NOs: 109 and 110, respectively.
• the T7-Prescission-Avi-BP_BPP_lib vector was used to generate the T7 Phylomer library T16, in which the library inserts were flanked at the N-terminus by binding protein and by a binding protein partner at the C-terminus.
• the library was generated as described in Example 1.
• T16 library inserts were generated from the fully sequenced genomes of eight archaebacterial genomes, namely Aeropyrum pernix, Archaeoglobus fulgidus, Haloarcula marismortui, Halobacterium salinarum, Methanocaldococcus jannaschii, Pyrococcus horikoshii, Sulfolobus solfataricus and Thermoplasma volcanium.
• the library inserts had varying insert lengths. Only inserts of the length 3n+l allowed in-frame translation of the C- terminal binding protein partner.
• the random fragment generation process also produced inserts with stop codons leading to non-readthrough into the DNA of the C- terminal binding protein partner.
• 500 ⁇ of the naive T16 library were amplified in 25 ml of the host strain BLT5615/pSUMO-avi3, which greatly reduced biotinylation of the avi-tag displayed on the T7 phage.
• 0.5 ml of the amplified, PEG-precipitated T16 library were further cleared of pre-biotinylated T7-phage by passing over a column containing 300 ul of Streptavidin Sepharose High Performance resin (GE Healthcare, cat#17-5113-0).
• the phage titer after clearing was 6.3 x 10 11 pfu/ml.
• M-280 paramagnetic Streptavidin beads (Invitrogen, cat#11206D) were washed with 1 ml of PBS-0.05%-Tween-20 (PBS-T) and buffer aspirated. 1 ml of 1% BSA-PBS-0.05% Tween were added to the beads and blocked overnight at 4°C with rotation. The blocking buffer was aspirated, the beads were washed once in 1 ml of PBS and resuspended in 90 ul of PBS.
• PBS-T PBS-0.05%-Tween-20
• the amplified starting phage library was named T16_Amp
• the bead-captured phage fraction was named T16_Acapt
• the non-captured phage fraction T16_Acycl.
• the starting phage library, T16_Amp and the fractions from the bead binding experiment T16_Acapt and T16-Acycl were analysed by 250 bp Illumina paired-end MiSeq.
• the samples were prepared for sequencing by the 2-step PCR described in the Illumina manual "16S Metagenomic Sequencing Library Preparation Guide”. Due to low phage numbers, the complete volume of T16_Acapt was amplified in 2.5 ml of BLT5615 host culture before sequencing.
• each phage fraction was used as a template for Amplicon PCR as described in the Illumina manual.
• the reactions were carried out with equimolar mixtures of four staggered forward and four staggered reverse primers as shown in SEQ ID NOs: 116-119.
• Oligonucleotides were synthesised by Sigma as desalted PCR primers.
• the PCR reaction was carried out with Herculase II Fusion DNA polymerase (Agilent
• the PCR reaction was carried out with Herculase II Fusion DNA polymerase (Agilent Cat#600675). Thermocycling conditions were: 2 min at 95°C + 8 cycles [20 sec at 95°C, 20 sec at 58°C, 1 min at 72°C] + 3 min at 72°C. The reactions were cleaned up by Agencourt AMPure XP PCR Purification beads (Beckman Coulter Cat#A63880) as described by the manufacturer.
• Samples were submitted to the Australian Genome Research Facility for sequencing by paired-end 250 bp Illumina MiSeq. Samples were loaded at 600 K/mm cluster density with 15% PhiX. The quality of the raw data was confirmed by FastQC. The paired-end sequencing reads were converted into Phylomer DNA and Phylomer peptide sequences by the in-house software PhySeq4NGS.
• the complexity of the Phylomer peptide library T16 was determined as 2.6 x 10 by phage titering before library amplification; the titer of the amplified library was 5.2 x 10 11 pfu/ml.
• NGS NGS was used to assess the genome coverage in the library.
• Table 10 shows the genomes covered in T16_Amp. Fold-difference: Observed percentage divided by expected percentage. ⁇ Expected representation is achieved if fold-difference is between 0.67 and 1.5. Over-representation if fold-difference >1.5; under-representation if fold-difference ⁇ 0.67.
• T16_Amp contained all eight archaebacterial genomes that were inserted into the library.
• the genomic fragments were pooled according to genome sizes to achieve equal coverage of genomic loci resulting in different expected percentage values for each genome.
• the observed proportion of genomic fragments from Methanocaldococcus jannaschii in the T16 library was 1.8- fold higher than expected, while the remaining seven genomes were in the expected range with four genomes slightly underrepresented.
• NGS also allowed the assessment of the library diversity. All peptide sequences of 6 aa and longer were clustered at 80% homology to work out the number of unique peptide sequences (Table 11); the copy numbers of all unique sequences are also shown in Table 11. The diversity is reflected in the "diversity number", which is calculated by dividing the number of unique sequences >5 aa by the number of peptide sequences >5 aa. The fact that 93% of all unique peptides occurred only once and a diversity number of 0.89 for T16_Amp indicated a very good diversity.
• Table 11 provides a summary of numbers of peptide sequences identified by NGS. The diversity number is calculated by dividing the number of unique sequences >5 aa by the number of peptide sequences >5 aa. Copy numbers for unique peptide sequence indicate the percentage of unique sequence that were identified once, twice etc.
• T16 library parameters are summarised in Table 12. All of the general peptide parameters were in the normal range for Phylomer libraries. The mean DNA insert length was determined as 222 bp, which translates into a peptide length of 74 aa. The mean observed peptide length, however, was only 20.5 aa indicating the presence of non-readthrough inserts caused by stop-codons in the randomly- amplified g fragments.
• Table 12 provides a summary of DNA insert lengths, display peptide lengths and percentage of annotated open reading frames (ORF).
• each library insert was separately assessed to determine whether the translated peptide was in-frame (IF) or out-of-frame (OOF) with the C-terminal binding protein partner. In the case of stop codons in the reading frame, inserts were classified as non-readthrough (nonRT).
• a sample of amplified T16_Amp library was subjected to a binding experiment with immobilised synthetic binding protein partner in order to gain insight into the actual proportion of cyclised Phylomers in the library. Cyclisation via binding and binding protein partner results in an occupied binding pocket of the binding protein so that the phage clones cannot bind to immobilised binding protein partner, while phage clones with non-cyclised Phylomers should be able to bind to immobilised binding protein partner(see Figure 16).
• the experiment was carried out with 1 x 10 pfu of phage library and a high amount of binding protein partner immobilised on magnetic beads that should have theoretically allowed the capture of lOOfold more uncyclised phage.

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