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US20220090054A1 - Chimeric proteins - Google Patents

Chimeric proteins Download PDF

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US20220090054A1
US20220090054A1 US17/407,074 US202117407074A US2022090054A1 US 20220090054 A1 US20220090054 A1 US 20220090054A1 US 202117407074 A US202117407074 A US 202117407074A US 2022090054 A1 US2022090054 A1 US 2022090054A1
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seq
repeat
peptide ligand
peptide
protein
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Laura Itzhaki
Alberto Perez Riba
Pamela Rowling
Grasilda Zenkeviciüte
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Cambridge Enterprise Ltd
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Cambridge Enterprise Ltd
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Priority claimed from GBGB1713316.6A external-priority patent/GB201713316D0/en
Priority claimed from GBGB1714038.5A external-priority patent/GB201714038D0/en
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Priority to US17/407,074 priority Critical patent/US20220090054A1/en
Assigned to CAMBRIDGE ENTERPRISE LIMITED reassignment CAMBRIDGE ENTERPRISE LIMITED CONFIRMATORY ASSIGNMENT Assignors: ZENKEVICIUTE, GRASILDA
Assigned to CAMBRIDGE ENTERPRISE LIMITED reassignment CAMBRIDGE ENTERPRISE LIMITED CONFIRMATORY ASSIGNMENT Assignors: ITZHAKI, Laura, ROWLING, Pamela, RIBA, ALBERTO PEREZ
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1044—Preparation or screening of libraries displayed on scaffold proteins
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics

Definitions

  • This invention relates to chimeric proteins and their production and uses.
  • tandem repeat proteins A priority area in medicine, particularly cancer research, is the expansion of the ‘druggable’ proteome, which is currently limited to narrow classes of molecular targets.
  • PPIs protein-protein interactions
  • the architecture of tandem repeat proteins has tremendous scope for rational design (Kobe & Kajava 2000, Longo & Blaber, 2014, Rowling et al., 2015).
  • the key features of tandem repeat proteins are relatively small size, modularity and extremely high stability (and therefore recombinant production) without the need of disulphide bonds.
  • Individual consensus-designed repeats are self-compatible and can be put together in any order; function is therefore also modular, which means that multiple functions can be independently designed and incorporated in a combinatorial fashion within a single molecule (WO2017106728).
  • Novel repeat protein functions e.g. DARPins (Tamaskovic et al., 2012), have been developed based on the natural type of PIP interface of these proteins i.e. spanning many repeat units to create an extended, high-affinity binding interface for the target. Mutations have been introduced into the surface residues in the tetratricopeptide (TPR) repeats of the cytosolic receptor peroxin 5 (Sampathkumar et al. (2008) J Mol. Biol, 381, 867-880). Binding of peptide ligands to peroxin 5 is shown to be mediated by residues located in several different TPR repeats.
  • TPR tetratricopeptide
  • chimeric proteins which comprise peptidyl ligands, such as short linear motifs (SLIMs), on scaffolds.
  • SLIMs short linear motifs
  • Such chimeric proteins i.e, modular binding proteins
  • ray be useful for example, as single- or multi-function protein therapeutics.
  • An aspect of the invention provides a chimeric protein comprising:
  • the scaffold is a continuous polypeptide strand such that the first end is the N terminus and the second end is the C terminus of the scaffold.
  • the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule,
  • One of the first or second target molecules may be an E3 ubiquitin ligase.
  • the ligands are different ligands (bind to different targets) and are not located in the same loop or at the same end of a scaffold.
  • Another aspect of the invention provides a method of producing a chimeric protein comprising;
  • Another aspect of the invention provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising; providing a nucleic acid encoding a scaffold comprising two or more repeat domains
  • one of the first or second target molecules is an E3 ubiquitin ligase.
  • the invention provides a chimeric protein, comprising
  • a tetratricopeptide (PR) scaffold comprising first and second ⁇ -helices linked by an inter-repeat loop, and.
  • each of the first and second ⁇ -helices comprises the amino acid sequence Y-X1X2X3X4; wherein Y is an amino acid sequence shown in Tables 4 to 6 and X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or optionally wherein X2 is P.
  • first and second ⁇ -helices each comprise the amino acid sequence:
  • X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or optionally wherein X2 is P.
  • the chimeric protein comprising third, fourth and fifth TPR repeat.
  • the invention also provides a chimeric protein comprising
  • a TPR scaffold comprising first and second ⁇ -helices linked by an inter-repeat loop, and,
  • heterologous peptide is located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.
  • the invention also provides a chimeric protein comprising
  • a TPR scaffold comprising first and second ⁇ -helices linked by an inter-repeat loop, and,
  • Another aspect of the invention provides a library comprising chimeric proteins, each chimeric protein in the library comprising;
  • Another aspect of the invention provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
  • the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (i) an inter-repeat loop; (ii) the N terminus or (iii) the C terminus of the chimeric protein, and
  • Another aspect of the invention provides a method of producing a library of chimeric proteins comprising;
  • Another aspect of the invention provides a method of screening a library comprising;
  • FIG. 1 shows the thermostability of consensus-designed tetratricopeptide (CTPR) proteins containing loop- or helix-grafted binding motifs: Thermal denaturation, monitored by circular dichroism, of 2-repeat RTPR (a CTPR in which lysine residues have been replaced with arginine residues) proteins: RTPR2 (in diamonds), RTPR2 containing a loop binding-module (circles) and RTPR2 containing a helix binding-module (squares). All samples are at 20 ⁇ M in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl.
  • CPR consensus-designed tetratricopeptide
  • FIG. 2 shows the thermostability of CTPR proteins of increasing length containing an increasing number of binding modules (alternating with blank modules): Thermal denaturation curves, monitored by circular dichroism, of TPR proteins containing 1, 2, 3 and 4 loops comprising a tankyrase-binding sequence: 1TBP-CTPR2, 2TBP-CTPR4, 3TBP-CTPR6, 4TBP-CTPR8. All samples are at 20 ⁇ M in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl.
  • FIGS. 3A and 3B show an example of helix grafting.
  • FIG. 3A (i) shows the crystal structures of SOS1 (son-of-sevenless homologue 1) bound to KRAS (Kirsten rat sarcoma) (PDB INVU, Margarit et al. Cell (2003) 112(5):685-95), and (ii) shows the SOS1 helix grafted onto a helix at the N-terminus of a CTPR2 protein (SEQ ID NO: 3098).
  • the modelled structure of SOS-RTPR2 is shown, and the sequence of the helix is given with the key KRAS-binding residues in grey and the residues that form the interface with the CTPR helices in black.
  • (iii) shows the modelled structure of SOS-TPR2 in complex with KRAS.
  • FIG. 3B shows binding of SOS-TPR2 to KRAS measured by competitive fluorescence polarization (FP)
  • FP competitive fluorescence polarization
  • FIGS. 4A and B show another example of helix grafting.
  • FIG. 4A shows the modelled structure of the Mdm2 (Mouse double minute 2 homolog) N-terminal domain in complex with the p53-TPR2 comprising the Mdm2-binding helix of p53 grafted onto a helix at the C-terminus of a CTPR2 protein.
  • FIG. 4B shows an ITC analysis of the interaction between p53-TPR2 and Mdm2 N-terminal domain. The N-terminal domain of Mdm2 was titrated into the cell containing 10 ⁇ M p53-TPR2.
  • FIGS. 5A and 5B show an example of single and multivalent loop-grafted CTPRs.
  • FIG. 5A shows an ITC analysis of the interaction between a series of tankyrase-binding loop-grafted CTPR2proteins (TBP-CTPR2) and the substrate-binding ARC4 (ankyrin-repeat cluster) domain of tankyrase. There is an enhancement of both binding affinity and dissociation constant with increasing number of binding modules.
  • FIG. 5B shows native gel analysis (using a native gel in Tris-Glycine buffer pH 8.0, 40 ⁇ M protein concentration) of multivalent TBP-CTPR proteins expressed as fusion constructs with the foldon trimerisation domain (Boudko ct al 2002; Meier et al. 2004).
  • 1TBP-CTPR2, 2TBP-CTPR4 and 4TBP-CTPR8 (all lacking the foldon domain) were purified and run as monomeric controls. Constructs having the foldon domain run at much higher molecular weights than their monomeric counterparts.
  • FIGS. 6A and 6B show an example of loop-grafted CTPRs comprising the 10-residue Skp2-binding sequence derived from p27 grafted into a loop of a CTPR protein (CTPR-p27).
  • FIG. 6A shows that HA-CTPR2-p27 is able to co-IP FLAG-Skp2 from HEK293T cells
  • FIG. 6B shows E. coli -expressed and purified TPR5-p27 inhibits p27 ubiquitination in vitro.
  • FIGS. 7A and 7B show another example of loop-grafted CTPRs.
  • FIG. 7A shows (left) ITC analysis of the interaction between the Keap1 (Kelch-like ECH-associated protein 1) KELCH domain and a CTPR2 protein containing a loop-grafted Keap1-binding sequence derived from the protein Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) (Nrf-CTPR2). No binding is observed for the blank CPTR2 protein (right).
  • FIG. 7B shows that three variants of Nrf-CTPR2 (Nrf-CTPR2 (i). Nrf-CTPR2 (ii). Nrf-CTPR2 (iii) can co-IP Keap1 from HEK293T cells.
  • FIG. 8 shows live-cell imaging of intracellular delivery of an RTPR achieved by resurfacing (by introducing Arginine residues at surface sites).
  • PC3 left
  • U2OS right
  • FITC FITC-labelled resurfaced TBP-RTPR2
  • Overlay of DIC differential interference contrast
  • confocal image Intracellular fluorescence was also observed at lower concentrations of protein.
  • FIGS. 9A and 9B show the induced degradation of the target protein beta-catenin by designed hetero-bifunctional RTPRs.
  • FIG. 9A shows the beta-catenin levels in cells transfected with either HA-tagged beta-catenin plasmid alone or HA-tagged beta-catenin plasmid together with one of two different hetero-bifunctional RTPR plasmids (LRH1-TPR-p27 and axin-TPR-p27, designed to bind simultaneously to beta-catenin and to E3 ligase SCF Skp2 ).
  • FIG. 9A shows the beta-catenin levels in cells transfected with either HA-tagged beta-catenin plasmid alone or HA-tagged beta-catenin plasmid together with one of two different hetero-bifunctional RTPR plasmids (LRH1-TPR-p27 and axin-TPR-p27, designed to bind simultaneously to beta-catenin and to E3
  • FIG. 9B shows a quantitative analysis of the beta-catenin levels in the presence of different hetero-bifunctional RTPRs designed to bind simultaneously to beta-catenin and to either E3 ligase SCF Skp2 or E3 ligase Mdm2.
  • the analysis was performed using densitometry of the bands detected by Western blots corresponding to HA-tagged beta-catenin normalised to actin bands using ImageJ. Negative controls used were single-function TPRs or blank (non-functional) TPRs.
  • FIGS. 10A-10D show examples of different chimeric protein formats.
  • a chimeric protein may comprise: two repeat domains with a helical target-binding peptide and a helical E3-binding peptide at the N and C termini ( FIG. 10A ): three repeat domains with a helical E3-binding peptide at the C terminus and a target peptide ligand in the first inter-repeat loop from the N terminus ( FIG. 10B ); three repeat domains with a helical target-binding peptide at the N terminus and an E3 peptide ligand in the second inter-repeat loop from the N terminus ( FIG. 10C ), four repeat domains with a target-peptide ligand and an E3 peptide ligand in the first and third inter-repeat loop from the N terminus ( FIG. 10D ).
  • FIG. 11 shows a schematic of a chimeric protein with four peptide ligands located in alternate inter-repeat loops. The binding sites am arrayed at 90° to each other.
  • FIG. 12 shows a schematic of a chimeric protein engineered so that peptide ligands in alternate inter-repeat loops bind adjacent epitopes on the target.
  • FIG. 13 shows the modelled structure of a hetero-bifunctional chimeric protein comprising TPR repeat domains, an LRH1-derived peptide ligand designed to bind target beta-catenin, and a p53-derived N-terminal peptide ligand designed to bind to the E3 ubiquitin ligase mdm2.
  • FIG. 14 shows a schematic of the combinatorial assembly of a module comprising a repeat domain and a terminal helical peptide ligand and a module comprising repeat domains and an inter-repeat loop peptide ligand to generate a chimeric protein.
  • FIG. 15 shows examples of different chimeric protein formals.
  • (i) shows the blank proteins;
  • (ii) shows binding peptides inserted into one or more inter-repeat loops.
  • (iii) shows helical binding peptides at one or both of the termini;
  • (iv) is a combination of loop and helical binding peptides;
  • (v) and (vi) show examples of how multivalency can be achieved.
  • FIG. 16 shows a schematic of the assembly of a chimeric protein by the progressive screening of chimeric proteins comprising modules with a diverse peptide ligand in addition to modules already identified in previous rounds of screening.
  • FIG. 17 shows the effect of designed multi-valent tankyrase-binding TPR proteins on Wnt signalling.
  • HEK293T cells were transfected with TPR-encoding plasmids using Lipofectamine2000.
  • the TPR proteins contained 1-4 copies of a tankyrase-binding peptide (TBP) grafted onto the inter-repeat loop(s).
  • TBP tankyrase-binding peptide
  • 2TBP-CTPR4 is a protein comprising 4 TPR modules with one TBP grafted onto the loop between the first and second TPR and one between the third and fourth TPR.
  • ‘Foldon’ indicates a trimeic TPR-foldon fusion protein.
  • FIG. 18 shows characterisation of the size and charge of liposome-encapsulated TPR proteins.
  • FIG. 19 shows the delivery of TPR proteins into cells by liposome encapsulation.
  • FITC dye-labelled liposomes stain the cell membrane upon membrane fusion (red panel), and RITC-labelled TPR protein cargo is then delivered into the cytoplasm.
  • the green panel and red-green merge show that the proteins have entered the cells and are spread diffusely in the cytoplasm.
  • FIG. 20 shows that liposome-encapsulated TPR proteins are not toxic to HEK293T cells at the concentrations used.
  • FIG. 21 shows the effect of designed TPR proteins delivered by liposome encapsulation.
  • the TPR proteins contained a tankyrase-binding peptide. Cells were treated with liposomes for 2 hr.
  • FIG. 22 shows the effect of designed TPR proteins delivered by liposome encapsulation.
  • Cells were treated with liposomes encapsulating 32 ⁇ g protein for variable times (2-8 h) indicated in the figure.
  • FIG. 23 shows the effect of designed hetero-bifunctional TPR proteins on KRAS levels in HEK 293T cells.
  • the TPR proteins contained a binding sequence for KRAS (a non-helical peptide sequence, referred to as KBL, grafted onto an inter-repeat loop of the RTPR) and a degron derived from p27 grafted onto another inter-repeat loop.
  • KBL non-helical peptide sequence
  • KBL non-helical peptide sequence
  • degron derived from p27 grafted onto another inter-repeat loop.
  • Cells were transiently transfected with 50 ng or 500 ng of TPR encoding plasmids, as indicated, and with KRAS plasmid or empty vector as control. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. In dark grey are cells treated transfected with single-function TPR plasmid (containing degron only).
  • FIG. 24 shows the effect of hetero-bifunctional TPR proteins targeting endogenous KRAS to the CMA (chaperone-mediated autophagy) pathway.
  • the TPR proteins contained a binding sequence for KRAS (either a grafted helix derived from son-of-sevenless-homolog I (SOS) or a non-helical peptide sequence (referred to as ‘KBL’) displayed in a loop of the RTPR) and targeted for degradation using two different chaperone-mediated autophagy peptides (referred to as ‘CMA_Q’ or ‘CMA_K’) at the N- or C-terminus of the construct.
  • KRAS grafted helix derived from son-of-sevenless-homolog I
  • KBL non-helical peptide sequence
  • FIG. 25 shows examples of variations in the linker sequence connecting a peptide ligand to an inter-repeat loop in order to optimise the binding affinity for the target.
  • the example shown is Nrf-TPR, a TPR protein designed to bind to Kep1 (see FIG. 7 of the original patent application).
  • Glycine residues were introduced into the linker to provide flexibility and increased spatial sampling. The introduction of this more flexible linker sequence was found to increase the binding affinity of the Nrf-TPR protein (labelled ‘Flexible’) when compared with the consensus-like linker sequence.
  • Altering the charge content of the linker sequence (‘labelled ‘Charged’) and altering the conformational properties (based on the predictions of the program CIDER (Holehouse et al. Biophys. J. 112, 16-21(2017)) of the loop by changing the amino acid composition of the linker sequence (labelled ‘CIDER-optimised’) also affected the Keap1-binding affinity.
  • FIG. 26 shows the schematic representation of a matrix of degradation-inducing chimeric proteins.
  • the matrix shown is for use in targeting 1-catenin for degradation.
  • These proteins comprise a scaffold (grey rectangles) onto which are grafted: (1) a target-binding peptide ligand and (2) a binding peptide for an E3 ubiquitin ligase or a component of another degradation pathway.
  • Each of the target-binding peptides is derived from a different protein that interacts with A-catenin (see Table 2).
  • Each of the degradation pathway-binding peptides (referred to as “degrons”) is derived from a substrate or binding partner of one of many different E3s or from a binding partner for one of a component of another cellular degradation pathway (including chaperone-mediated autophagy, selective autophagy and ESCRT (endosome-lysosome) pathways); ‘etc.’ denotes the fact that there are many such proteins that can be harnessed for degradation, as detailed further in Table 3
  • the schematic illustrates the combinational “plug-and-play” nature of these matrices, in terms of the ability to slot in any target-recruiting peptide and degradation-pathway-recruiting peptide.
  • the other factor that can be varied in the matrix arises from the fact that the two peptides can also be grafted onto different positions in the scaffold so as to present the target in different configurations with respect to the E3 or other degradation machinery.
  • the matrix Once the matrix is constructed, it can then be screened in cell-based assay in order to identify the best combination of two peptides and their positions within the scaffold that induces the greatest reduction in target protein levels.
  • the same panel of diverse degradation pathway components can be used for screen for degradation of any target
  • This invention relates to the chimeric proteins that comprise multiple repeat domains. These repeat domains are linked to each other in the polypeptide chain by inter-repeat loops.
  • One or more peptide ligands i.e., peptidyl binding motifs or binding domains
  • the peptide ligands may be to the same or different target molecules and the chimeric protein may be multi-functional and/or multi-valent.
  • the geometrical display of the grafted binding sites may be precisely and predictably tuned by adjusting the positions of the binding sites and the number and shape of the repeat domains. Chimeric proteins as described herein may be useful in a range of therapeutic and diagnostic applications.
  • a “repeat domain” is a repetitive structural element of 30 to 100 amino acids that forms a defined secondary structure. Multiple (two or more) repeat domains stack sequentially in a modular fashion to form a stable protein, which may for example have a solenoid or toroid structure. Repeat domains may be synthetic or may be naturally-occurring repeats from tandem repeat proteins, or variants thereof.
  • EGF repeats include: EGF repeats, cadherin repeats, leucine-rich repeats, HEAT repeats, ankyrin repeats, armadillo repeats, tetratricopeptide repeats, etc.
  • EGF repeats Whenever a linear solenoid domain structure participates in protein-protein interactions, frequently at least 3 or more repetitive subunits form the ligand-binding sites.
  • individual repeats might have a (limited) ability to fold on their own—they usually cannot perform the functions of the entire domain alone.
  • a repeat domain may have the structure of a solenoid repeat.
  • the structures of solenoid repeats are well known in the art (see for example Kobe & Kajava Trends in Biochemical Sciences 2000; 25(10):509-15).
  • a repeat domain may have an ⁇ / ⁇ or ⁇ /3 10 (helix-turn-helix or hth) structure, for example a tetratricopeptide repeat structure; ⁇ / ⁇ / ⁇ (helix-turn-helix-turn-helix or hth) structure, for example an armadillo repeat structure; a ⁇ / ⁇ / ⁇ / ⁇ structure; a ⁇ / ⁇ or 3 10 / ⁇ structure, for example a leucine rich repeat (LRR) structure; a ⁇ / ⁇ / ⁇ structure, for example, an IGF1RL, HPR or PelC repeat structure; or a ⁇ / ⁇ structure, for example a serralysin or EGF repeat structure.
  • LRR leucine rich repeat
  • a “scaffold” refers to two or more repeat domains
  • a “grafted scaffold” refers to a continuous polypeptide comprising a scaffold and a heterologous binding site (e.g., a peptide ligand).
  • Ankyrin repeat one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases.
  • Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops.
  • the loops between adjacent Ankyrin repeats are semi-structured and therefore are quite rigid.
  • the global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions.
  • the repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics.
  • the armadillo (Arm) repeat is an approximately 40 amino acid long tandemly repeated sequence motif first identified in the Drosophila melanogaster segment polarity gene armadillo involved in signal transduction through wingless.
  • Animal Arm-repeat proteins function in various processes, including intracellular signalling and cytoskeletal regulation, and include such proteins as beta-catenin, the junctional plaque protein plakoglobin, the adenomatous polyposis coli (APC) tumour suppressor protein, and the nuclear transport factor importin-alpha, amongst others [(PUBMED:9770300)].
  • Suitable repeat domains may include domains of the Ankyrin clan (Pfam: CL0465), such as ankyrin (PF00023), which may comprise a 30-34 amino-acid repeat composed of two beta strands and two alpha helices; domains of the leucino-rich repeat (LRR) clan (Pfam; CL0022), such as LRR1 (PF00560), which may comprise a 20-30 amino acid repeat composed of an ⁇ / ⁇ horseshoe fold; domains of the Pec Lyase-like (CL0268) clan, such as pec lyase C (PF00544), which may comprise a right handed beta helix; domains of the bet-Roll (CL0592) clan such as Hacmolysin-type calcium-binding repeat (PF000353), which may comprise short repeat units (e.g.
  • Consensus Sequence for ARM repeats include the following:
  • Suitable repeat domains may be identified using the PFAM database (see for example Finn at al Nucleic Acids Research (2016) Database Issue 44:D279-D285).
  • the repeat domain may have the structure of an ⁇ / ⁇ -solenoid repeat domain, such as a helix-turn-helix.
  • a helix-turn-helix domain comprises two antiparallel ⁇ -helices of 12-45 amino acids.
  • Suitable helix-turn-helix domains include tetratricopeptide-like repeat domains.
  • Tetratricopeptide-like repeats may include domains of the TPR clan (CL0020), for example and Arm domains (see for example Armadillo; PF00514; Huber et al Cell 1997; 90: 871-882), HEAT domains (Huntingtin, EF3, PP2A-TORI; PF02985; see for example Groves et al. Cell. 96 (1): 99-110), PPR domains (pentatricopeptide repeat PF01535; see for example Small (2000) Trends Biochem. Sci.
  • TALE domains TAL (transcription activator-like) effector; PF03377; see for example Zhang ct al Nature Biotechnology. 29 (2): 149-53) and TPR1 domains (tetratricopeptide repeat-1; PF00515; see for example Blatch et at BioEssays. 21 (11): 932-9).
  • helix-turn-helix domain may be synthetic, for example DHR1 to DHR83 as disclosed in Brunette et al., Nature 2015 528 580-584.
  • the helix-turn-helix scaffold may be a tetratricopeptide repeat domain (TPR) (D'Andrea & Regan, 2003) or a variant thereof.
  • TPR repeat domains may include naturally occurring or synthetic TPR domains. Suitable TPR repeat domains are well known in the art (see for example Parmeggiani et al., J. Mol. Biol. 427 563-575) and may have the amino acid sequence:
  • TPR repeat consensus sequences include the following:
  • TPR repeat domain sequences are shown in Tables 4-6 and 9 below.
  • Preferred TPR domains may include CTPR, RTPRa, RTPRb and KTPRb domains, for example a domain having a sequence shown in Table 4 or Table 6 or a variant of a sequence shown in Table 4 or Table 6.
  • a TPR repeat domain may be a human TPR repeat domain, preferably a TPR repeat domain from a human protein in blood. TPR repeat domains from human blood may have reduced immunogenicity in vivo. Suitable human blood TPR repeat domains may include repeat domains from IFIT1, IFTT2 or IFIT3. Other examples of human blood repeat domains identified in the plasma proteome database are shown in Table 5.
  • Suitable human blood repeat domains may be identified from the plasma proteome database (Nanjappa et al Nucl Acids Res 2014 January; 42(Database issue):D959-65) for example by searching for sequences with high sequence identity to the TPR repeat domain using standard sequence analysis tools (e.g. Altschul et al Nucleic Acids Res. 25:3389-34021; Altschul et al FEBS J. 272:5101-5109).
  • a variant of a reference repeat domain or binding site sequence set out herein may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence.
  • Particular amino acid sequence variants may differ from a repeat domain shown above by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.
  • Preferred variants of a TPR repeat domain may comprise one or more conserved residues, for example, 1, 2, 3, 4, 5, 6 or more preferably all of Leu at position 7, Gly or Ala at position 8, Tyr at position 11, Ala at position 20, Ala at position 27, Leu or lie at positions 28 and 30 and Pro at position 32.
  • BLAST which uses the method of Altachul et aL (1990) J. Mol. Biol. 215:405-410
  • FASTA which uses the method of Pearson and Lipman (1988) PNAS USA 85:2444
  • a repeat domain may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands.
  • aromatic residues in the repeat domain may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of repeat domain sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions.
  • a suitable TPR repeat domain for grafting hydrophobic peptide ligands may have the amino acid sequence:
  • lysine residues in the repeat domain may be replaced by arginine residues to prevent ubiquitination and subsequent degradation.
  • This may be particularly useful when the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC).
  • a suitable TPR repeat domain may have the amino acid sequence:
  • the chimeric protein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 repeat domains.
  • the chimeric protein comprises 2 to 5 repeat domains.
  • Chimeric proteins with fewer repeat domains may display increased cell penetration.
  • a chimeric protein with 2-3 repeat domains may be useful in binding intracellular target molecule.
  • Chimeric proteins with more repeat domains may display increased stability and functionality.
  • a chimeric protein with 4 or more repeat domains may be useful in binding extracellular target molecules.
  • a chimeric protein with 6 or more repeat domains may be useful in producing long linear molecules for targeting or assembling extracellular complexes in bi- or multivalent formats.
  • a chimeric protein may comprise:
  • the repeat domains of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the repeat domains.
  • a “binding domain” (“peptide ligand”) is a contiguous amino acid sequence that specifically binds to a target molecule.
  • Suitable peptide ligands that are capable of grafting onto a terminal helix or inter-repeat loop are well-known in the art and include peptide sequences selected from a library, antigen epitopes, natural protein-protein interactions (helical, extended or turn-like) and short linear motifs (SLiMs). Viral SLiMs (that hijack the host machinery) may be particularly useful because they may display high binding affinities (Davey et al (2011) Trends Biochem. Sci. 36, 159-169).
  • a suitable peptide ligand for a target molecule may be selected from a library, for example using phage or ribosome display, or identified or designed using rational approaches or computational design, for example using the crystal structure of a complex or an interaction.
  • peptide ligands may be identified in an amino acid sequence using standard sequence analysis tools (e.g. Davey et al Nucleic Acids Res. 2011 Jul. 1; 39 (Web Server issue): W56-W60).
  • Peptide ligands may be 5 to 25 amino acids in length, preferably 8 to 15 amino acids, although in some embodiments, longer peptide ligands may be employed.
  • the two or more peptide ligands are 40 angstroms apart from each other, they may be 35 angstroms, 30 angstroms, 25 angstroms, 20 angstroms, 15 angstroms but no less than 10 angstroms apart.
  • a person of skill in art can use a 3D structural software such as Chimera or Pymol to determine the minimum distances between positions for ideal positioning in three dimensional orientation.
  • the peptide ligands and the repeat domains of the chimeric protein are heterologous i.e. the peptide ligand is not associated with the repeat domain in naturally occurring proteins and the binding and repeat domains are artificially associated in the chimeric protein by recombinant means.
  • a chimeric protein described herein may comprise 1 to n+1 peptide ligands, where n is the number of repeat domains in the chimeric protein.
  • the number of peptide ligands is determined by the required functionality and valency of the chimeric protein.
  • one peptide ligand may be suitable for a mono-functional chimeric protein and two or more peptide ligands may be suitable for a bi-functional or multi-functional chimeric protein.
  • Chimeric proteins may be monovalent.
  • a target molecule may be bound by a single peptide ligand in a monovalent chimeric protein.
  • Chimeric proteins may be multivalent.
  • a target molecule may be bound by two or more of the same or different peptide ligands in a multivalent chimeric protein.
  • Chimeric proteins may be monospecific.
  • the peptide ligands in a monospecific chimeric protein may all bind to the same target molecule, more preferably the same site or epitope of the target molecule.
  • Chimeric proteins may be multi-specific.
  • the peptide ligands in a multi-specific chimeric protein may bind to different target molecules.
  • a bi-specific chimeric protein may comprise one or more peptide ligands that bind to a first target molecule and one or more peptide ligands that bind to a second target molecule and a tri-specific chimeric protein may comprise one or more peptide ligands that bind to a first target molecule, one or more peptide ligands that bind to a second target molecule and one or more peptide ligands that bind to third target molecule.
  • a bi-specific chimeric protein may bind to the two different target molecules concurrently. This may be useful in bringing the first and second target molecules into close proximity.
  • a chimeric protein which binds to a tumour specific antigen and a T cell antigen, such as CD3, may be useful in bringing T cells into proximity to tumour cells.
  • a T cell antigen such as CD3
  • the target molecules are from different biological pathways, this may be may be useful in achieving synergistic effects and also for minimising resistance.
  • a tri-specific chimeric protein may bind to three different target molecules concurrently.
  • one of the target molecules may be an E3 ubiquitin ligase.
  • tri-specific chimeric protein may binding to a first target molecule from a first biological pathway and a second target molecule from a second biological pathway as well as an E3 ubiquitin ligase. This may be useful in achieving synergistic effects and also for minimising resistance.
  • a peptide ligand may be located in an inter-repeat loop of the chimeric protein.
  • inter-repeat binding domain or “inter-repeat peptide ligand” may comprise 5 to 25 amino acid residues, preferably 8 to 15 amino acids. However, since there is no intrinsic restriction on the size of the inter-loop peptide ligand, longer sequences of more than 25 amino acid residues may be used in some embodiments.
  • an unstructured peptide ligand may be inserted into an inter-repeat loop.
  • One or more, two or more, three or more, four or more or five or more of the inter-repeat loops in the chimeric protein may comprise peptide ligands.
  • the peptide ligands may be located on consecutive inter-repeat loops or may have a different distribution in the inter-repeat loops of the chimeric protein.
  • inter-repeat loops comprising a peptide ligand may be separated in the modular protein by one or more, two or more, three or more or four or more inter-repeat loops which lack a peptide ligand.
  • a peptide ligand may be connected to an inter-repeat loop directly or via one or more additional residues or linkers. Additional residues or linkers may be useful for example when a peptide ligand requires conformational flexibility in order to bind to a target molecule, or when the amino acid residues that are adjacent to the minimal peptide ligand favourably influence the micro-environment of the binding interface.
  • Additional residues or linkers may be positioned at the N terminus of the peptide ligand, the C terminus of the peptide ligand, or both.
  • the sequence of an inter-repeat loop containing a peptide ligand may be [X 1-i ]-[X 1-n ]-[X 1-z ], where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue that is also denoted by X, [X 1-n ] is the peptide ligand, a is 1 to 100, [X 1-i ] is a linker and i is independently any number between 1 to 10.
  • D may be preferred at the first position of the linker [X 1-i ]
  • P may be preferred at the second position of linker [X 1-i ]
  • D may be preferred at the last position of the linker [X 1-z ] and/or P may be preferred at the penultimate position of linker [X 1-z ].
  • Examples of preferred inter-repeat loop sequences may include DP-[X 1-n ]-PX (SEQ ID NO:4); DPXX-[X 1-n ]-XXPX (SEQ ID NO:5); DPXX-[X 1-n ]-XPXX (SEQ ID NO:6); DPXX-[X 1-n ]-PXX (SEQ ID NO:7); PXX-[X 1-i ]-[X 1-n ]-[X 1-i ]-XXPX (SEQ ID NO:8), DPXX-[X 1-i ]-[X 1-n ]-[X 1-i ]-XPXX (SEQ ID NO:9), DPXX-[X 1-i ]-[X 1-n ]-[X 1-n ]-PXX (SEQ ID NO:10), DPXX-[X 1-i ]-[X 1-n ]-XPXX (SEQ ID NO:11), DPXX-[X
  • residues or linkers used to connect a peptide ligand to an inter-repeat loop depends on the peptide ligand and may be readily determined for any peptide ligand of interest using standard techniques.
  • small, non-hydrophobic amino acids such as glycine
  • proline residues may be used to increase rigidity, for example, when the peptide ligands are short.
  • an inter-repeat peptide ligand may be non-hydrophobic.
  • at least 40% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W).
  • the repeat domains may be modified to accommodate a hydrophobic peptide ligand, for example by replacing aromatic residues with charged or polar residues.
  • a peptide ligand may be located at one or both termini of the chimeric protein.
  • a peptide ligand may be located in a helical region of the scaffold in the chimeric protein.
  • a helical region or “helix” is a portion of a scaffold which assumes an ⁇ -helical structure.
  • the precise length of a helical peptide ligand is dependent on the length of the helical region of the scaffold. In general, the helical peptide ligand is no longer than the length of the helical region of the scaffold. However, if the helical region of the scaffold is located at one or other termini or is flanked by unstructured or loosely structured residues, then it may be possible to extend it to accommodate a longer helical peptide ligand.
  • a helical peptide ligand may comprise 3 to 25 amino acid residues, preferably 8 to 15 amino acids in length.
  • a helical peptide ligand may comprise 3-10 or 3-12 or 3-15 or 8-10 or 8-12 or 8-13 or 8-14 or 8-15 or 3-18 or 3-20 or 3-21 or 3-22 or 3-24 or 3-25 amino acids.
  • a helical peptide ligand may comprise 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 amino acid residues.
  • a peptide ligand located at the N or C terminus may comprise an ⁇ -helical structure and may comprise all or part of a half-repeat (i.e. all or part of a single ⁇ -helix) that stacks against an adjacent repeat domain.
  • the ⁇ -helix of the terminal peptide ligand makes stabilising interactions with an adjacent repeat domain and is stable and folded. Only a few of the positions that structurally define an ⁇ -helix are required for the correct interfacial interaction with the adjacent repeat domain.
  • a helical peptide ligand may be located at the N terminus of the protein.
  • the N terminal peptide ligand may be helical and may comprise all or part of the sequence X n -(X) 15 -X 1 X 2 XX (SEQ ID NO:17), preferably all or part of the sequence X-XYXXXIXXYXXXLXX-X 1 X 2 XX (SEQ ID NO:18), where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, X 1 is independently any amino acid, preferably D, and X 2 is independently any amino acid, preferably P, and n is 0 or any number.
  • the Y, I, and/or L residues in the N terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).
  • a helical peptide ligand may be located at the C terminus of the scaffold.
  • the C terminal peptide ligand may be helical and may comprise all or part of the sequence X n -(X) 15 -X 1 X 2 XX, preferably all or part of the sequence X 1 X 2 XX-XXAXXXLXX[A or V]XXXXX-X n , (SEQ ID NO:19) where X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, X 1 is independently any amino acid, preferably D, and X 2 is independently any amino acid, preferably P, and n is 0 or any number.
  • the A, L and/or V residues in the C terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).
  • the minimum length of the terminal peptide ligand is determined by the number of residues required to form a helix that binds to the target molecule. There is no intrinsic maximum length of the terminal peptide ligand and n may be any number.
  • residues of the peptide ligand that are in contact with the peptide ligand binding partner are those whose side chains are outward facing and are exposed to solvent. These residues are suitable for grafting to a helical portion of a scaffold.
  • the residues of the scaffold helix whose side chains face inwards and pack against the rest of the scaffold should not be substituted, and this way their interactions with the rest of the scaffold are maintained.
  • PDB codes from arty protein databank provide three dimensional co-ordinates that allow one of skill in the art to visualize the structure of the domain using programs such as PYMOL®, CHIMERA® and RASMOL®.
  • the inward-facing residues of the helix are undisturbed, and hence the resultant grafted scaffold will have a grafted helix that comprises a mixture of outward facing residues derived from the helix peptide and the native inward facing residues of the helix that were undisturbed.
  • the following example shows a nine-residue helix peptide ligand (X1-X2-X3-X4-X5-X6-X7-X8-X9).
  • a 3-dimensional view of the peptide ligand in complex with the target protein shows that residues X1, X2, X5, X8 and X9 (for example) of the peptide ligand interact with the target protein and thus are outward facing.
  • a helical portion of a given scaffold may be thirty amino acids in length (Y1-Y2-Y3- . . . -Y28-Y29-Y30).
  • a 3-dimensional view of the scaffold shows the helical region and that residues Y3, Y4, Y6, Y7, and Y10 (for example) are inward facing and thus interact with the rest of the scaffold.
  • residues Y3, Y4, Y6, Y7, and Y10 for example
  • residues Y3, Y4, Y6, Y7, and Y10 for example
  • residues Y3, Y4, Y6, Y7, and Y10 for example
  • peptide ligand residues X1, X2, X5, X8 and X9 are grafted to the scaffold replacing residues Y1, Y2, Y5, Y8 and Y9 with the corresponding outward facing residues peptide ligand residues X1, X2, X5, X8 and X9, thereby creating an isomorphic replacement.
  • the resultant grafted scaffold will have a grafted helix whose sequence would include the following residues:
  • the resulting grafted helix preserves the native hydrogen bonding within the scaffold and at the same time preserves the noncovalent interactions required for specific binding of the peptide ligand to its target protein.
  • the “peptide ligand” may also contain more than one consecutive set of outward facing residues to graft into the scaffold, in which case the grafted scaffold may contain invariant scaffold residues between the grafted peptide residues (e, g “X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9”).
  • a helical peptide ligand may comprise all or part of the sequence CX 1 X 2 C 2 X 3 X 4 C 3 X 5 X 6 C 4 , where X 1 to X 6 are independently any amino acid and, C 1 , C 2 , C 3 and C 4 are A, B, C and D, respectively.
  • a helical peptide ligand may be non-hydrophobic.
  • at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W).
  • a peptide ligand located at the N or C terminus may comprise a non-helical structure.
  • a peptide ligand that is an obligate N- or C-terminal domain (for example because the terminal amino or carboxylate group mediates the binding interaction) may be located at the beginning or end of the one or more repeat domains.
  • one or more positions in a peptide ligand may be diverse or randomised.
  • a chimeric protein comprising one or more diverse or randomised residues may form a library as described below.
  • the N and C terminal peptide ligands may be non-hydrophobic.
  • at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W).
  • the helix turn helix scaffold of the repeat domains may be modified, for example by replacing aromatic residues with charged or polar residues in order to accommodate a hydrophobic peptide ligand.
  • a chimeric protein as described herein may comprise peptide ligands in any arrangement or combination.
  • peptide ligands may be located at both the N and C terminus and optionally one or more inter-repeat loops of a chimeric protein; at the N terminus and optionally one or more loops of a chimeric protein; at the C terminus and optionally one or more loops of a chimeric protein; or in one or more inter-repeat loops of a chimeric protein.
  • the location of the peptide ligands within a chimeric protein may be determined by rational design, for example using modelling to identify the optimal arrangement for the presentation of two target molecules to each other (e.g. for substrate presentation to an E3 ubiquitin ligase); and/or by screening for example using populations of chimeric proteins with different arrangements of peptide ligands to identify the arrangement which confers the optimal interaction of target molecules.
  • Target proteins and peptide ligands that bind such proteins are described herein and are listed, without limitation, in the tables.
  • Suitable target molecules for chimeric proteins described herein include biological macromolecules, such as proteins.
  • the target molecule may be a receptor, enzyme, antigen, oligosaccharide, oligonucleotide, integral membrane protein, transcription factor, transcriptional regulator, G protein coupled receptor (GPCR) or any other target of interest.
  • GPCR G protein coupled receptor
  • Target molecules may include ⁇ -synuclein; ⁇ -amyloid; tau; superoxide dismutase; buntingtin; ⁇ -catenin; KRAS; components of superenhancers and other types of transcriptional regulators, such as N-Myc, C-Myc, Notch, aurora A, EWS-FLI1 (Ewing's sarcoma-friend leukemia integration 1), TEL-AML1, TAL1 (T-cell acute lymphocytic leukemia protein 1) and Sox2 ((sex determining region Y)-box 2); tankyrases; phosphatases such as PP2A; epigenetic writers, readers and erasers, such as histone deacetylases and histone methyltransferases; BRD4 and other bromodomain proteins; and kinases, such as PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1) and B
  • a chimeric protein may neutralise a biological activity of the target molecule, for example by inhibiting or antagonising its activity or binding to another molecule or by tagging it for ubiquitination and proteasomal degradation or for degradation via autophagy. In other embodiments, a chimeric protein may activate a biological activity of the target molecule.
  • the target molecule may be ⁇ -catenin.
  • Suitable peptide ligands that specifically bind to ⁇ -catenin are well-known in the art and include ⁇ -catenin-peptide ligands derived from axin (e.g. GAYPEYILDIHVYRVQLEL (SEQ ID NO:20) and variants thereof), Bcl-9 (e.g. SQEQLEHRYRSLITLYDIQLML (SEQ ID NO:21) and variants thereof), TCF7L2 (e.g. QELGDNDELMHFSYESTQD (SEQ ID NO:22) and variants thereof), ICAT (e.g.
  • YAYQRAIVEYMLRLMS (SEQ ID NO:23) and variants thereof), LRH-1 (e.g. YEQAIAAYLDALMC (SEQ ID NO:24) and variants thereof) or APC (e.g. SCSEELEALEALELDE (SEQ ID NO:25) and variants thereof).
  • the target molecule may be KRAS.
  • Suitable peptide ligands that specifically bind to KRAS are well-known in the art and include a KRAS-peptide ligand from SOS-1 (e.g. FEGIALTNYLKALEG (SEQ ID NO:26) and variants thereof) and KRAS-peptide ligands identified by phage display (see for example Sakramnto et al. Biochem. Biophys. Res. Comm. (2017) 484 605-611).
  • the target molecule may be tankyrase.
  • Suitable peptide ligands that specifically bind to tankyrase are well-known in the art and include tankyrase peptide ligands from Axin (e.g. REAGDGEE (SEQ ID NO:27) and HLQREAGDGEEFRS (SEQ ID NO:28) or variants thereof).
  • the target molecule may be EWS-FLI1.
  • Suitable peptide ligands that specifically bind to EWS-FLI1 are well-known in the art and include the ESAP1 peptide TMRGKKKRTRAN (SEQ ID NO:29) and variants thereof.
  • Other suitable sequences may be identified by phage display (see for example Erkizan et al. Cell Cycle (2011) 10, 3397-408).
  • the target molecule may be Aurora-A.
  • Suitable peptide ligands that specifically bind to Aurora-A are well-known in the art and include Aurora-A binding sequences from TPX2, such as SYSYDAPSDFINFSS (SEQ ID NO:30)(Bayliss et al. Mol. Cell (2003) 12, 851-62) and Aurora-A binding sequences from N-myc, such as N-myc residues 19-47 or 61-89 (see for example Richards et al. PNAS (2016) 113, 13726-31).
  • the target molecule may be N-Myc or C-Myc.
  • Suitable peptide ligands that specifically bind to N-myc or C-myc are well-known in the art and include helical binding sequences from Aurora-A (see for example Richards et al. PNAS (2016) 113, 13726-31).
  • the target molecule may be WDR5 (WD repeat-containing protein 5).
  • WDR5 WD repeat-containing protein 5
  • Suitable peptide ligands that specifically bind to WDR5 are well-known in the art and include the WDR5-interacting motif (WIN) of MLL1 (mixed lineage leukemia protein 1) (see for example Song & guitarist J. Biol. Chem. (2008) 283, 35258-64; Patel eat al. J. Biol. Chem. (2008) 283, 32158-61), e.g. EPPLNPHGSARAEVHLRKS (SEQ ID NO:31) and variants thereof.
  • WIN WDR5-interacting motif
  • MLL1 mixed lineage leukemia protein 1
  • EPPLNPHGSARAEVHLRKS SEQ ID NO:31
  • the target molecule may be BRD4 or a Bromodomain protein.
  • Suitable peptide ligands that specifically bind to BRD4 are well-known in the art and include sequences derived from histone protein ligands.
  • the target molecule may be a HDAC (histone deacetylase).
  • HDAC histone deacetylase
  • Suitable peptide ligands that specifically bind to HDAC are well-known in the art and include binding sequences derived from SMRT and other proteins that recruit HDACs to specific transcriptional regulatory complexes or binding sequences derived from histone proteins (see for example Watson et al. Nat. Comm. (2016) 7, 11262; Dowling et al. Biochem. (2008) 47, 13554-63).
  • the target molecule may be Notch.
  • Suitable peptide ligands that specifically bind to Notch are well-known in the art and include binding sequences from the N-terminus of MAML1 (mastermind like protein 1), e.g. SAVMERLRRRIELCRRHHST (SEQ ID NO:32) and variants thereof (see for example Moellering et al. Nature (2009) 462, 182-8).
  • the target molecule may be a Cdk (cyclin-dependent kinase).
  • Suitable peptide ligands that specifically bind to Cdks are well-known in the art and include substrate-based peptides, for example, Cdk2 sequences derived from cyclin A, such as TYTKKQVLRMEHLVLKVLTFDL (SEQ ID NO:33) and variants thereof (see for example Gondeau et al. J. Biol. Chem. (2005) 280, 13793-800; Mendoza et al. Cancer Res. (2003) 63, 1020-4).
  • the target molecule may be PLK1 (polo-like kinase 1).
  • PLK1 poly-like kinase 1
  • Suitable peptide ligands that specifically bind to PLK1 arE well-known in the art and include optimised substrate-derived sequences that bind to the substrate-binding PBD (polo-box domain), such as MAGPMQSEPLMGAKK (SEQ ID NO:34) and variants thereof.
  • the target molecule may be Tau.
  • Suitable peptide ligands that specifically bind to Tau are well-known in the art and include tau-binding sequences derived from alpha- and beta-tubulin, such as KDYEEVGVDSVE (SEQ ID NO:35) and YQQYQDATADEQG (SEQ ID NO:36) and variants thereof (see for example Maccioni et al. EMBO J. (1988) 7, 1957-63; Rivas at al. PNAS (1988) 85, 6092-6).
  • the target molecule may be BCR-ABL.
  • Suitable peptide ligands that specifically bind to BCR-ABL are well-known in the art and include optimized substrate-derived sequences, such as EAIYAAPFARKK (SEQ ID NO:37) and variants thereof.
  • the target molecule may be PP2A (protein phosphatase 2A).
  • PP2A protein phosphatase 2A
  • Suitable peptide ligands that specifically bind to PP2A are well-known in the art and include sequences that bind the B56 regulatory subunit, such as LQTIQEEE (SEQ ID NO:38) and variants thereof (see for example Hetz et al. Mol. Cell (2016), 63 686-95), some embodiments, the target molecule may be EED (Embryonic ectoderm development).
  • Suitable peptide ligands that specifically bind to EED are well-known in the art and include helical binding sequences from co-factor EZH2 (enhancer of zeste homolog 2), such as FSSNRQKILERTEILNQEWKQRRIQPV (SEQ ID NO:39) and variants thereof (see for example Kim at al. Nat. Chem. Biol. (2013) 9, 643-50.)
  • the target molecule may be MCL-1 (induced myeloid leukemia cell differentiation protein).
  • MCL-1 induced myeloid leukemia cell differentiation protein
  • Suitable peptide ligands that specifically bind to MCL-1 are well-known in the art and include sequences from BCL2, e.g. KALETLRRVGDGVQRNHETAF (SEQ ID NO:40) and variants thereof (see for example Stewart et al. Nat. Chem. Biol. (2010) 6, 595-601).
  • the target molecule may be RAS.
  • RAS peptide ligands are well-known in the art and include RAS-binding peptides identified by phage display, such as RRRRCPLYISYDPVCRRRR (SEQ ID NO:41) and variants thereof (see for example Sakamoto et al. BBRC (2017) 484, 605-11).
  • the target molecule may be GSK3 (glycogen synthase kinase 3).
  • GSK3 peptide ligands are well-known in the art and include substrate-competitive binding sequences such as KEAPPAPPQDP (SEQ ID NO:42), LSRRPDYR (SEQ ID NO:1436), RREGGMSRPADVDG (SEQ ID NO:44), and YRRAAVPPSPSLSRHSSPSQDEDEEE (SEQ ID NO:45) and variants thereof (see for example Ilouz et al. J. Biol. Chem. 281(2006), 30621-30630. Plotkin et al. J Pharmacol. Exp. Ther. (2003) 305, 974-980).
  • the target molecule may be CtBP (C-terminal binding protein).
  • CtBP peptide ligands are well-known in the art and include sequences identified from a cyclic peptide library screen, such as SGWTVVRMY (SEQ ID NO:46) and variants thereof (see for example Birts et al. Chem. Sci. (2013) 4, 3046-57).
  • Suitable peptide ligands for target molecules that may be used in a chimeric protein as described herein are shown in Tables 2 and 7.
  • a chimeric protein as described herein may comprise a peptide ligand for an E3 ubiquitin ligase.
  • E3 ubiquitin ligases include MDM2, SCF Skp2 , BTB-CUL3-RBX1, APC/C, SIAH, CHIP, Cul4-DDB1, SCF-family, ⁇ -TrCP, Fbw7 and Fbx4.
  • Suitable peptide ligands for E3 ubiquitin ligases are well known in the art and may be 5 to 20 amino acids.
  • a suitable peptide ligand for MDM2 may include a peptide ligand from p53 (e.g. FAAYWNLLSAYG)(SEQ ID NO:47) and or a variant thereof.
  • a suitable peptide ligand for SCF Skp2 may include a peptide ligand from p27 (e.g. AGSNEQEPKKRS)(SEQ ID NO:48) and variants thereof.
  • a suitable peptide ligand for Keap1-Cul3 may include a peptide ligand from Nrf2 (e.g.
  • a suitable peptide ligand for SPOP-Cul3 may be include a peptide ligand from Puc (e.g. LACDEVTSTTSSSTA (SEQ ID NO:50) or a variant thereof.
  • a suitable peptide ligand for APC/C may include the degrons termed ABBA (e.g. SLSSAFHVFEDGNXEN)(SEQ ID NO:51), KEN (e.g. SEDKENVPP) (SEQ ID NO:52), or DBOX (e.g. PRLPLGDVSNN)(SEQ ID NO:53) or a variant thereof.
  • a suitable peptide ligand for SIAH may include a peptide ligand from PHYL (e.g. LRPVAMVRPTV) (SEQ ID NO:54) or a variant thereof.
  • a suitable peptide ligand for CHIP (carboxyl terminus of Hsc70-interacting protein) may include peptide sequences such as ASRMEEVD (SEQ ID NO:55) (from Hsp90 C-terminus) and GPTIEEVD (SEQ II) NO:56) (from Hsp70 C-terminus) or a variant thereof.
  • a suitable peptide ligand for beta-TrCP may include a degron sequence motif (including phosphomimetic amino acids), such as DDGYFD (SEQ ID NO:57) or a variant thereof.
  • a suitable peptide ligand for Fbx4 may include sequences derived from TRF1, such as MPIFWKAHRMSKMGTG (SEQ ID NO:58) or a variant thereof (see for example Lee et al. Chembiochem (2013) 14, 445-451).
  • a suitable peptide ligand for FBw7 may include degron sequence motifs (including phosphomimetic amino acids), such as LPSGLLEPPQD (SEQ ID NO:59).
  • a suitable peptide ligand for DDB1-Cul4 may include sequences derived from HBx (hepatitis B virus X protein) and similar proteins from other viruses and from DCAFs (DDB1-CUL4-associated factors) including helical motifs such as ILPKVLHKRTLGL (SEQ ID NO:60), NFVSWHANRQLGM (SEQ ID NO:61), NTVEYFTSQQVTG (SEQ ID NO:62), and NITRDLIRRQIKE (SEQ ID NO:63) (see for example Li et al. Nat. Struct. Mol. Biol. (2010) 17, 105-111).
  • a chimeric protein comprising a peptide ligand for an E3 ubiquitin ligase may also comprise a peptide ligand for a target molecule, Without being bound to any one hypothesis, binding of the chimeric protein to both the target molecule and the E3 ubiquitin ligase may cause the target molecule to be ubiquitinated by the E3 ubiquitin ligase. Ubiquitinylated target molecules may then degraded by the proteasome. This allows the specific targeting of molecules for proteolysis by the chimeric protein.
  • the ubiquitination and subsequent degradation of a target protein has been shown for hetero-bifunctional small molecules (PROTACs; proteolysis targeting chimeras) that bind the target protein and a ubiquitin ligase simultaneously (see for example Bondeson et al. Nat. Chem. Biol. 2015; Deshaies 2015; Lu et al. 2015).
  • PROTACs proteolysis targeting chimeras
  • the chimeric protein may lack lysine residues, so that it avoids ubiquitination by the E3 ubiquitin ligase.
  • a suitable chimeric protein may comprise an N terminal peptide ligand that binds a target protein, such as ⁇ catenin, and a C terminal peptide ligand that binds an E3 ubiquitin ligase.
  • the N terminal peptide ligand may be a ⁇ catenin-binding sequence derived from Bcl9 and the C terminal peptide ligand may be an Mdm2-binding sequence derived from p53.
  • a chimeric protein may comprise a C terminal peptide ligand that binds a target protein, such as ⁇ catenin, and an N terminal peptide ligand that binds an E3 ubiquitin ligase (see FIG. 10A ).
  • Another suitable chimeric protein may comprise three repeat domains, a peptide ligand located in an inter-repeat loop that binds a target protein, such as ⁇ catenin, and a C terminal peptide ligand that binds an E3 ubiquitin ligase.
  • the inter-repeat loop peptide ligand may be derived from the phosphorylated region of APC (adenomentous polyposis col) and the C terminal peptide ligand may be an Mdm2-binding sequence derived from p53.
  • the chimeric protein may comprise a peptide ligand located in an inter-repeat loop that binds an E3 ubiquitin ligase, and a C terminal peptide ligand that binds a target protein, such as ⁇ catenin (See FIG. 10B ).
  • Another suitable chimeric protein may comprise three repeat domains, an N terminal peptide ligand that binds a target protein, such as ⁇ catenin, and a peptide ligand located in an inter module loop that binds an E3 ubiquitin ligase.
  • the N terminal peptide ligand may be a ⁇ catenin-binding sequence derived from LRH1 (liver receptor homolog 1) and the inter-module loop peptide ligand may be a sequence derived from the Skp2-targeting region of p27.
  • the chimeric protein may comprise an N terminal peptide ligand that binds an E3 ubiquitin ligase and a peptide ligand located in an inter-module loop that binds a target protein, such as ⁇ catenin (ee FIG. 10C ).
  • Another suitable chimeric protein may comprise four repeat domains, a first peptide ligand located in an inter-repeat loop that binds an E3 ubiquitin ligase and a second peptide ligand located in an inter-repeat loop that binds a target molecule.
  • the first and second inter-repeat loops may be separate by an inter-repeat loop lacking a peptide ligand.
  • the first peptide ligand may be located in the first inter-repeat loop inter-repeat loop from the N terminus and the second peptide ligand may be located in the third inter-repeat loop from the N terminus or vice versa.
  • a chimeric protein as described herein may comprise an amino acid shown in Table 8 or a variant thereof.
  • a chimeric protein as described herein may comprise a peptide ligand that binds to a component of a target-selective autophagy pathway, such as chaperone-mediated autophagy (CMA).
  • CMA chaperone-mediated autophagy
  • the chimeric protein and target molecules bound thereto are thus recognised by the autophagy pathway and the target molecules are subsequently degraded.
  • Suitable components of the CMA pathway include heat shock cognate protein of 70 kDa (hsc70, HSPA8, Gene ID: 3312).
  • Suitable peptide ligands are well known in the art (Dice J. F. (1990). Trends Biochem. Sci.
  • a chimeric protein may further comprise one or more additional domains which confer additional functionality, such as targeting domains, intracellular transport domains, stabilising domains or oligomerisation domains. Additional domains may for example be located at the N or C terminus of the chimeric protein or in a loop between repeats.
  • a targeting domain may be useful in targeting the chimeric protein to a particular destination in vivo, such as a target tissue, cell, membrane or intracellular organelle.
  • Suitable targeting domains include chimeric antigen receptors (CARs).
  • An intracellular transport domain may facilitate the passage of the chimeric protein through the cell membrane into cells, for example to bind intracellular target molecules.
  • Suitable intracellular transfer domains are well known in the art (see for example Bechara et al FEBS Letters 587 1 (2013) 1693-1702) and include cell-penetrating peptides (CPPs), such as Antennapedia (43-58), Tat (48-60), Cadherin (615-632) and poly-Arg.
  • a stabilising domain may increase the half-life of the chimeric protein in vivo.
  • Suitable stabilising domains are well known in the art and include Fc domains, serum albumin, unstructured peptides such as XTEN 98 or PAS 99 and polyethylene glycol (PEG).
  • An oligomerisation domain may facilitate the formation of multi-protein complexes, for example to increase avidity against multi-valent targets.
  • Suitable oligomerisation domains include the ‘foldon’ domain, the natural trimerisation domain of T4 fibritin (Meier et al., J. Mol. Biol. (2004) 344(4):1051-69).
  • a chimeric protein may further comprise a cytotoxic or therapeutic agent and/or or detectable label.
  • Suitable cytotoxic agents include, for example, chemotherapeutic agents, such as methotrexate, auristatin adriamicin, doxorubicin, melphalan, mitomycin C, ozogamicin, chlorambucil, maytansine, catansine, daunorubicin or other intercalating agents, enzymatically active toxins of bacterial, fungal, plant, or animal origin, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, ⁇ -amanitin, alpha-sarcin, Aleurites fordii proteins, tubulysins, dianthin proteins, Phytolaca americana proteins (PAPI, PAP11, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mit
  • Suitable cytotoxic agents may also include radioisotopes.
  • a variety of radionuclides are available for the production of radioconjugated chimeric proteins including, but not limited to, 90 Y, 125 I, 131 I, 123 I, 111 In, 131 In, 105 Rh, 153 Sm, 67 Cu, 67 Ga, 166 Ho, 177 Lu, 186 Re, 188 Re and 212 Bi.
  • Conjugates of a chimeric protein and one or more small anti-cancer molecules for example toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, may also be used.
  • Suitable therapeutic agents may include cytokines (e.g. IL2, IL12 and TNF), chemokines, pro-coagulant factors (e.g. tissue factor), enzymes, liposomes, and immune response factors.
  • cytokines e.g. IL2, IL12 and TNF
  • chemokines e.g. IL2, IL12 and TNF
  • pro-coagulant factors e.g. tissue factor
  • enzymes e.g. liposomes
  • liposomes e.g. liposomes, and immune response factors.
  • a detectable label may be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers. Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. Detectable labels may be attached to chimeric proteins using conventional chemistry known in the art.
  • the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents.
  • the label can also be bound to another specific binding member that binds the chimeric protein, or to a support.
  • a chimeric protein may be configured for display on a particle or molecular complex, such as a cell, ribosome or phage, for example for screening and selection.
  • a suitable chimeric protein may further comprise a display moiety, such as phage coat protein, to facilitate display on a particle or molecular complex.
  • the phage coat protein may be fused or covalently linked to the chimeric protein.
  • Chimeric proteins as described herein may be produced by recombinant means.
  • a method of producing a chimeric protein as described herein may comprise expressing a nucleic acid encoding the chimeric protein.
  • a nucleic acid may be expressed in a host cell and the expressed chimeric protein may then be isolated and/or purified from the cell culture.
  • the recombinant method may comprise;
  • Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule.
  • a method may comprise;
  • One of the first and second target molecules may be an E3 ubiquitin ligase.
  • a method may comprise;
  • nucleic acid encoding a chimeric protein as described herein is provided as an aspect of the invention.
  • the nucleic acid may be comprised within an expression vector.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
  • the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell.
  • Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters.
  • a vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts such as E. coli and/or in eukaryotic cells.
  • a host cell comprising a nucleic acid encoding a chimeric protein as described herein or vector containing such a nucleic acid is also provided as an aspect of the invention.
  • Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals.
  • the expression of proteins in prokaryotic cells is well established in the art.
  • a common bacterial host is E. coli .
  • a chimeric protein may also be produced by expression in eukaryotic cells in culture.
  • Mammalian cell lines available in the art for expression of a chimeric protein include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells (e.g. HEK293 cells), human embryonic retina cells (e.g. PerC6 cells) and many others.
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • baby hamster kidney cells NSO mouse melanoma cells
  • YB2/0 rat myeloma cells e.g. HEK293 cells
  • human embryonic retina cells e.g. PerC6 cells
  • the pRSET B (His-tag) constructs are transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies are grown in 2TY media containing ampicillin (50 micrograms/mL) at 37° C., 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures are then induced with IPTG (0.5 mM) for 16-20 h at 20° C. or 4 h at 37° C.
  • Cells are pelleted by centrifugation at 3000 g (4° C., 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris is pelleted by centrifugation at 15,000 g at 4° C. for 45 min.
  • lysis buffer 10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution
  • Ni-NTA beads 50% bed volume (GE Healthcare)(5 mL) are washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) before the supernatant of the cell lysate is bound to them for 1 hr at 4° C. in batch.
  • the loaded beads are washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non-specific interaction of lysate proteins with the beads.
  • Samples are eluted using phosphate buffer with 300 mM imidazole, and purified by size-exclusion chromatography using a HiLoad 16/60 SuperdexG75 column (GE Life-Science) pre-equilibrated in phosphate buffer (10 mM sodium phosphate. pH 7.4, 150 mM NaCl) and proteins separated in isocratic conditions. Purity is checked on NuPage protein gel (Invitrogen), and fractions found to be over 95% pure are pooled. Purified protein is flash-frozen and stored at ⁇ 80° C. until further use. Concentrations are determined by measuring absorbance at 280 nm and using a calculated extinction coefficient from ExPASy ProtParam (Gasteiger et al. 2005) for each variant. Molecular weight and purity is confirmed using mass spectrometry (MALDI).
  • MALDI mass spectrometry
  • chimeric proteins described herein are thermally very stable, with melting temperatures above 80° C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65° C. for 20 min. Very few of the E. coli proteins will remain folded at such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins are removed by centrifugation, leaving 80-90% pure sample of the desired protein. Constructs that fold reversibly can be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.
  • This approach allows the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is potentially scalable to industrial production and bioreactors.
  • Plasmids are transformed into E. coli C41 cells and plated overnight. 15 mis of 2TY medium (Roche) containing 50 micrograms/ml ampicillin is placed in each one multiple 50 ml tubes. Several colonies are picked from the plates and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells are grown at 37° C. until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells are pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAseI treatment is used. The lysate is spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.
  • the supernatant is added to 100 ⁇ l bed volume of pre-washed Ni-NTA agarose beads.
  • the subsequent affinity purification is performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used).
  • the first ish contained 10% BugBustar® solution and 30 mM imidazole in the chosen buffer.
  • the three successive ishes had 30 mM of imidazole in the chosen buffer. Beads are washed thoroughly to remove the detergent present in the BugBuster® solution.
  • Protein is eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole.
  • chosen buffer containing 300 mM imidazole.
  • Imidazole is removed using a NAP-5 disposable gel-filtration column (GE Healthcare).
  • Competition FP can be performed using 384-well black opaque optiplate microplates and a CLARIOstar microplate reader.
  • the grafted scaffold protein is titrated into a solution containing a mixture of FITC-labelled peptide ligand and target binding partner (target protein).
  • target protein target binding partner
  • the prepared plates are incubated for 30 minutes at room temperature before readings are taken.
  • the grafted scaffold is then titrated into the preformed FITC-peptide-target protein complex.
  • a decrease in polarisation with increasing concentrations of grafted scaffold indicates displacement of FITC-peptide upon binding of the grafted scaffold to its target.
  • ITC can be performed using a VP-ITC instrument (Microcal). Grafted scaffolds are dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl, 0.5 mM TCEP. Dialysed target protein (200 ⁇ M) is titrated into the sample cell containing the grafted scaffold at 20 ⁇ M. Injections of target protein into the cell are initiated with a 5 ⁇ L injection, followed by 29 injections of 10 ⁇ L. The reference power is set at 15 ⁇ Cal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data are fitted using the instrument software a one-site binding model.
  • HEK293T cells are cultured in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) supplemented with 10% fetal bovine serum and penicillin/streptomycin (LifeTech) at 37° C. with 5% CO 2 air supply.
  • HEK293T are seeded in 6-well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.
  • Plasmid encoding the target protein (1 ⁇ g) alone and with plasmid encoding one of various target-specific grafted scaffolds (1 ⁇ g) is transfected in HEK293T cells in 6-well plates using Lipofectamine2000. After 48 hours of transfection, the cells are lysed in 200 ⁇ L of Laemmli buffer. After sample is boiled at 95° C. for 20 min proteins are resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting is performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies.
  • DOTAP cationic
  • DOPE neutral
  • DiR aromatic
  • lipid cake is hydrated with 10 mM HEPES pH 7.4, containing 27 ⁇ M protein, so that the total lipid concentration is 4 mg/ml. This mixture is vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins are stored at 4° C. until further use.
  • EL empty liposomes without proteins
  • lipid cake is hydrated with 10 mM HEPES pH 7.4 without proteins.
  • ATP assay is used to investigate whether there is any cytotoxicity associated with EL and LFP.
  • 2 ⁇ 10 5 HEK 293T cells/well in 500 ⁇ L of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum are grown for 24 hours in a 24-well cell culture plate.
  • Cells are incubated with liposome (EL/LFP)-media (DMEM without FBS) mix, having different volumes (0-60 ⁇ L) of EL and LFP, for 15 minutes at 37° C.
  • Nano-Glo® HiBiT Lytic Detection System from Promega Corporation. It is based on the split NanoLuc assay, which consists of a large N-terminal fragment (LgBiT) and a small C-terminal region (SmBiT). Five of the SmBiT amino acids have been replaced to produce the HiBiT (VSGWRLFKKIS) (SEQ ID NO: 3102) fragment, which has greater affinity for the LgBiT fragment and maintains NanoLuc luciferase activity.
  • VSGWRLFKKIS HiBiT fragment
  • Either the HiBiT-tagged target DNA can be transient transfected or the endogenous target can be monitored by knock-in of the HiBiT tag sequence using CRISPR/Cas9 technology.
  • Subsequent introduction of the complementary polypeptide, LgBiT results in spontaneous and high affinity interaction between the HiBiT tag and LgBiT to reconstitute the luminescent NanoBit® enzyme. Detection of tagged protein levels is possible from live or lysed cells.
  • HEK293T cells are introduced into HEK293T cells by either DNA transient transfection or encapsulation within fusogenic liposomes.
  • HEK293T cells are seeded into either 24-well or 96-well plates After 24 hours, DNA encoding the HiBiT-tagged target protein (20 ng for 96-well plate; 100 ng for 24-well plate) is transiently transfected into cells.
  • Chimeric protein DNA 100 ng is either transiently transfected into cells at the same time as HiBiT-target DNA transfection or encapsulated into liposomes and introduced 24 hours into the cells after transfection. Cells are treated with chimeric protein-containing liposomes for 15 minutes before 2 hours of incubation.
  • Nano-Glo® HiBiT Lytic Buffer (LgBiT protein (1:100), Nano-Glo® HiBiT Lytic Substrate (1:50) 1 ⁇ PBS (1:1)) is added to the cells 24 hours after transient transfection or 2 hours after liposomal treatment.
  • the plates are shaken on an orbital shaker (1,000 rpm, 10 min) to ensure homogenous cell lysis and equilibration of LgBiT and HiBiT in the cell lysate.
  • the luminescence measurements are performed in white NunclonTM Delta 96-well plates at 25° C. using a CLARIOstar plate reader using a 460-480 emission filter.
  • the biophysical properties of a grafted scaffold may be assessed as follows: The molar ellipticity at 222 nm (a measure of helical structure content) is monitored as a function of increasing temperature. A decrease in the molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. This thermal unfolding experiment is used to determine the melting temperature of the scaffold and thereby to assess whether or not the grafting process has had a detrimental effect on the thermostability of the scaffold.
  • thermodynamic stability of the proteins is to measure chemical-induced denaturntion (either guanidine hydrochloride (GdnHCl) or urea) monitored by intrinsic protein fluorescence (tryptophan and tyrosine residues). Solutions are dispensed into Corning® 96-well, half-area, black polystyrene plates (CLS3993) with a Microlab ML510B dispenser (Hamilton) and measurements are carried out on a CLARIOstar Plate Reader (BMG Labtech). The buffer is added first into the wells, followed by 15 ⁇ l aliquots of protein stock. A stock solution of chemical denaturant (either 7 M GdnHCl or 9 M urea) is then dispensed into the wells to create a chemical-denaturant concentration gradient.
  • chemical-induced denaturntion either guanidine hydrochloride (GdnHCl) or urea
  • intrinsic protein fluorescence tryptophan and tyrosine residues
  • the helix of a given protein that interacts with its target binding partner is mapped onto the heptad distribution, and the stapled side of the peptide is set so as to form the hydrophobic interface with the rest of the scaffold protein.
  • the grafted scaffold may then be docked against the target protein using Haddock software (de Vries & Bonvin 2011; de Vries et al. 2010).
  • Haddock is a data-driven docking algorithm that uses known information about the interaction for its calculations.
  • the active (primary interaction residues) and the passive (5 ⁇ proximity to active) residues are extracted and inputted into the calculations.
  • the geometry of alpha-helices permits selection of amino acid positions of the scaffold that accommodate outward facing target binding residues of the peptide ligand.
  • a peptide ligand that binds to a given target protein is grafted onto the scaffold in a loop. Binding of the loop-grafted scaffold may be tested using ITC. ITC is particularly useful to assess these interactions, as it can measure the stoichiometry (n) of the interaction, and thus inform as to which loops (if there is more than one loop) are more or less accessible to the target protein, and can inform as to whether a multi-loop scaffold affords multivalency.
  • An advantage of a multivalent grafted scaffold is that one may achieve an avidity effect. This is particularly useful where a target molecule has multiple domains that can be bound by a peptide ligand. Binding of a multivalent grafted scaffold to such a target protein would produce an increased binding affinity and a decreased off rate according to the number of repeats in the grafted scaffold, thus achieving an avidity effect.
  • the function of a multi-valent grafted scaffold containing variable numbers of the peptide ligand binding motif that binds to a given target protein can be tested using the same assays as for the mono-valent grafted scaffold. The results are used to assess whether increased potency can be achieved by increasing the valency.
  • a peptide ligand that is known to bind the substrate recognition subunit of an E3 ligase (see Table 3 for such peptides and ligases) is inserted into the scaffold loop, Immunoprecipitation is used to confirm binding of the grafted scaffold to the E3 ligase. ITC analysis is used to assess the affinity of the interaction.
  • a bispecific grafted scaffold is constructed using a peptide ligand specific for a target protein (see Table 2) and a peptide ligand specific for an E3 ligase.
  • a plasmid encoding the hetero-bifunctional scaffold is transfected into HEK293T cells using Lipofectamine2000 together with HA-tagged ⁇ -catenin plasmid (using cells transfected with HA-tagged ⁇ -catenin plasmid alone as a control). After 48 hours of transfection, the cells are lysed, the sample is boiled and proteins are resolved by SDS-PAGE and immunoblotting is performed using anti-HA and anti-actin antibodies. Changes in target protein levels are evaluated by the densitometry of the bands corresponding to HA-target protein normalised to actin levels. In this way, different combinations of target protein binding peptides and E3 ligase peptide ligands can be compared for their abilities to reduce the levels of target protein.
  • a grafted scaffold protein is encapsulated within fusogenic liposomes made from cationic, neutral, and aromatic lipids, and then delivered into cells. Empty liposomes and liposomes encapsulating grafted scaffolds have been determined to be non-toxic to cells.
  • Chimeric proteins as described herein may be used to produce libraries. For example, where a given chimeric protein (grafted scaffold) is demonstrated to binds bispecifically to a target protein and to an E3 ligase may be further optimized by changing amino acid residues of the grafted scaffold and selecting for stronger or weaker binders.
  • Chimeric proteins which are demonstrated to bind may be further engineered to improve an activity or property or introduce a new activity or property, for example a binding property such as affinity and/or specificity, an in vivo property such as solubility, plasma stability, or cell penetration, or an activity such as increased neutralization of the target molecule and/or modulation of a specific activity of the target molecule or an analytical property.
  • Chimeric proteins may also be engineered to improve stability, solubility or expression level.
  • a library may be used to screen in order to identify and isolate chimeric proteins with specific binding activity.
  • a library may comprise chimeric proteins, each chimeric protein in the library comprising:
  • the residues at one or more positions in the peptide ligand of the chimeric proteins in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.
  • 1 to 12 positions within a helical peptide ligand at the N or C terminus of the chimeric proteins in the library may be diverse or randomised.
  • the non-constrained X n sequence of the peptide ligand may contain additional diversity.
  • 1 to n positions within an inter-repeat peptide ligand of the chimeric proteins in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.
  • peptide ligands may be screened individually and a chimeric protein progressively assembled from repeat domains comprising peptide ligands identified in different rounds of screening.
  • a library may comprise chimeric proteins, each chimeric protein in the library comprising:
  • At least one amino acid residue in the diverse peptide ligands in the library may be diverse.
  • a library may be produced by a method comprising:
  • the population of nucleic acids may be provided by a method comprising inserting a first population of nucleic acids encoding a diverse peptide ligand into a second population of nucleic acids encoding the two or more repeat domains linked by inter-repeat loops, optionally wherein the first and second nucleic acids are linked with a third population of nucleic acids encoding linkers of up to 10 amino acids.
  • the nucleic acids may be contained in vectors, for example expression vectors.
  • Suitable vectors include phage-based or phagemid-based phage display vectors.
  • the nucleic acids may be recombinantly expressed in a cell or in solution using a cell-free in vitro translation system such as a ribosome, to generate the library.
  • the library is expressed in a system in which the function of the chimeric protein enables isolation of its encoding nucleic acid.
  • the chimeric protein may be displayed on a particle or molecular complex to enable selection and/or screening.
  • the library of chimeric proteins may be displayed on beads, cell-free ribosomes, bacteriophage, prokaryotic cells or eukaryotic cells.
  • the encoded chimeric protein may be presented within an emulsion where activity of the chimeric protein causes an identifiable change.
  • the encoded chimeric protein may be expressed within or in proximity of a cell where activity of the chimeric protein causes a phenotypic change or changes in the expression of a reporter gene.
  • the nucleic acids are expressed in a prokaryotic cell, such as E coli .
  • the nucleic acids may be expressed in a prokaryotic cell to generate a library of recombine binding proteins that is displayed on the surface of bacteriophage.
  • Suitable prokaryotic phage display systems are well known in the art, and are described for example in Kontermann, R & Dubel, S, Antibody Engineering , Springer-Verlag New York, LLC; 2001, ISBN: 3540413545, WO92/01047, U.S. Pat. Nos.
  • Phage display systems allow the production of large libraries, for example libraries with 10 8 or more, 10 9 or more, or 10 10 or more members.
  • the cell may be a eukaryotic cell, such as a yeast, insect, plant or mammalian cell.
  • a diverse sequence as described herein is a sequence which varies between the members of a population i.e. the sequence is different in different members of the population.
  • a diverse sequence may be random i.e. the identity of the amino acid or nucleotide at each position in the diverse sequence may be randomly selected from the complete set of naturally occurring amino acids or nucleotides or a sub-set thereof.
  • Diversity may be introduced into the peptide ligand using approaches known to those skilled in the art, such as oligonucleotide-directed mutagenesis 22 , Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein).
  • Diverse sequences may be contiguous or may be distributed within the peptide ligand.
  • Suitable methods for introducing diverse sequences into peptide ligand are well-described in the art and include oligonucleotide-directed mutagenesis (see Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein).
  • diversification may be generated using oligonucleotide mixes created using partial or complete randomisation of nucleotides or created using codons mixtures, for example using trinucleotides.
  • a population of diverse oligonucleotides may be synthesised using high throughput gene synthesis methods and combined to create a precisely defined and controlled population of peptide ligands.
  • “doping” techniques in which the original nucleotide predominates with alternative nucleotide(s) present at lower frequency may be used.
  • the library is a display library.
  • the chimeric proteins in the library may be displayed on the surface of particles, or molecular complexes such as beads, for example, plastic or resin beads, ribosomes, cells or viruses, including replicable genetic packages, such as yeast, bacteria or bacteriophage (e.g. Fd, M13 or T7) particles, viruses, cells, including mammalian cells, or covalent, ribosomal or other in vitro display systems.
  • yeast bacteria or bacteriophage
  • the chimeric proteins in the library are displayed on the surface of a viral particle such as a bacteriophage.
  • a viral particle such as a bacteriophage.
  • Each chimeric protein in the library may further comprise a phage coat protein to facilitate display.
  • Each viral particle may comprise nucleic acid encoding the chimeric protein displayed on the particle.
  • Suitable viral particles include bacteriophage, for example filamentous bacteriophage such as M13 and Fd.
  • Phage display is described for example in WO92/01047 and US patents U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404.
  • Libraries as described herein may be screened for chimeric proteins which display binding activity, for example binding to a target molecule. Binding may be measured directly or may be measured indirectly through agonistic or antagonistic effects resulting from binding.
  • a method of screening may comprise;
  • the chimeric proteins in the library may comprise one peptide ligand with at least one diverse amino acid residue. Conveniently the chimeric proteins in the library comprise two repeat domains.
  • the library may be screened for peptide ligands that bind to a target molecule. Peptide ligands identified in this fashion can be assembled in a modular fashion to generate a chimeric protein as described herein that is multi-specific.
  • a first library may be screened for a first peptide ligand that binds to a first target molecule and a second library may be screened for a second peptide ligand that binds to a second target molecule.
  • the first and second peptide ligands are in different locations in the chimeric protein i.e. they are not both N terminal peptide ligands, C terminal peptide ligands or inter-repeat peptide ligands.
  • First and second peptide ligands that bind to the first and second target molecules, respectively, are identified from the first and second libraries. The identified first and second peptide ligands may then be incorporated into a chimeric protein that binds to the first and second target molecules.
  • a first library may comprise chimeric proteins in the library with a first diverse peptide ligand having at least one diverse amino acid residue.
  • a first peptide ligand that binds to a target molecule may be identified from the first library.
  • Chimeric proteins comprising the first peptide ligand may be used to generate a second library comprising a second diverse peptide ligand having at least one diverse amino acid residue.
  • the chimeric protein from the first library may be modified by addition of a second diverse peptide ligand at the N or C terminal or by the addition of additional repeat domains comprising the second diverse peptide ligand in an inter-repeat loop.
  • a second peptide ligand that binds to the same or a different target molecule may be identified from the second library.
  • Chimeric proteins comprising the first and second peptide ligands may be used to generate a third library comprising a third diverse peptide ligand having at least one diverse amino acid residue.
  • the chimeric protein from the second library may be modified by addition of a third diverse peptide ligand at the N or C terminal or by the addition of additional repeat domains comprising the third diverse peptide ligand in an inter-repeat loop.
  • a third peptide ligand that binds to the same target molecule as the first and/or second peptide ligands or a different target molecule may be identified from the third library. In this way, a chimeric protein containing multiple peptide ligands may be sequentially assembled (see FIG. 16 ).
  • a phage library of 10 8 -10 12 first peptide ligand variants may be combined with a phage library of 10 8 -10 12 second peptide ligand variants and a phage library of 10 8 -10 12 third peptide ligand variants.
  • a phage library of 10 8 -10 12 N terminal peptide ligand variants may be combined with a phage library of 10 8 -10 12 C terminal peptide ligand variants to generate a chimeric protein with N and C terminal peptide ligands.
  • Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cell phenotype.
  • the one or more identified or selected chimeric proteins may be recovered and subjected to further selection and/or screening.
  • the chimeric proteins in the library may comprise a first peptide ligand for a first target molecule, which has at least one diverse amino acid residue, and a second peptide ligand for a second target molecule, which has at least one diverse amino acid residue.
  • the library may be screened for peptide ligands that bind to the first and second target molecules.
  • the library may be screened for chimeric proteins comprising a first peptide ligand that binds to a first target molecule and a second peptide ligand that binds to a second target molecule.
  • Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cell phenotype.
  • the one or more identified or selected chimeric protein may be recovered and subjected to further selection and/or screening.
  • Chimeric proteins as described herein may be used to produce libraries comprising different combinations of peptide ligands grafted into an scaffold.
  • the combinations of ligands may comprise first peptide ligands that bind to a members of a protein degradation pathway, such as an E3 ubiquitin ligase, and second peptide ligands that bind to a target molecule.
  • a library may be screened in order to identify and isolate chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule, (ii) causing degradation of the target molecule in a cell through the protein degradation pathway.
  • a library may comprise chimeric proteins, each chimeric protein in the library comprising:
  • chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, the chimeric proteins in the library comprising different combinations of the first and second peptide ligands.
  • Suitable chimeric proteins, target molecules and members of protein degradation pathways and examples of peptide ligands thereto are described elsewhere herein.
  • each chimeric protein in a library of chimeric proteins may comprise:
  • the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands.
  • Different chimeric proteins in the library may comprise a peptide ligand for a different E3 ubiquitin ligase.
  • the chimeric proteins in the library may comprise peptide ligands for a panel of E3 ubiquitin ligases, each chimeric protein in the library comprising a peptide ligand for one of the E3 ubiquitin ligases in the panel.
  • E3 ubiquitin ligases are known in the art.
  • a suitable panel of E3 ubiquitin ligases may for example, comprise two, three, four, five or more of Mdm2, SCF (Skp2), Cul3-Keap1, Cul3-SPOP, APC/C, SIAH, SCF Fbw7 , SCF Fbw8 , Cul4-DDB1-Cdt2, DDB1-Cul4, DDB1-Cul5, SOCS box-Cul5-SPSB2, SOCS box-Cul5-SPSB4, CHIP, CRL4 (COPI/DET), UBRS, CRL2 (KLHDC2), GID4, TRIM21, Nedd4, Elongin C and p-TRP.
  • Examples of peptide ligands for E3 ubiquitin ligases are shown in Table 3.
  • the target molecule may be a target molecule as described above, for example, 1-catenin, KRAS, or myc.
  • the chimeric proteins in the library may comprise different peptide ligands for the target molecule i.e. different chimeric proteins in the library may comprise different peptide ligands for the same target molecule. Each chimeric protein in the library may comprise a different peptide ligand for the target molecule. Examples of peptide ligands target molecules are shown in Table 3.
  • the target molecule may be f-catenin, KRAS, or myc and the chimeric proteins in the library may comprise different peptide ligands for ⁇ -catenin, KRAS, or myc, respectively. Examples of different peptide ligands for ⁇ -catenin, KRAS, and myc are shown in Table 3.
  • a method of screening a library of chimeric proteins may comprise;
  • the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands,
  • the member of a protein degradation pathway may be an E3 ubiquitin ligase.
  • a method of screening a library of chimeric proteins may comprise;
  • the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands,
  • a method may further comprise identifying one or more combinations of first and second peptide ligands in chimeric proteins in the library which display the activity.
  • Binding of a chimeric protein may be determined by any suitable technique, described below and in the examples herein.
  • Suitable methods for determining binding of a chimeric protein to a target molecule include ELISA, bead-based binding assays (e.g. using streptavidin-coated beads in conjunction with biotinylated target molecules, surface plasmon resonance, flow cytometry, Western blotting, immunocytochemistry, immunoprecipitation, and affinity chromatography.
  • biochemical or cell-based assays such as fluorescence-based or luminescence-based reporter assays may be employed.
  • Isothermal Titration Calorimetry Cell transfection followed by assaying for expressed chimeric protein, Liposomal formulation and cytotoxicity assays, a dual-Luciferase Reporter Assay System such as TOPFLASH®, and a competition fluorescence polarisation (FP) assay to measure the binding of a chimeric protein to its targets.
  • FP competition fluorescence polarisation
  • binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme,
  • the library may be contacted with the target molecule under binding conditions for a time period sufficient for the target molecule to interact with the library and form a binding reaction complex with a least one member thereof.
  • Binding conditions are those conditions compatible with the known natural binding function of the target molecule. Those compatible conditions are buffer, pH and temperature conditions that maintain the biological activity of the target molecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction. Typically, those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the target molecule of interest.
  • the library may be contacted with the target molecule in the form of a heterogeneous or homogeneous admixture.
  • the members of the library can be in the solid phase with the target molecule present in the liquid phase.
  • the target molecule can be in the solid phase with the members of the library present in the liquid phase.
  • both the library members and the target molecule can be in the liquid phase.
  • Multiple rounds of panning may be performed in order to identify chimeric proteins which display the binding activity. For example, a population of chimeric proteins enriched for the binding activity may be recovered or isolated from the library and subjected to one or more further rounds of screening for the binding activity to produce one or further enriched populations. Chimeric proteins which display binding activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.
  • binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme.
  • a target molecule such as a ligand, receptor or enzyme.
  • the library may be screened by expressing the library in reporter cells and identifying one or more reporter cells with altered gene expression or phenotype. Suitable functional screening techniques for screening recombinant populations of chimeric proteins are well-known in the art.
  • chimeric proteins which display the improved property or activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.
  • a chimeric protein as described herein may be encapsulated in a liposome, for example for delivery into a cell.
  • Preferred liposomes include fusogenic liposomes.
  • Suitable fusogenic liposomes may comprise a cationic lipid, such as 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP), and a neutral lipid, such as dioleoylphosphatidylethanolamine (DOPE) for example in a 1:1 (w/w) ratio.
  • DOTAP 1, 2-dioleoyl-3-trimethylammoniumpropane
  • DOPE dioleoylphosphatidylethanolamine
  • a liposome may further comprise an aromatic lipid, such as DiO (3, 3′-dioctadecyloxacarbocyanine perchlorate), DiR (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanine iodide), N-(4,4-fluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionyl)-1,2-dibexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (BODIPY FL-DHPE), and 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diazas-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-C
  • a method described herein may comprise admixing a chimeric protein or encoding nucleic acid as described herein with a solution of lipids, for example in an organic solvent, such as chloroform, and evaporating the solvent to produce liposomes encapsulating the chimeric protein.
  • Liposome encapsulations comprising a chimeric protein as described herein are provided as an aspect of the invention.
  • a chimeric protein or encoding nucleic acid as described herein may be admixed with a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a chimeric protein or nucleic acid as described herein and a pharmaceutically acceptable excipient is provided as an aspect of the invention.
  • pharmaceutically acceptable refers to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • a subject e.g., human
  • Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
  • the pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the chimeric protein into association with a carrier which may constitute one or more accessory ingredients.
  • pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • compositions may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.
  • a chimeric protein, encoding nucleic acid or pharmaceutical composition comprising the chimeric protein or encoding nucleic acid may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g.
  • vaginal parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
  • compositions suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
  • compositions suitable for parenteral administration include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to cells, tissue or organs.
  • Suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection.
  • concentration of the active compound in the solution is from about 1 ng/ml to about 10 ⁇ g/ml, for example, from about 10 ng/ml to about 1 ⁇ g/mL
  • the formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • appropriate dosages of the chimeric protein can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of diagnostic benefit against any risk or deleterious side effects of the administration.
  • the selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the imaging agent, the amount of contrast required, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient.
  • the amount of imaging agent and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the imaging agent at a site, such as a tumour, a tissue of interest or the whole body, which allow for imaging without causing substantial harmful or deleterious side-effects.
  • Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.
  • Chimeric proteins described herein may be used in methods of diagnosis or treatment in human or animal subjects, e.g. human. Chimeric proteins for a target molecule may be used to treat disorders associated with the target molecule.
  • the pRSET B (His-tag) constructs were transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies were grown in 2TY media containing ampicillin (50 micrograms/mL) at 37° C., 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures were then induced with IPTG (0.5 mM) for 16-20 h at 20° C. or 4 h at 37′C.
  • Cells were pelleted by centrifugation at 3000 g (4′C, 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris was pelleted by centrifugation at 15,000 g at 4° C. for 45 min.
  • lysis buffer 10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution
  • Ni-NTA beads 50% bed volume (GE Healthcare)(5 mL) were washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) before the supernatant of the cell lysate was bound to them for 1 hr at 4° C. in batch.
  • the loaded beads were washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non-specific interaction of lysate proteins with the beads.
  • chimeric proteins described herein are thermally very stable, with melting temperatures above 80° C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65° C. for 20 min. Very few of the E. coli proteins survive such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins were removed by centrifugation, leaving 80-90% pure sample of the desired protein. All our constructs folded reversibly, and therefore could be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.
  • This approach allowed the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is scalable to industrial production and bioreactors.
  • Plasmids were transformed into E. coli C41 cells and plated overnight. 15 mis of 2TY medium (Roche) containing 50 micrograms/ml ampicillin was placed in multiple 50 ml tubes. Several colonies were picked and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells were grown at 37° C. until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells were pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAse I treatment was used. The lysate was spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.
  • the supernatant was added to 100 ⁇ l bed volume of pro-washed Ni-NTA agarose beads.
  • the subsequent affinity purification was performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used).
  • the first wash contained 10% BugBuster® solution and 30 mM imidazole in the chosen buffer. Here we used 50 mM sodium phosphate buffer pH 6.8, 150 mM NaCl. The three successive washes had 30 mM of imidazole in the chosen buffer. Beads were washed thoroughly to remove the detergent present in the BugBuster® solution.
  • Protein was eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole. The combination of Bugbuster® and imidazole and the repeat washes in small bead volumes yielded >95% pure protein. Imidazole was removed using a NAP-5 disposable gel-filtration column (GE Healthcare).
  • ITC was performed at 25° C. using a VP-ITC (Microcal).
  • 1TBP-CTPR2, 2TBP-CTPR4, 3TBP-CTPR6 and TNKS2 ARC4 were dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl, 0.5 mM TCEP.
  • Dialysed TNKS2 ARC4 200 ⁇ M was titrated into the sample cell containing 1TBP-CTPR2 at 20 ⁇ M. Similar experiments were performed for 2TBP-CTPR4 and 3TBP-CTPR6. Injections of TNKS2 ARC4 into the cell were initiated with a 5 ⁇ L injection, followed by 29 injections of 10 ⁇ L.
  • the reference power was set at 15 ⁇ Cal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data were fitted using the instrument software a one-site binding model.
  • HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) supplemented with 10/fetal bovine serum and penicillin/streptomycin (LifeTech) at 37° C. with 5% CO 2 air supply.
  • HEK293T were seeded in 6-well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.
  • HA- ⁇ -catenin (1 ⁇ g) alone and with various PROTACs (1 ⁇ g) was transfected in HEK293T cells in 6-well plates using Lipofectamine2000. After 48 hours of transfection, the cells were lysed in 200 ⁇ L of Laemmli buffer. After sample was boiled at 95° C. for 20 min proteins were resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting was performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies. Changes in ⁇ -catenin levels were evaluated by the densitometry of the bands corresponding to HA- ⁇ -catenin normalised to actin levels using ImageJ.
  • DOTAP cationic
  • DOPE neutral
  • DiR aromatic
  • lipid cake was hydrated with 10 mM HEPES pH 7.4, containing 27 ⁇ M protein, so that the total lipid concentration is 4 mg/ml. This mixture was vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins were stored at 4° C. until further use.
  • EL empty liposomes without proteins
  • lipid cake was hydrated with 10 mM HEPES pH 7.4 without proteins.
  • ATP assay was used to investigate whether there is any cytotoxicity associated with EL and LFP.
  • 2 ⁇ 10HEK 293T cells/well in 500 ⁇ L of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum were grown for 24 hours in a 24-well cell culture plate.
  • Cells were incubated with liposome (EL/LFP)-media (DMEM without FBS) mix, having different volumes (0-60 ⁇ L) of EL and LFP, for 15 minutes at 37° C.
  • EL/LFP liposome
  • DMEM liposome
  • DMEM without FBS liposome
  • 500 ⁇ L of CellTiter-Glo® Reagent was added and luminescence was measured using a microplate reader as par the manufacture's protocol. Untreated cells were used as control. Data were obtained from triplicate samples, and the standard deviations were calculated from two independent experiments.
  • the Wt pathway was activated by treating HEK293T cells with Wnt-conditioned media obtained from L-cells expressing Wnt3A for 8 days.
  • 10 5 HEK293T cells/well were seeded on a 24-well plate Nunclon Delta Surface plate (NUNC) and incubated overnight at 37° C., 5% CO 2 .
  • NUNC Nunclon Delta Surface plate
  • cells were transfected with 100 ng of TOPflash TCF7L2-firefly luciferase plasmid, 10 ng of CMV- Renilla plasmid (as internal control) and 100 ng of the corresponding TPR construct.
  • Plasmids were mixed with 0.5 ⁇ L of Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (invitrogen). Transfected cells were allowed to recover for 8 h, then they were treated with Wnt-conditioned media (1:2 final concentration) for a further 16 h.
  • the TOPflash assay was performed using the Dual-Luciferase Reporter Assay System (Promega) (Korinek et al., 1997 Science 275(5307):1784-7) following the manufacturer's instructions.
  • the activities of firefly and Renilla luciferases were measured sequentially from a single sample, using the CLARIOstar plate reader. Relative luciferase values were obtained from triplicate samples dividing the firefly luminescence activity by the CMV-induced Renilla activity, and standard deviation was calculated.
  • HEK 293T cells 10 5 HEK 293T cells in 500 ⁇ L of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum were grown overnight in each well of a 24-well cell culture plate.
  • DMEM Dulbecco's Modified Eagles Medium
  • For TOPFLASH reporter assays 100 ng/well of TOPFLASH plasmid and ng/well of CMV- Renilla plasmid (as internal control) were used to transfect cells in 24-well plates. Cells were transfected with the Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (Invitrogen). Transfected cells were allowed to recover for 8 hours, and Wnt signalling was activated by addition of Wnt3A-conditioned media obtained from L-cells.
  • Relative luciferase values were obtained from triplicate samples (from two independent experiments) by dividing the firefly luciferase values (from TOPFLASH) by the Renilla luciferase values (from CMV renilla), and standard deviations were calculated.
  • Nrf-TPR proteins were titrated into a solution containing a mixture of FITC-labelled Nrf2 peptide and Keap1 protein. The prepared plates were incubated for 30 minutes at room temperature before readings were take.
  • Tetratricopeptide repeat is a 34-residue motif that can be repeated in tandem to generate modular proteins. TPRs are used here as an example of helix-turn-helix tandem-repeats arrays, but any tandem repeat array may be used.
  • RTPR proteins comprising TPRs were derived from the consensus TPR sequence (CTPR). Two repeats were found to be sufficient to generate a highly stable mini-protein of 68 amino acids (RTPR2).
  • CTPR consensus TPR sequence
  • Two repeats were found to be sufficient to generate a highly stable mini-protein of 68 amino acids (RTPR2).
  • the molar ellipticity at 222 nm (a measure of helical secondary structure content) of three different RTPR modules was monitored as a function of increasing temperature. A decrease in the absolute molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. Even at the highest temperature recorded (85° C.), the RTPR2 protein without insertion was not fully denatured ( FIG. 1 ).
  • RTPR2 with a 20-residue unstructured loop between the two repeats showed a small shift to a lower melting temperature ( FIG. 1 ), but the protein remains fully folded up to 55° C. This is well above physiologically relevant temperatures.
  • RTPR2 with an additional N-terminal helix showed an increase in absolute molar ellipticity, indicating that the additional helical domain is folded.
  • the helix domain was capable of stabilising the RTPR2 module, shifting the transition midpoint to above 90° C. ( FIG. 1 ).
  • TBP-CTPR2 a two-repeat CTPR with a loop insertion that binds to the protein tankyrase (Guettler et al. 2011)
  • the TBP-CTPR2-containing proteins had two, four, six, and eight repeats, and they displayed one, two, three and four binding loops, respectively.
  • CTPR-mediated “stapling” (constraining) of binding helices therefore occurred through residues Tyr (i)-Ile (i+4)-Tyr (i+7)-Leu (i+11), fully stapling a 15-residue helix.
  • TPR repeat scaffolds are exceptional for display of binding helices, as they grow linearly in the opposite direction of the helix, thereby avoiding steric clashes with the target protein.
  • KRAS binding of the grafted scaffold can be assessed using the change in fluorescence polarisation of mant-GTP (2′-/3′-O-(N′-Methylanthraniloyl) guanosine-5′-O-triphosphate), a fluorescent analog of GTP ( FIG. 3B ).
  • the fluorescence of mant-GTP is dependent on the hydrophobicity of its environment (excitation at 360 nm, emission at 440 nm). An increase in fluorescence intensity and fluorescence polarization was observed previously upon binding to KRAS (Leshchiner et al. 2015).
  • SOS-TPR2 was then titrated into the preformed mant-GTP-KRAS complex.
  • degrons region within the substrate that is recognized by the E3 ubiquitin ligase
  • p53 binds to the Mdm2 E3 through an alpha helix ( FIG. 4A ).
  • Stapled versions of the p53 helix, as well as circular peptides and grafted coiled coils, have been developed by many groups, and the sequences have been optimised to give nanomolar affinities in some cases (se for example, Ji et al 2013; Lee et al 2014; Kritzer et al. 2006).
  • the p53 helix has a favourable geometry to be grafted onto the C-terminal solvating helix of the CTPR scaffold, and moreover the two helices have 30% sequence identity.
  • SLiM “3BP2” a sequence that binds to the substrate-binding ankyrin-repeat clusters (ARC) of the protein tankyrase, a multi-domain poly ADP-ribose polymerase that is upregulated in many cancers (Guettler et al. 2011) onto the CTPR scaffold. Granting SLiMs in folded domains led to an increase of proteolysis resistance; showing the potential to expand the interaction surface through further rational engineering, in silico methods and/or directed evolution; controlled geometric arrangement; and bi- or multivalency of interactions.
  • ARC substrate-binding ankyrin-repeat clusters
  • Multivalency in this system was increased further via oligomerisation of the binding modules by fusing them to the foldon domain of T4 fibritin ( FIG. 5B ).
  • This trimerisation domain comprises of a C-terminal helix, such as that of p53-CTPR, ending with the foldon domain, a short ⁇ -sheet to peptide capable of homo-trimerising.
  • the foldon domain has been shown to be highly stable and independently folded (Boudko et al 2002, Meier et al. 2004). In this way, multiple binding modules can be arranged with specified geometries to inhibit complex multivalent molecules that cannot be targeted with monovalent interactions due to their natural tendency to interact with other multivalent networks with high avidity.
  • Skp2 is the substrate recognition subunit of the SCF Skp2 ubiquitin ligase.
  • the Skp2-binding sequence that we inserted into the RTPR loop was based on the previously published degron peptide sequence derived from the substrate p27 that binds to Skp2 in complex with Cks1 (an accessory protein) (Hao et al, 2005). We used only 10 residues of this peptide. Although ideally the Skp2-binding sequence would include a phospho-threonine (as this residues makes some key contacts with Skp2 and Cks1), we instead explored whether we could replace the phospho-threonine with a phosphomimetic (glutamate) without affecting binding affinity.
  • Nrf-TPR a Loop Module Designed to Bind to E3 Ubiquitin Ligase Keap1-Cul3
  • Keap1 is the substrate recognition subunit of the Keap1-Cul3 ubiquitin ligase.
  • a Keap1-binding sequence that we inserted into the CTPR loop was based on the previously published degron peptide sequence derived from the Keap1 substrate Nrf2.
  • FIG. 8 shows that this approach was successful in delivering a fluorescent-labelled resurfaced TBP-RTPR2 protein into two different cell lines.
  • ⁇ -catenin The Wnt/ ⁇ -catenin signalling pathway is deregulated in many cancers and in neurodegenerative diseases, and therefore ⁇ -catenin is an important drug target.
  • Mdm2 and SCF 6 W We selected Mdm2 and SCF 6 W to test as E3 ubiquitin ligases, as we had successfully generated single-function TPRs to bind to them ( FIGS. 4 and 6 ).
  • a range of different factors contribute to efficient ubiquitination and target degradation by these hetero-bifunctional molecules, hence the power of screening different combinations of single-function modules and potentially also different lengths of intervening blank modules.
  • TPR proteins were designed to target either tankyrase ( FIG. 21 . FIG. 22 ) or KRAS ( FIG. 23 ). TPR proteins targeting tankyrase or were delivered into cells using liposome encapsulation, and the effect on Wnt signalling was assayed using a TOPFLASH assay. The results show that the TPR proteins are able to inhibit Wnt signalling.
  • KRAS we transfected KRAS plasmid alone or KRAS plasmid together with one of the TPR plasmids in HEK293T cells using Lipofoectamine2000. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. The results show that the designed hetero-bifunctional TPR is capable of reducing KRAS levels.
  • Hetero-bifunctional TPR proteins were designed to target endogenous KRAS for degradation via CMA ( FIG. 24 ).
  • TPR constructs or empty vector (light grey) were transiently transfected into either HEK293T or DLD1 (colorectal cancer cell line) using Lipofectamine2000. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. The designed hetero-bifunctional TPRs that resulted in reduction of KRAS levels compared to the empty vector control are shown in white.
  • the linker sequence connecting a peptide ligand to an inter-repeat loop was varied in order to optimise the binding affinity for the target for Nrf-TPR, a TPR protein designed to bind to the protein Keap1 (see FIG. 7 ).
  • Glycine residues were introduced into the linker to provide flexibility and increased spatial sampling.
  • the introduction of this more flexible linker sequence was found to increase the binding affinity of the Nrf-TPR protein (labelled ‘Flexible’) when compared with the consensus-like linker sequence altering the charge content of the linker sequence (‘labelled ‘Charged’) and altering the conformational properties (based on the predictions of the program CIDER (Holehouse et al. Biophys. J. 112, 16-21 (2017)) of the loop by changing the amino acid composition of the linker sequence (labelled ‘CIDER-optimised’) also affected the Keap1-binding affinity ( FIG. 25 ).
  • NNNAAAGAAAACNNN SEQ ID NO: 2978
  • APC/C KEN loop SEDKENV AGCGAGGATAAAGAAAATGTTCCGCCT PP (SEQ (SEQ ID NO: 2979) ID NO: 2835)
  • APC/C DBOX loop .R..L..[LIV NNNCGTNNNNNNCTGNNNN[CTG/ATT/GT consensus M].
  • APC/C Shugoshin 1 loop RLSLSPK CGCCTGAGCCTGAGCCCGAAAAAAAAC DBOX KN (SEQ (SEQ ID NO: 2981) ID NO: 2836)
  • APC/C Shugoshin 1 loop RSSLKKH CGCAGCAGCCTGAAAAAACATTGCAAC DBOX CN (SEQ (SEQ ID NO: 2982) ID NO: 2837)
  • APC/C Bcl-2- loop RSPLFIF CGCAGCCCGCTGTTTATTTTT SEQ ID NO: like (SEQ ID 2984) protein NO: 2839)
  • 11 DBOX
  • NN (SEQ ID NO: 3031) (SEQ ID NO: 2883) Cul4- Consensus loop [NQ] ⁇ 0,1 ⁇ .
  • ELYPLTSL RS (SEQ ID NO: 2938) ESCRT; Nef loop ExxxLL GAANNNNNNNNNCTGCTG (SEQ ID AP-1 NO: 3090) ESCRT; Env loop YxxL TATNNNNNNCTG (SEQ ID NO: 3091) AP-2 ESCRT; AP viral loop SREKPYK AGCCGCGAAAAACCGTATAAAGAAG adaptor EVTEDLL TGACCGAAGATCTGCTGCATCTGAAC HLNSLF AGCCTGTTT (SEQ ID NO: 3092) (SEQ ID NO: 2939) ESCRT; AP viral loop AAGAYDP GCGGCGGGCGCGTATGATCCGGCGCG adaptor ARKLLEQ CAAACTGCTGGAACAGTATGCGAAA YAKK AAA (SEQ ID NO: 3093) (SEQ ID NO: 2940) CMA Consensus loop KFERQ AAATTTGAACGCCAG (SEQ ID NO: (SEQ ID 3094) NO: 2941) CMA Consen
  • Axin-RTPR-SPOP MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGLACDEVTSTTSSSTA GGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 261) 7.
  • Axin-RTPR-p53 MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG DYQRAIEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 263) 10.
  • ICAT-RTPR-P27 MGSYAYQRAIVEYMLRLMSDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQ RAIEYYQRALELDPNNAGSNEQEPKKKRSPDAEAWYNGNAYYRQGDYQRAIEYYQRALELPENN (SEQ ID NO: 277 24.
  • ICAT-RTPR-p53 MGSYAYQRAIVEYMLRLMSDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQ RAIEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 278) 25.
  • LRH1-RTPR-p27 MQSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA IEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 283) 30.
  • LRH1-RTPR-p53 MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA IEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 284) 31.
  • APC-RTPR-Nrf2 MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPEGGSCEELEALEALELDEGGPNAEAWYNLGNAYYRQGDYQ RAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAYYRQ GDYQRAIEYYQRALELDPNN (SEQ ID NO: 287) 34.
  • APC-RTPR-p27 MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSCSEELEALEALELDEGGPNAEAWYNLGNAYYRQGDYQ RAIEYYQRALELDPNN (SEQ ID NO: 288) 35.
  • APC-RTPRT-p53 MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYESTQDGGPNAEAWYNLGNAYYRQG DYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 290) 37.
  • 1TBP-CTPR2 MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK ALELDPRS (SEQ ID NO: 291) 38.
  • SOS-RTPR-CMA_K MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQR AIEYYQRALELDPRSKFERQ (SEQ ID NO: 302) 49.
  • SOS-RTPR-CMA_Q MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSIPNPLLGLDKFERQ (SEQ ID NO: 303) 50.

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Abstract

This invention relates to modular proteins that interact with one or more target molecules. The chimeric proteins comprise two or more repeat domains, such as tetratricopeptide repeat domains; inter-repeat loops linking the repeat domains; and one or more peptide ligands. Each peptide ligand is located in an inter-repeat loop or at the N or C terminus of the chimeric protein. The peptide ligands may include heterologous peptidyl binding motifs, such as short linear motifs (SLiMs). Chimeric proteins with various configurations and methods for their production and use are provided.

Description

  • This application is a continuation of U.S. application Ser. No. 16/282,155, filed Feb. 21, 2019, which is a continuation-in-part application of PCT/EP2018A68580, filed Jul. 9, 2018, which claims the benefit of GB1714038.5, filed Sep. 1, 2017, and GB1713316.6, filed Aug. 18,2017, each of which is incorporated herein by reference in its entirety. All publications cited herein, are incorporated by reference herein in their entirety.
  • All publications cited herein are incorporated by reference herein in their entirety.
    • SEQUENCE LISTING
  • The instant application contains a Sequence Listing which has been submitted electronically in text format and is hereby incorporated by reference in its entirety. Said text copy, created on Mar. 17, 2020, is named 119691-10101 revised seq listing.txt and is 1,111 kb in size.
    • FIELD
  • This invention relates to chimeric proteins and their production and uses.
    • BACKGROUND
  • A priority area in medicine, particularly cancer research, is the expansion of the ‘druggable’ proteome, which is currently limited to narrow classes of molecular targets. For example, protein-protein interactions (PPIs) are fundamental to all biological processes and represent a large proportion of potential drug targets, but they are not readily amenable to conventional small molecule inhibition. The architecture of tandem repeat proteins has tremendous scope for rational design (Kobe & Kajava 2000, Longo & Blaber, 2014, Rowling et al., 2015). The key features of tandem repeat proteins are relatively small size, modularity and extremely high stability (and therefore recombinant production) without the need of disulphide bonds. Individual consensus-designed repeats are self-compatible and can be put together in any order; function is therefore also modular, which means that multiple functions can be independently designed and incorporated in a combinatorial fashion within a single molecule (WO2017106728).
  • Novel repeat protein functions, e.g. DARPins (Tamaskovic et al., 2012), have been developed based on the natural type of PIP interface of these proteins i.e. spanning many repeat units to create an extended, high-affinity binding interface for the target. Mutations have been introduced into the surface residues in the tetratricopeptide (TPR) repeats of the cytosolic receptor peroxin 5 (Sampathkumar et al. (2008) J Mol. Biol, 381, 867-880). Binding of peptide ligands to peroxin 5 is shown to be mediated by residues located in several different TPR repeats. The interactions of TPR containing protein kinesin-1 with different cargo proteins has also been reported (Zhu et al PLoS One 2012 7 3 e33943). The specificity and stability of ankyrin repeat proteins has been modified through the introduction of mutations into ankyrin repeat sequences (Li et al (2006) Biochemistry 45 15168-15178).
    • SUMMARY OF THE INVENTION
  • The present inventors have found that chimeric proteins which comprise peptidyl ligands, such as short linear motifs (SLIMs), on scaffolds. Such chimeric proteins (i.e, modular binding proteins), ray be useful for example, as single- or multi-function protein therapeutics.
  • An aspect of the invention provides a chimeric protein comprising:
      • a scaffold comprising a first end and a second end, and two or more repeat domains linked by inter-repeat loops between the ends; and
      • one or more peptide ligands, wherein a single peptide ligand is located in the scaffold in (i), an inter-repeat loop, (ii) at the first end, or (iii) at the second end of the scaffold, thereby forming a chimeric protein (a grafted scaffold).
  • In a preferred embodiment, the scaffold is a continuous polypeptide strand such that the first end is the N terminus and the second end is the C terminus of the scaffold.
  • In some preferred embodiments, the chimeric protein may comprise a first peptide ligand that binds a first target molecule and a second peptide ligand that binds a second target molecule, One of the first or second target molecules may be an E3 ubiquitin ligase. Where a chimeric protein comprises two or more peptide ligands, the ligands are different ligands (bind to different targets) and are not located in the same loop or at the same end of a scaffold.
  • Another aspect of the invention provides a method of producing a chimeric protein comprising;
      • inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding a scaffold comprising two or more repeat domains linked by inter-repeat loops, to produce a chimeric nucleic acid encoding a chimeric protein as described herein; and
      • expressing the chimeric nucleic acid to produce the chimeric protein.
  • Another aspect of the invention provides a method of producing a chimeric protein that binds to a first target molecule and a second target molecule comprising; providing a nucleic acid encoding a scaffold comprising two or more repeat domains
      • linked by inter-repeat loops, and
      • incorporating into the nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising the first and second peptide ligands, wherein the peptide ligands are independently located in an inter-repeat loop or at the N or C terminus of the chimeric protein; and
      • expressing the nucleic acid to produce the protein.
  • In some preferred embodiments, one of the first or second target molecules is an E3 ubiquitin ligase.
  • In another aspect, the invention provides a chimeric protein, comprising
  • (i) a tetratricopeptide (PR) scaffold comprising first and second α-helices linked by an inter-repeat loop, and.
  • (ii) a first heterologous peptide that binds to a target protein, and
  • (iii) a second heterologous peptide that binds to an E3 ubiquitin ligase, wherein the first and second heterologous peptides are, independently, located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.
  • In a preferred embodiment, each of the first and second α-helices comprises the amino acid sequence Y-X1X2X3X4; wherein Y is an amino acid sequence shown in Tables 4 to 6 and X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or optionally wherein X2 is P.
  • In another preferred embodiment, the first and second α-helices each comprise the amino acid sequence:
  • (SEQ ID NO: 1)
    AEAWYNLGNAYYKQGDYQKAIEYYQKALEL-X1X2X3X4; or
    (SEQ ID NO: 307)
    AEALNNLGNVYREQGDYNKAIEYYQKALEL-XLX2X3X4; or
    (SEQ ID NO: 2)
    AEAWYNLGNAYYRQGDYQRAIEYYQRALEL-X1X2X3X4; or
    (SEQ ID NO: 3)
    AEALNNLGNVYREQGDYNQRAIEYYQRALEL-X1X2X3X4; or
    (SEQ ID NO: 308)
    AEALRNLGRVYRRQGRYQRAIEYYRRALEL-X1X2X3X4,
  • wherein X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or optionally wherein X2 is P.
  • In another preferred embodiment, the chimeric protein comprising third, fourth and fifth TPR repeat.
  • The invention also provides a chimeric protein comprising
  • (i) a TPR scaffold comprising first and second α-helices linked by an inter-repeat loop, and,
  • (ii) a heterologous peptide ligand that binds an E3 ligase,
  • wherein the heterologous peptide is located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.
  • The invention also provides a chimeric protein comprising
  • (i) a TPR scaffold comprising first and second α-helices linked by an inter-repeat loop, and,
  • (ii) a heterologous peptide ligand that binds a target protein,
      • wherein the heterologous peptide ligand is located in an inter-repeat loop or at the N or at the C terminus of the chimeric protein.
  • Another aspect of the invention provides a library comprising chimeric proteins, each chimeric protein in the library comprising;
      • (i) two or more repeat domains,
      • (ii) inter-repeat loops linking the repeat domains; and
      • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
        • wherein at least one amino acid residue in the peptide ligands in the library is diverse.
  • Another aspect of the invention provides a library comprising a first and a second sub-library of chimeric proteins, each chimeric protein in the first and second sub-libraries comprising;
      • (i) two or more repeat domains,
      • (ii) inter-repeat loops linking the repeat domains; and
      • (iii) a peptide ligand comprising at least one diverse amino acid residue,
  • wherein the peptide ligand in the chimeric proteins in the first sub-library binds to a first target molecule and is located in one of (i) an inter-repeat loop; (ii) the N terminus or (iii) the C terminus of the chimeric protein, and
      • the peptide ligand in the chimeric proteins in the second sub-library binds to a second target molecule and is located in another of (i) an inter-repeat loop; (ii) the N terminus or (iii) the C terminus of the chimeric protein.
  • Another aspect of the invention provides a method of producing a library of chimeric proteins comprising;
      • (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
        • (i) two or more repeat domains,
        • (ii) inter-repeat loops linking the repeat domains; and
        • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
        • wherein the peptide ligands in the population an diverse, and
      • (b) expressing the population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.
  • Another aspect of the invention provides a method of screening a library comprising;
      • (a) providing a library of chimeric proteins, each chimeric protein in the library comprising;
        • (i) two or more repeat domains,
        • (ii) inter-repeat loops linking the repeat domains; and
        • (iii) a peptide ligand located in the inter-repeat loop, at the N terminus or at the C terminus of the protein.
        • wherein at least one amino acid residue in the peptide ligands in the library is diverse,
      • (b) screening the library for chimeric proteins which display a binding activity, and
      • (c) identifying one or more chimeric proteins in the library which display the binding activity.
  • Other aspects and embodiments of the invention are described in more detail below.
    • Other Embodiments
  • From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
  • The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
  • All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference
    • BRIEF DESCRIPTION OF FIGURES
  • FIG. 1 shows the thermostability of consensus-designed tetratricopeptide (CTPR) proteins containing loop- or helix-grafted binding motifs: Thermal denaturation, monitored by circular dichroism, of 2-repeat RTPR (a CTPR in which lysine residues have been replaced with arginine residues) proteins: RTPR2 (in diamonds), RTPR2 containing a loop binding-module (circles) and RTPR2 containing a helix binding-module (squares). All samples are at 20 μM in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl.
  • FIG. 2 shows the thermostability of CTPR proteins of increasing length containing an increasing number of binding modules (alternating with blank modules): Thermal denaturation curves, monitored by circular dichroism, of TPR proteins containing 1, 2, 3 and 4 loops comprising a tankyrase-binding sequence: 1TBP-CTPR2, 2TBP-CTPR4, 3TBP-CTPR6, 4TBP-CTPR8. All samples are at 20 μM in 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl.
  • FIGS. 3A and 3B show an example of helix grafting. FIG. 3A (i) shows the crystal structures of SOS1 (son-of-sevenless homologue 1) bound to KRAS (Kirsten rat sarcoma) (PDB INVU, Margarit et al. Cell (2003) 112(5):685-95), and (ii) shows the SOS1 helix grafted onto a helix at the N-terminus of a CTPR2 protein (SEQ ID NO: 3098). The modelled structure of SOS-RTPR2 is shown, and the sequence of the helix is given with the key KRAS-binding residues in grey and the residues that form the interface with the CTPR helices in black. (iii) shows the modelled structure of SOS-TPR2 in complex with KRAS.
  • FIG. 3B shows binding of SOS-TPR2 to KRAS measured by competitive fluorescence polarization (FP) The complex between mant-GTP and KRAS was pre-formed, and 0.1-300 μM SOS-RTPR2 was then titrated in to the complex, displacing the mant-GTP from KRAS resulting in a decrease in FP. EC50 is 3 μM.
  • FIGS. 4A and B show another example of helix grafting. FIG. 4A shows the modelled structure of the Mdm2 (Mouse double minute 2 homolog) N-terminal domain in complex with the p53-TPR2 comprising the Mdm2-binding helix of p53 grafted onto a helix at the C-terminus of a CTPR2 protein. FIG. 4B shows an ITC analysis of the interaction between p53-TPR2 and Mdm2 N-terminal domain. The N-terminal domain of Mdm2 was titrated into the cell containing 10 μM p53-TPR2.
  • FIGS. 5A and 5B show an example of single and multivalent loop-grafted CTPRs. FIG. 5A shows an ITC analysis of the interaction between a series of tankyrase-binding loop-grafted CTPR2proteins (TBP-CTPR2) and the substrate-binding ARC4 (ankyrin-repeat cluster) domain of tankyrase. There is an enhancement of both binding affinity and dissociation constant with increasing number of binding modules. FIG. 5B shows native gel analysis (using a native gel in Tris-Glycine buffer pH 8.0, 40 μM protein concentration) of multivalent TBP-CTPR proteins expressed as fusion constructs with the foldon trimerisation domain (Boudko ct al 2002; Meier et al. 2004). 1TBP-CTPR2, 2TBP-CTPR4 and 4TBP-CTPR8 (all lacking the foldon domain) were purified and run as monomeric controls. Constructs having the foldon domain run at much higher molecular weights than their monomeric counterparts.
  • FIGS. 6A and 6B show an example of loop-grafted CTPRs comprising the 10-residue Skp2-binding sequence derived from p27 grafted into a loop of a CTPR protein (CTPR-p27). FIG. 6A shows that HA-CTPR2-p27 is able to co-IP FLAG-Skp2 from HEK293T cells FIG. 6B shows E. coli-expressed and purified TPR5-p27 inhibits p27 ubiquitination in vitro.
  • FIGS. 7A and 7B show another example of loop-grafted CTPRs. FIG. 7A shows (left) ITC analysis of the interaction between the Keap1 (Kelch-like ECH-associated protein 1) KELCH domain and a CTPR2 protein containing a loop-grafted Keap1-binding sequence derived from the protein Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) (Nrf-CTPR2). No binding is observed for the blank CPTR2 protein (right). FIG. 7B shows that three variants of Nrf-CTPR2 (Nrf-CTPR2 (i). Nrf-CTPR2 (ii). Nrf-CTPR2 (iii) can co-IP Keap1 from HEK293T cells.
  • FIG. 8 shows live-cell imaging of intracellular delivery of an RTPR achieved by resurfacing (by introducing Arginine residues at surface sites). PC3 (left) and U2OS (right) cells incubated with 10 μM FITC-labelled resurfaced TBP-RTPR2 for 3 hours at 37° C., 5% CO2. Overlay of DIC (differential interference contrast) and confocal image. Intracellular fluorescence was also observed at lower concentrations of protein.
  • FIGS. 9A and 9B show the induced degradation of the target protein beta-catenin by designed hetero-bifunctional RTPRs. FIG. 9A shows the beta-catenin levels in cells transfected with either HA-tagged beta-catenin plasmid alone or HA-tagged beta-catenin plasmid together with one of two different hetero-bifunctional RTPR plasmids (LRH1-TPR-p27 and axin-TPR-p27, designed to bind simultaneously to beta-catenin and to E3 ligase SCFSkp2). FIG. 9B shows a quantitative analysis of the beta-catenin levels in the presence of different hetero-bifunctional RTPRs designed to bind simultaneously to beta-catenin and to either E3 ligase SCFSkp2 or E3 ligase Mdm2. The analysis was performed using densitometry of the bands detected by Western blots corresponding to HA-tagged beta-catenin normalised to actin bands using ImageJ. Negative controls used were single-function TPRs or blank (non-functional) TPRs.
  • FIGS. 10A-10D show examples of different chimeric protein formats. A chimeric protein may comprise: two repeat domains with a helical target-binding peptide and a helical E3-binding peptide at the N and C termini (FIG. 10A): three repeat domains with a helical E3-binding peptide at the C terminus and a target peptide ligand in the first inter-repeat loop from the N terminus (FIG. 10B); three repeat domains with a helical target-binding peptide at the N terminus and an E3 peptide ligand in the second inter-repeat loop from the N terminus (FIG. 10C), four repeat domains with a target-peptide ligand and an E3 peptide ligand in the first and third inter-repeat loop from the N terminus (FIG. 10D).
  • FIG. 11 shows a schematic of a chimeric protein with four peptide ligands located in alternate inter-repeat loops. The binding sites am arrayed at 90° to each other.
  • FIG. 12 shows a schematic of a chimeric protein engineered so that peptide ligands in alternate inter-repeat loops bind adjacent epitopes on the target.
  • FIG. 13 shows the modelled structure of a hetero-bifunctional chimeric protein comprising TPR repeat domains, an LRH1-derived peptide ligand designed to bind target beta-catenin, and a p53-derived N-terminal peptide ligand designed to bind to the E3 ubiquitin ligase mdm2.
  • FIG. 14 shows a schematic of the combinatorial assembly of a module comprising a repeat domain and a terminal helical peptide ligand and a module comprising repeat domains and an inter-repeat loop peptide ligand to generate a chimeric protein.
  • FIG. 15 shows examples of different chimeric protein formals. (i) shows the blank proteins; (ii) shows binding peptides inserted into one or more inter-repeat loops. (iii) shows helical binding peptides at one or both of the termini; (iv) is a combination of loop and helical binding peptides; (v) and (vi) show examples of how multivalency can be achieved.
  • FIG. 16 shows a schematic of the assembly of a chimeric protein by the progressive screening of chimeric proteins comprising modules with a diverse peptide ligand in addition to modules already identified in previous rounds of screening.
  • FIG. 17 shows the effect of designed multi-valent tankyrase-binding TPR proteins on Wnt signalling. HEK293T cells were transfected with TPR-encoding plasmids using Lipofectamine2000. The TPR proteins contained 1-4 copies of a tankyrase-binding peptide (TBP) grafted onto the inter-repeat loop(s). For example, 2TBP-CTPR4 is a protein comprising 4 TPR modules with one TBP grafted onto the loop between the first and second TPR and one between the third and fourth TPR. ‘Foldon’ indicates a trimeic TPR-foldon fusion protein.
  • FIG. 18 shows characterisation of the size and charge of liposome-encapsulated TPR proteins.
  • FIG. 19 shows the delivery of TPR proteins into cells by liposome encapsulation. FITC dye-labelled liposomes stain the cell membrane upon membrane fusion (red panel), and RITC-labelled TPR protein cargo is then delivered into the cytoplasm. The green panel and red-green merge show that the proteins have entered the cells and are spread diffusely in the cytoplasm.
  • FIG. 20 shows that liposome-encapsulated TPR proteins are not toxic to HEK293T cells at the concentrations used.
  • FIG. 21 shows the effect of designed TPR proteins delivered by liposome encapsulation. The TPR proteins contained a tankyrase-binding peptide. Cells were treated with liposomes for 2 hr.
  • FIG. 22 shows the effect of designed TPR proteins delivered by liposome encapsulation. Cells were treated with liposomes encapsulating 32 μg protein for variable times (2-8 h) indicated in the figure.
  • FIG. 23 shows the effect of designed hetero-bifunctional TPR proteins on KRAS levels in HEK 293T cells. The TPR proteins contained a binding sequence for KRAS (a non-helical peptide sequence, referred to as KBL, grafted onto an inter-repeat loop of the RTPR) and a degron derived from p27 grafted onto another inter-repeat loop. Cells were transiently transfected with 50 ng or 500 ng of TPR encoding plasmids, as indicated, and with KRAS plasmid or empty vector as control. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. In dark grey are cells treated transfected with single-function TPR plasmid (containing degron only).
  • FIG. 24 shows the effect of hetero-bifunctional TPR proteins targeting endogenous KRAS to the CMA (chaperone-mediated autophagy) pathway. The TPR proteins contained a binding sequence for KRAS (either a grafted helix derived from son-of-sevenless-homolog I (SOS) or a non-helical peptide sequence (referred to as ‘KBL’) displayed in a loop of the RTPR) and targeted for degradation using two different chaperone-mediated autophagy peptides (referred to as ‘CMA_Q’ or ‘CMA_K’) at the N- or C-terminus of the construct. Constructs or empty vector (light grey) were transiently transfected into either HEK293T or DLD1 (colorectal cancer cell line). 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. Those constructs that resulted in significant reduction in KRAS compared to the empty vector control are shown in white.
  • FIG. 25 shows examples of variations in the linker sequence connecting a peptide ligand to an inter-repeat loop in order to optimise the binding affinity for the target. The example shown is Nrf-TPR, a TPR protein designed to bind to Kep1 (see FIG. 7 of the original patent application). Glycine residues were introduced into the linker to provide flexibility and increased spatial sampling. The introduction of this more flexible linker sequence was found to increase the binding affinity of the Nrf-TPR protein (labelled ‘Flexible’) when compared with the consensus-like linker sequence. Altering the charge content of the linker sequence (‘labelled ‘Charged’) and altering the conformational properties (based on the predictions of the program CIDER (Holehouse et al. Biophys. J. 112, 16-21(2017)) of the loop by changing the amino acid composition of the linker sequence (labelled ‘CIDER-optimised’) also affected the Keap1-binding affinity.
  • FIG. 26 shows the schematic representation of a matrix of degradation-inducing chimeric proteins. The matrix shown is for use in targeting 1-catenin for degradation. These proteins comprise a scaffold (grey rectangles) onto which are grafted: (1) a target-binding peptide ligand and (2) a binding peptide for an E3 ubiquitin ligase or a component of another degradation pathway. Each of the target-binding peptides is derived from a different protein that interacts with A-catenin (see Table 2). Each of the degradation pathway-binding peptides (referred to as “degrons”) is derived from a substrate or binding partner of one of many different E3s or from a binding partner for one of a component of another cellular degradation pathway (including chaperone-mediated autophagy, selective autophagy and ESCRT (endosome-lysosome) pathways); ‘etc.’ denotes the fact that there are many such proteins that can be harnessed for degradation, as detailed further in Table 3 The schematic illustrates the combinational “plug-and-play” nature of these matrices, in terms of the ability to slot in any target-recruiting peptide and degradation-pathway-recruiting peptide. The other factor that can be varied in the matrix arises from the fact that the two peptides can also be grafted onto different positions in the scaffold so as to present the target in different configurations with respect to the E3 or other degradation machinery. Once the matrix is constructed, it can then be screened in cell-based assay in order to identify the best combination of two peptides and their positions within the scaffold that induces the greatest reduction in target protein levels. The same panel of diverse degradation pathway components can be used for screen for degradation of any target
    •  DETAILED DESCRIPTION
  • This invention relates to the chimeric proteins that comprise multiple repeat domains. These repeat domains are linked to each other in the polypeptide chain by inter-repeat loops. One or more peptide ligands (i.e., peptidyl binding motifs or binding domains), are located in one or more of the inter-repeat loops and/or in N or C terminal helices of the chimeric protein. The peptide ligands may be to the same or different target molecules and the chimeric protein may be multi-functional and/or multi-valent. The geometrical display of the grafted binding sites may be precisely and predictably tuned by adjusting the positions of the binding sites and the number and shape of the repeat domains. Chimeric proteins as described herein may be useful in a range of therapeutic and diagnostic applications.
  • A “repeat domain” is a repetitive structural element of 30 to 100 amino acids that forms a defined secondary structure. Multiple (two or more) repeat domains stack sequentially in a modular fashion to form a stable protein, which may for example have a solenoid or toroid structure. Repeat domains may be synthetic or may be naturally-occurring repeats from tandem repeat proteins, or variants thereof.
  • Due to the identical form of their building blocks, solenoid domains can only assume a limited number of shapes. Two main topologies are possible: linear (or open, generally with some degree of helical curvature) and circular (or closed). Patthy, László (2007). Protein Evolution. Wiley-Blackwell. ISBN 978-1-4051-5166-5.
  • If the two terminal repeats in a solenoid do not physically interact, it leads to an open or linear structure. Members of this group are frequently rod- or crescent-shaped. The number of individual repeats can range from 2 to over 50. A clear advantage of this topology is that both the N- and C-terminal ends are free to add new repeats and folds, or even remove existing ones during evolution without any gross impact on the structural stability of the entire domain. Kinch L N, Grishin N V (June 2002). Curr. Opin. Struct. Biol. 12 (3): 400-8. doi:10.1016/s0959-440x(02)00338-x. PMID 12127461. This type of domain is extremely common among extracellular segments of receptors or cell adhesion molecules. A non-exhaustive list of examples include: EGF repeats, cadherin repeats, leucine-rich repeats, HEAT repeats, ankyrin repeats, armadillo repeats, tetratricopeptide repeats, etc. Whenever a linear solenoid domain structure participates in protein-protein interactions, frequently at least 3 or more repetitive subunits form the ligand-binding sites. Thus—while individual repeats might have a (limited) ability to fold on their own—they usually cannot perform the functions of the entire domain alone.
  • In the case when the N- and C-terminal repeats lie in close physical contact in a solenoid domain, the result is a topologically compact, closed structure. Such domains typically display a high rotational symmetry (unlike open solenoids that only have translational symmetries), and assume a wheel-like shape. Because of the limitations of this structure, the number of individual repeats is not arbitrary. In the case of WD40 repeats (perhaps the largest family of closed solenoids) the number of repeats can range from 4 to 10 (more usually between 5 and 7). (Vogel C, Berzuini C, Bashton M, Gough J, Teichmann S A (February 2004). J. Mol. Biol. 336 (3): 809-23). Kelch repeats, beta-barrels and beta-trefoil repeats are further examples for this architecture.
  • A repeat domain may have the structure of a solenoid repeat. The structures of solenoid repeats are well known in the art (see for example Kobe & Kajava Trends in Biochemical Sciences 2000; 25(10):509-15). For example, a repeat domain may have an α/α or α/310 (helix-turn-helix or hth) structure, for example a tetratricopeptide repeat structure; α/α/α (helix-turn-helix-turn-helix or hthth) structure, for example an armadillo repeat structure; a β/β/α/α structure; a α/β or 310/β structure, for example a leucine rich repeat (LRR) structure; a β/β/β structure, for example, an IGF1RL, HPR or PelC repeat structure; or a β/β structure, for example a serralysin or EGF repeat structure.
  • A “scaffold” refers to two or more repeat domains, and a “grafted scaffold” refers to a continuous polypeptide comprising a scaffold and a heterologous binding site (e.g., a peptide ligand).
  • Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops. The loops between adjacent Ankyrin repeats are semi-structured and therefore are quite rigid. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics.
  • The armadillo (Arm) repeat is an approximately 40 amino acid long tandemly repeated sequence motif first identified in the Drosophila melanogaster segment polarity gene armadillo involved in signal transduction through wingless. Animal Arm-repeat proteins function in various processes, including intracellular signalling and cytoskeletal regulation, and include such proteins as beta-catenin, the junctional plaque protein plakoglobin, the adenomatous polyposis coli (APC) tumour suppressor protein, and the nuclear transport factor importin-alpha, amongst others [(PUBMED:9770300)].
  • Suitable repeat domains may include domains of the Ankyrin clan (Pfam: CL0465), such as ankyrin (PF00023), which may comprise a 30-34 amino-acid repeat composed of two beta strands and two alpha helices; domains of the leucino-rich repeat (LRR) clan (Pfam; CL0022), such as LRR1 (PF00560), which may comprise a 20-30 amino acid repeat composed of an α/β horseshoe fold; domains of the Pec Lyase-like (CL0268) clan, such as pec lyase C (PF00544), which may comprise a right handed beta helix; domains of the bet-Roll (CL0592) clan such as Hacmolysin-type calcium-binding repeat (PF000353), which may comprise short repeat units (e.g. 9-mers) that form a beta-roll made up of a super-helix of beta-strand-turns of two short strands each, stabilised by Ca2+ ions; domains of the PSI clan (CL0630), such as trefoil (PF00088); and domains of the tetratricopeptide clan (CL0020), such as TPR-1 (PR00515), which may comprise a 24 to 30, or 24 to 40, or 24 to 90 amino acid repeat composed of a helix-turn-helix.
  • Consensus Sequences for ANK Repeats
  • SMART database, see Table 10) include the following:
  • (SEQ ID NO: 722)
    O04242/1-30 NGHTALHIAASK------------------GDEQCVKLLLEHGA-------DPNA
    CONSENSUS/80% .t.sslhhsh.t..................tp.phhphllp.t.......pht.
    CONSENSUS/65% pstosLphAstp..................sphphlphL1ptss......shsh
    CONSENSUS/50% sGpTsLHhAsps..................sshcllchL1spus......slst
  • Consensus Sequence for ARM repeats (SMART database, see Table 11) include the following:
  • (SEQ ID NO: 1158)
    IMO2HUMANb PND-KIQAVIDAG--VCRRLVELLM------------------
    HNDYKVVSPALRA
    CONSENSUS/80% pt...h..hhp.t..hl..lhphlt..................p.pl.t.shhs
    CONSENSUS/65% ssp.ptphlhpts..slshLlpLLp..................pts.plhptsshs
    CONSENSUS/50% ssc.sppsllcsG..slstLlpLLs..................sscsclppsAstA
    (SEQ. ID NO: 3100)
    IMO2HUMANb VGNIVT
    CONSENSUS/80% ltpls.
    CONSENSUS/65% LpNlst
    CONSENSUS/50% LsNlus
  • Suitable repeat domains may be identified using the PFAM database (see for example Finn at al Nucleic Acids Research (2016) Database Issue 44:D279-D285).
  • In some preferred embodiments, the repeat domain may have the structure of an α/α-solenoid repeat domain, such as a helix-turn-helix. A helix-turn-helix domain comprises two antiparallel α-helices of 12-45 amino acids.
  • Suitable helix-turn-helix domains include tetratricopeptide-like repeat domains. Tetratricopeptide-like repeats may include domains of the TPR clan (CL0020), for example and Arm domains (see for example Armadillo; PF00514; Huber et al Cell 1997; 90: 871-882), HEAT domains (Huntingtin, EF3, PP2A-TORI; PF02985; see for example Groves et al. Cell. 96 (1): 99-110), PPR domains (pentatricopeptide repeat PF01535; see for example Small (2000) Trends Biochem. Sci. 25 (2): 46-7), TALE domains (TAL (transcription activator-like) effector; PF03377; see for example Zhang ct al Nature Biotechnology. 29 (2): 149-53) and TPR1 domains (tetratricopeptide repeat-1; PF00515; see for example Blatch et at BioEssays. 21 (11): 932-9).
  • Other suitable helix-turn-helix domain may be synthetic, for example DHR1 to DHR83 as disclosed in Brunette et al., Nature 2015 528 580-584.
  • In some preferred embodiments, the helix-turn-helix scaffold may be a tetratricopeptide repeat domain (TPR) (D'Andrea & Regan, 2003) or a variant thereof. TPR repeat domains may include naturally occurring or synthetic TPR domains. Suitable TPR repeat domains are well known in the art (see for example Parmeggiani et al., J. Mol. Biol. 427 563-575) and may have the amino acid sequence:
  • (SEQ ID NO: 1)
    AEAWYNLGNAYYKQGDYQKAIEYYQKALEL-X1X2X3X4,
      • wherein X1-4 are independently any amino acid, preferably X1 and X2 being D and P respectively, or may be a variant of this sequence.
  • Additional TPR repeat consensus sequences (SMART database, see Table 9) include the following:
  • S75991
    (SEQ ID NO: 309)
    ALTLNNIGTI YYAREDYDQA LNYYEQALSL SRAV 
    CONSENSUS/80% XXhhXthuXh hXXXtphppA htXhppsltht XpX
    CONSENSUS/65% spshhphGth hhphsphppA lphappAlpl pspX
    CONSENSUS/50% spsatslGps atptucaccA lcsap+ALcl sPss
  • Other TPR repeat domain sequences are shown in Tables 4-6 and 9 below.
  • The grouping of amino acids to classes and class abbreviation (the key) used within consensus sequences are shown below.
  • Class Key Residues
    alcohol o S, T
    aliphatic l I, L, V
    any . A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,
    V, W, Y
    aromatic a F, H, W, Y
    charged c D, E, H, K, R
    hydrophobic h A, C, F, G, H, I, K, L, M, R, T, V, W, Y
    negative − D, E
    polar p C, D, E, H, K, N, Q, R, S, T
    positive + H, K, R
    small s A, C, D, G, N, P, S, T, V
    tiny u A, G, S
    turnlike t A, C, D, E, G, H, K, N, Q, R, S, T
  • Preferred TPR domains may include CTPR, RTPRa, RTPRb and KTPRb domains, for example a domain having a sequence shown in Table 4 or Table 6 or a variant of a sequence shown in Table 4 or Table 6.
  • In some embodiments, a TPR repeat domain may be a human TPR repeat domain, preferably a TPR repeat domain from a human protein in blood. TPR repeat domains from human blood may have reduced immunogenicity in vivo. Suitable human blood TPR repeat domains may include repeat domains from IFIT1, IFTT2 or IFIT3. Other examples of human blood repeat domains identified in the plasma proteome database are shown in Table 5.
  • Suitable human blood repeat domains may be identified from the plasma proteome database (Nanjappa et al Nucl Acids Res 2014 January; 42(Database issue):D959-65) for example by searching for sequences with high sequence identity to the TPR repeat domain using standard sequence analysis tools (e.g. Altschul et al Nucleic Acids Res. 25:3389-34021; Altschul et al FEBS J. 272:5101-5109).
  • A variant of a reference repeat domain or binding site sequence set out herein may comprise an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a repeat domain shown above by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids. Preferred variants of a TPR repeat domain may comprise one or more conserved residues, for example, 1, 2, 3, 4, 5, 6 or more preferably all of Leu at position 7, Gly or Ala at position 8, Tyr at position 11, Ala at position 20, Ala at position 27, Leu or lie at positions 28 and 30 and Pro at position 32.
  • Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altachul et aL (1990) J. Mol. Biol. 215:405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85:2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 3389-3402) may be used.
  • Sequence comparison may be made over the full-length of the relevant sequence described herein.
  • For example, a repeat domain may comprise one or more point mutations to facilitate grafting of hydrophobic peptide ligands. For example, aromatic residues in the repeat domain may be substituted for polar or charged residues. Suitable substitutions may be identified in a rational manner, for example using Hidden Markov plots of repeat domain sequences to identify non-aromatic residues that are found in nature in consensus aromatic positions. A suitable TPR repeat domain for grafting hydrophobic peptide ligands may have the amino acid sequence:
  • (SEQ ID NO: 2)
    AEAWYNEGNAYYRQGDYQRAIEYYQRALEL-X1X2X3X4, 
      • wherein X1-4 are independently any amino acid, preferably X1 and X2 being D and P
  • In some embodiments, lysine residues in the repeat domain may be replaced by arginine residues to prevent ubiquitination and subsequent degradation. This may be particularly useful when the chimeric protein comprises an E3 ubiquitin ligase-peptide ligand, for example in a proteolysis targeting chimera (PROTAC). For example, a suitable TPR repeat domain may have the amino acid sequence:
  • (SEQ ID NO: 307)
    AEALMNLGNVYREQGDYQRAIEWQRALEL-X1X2X3X4, 
      • wherein X1-4 are independently any amino acid, preferably X1 and X2 being D and P respectively.
  • In preferred embodiments, the chimeric protein may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 repeat domains. Preferably, the chimeric protein comprises 2 to 5 repeat domains. Chimeric proteins with fewer repeat domains may display increased cell penetration. For example, a chimeric protein with 2-3 repeat domains may be useful in binding intracellular target molecule. Chimeric proteins with more repeat domains may display increased stability and functionality. For example, a chimeric protein with 4 or more repeat domains may be useful in binding extracellular target molecules. A chimeric protein with 6 or more repeat domains may be useful in producing long linear molecules for targeting or assembling extracellular complexes in bi- or multivalent formats.
  • In other embodiments, sufficient stability and functionality may be conferred by a single repeat domain with N and C terminal peptide ligands. For example, a chimeric protein may comprise:
      • (i) a repeat domain, and
      • (ii) peptide ligands at de N and C terminal of the repeat domain.
  • The repeat domains of a chimeric protein may lack binding activity i.e. the binding activity of the chimeric protein is mediated by the peptide ligands and not by residues within the repeat domains.
  • A “binding domain” (“peptide ligand”) is a contiguous amino acid sequence that specifically binds to a target molecule. Suitable peptide ligands that are capable of grafting onto a terminal helix or inter-repeat loop are well-known in the art and include peptide sequences selected from a library, antigen epitopes, natural protein-protein interactions (helical, extended or turn-like) and short linear motifs (SLiMs). Viral SLiMs (that hijack the host machinery) may be particularly useful because they may display high binding affinities (Davey et al (2011) Trends Biochem. Sci. 36, 159-169).
  • A suitable peptide ligand for a target molecule may be selected from a library, for example using phage or ribosome display, or identified or designed using rational approaches or computational design, for example using the crystal structure of a complex or an interaction. In some embodiments, peptide ligands may be identified in an amino acid sequence using standard sequence analysis tools (e.g. Davey et al Nucleic Acids Res. 2011 Jul. 1; 39 (Web Server issue): W56-W60).
  • Peptide ligands may be 5 to 25 amino acids in length, preferably 8 to 15 amino acids, although in some embodiments, longer peptide ligands may be employed.
  • Generally in chimeric proteins of the invention, the two or more peptide ligands are 40 angstroms apart from each other, they may be 35 angstroms, 30 angstroms, 25 angstroms, 20 angstroms, 15 angstroms but no less than 10 angstroms apart. A person of skill in art can use a 3D structural software such as Chimera or Pymol to determine the minimum distances between positions for ideal positioning in three dimensional orientation.
  • The peptide ligands and the repeat domains of the chimeric protein are heterologous i.e. the peptide ligand is not associated with the repeat domain in naturally occurring proteins and the binding and repeat domains are artificially associated in the chimeric protein by recombinant means.
  • A chimeric protein described herein may comprise 1 to n+1 peptide ligands, where n is the number of repeat domains in the chimeric protein. The number of peptide ligands is determined by the required functionality and valency of the chimeric protein. For example, one peptide ligand may be suitable for a mono-functional chimeric protein and two or more peptide ligands may be suitable for a bi-functional or multi-functional chimeric protein.
  • Chimeric proteins may be monovalent. A target molecule may be bound by a single peptide ligand in a monovalent chimeric protein. Chimeric proteins may be multivalent. A target molecule may be bound by two or more of the same or different peptide ligands in a multivalent chimeric protein.
  • Chimeric proteins may be monospecific. The peptide ligands in a monospecific chimeric protein may all bind to the same target molecule, more preferably the same site or epitope of the target molecule.
  • Chimeric proteins may be multi-specific. The peptide ligands in a multi-specific chimeric protein may bind to different target molecules. For example, a bi-specific chimeric protein may comprise one or more peptide ligands that bind to a first target molecule and one or more peptide ligands that bind to a second target molecule and a tri-specific chimeric protein may comprise one or more peptide ligands that bind to a first target molecule, one or more peptide ligands that bind to a second target molecule and one or more peptide ligands that bind to third target molecule.
  • A bi-specific chimeric protein may bind to the two different target molecules concurrently. This may be useful in bringing the first and second target molecules into close proximity.
  • When the target molecules are located on different cells, concurrent binding of the target molecules to the chimeric protein may bring the cells into close proximity, for example to promote or enhance the interaction of the cells. For example, a chimeric protein which binds to a tumour specific antigen and a T cell antigen, such as CD3, may be useful in bringing T cells into proximity to tumour cells. When the target molecules are from different biological pathways, this may be may be useful in achieving synergistic effects and also for minimising resistance.
  • A tri-specific chimeric protein may bind to three different target molecules concurrently. In some embodiments, one of the target molecules may be an E3 ubiquitin ligase. For example, tri-specific chimeric protein may binding to a first target molecule from a first biological pathway and a second target molecule from a second biological pathway as well as an E3 ubiquitin ligase. This may be useful in achieving synergistic effects and also for minimising resistance.
  • A peptide ligand may be located in an inter-repeat loop of the chimeric protein.
  • An “inter-repeat binding domain” or “inter-repeat peptide ligand” may comprise 5 to 25 amino acid residues, preferably 8 to 15 amino acids. However, since there is no intrinsic restriction on the size of the inter-loop peptide ligand, longer sequences of more than 25 amino acid residues may be used in some embodiments.
  • In some embodiments, an unstructured peptide ligand may be inserted into an inter-repeat loop.
  • One or more, two or more, three or more, four or more or five or more of the inter-repeat loops in the chimeric protein may comprise peptide ligands. The peptide ligands may be located on consecutive inter-repeat loops or may have a different distribution in the inter-repeat loops of the chimeric protein. For example, inter-repeat loops comprising a peptide ligand may be separated in the modular protein by one or more, two or more, three or more or four or more inter-repeat loops which lack a peptide ligand.
  • A peptide ligand may be connected to an inter-repeat loop directly or via one or more additional residues or linkers. Additional residues or linkers may be useful for example when a peptide ligand requires conformational flexibility in order to bind to a target molecule, or when the amino acid residues that are adjacent to the minimal peptide ligand favourably influence the micro-environment of the binding interface.
  • Additional residues or linkers may be positioned at the N terminus of the peptide ligand, the C terminus of the peptide ligand, or both. For example, the sequence of an inter-repeat loop containing a peptide ligand may be [X1-i]-[X1-n]-[X1-z], where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue that is also denoted by X, [X1-n] is the peptide ligand, a is 1 to 100, [X1-i] is a linker and i is independently any number between 1 to 10. In some embodiments, D may be preferred at the first position of the linker [X1-i] P may be preferred at the second position of linker [X1-i], D may be preferred at the last position of the linker [X1-z] and/or P may be preferred at the penultimate position of linker [X1-z]. Examples of preferred inter-repeat loop sequences may include DP-[X1-n]-PX (SEQ ID NO:4); DPXX-[X1-n]-XXPX (SEQ ID NO:5); DPXX-[X1-n]-XPXX (SEQ ID NO:6); DPXX-[X1-n]-PXXX (SEQ ID NO:7); PXX-[X1-i]-[X1-n]-[X1-i]-XXPX (SEQ ID NO:8), DPXX-[X1-i]-[X1-n]-[X1-i]-XPXX (SEQ ID NO:9), DPXX-[X1-i]-[X1-n]-[X1-n]-PXXX (SEQ ID NO:10), DPXX-[X1-i]-[X1-n]-XPXX (SEQ ID NO:11), DPXX-[X1-i]-[X1-n]-XPXX (SEQ ID NO:12), DPXX-[X1-i]-[X1-n]-XPXX (SEQ ID NO:13), DPXX-[X1-n]-[X1-i]-XXPX (SEQ ID NO:14), DPXX-[X1-n]-[X1-i]-XPXX (SEQ ID NO:15) and DPXX-[X1-n]-[X1-i]-PXXX (SEQ ID NO:16).
  • The precise sequence of the residues or linkers used to connect a peptide ligand to an inter-repeat loop depends on the peptide ligand and may be readily determined for any peptide ligand of interest using standard techniques. For example, small, non-hydrophobic amino acids, such as glycine, may be used to provide flexibility and increased spatial sampling, for example when a peptide ligand needs to adopt a specific conformation, or proline residues may be used to increase rigidity, for example, when the peptide ligands are short.
  • In some preferred embodiments, an inter-repeat peptide ligand may be non-hydrophobic. For example, at least 40% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W). Alternatively, the repeat domains may be modified to accommodate a hydrophobic peptide ligand, for example by replacing aromatic residues with charged or polar residues.
  • A peptide ligand may be located at one or both termini of the chimeric protein.
  • A peptide ligand may be located in a helical region of the scaffold in the chimeric protein. A helical region or “helix” is a portion of a scaffold which assumes an α-helical structure.
  • The precise length of a helical peptide ligand is dependent on the length of the helical region of the scaffold. In general, the helical peptide ligand is no longer than the length of the helical region of the scaffold. However, if the helical region of the scaffold is located at one or other termini or is flanked by unstructured or loosely structured residues, then it may be possible to extend it to accommodate a longer helical peptide ligand.
  • A helical peptide ligand may comprise 3 to 25 amino acid residues, preferably 8 to 15 amino acids in length. In some embodiments, a helical peptide ligand may comprise 3-10 or 3-12 or 3-15 or 8-10 or 8-12 or 8-13 or 8-14 or 8-15 or 3-18 or 3-20 or 3-21 or 3-22 or 3-24 or 3-25 amino acids. In some embodiments, a helical peptide ligand may comprise 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 amino acid residues.
  • In some embodiments, a peptide ligand located at the N or C terminus may comprise an α-helical structure and may comprise all or part of a half-repeat (i.e. all or part of a single α-helix) that stacks against an adjacent repeat domain. The α-helix of the terminal peptide ligand makes stabilising interactions with an adjacent repeat domain and is stable and folded. Only a few of the positions that structurally define an α-helix are required for the correct interfacial interaction with the adjacent repeat domain. The residues in some of these positions are defined (Tyr (i)-Ile (1+4)-Tyr (i+7)-Leu (i+11) for the N-terminal α-helix and Ala (i)-Lau(i+4)-Ala/Val (i+7) for the C-terminal helix), but the remaining positions of the α-helix may be modified to form a helical peptide ligand.
  • A helical peptide ligand may be located at the N terminus of the protein. The N terminal peptide ligand may be helical and may comprise all or part of the sequence Xn-(X)15-X1X2XX (SEQ ID NO:17), preferably all or part of the sequence X-XYXXXIXXYXXXLXX-X1X2XX (SEQ ID NO:18), where each residue denoted by X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, X1 is independently any amino acid, preferably D, and X2 is independently any amino acid, preferably P, and n is 0 or any number. In some embodiments, the Y, I, and/or L residues in the N terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).
  • A helical peptide ligand may be located at the C terminus of the scaffold. The C terminal peptide ligand may be helical and may comprise all or part of the sequence Xn-(X)15-X1X2XX, preferably all or part of the sequence X1X2XX-XXAXXXLXX[A or V]XXXXX-Xn, (SEQ ID NO:19) where X is independently any amino acid and may be the same amino acid or a different amino acid to any other residue in the sequence that is also denoted by X, X1 is independently any amino acid, preferably D, and X2 is independently any amino acid, preferably P, and n is 0 or any number. In some embodiments, the A, L and/or V residues in the C terminal peptide ligand may be substituted for an amino acid residue with similar properties (i.e. a conservative substitution).
  • The minimum length of the terminal peptide ligand is determined by the number of residues required to form a helix that binds to the target molecule. There is no intrinsic maximum length of the terminal peptide ligand and n may be any number.
  • It is within the skill in the art to graft selected residues of a peptide ligand into a helix portion of a scaffold containing a helix, and the invention contemplates this variation of grafting as an equivalent to grafting a peptide ligand itself. The residues of the peptide ligand that are in contact with the peptide ligand binding partner (the target protein) are those whose side chains are outward facing and are exposed to solvent. These residues are suitable for grafting to a helical portion of a scaffold. The residues of the scaffold helix whose side chains face inwards and pack against the rest of the scaffold should not be substituted, and this way their interactions with the rest of the scaffold are maintained. It is within the skill in the art to visualize the scaffold structure to identify which of the residues of the helix selected for grafting are facing outwards. PDB codes from arty protein databank provide three dimensional co-ordinates that allow one of skill in the art to visualize the structure of the domain using programs such as PYMOL®, CHIMERA® and RASMOL®. At the same time, it is well within the skill in the art to identify residues of the helix peptide ligand that face outwards form noncovalent interactions (hydrogen bonds and/or Van der Waals and/or hydrophobic interactions) with its binding partner, using a program such as PYMOL®, CHIMERA® and RASMOL® to visualize a peptide ligand complexed with its binding partner. Helix grafting is performed by selectively replacing the outward-facing residues of the helix with corresponding outward-facing residues of the peptide ligand. The inward-facing residues of the helix are undisturbed, and hence the resultant grafted scaffold will have a grafted helix that comprises a mixture of outward facing residues derived from the helix peptide and the native inward facing residues of the helix that were undisturbed.
  • For instance, the following example shows a nine-residue helix peptide ligand (X1-X2-X3-X4-X5-X6-X7-X8-X9). A 3-dimensional view of the peptide ligand in complex with the target protein (using one of the above-noted programs) shows that residues X1, X2, X5, X8 and X9 (for example) of the peptide ligand interact with the target protein and thus are outward facing. Similarly, a helical portion of a given scaffold may be thirty amino acids in length (Y1-Y2-Y3- . . . -Y28-Y29-Y30). A 3-dimensional view of the scaffold shows the helical region and that residues Y3, Y4, Y6, Y7, and Y10 (for example) are inward facing and thus interact with the rest of the scaffold. One of skill in the art would recognize Y1, Y2, Y5, Y8 and Y9 as outward facing, thus identifying these residues as scaffold helical residues that may be replaced with peptide ligand outward facing residues. Therefore, peptide ligand residues X1, X2, X5, X8 and X9 are grafted to the scaffold replacing residues Y1, Y2, Y5, Y8 and Y9 with the corresponding outward facing residues peptide ligand residues X1, X2, X5, X8 and X9, thereby creating an isomorphic replacement. The resultant grafted scaffold will have a grafted helix whose sequence would include the following residues:

  • X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9 (Y10-30)
  • The resulting grafted helix preserves the native hydrogen bonding within the scaffold and at the same time preserves the noncovalent interactions required for specific binding of the peptide ligand to its target protein.
  • The “peptide ligand” may also contain more than one consecutive set of outward facing residues to graft into the scaffold, in which case the grafted scaffold may contain invariant scaffold residues between the grafted peptide residues (e, g “X1 X2 Y3 Y4 X5 Y6 Y7 X8 X9”).
  • A helical peptide ligand may comprise all or part of the sequence CX1X2C2X3X4C3X5X6C4, where X1 to X6 are independently any amino acid and, C1, C2, C3 and C4 are A, B, C and D, respectively.
  • In some embodiments, a helical peptide ligand may be non-hydrophobic. For example, at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W).
  • In other embodiments, a peptide ligand located at the N or C terminus may comprise a non-helical structure. For example, a peptide ligand that is an obligate N- or C-terminal domain (for example because the terminal amino or carboxylate group mediates the binding interaction) may be located at the beginning or end of the one or more repeat domains.
  • In some embodiments, one or more positions in a peptide ligand may be diverse or randomised. A chimeric protein comprising one or more diverse or randomised residues may form a library as described below.
  • In some embodiments, the N and C terminal peptide ligands may be non-hydrophobic. For example, at least 20% of the amino acids in the peptide ligand may be charged (e.g. D, E, R or K) or polar (e.g. Q, N, H, T, Y, C or W). Alternatively, the helix turn helix scaffold of the repeat domains may be modified, for example by replacing aromatic residues with charged or polar residues in order to accommodate a hydrophobic peptide ligand.
  • A chimeric protein as described herein may comprise peptide ligands in any arrangement or combination. For example, peptide ligands may be located at both the N and C terminus and optionally one or more inter-repeat loops of a chimeric protein; at the N terminus and optionally one or more loops of a chimeric protein; at the C terminus and optionally one or more loops of a chimeric protein; or in one or more inter-repeat loops of a chimeric protein.
  • The location of the peptide ligands within a chimeric protein may be determined by rational design, for example using modelling to identify the optimal arrangement for the presentation of two target molecules to each other (e.g. for substrate presentation to an E3 ubiquitin ligase); and/or by screening for example using populations of chimeric proteins with different arrangements of peptide ligands to identify the arrangement which confers the optimal interaction of target molecules.
  • Target Proteins and Targeting Peptide Ligands
  • Target proteins and peptide ligands that bind such proteins are described herein and are listed, without limitation, in the tables.
  • Suitable target molecules for chimeric proteins described herein include biological macromolecules, such as proteins. The target molecule may be a receptor, enzyme, antigen, oligosaccharide, oligonucleotide, integral membrane protein, transcription factor, transcriptional regulator, G protein coupled receptor (GPCR) or any other target of interest. Proteins that are difficult to target with small molecules, such as PPIs, proteins that accumulate in neurodegenerative diseases and proteins overexpressed in disease conditions, such as cancer, may be particularly suitable target molecules. Target molecules may include α-synuclein; β-amyloid; tau; superoxide dismutase; buntingtin; β-catenin; KRAS; components of superenhancers and other types of transcriptional regulators, such as N-Myc, C-Myc, Notch, aurora A, EWS-FLI1 (Ewing's sarcoma-friend leukemia integration 1), TEL-AML1, TAL1 (T-cell acute lymphocytic leukemia protein 1) and Sox2 ((sex determining region Y)-box 2); tankyrases; phosphatases such as PP2A; epigenetic writers, readers and erasers, such as histone deacetylases and histone methyltransferases; BRD4 and other bromodomain proteins; and kinases, such as PLK1 (polo-like kinase 1), c-ABL (Abelson murine leukemia viral oncogene homolog 1) and BCR (breakpoint cluster region)-ABL.
  • In some embodiments, a chimeric protein may neutralise a biological activity of the target molecule, for example by inhibiting or antagonising its activity or binding to another molecule or by tagging it for ubiquitination and proteasomal degradation or for degradation via autophagy. In other embodiments, a chimeric protein may activate a biological activity of the target molecule.
  • In some embodiments, the target molecule may be β-catenin. Suitable peptide ligands that specifically bind to β-catenin are well-known in the art and include β-catenin-peptide ligands derived from axin (e.g. GAYPEYILDIHVYRVQLEL (SEQ ID NO:20) and variants thereof), Bcl-9 (e.g. SQEQLEHRYRSLITLYDIQLML (SEQ ID NO:21) and variants thereof), TCF7L2 (e.g. QELGDNDELMHFSYESTQD (SEQ ID NO:22) and variants thereof), ICAT (e.g. YAYQRAIVEYMLRLMS (SEQ ID NO:23) and variants thereof), LRH-1 (e.g. YEQAIAAYLDALMC (SEQ ID NO:24) and variants thereof) or APC (e.g. SCSEELEALEALELDE (SEQ ID NO:25) and variants thereof).
  • In some embodiments, the target molecule may be KRAS. Suitable peptide ligands that specifically bind to KRAS are well-known in the art and include a KRAS-peptide ligand from SOS-1 (e.g. FEGIALTNYLKALEG (SEQ ID NO:26) and variants thereof) and KRAS-peptide ligands identified by phage display (see for example Sakramnto et al. Biochem. Biophys. Res. Comm. (2017) 484 605-611).
  • In some embodiments, the target molecule may be tankyrase. Suitable peptide ligands that specifically bind to tankyrase are well-known in the art and include tankyrase peptide ligands from Axin (e.g. REAGDGEE (SEQ ID NO:27) and HLQREAGDGEEFRS (SEQ ID NO:28) or variants thereof).
  • In some embodiments, the target molecule may be EWS-FLI1. Suitable peptide ligands that specifically bind to EWS-FLI1 are well-known in the art and include the ESAP1 peptide TMRGKKKRTRAN (SEQ ID NO:29) and variants thereof. Other suitable sequences may be identified by phage display (see for example Erkizan et al. Cell Cycle (2011) 10, 3397-408).
  • In some embodiments, the target molecule may be Aurora-A. Suitable peptide ligands that specifically bind to Aurora-A are well-known in the art and include Aurora-A binding sequences from TPX2, such as SYSYDAPSDFINFSS (SEQ ID NO:30)(Bayliss et al. Mol. Cell (2003) 12, 851-62) and Aurora-A binding sequences from N-myc, such as N-myc residues 19-47 or 61-89 (see for example Richards et al. PNAS (2016) 113, 13726-31).
  • In some embodiments, the target molecule may be N-Myc or C-Myc. Suitable peptide ligands that specifically bind to N-myc or C-myc are well-known in the art and include helical binding sequences from Aurora-A (see for example Richards et al. PNAS (2016) 113, 13726-31).
  • In some embodiments, the target molecule may be WDR5 (WD repeat-containing protein 5). Suitable peptide ligands that specifically bind to WDR5 are well-known in the art and include the WDR5-interacting motif (WIN) of MLL1 (mixed lineage leukemia protein 1) (see for example Song & Kingston J. Biol. Chem. (2008) 283, 35258-64; Patel eat al. J. Biol. Chem. (2008) 283, 32158-61), e.g. EPPLNPHGSARAEVHLRKS (SEQ ID NO:31) and variants thereof.
  • In some embodiments, the target molecule may be BRD4 or a Bromodomain protein. Suitable peptide ligands that specifically bind to BRD4 are well-known in the art and include sequences derived from histone protein ligands.
  • In some embodiments, the target molecule may be a HDAC (histone deacetylase). Suitable peptide ligands that specifically bind to HDAC are well-known in the art and include binding sequences derived from SMRT and other proteins that recruit HDACs to specific transcriptional regulatory complexes or binding sequences derived from histone proteins (see for example Watson et al. Nat. Comm. (2016) 7, 11262; Dowling et al. Biochem. (2008) 47, 13554-63).
  • In some embodiments, the target molecule may be Notch. Suitable peptide ligands that specifically bind to Notch are well-known in the art and include binding sequences from the N-terminus of MAML1 (mastermind like protein 1), e.g. SAVMERLRRRIELCRRHHST (SEQ ID NO:32) and variants thereof (see for example Moellering et al. Nature (2009) 462, 182-8).
  • In some embodiments, the target molecule may be a Cdk (cyclin-dependent kinase). Suitable peptide ligands that specifically bind to Cdks are well-known in the art and include substrate-based peptides, for example, Cdk2 sequences derived from cyclin A, such as TYTKKQVLRMEHLVLKVLTFDL (SEQ ID NO:33) and variants thereof (see for example Gondeau et al. J. Biol. Chem. (2005) 280, 13793-800; Mendoza et al. Cancer Res. (2003) 63, 1020-4).
  • In some embodiments, the target molecule may be PLK1 (polo-like kinase 1). Suitable peptide ligands that specifically bind to PLK1 arE well-known in the art and include optimised substrate-derived sequences that bind to the substrate-binding PBD (polo-box domain), such as MAGPMQSEPLMGAKK (SEQ ID NO:34) and variants thereof.
  • In some embodiments, the target molecule may be Tau. Suitable peptide ligands that specifically bind to Tau are well-known in the art and include tau-binding sequences derived from alpha- and beta-tubulin, such as KDYEEVGVDSVE (SEQ ID NO:35) and YQQYQDATADEQG (SEQ ID NO:36) and variants thereof (see for example Maccioni et al. EMBO J. (1988) 7, 1957-63; Rivas at al. PNAS (1988) 85, 6092-6).
  • In some embodiments, the target molecule may be BCR-ABL. Suitable peptide ligands that specifically bind to BCR-ABL are well-known in the art and include optimized substrate-derived sequences, such as EAIYAAPFARKK (SEQ ID NO:37) and variants thereof.
  • In some embodiments, the target molecule may be PP2A (protein phosphatase 2A). Suitable peptide ligands that specifically bind to PP2A are well-known in the art and include sequences that bind the B56 regulatory subunit, such as LQTIQEEE (SEQ ID NO:38) and variants thereof (see for example Hetz et al. Mol. Cell (2016), 63 686-95), some embodiments, the target molecule may be EED (Embryonic ectoderm development).
  • Suitable peptide ligands that specifically bind to EED are well-known in the art and include helical binding sequences from co-factor EZH2 (enhancer of zeste homolog 2), such as FSSNRQKILERTEILNQEWKQRRIQPV (SEQ ID NO:39) and variants thereof (see for example Kim at al. Nat. Chem. Biol. (2013) 9, 643-50.)
  • In some embodiments, the target molecule may be MCL-1 (induced myeloid leukemia cell differentiation protein). Suitable peptide ligands that specifically bind to MCL-1 are well-known in the art and include sequences from BCL2, e.g. KALETLRRVGDGVQRNHETAF (SEQ ID NO:40) and variants thereof (see for example Stewart et al. Nat. Chem. Biol. (2010) 6, 595-601).
  • In some embodiments, the target molecule may be RAS. Suitable RAS peptide ligands are well-known in the art and include RAS-binding peptides identified by phage display, such as RRRRCPLYISYDPVCRRRR (SEQ ID NO:41) and variants thereof (see for example Sakamoto et al. BBRC (2017) 484, 605-11).
  • In some embodiments, the target molecule may be GSK3 (glycogen synthase kinase 3). Suitable GSK3 peptide ligands are well-known in the art and include substrate-competitive binding sequences such as KEAPPAPPQDP (SEQ ID NO:42), LSRRPDYR (SEQ ID NO:1436), RREGGMSRPADVDG (SEQ ID NO:44), and YRRAAVPPSPSLSRHSSPSQDEDEEE (SEQ ID NO:45) and variants thereof (see for example Ilouz et al. J. Biol. Chem. 281(2006), 30621-30630. Plotkin et al. J Pharmacol. Exp. Ther. (2003) 305, 974-980).
  • In some embodiments, the target molecule may be CtBP (C-terminal binding protein). Suitable CtBP peptide ligands are well-known in the art and include sequences identified from a cyclic peptide library screen, such as SGWTVVRMY (SEQ ID NO:46) and variants thereof (see for example Birts et al. Chem. Sci. (2013) 4, 3046-57).
  • Examples of suitable peptide ligands for target molecules that may be used in a chimeric protein as described herein are shown in Tables 2 and 7.
  • E3 Ligase Peptide Ligand
  • In some preferred embodiments, a chimeric protein as described herein may comprise a peptide ligand for an E3 ubiquitin ligase. Examples of suitable E3 ubiquitin ligases include MDM2, SCFSkp2, BTB-CUL3-RBX1, APC/C, SIAH, CHIP, Cul4-DDB1, SCF-family, β-TrCP, Fbw7 and Fbx4.
  • E3 Ligase Peptide Ligands
  • Suitable peptide ligands for E3 ubiquitin ligases (degrons) are well known in the art and may be 5 to 20 amino acids. For example, a suitable peptide ligand for MDM2 may include a peptide ligand from p53 (e.g. FAAYWNLLSAYG)(SEQ ID NO:47) and or a variant thereof. A suitable peptide ligand for SCFSkp2 may include a peptide ligand from p27 (e.g. AGSNEQEPKKRS)(SEQ ID NO:48) and variants thereof. A suitable peptide ligand for Keap1-Cul3 may include a peptide ligand from Nrf2 (e.g. DPETGEL)(SEQ ID NO:49) or a variant thereof. A suitable peptide ligand for SPOP-Cul3 may be include a peptide ligand from Puc (e.g. LACDEVTSTTSSSTA (SEQ ID NO:50) or a variant thereof. A suitable peptide ligand for APC/C may include the degrons termed ABBA (e.g. SLSSAFHVFEDGNXEN)(SEQ ID NO:51), KEN (e.g. SEDKENVPP) (SEQ ID NO:52), or DBOX (e.g. PRLPLGDVSNN)(SEQ ID NO:53) or a variant thereof. In some instances, a combination of these degrons for may be used (mimicking the bipartite or tripartite degrons found in some natural substrates). A suitable peptide ligand for SIAH may include a peptide ligand from PHYL (e.g. LRPVAMVRPTV) (SEQ ID NO:54) or a variant thereof. A suitable peptide ligand for CHIP (carboxyl terminus of Hsc70-interacting protein) may include peptide sequences such as ASRMEEVD (SEQ ID NO:55) (from Hsp90 C-terminus) and GPTIEEVD (SEQ II) NO:56) (from Hsp70 C-terminus) or a variant thereof. A suitable peptide ligand for beta-TrCP may include a degron sequence motif (including phosphomimetic amino acids), such as DDGYFD (SEQ ID NO:57) or a variant thereof. A suitable peptide ligand for Fbx4 may include sequences derived from TRF1, such as MPIFWKAHRMSKMGTG (SEQ ID NO:58) or a variant thereof (see for example Lee et al. Chembiochem (2013) 14, 445-451). A suitable peptide ligand for FBw7 may include degron sequence motifs (including phosphomimetic amino acids), such as LPSGLLEPPQD (SEQ ID NO:59). A suitable peptide ligand for DDB1-Cul4 may include sequences derived from HBx (hepatitis B virus X protein) and similar proteins from other viruses and from DCAFs (DDB1-CUL4-associated factors) including helical motifs such as ILPKVLHKRTLGL (SEQ ID NO:60), NFVSWHANRQLGM (SEQ ID NO:61), NTVEYFTSQQVTG (SEQ ID NO:62), and NITRDLIRRQIKE (SEQ ID NO:63) (see for example Li et al. Nat. Struct. Mol. Biol. (2010) 17, 105-111).
  • E3 Ligases and E3 Ligase Peptide Ligands
  • Examples of suitable peptide ligands for E3 ubiquitin ligases that may be used in a chimeric protein as described herein are shown in Table 3.
  • A chimeric protein comprising a peptide ligand for an E3 ubiquitin ligase may also comprise a peptide ligand for a target molecule, Without being bound to any one hypothesis, binding of the chimeric protein to both the target molecule and the E3 ubiquitin ligase may cause the target molecule to be ubiquitinated by the E3 ubiquitin ligase. Ubiquitinylated target molecules may then degraded by the proteasome. This allows the specific targeting of molecules for proteolysis by the chimeric protein. The ubiquitination and subsequent degradation of a target protein has been shown for hetero-bifunctional small molecules (PROTACs; proteolysis targeting chimeras) that bind the target protein and a ubiquitin ligase simultaneously (see for example Bondeson et al. Nat. Chem. Biol. 2015; Deshaies 2015; Lu et al. 2015).
  • In some embodiments, the chimeric protein may lack lysine residues, so that it avoids ubiquitination by the E3 ubiquitin ligase.
  • Examples of chimeric proteins that bind E3 ubiquitin ligase and a target molecule are shown in Tables 1 and 8.
  • A suitable chimeric protein may comprise an N terminal peptide ligand that binds a target protein, such as β catenin, and a C terminal peptide ligand that binds an E3 ubiquitin ligase. For example, the N terminal peptide ligand may be a β catenin-binding sequence derived from Bcl9 and the C terminal peptide ligand may be an Mdm2-binding sequence derived from p53. Alternatively, a chimeric protein may comprise a C terminal peptide ligand that binds a target protein, such as β catenin, and an N terminal peptide ligand that binds an E3 ubiquitin ligase (see FIG. 10A).
  • Another suitable chimeric protein may comprise three repeat domains, a peptide ligand located in an inter-repeat loop that binds a target protein, such as β catenin, and a C terminal peptide ligand that binds an E3 ubiquitin ligase. For example, the inter-repeat loop peptide ligand may be derived from the phosphorylated region of APC (adenomentous polyposis col) and the C terminal peptide ligand may be an Mdm2-binding sequence derived from p53. Alternatively, the chimeric protein may comprise a peptide ligand located in an inter-repeat loop that binds an E3 ubiquitin ligase, and a C terminal peptide ligand that binds a target protein, such as β catenin (See FIG. 10B).
  • Another suitable chimeric protein may comprise three repeat domains, an N terminal peptide ligand that binds a target protein, such as β catenin, and a peptide ligand located in an inter module loop that binds an E3 ubiquitin ligase. For example, the N terminal peptide ligand may be a β catenin-binding sequence derived from LRH1 (liver receptor homolog 1) and the inter-module loop peptide ligand may be a sequence derived from the Skp2-targeting region of p27. Alternatively, the chimeric protein may comprise an N terminal peptide ligand that binds an E3 ubiquitin ligase and a peptide ligand located in an inter-module loop that binds a target protein, such as β catenin (ee FIG. 10C).
  • Another suitable chimeric protein may comprise four repeat domains, a first peptide ligand located in an inter-repeat loop that binds an E3 ubiquitin ligase and a second peptide ligand located in an inter-repeat loop that binds a target molecule. The first and second inter-repeat loops may be separate by an inter-repeat loop lacking a peptide ligand. For example, the first peptide ligand may be located in the first inter-repeat loop inter-repeat loop from the N terminus and the second peptide ligand may be located in the third inter-repeat loop from the N terminus or vice versa.
  • In some preferred embodiments, a chimeric protein as described herein may comprise an amino acid shown in Table 8 or a variant thereof.
  • In other preferred embodiments, a chimeric protein as described herein may comprise a peptide ligand that binds to a component of a target-selective autophagy pathway, such as chaperone-mediated autophagy (CMA). The chimeric protein and target molecules bound thereto are thus recognised by the autophagy pathway and the target molecules are subsequently degraded. Suitable components of the CMA pathway include heat shock cognate protein of 70 kDa (hsc70, HSPA8, Gene ID: 3312). Suitable peptide ligands are well known in the art (Dice J. F. (1990). Trends Biochem. Sci. 15, 305-309) and include Lys-Phe-Glu-Arg-Gln (KFERQ (SEQ ID NO:64)) and variants thereof, such as CMA_Q and CMA_K, as described herein. These domains have been demonstrated to be capable of targeting heterologous proteins to the autophagy pathway (Fan, X. et al; (2014) Nature Neuroscience 17, 471-480).
  • In addition to repeat domains and peptide ligands, a chimeric protein may further comprise one or more additional domains which confer additional functionality, such as targeting domains, intracellular transport domains, stabilising domains or oligomerisation domains. Additional domains may for example be located at the N or C terminus of the chimeric protein or in a loop between repeats.
  • A targeting domain may be useful in targeting the chimeric protein to a particular destination in vivo, such as a target tissue, cell, membrane or intracellular organelle. Suitable targeting domains include chimeric antigen receptors (CARs).
  • An intracellular transport domain may facilitate the passage of the chimeric protein through the cell membrane into cells, for example to bind intracellular target molecules. Suitable intracellular transfer domains are well known in the art (see for example Bechara et al FEBS Letters 587 1 (2013) 1693-1702) and include cell-penetrating peptides (CPPs), such as Antennapedia (43-58), Tat (48-60), Cadherin (615-632) and poly-Arg.
  • A stabilising domain may increase the half-life of the chimeric protein in vivo. Suitable stabilising domains are well known in the art and include Fc domains, serum albumin, unstructured peptides such as XTEN98 or PAS99 and polyethylene glycol (PEG).
  • An oligomerisation domain may facilitate the formation of multi-protein complexes, for example to increase avidity against multi-valent targets. Suitable oligomerisation domains include the ‘foldon’ domain, the natural trimerisation domain of T4 fibritin (Meier et al., J. Mol. Biol. (2004) 344(4):1051-69).
  • In addition to repeat domains, peptide ligands and optionally one or more additional domains, a chimeric protein may further comprise a cytotoxic or therapeutic agent and/or or detectable label.
  • Suitable cytotoxic agents include, for example, chemotherapeutic agents, such as methotrexate, auristatin adriamicin, doxorubicin, melphalan, mitomycin C, ozogamicin, chlorambucil, maytansine, catansine, daunorubicin or other intercalating agents, enzymatically active toxins of bacterial, fungal, plant, or animal origin, such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, α-amanitin, alpha-sarcin, Aleurites fordii proteins, tubulysins, dianthin proteins, Phytolaca americana proteins (PAPI, PAP11, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, pyrrolobenzodiazepines, and the tricothecenes and fragments of any of these. Suitable cytotoxic agents may also include radioisotopes. A variety of radionuclides are available for the production of radioconjugated chimeric proteins including, but not limited to, 90Y, 125I, 131I, 123I, 111In, 131In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re, 188Re and 212Bi. Conjugates of a chimeric protein and one or more small anti-cancer molecules, for example toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC1065, and the derivatives of these toxins that have toxin activity, may also be used.
  • Suitable therapeutic agents may include cytokines (e.g. IL2, IL12 and TNF), chemokines, pro-coagulant factors (e.g. tissue factor), enzymes, liposomes, and immune response factors.
  • A detectable label may be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers. Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. Detectable labels may be attached to chimeric proteins using conventional chemistry known in the art.
  • There are numerous methods by which the label can produce a signal detectable by external means, for example, by visual examination, electromagnetic radiation, heat, and chemical reagents. The label can also be bound to another specific binding member that binds the chimeric protein, or to a support.
  • In some embodiments, a chimeric protein may be configured for display on a particle or molecular complex, such as a cell, ribosome or phage, for example for screening and selection. A suitable chimeric protein may further comprise a display moiety, such as phage coat protein, to facilitate display on a particle or molecular complex. The phage coat protein may be fused or covalently linked to the chimeric protein.
  • Providing a Chimeric Protein According to the Invention
  • Chimeric proteins as described herein may be produced by recombinant means. For example, a method of producing a chimeric protein as described herein may comprise expressing a nucleic acid encoding the chimeric protein. A nucleic acid may be expressed in a host cell and the expressed chimeric protein may then be isolated and/or purified from the cell culture.
  • In some embodiments, the recombinant method may comprise;
      • inserting a first nucleic acid encoding a peptide ligand into a second nucleic acid encoding two or more repeat domains, e.g, a TPR repeat as described herein, e.g., CTPR or RTPR2, to produce a chimeric nucleic acid encoding a chimeric protein comprising a peptide ligand. The first nucleic acid may be inserted into an inter-repeat loop (for example, the RTPR2 scaffold contains a 20 amino acid loop and the first peptide ligand may be inserted anywhere between codons encoding two loop amino acids. Alternatively, the first nucleic acid may be inserted into the second nucleic acid at the codon encoding the N-terminus or the C-terminus of the scaffold such that the peptide is in-frame with the scaffold, thereby forming a chimeric nucleic acid encoding a chimeric protein (a grafted scaffold). and,
      • expressing the chimeric nucleic acid to produce the chimeric protein.
  • Methods described herein may be useful in producing a chimeric protein that binds to a first target molecule and a second target molecule. For example, a method may comprise;
      • providing a nucleic acid encoding two or more repeat domains linked by inter-repeat loops, each repeat domain; and
      • incorporating into the nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a first target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to a second target molecule to generate a nucleic acid encoding a chimeric protein comprising the first and second peptide ligands, wherein the first nucleotide sequence encoding the first peptide ligand is located in an inter-repeat loop or at the N or C terminus of the grafted scaffold and the second nucleotide sequence encoding the second peptide ligand is located in a different inter-repeat loop than the first peptide ligand or is located at the N or C terminus wherein the first peptide ligand is not located; and
      • expressing the nucleic acid to produce the chimeric protein.
  • One of the first and second target molecules may be an E3 ubiquitin ligase. For example, a method may comprise;
      • providing a nucleic acid encoding two or more repeat domains linked by inter-repeat loops between the repeat domains; and
      • incorporating into the nucleic acid a first nucleotide sequence encoding a first peptide ligand that binds to a target molecule and a second nucleotide sequence encoding a second peptide ligand that binds to an E3 ubiquitin ligase to generate a nucleic acid encoding a chimeric protein comprising the first and second peptide ligands, wherein the first and second peptide ligands are located (i) in different inter-repeat loops or (ii) the first ligand is located in an inter-repeat loop while the second peptide ligand is located at the N or C terminus of the scaffold, or (iii) the first and second peptide ligands are located at the N and C termini of the scaffold, respectively; and
      • expressing the nucleic acid to produce the protein.
  • An isolated nucleic acid encoding a chimeric protein as described herein is provided as an aspect of the invention. The nucleic acid may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell.
  • Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts such as E. coli and/or in eukaryotic cells.
  • Many techniques and protocols that are suitable for the expression of recombinant chimeric proteins in cell culture and their subsequent isolation and purification are known in the art (see for example Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed RS Tuan (March 1997) Humans Press Inc).
  • A host cell comprising a nucleic acid encoding a chimeric protein as described herein or vector containing such a nucleic acid is also provided as an aspect of the invention. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of proteins in prokaryotic cells is well established in the art. A common bacterial host is E. coli. A chimeric protein may also be produced by expression in eukaryotic cells in culture. Mammalian cell lines available in the art for expression of a chimeric protein include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells (e.g. HEK293 cells), human embryonic retina cells (e.g. PerC6 cells) and many others.
  • The following procedures and assays may be used according to the invention.
    • Preparation of Grafted Scaffold Protein
  • Large-Scale Protein Purification (His-Tagged) from E. coli
  • The pRSET B (His-tag) constructs are transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies are grown in 2TY media containing ampicillin (50 micrograms/mL) at 37° C., 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures are then induced with IPTG (0.5 mM) for 16-20 h at 20° C. or 4 h at 37° C. Cells are pelleted by centrifugation at 3000 g (4° C., 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris is pelleted by centrifugation at 15,000 g at 4° C. for 45 min. Ni-NTA beads 50% bed volume (GE Healthcare)(5 mL) are washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) before the supernatant of the cell lysate is bound to them for 1 hr at 4° C. in batch. The loaded beads are washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non-specific interaction of lysate proteins with the beads. Samples are eluted using phosphate buffer with 300 mM imidazole, and purified by size-exclusion chromatography using a HiLoad 16/60 SuperdexG75 column (GE Life-Science) pre-equilibrated in phosphate buffer (10 mM sodium phosphate. pH 7.4, 150 mM NaCl) and proteins separated in isocratic conditions. Purity is checked on NuPage protein gel (Invitrogen), and fractions found to be over 95% pure are pooled. Purified protein is flash-frozen and stored at −80° C. until further use. Concentrations are determined by measuring absorbance at 280 nm and using a calculated extinction coefficient from ExPASy ProtParam (Gasteiger et al. 2005) for each variant. Molecular weight and purity is confirmed using mass spectrometry (MALDI).
  • Large-Scale Protein Purification (Heat Treatment) from E. coli
  • Many of the chimeric proteins described herein are thermally very stable, with melting temperatures above 80° C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65° C. for 20 min. Very few of the E. coli proteins will remain folded at such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins are removed by centrifugation, leaving 80-90% pure sample of the desired protein. Constructs that fold reversibly can be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.
  • This approach allows the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is potentially scalable to industrial production and bioreactors.
  • Small-Scale Purification of His-Tagged Proteins for Higher-Throughput Testing
  • Plasmids are transformed into E. coli C41 cells and plated overnight. 15 mis of 2TY medium (Roche) containing 50 micrograms/ml ampicillin is placed in each one multiple 50 ml tubes. Several colonies are picked from the plates and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells are grown at 37° C. until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells are pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAseI treatment is used. The lysate is spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.
  • The supernatant is added to 100 μl bed volume of pre-washed Ni-NTA agarose beads. The subsequent affinity purification is performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used). The first ish contained 10% BugBustar® solution and 30 mM imidazole in the chosen buffer. Here we used 50 mM sodium phosphate buffer pH 6.8, 150 mM NaCl. The three successive ishes had 30 mM of imidazole in the chosen buffer. Beads are washed thoroughly to remove the detergent present in the BugBuster® solution. Protein is eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole. The combination of Bugbuster® and imidazole and the repeat washes in small bead volumes yielded >95% pure protein. Imidazole is removed using a NAP-5 disposable gel-filtration column (GE Healthcare).
  • Measuring Binding of Grafted Scaffold Protein to Target Protein
  • Competition Fluorescence Polarisation (FP) Assay
  • To measure the binding of a grafted scaffold to a target protein, Competition FP can be performed using 384-well black opaque optiplate microplates and a CLARIOstar microplate reader. The grafted scaffold protein is titrated into a solution containing a mixture of FITC-labelled peptide ligand and target binding partner (target protein). The prepared plates are incubated for 30 minutes at room temperature before readings are taken. The grafted scaffold is then titrated into the preformed FITC-peptide-target protein complex. A decrease in polarisation with increasing concentrations of grafted scaffold indicates displacement of FITC-peptide upon binding of the grafted scaffold to its target.
  • Isotheral Titration Calorimetry (ITC)
  • ITC can be performed using a VP-ITC instrument (Microcal). Grafted scaffolds are dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl, 0.5 mM TCEP. Dialysed target protein (200 μM) is titrated into the sample cell containing the grafted scaffold at 20 μM. Injections of target protein into the cell are initiated with a 5 μL injection, followed by 29 injections of 10 μL. The reference power is set at 15 μCal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data are fitted using the instrument software a one-site binding model.
  • Cell Culture and Cell Transfection
  • HEK293T cells are cultured in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) supplemented with 10% fetal bovine serum and penicillin/streptomycin (LifeTech) at 37° C. with 5% CO2 air supply.
  • HEK293T are seeded in 6-well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.
  • Western Blot Assay of Target-Protein Engagement and of Target-Protein Levels
  • Plasmid encoding the target protein (1 μg) alone and with plasmid encoding one of various target-specific grafted scaffolds (1 μg) is transfected in HEK293T cells in 6-well plates using Lipofectamine2000. After 48 hours of transfection, the cells are lysed in 200 μL of Laemmli buffer. After sample is boiled at 95° C. for 20 min proteins are resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting is performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies. Changes in target protein levels upon co-transfection with bifunctional grafted scaffolds are evaluated by the densitometry of the bands corresponding to the target protein normalised to actin levels using ImageJ. Co-immunoprecipitation can also be used to show that the grafted scaffold binds to the target protein and/or to the desired component of the degradation machinery.
  • Liposomal Formulation and Cytotoxicity Assay
  • To make liposomal formulations of proteins (LFP), lipids (DOTAP (cationic): DOPE (neutral): DiR (aromatic)=1:1:0.1 w/w) are dissolved in chloroform, and solvent is evaporated under vacuum overnight. Resulting mixed lipid cake is hydrated with 10 mM HEPES pH 7.4, containing 27 μM protein, so that the total lipid concentration is 4 mg/ml. This mixture is vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins are stored at 4° C. until further use. To make empty liposomes (EL, empty liposomes without proteins), lipid cake is hydrated with 10 mM HEPES pH 7.4 without proteins.
  • An ATP assay is used to investigate whether there is any cytotoxicity associated with EL and LFP. In a typical procedure, 2×105HEK 293T cells/well in 500 μL of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum are grown for 24 hours in a 24-well cell culture plate. Cells are incubated with liposome (EL/LFP)-media (DMEM without FBS) mix, having different volumes (0-60 μL) of EL and LFP, for 15 minutes at 37° C. After washing twice with 1×PBS, 500 μL of CellTiter-Glo® Reagent (Promega) is added and luminescence is measured using a microplate reader as par the manufacture's protocol. Untreated cells are used as control. Data are obtained from triplicate samples, and the standard deviations are calculated from two independent experiments.
    • HiBit Split-Luciferase Assay
  • An alternative method for measuring target protein levels is the Nano-Glo® HiBiT Lytic Detection System from Promega Corporation. It is based on the split NanoLuc assay, which consists of a large N-terminal fragment (LgBiT) and a small C-terminal region (SmBiT). Five of the SmBiT amino acids have been replaced to produce the HiBiT (VSGWRLFKKIS) (SEQ ID NO: 3102) fragment, which has greater affinity for the LgBiT fragment and maintains NanoLuc luciferase activity. Either the HiBiT-tagged target DNA can be transient transfected or the endogenous target can be monitored by knock-in of the HiBiT tag sequence using CRISPR/Cas9 technology. Subsequent introduction of the complementary polypeptide, LgBiT, results in spontaneous and high affinity interaction between the HiBiT tag and LgBiT to reconstitute the luminescent NanoBit® enzyme. Detection of tagged protein levels is possible from live or lysed cells.
  • Protein is introduced into HEK293T cells by either DNA transient transfection or encapsulation within fusogenic liposomes. HEK293T cells are seeded into either 24-well or 96-well plates After 24 hours, DNA encoding the HiBiT-tagged target protein (20 ng for 96-well plate; 100 ng for 24-well plate) is transiently transfected into cells. Chimeric protein DNA (100 ng) is either transiently transfected into cells at the same time as HiBiT-target DNA transfection or encapsulated into liposomes and introduced 24 hours into the cells after transfection. Cells are treated with chimeric protein-containing liposomes for 15 minutes before 2 hours of incubation.
  • Nano-Glo® HiBiT Lytic Buffer (LgBiT protein (1:100), Nano-Glo® HiBiT Lytic Substrate (1:50) 1×PBS (1:1)) is added to the cells 24 hours after transient transfection or 2 hours after liposomal treatment. The plates are shaken on an orbital shaker (1,000 rpm, 10 min) to ensure homogenous cell lysis and equilibration of LgBiT and HiBiT in the cell lysate. The luminescence measurements are performed in white Nunclon™ Delta 96-well plates at 25° C. using a CLARIOstar plate reader using a 460-480 emission filter.
  • Determining Properties of a Grafted Scaffold
  • The biophysical properties of a grafted scaffold may be assessed as follows: The molar ellipticity at 222 nm (a measure of helical structure content) is monitored as a function of increasing temperature. A decrease in the molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. This thermal unfolding experiment is used to determine the melting temperature of the scaffold and thereby to assess whether or not the grafting process has had a detrimental effect on the thermostability of the scaffold.
  • An alternative method to determine the thermodynamic stability of the proteins is to measure chemical-induced denaturntion (either guanidine hydrochloride (GdnHCl) or urea) monitored by intrinsic protein fluorescence (tryptophan and tyrosine residues). Solutions are dispensed into Corning® 96-well, half-area, black polystyrene plates (CLS3993) with a Microlab ML510B dispenser (Hamilton) and measurements are carried out on a CLARIOstar Plate Reader (BMG Labtech). The buffer is added first into the wells, followed by 15 μl aliquots of protein stock. A stock solution of chemical denaturant (either 7 M GdnHCl or 9 M urea) is then dispensed into the wells to create a chemical-denaturant concentration gradient.
  • Preparation of a Helix-Grafted Scaffold that Binds to a Target Protein
  • First, the helix of a given protein that interacts with its target binding partner is mapped onto the heptad distribution, and the stapled side of the peptide is set so as to form the hydrophobic interface with the rest of the scaffold protein. The grafted scaffold may then be docked against the target protein using Haddock software (de Vries & Bonvin 2011; de Vries et al. 2010). Haddock is a data-driven docking algorithm that uses known information about the interaction for its calculations. The active (primary interaction residues) and the passive (5 Å proximity to active) residues are extracted and inputted into the calculations. Docking is not necessary to validate helical grafted scaffold, and inspection of the structure of the helix-target protein structure and of the scaffold structure may be sufficient: The geometry of alpha-helices permits selection of amino acid positions of the scaffold that accommodate outward facing target binding residues of the peptide ligand.
  • Preparation of a Scaffold Scaffold with a Singe Binding Function Grafted onto an Inter-Repeat Loop
  • First, a peptide ligand that binds to a given target protein is grafted onto the scaffold in a loop. Binding of the loop-grafted scaffold may be tested using ITC. ITC is particularly useful to assess these interactions, as it can measure the stoichiometry (n) of the interaction, and thus inform as to which loops (if there is more than one loop) are more or less accessible to the target protein, and can inform as to whether a multi-loop scaffold affords multivalency. An advantage of a multivalent grafted scaffold is that one may achieve an avidity effect. This is particularly useful where a target molecule has multiple domains that can be bound by a peptide ligand. Binding of a multivalent grafted scaffold to such a target protein would produce an increased binding affinity and a decreased off rate according to the number of repeats in the grafted scaffold, thus achieving an avidity effect.
  • Introducing Multivalency into a Single Binding Function Scaffold
  • The function of a multi-valent grafted scaffold containing variable numbers of the peptide ligand binding motif that binds to a given target protein can be tested using the same assays as for the mono-valent grafted scaffold. The results are used to assess whether increased potency can be achieved by increasing the valency.
  • Preparing a Loop-Grafted Scaffold Using a Peptide Ligand that Binds to an E3 Ubiquitin Ligase
  • A peptide ligand that is known to bind the substrate recognition subunit of an E3 ligase (see Table 3 for such peptides and ligases) is inserted into the scaffold loop, Immunoprecipitation is used to confirm binding of the grafted scaffold to the E3 ligase. ITC analysis is used to assess the affinity of the interaction.
  • Preparation of Hetero-Bifunctional Scaffolds that Direct Target Proteins for Ubiquitination and Subsequent Degradation
  • A bispecific grafted scaffold is constructed using a peptide ligand specific for a target protein (see Table 2) and a peptide ligand specific for an E3 ligase.
  • To test whether these bispecific grafted scaffolds are capable of directing the target protein for ubiquitination and degradation, a plasmid encoding the hetero-bifunctional scaffold is transfected into HEK293T cells using Lipofectamine2000 together with HA-tagged β-catenin plasmid (using cells transfected with HA-tagged β-catenin plasmid alone as a control). After 48 hours of transfection, the cells are lysed, the sample is boiled and proteins are resolved by SDS-PAGE and immunoblotting is performed using anti-HA and anti-actin antibodies. Changes in target protein levels are evaluated by the densitometry of the bands corresponding to HA-target protein normalised to actin levels. In this way, different combinations of target protein binding peptides and E3 ligase peptide ligands can be compared for their abilities to reduce the levels of target protein.
  • Delivering a Grafted Scaffold Protein into Cells
  • A grafted scaffold protein is encapsulated within fusogenic liposomes made from cationic, neutral, and aromatic lipids, and then delivered into cells. Empty liposomes and liposomes encapsulating grafted scaffolds have been determined to be non-toxic to cells.
  • Libraries
  • Chimeric proteins as described herein may be used to produce libraries. For example, where a given chimeric protein (grafted scaffold) is demonstrated to binds bispecifically to a target protein and to an E3 ligase may be further optimized by changing amino acid residues of the grafted scaffold and selecting for stronger or weaker binders.
  • Chimeric proteins which are demonstrated to bind may be further engineered to improve an activity or property or introduce a new activity or property, for example a binding property such as affinity and/or specificity, an in vivo property such as solubility, plasma stability, or cell penetration, or an activity such as increased neutralization of the target molecule and/or modulation of a specific activity of the target molecule or an analytical property. Chimeric proteins may also be engineered to improve stability, solubility or expression level.
  • Alternatively, a library may be used to screen in order to identify and isolate chimeric proteins with specific binding activity.
  • A library may comprise chimeric proteins, each chimeric protein in the library comprising:
      • (i) two or more repeat domains,
      • (ii) inter-repeat loops linking the repeat domains; and
      • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
      • wherein at least one amino acid residue in the peptide ligands in the library is diverse.
  • The residues at one or more positions in the peptide ligand of the chimeric proteins in the library may be diverse or randomised i.e. the residue located at the one or more positions may be different in different molecules in a population.
  • For example, 1 to 12 positions within a helical peptide ligand at the N or C terminus of the chimeric proteins in the library may be diverse or randomised. In addition, the non-constrained Xn sequence of the peptide ligand may contain additional diversity. Alternatively or additionally, 1 to n positions within an inter-repeat peptide ligand of the chimeric proteins in the library may be diverse or randomised, where n is the number of amino acids in the peptide ligand.
  • In some embodiments, peptide ligands may be screened individually and a chimeric protein progressively assembled from repeat domains comprising peptide ligands identified in different rounds of screening. For example, a library may comprise chimeric proteins, each chimeric protein in the library comprising:
      • (i) two or more repeat domains,
      • (ii) inter-repeat loops linking the repeat domains; and
      • (iii) one or more constant peptide ligands having the same amino acid sequence in each chimeric protein in the library and one or more diverse peptide ligands, preferably one diverse peptide ligand, having a different amino acid sequence in each chimeric protein in the library,
      • the peptide ligands being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
  • At least one amino acid residue in the diverse peptide ligands in the library may be diverse.
  • A library may be produced by a method comprising:
      • (a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
        • (i) two or more repeat domains,
        • (ii) inter-repeat loops linking the two or more repeat domains; and
        • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein, wherein one or more residues of a peptide ligand in each chimeric protein is diverse in the library, and
      • (b) expressing the population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.
  • The population of nucleic acids may be provided by a method comprising inserting a first population of nucleic acids encoding a diverse peptide ligand into a second population of nucleic acids encoding the two or more repeat domains linked by inter-repeat loops, optionally wherein the first and second nucleic acids are linked with a third population of nucleic acids encoding linkers of up to 10 amino acids.
  • The nucleic acids may be contained in vectors, for example expression vectors. Suitable vectors include phage-based or phagemid-based phage display vectors.
  • The nucleic acids may be recombinantly expressed in a cell or in solution using a cell-free in vitro translation system such as a ribosome, to generate the library. In some preferred embodiments, the library is expressed in a system in which the function of the chimeric protein enables isolation of its encoding nucleic acid. For example, the chimeric protein may be displayed on a particle or molecular complex to enable selection and/or screening. In some embodiments, the library of chimeric proteins may be displayed on beads, cell-free ribosomes, bacteriophage, prokaryotic cells or eukaryotic cells. Alternatively, the encoded chimeric protein may be presented within an emulsion where activity of the chimeric protein causes an identifiable change. Alternatively, the encoded chimeric protein may be expressed within or in proximity of a cell where activity of the chimeric protein causes a phenotypic change or changes in the expression of a reporter gene.
  • Preferably, the nucleic acids are expressed in a prokaryotic cell, such as E coli. For example, the nucleic acids may be expressed in a prokaryotic cell to generate a library of recombine binding proteins that is displayed on the surface of bacteriophage. Suitable prokaryotic phage display systems are well known in the art, and are described for example in Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN: 3540413545, WO92/01047, U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404. Phage display systems allow the production of large libraries, for example libraries with 108 or more, 109 or more, or 1010 or more members.
  • In other embodiments, the cell may be a eukaryotic cell, such as a yeast, insect, plant or mammalian cell.
  • A diverse sequence as described herein is a sequence which varies between the members of a population i.e. the sequence is different in different members of the population. A diverse sequence may be random i.e. the identity of the amino acid or nucleotide at each position in the diverse sequence may be randomly selected from the complete set of naturally occurring amino acids or nucleotides or a sub-set thereof. Diversity may be introduced into the peptide ligand using approaches known to those skilled in the art, such as oligonucleotide-directed mutagenesis22 , Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein).
  • Diverse sequences may be contiguous or may be distributed within the peptide ligand. Suitable methods for introducing diverse sequences into peptide ligand are well-described in the art and include oligonucleotide-directed mutagenesis (see Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein). For example, diversification may be generated using oligonucleotide mixes created using partial or complete randomisation of nucleotides or created using codons mixtures, for example using trinucleotides. Alternatively, a population of diverse oligonucleotides may be synthesised using high throughput gene synthesis methods and combined to create a precisely defined and controlled population of peptide ligands. Alternatively, “doping” techniques in which the original nucleotide predominates with alternative nucleotide(s) present at lower frequency may be used.
  • Preferably, the library is a display library. The chimeric proteins in the library may be displayed on the surface of particles, or molecular complexes such as beads, for example, plastic or resin beads, ribosomes, cells or viruses, including replicable genetic packages, such as yeast, bacteria or bacteriophage (e.g. Fd, M13 or T7) particles, viruses, cells, including mammalian cells, or covalent, ribosomal or other in vitro display systems. Techniques for the production of display libraries, such as phage display libraries are well known in the art. Each particle or molecular complex may comprise nucleic acid that encodes the chimeric protein that is displayed by the particle.
  • In some preferred embodiments, the chimeric proteins in the library are displayed on the surface of a viral particle such as a bacteriophage. Each chimeric protein in the library may further comprise a phage coat protein to facilitate display. Each viral particle may comprise nucleic acid encoding the chimeric protein displayed on the particle. Suitable viral particles include bacteriophage, for example filamentous bacteriophage such as M13 and Fd.
  • Suitable methods for the generation and screening of phage display libraries are well known in the art. Phage display is described for example in WO92/01047 and US patents U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404.
  • Libraries as described herein may be screened for chimeric proteins which display binding activity, for example binding to a target molecule. Binding may be measured directly or may be measured indirectly through agonistic or antagonistic effects resulting from binding. A method of screening may comprise;
      • (a) providing a library of chimeric proteins, each chimeric protein in the library comprising;
        • (i) two or more repeat domains,
        • (ii) inter-repeat loops linking the repeat domains; and
        • (iii) one or more peptide ligands, each the peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein,
        • wherein one or more residues of the one or more peptide ligands are diverse in the library,
      • (b) screening the library for chimeric proteins which display a binding activity, and
      • (c) identifying one or more chimeric proteins in the library which display the binding activity.
  • In some embodiments, the chimeric proteins in the library may comprise one peptide ligand with at least one diverse amino acid residue. Conveniently the chimeric proteins in the library comprise two repeat domains. The library may be screened for peptide ligands that bind to a target molecule. Peptide ligands identified in this fashion can be assembled in a modular fashion to generate a chimeric protein as described herein that is multi-specific.
  • For example, a first library may be screened for a first peptide ligand that binds to a first target molecule and a second library may be screened for a second peptide ligand that binds to a second target molecule. The first and second peptide ligands are in different locations in the chimeric protein i.e. they are not both N terminal peptide ligands, C terminal peptide ligands or inter-repeat peptide ligands. First and second peptide ligands that bind to the first and second target molecules, respectively, are identified from the first and second libraries. The identified first and second peptide ligands may then be incorporated into a chimeric protein that binds to the first and second target molecules.
  • A first library may comprise chimeric proteins in the library with a first diverse peptide ligand having at least one diverse amino acid residue. A first peptide ligand that binds to a target molecule may be identified from the first library. Chimeric proteins comprising the first peptide ligand may be used to generate a second library comprising a second diverse peptide ligand having at least one diverse amino acid residue. For example, the chimeric protein from the first library may be modified by addition of a second diverse peptide ligand at the N or C terminal or by the addition of additional repeat domains comprising the second diverse peptide ligand in an inter-repeat loop. A second peptide ligand that binds to the same or a different target molecule may be identified from the second library. Chimeric proteins comprising the first and second peptide ligands may be used to generate a third library comprising a third diverse peptide ligand having at least one diverse amino acid residue. For example, the chimeric protein from the second library may be modified by addition of a third diverse peptide ligand at the N or C terminal or by the addition of additional repeat domains comprising the third diverse peptide ligand in an inter-repeat loop. A third peptide ligand that binds to the same target molecule as the first and/or second peptide ligands or a different target molecule may be identified from the third library. In this way, a chimeric protein containing multiple peptide ligands may be sequentially assembled (see FIG. 16).
  • The use of separate libraries for each peptide ligand allows large numbers of different variants of each peptide ligand to be screened independently and then combined. For example, a phage library of 108-1012 first peptide ligand variants may be combined with a phage library of 108-1012 second peptide ligand variants and a phage library of 108-1012 third peptide ligand variants. In some embodiments, a phage library of 108-1012 N terminal peptide ligand variants may be combined with a phage library of 108-1012 C terminal peptide ligand variants to generate a chimeric protein with N and C terminal peptide ligands.
  • Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cell phenotype. The one or more identified or selected chimeric proteins may be recovered and subjected to further selection and/or screening.
  • In other embodiments, the chimeric proteins in the library may comprise a first peptide ligand for a first target molecule, which has at least one diverse amino acid residue, and a second peptide ligand for a second target molecule, which has at least one diverse amino acid residue. The library may be screened for peptide ligands that bind to the first and second target molecules. For example, the library may be screened for chimeric proteins comprising a first peptide ligand that binds to a first target molecule and a second peptide ligand that binds to a second target molecule.
  • Screening a library for binding activity may comprise providing a target molecule and identifying or selecting members of the library that bind to the target, or expressing the library in a population of cells and identifying or selecting members of the library that elicit a cell phenotype. The one or more identified or selected chimeric protein may be recovered and subjected to further selection and/or screening.
  • Chimeric proteins as described herein may be used to produce libraries comprising different combinations of peptide ligands grafted into an scaffold. The combinations of ligands may comprise first peptide ligands that bind to a members of a protein degradation pathway, such as an E3 ubiquitin ligase, and second peptide ligands that bind to a target molecule. A library may be screened in order to identify and isolate chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule, (ii) causing degradation of the target molecule in a cell through the protein degradation pathway.
  • A library may comprise chimeric proteins, each chimeric protein in the library comprising:
  • (i) a scaffold;
  • (ii) a first peptide ligand for a member of a protein degradation pathway and
  • (iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain.
  • wherein different chimeric proteins in the library comprise different first peptide ligands for different members of the protein degradation pathway and different second peptide ligands for the target molecule, the chimeric proteins in the library comprising different combinations of the first and second peptide ligands.
  • Suitable chimeric proteins, target molecules and members of protein degradation pathways and examples of peptide ligands thereto are described elsewhere herein.
  • Preferably, the member of a protein degradation pathway is an E3 ubiquitin ligase. For example, each chimeric protein in a library of chimeric proteins may comprise:
  • (i) a scaffold;
  • (ii) a first peptide ligand for an E3 ubiquitin ligase and
  • (iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,
  • wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands.
  • Different chimeric proteins in the library may comprise a peptide ligand for a different E3 ubiquitin ligase. For example, the chimeric proteins in the library may comprise peptide ligands for a panel of E3 ubiquitin ligases, each chimeric protein in the library comprising a peptide ligand for one of the E3 ubiquitin ligases in the panel.
  • Numerous E3 ubiquitin ligases are known in the art. A suitable panel of E3 ubiquitin ligases may for example, comprise two, three, four, five or more of Mdm2, SCF (Skp2), Cul3-Keap1, Cul3-SPOP, APC/C, SIAH, SCFFbw7, SCFFbw8, Cul4-DDB1-Cdt2, DDB1-Cul4, DDB1-Cul5, SOCS box-Cul5-SPSB2, SOCS box-Cul5-SPSB4, CHIP, CRL4 (COPI/DET), UBRS, CRL2 (KLHDC2), GID4, TRIM21, Nedd4, Elongin C and p-TRP. Examples of peptide ligands for E3 ubiquitin ligases are shown in Table 3.
  • The target molecule may be a target molecule as described above, for example, 1-catenin, KRAS, or myc. The chimeric proteins in the library may comprise different peptide ligands for the target molecule i.e. different chimeric proteins in the library may comprise different peptide ligands for the same target molecule. Each chimeric protein in the library may comprise a different peptide ligand for the target molecule. Examples of peptide ligands target molecules are shown in Table 3. For example, the target molecule may be f-catenin, KRAS, or myc and the chimeric proteins in the library may comprise different peptide ligands for β-catenin, KRAS, or myc, respectively. Examples of different peptide ligands for β-catenin, KRAS, and myc are shown in Table 3.
  • A method of screening a library of chimeric proteins may comprise;
  • (a) providing a library of chimeric proteins, each chimeric protein in the library comprising:
  • (i) a scaffold;
  • (ii) a first peptide ligand for a member of a protein degradation pathway and
  • (iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,
  • wherein the chimeric proteins in the library comprise first peptide ligands for different members of a protein degradation pathway and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands,
  • (b) screening the library for chimeric proteins which display an activity selected from (i) binding to the member of a protein degradation pathway and the target molecule and (ii) causing degradation of the target molecule in a cell through the protein degradation pathway,
  • (c) identifying one or more chimeric proteins in the library which display the activity.
  • In some embodiments, the member of a protein degradation pathway may be an E3 ubiquitin ligase. A method of screening a library of chimeric proteins may comprise;
  • (a) providing a library of chimeric proteins, each chimeric protein in the library comprising:
  • (i) a scaffold;
  • (ii) a first peptide ligand for an E3 ubiquitin ligase and
  • (iii) a second peptide ligand for a target molecule, the peptide ligands being located at and of the scaffold of the chimeric domain,
  • wherein the chimeric proteins in the library comprise first peptide ligands for different E3 ubiquitin ligases and different second peptide ligands for the target molecule, the chimeric proteins comprising different combinations of the first and second peptide ligands,
  • (b) screening the library for chimeric proteins which display an activity selected from (i) binding to an E3 ubiquitin ligase and the target molecule, (ii) causing ubiquitination of the target molecule by an E3 ubiquitin ligase in a cell and (iii) causing degradation of the target molecule in a cell,
  • (c) identifying one or more chimeric proteins in the library which display the activity.
  • A method may further comprise identifying one or more combinations of first and second peptide ligands in chimeric proteins in the library which display the activity.
  • Determination of Binding of a Chimeric Protein
  • Binding of a chimeric protein may be determined by any suitable technique, described below and in the examples herein.
  • Suitable methods for determining binding of a chimeric protein to a target molecule are well known in the art and include ELISA, bead-based binding assays (e.g. using streptavidin-coated beads in conjunction with biotinylated target molecules, surface plasmon resonance, flow cytometry, Western blotting, immunocytochemistry, immunoprecipitation, and affinity chromatography. Alternatively, biochemical or cell-based assays, such as fluorescence-based or luminescence-based reporter assays may be employed. For example, Isothermal Titration Calorimetry, Cell transfection followed by assaying for expressed chimeric protein, Liposomal formulation and cytotoxicity assays, a dual-Luciferase Reporter Assay System such as TOPFLASH®, and a competition fluorescence polarisation (FP) assay to measure the binding of a chimeric protein to its targets.
  • In some embodiments, binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme,
  • Where a library is in use, the library may be contacted with the target molecule under binding conditions for a time period sufficient for the target molecule to interact with the library and form a binding reaction complex with a least one member thereof. Binding conditions are those conditions compatible with the known natural binding function of the target molecule. Those compatible conditions are buffer, pH and temperature conditions that maintain the biological activity of the target molecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction. Typically, those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the target molecule of interest. The library may be contacted with the target molecule in the form of a heterogeneous or homogeneous admixture. Thus, the members of the library can be in the solid phase with the target molecule present in the liquid phase. Alternatively, the target molecule can be in the solid phase with the members of the library present in the liquid phase. Still further, both the library members and the target molecule can be in the liquid phase.
  • Multiple rounds of panning may be performed in order to identify chimeric proteins which display the binding activity. For example, a population of chimeric proteins enriched for the binding activity may be recovered or isolated from the library and subjected to one or more further rounds of screening for the binding activity to produce one or further enriched populations. Chimeric proteins which display binding activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.
  • In some embodiments, binding may be determined by detecting agonism or antagonism resulting from the binding of a chimeric protein to a target molecule, such as a ligand, receptor or enzyme. For example, the library may be screened by expressing the library in reporter cells and identifying one or more reporter cells with altered gene expression or phenotype. Suitable functional screening techniques for screening recombinant populations of chimeric proteins are well-known in the art.
  • Further rounds of screening may be employed to identify chimeric proteins which display the improved property or activity. For example, a population of chimeric proteins enriched for binding to the target molecule may be recovered or isolated from the library and subjected to one or more further rounds of screening for the improved or new property or activity to produce one or further enriched populations. Optionally, this may be repeated one or more times. Chimeric proteins which display the improved property or activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.
  • A chimeric protein as described herein may be encapsulated in a liposome, for example for delivery into a cell. Preferred liposomes include fusogenic liposomes. Suitable fusogenic liposomes may comprise a cationic lipid, such as 1, 2-dioleoyl-3-trimethylammoniumpropane (DOTAP), and a neutral lipid, such as dioleoylphosphatidylethanolamine (DOPE) for example in a 1:1 (w/w) ratio. Optionally, a liposome may further comprise an aromatic lipid, such as DiO (3, 3′-dioctadecyloxacarbocyanine perchlorate), DiR (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethylindotricarbocyanine iodide), N-(4,4-fluoro-5,7-dimethyl-4-bora-3a,4a-diaza-sindacene-3-propionyl)-1,2-dibexadecanoyl-sn-glycero-3-phosphoethanolamine (triethylammonium salt) (BODIPY FL-DHPE), and 2-(4,4-difluoro-5-methyl-4-bora-3a,4a-diazas-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-C12HPC) for example in a 0.1:1:1 (w/w) ratio relative to the neutral and cationic lipid. Suitable techniques for the encapsulation of proteins in liposomes and their delivery into cells are established in the art (s for example, Kube et al Langmuir (2017) 33 1051-1059; Kolašinac et al (2018) Int. J. Mol. Sci. 19 346).
  • A method described herein may comprise admixing a chimeric protein or encoding nucleic acid as described herein with a solution of lipids, for example in an organic solvent, such as chloroform, and evaporating the solvent to produce liposomes encapsulating the chimeric protein. Liposome encapsulations comprising a chimeric protein as described herein are provided as an aspect of the invention.
  • A chimeric protein or encoding nucleic acid as described herein may be admixed with a pharmaceutically acceptable excipient. A pharmaceutical composition comprising a chimeric protein or nucleic acid as described herein and a pharmaceutically acceptable excipient is provided as an aspect of the invention.
  • The term“pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.
    • Pharmaceutical Compositions and Formulations
  • The pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the chimeric protein into association with a carrier which may constitute one or more accessory ingredients. In general, pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Pharmaceutical compositions may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.
  • Dosage and Mode of Administration
  • A chimeric protein, encoding nucleic acid or pharmaceutical composition comprising the chimeric protein or encoding nucleic acid may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.
  • Pharmaceutical compositions suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.
  • Pharmaceutical compositions suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to cells, tissue or organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example, from about 10 ng/ml to about 1 μg/mL The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • It will be appreciated that appropriate dosages of the chimeric protein, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of diagnostic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the imaging agent, the amount of contrast required, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of imaging agent and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the imaging agent at a site, such as a tumour, a tissue of interest or the whole body, which allow for imaging without causing substantial harmful or deleterious side-effects.
  • Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.
  • Chimeric proteins described herein may be used in methods of diagnosis or treatment in human or animal subjects, e.g. human. Chimeric proteins for a target molecule may be used to treat disorders associated with the target molecule.
  • Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.
  • It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.
  • Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.
  • All documents and sequence database entries mentioned in this specification are incorporated herein by reference in their entirety for all purposes.
  • “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.
  • Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.
    • Experiments
  • 1. Methods
  • 1.1 Large-Scale Protein Purification (his-Tamed) from E. coli
  • The pRSET B (His-tag) constructs were transformed into chemically competent E. coli C41 cells by heat shock and plated on LB-Amp plates. Colonies were grown in 2TY media containing ampicillin (50 micrograms/mL) at 37° C., 220 rpm until the optical density (O.D.) at 600 nm reached 0.6. Cultures were then induced with IPTG (0.5 mM) for 16-20 h at 20° C. or 4 h at 37′C. Cells were pelleted by centrifugation at 3000 g (4′C, 10 min) and resuspended in lysis buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl, 1 tablet of SIGMAFAST protease inhibitor cocktail (EDTA-free per 100 mL of solution), then lysed on a Emulsiflex C5 homogenizer at 15000 psi. Cell debris was pelleted by centrifugation at 15,000 g at 4° C. for 45 min. Ni-NTA beads 50% bed volume (GE Healthcare)(5 mL) were washed once with phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) before the supernatant of the cell lysate was bound to them for 1 hr at 4° C. in batch. The loaded beads were washed three times with phosphate buffer (40 mL) containing 30 mM of imidazole to prevent non-specific interaction of lysate proteins with the beads. Samples were eluted using phosphate buffer with 300 mM imidazole, and purified by size-exclusion chromatography using a HiLoad 16/60 SuperdexG75 column (GE Life-Science) pre-equilibrated in phosphate buffer (10 mM sodium phosphate, pH 7.4, 150 mM NaCl) and proteins separated in isocratic conditions, Purity was checked on NuPage protein gel (Invitrogen), and fractions found to be over 95% pure were pooled. Purified protein was flash-frozen and stored at −80° C. until further use. Concentrations were determined by measuring absorbance at 280 nm and using a calculated extinction coefficient from ExPASy ProtParam (Gasteiger et al. 2005) for each variant. Molecular weight and purity was confirmed using mass spectrometry (MALDI.
  • 1.2 Large-Scale Protein Purification (Heat Treatment from E. coli
  • All chimeric proteins described herein are thermally very stable, with melting temperatures above 80° C. This means that the chimeric proteins could be separated from E. coli proteins by incubating the cell lysates at 65° C. for 20 min. Very few of the E. coli proteins survive such temperatures, and therefore, they will unfold and aggregate. Aggregated proteins were removed by centrifugation, leaving 80-90% pure sample of the desired protein. All our constructs folded reversibly, and therefore could be further purified by methods such as acetone or salt precipitation to remove DNA and other contaminants.
  • This approach allowed the production of large amounts of functional proteins without expensive affinity purification methods such as antibodies or His tags and is scalable to industrial production and bioreactors.
  • 1.3 Small-Scale Purification of His-Tagged Proteins for Higher-Throughput Testing
  • Plasmids were transformed into E. coli C41 cells and plated overnight. 15 mis of 2TY medium (Roche) containing 50 micrograms/ml ampicillin was placed in multiple 50 ml tubes. Several colonies were picked and resuspended in each 15 ml culture. For sufficient aeration it is important to only loosely tighten the lids of the 50 ml tubes. Cells were grown at 37° C. until OD600 of 0.6 and then induced with 0.5 mM IPTG overnight. Cells were pelleted at 3000 g (Eppendorf Centrifuge 5804) and then resuspended in 1 ml of BugBuster® cell lysis reagent. Alternatively, sonication in combination with lysozyme and DNAse I treatment was used. The lysate was spun at 12000 g for 1 minute to pellet any insoluble protein and cell debris.
  • The supernatant was added to 100 μl bed volume of pro-washed Ni-NTA agarose beads. The subsequent affinity purification was performed in batch, by washing the beads 4 times with 1 ml of buffer each time (alternatively, Qiagen Ni-NTA Spin Columns can be used). The first wash contained 10% BugBuster® solution and 30 mM imidazole in the chosen buffer. Here we used 50 mM sodium phosphate buffer pH 6.8, 150 mM NaCl. The three successive washes had 30 mM of imidazole in the chosen buffer. Beads were washed thoroughly to remove the detergent present in the BugBuster® solution. Protein was eluted from the beads in a single step using 1 ml of chosen buffer containing 300 mM imidazole. The combination of Bugbuster® and imidazole and the repeat washes in small bead volumes yielded >95% pure protein. Imidazole was removed using a NAP-5 disposable gel-filtration column (GE Healthcare).
  • 1.4 Competition Fluorescence Polarization (FP)
  • To assay the binding of the designed SOS-TPR protein to KRAS, Competition FP was performed using purified KRAS Q61H mutant and (2′-(or-3′)-O-(N-Methylandaniloyl) Guanosine 5′-Triphosphate, a fluorescent version of GTP, also known as mant-GTP. SOS-TPR was titrated using a 2-fold serial dilution against a 1:1 complex of KRAS Q61H and mant-GTP (1 μM) in a black 96-wellplate (CLS3993SIGMA). Plates were prepared under reduced light conditions and incubated at room temperature. Readings were taken on the CLARIOstar microplate reader, using an excitation filter at 360 nm and emission filter at 440 nm.
  • 1.5 Isothermal Titration Calorimetry (TC)
  • ITC was performed at 25° C. using a VP-ITC (Microcal). 1TBP-CTPR2, 2TBP-CTPR4, 3TBP-CTPR6 and TNKS2 ARC4 were dialysed into 10 mM sodium phosphate buffer pH 7.4, 150 mM NaCl, 0.5 mM TCEP. Dialysed TNKS2 ARC4 (200 μM) was titrated into the sample cell containing 1TBP-CTPR2 at 20 μM. Similar experiments were performed for 2TBP-CTPR4 and 3TBP-CTPR6. Injections of TNKS2 ARC4 into the cell were initiated with a 5 μL injection, followed by 29 injections of 10 μL. The reference power was set at 15 μCal/s with an initial delay of 1000 s and a stirring speed of 485 rpm. Data were fitted using the instrument software a one-site binding model.
  • 1.6 Cell Culture
  • HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium (Sigma Aldrich) supplemented with 10/fetal bovine serum and penicillin/streptomycin (LifeTech) at 37° C. with 5% CO2 air supply.
  • 1.7 Cell Transfection
  • HEK293T were seeded in 6-well tissue culture plates (500,000 cells per well) and transfected the next day using the Lipofectamine2000 transfection reagent (Invitrogen) according to the manufacturer's protocol.
  • 1.8 β-Catenin Levels Western Blot Assay
  • HA-β-catenin (1 μg) alone and with various PROTACs (1 μg) was transfected in HEK293T cells in 6-well plates using Lipofectamine2000. After 48 hours of transfection, the cells were lysed in 200 μL of Laemmli buffer. After sample was boiled at 95° C. for 20 min proteins were resolved by SDS-PAGE and transferred to a PVDF membrane, and immunoblotting was performed using anti-HA (C29F4, Cell Signaling Technologies) and anti-actin (A2066, Sigma-Aldrich) antibodies. Changes in β-catenin levels were evaluated by the densitometry of the bands corresponding to HA-β-catenin normalised to actin levels using ImageJ.
  • 1.9 Liposomal Formulation and Cytotoxicity Assay
  • To make liposomal formulations of proteins (LFP), lipids (DOTAP (cationic): DOPE (neutral): DiR (aromatic)=1:1:0.1 w/w) were dissolved in chloroform, and solvent was evaporated under vacuum overnight. Resulting mixed lipid cake was hydrated with 10 mM HEPES pH 7.4, containing 27 μM protein, so that the total lipid concentration is 4 mg/ml. This mixture was vortexed for 2 minutes and then sonicated for 20 minutes at room temperature. Liposomes encapsulating proteins were stored at 4° C. until further use. To make empty liposomes (EL, empty liposomes without proteins), lipid cake was hydrated with 10 mM HEPES pH 7.4 without proteins.
  • An ATP assay was used to investigate whether there is any cytotoxicity associated with EL and LFP. In a typical procedure, 2×10HEK 293T cells/well in 500 μL of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum were grown for 24 hours in a 24-well cell culture plate. Cells were incubated with liposome (EL/LFP)-media (DMEM without FBS) mix, having different volumes (0-60 μL) of EL and LFP, for 15 minutes at 37° C. After washing twice with 1×PBS, 500 μL of CellTiter-Glo® Reagent (Promega) was added and luminescence was measured using a microplate reader as par the manufacture's protocol. Untreated cells were used as control. Data were obtained from triplicate samples, and the standard deviations were calculated from two independent experiments.
  • 1.10 TOPFLASH Assay
  • The Wt pathway was activated by treating HEK293T cells with Wnt-conditioned media obtained from L-cells expressing Wnt3A for 8 days. To perform the assay, 105 HEK293T cells/well were seeded on a 24-well plate Nunclon Delta Surface plate (NUNC) and incubated overnight at 37° C., 5% CO2. The following day, cells were transfected with 100 ng of TOPflash TCF7L2-firefly luciferase plasmid, 10 ng of CMV-Renilla plasmid (as internal control) and 100 ng of the corresponding TPR construct. Plasmids were mixed with 0.5 μL of Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (invitrogen). Transfected cells were allowed to recover for 8 h, then they were treated with Wnt-conditioned media (1:2 final concentration) for a further 16 h. The TOPflash assay was performed using the Dual-Luciferase Reporter Assay System (Promega) (Korinek et al., 1997 Science 275(5307):1784-7) following the manufacturer's instructions. The activities of firefly and Renilla luciferases were measured sequentially from a single sample, using the CLARIOstar plate reader. Relative luciferase values were obtained from triplicate samples dividing the firefly luminescence activity by the CMV-induced Renilla activity, and standard deviation was calculated.
  • 1.11 TOPFLASH Assay Using Liposome Encapsulation to Deliver Designed TPR Proteins into the Cell
  • 105 HEK 293T cells in 500 μL of Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum were grown overnight in each well of a 24-well cell culture plate. For TOPFLASH reporter assays, 100 ng/well of TOPFLASH plasmid and ng/well of CMV-Renilla plasmid (as internal control) were used to transfect cells in 24-well plates. Cells were transfected with the Lipofectamine 2000 transfection reagent according to the manufacturer's protocol (Invitrogen). Transfected cells were allowed to recover for 8 hours, and Wnt signalling was activated by addition of Wnt3A-conditioned media obtained from L-cells. 16 hours post Wnt pathway activation, proteins were delivered into the cells by liposomal treatment. Cells were incubated with liposome (LFP)-media (DMEM without FBS) mix for 15 minutes at 37° C. followed by one PBS wash. Wnt3A conditioned media was replaced and cells were incubated for variable time durations (2-8 hours). Following incubation, TOPFLASH assays were performed using the Dual-Luciferase Reporter Assay System (Promega)(Korinek et al., 1997) following the manufacturer's instructions. Relative luciferase values were obtained from triplicate samples (from two independent experiments) by dividing the firefly luciferase values (from TOPFLASH) by the Renilla luciferase values (from CMV renilla), and standard deviations were calculated.
  • 1.12. Competition Fluorescence Polarisation (F) Assay to Measure the Binding of Designed Nrf-TPR Proteins to Keap1
  • To measure the binding of the designed Nrf-TPR proteins to Keap1, Competition FP was performed using 384-well black opaque optiplate microplates and a CLARIOstar microplate reader. Nrf-TPR proteins were titrated into a solution containing a mixture of FITC-labelled Nrf2 peptide and Keap1 protein. The prepared plates were incubated for 30 minutes at room temperature before readings were take.
  • 2. Results
  • Tetratricopeptide repeat (TPR) is a 34-residue motif that can be repeated in tandem to generate modular proteins. TPRs are used here as an example of helix-turn-helix tandem-repeats arrays, but any tandem repeat array may be used.
  • RTPR proteins comprising TPRs were derived from the consensus TPR sequence (CTPR). Two repeats were found to be sufficient to generate a highly stable mini-protein of 68 amino acids (RTPR2). The biophysical properties of two types of engineering strategy; loop insertions and terminal helix grafting, were assessed. The molar ellipticity at 222 nm (a measure of helical secondary structure content) of three different RTPR modules was monitored as a function of increasing temperature. A decrease in the absolute molar ellipticity with increasing temperature indicates a loss of structure and the unfolding of the protein. Even at the highest temperature recorded (85° C.), the RTPR2 protein without insertion was not fully denatured (FIG. 1). RTPR2 with a 20-residue unstructured loop between the two repeats showed a small shift to a lower melting temperature (FIG. 1), but the protein remains fully folded up to 55° C. This is well above physiologically relevant temperatures. RTPR2 with an additional N-terminal helix showed an increase in absolute molar ellipticity, indicating that the additional helical domain is folded. Moreover, unlike the loop insertion, the helix domain was capable of stabilising the RTPR2 module, shifting the transition midpoint to above 90° C. (FIG. 1). These results showed that the two engineering strategies generated folded and stable modular mini-proteins capable of withstanding high temperatures.
  • A key feature of the TPR scaffold was its modular nature. This modularity allowed display any number of binding modules in tandem to obtain bi- and multi-valent and multi-functional molecules against one, two or more targets. The stability of these proteins was shown to be modular. The stabilities of proteins comprising TBP-CTPR2 (a two-repeat CTPR with a loop insertion that binds to the protein tankyrase (Guettler et al. 2011)) repeated in tandem were measured. The TBP-CTPR2-containing proteins had two, four, six, and eight repeats, and they displayed one, two, three and four binding loops, respectively. The helical content of the proteins, monitored by molar ellipticity at 222 nm, was found to increase in proportion to the number of repeats, as did the stability, indicating that they were behaving like classic helical repeat proteins (FIG. 2). These results demonstrate that bi- or multi-functional chimeric proteins have a high thermostability.
  • 2.1. Demonstration of Proteins with a Single Binding Function Grafted onto an Alpha-Helix
  • 2.1.1 SOS1-TPR, a Helix-Grafted Binding Module Designed to Bind to Oncoprotein KRAS
  • First, we mapped the helix of SOS1 that interacts with KRAS (Margarit et al. 2003 Cell 112 5 685-695) onto the heptad distribution. We matched the heptad positions with the stapled SOS1 helical peptide produced by Leshchiner et al. (PNAS 2015 112 (6) 1761-1766) and set the stapled side of the peptide to form the hydrophobic interface with the rest of the TPR protein (FIG. 3A). The length of the helix is important. An N-terminal solvating CTPR helix ends in the sequence DPNN, which forms a short loop that leads into the next repeat. CTPR-mediated “stapling” (constraining) of binding helices therefore occurred through residues Tyr (i)-Ile (i+4)-Tyr (i+7)-Leu (i+11), fully stapling a 15-residue helix.
  • We created a hydrophobic interface between the grafted helix and the adjacent repeat and allowed the formation of the DPNN loop at the C-terminal end of the grafted helix. We then grafted the final sequence onto the crystal structure of a CTPR B helix for further validation of the interaction. Our designed KRAS-binding protein, SOS1-TPR, was docked against KRAS using the Haddock software (de Vries & Bonvin 2011; de Vries et al. 2010). Haddock is a data-driven docking algorithm that uses known information about the interaction for its calculations. The crystal structure of SOS1-KRAS (PDB: INVU) (Margarit et al. 2003) was originally used to design the stapled peptide. The active (primary interaction residues) and the passive (5 Å proximity to active) residues were extracted and inputted into the calculations.
  • Docking is not necessary to validate helical grafted scaffold. The geometry of α-helices permits selection of amino acid positions of the scaffold that accommodate outward facing target binding residues of the peptide ligand. TPR repeat scaffolds are exceptional for display of binding helices, as they grow linearly in the opposite direction of the helix, thereby avoiding steric clashes with the target protein.
  • KRAS binding of the grafted scaffold can be assessed using the change in fluorescence polarisation of mant-GTP (2′-/3′-O-(N′-Methylanthraniloyl) guanosine-5′-O-triphosphate), a fluorescent analog of GTP (FIG. 3B). The fluorescence of mant-GTP is dependent on the hydrophobicity of its environment (excitation at 360 nm, emission at 440 nm). An increase in fluorescence intensity and fluorescence polarization was observed previously upon binding to KRAS (Leshchiner et al. 2015). SOS-TPR2 was then titrated into the preformed mant-GTP-KRAS complex. There was a clear decrease in polarisation with increasing concentrations of SOS-TPR2, indicating displacement of mant-GTP upon binding of SOS-TRP2 to KRAS (FIG. 3B). Fitting the data gave an EC50 of 3.4 μM. In contrast, a blank protein, CTPR3, had no effect on the fluorescence polarisation.
  • 2.1.2 p53-TR, a Helix-Granted Binding Module Designed to Bind to Mdm2
  • Many degrons (region within the substrate that is recognized by the E3 ubiquitin ligase) are unstructured. However, p53 binds to the Mdm2 E3 through an alpha helix (FIG. 4A). Stapled versions of the p53 helix, as well as circular peptides and grafted coiled coils, have been developed by many groups, and the sequences have been optimised to give nanomolar affinities in some cases (se for example, Ji et al 2013; Lee et al 2014; Kritzer et al. 2006). The p53 helix has a favourable geometry to be grafted onto the C-terminal solvating helix of the CTPR scaffold, and moreover the two helices have 30% sequence identity.
  • Proof of binding of p53-CTPR2 to Mdm2 (N-terminal domain) was obtained using isothermal titration calorimetry (ITC). Mdm2 was titrated into a solution containing 10 μM of p53-TPR2. ITC measures the heat released upon binding. A high-affinity interaction was observed with a dissociation constant of approximately 50 nM (FIG. 4B).
  • 2.2. Demonstration of proteins with a single Binding Function Grafted onto an Inter-Repeat Loop
  • 2.2.1 TPB2-TPR a Loop Module Designed to Bind to Oncoprotein Tankyrase
  • First, we introduced the SLiM “3BP2”, a sequence that binds to the substrate-binding ankyrin-repeat clusters (ARC) of the protein tankyrase, a multi-domain poly ADP-ribose polymerase that is upregulated in many cancers (Guettler et al. 2011) onto the CTPR scaffold. Granting SLiMs in folded domains led to an increase of proteolysis resistance; showing the potential to expand the interaction surface through further rational engineering, in silico methods and/or directed evolution; controlled geometric arrangement; and bi- or multivalency of interactions.
  • We tested the binding of 1TBP-CTPR2, 2TBP-CTPR4 and 3TBP-CTPR6 to the ARC4 domain of tankyrase using ITC (FIG. 5A). This technique is particularly useful for these interactions, as it can measure the stoichiometry (n) of the interaction. We showed that n increased with the number of binding loops, meaning that there were as many tankyrase molecules bound to one TBP-CTPR as loops in the protein. Thus, all loops are accessible to the binding partner. Moreover, the binding affinity increases and the off rate decreases with the number of repeats indicative of an avidity effect. This type of multivalent molecule would be particularly useful for full-length tankyrase, as it has four ARC domains capable of binding the 3BP2 peptide.
  • Multivalency in this system was increased further via oligomerisation of the binding modules by fusing them to the foldon domain of T4 fibritin (FIG. 5B). This trimerisation domain comprises of a C-terminal helix, such as that of p53-CTPR, ending with the foldon domain, a short β-sheet to peptide capable of homo-trimerising. The foldon domain has been shown to be highly stable and independently folded (Boudko et al 2002, Meier et al. 2004). In this way, multiple binding modules can be arranged with specified geometries to inhibit complex multivalent molecules that cannot be targeted with monovalent interactions due to their natural tendency to interact with other multivalent networks with high avidity.
  • 2.2.2 Effect of Introducing Multivalency into a Single Binding Function TPR
  • We tested the function of multi-valent CTPR proteins containing variable numbers of the “3BP2” motif that binds to the protein tankyrase. (1TBP-CTPR2, 2TBP-CTPR4 and 3TBP-CTPR6 etc.). Multi-valency was increased further via oligomerisation of the TPRs by fusing them to the foldon domain of T4 fibritin (1TBP-CTPR2-Foldon, 2TBP-CTPR4-Foldon etc). The inhibitory effect of the TBP-grafted TPRs was assayed using a beta-catenin reporter gene assay (TOPFLASH assay). Increasing the number of functional units increased the inhibitory effect of the proteins, as mentioned using a Wnt signalling assay (FIG. 17).
  • 2.2.3 Skp2-RTPR, A Loop Module Designed to Bind to E3 Ubiquitin Ligase SCF
  • Skp2 is the substrate recognition subunit of the SCFSkp2 ubiquitin ligase. The Skp2-binding sequence that we inserted into the RTPR loop was based on the previously published degron peptide sequence derived from the substrate p27 that binds to Skp2 in complex with Cks1 (an accessory protein) (Hao et al, 2005). We used only 10 residues of this peptide. Although ideally the Skp2-binding sequence would include a phospho-threonine (as this residues makes some key contacts with Skp2 and Cks1), we instead explored whether we could replace the phospho-threonine with a phosphomimetic (glutamate) without affecting binding affinity. We found using co-immunoprecipitation that the resulting p27-TPR protein was able to bind to Skp2 (FIG. 6A) and that it was able to inhibit the ubiquitination of p27 in vitro with a high efficiency indicating a dissociation constant of the order of 30 nM (FIG. 6B). As the peptide adopts a turn-like conformation in its Skp2/Cks1-bound state, constraining it within the RTPR scaffold leads to a large enhancement in binding affinity that outweighs any loss in affinity arising from replacing the phosphothreonine with a phosphomimetic.
  • 2.2.4 Nrf-TPR, a Loop Module Designed to Bind to E3 Ubiquitin Ligase Keap1-Cul3
  • Keap1 is the substrate recognition subunit of the Keap1-Cul3 ubiquitin ligase. A Keap1-binding sequence that we inserted into the CTPR loop was based on the previously published degron peptide sequence derived from the Keap1 substrate Nrf2. We found using co-immunoprecipitation that the resulting Nrf-TPR protein was able to bind to Keap1 (FIG. 7A) and that the interaction had a high affinity in the low nanomolar range as measured by ITC analysis (FIG. 7B).
  • 2.3. Engineering the RTPR Scaffold for Delivery into the Cell
  • Combining our RTPR sequences with an alternative consensus TPR sequence (Parmeggiani et at. 2015) we included additional solvent-exposed Arginine residues, as such ‘resurfacing’ or ‘supercharging’ has been shown previously to facilitate the entry of proteins into cells (Chapman & McNaughton 2016; Thompson et al, 2012). FIG. 8 shows that this approach was successful in delivering a fluorescent-labelled resurfaced TBP-RTPR2 protein into two different cell lines.
  • 2.4. Design of Hetero-Bifunctional TPRs to Direct Proteins for Ubiquitination and Subsequent Degradation
  • The Wnt/β-catenin signalling pathway is deregulated in many cancers and in neurodegenerative diseases, and therefore β-catenin is an important drug target. There are a large number of known binding sequences (both helical and non-helical) for β-catenin that appear suitable for grafting onto the TPR scaffold, and therefore we chose it as the first target for our design of hetero-bifunctional TPRs to induce protein degradation. We selected Mdm2 and SCF6W to test as E3 ubiquitin ligases, as we had successfully generated single-function TPRs to bind to them (FIGS. 4 and 6). We generated structural models of some of the hetero-bifunctional molecules and used these as a crude assessment of whether the resulting presentation of β-catenin to the E3 looked appropriate. We then generated a small library of plasmids encoding proteins comprising three or four TPRs functionalized with different combinations of the β-catenin-binding module and the two E3 ligase-binding modules.
  • We transfected HA-tagged β-catenin plasmid alone or HA-tagged β-catenin plasmid together with one of the various hetero-bifunctional TPR plasmids in HEK293T cells using Lipofectamine2000. After 48 hours of transfection, the cells were lysed, the sample was boiled and proteins were resolved by SDS-PAGE and immunoblotting was performed using anti-HA and anti-actin antibodies, Changes in β-catenin levels were evaluated by the densitometry of the bands corresponding to HA-β-catenin normalised to actin levels (FIG. 9). The results show that a number of the hetero-bifunctional molecules are capable of reducing β-catenin levels by up to 70%. In contrast, neither a blank TPR nor single-function TPRs have any effect on β-catenin levels.
  • A range of different factors contribute to efficient ubiquitination and target degradation by these hetero-bifunctional molecules, hence the power of screening different combinations of single-function modules and potentially also different lengths of intervening blank modules.
  • 2.5 Using a Delivery Vehicle to Introduce the Modular TPR Proteins into Cells
  • We encapsulated the designed TPR proteins within fusogenic liposomes made from cationic, neutral, and aromatic lipids, and we showed that they were thereby delivered into cells (FIGS. 18 and 19). Empty liposomes and liposomes encapsulating TPR proteins are not toxic to the cell (FIG. 20).
  • 2.6 Further Examples of Hetero-Bifunctional TPRs to Direct Proteins for Ubiquitination and Subsequent Degradation
  • TPR proteins were designed to target either tankyrase (FIG. 21. FIG. 22) or KRAS (FIG. 23). TPR proteins targeting tankyrase or were delivered into cells using liposome encapsulation, and the effect on Wnt signalling was assayed using a TOPFLASH assay. The results show that the TPR proteins are able to inhibit Wnt signalling. For KRAS, we transfected KRAS plasmid alone or KRAS plasmid together with one of the TPR plasmids in HEK293T cells using Lipofoectamine2000. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. The results show that the designed hetero-bifunctional TPR is capable of reducing KRAS levels.
  • 2.7 Hetero-Bifunctional TPRs to Direct KRAS for Degradation Via Chaperone-Mediated Autophagy (CMA)
  • Hetero-bifunctional TPR proteins were designed to target endogenous KRAS for degradation via CMA (FIG. 24). TPR constructs or empty vector (light grey) were transiently transfected into either HEK293T or DLD1 (colorectal cancer cell line) using Lipofectamine2000. 24 hours post transfection the cells were lysed, and KRAS levels were evaluated by western blot. The designed hetero-bifunctional TPRs that resulted in reduction of KRAS levels compared to the empty vector control are shown in white.
  • 2.8 Variations in the Linker Sequence Connecting a Peptide Ligand to an Inter-Repeat Loop
  • The linker sequence connecting a peptide ligand to an inter-repeat loop was varied in order to optimise the binding affinity for the target for Nrf-TPR, a TPR protein designed to bind to the protein Keap1 (see FIG. 7). Glycine residues were introduced into the linker to provide flexibility and increased spatial sampling. The introduction of this more flexible linker sequence was found to increase the binding affinity of the Nrf-TPR protein (labelled ‘Flexible’) when compared with the consensus-like linker sequence altering the charge content of the linker sequence (‘labelled ‘Charged’) and altering the conformational properties (based on the predictions of the program CIDER (Holehouse et al. Biophys. J. 112, 16-21 (2017)) of the loop by changing the amino acid composition of the linker sequence (labelled ‘CIDER-optimised’) also affected the Keap1-binding affinity (FIG. 25).
  • TABLE 1
    Degron β-catenin-
    sequence Targeted binding
    Targeted derived protein for sequence
    Ubiquitin Ligase from Degradation derived from: Scaffold
    Mdm2 p53 β-catenin axin RTPR
    Mdm2 p53 β-catenin Bcl-9 RTPR
    Mdm2 p53 β-catenin TCF-4 RTPR
    Mdm2 p53 β-catenin ICAT RTPR
    Mdm2 p53 β-catenin LRH-1 RTPR
    Mdm2 p53 β-catenin APC RTPR
    SCFskp2 p27 β-catenin axin RTPR
    SCFskp2 p27 β-catenin Bcl-9 RTPR
    SCFskp2 p27 β-catenin TCF-4 RTPR
    SCFskp2 p27 β-catenin ICAT RTPR
    SCFskp2 p27 β-catenin LRH-1 RTPR
    SCFskp2 p27 β-catenin APC RTPR
    BTB-CUL3-RBX1 Nrf2 β-catenin Bcl-9 RTPR
    RTB-CUL3-RBX1 SPOP β-catenin Bcl-9 RTPR
    APC/C ABBA β-catenin Bcl-9 RTPR
    APC/C KEN β-catenin Bcl-9 RTPR
    APC/C DBOX β-catenin Bcl-9 RTPR
    SIAH PHYL β-catenin Bcl-9 RTPR
    BTB-CUL3-RBX1 Nrf2 β-catenin axin RTPR
    BTB-CUL3-RBX1 SPOP β-catenin axin RTPR
    APC/C ABBA β-catenin axin RTPR
    APC/C KEN β-catenin axin RTPR
    APC/C DBOX β-catenin axin RTPR
    SIAH PHYL β-catenin axin RTPR
    BTB-CUL3-RBX1 Nrf2 β-catenin TCF-4 RTPR
    BTB-CUL3-RBX1 Nrf2 β-catenin APC RTPR
  • TABLE 2
    Target protein Grafting
    and binding site in DNA sequence optimised for
    partner scaffold Amino acid sequence E. coli expression
    β-catenin
    axin helix GAYPEYILDIHVYRVQL GGTGCATATCCGGAATACATC
    EL (SEQ ID NO: 20) CTGGATATTCATGTTTATCGTG
    TTCAGCTGGAACTG (SEQ ID
    NO: 65)
    Bcl-9 helix SQEQLEHRYRSLITLYD AGCCAAGAACAGCTGGAACAT
    IQLML (SEQ ID NO: 21) CGTTATCGTAGCCTGATTACCC
    TGTATGATATTCAGCTGATGCT
    G (SEQ ID NO: 67)
    TCF-4 loop QELGDNDELMHFSYES CAAGAACTGGGCGATAATGAT
    TQD (SEQ ID NO: 22) GAACTGATGCACTTTAGCTAT
    GAAAGCACCCAGGAT (SEQ ID
    NO: 69)
    ICAT helix YAYQRAIVEYMLRLMS TATGCATATCAGCGTGCCATC
    (SEQ ID NO: 23) GTTGAATATATGCTGCGTCTG
    ATGAGC (SEQ ID NO: 71)
    LRH-1 helix YEQAIAAYLDALMC TATGAACAGGCAATTGCAGCA
    (SEQ ID NO: 24) TATCTGGATGCACTGATGTGT
    (SEQ ID NO: 73)
    APC loop SCSEELEALEALELDE AGCTGTAGCGAAGAACTGGAA
    (SEQ ID NO: 25) GCCCTGGAAGCATTAGAACTG
    GATGAA (SEQ ID NO: 75)
    α-catenin helix RSKKAHVLAASVEQAT CGCAGCAAAAAAGCGCATGTG
    QNFLEKGEQIAKESQ CTGGCGGCGAGCGTGGAACAG
    (SEQ ID NO: 1327) GCGACCCAGAACTTTCTGGAA
    AAAGGCGAACAGATTGCGAA
    AGAAAGCCAG (SEQ ID NO:
    2136)
    α-catenin helix RTLTVERLLEPLVTQVT CGCACCCTGACCGTGGAACGC
    TLV (SEQ ID NO: 1328) CTGCTGGAACCGCTGGTGACC
    CAGGTGACCACCCTGGTG
    (SEQ ID NO: 2137)
    APC Membrance loop RREQLEAQEARAREAH CGCCGCGAACAGCTGGAAGCG
    recruitment AREAHAREAYTREAYG CAGGAAGCGCGCGCGCGCGA
    protein REAYAREAHTWEAHG AGCGCATGCGCGCGAAGCGCA
    REARTREAQA (SEQ ID TGCGCGCGAAGCGTATACCCG
    NO: 1329) CGAAGCGTATGGCCGCGAAGC
    GTATGCGCGCGAAGCGCATAC
    CTGGGAAGCGCATGGCCGCGA
    AGCGCGCACCCGCGAAGCGCA
    GGCG (SEQ ID NO: 2138)
    SOX loop D..EFDQYL (SEQ ID NO: GATNNNNNNGAATTTGATCAG
    1330) TATCTG (SEQ ID NO: 2139)
    kindlin 2 loop QALLDKAKINQ CAGGCGCTGCTGGATAAAGCG
    GWLDSSRSLMEQDKEN AAAATTAACCAGGGCTGGCTG
    EALLRF (SEQ ID NO: GATAGCAGCCGCAGCCTGATG
    1331) GAACAGGATAAAGAAAACGA
    AGCGCTGCTGCGCTTT (SEQ ID
    NO: 2140)
    KRAS
    SOS1 helix FEGIALTNYLKALEG TTTGAAGGTATTGCACTGACC
    (SEQ ID NO: 1332) AATTATCTGAAAGCACTGGAA
    GGT (SEQ ID NO: 2141)
    phage-display loop PLYISY (SEQ ID NO: CCCCTGTACATCAGCTAC (SEQ
    library peptide 1333) ID NO: 2142)
    KR-pep1
    Synthetic peptide helix SIEDLHEYWARLWNYL AGCATTGAAGATCTGCATGAA
    225-1 YVA (SEQ ID NO: 1334) TATTGGGCGCGCCTGTGGAAC
    TATCTGTATGTGGCG (SEQ ID
    NO: 2143)
    Synthetic peptide helix QASLEELHEYWARLW CAGGCGAGCCTGGAAGAACTG
    225-15a NYRVA (SEQ ID NO: CATGAATATTGGGCGCGCCTG
    1335) TGGAACTATCGCGTGGCG
    (SEQ ID NO: 2144)
    Synthetic peptide helix NASIKQLHAYWQRLYA AACGCGAGCATTAAACAGCTG
    225-15b YLAAVA (SEQ ID NO: CATGCGTATTGGCAGCGCCTG
    1336) TATGCGTATCTGGCGGCGGTG
    GCG (SEQ ID NO: 2145)
    phage-display loop CMWWREICPVWW TGCATGTGGTGGCGCGAAATT
    library peptide (SEQ ID NO: 1337) TGCCCGGTGTGGTGG (SEQ ID
    KR-pep3 NO: 2146)
    Raf-S loop FARKTFLKLAF (SEQ ID TTTGCGCGCAAAACCTTTCTG
    NO: 1338) AAACTGGCGTTT (SEQ ID NO:
    2147)
    NF1 loop ARRFFLDIAD (SEQ ID GCGCGCCGCTTCTTTCTGGATA
    NO: 1339) TTGCGGAT (SEQ ID NO: 2148)
    RasIn peptide 2 loop FRWP..RL.. (SEQ ID NO: TTTCGCTGGCCGNNNNNNCGC
    1340) CTGNNNNNN (SEQ ID NO: 2149)
    RasIn peptide 1 loop t.VFXh.p (SEQ ID NO: AGCATTGTGTTTGGCGCGCAT
    1341) GAT (SEQ ID NO: 2150)
    NF1 monobody loop YGHGQVYYY (SEQ ID TATGGCCATGGCCAGGTGTAT
    peptide (74-84) NO: 1342) TATTAT (SEQ ID NO: 2151)
    farnesyl loop ENPKQN (SEQ ID NO: GAAAACCCGAAACAGAAC
    transferase 1 1343) (SEQ ID NO: 2152)
    farnesyl loop DAYECLDASRPW (SEQ GATGCGTATGAATGCCTGGAT
    transferase 2 ID NO: 1344) GCGAGCCGCCCGTGG (SEQ ID
    NO: 2153)
    farnesyl loop KSRDFYH (SEQ ID NO: AAATCCCGCGATTTCTATCAT
    transferase 3 1345) (SEQ ID NO: 2154)
    c-Myc
    Aurora A helix AGVEHQLRREVEIQSH GCGGGCGTGGAACATCAGCTG
    (SEQ ID NO: 1346) CGCCGCGAAGTGGAAATTCAG
    AGCCAT (SEQ ID NO: 2155)
    Aurora A loop WSVHAPSSRRTTpLAGT TGGAGCGTGCATGCGCCGAGC
    LDYLPPEMI (SEQ ID AGCCGCCGCACCGAACTGGCG
    NO: 1347) GGCACCCTGGATTATCTGCCG
    CCGGAAATGATT (SEQ ID NO:
    2156)
    Aurora A helix TYQETY (SEQ ID NO: ACCTATCAGGAAACCTAT
    1348) (SEQ ID NO: 2157)
    Omomyc helix QAEEQKLSEEDLLR CAGGCGGAAGAACAGAAACT
    KRREQLKHKLEQLRNS GAGCGAAGAAGATCTGCTGCG
    CA (SEQ ID NO: 1349) CAAACGCCGCGAACAGCTGAA
    ACATAAACTGGAACAGCTGCG
    CAACAGCTGCGCG (SEQ ID
    NO: 2158)
    Myc H1 F8A NELKRSFAALRDQI AACGAACTGAAACGCAGCTTT
    (SEQ ID NO: 1350) GCGGCGCTGCGCGATCAGATT
    (SEQ ID NO: 2159)
    Myc H1 F8A NELKRAFAALRDQI AACGAACTGAAACGCGCGTTT
    S6A (SEQ ID NO: 1351) GCGGCGCTGCGCGATCAGATT
    (SEQ ID NO: 2160)
    MIP helix IREKNHYHRQEVDDLR ATTCGCGAAAAAAACCATTAT
    RQNALLEQQVRAL CATCGCCAGGAAGTGGATGAT
    (SEQ ID NO: 1352) CTGCGCCGCCAGAACGCGCTG
    CTGGAACAGCAGGTGCGCGCG
    CTG (SEQ ID NO: 2161)
    PIN1 loop FNHITNASQWE (SEQ ID TTTAACCATATTACCAACGCG
    NO: 1353) AGCCAGTGGGAA (SEQ ID NO:
    2162)
    PIN2 loop GDLGAFSRGQM (SEQ GGCGATCTGGGCGCGTTTAGC
    ID NO: 1354) CGCGGCCAGATG (SEQ ID NO:
    2163)
    9E10 paratrope loop RSEFYYYGNTYYYSAM CGCAGCGAATTTTATTATTATG
    D (SEQ ID NO: 1355) GCAACACCTATTATTATAGCG
    CGATGGAT (SEQ ID NO: 2164)
    BIN1 loop QHDYTATDE (SEQ ID CAGCATGATTATACCGCGACC
    NO: 1356) GATGAA (SEQ ID NO: 2165)
    BIN1 loop QNPEEQDEGW (SEQ ID CAGAACCCGGAAGAACAGGA
    NO: 1357) TGAAGGCTGG (SEQ ID NO:
    2166)
    BIN1 loop EKCRGVFPENF (SEQ ID GAAAAGTGCCGCGGCGTGTTT
    NO: 1358) CCGGAAAACTTT (SEQ ID NO:
    2167)
    BRD4
    JMJD6 loop KWTLERLKRKYRN AAATGGACCCTGGAACGTCTG
    (SEQ ID NO: 1522) AAACGTAAATACCGTAAC
    (SEQ ID NO: 2303)
    murine leukemia loop TWRVQRSQNPLKIRLT ACCTGGCGTGTTCAGCGTTCTC
    virus integrase R (SEQ ID NO: 1523) AGAACCCGCTGAAAATCCGTC
    TGACCCGT (SEQ ID NO: 2304)
    EWS-FLI1
    ESAP1 loop TMRGKKKRTRAN (SEQ ACCATGCGCGGCAAAAAAAA
    ID NO: 1524) ACGCACCCGCGCGAAC (SEQ
    ID NO: 2305)
    Aurora A
    TPX2 loop SYSYDAPSDFINFSS AGCTATAGCTATGATGCGCCG
    (SEQ ID NO: 1525) AGCGATTTTATTAACTTTAGCA
    GC (SEQ ID NO: 2306)
    TPX2 loop SYSYDAPSDFINFSSLD AGCTATAGCTATGATGCGCCG
    DEGDTQNIDSWFEEKA AGCGATTTTATTAACTTTAGCA
    NLEN (SEQ ID NO: 1526) GCCTGGATGATGAAGGCGATA
    CCCAGAACATTGATAGCTGGT
    TTGAAGAAAAAGCGAACCTGG
    AAAAC (SEQ ID NO: 2307)
    TPX3 loop MSQVKSSYSYDAPSDFI ATGAGCCAGGTGAAGTCATCT
    NFSSLDD (SEQ ID NO: TATTCCTATGATGCCCCCAGC
    1527) GATTTCATCAATTTTTCATCCT
    TGGATGATGAA (SEQ ID NO:
    2308)
    N-myc helix MALSPSRGFAEHSSEPP ATGGCGCTGAGCCCGAGCCGC
    SWVTIMLYENELWI GGCTTTGCGGAACATAGCAGC
    (SEQ ID NO: 1528) GAACCGCCGAGCTGGGTGACC
    ATTATGCTGTATGAAAACGAA
    CTGTGGATT (SEQ ID NO: 2309)
    N-myc loop LEFDSLQPCFYPDEDDF CTGGAATTTGATAGCCTGCAG
    YFGGPDSTPPGE (SEQ CCGTGCTTTTATCCGGATGAA
    ID NO: 1529) GATGATTTTTATTTTGGCGGCC
    CGGATAGCACCCCGCCGGGCG
    AA (SEQ ID NO: 2310)
    CK2alpha
    CK2beta loop RLYGFKIHPMAYQLQ CGCCTGTATGGCTTTAAAATTC
    (SEQ ID NO: 1530) ATCCGATGGCGTATCAGCTGC
    AG (SEQ ID NO: 2311)
    WDR5
    MLL1 loop EPPLNPHGSARAEVHLR GAACCGCCGCTGAACCCGCAT
    KS (SEQ ID NO: 1531) GGCAGCGCGCGCGCGGAAGTG
    CATCTGCGCAAAAGC (SEQ ID
    NO: 2312)
    Notch
    MAML1 helix SAVMERLRRRIELCRRH AGCGCGGTGATGGAACGCCTG
    HST (SEQ ID NO: 1532) CGCCGCCGCATTGAACTGTGC
    CGCCGCCATCATAGCACC
    (SEQ ID NO: 2313)
    Cdk2
    cyclin A helix TYTKKQVLRMEHLVLK ACCTATACCAAAAAACAGGTG
    VLTFDL (SEQ ID NO: CTGCGCATGGAACATCTGGTG
    1533) CTGAAAGTGCTGACCTTT (SEQ
    ID NO: 2314)
    aptmer library LVCKSYRLDWEAGALF CTGGTGTGCAAAAGCTATCGC
    RSLF (SEQ ID NO: 1534) CTGGATTGGGAAGCGGGCGCG
    CTGTTTCGCAGCCTGTTT (SEQ
    ID NO: 2315)
    aptmer library YSFVHHGFFNFRVSWR TATAGCTTTGTGCATCATGGCT
    EMLA (SEQ ID NO: TTTTTAACTTTCGCGTGAGCTG
    1535) GCGCGAAATGCTGGCG (SEQ
    ID NO: 2316)
    peptide loop TAALS (SEQ ID NO: ACCGCGGCGCTGAGC (SEQ ID
    1536) NO: 2317)
    peptide loop TALLS (SEQ ID NO: ACCGCGCTGCTGAGC (SEQ ID
    1537) NO: 2318)
    peptide loop LAALS (SEQ ID NO: CTGGCGGCGCTGAGC (SEQ ID
    1538) NO: 2319)
    peptide loop DAALT (SEQ ID NO: GATGCGGCGCTGACC (SEQ ID
    1539) NO: 2320)
    peptide loop YAALQ (SEQ ID NO: TATGCGGCGCTGCAG (SEQ ID
    1540) NO: 2321)
    peptide loop SKL.RFTGCSC (SEQ ID AGCAAACTGNNNCGCTTTACC
    NO: 1541) GGCTGCAGCTGC (SEQ ID NO:
    2322)
    RXL peptide loop PVKRRLFL (SEQ ID NO: CCGGTGAAACGCCGCCTGTTT
    1542) CTG (SEQ ID NO: 2323)
    p21 loop GRKRRQTSMTDFYHSK GGCCGCAAACGCCGCCAGACC
    RRLIFSKRKP (SEQ ID AGCATGACCGATTTTTATCAT
    NO: 1543) AGCAAACGCCGCCTGATTTTT
    AGCAAACGCAAACCG (SEQ ID
    NO: 2324)
    PLK1
    peptide loop MAGPMQTSpTPKNAGK ATGGCGGGCCCGATGCAGACC
    K (SEQ ID NO: 1544) AGCACCCCGAAAAACGCGGGC
    AAAAAA (SEQ ID NO: 2325)
    PBIP1 loop FDPPLHSpTA (SEQ ID TTTGATCCGCCGCTGCATAGC
    NO: 1545) ACCGCG (SEQ ID NO: 2326)
    designed peptide loop PLHSpTAI (SEQ ID NO: CCGCTGCATAGCACCGCGATT
    1546) (SEQ ID NO: 2327)
    designed peptide loop MDSpTPL (SEQ ID NO: ATGGATAGCACCCCGCTG
    1547) (SEQ ID NO: 2328)
    Emi2 loop FSQHKpTI (SEQ ID NO: TTTAGCCAGCATAAAACCAGC
    1548) ATT (SEQ ID NO: 2329)
    HEF1 loop LHYPSpTTALQE (SEQ CTGCATTATCCGAGCACCACC
    ID NO: 1549) GCGCTGCAGGAA (SEQ ID NO:
    2330)
    cdc-25 loop LLCSpTPNGL (SEQ ID CTGCTGTGCAGCACCCCGAAC
    NO: 1550) GGCCTG (SEQ ID NO: 2331)
    BCR-ABL
    optimised loop EAIYAAPFAKKK (SEQ GAAGCGATTTATGCGGCGCCG
    substrate peptide ID NO: 1551) TTTGCGAAAAAAAAA (SEQ ID
    NO: 2332)
    proline-rich helix APSYPPPPP (SEQ ID NO: GCGCCGAGCTATCCGCCGCCG
    peptide 1552) CCGCCG (SEQ ID NO: 2333)
    PP2A
    optimised loop LQTIQEEE (SEQ ID NO: CTGCAGACCATTCAGGAAGAA
    substate peptide 1553) GAA (SEQ ID NO: 2334)
    PP1c
    consensus loop RV.F CGCGTGNNNTTT (SEQ ID NO:
    sequence 2335)
    consensus loop SILK (SEQ ID NO: 1554) AGCATTCTGAAA (SEQ ID NO:
    sequence 2336)
    KNL1 loop SRRVSFADTIKVFQT AGCCGCCGCGTGAGCTTTGCG
    (SEQ ID NO: 1555) GATACCATTAAAGTGTTTCAG
    ACC (SEQ ID NO: 2337)
    EED
    (Embryonic
    ectoderm
    development)
    EZH2 helix FSSNRQKILERTEILNQE TTTAGCAGCAACCGCCAGAAA
    WKQRRIQPV (SEQ ID ATTCTGGAACGCACCGAAATT
    NO: 1556) CTGAACCAGGAATGGAAACAG
    CGCCGCATTCAGCCGGTG
    (SEQ ID NO: 2338)
    MCL-1
    EZH2 helix KALETLRRVGDGVQRN AAAGCGCTGGAAACCCTGCGC
    HETAF (SEQ ID NO: CGCGTGGGCGATGGCGTGCAG
    1557) CGCAACCATGAAACCGCGTTT
    (SEQ ID NO: 2339)
    NOXA BH3 helix AELEVESATQLRRFGD GCGGAACTGGAAGTGGAAAG
    KLNFRQKLL (SEQ ID CGCGACCCAGCTGCGCCGCTT
    NO: 1558) TGGCGATAAACTGAACTTTCG
    CCAGAAACTGCTG (SEQ ID NO:
    2340)
    MCL-1 BH3 helix KALETLR.VGD.VQRNH AAAGCGCTGGAAACCCTGCGC
    ETAF (SEQ ID NO: 1559) NNNGTGGGCGATNNNGTGCAG
    CGCAACCATGAAACCGCGTTT
    (SEQ ID NO: 2341)
    GSK3
    Substrate- loop KEAPPAPPQDP (SEQ ID AAAGAAGCGCCGCCGGCGCCG
    competitive NO: 1723) CCGCAGGATCCG (SEQ ID NO:
    binding peptide 2477)
    Substrate- loop LSRRPDYR (SEQ ID NO: CTGAGCCGCCGCCCGGATTAT
    competitive 1724) CGC (SEQ ID NO: 2478)
    binding peptide
    Substrate- loop RREGGMSRPADVDG CGCCGCGAAGGCGGCATGAGC
    competitive (SEQ ID NO: 1725) CGCCCGGCGGATGTGGATGGC
    binding peptide (SEQ ID NO: 2479)
    Substrate- loop YRRAAVPPSPSLSRHSS TATCGCCGCGCGGCGGTGCCG
    competitive PSQDEDEEE (SEQ ID CCGAGCCCGAGCCTGAGCCGC
    binding peptide NO: 1726) CATAGCAGCCCGAGCCAGGAT
    GAAGATGAAGAAGAA (SEQ ID
    NO: 2480)
    CtBP
    From cyclic loop SGWTVVRMY (SEQ ID AGCGGCTGGACCGTGGTGCGC
    peptide library NO: 1890) ATGTAT (SEQ ID NO: 2616)
    tankyrase
    consensus loop REAGDGEE (SEQ ID CGTGAAGCCGGTGATGGTGAA
    substrate peptide NO: 1891) GAA (SEQ ID NO: 2617)
    consensus loop HLQREAGDGEEFRS CATCTGCAGCGTGAAGCCGGT
    substrate peptide (SEQ ID NO: 1892) GATGGTGAAGAATTTCGTAGC
    (SEQ ID NO: 2618)
    Bcl-2 and BCL-XL
    Bim BH3 helix IWIAQELRRIGDEFNAY ATTTGGATTGCGCAGGAACTG
    YARR (SEQ ID NO: CGCCGCATTGGCGATGAATTT
    2056) AACGCGTATTATGCGCGCCGC
    (SEQ ID NO: 2754)
    Bak BH3 helix GQVGRQLAIIGDDINR GGCCAGGTGGGCCGCCAGCTG
    (SEQ ID NO: 2057) GCGATTATTGGCGATGATATT
    AACCGC (SEQ ID NO: 2755)
    Bad BH3 helix NLWAAQRYGRELRRM AACCTGTGGGCGGCGCAGCGC
    SDEFVDSFKK (SEQ ID TATGGCCGCGAACTGCGCCGC
    NO: 2058) ATGAGCGATGAATTTGTGGAT
    AGCTTTAAAAAA (SEQ ID NO:
    2756)
    Jun
    library-selected helix SIAATLEKEEANLEKM AGCATCGCCGCCACCCTGGAG
    peptide NKKLAAEIESLLKEKDK AAGGAGGAGGCCAACCTGGA
    LESVLNYHE (SEQ ID GAAGATGAACAAGAAGCTGG
    NO: 2059) CCGCCGAGATCGAGAGCCTGC
    TGAAGGAGAAGGACAAGCTG
    GAGAGCGTGCTGAACTACCAC
    GAG (SEQ ID NO: 2757)
    library-selected helix VQEIEQEIQELEKRIKQI GTTCAGGAAATCGAACAGGAA
    peptide QQEFQEIEQQIALL (SEQ ATCCAGGAACTGGAAAAACGT
    ID NO: 2060) ATCAAACAGATCCAGCAGGAA
    TTCCAGGAAATCGAACAGCAG
    ATCGCGCTGCTG (SEQ ID NO:
    2758)
    BFL1
    NOXA BH3 helix ATQLRRFGDKLNFRQ GCGACCCAGCTGCGCCGCTTT
    (SEQ ID NO: 2061) GGCGATAAACTGAACTTTCGC
    CAG (SEQ ID NO: 2759)
    BAX
    Bcl-2 BH3 helix and EIVAKYIHYKLSQRGYE GAAATTGTGGCGAAATATATT
    loop WDA (SEQ ID NO: 2062) CATTATAAACTGAGCCAGCGC
    GGCTATGAATGGGATGCG
    (SEQ ID NO: 2760)
    eIF4E
    eIF4G helix KKRYDREFLLGFQF AAAAAACGCTATGATCGCGAA
    (SEQ ID NO: 2063) TTTCTGCTGGGCTTTCAGTTT
    (SEQ ID NO: 2761)
    eIF4G helix and GKKRYDREFLLGFQFIF GGCAAAAAACGCTATGATCGC
    loop ASMQKPEGLPHISDVVL GAATTTCTGCTGGGCTTTCAGT
    (SEQ ID NO: 2064) TTATTTTTGCGAGCATGCAGA
    AACCGGAAGGCCTGCCGCATA
    TTAGCGATGTGGTGCTG (SEQ
    ID NO: 2762)
    optimised helix and TKLIYERAFMKNLRGSP ACCAAACTGATTTATGAACGC
    peptide loop LSQTPPSNVPSCLLRGT GCGTTTATGAAAAACCTGCGC
    (SEQ ID NO: 2065) GGCAGCCCGCTGAGCCAGACC
    CCGCCGAGCAACGTGCCGAGC
    TGCCTGCTGCGCGGCACC (SEQ
    ID NO: 2763)
    Fos
    library-selected helix AIARLEERVKTLKAEIY GCGATTGCGCGCCTGGAAGAA
    peptide ELRSKANMLREQIAQL CGCGTGAAAACCCTGAAAGCG
    GAP (SEQ ID NO: 2066) GAAATTTATGAACTGCGCAGC
    AAAGCGAACATGCTGCGCGAA
    CAGATTGCGCAGCTGGGCGCG
    CCG (SEQ ID NO: 2764)
    library-selected helix AIARLEERVKTLKAEIY GCGATTGCGCGCCTGGAAGAA
    peptide ELQSEANMLREQIAQL CGCGTGAAAACCCTGAAAGCG
    GAP (SEQ ID NO: 2067) GAAATTTATGAACTGCGCAGC
    AAAGCGAACATGCTGCGCGAA
    CAGATTGCGCAGCTGGGCGCG
    CCG (SEQ ID NO: 2765)
    HDAC4
    SMRT loop HIRGSITQGIPRSYV CACATCCGTGGTTCTATCACCC
    corepressor (SEQ ID NO: 2068) AGGGTATCCCGCGTTCTTACG
    TT (SEQ ID NO: 2766)
    BCL6
    SMRT and N-CoR loop GRSIHEIPR (SEQ ID NO: GGCCGCAGCATTCATGAAATT
    corepressors 2069) CCGCGC (SEQ ID NO: 2767)
    SMRT and N-CoR loop GLVATVKEAGRSIHEIP GGCCTGGTGGCGACCGTGAAA
    corepressors REEL (SEQ ID NO: 2070) GAAGCGGGCCGCAGCATTCAT
    GAAATTCCGCGCGAAGAACTG
    (SEQ ID NO: 2768)
    Tau
    alpha-tubulin loop KDYEEVGVDSVE (SEQ AAAGATTATGAAGAAGTGGGC
    ID NO: 2071) GTGGATAGCGTGGAA (SEQ ID
    NO: 2769)
    beta-tubulin loop YQQYQDATADEQG TATCAGCAGTATCAGGATGCG
    (SEQ ID NO: 2072) ACCGCGGATGAACAGGGC
    (SEQ ID NO: 2770)
    PD-L1
    HIP1R loop DAVRRIEDMMNQARH GATGCGGTGCGCCGCATTGAA
    ASSGV (SEQ ID NO: GATATGATGAACCAGGCGCGC
    2073) CATGCGAGCAGCGGCGTG
    (SEQ ID NO: 2771)
    KDM4A
    library-selected loop YVYNTRSGWRWYT TACGTTTACAACACCCGTTCTG
    peptide (SEQ ID NO: 2074) GTTGGCGTTGGTACACC (SEQ
    ID NO: 2772)
    EGFR
    EGFR helix VRKRTLRRLLQERELV GTGCGCAAACGCACCCTGCGC
    (juxtamembrane E (SEQ ID NO: 2075) CGCCTGCTGCAGGAACGCGAA
    coiled-coil CTGGTGGAA (SEQ ID NO: 2773)
    domain)
    RAB25
    RFIP1 helix RQVRELENYIDRLVRV CGCCAGGTGCGCGAACTGGAA
    MEETPNILRIPR (SEQ ID AACTATATTGATCGCCTGGTG
    NO: 2076) CGCGTGATGGAAGAAACCCCG
    AACATTCTGCGCATTCCGCGC
    (SEQ ID NO: 2774)
    GPCRs and other
    transmembrane
    proteins
    PAR1 pepducin N-term pal-KKSRALF-NH2 (pal- = synthetic peptide
    palmitoyl; -NH2 = amino
    group) (SEQ ID NO: 2077) 
    PAR1 pepducin N-term pal-RCLSSSAVANRS- synthetic peptide
    NH2 (SEQ ID NO: 2078)
    PAR1 pepducin N-term pal-RSLSSSAVANRS- synthetic peptide
    NH2 (SEQ ID NO: 2079)
    PAR1 pepducin N-term pal-AVANRSKKSRALF- synthetic peptide
    NH2 (SEQ ID NO: 2080)
    PAR1 pepducin N-term pal- synthetic peptide
    RCESSSAEANRSKKERE
    LF-NH2 (SEQ ID NO:
    2081)
    PAR1 pepducin N-term pal-ASSESQRYVYSIL- synthetic peptide
    NH2 (SEQ ID NO: 2082)
    PAR1 pepducin N-term pal-ASSASQEYVYSIL- synthetic peptide
    NH2 (SEQ ID NO: 2083)
    PAR2 pepducin N-term pal- synthetic peptide
    RSSAMDENSEKKRKSAI
    K-NH2 (SEQ ID NO:
    2084)
    PAR2 pepducin N-term pal- synthetic peptide
    GDENSEKKRKQAIK-
    NH2 (SEQ ID NO: 2085)
    PAR4 pepducin N-term pal-SGRRYGHALR-NH2 synthetic peptide
    (SEQ ID NO: 2086)
    PAR4 pepducin N-term pal-ATGAPRLPST-NH2 synthetic peptide
    (SEQ ID NO: 2087)
    PAR4 pepducin N-term pal-RLAHGYRRGS-NH2 synthetic peptide
    (SEQ ID NO: 2088)
    CXCR1/2 N-term pal-RTLFKAHMGQKHR- synthetic peptide
    pepducin NH2 (SEQ ID NO: 2089)
    CXCR1/2 N-term pal-LCA-YSRVGRSVTD- synthetic peptide
    pepducin NH2 (LCA- = lithocholic
    acid) (SEQ ID NO: 2090)
    CXCR4 N-term pal-HSKGHQKRKALK- synthetic peptide
    pepducin NH2 (SEQ ID NO: 2091)
    CXCR4 N-term pal-MGYQKKLRSMTD- synthetic peptide
    pepducin NH2 (SEQ ID NO: 2092)
    CXCR4 N-term pal- synthetic peptide
    pepducin MGYQKKLRSMTDKYR
    L-NH2 (SEQ ID NO:
    2093)
    S1P3 pepducin N-term myristoyl- synthetic peptide
    GMRPYDANKR-NH2
    (SEQ ID NO: 2094)
    S1P3 pepducin N-term myristoyl-GRPYDAN- synthetic peptide
    NH2 (SEQ ID NO: 2095)
    FRP2 pepducin N-term pal- synthetic peptide
    KIHKKGMIKSSRPLRV-
    NH2 (SEQ ID NO: 2096)
    FRP2 pepducin N-term pal-KIHKKGMIKS-NH2 synthetic peptide
    (SEQ ID NO: 2097)
    FRP2 pepducin N-term pal-KIHKKGMIKSSR- synthetic peptide
    NH2 (SEQ ID NO: 2098)
    LGR7 pepducin N-term pal- synthetic peptide
    KRKALKALILNEKKVQ-
    H (-H = hydrogen) (SEQ
    ID NO: 2099)
    SMO pepducin N-term pal-TFVADWRNSNRY-H synthetic peptide
    (SEQ ID NO: 2100)
    SMO pepducin N-term pal- synthetic peptide
    TWAWHTSFKALGTTY
    QPLSGKTS-H (SEQ ID
    NO: 2101)
    SMO pepducin N-term pal- synthetic peptide
    RGVMTLFSIKSNHPGLL
    SEKAASKINETMLR-H
    (SEQ ID NO: 2102)
    IGF1R pepducin N-term pal-RNNSRLGNGVLY- synthetic peptide
    NH2 (SEQ ID NO: 2103)
    CD226 pepducin N-term pal-RRERRDLFTE-NH2 synthetic peptide
    (SEQ ID NO: 2104)
    TRPV1 N-term pal-MGETVNKIAQES- synthetic peptide
    TRPducin NH2 (SEQ ID NO: 2105)
    Nrp1/2
    paratrope loop RASQYFSSYLA (SEQ ID CGCGCGAGCCAGTATTTTAGC
    NO: 2106) AGCTATCTGGCG (SEQ ID NO:
    2775)
    paratrope helix AREDFRNRRLWYVMD GCGCGCGAAGATTTTCGCAAC
    Y (SEQ ID NO: 2107) CGCCGCCTGTGGTATGTGATG
    GATTAT (SEQ ID NO: 2776)
    IL18
    paratrope helix KASGYSFTDYFIY (SEQ AAAGCGAGCGGCTATAGCTTT
    ID NO: 2108) ACCGATTATTTTATTTAT (SEQ
    ID NO: 2777)
    IL15
    paratrope loop YRDRRRPS (SEQ ID NO: TATCGCGATCGCCGCCGCCCG
    2109) AGC (SEQ ID NO: 2778)
    Thyroid
    stimulating
    hormone receptor
    paratrope loop SGSSSDIGSNYVS (SEQ AGCGGCAGCAGCAGCGATATT
    ID NO: 2110) GGCAGCAACTATGTGAGC
    (SEQ ID NO: 2779)
    EGF receptor
    paratrope loop QQWSSHIFT (SEQ ID CAGCAGTGGAGCAGCCATATT
    NO: 2111) TTTACC (SEQ ID NO: 2780)
    paratrope helix ASRDYDYAGRYFDY GCGAGCCGCGATTATGATTAT
    (SEQ ID NO: 2112) GCGGGCCGCTATTTTGATTAT
    (SEQ ID NO: 2781)
    IL23
    paratrope loop QNGHSFPFT (SEQ ID CAGAACGGCCATAGCTTTCCG
    NO: 2113) TTTACC (SEQ ID NO: 2782)
    paratrope helix YINPYNDGTK (SEQ ID TATATTAACCCGTATAACGAT
    NO: 2114) GGCACCAAA (SEQ ID NO: 2783)
    paratrope helix ARNWDVAY (SEQ ID GCGCGCAACTGGGATGTGGCG
    NO: 2115) TAT (SEQ ID NO: 2784)
    Lymphocyte
    function-
    associated
    antigen 1
    (LFA-1)
    paratrope helix SGYSFTGHWMN (SEQ AGCGGCTATAGCTTTACCGGC
    ID NO: 2116) CATTGGATGAAC (SEQ ID NO:
    2785)
    paratrope helix MIHPSDSETR (SEQ ID ATGATTCATCCGAGCGATAGC
    NO: 2117) GAAACCCGC (SEQ ID NO: 2786)
    paratrope helix ARGIYFYGTTYFDY GCGCGCGGCATTTATTTTTATG
    (SEQ ID NO: 2118) GCACCACCTATTTTGATTAT
    (SEQ ID NO: 2787)
    C3b
    paratrope helix SGFSFTSSVS (SEQ ID AGCGGCTTTAGCTTTACCAGC
    NO: 2119) AGCGTGAGC (SEQ ID NO: 2788)
    paratrope helix LIYPYNGFN (SEQ ID CTGATTTATCCGTATAACGGCT
    NO: 2120) TTAAC (SEQ ID NO: 2789)
    FGF receptor 3
    paratrope helix AASGFTFTSTGIS (SEQ GCGGCGAGCGGCTTTACCTTT
    ID NO: 2121) ACCAGCACCGGCATTAGC
    (SEQ ID NO: 2790)
    paratrope helix ARTYGIYDLYVDYTE GCGCGCACCTATGGCATTTAT
    (SEQ ID NO: 2122) GATCTGTATGTGGATTATACC
    GAA (SEQ ID NO: 2791)
    IL2
    paratrope helix SRDYGYYFD (SEQ ID AGCCGCGATTATGGCTATTAT
    NO: 2123) TTTGAT (SEQ ID NO: 2792)
    paratrope helix GYSFTRYWMH (SEQ ID GGCTATAGCTTTACCCGCTATT
    NO: 2124) GGATGCAT (SEQ ID NO: 2793)
    HER2
    paratrope loop QWWWWPST (SEQ ID CAGTGGTGGTGGTGGCCGAGC
    NO: 2125) ACC (SEQ ID NO: 2794)
    paratrope helix ASGFSIWWSWIH (SEQ GCGAGCGGCTTTAGCATTTGG
    ID NO: 2126) TGGAGCTGGATTCAT (SEQ ID
    NO: 2795)
    membrane-type
    serine protease 1
    paratrope loop YDNNQRPS (SEQ ID TATGATAACAACCAGCGCCCG
    NO: 2127) AGC (SEQ ID NO: 2796)
    paratrope helix TFHIRRYRSGYYDKMD ACCTTTCATATTCGCCGCTATC
    H (SEQ ID NO: 2128) GCAGCGGCTATTATGATAAAA
    TGGATCAT (SEQ ID NO: 2797)
    beta-secretase
    paratrope helix ARGPFSPWVMDY (SEQ GCGCGCGGCCCGTTTAGCCCG
    ID NO: 2129) TGGGTGATGGATTAT (SEQ ID
    NO: 2798)
    VEGF-R
    paratrope helix TRHDGTNFD (SEQ ID ACCCGCCATGATGGCACCAAC
    NO: 2130) TTTGAT (SEQ ID NO: 2799)
    paratrope helix QQAKAFPPT (SEQ ID CAGCAGGCGAAAGCGTTTCCG
    NO: 2131) CCGACC (SEQ ID NO: 2800)
    lrp5/6 receptor
    paratrope helix SGHVNAVKNYGY (SEQ AGCGGCCATGTGAACGCGGTG
    ID NO: 2132) AAAAACTATGGCTAT (SEQ ID
    NO: 2801)
    hepsin protease
    paratrope helix WINTETGS (SEQ ID NO: TGGATTAACACCGAAACCGGC
    2133) AGC (SEQ ID NO: 2802)
    Factor D
    paratrope helix WINTYTGE (SEQ ID NO: TGGATTAACACCTATACCGGC
    2134) GAA (SEQ ID NO: 2803)
    paratrope helix GYTFTNYGMN (SEQ ID GGCTATACCTTTACCAACTAT
    NO: 2135) GGCATGAAC (SEQ ID NO: 2804)
  • TABLE 3
    Degron
    sequence Grafting
    Ubiquitin derived site in Amino acid
    Ligase from scaffold sequence DNA seqeunce E. Coli codon optimised 5′ to 3′
    Mdm2 Consensus helix F[∨P]{3} TTT[NNNNNNNNN(EXCEPT CCN)]TGG[NNN
    W[∨P]{2,3} {2,3}(∨CCN)][GTG/ATT/CTG](SEQ ID NO:
    [VIL] 2945)
    Mdm2 p53 helix FAAYWN TTTGCAGCCTATTGGAATCTGCTGAGCGCAT
    LLSAYG ATGGT (SEQ ID NO: 2946)
    NO: 2805)
    Mdm2 p53 helix RFMDYW CGCTTCATGGATTATTGGGAAGGTCTG (SEQ
    EGL (SEQ ID NO: 2947)
    2806)
    Mdm2 p53 helix TSFAEYW ACCAGCTTTGCCGAGTATTGGGCCCTGCTGG
    ALLAENL CCGAGAATCTG (SEQ ID NO: 2948)
    (SEQ ID
    NO: 2807)
    Mdm2 p53 helix EAQWAA GAAGCGCAGTGGGCGGCGCTG (SEQ ID NO:
    L (SEQ ID 2949)
    NO: 2808)
    Mdm2 p53 helix FEAQWA TTTGAAGCGCAGTGGGCGGCGCTG (SEQ ID
    AL (SEQ NO: 2950)
    ID NO:
    2809)
    Mdm2 p63 helix FQHIWDF TTTCAGCATATTTGGGATTTTCTG (SEQ ID
    L (SEQ ID NO: 2951)
    NO: 2810)
    Mdm2 p73 helix FEHLWSS TTTGAACATCTGTGGAGCAGCCTG (SEQ ID
    L (SEQ ID NO: 2952)
    NO: 2811)
    SCF(Sk Consensus loop .[DE].pTP. NNN[GAT/GAA]NNNACCCCGNNNAAA (SEQ
    p2) K ID NO: 2953)
    SCF(Sk p27 loop AGSNEQE GCAGGTAGCAATGAACAAGAACCGAAAAA
    p2) PKKRS ACGTAGT (SEQ ID NO: 2954)
    (SEQ ID
    NO: 2812)
    Cul3- Consensus loop [DNS].[DE [GAC/AAC/AGC]NNN[GAC/GAA/AGC][ACC/A
    Keap1 S][TNS] AC/AGC]GGCGAA (SEQ ID NO: 2955)
    GE
    Cul3- Nrf2 loop DPETGEL GATCCGGAAACCGGTGAACTG (SEQ ID NO:
    Keap1 (SEQ ID 2956)
    NO: 2813)
    Cul3- Sequestosome- loop DPSTGEL GATCCGAGCACCGGCGAACTG (SEQ ID NO:
    Keap1 1 (SEQ ID 2957)
    NO: 2814)
    Cul3- IKKB loop NQETGE AACCAGGAAACCGGCGAA (SEQ ID NO:
    Keap1 (SEQ ID 2958)
    NO: 2815)
    Cul3- APC loop SPETGE AGCCCGGAAACCGGCGAA (SEQ ID NO: 2959)
    KEAP1 membrane (SEQ ID
    recruitment NO: 2816)
    protein 1
    Cul3- Prothymosin loop NEENGE AACGAAGAAAACGGCGAA (SEQ ID NO:
    alpha (SEQ ID 2960)
    NO: 2817)
    Cul3- Nucleosome- loop DPENGE GATCCGGAAAACGGCGAA (SEQ ID NO: 2961)
    KEAP1 remodeling (SEQ ID
    factor NO: 2818)
    subunit
    BPTF
    Cul3- Serine/ loop NVESGE AACGTGGAAAGCGGCGAA (SEQ ID NO:
    KEAP1 threonine- (SEQ ID 2962)
    protein NO: 2819)
    phosphatase
    PGAM5,
    mitochondrial
    Cul3- Nuclear loop DEET GE GATGAAGAAACCGGCGAA (SEQ ID NO:
    KEAP1 factor (SEQ ID 2963)
    erythroid NO: 2820)
    2-related
    factor 2
    Cul3- Partner loop DEETGE GATGAAGAAACCGGCGAA (SEQ ID NO:
    KEAP1 and (SEQ ID 2964)
    localizer NO: 2821)
    of
    BRCA2
    Cul3- Consensus loop QD.DLGV CAGGATNNNGATCTGGGTGTG (SEQ ID NO:
    KEAP1_2 (SEQ ID 2965)
    NO: 2822)
    Cul3- Consensus loop [AVP].[ST] [GCG/GTG/CCG]NNN[AGC/ACC][AGC/ACC][A
    SPOP [ST][ST] GC/ACC]
    Cul3- Map loop ELDSPSS GAACTGGATAGCCCGAGCAGCACCAGCAGC
    SPOP kinase TSSSS AGCAGC (SEQ ID NO: 2966)
    phosphatase (SEQ ID
    NO: 2823)
    Cul3- SBC loop LACDEVT CTGGCATGTGATGAAGTTACCAGCACCACC
    SPOP STTSSST AGTAGCAGCACCGCA (SEQ ID NO: 2967)
    A (SEQ ID
    NO: 2824)
    Cul3- Androgen loop ASSTT GCGAGCAGCACCACC (SEQ ID NO: 2968)
    SPOP receptor (SEQ ID
    NO: 2825)
    Cul3- Map loop DEVTSTT GATGAAGTGACCAGCACCACCAGCAGCAGC
    SPOP kinase SSST ACC (SEQ ID NO: 2969)
    phosphatase (SEQ ID
    NO: 2826)
    Cul3- Consensus loop E.EE.E[AV] GAANNNGAAGAANNNGAA[GCG/GTG]GATA
    KELCH DQH ACCAT (SEQ ID NO: 2970)
    (SEQ ID
    NO: 2827)
    Cul3- Serine/ helix/ EPEEPEA GAACCGGAAGAACCGGAAGCGGATCAGCA
    KELCH threonine- loop DQH (SEQ T (SEQ ID NO: 2971)
    protein ID NO:
    kinase 2828)
    WNK1
    Cul3- Serine/ helix/ ECEETEV GAATGCGAAGAAACCGAAGTGGATCAGCAT
    KELCH threonine- loop DQH (SEQ (SEQ ID NO: 2972)
    protein ID NO:
    kinase 2829)
    WNK3
    Cul3- Serine/ helix/ EPEEPEA GAACCGGAAGAACCGGAAGCGGATCAGCA
    KELCH threonine- loop DQH (SEQ T (SEQ ID NO: 2973)
    protein ID NO:
    kinase 2830)
    WNK4
    Cul3- Nuclear helix/ ILWRQDI ATTCTGTGGCGCCAGGATATTGATCTGGGC
    KELCH factor loop DLGV GTG (SEQ ID NO: 2974)
    erythroid (SEQ ID
    2-related NO: 2831)
    KELCH Consensus loop [AP]P[MV] [GCG/CCG]CCG[ATG/GTG][ATT/ATG]GTG
    actinfilin [IM]V (SEQ ID NO: 3099)
    APC/C ABBA loop [FIVL].[IL [TTT/ATT/GTG/CTG]NNN[ATT/CTG/ATG/GTG/
    MVP][FH CCG][TTT/CAT/TAT][NNN{0,3}][GAT/GAA/AG
    Y].[DE]. C/ACC]
    {O,3}{DES
    T}
    APC/C ABBA loop SLSSAFH AGCCTGAGCAGCGCGTTTCATGTGTTTGAA
    VFEDGN GATGGCAACAAAGAAAAC (SEQ ID NO:
    KEN (SEQ 2975)
    ID NO:
    2832)
    APC/C Cyclin- loop FTIHVD TTTACCATTCATGTGGAT (SEQ ID NO: 2976)
    A2:ABBA (SEQ ID
    NO: 2833)
    APC/C ABOX loop QRVL CAGCGTGTTCTG (SEQ ID NO: 2977)
    (SEQ ID
    NO: 2834)
    APC/C Consensus loop .KEN. NNNAAAGAAAACNNN (SEQ ID NO: 2978)
    APC/C KEN loop SEDKENV AGCGAGGATAAAGAAAATGTTCCGCCT
    PP (SEQ (SEQ ID NO: 2979)
    ID NO:
    2835)
    APC/C DBOX loop .R..L..[LIV NNNCGTNNNNNNCTGNNNNNN[CTG/ATT/GT
    consensus M]. G/ATG]NNN (SEQ ID NO: 2980)
    APC/C Shugoshin 1: loop RLSLSPK CGCCTGAGCCTGAGCCCGAAAAAAAAC
    DBOX KN (SEQ (SEQ ID NO: 2981)
    ID NO:
    2836)
    APC/C Shugoshin 1: loop RSSLKKH CGCAGCAGCCTGAAAAAACATTGCAAC
    DBOX CN (SEQ (SEQ ID NO: 2982)
    ID NO:
    2837)
    APC/C Shugoshin 1: loop HLSLKDI CATCTGAGCCTGAAAGATATTACCAAC (SEQ
    DBOX TN (SEQ ID NO: 2983)
    ID NO:
    2838)
    APC/C Bcl-2- loop RSPLFIF CGCAGCCCGCTGTTTATTTTT (SEQ ID NO:
    like (SEQ ID 2984)
    protein NO: 2839)
    11:
    DBOX
    APC/C Bcl-2- loop RSSLLSR CGCAGCAGCCTGCTGAGCCGC (SEQ ID NO:
    like (SEQ ID 2985)
    protein NO: 2840)
    11:
    DBOX
    APC/C Securin: loop RKALGT CGCAAAGCGCTGGGCACCGTG (SEQ ID NO:
    DBOX V (SEQ ID 2986)
    NO: 2841)
    APC/C Securin- loop RKALGT CGCAAAGCGCTGGGCACCGTG (SEQ ID NO:
    2:DBOX V (SEQ ID 2987)
    NO: 2842)
    APC/C Ski-like loop RLCLPQV CGCCTGTGCCTGCCGCAGGTG (SEQ ID NO:
    protein: (SEQ ID 2988)
    DBOX NO: 2843)
    APC/C Aurora loop RLPLAQV CGCCTGCCGCTGGCGCAGGTG (SEQ ID NO:
    kinase B: (SEQ ID 2989)
    DBOX NO: 2844)
    APC/C Serine/ loop NRKPLTV AACAGGAAGCCCCTGACCGTGCTGAAC
    threonine- LN (SEQ (SEQ ID NO: 2990)
    protein ID NO:
    kinase 2845)
    PLK1:
    DBOX
    APC/C Cyclin- loop RAALAV CGCGCGGCGCTGGCGGTGCTG (SEQ ID NO:
    A2: L (SEQ ID 2991)
    DBOX NO: 2846)
    APC/C G2/mitotic- loop PRTALGD CCGCGCACCGCGCTGGGCGATATTGGC (SEQ
    specific IG (SEQ ID NO: 2992)
    cyclin- ID NO:
    B1: 2847)
    DBOX
    APC/C G2/mitotic- loop RSAFEDL CGCAGCGCGTTTGAAGATCTGACCAAC (SEQ
    specific TN (SEQ ID NO: 2993)
    cyclin- ID NO:
    B3: 2848)
    DBOX
    APC/C S-phase loop HRKHLQ CATCGCAAACATCTGCAGGAAATTCCG (SEQ
    kinase- EIP (SEQ ID NO: 2994)
    associated ID NO:
    protein 2849)
    2:DBOX
    APC/C Nuclear loop RSGLQLS CGCAGCGGCCTGCAGCTGAGC (SEQ ID NO:
    autoantigen (SEQ ID 2995)
    Sp- NO: 2850)
    100:
    DBOX
    APC/C Nucleolar loop RRGLILA CGCCGCGGCCTGATTCTGGCG (SEQ ID NO:
    and (SEQ ID 2996)
    spindle- NO: 2851)
    associated
    protein
    1: DBOX
    APC/C BRCA1- loop RHCLPTL CGCCATTGCCTGCCGACCCTG (SEQ ID NO:
    A (SEQ ID 2997)
    complex NO: 2852)
    subunit
    RAP80:
    DBOX
    APC/C BARD1: loop RNLLHD CGCAACCTGCTGCATGATAAC (SEQ ID NO:
    DBOX N (SEQ ID 2998)
    NO: 2853)
    APC/C BARD1: loop RAALDRL CGCGCGGCGCTGGATCGCCTG (SEQ ID NO:
    DBOX (SEQ ID 2999)
    NO: 2854)
    APC/C E3 loop RKKL CGCAAAAAACTG (SEQ ID NO: 3000)
    ubiquitin (SEQ ID
    ligase NO: 2855)
    RNF157:
    DBOX
    APC/C E3 loop RRRL CGCCGCCGCCTG (SEQ ID NO: 3001)
    ubiquitin (SEQ ID
    ligase NO: 2856)
    RNF157:
    DBOX
    APC/C Nuclear- loop RARLCSS CGCGCGCGCCTGTGCAGCAGC (SEQ ID NO:
    interacting (SEQ ID 3002)
    partner NO: 2857)
    of ALK:
    DBOX
    APC/C Nuclear- loop RLPLVPE CGCCTGCCGCTGGTGCCGGAA (SEQ ID NO:
    interacting (SEQ ID 3003)
    partner NO: 2858)
    of ALK:
    DBOX
    APC/C Tribbles loop RKKLVLE CGCAAAAAACTGGTGCTGGAA (SEQ ID NO:
    homolog (SEQ ID 3004)
    3: DBOX NO: 2859)
    APC/C Anillin: loop RENLQRK CGCGAAAACCTGCAGCGCAAA (SEQ ID NO:
    DBOX (SEQ ID 3005)
    NO: 2860)
    APC/C Anillin: loop RQPLSEA CGCCAGCCGCTGAGCGAAGCG (SEQ ID NO:
    DBOX (SEQ ID 3006)
    NO: 2861)
    APC/C Ninein- loop RTQLETK CGCACCCAGCTGGAAACCAAA (SEQ ID NO:
    like (SEQ ID 3007)
    protein: NO: 2862)
    DBOX
    APC/C Dual loop RNSLRQT CGCAACAGCCTGCGCCAGACC (SEQ ID NO:
    specificity (SEQ ID 3008)
    protein NO: 2863)
    kinase
    TTK:
    DBOX
    APC/C Inactive loop RYGLHPD CGCTATGGCCTGCATCCGGAT (SEQ ID NO:
    serine/ (SEQ ID 3009)
    threonine- NO: 2864)
    protein
    kinase
    TEX14:
    DBOX
    APC/C DBOX loop PRLPLGD CCGCGTCTGCCGCTGGGTGATGTTAGCAAT
    VSNN AAT (SEQ ID NO: 3010)
    (SEQ ID
    NO: 2865)
    APC/C Bub1b loop AKENE GCGAAAGAAAACGAA (SEQ ID NO: 3011)
    (SEQ ID
    NO: 2866)
    APC/C Bub1b loop SKENV AGCAAAGAAAACGTG (SEQ ID NO: 3012)
    (SEQ ID
    NO: 2867)
    APC/ Consensus loop .[ILM]R$ NNN[ATT/CTG/ATG]CTG
    CTPR1
    SCFFbw7_1 Consensus loop [LIVMP]. [CTG/ATT/GTT/ATG/CCG][NNN{0,2}]ACCCC
    {0,2}pTP.. GNNNNNN[AGC/ACC](SEQ ID NO: 3013)
    [pSpT]
    SCFFbw7_1 Neurogenic loop PFLpTPpS CCGTTTCTGACCCCGAGCCCGGAA (SEQ ID
    locus PE (SEQ NO: 3014)
    notch ID NO:
    homolog 2868)
    protein 1
    SCFFbw7_1 Uracil- loop PGpTPPSp CCGGGCACCCCGCCGAGCAGC (SEQ ID NO:
    DNA S (SEQ ID 3015)
    glycosylase NO: 2869)
    SCFFbw7_1 G1/S- loop LLpTPPQp CTGCTGACCCCGCCGCAGAGC (SEQ ID NO:
    specific S (SEQ ID 3016)
    cyclin-E1 NO: 2870)
    SCFFbw7_2 Consensus loop [LIVMP]. [CTG/ATT/GTT/ATG/CCG][NNN{0,2}]ACCCC
    {0,2}pTP..E GNNNNNNGAA (SEQ ID NO: 3017)
    SCFFbw7_2 Neurogenic loop PFLpTPSP CCGTTTCTGACCCCGAGCCCGGAA (SEQ ID
    locus E (SEQ ID NO: 3018)
    notch NO: 2871)
    homolog
    protein 1
    SCFFbw7 G1/S- loop SLIPpTPD AGCCTGATTCCGACCCCGGATAAA (SEQ ID
    specific K (SEQ ID NO: 3019)
    cyclin-E1 NO: 2872)
    SCFFbw7 cyclin- loop PEQTSEP CCGGAACAGACCAGCGAACCGACCGATGTT
    D3 TDVAI GCAATT (SEQ ID NO: 3020)
    (SEQ ID
    NO: 2873)
    SCFFbw7 Sterol loop SDSEPD AGCGATAGCGAACCGGAT (SEQ ID NO: 3021)
    regulatory (SEQ ID
    element- NO: 2874)
    binding
    protein 1
    SCFFbw7 SV40 loop TPxxE ACCCCGNNNNNNGAA (SEQ ID NO: 3022)
    SCFFbw7 cyclin E1 loop SLIPEPDR AGCCTGATTCCGGAACCGGATCGT (SEQ ID
    (SEQ ID NO: 3023)
    NO: 2875)
    SCFFbw7 Nuclear loop pSGVETp AGCGGCGTGGAAACCAGCTTT (SEQ ID NO:
    factor SF (SEQ 3024)
    NF- ID NO:
    kappa-B 2876)
    p105
    subunit
    SCFFbw7 E3 loop LKLKKSL CTGAAACTGAAAAAAAGCCTG (SEQ ID NO:
    ubiquitin (SEQ ID 3025)
    ligase NO: 2877)
    RNF157
    SCFFbw7 NF- loop pSGLDpS AGCGGCCTGGATAGC (SEQ ID NO: 3026)
    kappa-B (SEQ ID
    inhibitor NO: 2878)
    alpha
    SCFFbw7 NF- loop DpSGIEpS GATAGCGGCATTGAAAGC (SEQ ID NO: 3027)
    kappa-B (SEQ ID
    inhibitor NO: 2879)
    epsilon
    SCFFbw7 Program loop pSSRDSG AGCAGCCGCGATAGCGGCCGCGGCGATAGC
    med cell RGDSD (SEQ ID NO: 3028)
    death (SEQ ID
    protein 4 NO: 2880)
    SCFFbw7 NF- loop DpSGLGp GATAGCGGCCTGGGCAGC (SEQ ID NO: 3029)
    kappa-B S (SEQ ID
    inhibitor NO: 2881)
    beta
    SCFFbw8 myc loop EPPLEP GAACCGCCTCTGGAACCG (SEQ ID NO: 3030)
    (SEQ ID
    NO: 2882)
    SCF_TI Consensus loop .[VLIA][V NNN[GTG/CTG/ATT/GCG][GTG/CTG/ATT]GG
    R1 LI]GWPP TTATCCGCCG[GTG/CTG/ATT]NNNNNNCGTN
    [VLI]...R. NN (SEQ ID NO: 3031)
    (SEQ ID
    NO: 2883)
    Cul4- Consensus loop [NQ]{0,1}. [AAC/CAG{0,1}]NNNNNN[ATT/CTG/ATG/GTG]
    DDB1- .[ILMV][S [AGC/ACC][GAC/GAA/AAC][TTT/TAT][TTT/T
    Cdt2_1 T][DEN][F AT][NNN{0,3}][AAA/CGT{2,3}][NNN
    Y][FY].{2, (∨GAA/GAT)]
    3}[KR]{2,
    3}[{circumflex over ( )}DE]
    Cul4- Consensus loop [NQ]{0,1}. [AAC/CAG{0,1}]NNNNNN[ATT/CTG/ATG/GTG]
    DDB1- .[ILMV] [ACC][GAC/GAA/AAC][CAT/ATG/TTT/TAT][T
    Cdt2_2 T[DEN][H TT/TAT/ATG][NNN{2,3}][AAA/CGT{2,3}][NNN
    MFY][F (∨GAA/GAT)]
    MY].{2,3}
    [KR]{2,3}
    [{circumflex over ( )}DE]
    Cul4- PIP loop QRRMTD CAGCGTCGTATGACCGATTTTTATGCACGTC
    DDB1- FYARRR GTCGT (SEQ ID NO: 3032)
    Cdt2 (SEQ ID
    NO: 2884)
    DDB1- paramoxyvirus helix TVAYFTL ACCGTTGCATATTTTACCCTGCAGCAGGTTT
    CUL4 SV5-V QQVYG ATGGT (SEQ ID NO: 3033)
    protein (SEQ ID
    NO: 2885)
    DDB1- Hepatitis helix ILPAVLH ATTCTGCCTGCAGTTCTGCATCTGCGTACCG
    CUL4 B virus X LRTVYG TTTATGGT (SEQ ID NO: 3034)
    protein (SEQ ID
    NO: 2886)
    DDB1- Woodchuck helix NFVAWH AATTTTGTTGCATGGCATGCACTGCGTCAGG
    CUL4 Hepatitis ALRQVY TTTATGGT (SEQ ID NO: 3035)
    virus X G (SEQ ID
    protein NO: 2887)
    DDB1- DCAF9 helix NITADLIL AACATTACCGCAGATCTGATTCTGCGTCAG
    CUL5 RQVYG GTTTATGGT (SEQ ID NO: 3036)
    (SEQ ID
    NO: 2888)
    Unknown Bonger loop RRRG CGTCGTCGTGGT (SEQ ID NO: 3037)
    (SEQ ID
    NO: 2889)
    SOCS iNOS loop DINN GACATCAACAAC (SEQ ID NO: 3038)
    box- (SEQ ID
    Cul5- NO: 2890)
    SPSB2
    SCF_ Consensus loop DpSG.{2,3} GATAGCGGC[NNN{2,3}][AGC/ACC](SEQ ID
    TRCP1_1 [pSpt] NO: 3039)
    SCF_ SETBP1 loop DSGIGT GATAGCGGCATTGGCACC (SEQ ID NO: 3040)
    TRCP1_1 (SEQ ID
    NO: 2891)
    β-TRCP β-catenin loop DEGNYE GATGAAGGCAACTATGAA (SEQ ID NO: 3041)
    (SEQ ID
    NO: 2892)
    β-TRCP Vpu loop DSGxxS GATAGCGGCNNNNNNAGC (SEQ ID NO:
    (SEQ ID 3042)
    NO: 2893)
    β-TRCP RE1- loop SEGSDDS AGCGAAGGCAGCGATGATAGCGGCCTG
    sliencing GL (SEQ (SEQ ID NO: 3043)
    transcript ID NO:
    ion factor 2894)
    β-TRCP Prolactin loop TDSGRGS ACCGATAGCGGCCGCGGCAGC (SEQ ID NO:
    receptor (SEQ ID 3044)
    NO: 2895)
    β-TRCP Protein loop DSGYNT GATAGCGGCTATAACACC (SEQ ID NO: 3045)
    aurora (SEQ ID
    borealis NO: 2896)
    β-TRCP Vaccinia loop YSGNLEp TATAGCGGCAACCTGGAAAGC (SEQ ID NO:
    virus NO: 2897) 3046)
    SCFFbw2 G1/S- loop pSQTSTPT AGCCAGACCAGCACCCCGACCGATGTGACC
    specific DVTAIHL GCGATTCATCTG (SEQ ID NO: 3047)
    cyclin- (SEQ ID
    D3 NO: 2898)
    SCFFbw3 G1/S- loop PTDVTAI CCGACCGATGTGACCGCGATT (SEQ ID NO:
    specific (SEQ ID 3048)
    cyclin- NO: 2899)
    D3
    SCFFbw4 cyclin D1 loop EEEVSLA GAAGAAGAAGTTAGCCTGGCAAGCGAACCG
    SEPTDVR ACCGATGTTCGTGAT (SEQ ID NO: 3049)
    D (SEQ ID
    NO: 2900)
    OPDH consensus loop [IL]ApT.{6, [ATT/CTG]GCGACC[NNN{6,8}][TTT/CTG/ATT/
    VHL 1 8}[FLIVM]. GTG/ATG]NNN[TTT/CTG/ATT/GTG/ATG]
    [FLIVM] (SEQ ID NO: 3050)
    SCF coil consensus loop [RK][RK]. [CGT/AAA][CGT/AAA]NNNAGCCTGNNNTTT
    SL.F[FLM]. [TTT/CTA/ATG]NNN[CGT/AAA]CGT[CGT/AAA/
    [RK]R[HR CAT]NNN[CGT/AAA](SEQ ID NO: 3051)
    K].[RK]
    (SEQ ID
    NO: 2901)
    CHIP Hsp90 loop or ASRMEE GCAAGCCGTATGGAAGAAGTTGAT (SEQ ID
    C- VD (SEQ NO: 3052)
    terminus ID NO:
    2902)
    CHIP Hsp70 loop or GPTIEEV GGTCCGACCATTGAAGAAGTTGAT (SEQ ID
    C- D (SEQ ID NO: 3053)
    terminus NO: 2903)
    SOCS VASA loop DINNNNN GACATCAACAACAACAACAACATCGTTGAA
    box- IVEDVER GACGTTGAACGTAAACGTGAATTCTACATC
    Cul5- KREFYI (SEQ ID NO: 3054)
    SPSB4 (SEQ ID
    NO: 2904)
    UBR5 PAM2 loop SKLSVNA TCTAAACTGTCTGTTAACGCGCCGGAATTCT
    PEFYPSG ACCCGTCTGGT (SEQ ID NO: 3055)
    (SEQ ID
    NO: 2905)
    CRL2(K Usp1 C- IGLLGG ATCGGTCTGCTGGGTGGT (SEQ ID NO: 3056)
    LHDC2) terminus (SEQ ID
    NO: 2906)
    GID4 Pro/N- N- PGLW CCGGGTCTGTGG (SEQ ID NO: 3057)
    degron terminus (SEQ ID
    NO: 2907)
    TRIM21 Fc loop WxW TGGNNNTGG
    fragment
    TRIM21 Fc loop HNH CATAACCAT
    fragment
    Nedd4 PPxY loop TAPPPAY ACCGCGCCGCCGCCGGCGTATGCGACCCTG
    motif ATLG GGC (SEQ ID NO: 3058)
    (SEQ ID
    NO: 2908)
    Elongin Vif loop SLxxxLxx AGCCTGNNNNNNNNNCTGNNNNNNNNNAT
    C xI (SEQ ID T (SEQ ID NO: 3059)
    NO: 2909)
    Unknown ID2 loop SRTPLTT AGCCGCACCCCGCTGACCACCCTGAAC (SEQ
    LN (SEQ ID NO: 3060)
    ID NO:
    2910)
    Unknown ZAP70 loop DGYTPEP GATGGCTATACCCCGGAACCG (SEQ ID NO:
    (SEQ ID 3061)
    NO: 2911)
    Unknown SH3R1 loop RPTAAVT CGCCCGACCGCGGCGGTGACCCCGATT (SEQ
    PI (SEQ ID ID NO: 3062)
    NO: 2912)
    Unknown ETV1 loop DEQFVPD GATGAACAGTTTGTGCCGGAT (SEQ ID NO:
    (SEQ ID 3063)
    NO: 2913)
    Unknown EPAS1 loop LAPYIPM CTGGCGCCGTATATTCCGATGGATGGCGAA
    DGEDFQL GATTTTCAGCTG (SEQ ID NO: 3064)
    (SEQ ID
    NO: 2914)
    Unknown hantavirus loop YVGLVW TATGTGGGCCTGGTGTGGGGCGTGCTGCTG
    GVLLTTE ACCACCGAACTGATTGTGTGGGCGGCGAGC
    LIVWAAS GCG (SEQ ID NO: 3065)
    A (SEQ ID
    NO: 2915)
    CRL4_ SETD8 loop PKTPPSS CCGAAAACCCCGCCGAGCAGCTGCGATAGC
    CDT2 1 CDSTN ACCAAC (SEQ ID NO: 3066)
    (SEQ ID
    NO: 2916)
    CBL Consensus loop [DN].pY[S [GAT/AAC]NNNTAT[AGC/ACC]NNNCCG (SEQ
    (PTK) T].P ID NO: 3067)
    CBL Consensus loop DpYR GATTATCGT
    (met)
    CBL SH2B loop RAIDNQY CGCGCGATTGATAACCAGTATACCCCGCTG
    adapter TPL (SEQ (SEQ ID NO: 3068)
    protein 3 ID NO:
    2917)
    CBL Protein loop IRGSNEY ATTCGCGGCAGCAACGAATATACCGAAGGC
    sprouty TEGPS CCGAGC (SEQ ID NO: 3069)
    homolog (SEQ ID
    1 NO: 2918)
    CBL Protein loop IRNTNEY ATTCGCAACACCAACGAATATACCGAAGGC
    sprouty TEGPT CCGACC (SEQ ID NO: 3070)
    homolog (SEQ ID
    2 NO: 2919)
    CBL Protein loop HVENDYI CATGTGGAAAACGATTATATTGATAACCCG
    sprouty DNPS AGC (SEQ ID NO: 3071)
    homolog (SEQ ID
    4 NO: 2920)
    CBL Tyrosine- loop SFNPYEP AGCTTTAACCCGTATGAACCGGAACTGGCG
    protein ELA (SEQ (SEQ ID NO: 3072)
    kinase ID NO:
    SYK 2921)
    CBL Tyrosine- loop TLNSDGp ACCCTGAACAGCGATGGCTATACCCCGGAA
    protein YTPEPA CCGGCG (SEQ ID NO: 3073)
    kinase (SEQ ID
    ZAP-70 NO: 2922)
    CBL Plexin- loop IPFLDYR ATTCCGTTTCTGGATTATCGCACCTATGCGG
    A3 TYAV TG (SEQ ID NO: 3074)
    (SEQ ID
    NO: 2923)
    CBL Plexin- loop IPFLDYR ATTCCGTTTCTGGATTATCGCACCTATGCGA
    A1 TYAM TG (SEQ ID NO: 3075)
    (SEQ ID
    NO: 2924)
    CBL Platelet- loop SIFDNLY AGCATTTTTAACAGCCTGTATACCACCCTGA
    derived TTLSD GCGAT (SEQ ID NO: 3076)
    growth (SEQ ID
    factor NO: 2925)
    receptor
    alpha
    CBL Platelet- loop SIFNSLYT AGCATTTTTAACAGCCTGTATACCACCCTGA
    derived TLSD GCGAT (SEQ ID NO: 3077)
    growth NO: 2926)
    factor
    receptor
    beta
    CBL Tumor loop KGDGGL AAAGGCGATGGCGGCCTGTATAGCAGCCTG
    necrosis YSSLPP CCGCCG (SEQ ID NO: 3078)
    factor (SEQ ID
    receptor NO: 2927)
    superfamily
    member
    16
    CRL4(C Trib1 loop SDQIVPE TCTGACCAGATCGTTCCGGAATAC (SEQ ID
    OP1/DET) Y(SEQ ID NO: 3079)
    NO: 2928)
    SH3RF1 E3 loop RPTAAVT CGCCCGACCGCGGCGGTGACCCCGATT (SEQ
    ubiquitin- PI (SEQ ID ID NO: 3080)
    protein NO: 2929)
    ligase
    SH3RF1
    COP-1 Consensus loop [D,E][D,E] [GAA/GAC][GAA/GAC][NNNNNN/NNNNNNN
    .{2,3}VP NN]GTGCCG[GAA/GAC](SEQ ID NO: 3081)
    [DE]
    COP-1 Tribbles loop SDQIVPE AGCGATCAGATTGTGCCGGAATAT (SEQ ID
    homolog Y (SEQ ID NO: 3082)
    1 NO: 2930)
    SIAH Consensus loop .P.A.V.P NNNCCGNNNGCGNNNGTGNNNCCG[NNN
    [{circumflex over ( )}P](SEQ ID EXCEPT CCN](SEQ ID NO: 3083)
    NO: 2931)
    SIAH AF4/FM loop KPTAYVR AAACCGACCGCGTATGTGCGCCCGATG (SEQ
    R2 (beta PM (SEQ ID NO: 3084)
    family sttrand) ID NO:
    member 2932)
    4
    SIAH calcyclin- loop KPAAVV AAACCGGCGGCGGTGGTGGCGCCGATT
    binding API (SEQ (SEQ ID NO: 3085)
    protein ID NO:
    2933)
    SIAH POU loop APTAVVL GCGCCGACCGCGGTGGTGCTGCCGCAT (SEQ
    domain PH (SEQ ID NO: 3086)
    class 2- ID NO:
    associating 2934)
    factor 1
    SIAH Retrotrans loop PPRALVL CCGCCGCGCGCGCTGGTGCTGCCGCAT (SEQ
    poson- PH (SEQ ID NO: 3087)
    derived ID NO:
    protein 2935)
    PEG10
    ERAD- CL1 amphipathic ACKNWF GCGTGCAAAAACTGGTTTAGCAGCCTGAGC
    C helix SSLSHFVI CATTTTGTGATTCATCTG (SEQ ID NO: 3088)
    extension HL (SEQ
    ID NO:
    2936)
    Nend_Nb N- ΛM{0,1}[F [ATG{0,1}][TTT/CTG/TAT/TGG/ATT][NNN∨CC
    ox 2 terminal LYIW][∨P] G]
    extension
    Nend_U N- ΛM{0,1}[R [ATG{0,1}][AAA/CGT][NNN∨CCG]
    BRbox 1 terminal K][∨P]
    extension
    Nend_U N- ΛM{0,1}[E [ATG{0,1}][GAT/GAA]
    BRbox 2 terminal D]
    extension
    Nend_U N- ΛM{0,1}[N [ATG{0,1}][CAG/AAC]
    BRbox 3 terminal Q]
    extension
    Nend_U N- ΛM{0,1}[C] [ATG{0,1}][TGC]
    BRbox 4 terminal
    extension
    Other
    degradation
    pathways:
    pathway;
    protein to Degron Intro-
    which the sequence duction
    degron derived to Amino acid
    binds from scaffold sequence DNA sequence E. Coli codon optimised 5′ to 3′
    ESRCT; Consensus, loop LYPxxxL GAACTGTACCCGCTGACCTCTCTGCG
    ALIX e.g. HIV (SEQ ID TTCT (SEQ ID NO: 3089)
    Gag NO: 2937),
    e.g.
    ELYPLTSL
    RS (SEQ ID
    NO: 2938)
    ESCRT; Nef loop ExxxLL GAANNNNNNNNNCTGCTG (SEQ ID
    AP-1 NO: 3090)
    ESCRT; Env loop YxxL TATNNNNNNCTG (SEQ ID NO: 3091)
    AP-2
    ESCRT; AP viral loop SREKPYK AGCCGCGAAAAACCGTATAAAGAAG
    adaptor EVTEDLL TGACCGAAGATCTGCTGCATCTGAAC
    HLNSLF AGCCTGTTT (SEQ ID NO: 3092)
    (SEQ ID
    NO: 2939)
    ESCRT; AP viral loop AAGAYDP GCGGCGGGCGCGTATGATCCGGCGCG
    adaptor ARKLLEQ CAAACTGCTGGAACAGTATGCGAAA
    YAKK AAA (SEQ ID NO: 3093)
    (SEQ ID
    NO: 2940)
    CMA Consensus loop KFERQ AAATTTGAACGCCAG (SEQ ID NO:
    (SEQ ID 3094)
    NO: 2941)
    CMA Consensus loop QRFFE CAGCGCTTTTTTGAA (SEQ ID NO:
    (SEQ ID 3095)
    NO: 2942)
    Autophagy; Consensus loop W/F/Y]xx[L/ (TGG/TTC/TAT)NNNNNN(CTG/ATC/GTG)
    LC3/Atg8 LIR (LC3- I/V]
    family interacting)/
    AIM
    (Atg8
    family-
    interacting)
    motif
    Autophagy; CCPG1 loop TASDDSDI ACCGCGTCTGACGACTCTGACATCGT
    FIP200 VTLEPPK TACCCTGGAACCGCCGAAA (SEQ ID
    (SEQ ID NO: 3096)
    NO: 2943)
    Autophagy; DVL loop EVRDRM GAAGTTCGTGACCGTATGTGGCTGAA
    LC3 WLKITI AATCACCATC (SEQ ID NO: 3097)
    (SEQ ID
    NO: 2944)
  • KEY
    • . any amino acid
    • allowed amino acid at the
    • [X] position
    • p phosphorylated amino acid
    • $ C-terminal of chain
    • {circumflex over ( )}X N-terminal of chain where x & y are the minimum, maximum of X
    • X{x,y} aminoacids at the position amino acid not allowed at
    • [vX] the position
    • NNN any codon one of these codons at the
    • [NNN/NNN/NNN] position
    • [vNNN] any codon except this codon, where x & y are the maximum & minimum of
    • [NNN{x,y}] codons
  • TABLE 4
    Multiple Alignment of DNA sequences of all CTPR and RTPR used in hetero-
    bifunctional CTPRs and RTPRs
    CLUSTAL multiple sequence alignment by MUSCLE (3.8)
    RTPRc GCAGAAGCACTGCGTAATCTGGGTCGTGTTTATCGTCGTCAGGGTCGTTATCAGCGTGCA
    RTPRa-ii-H GCCGAAGCTTGGTATAATCTGGGGAATGCCTATTACAGACAGGGGGATTATCAGCGCGCC
    RTPRa-i-E GCAGAAGCATGGTATAATCTGGGTAATGCATATTATCGCCAGGGTGATTATCAGCGTGCC
    RTPRa-iii-E GCAGAAGCATGGTATAATCTGGGCAATGCATATTATCGTCAGGGTGATTATCAGCGTGCC
    CTPRa-E GCAGAAGCATGGTATAATCTGGGTAATGCATATTACAAACAGGGCGATTATCAGAAAGCC
    CTPRb-E GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGATTATCAGAAAGCC
    RTPRb-E GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTOAACAGGGCGATTATCAGCGTGCC
    RTPRa-ii-E GCCGAGGCCTGGTATAACCTTGGCAACGCCTATTATCGTCAAGGCGACTACCAGAGAGCA
    RTPRc-H GCCGAGGCTCTGAGAAATCTGGGCAGAGTGTACAGACGGCAGGGCAGATACCAGCGGGCC
    CTPRb-H GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGACTACCAGAAGGCC
    RTPRb-H GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGACTACCAGCGGGCC
    RTPRa-iv-E GCCGAGGCCTGGTACAACCTGGGTAACGCCTATTATCGCCAAGGCGACTACCAGCGTGCA
    CTPRa-H GCCGAGGCCTGGTACAATCTGGGCAACGCCTACTACAAGCAGGGCGACTACCAGAAGGCC
    RTPRa-i-H GCCGAGGCCTGGTACAACCTGGGCAACGCCTACTACCGGCAGGGCGACTACCAGCGGGCC
    ** ** **   *   ** ** **    *  **       ** **    ** ***   **
    RTPRc ATTGAATATTATCGTCGCGCACTGGAATTAGATCCGVNNNNN (SEQ ID NO: 148)
    RTPRa-ii-H ATTGAATATTATCAGCOGGCTCTGGAACTGGATCCTNNNNNN (SEQ ID NO: 149)
    RTPRa-i-E ATTGAATATTATCAACGTGCACTGGAACTGGACCCGNNNNNN (SEQ ID NO: 150)
    RTPRa-iii-E ATCGAATATTATCAACGTGCACTGGAACTGGACCCGNNNNNN (SEQ ID NO: 151)
    CTPRa-E ATCGAGTATTATCAAAAAGCACTGGAACTGGACCCGNNNNNN (SEQ ID NO: 152)
    CTPRb-E ATCGAATATTATCAAAAAGCGCTGGAACTGGACCCGNNNNNN (SEQ ID NO: 153)
    RTPRb-E ATTGAATATTATCAACGTGCGCTGGAATTAGATCCGNNNNNN (SEQ ID NO: 154)
    RTPRa-ii-E ATCGAATATTACCAGCGTGCGTTAGAATTAGATCCGNNNNNN (SEQ ID NO: 155)
    RTPRc-H ATCGAGTATTACCGCAGAGCCCTGGAACTGGACCCCNNNNNN (SEQ ID NO: 156)
    CTPRb-H ATCGAGTATTATCAGAAGGCCCTGGAACTGGACCCCNNNNNN (SEQ ID NO: 157)
    RTPRb-H ATCGAGTATTATCAGAGAGCCCTGGAACTGGACCCCNNNNNN (SEQ ID NO: 158)
    RTPRa-iv-E ATTGAGTACTACCAACGTGCCCTGGAACTGGACCCTNNNNNN (SEQ ID NO: 159)
    CTPRa-H ATCGAGTATTATCAGAAGGCCCTGGAACTGGACCCCNNNNNN (SEQ ID NO: 160)
    RTPRa-i-H ATCGAGTACTACCAGAGAGCCCTGGAACTGGACCCTNNNNNN (SEQ ID NO: 161)
    ** ** ** ** *     **  * *** * ** ** ******
  • TABLE 5
    089 AEAYSNLGNVYKER 118 NP_858058.1
    GQLQEAIEHYRHALRL (SEQ ID NO: 162)
    191 AVAWSNLGCVFNAQ 220 (SEQ ID NO: 163)
    GEIWLAIHHFEKAVTL
    327 ADSLNNLANIKREQ 356 (SEQ ID NO: 164)
    GNIEEAVRLYRKALEV
    264 NLACVYYEQGLIDL 288 (SEQ ID NO: 165)
    AIDTYRRAIEL
    079 AEAYSNLGNVYKER 108 NP_858059.1
    GQLQEAIEHYRHALRL (SEQ ID NO: 166)
    181 AVAWSNLGCVFNAQ 210 (SEQ ID NO: 167)
    GEIWLAIHHFEKAVTL
    317 ADSLNNLANIKREQ 346 (SEQ ID NO: 168)
    GNIEEAVRLYRKALEV
    254 NLACVYYEQGLIDL 278 (SEQ ID NO: 169)
    AIDTYRRAIEL
    079 AEAYSNLGNVYKER 108 NP_003596.2
    GQLQEAIEHYRHALRL (SEQ ID NO: 170)
    812 ESFYNLGRGLHQLG 840 NP_036218.1
    LIHLAIHYYQKALEL (SEQ ID NO: 171)
    637 AWYGLGMIYYKQEK 664 NP_001247.2
    FSLAEMHFQKALDI (SEQ ID NO: 172)
    568 EAWCAAGNCFSLQR 596 (SEQ ID NO: 173)
    EHDIAIKFFQRAIQV
    275 AQSCYSLGNTYTLL 304 (SEQ ID NO: 174)
    QDYEKAIDYHLKHLAI
    058 YSQLGNAYFYLHDY 084 (SEQ ID NO: 175)
    AKALEYHHHDLTL
    315 GRACWSLGNAYTAL 344 (SEQ ID NO: 176)
    GNHDQAMHFAEKHLEI
    247 NMGNIYLKQRNYSK 270 NP_783195.2
    AIKFYRMALD (SEQ ID NO: 177)
    495 ALTNKGNTVFANGD 520 (SEQ ID NO: 178)
    YEKAAEFYKEAL
    238 NMGNIYLKQRNYSK 261 NP_006522.2
    AIKFYRMALD (SEQ ID NO: 179)
    486 ALTNKGNTVFANGD 511 (SEQ ID NO: 180)
    YEKAAEFYKEAL
    715 AQAWMNMGGIQHIK 744 NP_787057.2
    GKYVSARAYYERALQL (SEQ ID NO: 181)
    575 AEILSPLGALYYNT 604 (SEQ ID NO: 182)
    GRYEEALQIYQEAAAL
    114 AQAAKNKGNKYFKA 143 NP_055635.3
    GKYEQAIQCYTEAISL (SEQ ID NO: 183)
    610 YNLGKLYHEQGHYE 634 NP_689801.1
    EALSVYKEAIQ (SEQ ID NO: 184)
    586 AQAWMNMGGIQHIK 615 NP_114126.2
    GKYVSARAYYERALQL (SEQ ID NO: 185)
    446 AEILSPLGALYYNT 472 (SEQ ID NO: 186)
    GRYEEALQIYQEA
    018 AETFKEQGNAYYAK 47 NP_003306.1
    KDYNEAYNYYTKAIDM (SEQ ID NO: 187)
    300 AKAYARIGNSYFKE 327 NP_006810.1
    EKYKDAIHFYNKSL (SEQ ID NO: 188)
    231 LGNDAYKKKDFDTA 254 (SEQ ID NO: 189)
    LKHYDKAKEL
    365 NKGNECFQKGDYPQ 387 (SEQ ID NO: 190)
    AMKHYTEAI
    028 AETFKEQGNAYYAK 057 AAH11837.2
    KDYNEAYNYYTKAIDM (SEQ ID NO: 191)
    228 AYSNLGNAHVFLGR 255 NP_056412.2
    FDVAAEYYKKTLQL (SEQ ID NO: 192)
    266 AQACYSLGNTYTLL 293 (SEQ ID NO: 193)
    QDYERAAEYHLRHL
    28 AEELKTQANDYFKAK 57 NP_006238.1
    DYENAIKFYSQAIEL (SEQ ID NO: 194)
    318 DAYKSLGQAYRELG 346 NP_078801.2
    NFEAATESFQKALLL (SEQ ID NO: 195)
    1262 ETLKNLAVLSYEG 1290 NP_694972.3
    GDFEKAAELYKRAMEI (SEQ ID NO: 196)
    140 GNKYFKQGKYDEAI 161 NP_078880.1
    DCYTKGMD (SEQ ID NO: 197)
    289 GNGFFKEGKYERAI 309 (SEQ ID NO: 198)
    ECYTRGI
    600 CWESLGEAYLSRGG 627 NP_055454.1
    YTTALKSFTKASEL (SEQ ID NO: 199)
    172 KATYRAGIAFYHLG 197 NP_689692.2
    DYARALRYLQEA (SEQ ID NO: 200)
    174 LGKIHLLEGDLDKA 196 NP_149017.2
    IEVYKKAVE (SEQ ID NO: 201)
    158 LGDLFSKAGDFPRA 181 NP_038460.3
    AEAYQKQLRF (SEQ ID NO: 202)
    384 AYFNAGNIYFHHRQ 411 NP_001007796.1
    FSQAS DYFSKALKF
    (SEQ ID NO: 203)
    104 EAWNQLGEVYWKKG 130 NP_612385.1
    DVAAAHTCFSGAL (SEQ ID NO: 204)
    814 EAWQGLGEVLQAQG 842 NP_065191.2
    QNEAAVDCFLTALEL (SEQ ID NO: 205)
    446 AKLWNNVGRALENE 472 NP_861448.1
    KNFERALKYFLQA (SEQ ID NO: 206)
    597 ADLWYNLAIVHIEL 626 (SEQ ID NO: 207)
    KEPNEALKNFNRALEL
    251 YRRKGDLDKAIELF 269 NP_001540.2
    QRVLE (SEQ ID NO: 208)
    251 YRRKGDLDKAIELF 269 NP_001026853.1
    QRVLE
    (SEQ ID NO: 209)
    079 AKTYKDEGNDYFKE 106 NP_004614.2
    KDYKKAVISYTEGL (SEQ ID NO: 210)
    501 AKVHYNIGKNLADK 530 NP_116202.2
    GNQTAAIRYYREAVRL (SEQ ID NO: 211)
    482 AKVHYNIGKNLADK 511 NP_001073137.1
    GNQTAAIRYYREAVRL
    (SEQ ID NO: 212)
    200 GNELVKKGNHKKAI 220 NP_006800.2
    EKYSESL (SEQ ID NO: 213)
    123 GNEQFKKGDYIEAE 145 NP_003305.1
    SSYSRALEM (SEQ ID NO: 214)
    438 ESLSLLGFVYKLEG 466 NP_001001887.1
    NMNEALEYYERALRL
    (SEQ ID NO: 215)
    438 ESLSLLGFVYKLEG 466 NP_001539.3
    NMNEALEYYERALRL (SEQ ID NO: 216)
    375 AKTKNNLASAYLKQ 402 NP_803136.2
    NKYQQAEELYKEIL (SEQ ID NO: 217)
    564 WFSLGCAYLALEDY 590 NP_060205.3
    QGSAKAFQRCVTL (SEQ ID NO: 218)
    306 AESCYQLARSFHVQ 335 NP_055448.1
    EDYDQAFQYYYQATQF (SEQ ID NO: 219)
  • TABLE 6
    CTPRa E. coli expression codon optimised
    GCAGAAGCATGGTATAATCTGGGTAATGCATATTACAAACAGGGCGA
    TTATCAGAAAGCCATCGAGTATTATCAAAAAGCACTGGAACTGGACC
    CGNNNNNN
    AEAWYNLGNAYYKQGDYQKAIEYYQKALELDPXX
    (SEQ ID NO: 220)
    CTPRa H. Sapiens expression codon optimised
    GCCGAGGCCTGGTACAATCTGGGCAACGCCTACTACAAGCAGGGCGA
    CTACCAGAAGGCCATCGAGTATTATCAGAAGGCCCTGGAACTGGACC
    CCNNNNNN
    AEAWYNLGNAYYKQGDYQKAIEYYQKALELDPXX
    (SEQ ID NO: 220)
    RTPRa-i H. sapiens expression codon optimised
    GCCGAGGCCTGGTACAACCTGGGCAACGCCTACTACCGGCAGGGCGA
    CTACCAGCGGGCCATCGAGTACTACCAGAGAGCCCTGGAACTGGACC
    CTNNNNNN
    AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 221)
    RTPRa-ii H. sapiens expression codon optimised
    GCCGAAGCTTGGTATAATCTGGGGAATGCCTATTACAGACAGGGGGA
    TTATCAGCGCGCCATTGAATATTATCAGCGGGCTCTGGAACTGGATC
    CTNNNNNN
    AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 221)
    RTPRa-i E. coli expression codon optimised
    GCAGAAGCATGGTATAATCTGGGTAATGCATATTATCGCCAGGGTGA
    TTATCAGCGTGCCATTGAATATTATCAACGTGCACTGGAACTGGACC
    CGNNNNNN
    AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 221)
    RTPRa-ii E. coli expression codon optimised
    GCCGAGGCCTGGTATAACCTTGGCAACGCCTATTATCGTCAAGGCGA
    CTACCAGAGAGCAATCGAATATTACCAGCGTGCGTTAGAATTAGATC
    CGNNNNNN
    AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 221)
    RTPRa-iii E. coli expression codon optimised
    GCAGAAGCATGGTATAATCTGGGCAATGCATATTATCGTCAGGGTGA
    TTATCAGCGTGCCATCGAATATTATCAACGTGCACTGGAACTGGACC
    CGNNNNNN
    AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 221)
    RTPRa-iv E. coli expression codon optimised
    GCCGAGGCCTGGTACAACCTGGGTAACGCCTATTATCGCCAAGGCGA
    CTACCAGCGTGCAATTGAGTACTACCAACGTGCCCTGGAACTGGACC
    CTNNNNNN
    AEAWYNLGNAYYRQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 221)
    CTPRb E. coli expression codon optimised
    GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGA
    TTATCAGAAAGCCATCGAATATTATCAAAAAGCGCTGGAACTGGACC
    CGNNNNNN
    AEALNNLGNVYREQGDYQKAIEYYQKALEL-DPXX
    (SEQ ID NO: 222)
    CTPRb H. sapiens expression codon optimised
    GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGA
    CTACCAGAAGGCCATCGAGTATTATCAGAAGGCCCTGGAACTGGACC
    CCNNNNNN
    AEALNNLGNVYREQGDYQKAIEYYQKALEL-DPXX
    (SEQ ID NO: 222)
    RTPRb E. coli expression codon optimised
    GCAGAAGCACTGAATAATCTGGGTAATGTTTATCGTGAACAGGGCGA
    TTATCAGCGTGCCATTGAATATTATCAACGTGCGCTGGAATTAGATC
    CGNNNNNN
    AEALNNLGNVYREQGDYQRAIEYYQRALEL-DPXX
    (SEQ ID NO: 223)
    RTPRb H. sapiens expression codon optimised
    GCCGAGGCTCTGAACAACCTGGGCAACGTGTACAGAGAGCAGGGCGA
    CTACCAGCGGGCCATCGAGTATTATCAGAGAGCCCTGGAACTGGACC
    CCNNNNNN
    AEALNNLGNVYREQGDYQRAIEYYQRALELDPXX
    (SEQ ID NO: 223)
    RTPRc E. coli expression codon optimised
    GCAGAAGCACTGCGTAATCTGGGTCGTGTTTATCGTCGTCAGGGTCG
    TTATCAGCGTGCAATTGAATATTATCGTCGCGCACTGGAATTAGATC
    CGNNNNNN
    AEALRNLGRVYRRQGRYQRAIEYYRRALELDPXX
    (SEQ ID NO: 225)
    RTPRc H. Sapiens expression codon optimised
    GCCGAGGCTCTGAGAAATCTGGGCAGAGTGTACAGACGGCAGGGCAG
    ATACCAGCGGGCCATCGAGTANTACCGCAGAGCCCTGGAACTGGACC
    CCNNNNNN
    AEALRNLGRVYRRQGRYQRAIEYYRRALELDPXX
    (SEQ ID NO: 225)
  • TABLE 7
    RSCB therapeutic
    Protein Target Paratope struct no area
    Nrp1/2 RASQYFSSYLA loop 2qqn anti-
    (SEQ ID NO: 226) angiogenic
    Nrp1/2 AREDFRNRRLWYVMDY helix 2qql anti-
    (SEQ ID NO: 227) angiogenic
    IL18 KASGYSFTDYFIY helix 2yxt anti-
    (SEQ ID NO: 228) inflammatory
    IL15 YRDRRRPS loop 2xqb anti-
    (SEQ ID NO: 229) inflammatory
    Thyroid stimulating SGSSSDIGSNYVS loop 2xwt
    hormone receptor (SEQ ID NO: 230)
    EGF receptor QQWSSHIFT loop 3C09 cancer
    (SEQ ID NO: 231)
    EGF receptor ASRDYDYAGRYFDY helix 3C09 cancer
    (SEQ ID NO: 232)
    IL23 QNGHSFPFT loop 3d85 anti-
    (SEQ ID NO: 233) inflammatory
    IL23 YINPYNDGTK helix 3d85 anti-
    (SEQ ID NO: 234) inflammatory
    IL23 ARNWDVAY helix 3d85 anti-
    (SEQ ID NO: 235) inflammatory
    Lymphocyte function- SGYSFTGHWMN helix 3eoa auto-immune
    associated antigen (SEQ ID NO: 236)
    1 (LFA-1)
    Lymphocyte function- MIHPSDSETR helix 3eoa auto-immune
    associated antigen (SEQ ID NO: 237)
    1 (LFA-1)
    Lymphocyte function- ARGIYFYGTTYFDY helix 3eoa auto-immune
    associated antigen (SEQ ID NO: 238)
    1 (LFA-1)
    C3b SGFSFTSSVS helix 3g6j anti-
    (SEQ ID NO: 239) inflammatory
    C3b LIYPYNGFN helix 3g6j anti-
    (SEQ ID NO: 240) inflammatory
    FGF receptor
     3 AASGFTFTSTGIS helix 3grw multiple
    (SEQ ID NO: 241) myeloma
    FGF receptor
     3 ARTYGIYDLYVDYTE helix 3grw multiple
    (SEQ ID NO: 242) myeloma
    IL2 SRDYGYYFD helix 3iu3 anti-
    (SEQ ID NO: 243) inflammatory
    IL2 GYSFTRYWMH helix 3iu3 anti-
    (SEQ ID NO: 244) inflammatory
    HER2 QWWWWPST loop 3n85 breast cancer
    (SEQ ID NO: 245)
    HER2 ASGFSIWWSWIH helix 3n85 breast cancer
    (SEQ ID NO: 246)
    membrane-type YDNNQRPS loop 3nps metastasis of
    serine protease 1 (SEQ ID NO: 247) carcinomas
    membrane-type TFHIRRYRSGYY helix 3nps metastasis of
    serine protease 1 DKMDH carcinomas
    (SEQ ID NO: 248)
    beta-secretase ARGPFSPWVMDY helix 3r1g Alzheimer's
    (SEQ ID NO: 249) disease
    VEGF-R TRHDGTNFD helix 3s35 anti-
    (SEQ ID NO: 250) angiogenic
    VEGF-R QQAKAFPPT loop 3s37 anti-
    (SEQ ID NO: 251) angiogenic
    irp5/6 receptor SGHVNAVKNYGY helix 3sob bone-loss
    (SEQ ID NO: 252)
    hepsin protease WINTETGS Helix 3t2n prostrate
    (SEQ ID NO: 253) cancer
    Factor D WINTYTGE helix 4d9r anti-
    (SEQ ID NO: 254) inflammatory
    Factor D GYTFTNYGMN helix 4d9r anti-
    (SEQ ID NO: 255) inflammatory
  • TABLE 8
    1. Axin-RTPR-ABRA
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPGGSLSSAFHVFEDGNKENGGPNAEANWYNLGNAYYRQGDYQRAIEYYQRAELDPNN
    (SEQ ID NO: 256)
    2. Axin-RTPR-DBOX
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPGGPRLPLGDVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 257)
    3. Axin-RTPR-KEN
    MGSGAYPEYILDIEVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYKLGNAYYRQG
    DYQRAIEYYQRALELDPGGPRLPLGDVENNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 258)
    4. Axin-RTPR-Nrf2
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPGGPRLPLGDVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 259)
    5. Axin-RTPR-SIAH
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPGGLRPVAMVRPTVGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 20)
    6. Axin-RTPR-SPOP
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGLACDEVTSTTSSSTA
    GGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 261)
    7. Axin-RTPR-p27
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 262)
    8. Axin-RTPR-p53
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 263)
    10. Bc19-RTPR-ABBA
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYY
    RQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID ND: 264)
    11. Bc19-RTPR-DBOX-v1
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEANYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYY
    RQGDYQRAIEYYQRALELDPGGPRLPLGDVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 265)
    12. Bc19-RTPR-DBOX-v2
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGPRLPLGDVSNNG
    GPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 266)
    13. Bc19-RTPR-KEN
    MGSGAYPEYILDIHVYRVQLELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPGGSLSSAFHVFEDGNKENGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 267)
    14. Bc19-RTPR-Nrf2
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYY
    RQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 268)
    15. Bc19-RTPR-p27
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYY
    RQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYMLGNAYYRQGDYQRAIEYQRALELDPNN
    (SEQ ID NO: 269)
    16. Bc19-RTPR-p53
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYY
    RQGDYQRAIEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 270)
    17. Bc19-RTPR-SIAH
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYY
    RQGDYQRAIEYYQRALELDPGGLRPVAMVRPTVGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 271)
    18. Bc19-RTPR-SPOP
    MGSSQEQLEHRYRSLITLYDIQLMLDPNNAEAWYNLGNAYYRQGDYQRATEYYQRALELDPGGLACDEVTSTTSS
    STAGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 273)
    19. TCF7L2-RTPR-Nrf2
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYESTQDGGPNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAY
    YRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 273)
    20. TCF7L2-RTPR-p27
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYESTQDGGPNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLG
    NAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 274)
    21. p27-RTPR-TCF7L2
    MRGSHHHHHHGLVPRGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLGNA
    YYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYEST
    QDGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRS (SEQ ID NO: 275)
    22. TCF7L2-RTPR-p53
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYESTQDGGPNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLSAYG
    (SEQ ID NO: 276)
    23. ICAT-RTPR-P27
    MGSYAYQRAIVEYMLRLMSDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQ
    RAIEYYQRALELDPNNAGSNEQEPKKKRSPDAEAWYNGNAYYRQGDYQRAIEYYQRALELPENN
    (SEQ ID NO: 277
    24. ICAT-RTPR-p53
    MGSYAYQRAIVEYMLRLMSDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQ
    RAIEYYQRALELDPNNFAAYWNLLSAYG (SEQ ID NO: 278)
    25. LRH1-RTPR-AABA
    MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPGGSEDKENVPPGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 279)
    26. LRH1-RTPR-DBOX
    MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPGGPRLPLGDVSNNGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 280)
    27. LRH1-RTPR-KEN
    MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPGGSEDKENVPPGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 281)
    28. LRH1-RTPR-Nrf2
    MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 282
    29. LRH1-RTPR-p27
    MQSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 283)
    30. LRH1-RTPR-p53
    MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPNNFAAYWNLLSAYG
    (SEQ ID NO: 284)
    31. LRH1-RTPR-SIAH
    MGSYEQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPGGLRPVAMVRPTVGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 285)
    32. LRH1-RTPR-SPOP
    MGSYDQAIAAYLDALMCDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRA
    IEYYQRALELDPGGLACDEVTSTTSSSTAGGPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN
    (SEQ ID NO: 286)
    33. APC-RTPR-Nrf2
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPEGGSCEELEALEALELDEGGPNAEAWYNLGNAYYRQGDYQ
    RAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGDPETGELGGPNAEAWYNLGNAYYRQ
    GDYQRAIEYYQRALELDPNN (SEQ ID NO: 287)
    34. APC-RTPR-p27
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGSCSEELEALEALELDEGGPNAEAWYNLGNAYYRQGDYQ
    RAIEYYQRALELDPNN (SEQ ID NO: 288)
    35. p27-RTPR-APC
    MRGSHHHHHHGLVPRGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWYNLGNA
    YYRQGDYQRAIEYYQRALELDPRSAEAWYNLGNAYYRQGDYQRAIEVYQRALELDPGGSCSEELEALEALELDEG
    GPNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRS (SEQ ID NO: 289)
    36. APC-RTPRT-p53
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPGGQELGDNDELMHFSYESTQDGGPNAEAWYNLGNAYYRQG
    DYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNFAAYWNLLSAYG
    (SEQ ID NO: 290)
    37. 1TBP-CTPR2
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRS (SEQ ID NO: 291)
    38. 2TBP-CTPR4
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEMWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
    EYYQKALELDPRS (SEQ ID NO: 292)
    39. 3TBP-CTPR6
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
    EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGD
    YQKAIEYYQKALELDPRS (SEQ ID NO: 293)
    40. 4TBP-CTPR8
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
    EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGD
    YQKAIEYYQKALELDPRSAEAWYNDGNAYYKQGDXQIKIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAY
    YKQGDYQKAIEYYQKALELDPRS (SEQ ID NO: 294)
    41. 1TBP-CTPR2-Foldon (Foldon sequence in bold)
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAKASLNLANADIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLRS (SEQ ID NO: 295)
    42. 2TBP-CTPR4-Foldon (Foldon sequence in bold)
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAEAWYNLGNAYYKQGDYQKAIEYTQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
    EYYQKALELDPRSAKASLNLANADIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLRS
    (SEQ ID NO: 296)
    43. 3TBP-CTPR6-Foldon (Foldon sequence in bold)
    MGSAEAWYNDGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
    EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGP
    YQKAIEYYQKALELDPRSAKASLNLANADIKTLQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLRS
    (SEQ ID NO: 297)
    44. 4TBP-CTPR8-Foldon (Foldon sequence in bold)
    MGSAEAWYNLGNAYYKQGDYQKAIEYYQNKALELPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAIEYYQK
    ALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGDYQKAI
    EYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIETDDKALELDPNNREAGDGEEDPRSAEAWYNLGNAYYKQGD
    YQKAIEYYQKALELDPRSAEAWYNLGNAYYKQGDYQKAIEYYQKALELDPNNREAGDGEEDPRSAEAWYNLGNAY
    YKQGDYQKAIEYYQKALELDPRSAKASLNLANADIKTIQEAGYIPEAPRDGQAYVRKDGEWVLLSTFLRS
    (SEQ ID NO: 298)
    45. KBL-RTPR-CMA_Q
    MGSIPNPLLGLDGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISYDPAEAWYNLGNAYYRQGDYQR
    AIEYYQRALELDPRSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNQRFFE (SEQ ID NO: 299)
    46. CMA_Q-KBL-RTPR
    MGSQRFFEGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISYDPAEAWYNLGNAYYRQGDYQRAIEY
    YQRALELDPRSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 300)
    47. CKA_K-KBL-RTPR
    MGSRFERQGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISYDPAEAWYNLGNAYYRQGDYQRAIEY
    YQRALELDPRSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNN (SEQ ID NO: 301)
    48. SOS-RTPR-CMA_K
    MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQR
    AIEYYQRALELDPRSKFERQ (SEQ ID NO: 302)
    49. SOS-RTPR-CMA_Q
    MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPRSIPNPLLGLDKFERQ
    (SEQ ID NO: 303)
    50. SOS-RTPR-p27
    MGSFEGIALTNYLKALEGDPNNAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAEAWYNLGNAYYRQGDYQR
    AIEYYQRALELDPRSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSPDAEAWINLGNAYY
    RQGDYQRAIEYYQRALELDPRS (SEQ, ID NO:304)
    51. KBL-RTPR-p27
    MGSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNPLYISYDPAEAWYNLGNAYYRQGDYQRAIEYYQRALEL
    DPRSAEAWYNLGNAYYRQGDYQRAIEYYQRALELDPNNAGSNEQEPKKRSAEAWYNLGNAYYRQGDYQRAIEYYQ
    RALELDPNN (SEQ ID NO: 305)

  • TABLE 9-11
    Abbreviations
    Group name Amino acids Displayed as
    Default X .
    Single X
    Figure US20220090054A1-20220324-P00019
    Alanine A A
    Cysteine C C
    Aspartic Acid D D
    Glutamic Acid E E
    Phenylalanine F F
    Glycine G G
    Histidine H H
    Isoleucine I I
    Lysine K K
    Leucine L L
    Methionine M M
    Asparagine N N
    Proline P P
    Glutamine Q Q
    Arginine R R
    Serine S S
    Threonine T T
    Valine V V
    Tryptophan W W
    Tyrosine Y Y
    Negative D, E −
    Ser/Thr S, T *
    Aliphatic I, L, V l
    Positive H, K, R +
    Tiny A, G, S t
    Aromatic F, H, W, Y a
    Charged D, E, H, K, R c
    Small A, C, D, G, N, P, S, T, V s
    Polar C, D, E, H, K, N, Q, R, S, T p
    Big E, F, H, I, K, L, M, Q, R, W, Y B
    Hydrophobic A, C, F, G, H, I, L, M, T, V, W, Y h
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Claims (17)

1-15. (canceled)
16. A chimeric protein comprising two or more repeat domains linked by inter-repeat loops; and one or more heterologous peptide ligands that bind to a target molecule, each said peptide ligand being located in an inter-repeat loop or at the N or C terminus of the chimeric protein.
17. A chimeric protein according to claim 16 wherein the repeat domains are helix-turn-helix repeat domains.
18. A chimeric protein according to claim 17 wherein the repeat domains are tetratricopeptide (TPR) repeat domains.
19. A chimeric protein according to claim 18 wherein the repeat domains have the amino acid sequence Y-X1X2X3X4;
wherein Y is an amino acid sequence shown in any of Tables 4 to 6 or a variant thereof and X1, X2, X3, X4 are independently any amino acid.
20. A chimeric protein according to claim 19 wherein the repeat domains have the amino acid sequence;
AEAWYNL GNAYYKQGDYQKAIEYYQKALEL-X1X2X3X4; or AEALNNLGNVYREQGDYQKAIEYYQKALEL-X1X2X3X4 or AEAWYNLGNAYYRQGDYQRAIEYYQRALEL-X1X2X3X4; or AEALNNLGNVYREQGDYQRAIEYYQRALEL-X1X2X3X4; or AEALRNLGRVYRRQGRYQRAIEYYRRALEL-X1X2X3X4
wherein X1, X2, X3, X4 are independently any amino acid, and optionally wherein X1 is D and/or wherein X2 is P.
21. A chimeric protein according to claim 16 comprising 2-5 repeat domains.
22. A chimeric protein according to claim 16 wherein the peptide ligands are locating in one or more inter-repeat loops, and optionally are connected to the inter-repeat loops by a linker, and optionally wherein the peptide ligands are non-hydrophobic.
23. A chimeric protein according to claim 16 wherein a peptide ligand is located at the N terminus, the C terminus or at both the N and C termini, thereby to provide an N terminal peptide ligand and/or a C terminal peptide ligand.
24. A chimeric protein according to claim 23 wherein the N terminal peptide ligand, the C terminal peptide ligand or both of the N and C terminal peptide ligands comprises an α helix.
25. A chimeric protein according to claim 24 wherein the N terminal peptide ligand comprises the sequence Xn-XYXXXIXXYXXXLXX-X1X2XX, where residues denoted by X are independently any amino acid, X1 and X2 are independently any amino acid and n is 0 or any number, and optionally wherein X1 is D and/or wherein X2 is P.
26. A chimeric protein according to claim 24 wherein the C terminal peptide ligand comprises the sequence X1X2XX-XXAXXXLXX[AV]XXXXX-Xn, where residues denoted by X are independently any amino acid, X1, and X2 are independently any amino acid n is 0 or any number, and optionally wherein X1 is D and/or wherein X2 is P.
27. A chimeric protein according to claim 16 wherein the target molecule is β-catenin, KRAS, tankyrase, c-myc, n-myc, ras, notch and aurora A, α-synuclein, β-amyloid, tau, superoxide dismutase, huntingtin, oncogenic histone deacetylase, or oncogenic histone methyltransferase.
28. A chimeric protein according to claim 16 comprising a first peptide ligand that binds a first target molecule and a second peptide ligand that binds an E3 ubiquitin ligase.
29. A chimeric protein according to claim 28 comprising
(i) an N terminal peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or
(ii) an inter-repeat peptide ligand that binds a target protein and a C terminal peptide ligand that binds an E3 ubiquitin ligase, or
(iii) an inter-repeat peptide ligand that binds a target protein and an N terminal peptide ligand that binds an E3 ubiquitin ligase, or
(iv) a C terminal domain that binds a target protein and an N terminal peptide ligand that binds an E1 ubiquitin ligase, or
(v) an inter-repeat binding domain that binds an E3 ubiquitin ligase and an N terminal binding domain that binds a target protein, or
(vi) an inter-repeat binding domain that binds an E3 ubiquitin ligase and a C terminal binding domain that binds a target protein.
30. A method of producing a library of chimeric proteins comprising;
(a) providing a population of nucleic acids encoding a diverse population of chimeric proteins comprising
(i) two or more repeat domains
(ii) inter-repeat loops linking said repeat domains; and
(iii) one or more binding domains, each said binding domain being located in an inter-repeat loop or comprise the N and/or C terminus of the chimeric protein,
wherein the binding domains in said population are diverse, and
(b) expressing said population of nucleic acids to produce the diverse population, thereby producing a library of chimeric proteins.
31. A method of screening a library comprising;
(a) providing a library produced according to the method of claim 30, wherein at least one amino acid residue in the binding domains in said library is diverse,
(b) screening the library for chimeric proteins which display a binding activity, and
(c) identifying one or more chimeric proteins in the library which display the binding activity.
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