Polypeptide screening

Abstract

The invention relates to a method for selecting a polypeptide displayed on a genetic display system according to an activity, comprising the steps of: displaying the polypeptide on the genetic display system, screening the system for binding to the target, and selecting members which bind to the target; cloning nucleic acid encoding the polypeptide from the display system members and expressing the nucleic acid in an expression system to produce the polypeptide, or sequencing the polypeptide from the display system members and using synthetic DNA based clone and express a polypeptide or repertoire of polypeptides; and determining the activity of the polypeptide(s) in an assay.

Description

[0001] Polypeptide Screening

[0002] The present invention concerns methods for identification of polypeptide ligands having a desired binding activity. In particular, the invention concerns the identification of polypeptides on a genetic display system which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. Isolation of the polypeptides from the genetic display system and expression in a polypeptide expression system permits high-throughput assessment of polypeptide function.

[0003] Cyclic peptides are able to bind with high affinity and target specificity to protein targets and hence are an attractive molecule class for the development of therapeutics. In fact, several cyclic peptides are successfully used in the clinic, as for example the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the anti-cancer drug ocreotide (Driggers, et al., Nat Rev Drug Discov 2008, 7 (7), 608-24). Good binding properties result from a relatively large interaction surface formed between the peptide and the target as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 A2; Wu, B., et al., Science 330 (6007), 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin ocVb3 (355 A2) (Xiong, J. P., et al., Science 2002, 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A2; Zhao, G., et al., J Struct Biol 2007 , 160 ( ), 1-10).

[0004] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, leading to a smaller loss of entropy upon binding to targets and resulting in a higher binding affinity. The reduced flexibility also leads to locking target-specific conformations, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent and selective inhibitor of matrix metalloproteinase 8, MMP-8) which lost its selectivity over other MMPs when its ring was opened (Cherney, R. J., et al., J Med Chem 1998, 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in multicyclic peptides having more than one peptide ring as for example in vancomycin, nisin or actinomycin.

[0005] Different research teams have previously tethered polypeptides with cysteine residues to a synthetic molecular structure (Kemp, D. S. and McNamara, P. E., J. Org. Chem, 1985; Timmerman, P. et al., ChemBioChem, 2005). Meloen and co-workers had used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclisation of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman, P. et al., ChemBioChem, 2005). Methods for the generation of candidate drug compounds wherein said compounds are generated by linking cysteine containing polypeptides to a molecular scaffold as for example tris(bromomethyl)benzene are disclosed in WO 2004 / 077062 and WO 2006 / 078161 .

[0006] W02004 / 077062 discloses a method of selecting a candidate drug compound. In particular, this document discloses various scaffold molecules comprising first and second reactive groups, and contacting said scaffold with a further molecule to form at least two linkages between the scaffold and the further molecule in a coupling reaction.

[0007] W02006 / 078161 discloses binding compounds, immunogenic compounds and peptidomimetics. This document discloses the artificial synthesis of various collections of peptides taken from existing proteins. These peptides are then combined with a constant synthetic peptide having some amino acid changes introduced in order to produce combinatorial libraries. By introducing this diversity via the chemical linkage to separate peptides featuring various amino acid changes, an increased opportunity to find the desired binding activity is provided. Figure 1 of this document shows a schematic representation of the synthesis of various loop peptide constructs. The constructs disclosed in this document rely on -SH functionalised peptides, typically comprising cysteine residues, and (hetero)aromatic groups on the scaffold, typically comprising benzylic halogen substituents such as bis- or tris-bromomethylbenzene. Such groups react to form a thioether linkage between the peptide and the scaffold.

[0008] Heinis et al. developed a phage display-based combinatorial approach to generate and screen large libraries of bicyclic peptides to targets of interest (Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7; see also international patent application W02009 / 098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) were displayed on phage and cyclised by covalently linking the cysteine side chains to a small molecule (tris-(bromomethyl)benzene). Bicyclic peptides isolated in selections for affinity to the human proteases cathepsin G and plasma Kallikrein (PK) had nanomolar inhibitory constants. The best inhibitor, PK15, inhibits human PK (hPK) with a Ki of 3 nM. Similarities in the amino acid sequences of several isolated bicyclic peptides suggested that both peptide loops contribute to the binding. PK15 did not inhibit rat PK (81% sequence identity) nor the homologous human serine proteases factor Xia (hfXIa; 69% sequence identity) or thrombin (36% sequence identity) at the highest concentration tested (10 pM) (Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7). This finding suggested that the bicyclic inhibitor possesses high affinity for its target, and is highly specific.

[0009] WO2014 / 140342 describes the conjugation of phage polypeptides to scaffold molecules on a solid phase, rather than in solution as described in Heinis et al. This increases the overall efficiency of phage selection of bicyclic polypeptides. However, high-throughput binding assays performed on phage do not provide sufficient correlation to slower, but more accurate, assays performed later in the discovery workflow, such as Surface Plasmon Resonance (SPR) assays which are performed using chemically synthesized, scaffold-bound bicyclic polypeptides (Bicycles®). Accordingly, a significant number of Bicycles® recovered from high- throughput phage screens do not display desired binding affinities.

[0010] Summary of the Invention

[0011] In a first aspect, the invention provides a method for selecting a polypeptide displayed on a genetic display system according to an activity of the polypeptide, comprising the steps of: a. displaying the polypeptide on the genetic display system, screening the system for binding to the target, and selecting members which bind to the target; b. isolating nucleic acid encoding the polypeptide selected in (a) directly from the display system members or synthesizing nucleic acid with the sequence encoding the polypeptide selected in (a), and expressing the nucleic acid in an expression system to produce the polypeptide; c. determining the activity of the polypeptide in an assay.

[0012] In one embodiment, nucleic acid encoding the polypeptide selected in part (a) above is cloned from the genetic display assay member and expressed in an expression system. Alternatively, the selected genetic display assay member is sequenced, and the sequence information is used to design synthetic DNA used to express a polypeptide or a repertoire of polypeptides, the activity of which is determined in step (c).

[0013] In embodiments, the selected polypeptide comprises reactive groups, and said polypeptide is covalently bound to a molecular scaffold by reaction with said reactive groups such that two or more peptide loops are subtended between attachment points to the scaffold.

[0014] In Heinis et al., binding of polypeptides to targets is assessed on scaffold-modified phage, in which the polypeptides displayed on phage are exposed to the scaffold in solution, screened against the target in multiple rounds of selection, and the phage which bind to the target are identified. In some experiments, Heinis et al carried out testing for biological activity on expressed fusion proteins, which still comprised the phage coat proteins in which the polypeptide was displayed. Affinity testing, however, was not performed until the candidate polypeptides had been chemically synthesized and modified with scaffold.

[0015] The present system, using cloning and expression of polypeptides identified in a genetic display assay, provides a read-out of the activity of the polypeptide at an earlier stage of the procedure, and using high-throughput assay technologies can analyse thousands of polypeptides simultaneously. Polypeptides can be analysed individually or in a bulk assay, and results deconvoluted for example using polypeptide mass as determined by mass spectrometry.

[0016] The approach can also be applied to affinity maturation, in which a parent polypeptide sequence identified by genetic display and new daughter sequences that are closely related (where some residues are retained and some modified compared to the parent) may be cloned, expressed and subjected to analysis for activity by the methods described herein.

[0017] In embodiments, therefore, the polypeptide of the present invention may be a polypeptide as described in Heinis et al., wherein the polypeptide is covalently bound to a molecular scaffold such that two or more peptide loops are subtended between attachment points to the scaffold. For example the polypeptide comprises the sequence (X)IY(X)mY(X)nY(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 1 and 20 defining the length of intervening polypeptide segments and I and o are numbers between 0 and 20 defining the length of the flanking polypeptide segments.

[0018] Preferably, I and o are independently between 0 and 15. In embodiments, m and n are independently between 2 and 10, preferably between 4 and 8, for example 4, 5, 6, 7 or 8.

[0019] In embodiments, the polypeptide comprises a sequence Y(X)mY(X)nY, wherein m is 4, 5, 6, 7, or 8 and n is 4, 5, 6, 7, 8; preferably wherein m is 5, 6 or 7, and n is 5, 6, or 7. Y is a reactive group, and is preferably Cysteine (C). X is any amino acid.

[0020] In some embodiments, the peptide ligand of the invention may comprise a polypeptide with the sequence C(X)6C(X)6C, wherein C is Cysteine and X represents any amino acid.

[0021] The polypeptide is preferably a polypeptide which comprises at least three reactive groups, separated by at least two sequences which can form the “loops” of the polypeptide once conjugated to the molecular scaffold. The loops may be any suitable length, such as two, three, four, five, six, seven or more amino acids long. The loops may be the same length, or different. Preferably, at least two loops are provided. In some embodiments, three, four, five, six or more loops may be present.

[0022] Reactive groups in the polypeptide are capable of forming covalent linkages with the scaffold. Examples include amino acids with thiol groups, and also amino groups, carboxyl groups, guanidinium groups, phenolic groups or hydroxyl groups. The reactive groups of the polypeptides can be selected from azide, keto-carbonyl, alkyne, vinyl, or aryl halide groups, or can be the amino or carboxy termini of the polypeptide. Most commonly, reactive groups comprise cysteine residues. The scaffold may be any structure which provides multiple attachment points for the reactive groups of the polypeptide. Exemplary scaffolds are described below. Scaffold molecules are conjugated to the polypeptide whilst the polypeptides are incorporated into the genetic display system, such that the genetic display system displays the polypeptide ligand including the molecular scaffold. Excess scaffold is removed.

[0023] In embodiments, the polypeptide ligands are multispecific. In a first configuration, for example, the polypeptide loops formed by the interaction of the polypeptide with the molecular scaffold are capable of binding to more than one target. Within this configuration, in one embodiment loops may be selected individually for binding to the desired targets, and then combined. In another embodiment, the loops are selected together, as part of a single structure, for binding to different desired targets.

[0024] In a second configuration, a functional group may be attached to the N or C terminus, or both, of the polypeptide. The functional group may take the form of a binding group, such as a polypeptide, including an antibody domain, an Fc domain or a further structured peptide as described above, capable of binding to a target. It may moreover take the form of a reactive group, capable of chemical bonding with a target. Moreover, it can be an effector group, including large plasma proteins, such as serum albumin, and a cell penetrating peptide.

[0025] In a third configuration, a functional group may be attached to the molecular scaffold itself. Examples of functional groups are as for the preceding configuration.

[0026] In further embodiments, the polypeptide ligand comprises a polypeptide linked to a molecular scaffold at n attachment points, wherein said polypeptide is cyclised and forms n separate loops subtended between said n attachment points on the molecular scaffold, wherein n is greater than or equal to 2.

[0027] The polypeptide is preferably cyclised by N- to C-terminal fusion, and can be cyclised before or after attachment to the molecular scaffold. Attachment before cyclisation is preferred.

[0028] Several methods are known in the art for peptide cyclisation. For example, the polypeptide is cyclised by N-C crosslinking, using a crosslinking agent such as EDC.

[0029] In another embodiment, the peptide can be designed to comprise a protected N“ or C“ derivatised amino acid, and cyclised by deprotection of the protected N“ or C“ derivatised amino acid to couple said amino acid to the opposite terminus of the polypeptide.

[0030] In a preferred embodiment, the polypeptide is cyclised by enzymatic means. For example, the enzyme is a transglutaminase, for instance a microbial transglutaminase, such as Streptomyces mobaraensis transglutaminase. In order to take advantage of enzymatic cyclisation, it may be necessary to incorporate an N- and / or C-terminal substrate sequence for the enzyme in the polypeptide. Some or all of the substrate sequence(s) can be eliminated during the enzymatic reaction, meaning that the cyclised polypeptide may not comprise the substrate sequences in its final configuration.

[0031] The affinity of such polypeptides for target when displayed on phage and modified with scaffold shows inconsistent correlation with the affinity of the same polypeptide for target when chemically synthesised and modified with scaffold. There are several factors that are suspected to contribute to this unsatisfactory correlation.

[0032] Firstly, each phage displays multiple copies of the same Bicycle (Ledsgaard et al., 2018, Basics of Antibody Phage Display Technology. Toxins [Online], 10(6), p.236). This produces an avidity effect upon binding to the target material. While affinity is the strength of an interaction between two molecules, avidity is the observed increase in affinity, due to multivalent binding (Vauquelin and Charlton, 2013, Exploring avidity: understanding the potential gains in functional affinity and target residence time of bivalent and heterobivalent ligands. British Journal of Pharmacology [Online], 168(8), pp.1771-1785). This means the affinity of each individual interaction is the same, but the overall molecule binding is stronger due to multiple binding sites per molecule / ligand. This avidity can reduce correlation between the binding affinity of a Bicycle and the phage screening signal it produces. Weak binders can still appear as positive hits due to the avidity effect.

[0033] Secondly, the phage concentration is not constant between samples (due to e.g. differential expression levels of different clones by E. coli host), and the output signal is not normalized to account for this. In practice, this could result in a sample with a higher concentration of a weak binder producing a higher signal than a stronger binder of a lower concentration.

[0034] Thirdly there may be specific or non-specific interactions between the phage and the target or assay components that give rise to a higher or lower signal than might otherwise have been observed.

[0035] The same issues are observed when screening for other characteristics, including cell reporter functionality, cell internalisation, uptake / efflux assays, enzyme inhibition activity, activity in cell killing, cell viability modification and cell cycle inhibition, and ion flux assays. Accordingly, an assay as referred to herein can be a binding assay or an activity assay, including an assay as set out above. Cloning and expressing the polypeptide provides a high-throughput solution to screening for activity, without the drawbacks of chemical synthesis or the presence of artefacts from the genetic display system. Additionally, it provides the potential to produce a greater yield of polypeptide versus directly cleaving the fused polypeptide from its fusion partner in genetic display system thus allowing interrogation with a broader variety of assay techniques. Preferably, in order to further enhance throughput, expression is carried out in a cell-free expression system (CFES). Additionally, during the cloning procedure, one or more polypeptide tags may be added to the expression system, such that the polypeptide is expressed with one or more tags.

[0036] The invention envisages the screening of a plurality of polypeptide variants which have sequences which may differ in one or more positions. For example, if a polypeptide has a general formula (X)IY(X)mY(X)nY(X)o as set forth above, variants can be created which differ in the sequence of amino acids represented by (X)l, (X)m, (X)n and (X)o; preferably, the differences are in sequences (X)m and (X)n, which represent the polypeptide “loops” subtended between attachment points to the scaffold.

[0037] In a preferred embodiment, repertoires of polypeptide variants are provided in the form of a nucleic acid library, and incorporated as part of a genetic display system. Applicable systems include phage display, bacterial display, yeast display, ribosome or polysome display, mRNA display and in vitro expression in artificial microcapsules. The preferred technique is phage display using a filamentous bacteriophage.

[0038] Cloning of polypeptides from the display system is advantageously carried out by PCR. The cloning preferably avoids including in the polypeptide any substantial portion of the genetic display system, such as a portion of the phage coat protein to which the polypeptide is fused for the purposes of phage display.

[0039] In a further aspect, the invention provides a method comprising the steps of identifying a polypeptide according to the previous aspect, and modifying the polypeptide. Preferred modifications suitable for polypeptides according to the present invention are set forth below.

[0040] Brief Description of the Figures

[0041] Figure 1 : Schematic of insert construct design. A) non-conjugated construct. B) DHFR construct.

[0042] Figure 2: Sample vector plasmid from overnight digest with restriction endonuclease. A 1% agarose gel stained with SYBR Gold II was used. A) Vector plasmid containing DHFR.

[0043] Lane 1 : DNA ladder, 5 pL loaded - Distinguishable bands annotated (kBP). Lane 2: Uncut plasmid.,

[0044] Lane 3: Digested (linearized) plasmid.

[0045] B) Vector Plasmid with just the tag regions, no DHFR.

[0046] Lane M: DNA ladder, 5 pL loaded - Distinguishable bands annotated (kBP). Lane 1 : Uncut plasmid,

[0047] Lane 2: Digested (linearized) plasmid.

[0048] Figure 3: Clone sequence following Gibson assembly. Sanger sequencing was used. Bicycle (BCY) sequence is annotated.

[0049] Figure 4: Coomassie stained SDS-PAGE gel of the different fractions from purification of peptide 121-83-01. 5 pL of sample and dye was loaded per well. Post E: Mix of resin and any remaining elution solution. FT: Flow through fraction, W1 : First wash fraction, W2: Second wash fraction, E1 : First elution fraction, E2: Second elution fraction. Ladder units are kDa.

[0050] Figure 5: Blot of peptide 121-83-01 from CFES, detected using HRP conjugated secondary antibody. Ladder was positioned based on the corresponding light membrane image (not included) and units are kDa

[0051] Figure 6: Coomassie stained SDS-PAGE gel of the elution fractions from cyclisation reactions. Increasing concentrations of scaffolds were tested on three different peptides. Ladder units are kDa.

[0052] Figure 7: Deconvoluted LC-MS spectrum of 141-09-00 scaffolded with 19.2 uM TCTZ. Derived from raw mass spectrum.

[0053] Figure 8: Mass Spectrograph of peptide 121-83-01 with OX scaffold. Run on MALDI-TOF.

[0054] Figure 9: Mass Spectrograph of peptide 121-83-01 with 10X TATB scaffold. Run on MALDI- TOF.

[0055] Figure 10: Mass Spectrograph of peptide 121-83-01 with 20X TATB scaffold. Run on MALDI- TOF.

[0056] Figure 11 : Mass Spectrograph of peptide 141-03-00 with OX scaffold. Run on MALDI-TOF. Figure 12: Mass Spectrograph of peptide 141-03-00 with 24X TCTZ scaffold. Run on MALDI- TOF.

[0057] Figure 13: Mass Spectrograph of peptide 141-03-00 with 48X TCTZ scaffold. Run on MALDI- TOF.

[0058] Figure 14: Mass Spectrograph of peptide 141-09-00 with OX scaffold. Run on MALDI-TOF.

[0059] Figure 15: Mass Spectrograph of peptide 141-09-00 with 24X TCTZ scaffold. Run on MALDI- TOF.

[0060] Figure 16: Mass Spectrograph of peptide 141-09-00 with 48X TCTZ scaffold. Run on MALDI- TOF.

[0061] Figure 17A: Binding data comparing expressed and synthesised polypeptides by SPR and ELISA.

[0062] Figure 17B: Cell binding data comparing expressed polypeptides with positive and negative controls; fluorescently labelled polypeptides binding to U87MG cells.

[0063] Detailed Description of the Invention

[0064] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as in the arts of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001 , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4thed., John Wiley & Sons, Inc.; Patil and Sivaram, A Complete Guide to Gene Cloning: From Basic to Advanced, Springer: ISBN978-3-030-96853-3, 28 April 2023; Jaroszewicz et al., Phage display and other peptide display technologies, FEMS Microbiology Reviews, fuab052, 46, 2022, 1-25, as well as resources on Addgene.org), which are incorporated herein by reference.

[0065] A (poly)peptide ligand or (poly)peptide conjugate, as referred to herein, refers to a polypeptide covalently bound to a molecular scaffold. Typically, such polypeptides comprise two or more reactive groups which are capable of forming covalent bonds to the scaffold, and a sequence subtended between said reactive groups which is referred to as the loop sequence, since it forms a loop when the peptide is bound to the scaffold. In the present case, the polypeptides comprise at least three reactive groups, and form at least two loops on the scaffold.

[0066] The reactive groups are groups capable of forming a covalent bond with the molecular scaffold. Typically, the reactive groups are present on amino acid side chains on the peptide. Examples are amino-containing groups such as cysteine, lysine and selenocysteine.

[0067] Specificity, in the context herein, refers to the ability of a ligand to bind or otherwise interact with its cognate target to the exclusion of entities which are similar to the target. For example, specificity can refer to the ability of a ligand to inhibit the interaction of a human enzyme, but not a homologous enzyme from a different species. Using the approach described herein, specificity can be modulated, that is increased or decreased, so as to make the ligands more or less able to interact with homologues or paralogues of the intended target. Specificity is not intended to be synonymous with activity, affinity or avidity, and the potency of the action of a ligand on its target (such as, for example, binding affinity or level of inhibition) are not necessarily related to its specificity.

[0068] Binding activity, as used herein, refers to quantitative binding measurements taken from binding assays, for example as described herein. Therefore, binding activity refers to the amount of peptide ligand which is bound at a given target concentration.

[0069] Multispecificity is the ability to bind to two or more targets. Typically, binding peptides are capable of binding to a single target, such as an epitope in the case of an antibody, due to their conformational properties. However, peptides can be developed which can bind to two or more targets; dual specific antibodies, for example. In the present invention, the peptide ligands can be capable of binding to two or more targets and can therefore be multispecific. Preferably, they bind to two targets, and are dual specific. The binding may be independent, which would mean that the binding sites for the targets on the peptide are not structurally hindered by the binding of one or other of the targets. In this case both targets can be bound independently. More generally it is expected that the binding of one target will at least partially impede the binding of the other.

[0070] A target is a molecule or part thereof which the peptide ligands bind to.

[0071] The molecular scaffold is any molecule which is able to connect the peptide at multiple points to impart one or more structural features to the peptide. It is not a cross-linker, in that it does not merely replace a disulphide bond; instead, it provides two or more attachment points for the peptide. Preferably, the molecular scaffold comprises at least three attachment points for the peptide, referred to as scaffold reactive groups. These groups are capable of reacting to the reactive groups on the peptide to form a covalent bond. Preferred structures for molecular scaffolds are described below.

[0072] Screening for binding activity (or any other desired activity) is conducted according to methods well known in the art, for instance from phage display technology. For example, targets immobilised to a solid phase can be used to identify and isolate binding members of a repertoire. Screening allows selection of members of a repertoire according to desired characteristics.

[0073] The term library refers to a mixture of heterogeneous polypeptides or nucleic acids. The library is composed of members, which are not identical. To this extent, library is synonymous with repertoire. Sequence differences between library members are responsible for the diversity present in the library. The library may take the form of a simple mixture of polypeptides or nucleic acids, or may be in the form of organisms or cells, for example bacteria, viruses, animal or plant cells and the like, transformed with a library of nucleic acids. Preferably, each individual organism or cell contains only one or a limited number of library members.

[0074] Preferably the library comprises 104, 105, 106, 107, 108, 109, 1010or more polypeptide variants.

[0075] In one embodiment, a library of nucleic acids encodes a repertoire of polypeptides. Each nucleic acid member of the library preferably has a sequence related to one or more other members of the library. By related sequence is meant an amino acid sequence having at least 50% identity, for example at least 60% identity, for example at least 70% identity, for example at least 80% identity, for example at least 90% identity, for example at least 95% identity, for example at least 98% identity, for example at least 99% identity to at least one other member of the library. Identity can be judged across a contiguous segment of at least 3 amino acids, for example at least 4, 5, 6, 7, 8, 9 or 10 amino acids, for example least 12 amino acids, for example least 14 amino acids, for example least 16 amino acids, for example least 17 amino acids or the full length of the reference sequence.

[0076] As those skilled in the art will appreciate, related sequences include deletion variants of such sequences wherein one or more, such as at least 1 , 2, 3, 4 or 5 nucleotides are deleted. Deletion may occur at either end of the reference sequence or within the reference sequence. Related nucleic acid sequences include addition variants of such sequences wherein one or more, such as at least 1 , 2, 3, 4, or 5 nucleotides are added or introduced into the reference sequence. Addition may occur at either end of the reference sequence or within the reference sequence.

[0077] Related nucleic acid sequences include sequences wherein one or more nucleotides such as at least 1 , 2, 3, 4, or 5 nucleotides in the reference sequence are exchanged for one or more alternative nucleotides. Variants of nucleic acid sequences include sequences encoding naturally occurring amino acids and / or unnatural amino acids.

[0078] A repertoire is a collection of variants, in this case polypeptide variants, which differ in their sequence. Typically, the location and nature of the reactive groups will not vary, but the sequences forming the loops between them can be randomised. Repertoires differ in size, but should be considered to comprise at least 102members. Preferably, the repertoire comprises 104, 105, 106, 107, 108, 109, 1010or more polypeptide variants. Repertoires of 1011or more members can be constructed.

[0079] (i) Molecular scaffold

[0080] Molecular scaffolds are described in, for example, W02009098450 and references cited therein, particularly W02004077062 and W02006078161 .

[0081] As noted in the foregoing documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0082] In one embodiment the molecular scaffold may be, or may be based on, natural monomers such as nucleosides, sugars, or steroids. For example the molecular scaffold may comprise a short polymer of such entities, such as a dimer or a trimer.

[0083] In one embodiment the molecular scaffold is a compound of known toxicity, for example of low toxicity. Examples of suitable compounds include cholesterols, nucleotides, steroids, or existing drugs such as tamazepam.

[0084] In one embodiment the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0085] In one embodiment the molecular scaffold comprises reactive groups that are capable of reacting with functional group(s) of the polypeptide to form covalent bonds. The molecular scaffold may comprise chemical groups as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, acyl halides, and a,|3-unsaturated sulfinyls, sulfonyls and carbonyls.

[0086] In one embodiment, the molecular scaffold may comprise or may consist of tris(bromomethyl)benzene, especially 1 ,3,5-Tris(bromomethyl)benzene (‘TBMB’), or a derivative thereof.

[0087] In one embodiment, the molecular scaffold is 2,4,6-Tris(bromomethyl)mesitylene. It is similar to 1 ,3,5-Tris(bromomethyl)benzene but contains additionally three methyl groups attached to the benzene ring. This has the advantage that the additional methyl groups may form further contacts with the polypeptide and hence add additional structural constraint.

[0088] Other molecular scaffolds include 1 ,3,5-triacryloyl-1 ,3,5-triazinane (TATA), 1 -[3,5-bis(2- bromoacetyl)-1 ,3,5-triazinan-1-yl]-2-bromoethanone (TATB), N,N’,N”-(benzene-1 ,3,5-triyl)- tris(2-bromoacetamide) (TBAB) 1 ,1',1"-(1 ,4,7-triazonane-1 ,4,7-triyl)tris(2-chloroethan-1-one) (TCAZ), 1 ,1 ',1"-(1 ,4,7-triazonane-1 ,4,7-triyl)tris(2-bromothan-1-one) (TBAZ), 2,4,6- tris(bromomethyl)-s-triazine (TBMT), 2,4,6-tris(chloromethyl)-1 ,3,5-triazine (TCTZ), 1 ,T,1"-(1 H,4H-3a,6a-(methaniminomethano)pyrrolo[3,4-c]pyrrole-2,5,8(3H,6H)-triyl)tris[2-chloro- ethanone] (TBCU), 1 ,3,5-tri(ethenesulfonyl)-1 ,3,5-triazinane (TSTA), 2-bromo-1-[7,10-bis(2- bromoacetyl)-3,7,10-triazatricyclo[3.3.3.0A1 ,5]undecan-3-yl]ethenone (TBCU) and [3,5-bis[4- (bromomethyl)benzoyl]-1 ,3,5-triazinan-1-yl]-[4-(bromomethyl)phenyl]methanone (TBPM). See Chen et al., ChemBioChem 2012, 13, 1032 - 1038.

[0089] The molecular scaffold of the invention contains chemical groups that allow functional groups of the polypeptide of the encoded library of the invention to form covalent links with the molecular scaffold. Said chemical groups are selected from a wide range of functionalities including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides, acyl halides, a,|3-unsaturated sulfinyls, a,|3-unsaturated sulfonyls and a,|3-unsaturated carbonyls.

[0090] In one embodiment, the molecular scaffold is 1 , 1 ', 1 "-(1 ,3,5-triazinane-1 ,3,5-triyl)triprop-2-en- 1-one (also known as triacryloylhexahydro-s-triazine (TATA)):

[0091]

[0092] Thus, following cyclisation with a peptide (e.g. by reaction with cysteine residues in the peptide), the molecular scaffold typically forms a tri-substituted 1 ,1',1"-(1 ,3,5-triazinane- 1 ,3,5-triyl)tripropan-1 -one derivative of TATA having the following structure: wherein * denotes the point of attachment of the peptide (e.g. of the cysteine residues).

[0093] In an alternative embodiment, the molecular scaffold is 1 ,3,5-tris(bromoacetyl) hexahydro-1 , 3, 5-triazine (TATB): Thus, following cyclisation with a peptide (e.g. by reaction with cysteine residues in the peptide), the molecular scaffold typically forms a tri-substituted 1 ,3,5-tris(bromoacetyl) hexahydro-1 , 3, 5-triazine derivative of TATB having the following structure:

[0094] wherein * denotes the point of attachment of the peptide (e.g. of the cysteine residues).

[0095] In an alternative embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)-s-triazine (TBMT): or 2,4,6-tris(chloromethyl)-1 ,3,5-triazine (TCTZ):

[0096] Thus, following cyclisation with a peptide (e.g. by reaction with cysteine residues in the peptide), the molecular scaffold typically forms a tri-substituted 2,4,6-tris(bromomethyl)-s- triazine derivative of TBMT or a tri-substituted 2,4,6-tris(chloromethyl)-s-triazine derivative of TCTZ having the following structure: wherein * denotes the point of attachment of the peptide (e.g. of the cysteine residues).

[0097] Similarly, other scaffold molecules will be modified by the attachment of the polypeptide, typically in a nucleophilic substitution reaction.

[0098] (ii) Polypeptide

[0099] The reactive groups of the polypeptides can be provided by side chains of natural or nonnatural amino acids. The reactive groups of the polypeptides can be selected from thiol groups, amino groups, carboxyl groups, guanidinium groups, phenolic groups or hydroxyl groups. The reactive groups of the polypeptides can be selected from azide, keto-carbonyl, alkyne, vinyl, or aryl halide groups. The reactive groups of the polypeptides for linking to a molecular scaffold can be the amino or carboxy termini of the polypeptide.

[0100] In some embodiments each of the reactive groups of the polypeptide for linking to a molecular scaffold are of the same type. For example, each reactive group may be a cysteine residue, homocysteine (hCys, (S)-2-Amino-4-sulfanylbutanoic acid), pCys ((R)-3- amino-3-mercaptopropanoic acid), cysteamine (Cystam) or penicillamine (Pen, (R)-2-amino- 3-mercapto-3-methylbutanoic acid), Dap ((S)-2,3-diaminopropanoic acid) or N-alkyl-Dap (e.g. N-methyl-Dap, (S)-2-amino-3-(methylamino)propanoic acid. Further details are provided in W02009098450.

[0101] In some embodiments the reactive groups for linking to a molecular scaffold may comprise two or more different types, or may comprise three or more different types. For example, the reactive groups may comprise two cysteine residues and one lysine residue, or may comprise one cysteine residue, one lysine residue and one N-terminal amine.

[0102] Cysteine can be employed because it has the advantage that its reactivity is most different from all other amino acids. Scaffold reactive groups that could be used on the molecular scaffold to react with thiol groups of cysteines are alkyl halides (or also named halogenoalkanes or haloalkanes). Examples are bromomethylbenzene (the scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive goups that are used to couple selectively compounds to cysteines in proteins are maleimides. Examples of maleimides which may be used as molecular scaffolds in the invention include: tris-(2- maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene.

[0103] Selenocysteine is also a natural amino acid which has a similar reactivity to cysteine and can be used for the same reactions. Thus, wherever cysteine is mentioned, it is typically acceptable to substitute selenocysteine unless the context suggests otherwise.

[0104] Lysines (and primary amines of the N-terminus of peptides) are also suited as reactive groups to modify peptides on phage by linking to a molecular scaffold. However, they are more abundant in phage proteins than cysteines and there is a higher risk that phage particles might become cross-linked or that they might lose their infectivity. Nevertheless, it has been found that lysines are especially useful in intramolecular reactions (e.g. when a molecular scaffold is already linked to the phage peptide) to form a second or consecutive linkage with the molecular scaffold. In this case the molecular scaffold reacts preferentially with lysines of the displayed peptide (in particular lysines that are in close proximity). Scaffold reactive groups that react selectively with primary amines are succinimides, aldehydes or alkyl halides. In the bromomethyl group that is used in a number of the accompanying examples, the electrons of the benzene ring can stabilize the cationic transition state. This particular aryl halide is therefore 100-1000 times more reactive than alkyl halides. Examples of succinimides for use as molecular scaffold include tris- (succinimidyl aminotriacetate), 1 ,3,5-Benzenetriacetic acid. Examples of aldehydes for use as molecular scaffold include Triformylmethane. Examples of alkyl halides for use as molecular scaffold include 1 ,3,5-Tris(bromomethyl)-2,4,6-trimethylbenzene, 1 ,3,5- Tris(bromomethyl) benzene, 1 ,3,5-Tris(bromomethyl)-2,4,6-triethylbenzene.

[0105] The amino acids with reactive groups for linking to a molecular scaffold may be located at any suitable positions within the polypeptide. In order to influence the particular structures or loops created, the positions of the amino acids having the reactive groups may be varied by the skilled operator, e.g. by manipulation of the nucleic acid encoding the polypeptide in order to mutate the polypeptide produced. By such means, loop length can be manipulated in accordance with the present teaching.

[0106] (iii) Reactive groups of the polypeptide

[0107] The molecular scaffold of the invention may be bonded to the polypeptide via functional or reactive groups on the polypeptide. These are typically formed from the side chains of particular amino acids found in the polypeptide polymer. Such reactive groups may be a cysteine side chain, a lysine side chain, or an N-terminal amine group or any other suitable reactive group. Again, details may be found in W02009098450.

[0108] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartate or glutamate, the guanidinium group of arginine, the phenolic group of tyrosine or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups including an azide, a keto-carbonyl, an alkyne, a vinyl, or an aryl halide group. The amino and carboxyl group of the termini of the polypeptide can also serve as reactive groups to form covalent bonds to a molecular scaffold / molecular core.

[0109] In some embodiments, the polypeptide may form alkylamino linkages to the scaffold. Preferred reactive groups include 2,3-diaminopropionic acid (Dap), p-N-alkyl-2,3- diaminopropionic acid (N-AIkDap) or |3-N-haloalkyl-2,3-diaminopropionic acid (N-AIkDap). See WO2018 / 115203.

[0110] The polypeptides of the invention contain at least three reactive groups. Said polypeptides can also contain four or more reactive groups. The more reactive groups are used, the more loops can be formed in the molecular scaffold.

[0111] In a preferred embodiment, polypeptides with three reactive groups are generated. Reaction of said polypeptides with a molecular scaffold / molecular core having a three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is favourable for several reasons. The nucleic acids of the compound libraries encode only the primary sequences of the polypeptide but not the isomeric state of the molecules that are formed upon reaction of the polypeptide with the molecular core. If only one product isomer can be formed, the assignment of the nucleic acid to the product isomer is clearly defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer that was isolated in a screening or selection process. The formation of a single product isomer is also advantageous if a specific member of a library of the invention is synthesized. In this case, the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.

[0112] In another embodiment of the invention, polypeptides with four reactive groups are generated. Reaction of said polypeptides with a molecular scaffold / molecular core having a tetrahedral symmetry generates two product isomers. Even though the two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomer can be determined by chemically synthesizing both isomers, separating the two isomers and testing both isomers for binding to a target ligand.

[0113] In one embodiment of the invention, at least one of the reactive groups of the polypeptides is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the directing of said orthogonal reactive groups to specific sites of the molecular core.

[0114] Linking strategies involving orthogonal reactive groups may be used to limit the number of product isomers formed. In other words, by choosing distinct or different reactive groups for one or more of the at least three bonds to those chosen for the remainder of the at least three bonds, a particular order of bonding or directing of specific reactive groups of the polypeptide to specific positions on the molecular scaffold may be usefully achieved.

[0115] In another embodiment, the reactive groups of the polypeptide of the invention are reacted with molecular linkers wherein said linkers are capable to react with a molecular scaffold so that the linker will intervene between the molecular scaffold and the polypeptide in the final bonded state.

[0116] In some embodiments, amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid. Excluded from these exchangeable amino acids are the ones harbouring functional groups for cross-linking the polypeptides to a molecular core, such that the loop sequences alone are exchangeable. The exchangeable polypeptide sequences have either random sequences, constant sequences or sequences with random and constant amino acids. The amino acids with reactive groups are either located in defined positions within the polypeptide, since the position of these amino acids determines loop size.

[0117] In one embodiment, a polypeptide with three reactive groups has the sequence (X)iY(X)mY(X)nY(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 2 and 20 defining the length of intervening polypeptide segments, which may be the same or different, and I and o are numbers between 0 and 20 defining the length of flanking polypeptide segments.

[0118] Preferably, I and o are independently between 0 and 15. In embodiments, m and n are independently between 2 and 10, preferably between 4 and 9, for example 4, 5, 6, 7 or 8.

[0119] In embodiments, the polypeptide comprises a sequence Y(X)mY(X)nY, wherein m is 4, 5, 6, 7, or 8 and n is 4, 5, 6, 7, 8; preferably wherein m is 5, 6 or 7, and n is 5, 6, or 7. Y is a reactive group, and is preferably Cysteine (C). X is any amino acid. As used herein, “X” or “Y” refer to any reactive group or amino acid, and when referring to a plurality of such entities, as in (X)m where m=2 or more, independently refer to multiple amino acids or reactive groups which may not be the same. Thus, the expression (X)m where m is 2 may refer to two different amino acids. Alternatively, it may refer to two residues of the same amino acid.

[0120] Alternatives to thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions. Alternatively these techniques may be used in modification or attachment of further moieties (such as small molecules of interest which are distinct from the molecular scaffold) to the polypeptide after they have been selected or isolated according to the present invention - in this embodiment then clearly the attachment need not be covalent and may embrace non-covalent attachment. These methods may be used instead of (or in combination with) the thiol mediated methods by producing phage that display proteins and peptides bearing unnatural amino acids with the requisite chemical reactive groups, in combination small molecules that bear the complementary reactive group, or by incorporating the unnatural amino acids into a chemically or recombinantly synthesised polypeptide when the molecule is being made after the selection / isolation phase. Further details can be found in W02009098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.

[0121] (iv) Combination of loops to form multispecific molecules

[0122] Loops from peptide ligands, or repertoires of peptide ligands, are advantageously combined by sequencing and de novo synthesis of a polypeptide incorporating the combined loops. Alternatively, nucleic acids encoding such polypeptides can be synthesised.

[0123] Where repertoires are to be combined, particularly single loop repertoires, the nucleic acids encoding the repertoires are advantageously digested and re-ligated, to form a novel repertoire having different combinations of loops from the constituent repertoires. Phage vectors can include polylinkers and other sites for restriction enzymes which can provide unique points for cutting and relegation the vectors, to create the desired multispecific peptide ligands. Methods for manipulating phage libraries are well known in respect of antibodies, and can be applied in the present case also.

[0124] (v) Attachment of Effector Groups and Functional Groups Effector and / or functional groups can be attached, for example, to the N or C termini of the polypeptide, or to the molecular scaffold.

[0125] Appropriate effector groups include antibodies and parts or fragments thereof. For instance, an effector group can include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, in addition to the one or more constant region domains. An effector group may also comprise a hinge region of an antibody (such a region normally being found between the CH1 and CH2 domains of an IgG molecule).

[0126] In a further preferred embodiment of this aspect of the invention, an effector group according to the present invention is an Fc region of an IgG molecule. Advantageously, a peptide ligand-effector group according to the present invention comprises or consists of a peptide ligand Fc fusion having a tp half-life of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more. Most advantageously, the peptide ligand according to the present invention comprises or consists of a peptide ligand Fc fusion having a tp half-life of a day or more.

[0127] Functional groups include, in general, binding groups, drugs, reactive groups for the attachment of other entities, metal chelators, chromophores, functional groups which aid uptake of the macrocyclic peptides into cells, fluorophores e.g. GFP and mCherry, and the like. A polypeptide may also be attached to a second polypeptide, which may be the same or different. The polypeptides may be bound to scaffolds as described herein, which again may the same or different.

[0128] The ability of peptides to penetrate into cells will allow peptides against intracellular targets to be effective. Targets that can be accessed by peptides with the ability to penetrate into cells include transcription factors, intracellular signalling molecules such as tyrosine kinases and molecules involved in the apoptotic pathway. Functional groups which enable the penetration of cells include peptides or chemical groups which have been added either to the peptide or the molecular scaffold. Peptides such as those derived from such as VP22, HIV- Tat, a homeobox protein of Drosophila (Antennapedia), e.g. as described in Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p821 "Cell-penetrating peptides in drug development: enabling intracellular targets" and “Intracellular delivery of large molecules and small peptides by cell penetrating peptides” by Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examples of short peptides which have been shown to be efficient at translocation through plasma membranes include the 16 amino acid penetratin peptide from Drosophila Antennapedia protein (Derossi et al (1994) J Biol. 11

[0129] Chem. Volume 269 p10444 ‘’The third helix of the Antennapedia homeodomain translocates through biological membranes”), the 18 amino acid ‘model amphipathic peptide’ (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127 “Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically”) and arginine rich regions of the HIV TAT protein. Non peptidic approaches include the use of small molecule mimics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153 ‘Small-molecule mimics of an a-helix for efficient transport of proteins into cells’. Other chemical strategies to add guanidinium groups to molecules also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585 “Guanidinylated Neomcyin Delivers Large Bioactive Cargo into cells through a heparin Sulphate Dependent Pathway”). Small molecular weight molecules such as steroids may be added to the molecular scaffold to enhance uptake into cells.

[0130] One class of functional groups which may be attached to peptide ligands includes antibodies and binding fragments thereof, such as Fab, Fv or single domain fragments. In particular, antibodies which bind to proteins capable of increasing the half life of the peptide ligand in vivo may be used.

[0131] RGD peptides, which bind to integrins which are present on many cells, may also be incorporated.

[0132] In one embodiment, a peptide ligand-effector group according to the invention has a tp halflife selected from the group consisting of: 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more. Advantageously a peptide ligand-effector group or composition according to the invention will have a tp half life in the range 12 to 60 hours. In a further embodiment, it will have a t half-life of a day or more. In a further embodiment still, it will be in the range 12 to 26 hours.

[0133] Functional groups include drugs, such as cytotoxic agents for cancer therapy. These include Alkylating agents such as Cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; Anti-metabolites including purine analogs azathioprine and mercaptopurine)) or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as Vincristine, Vinblastine, Vinorelbine and Vindesine; Podophyllotoxin and its derivatives etoposide and teniposide; Taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors including camptothecins: irinotecan and topotecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Further agents can include antitumour antibiotics which include the immunosuppressant dactinomycin (which is used in kidney transplantations), doxorubicin, epirubicin, bleomycin and others. Cytotoxic agents include cytotoxic peptides, such as MMAE and DM-1.

[0134] Possible effector groups also include enzymes, for instance such as carboxypeptidase G2 for use in enzyme / prodrug therapy, where the peptide ligand replaces antibodies in ADEPT.

[0135] Effector groups can include cytotoxic groups, metal chelators, chromophores, fluorophores, as well as one or more additional Bicycles®, which may be the same or different.

[0136] Any suitable linker may be used to attach such groups to the expressed peptide. Linkers may comprise or consist of amino acids or other polymers e.g. PEG.

[0137] (vi) Peptide modification

[0138] To develop the bicyclic peptides (Bicycles; peptides conjugated to molecular scaffolds) into a suitable drug-like molecule, whether that be for injection, inhalation, nasal, ocular, oral or topical administration, a number of properties need considered. Often, the following considerations apply in designing a lead Bicycle®:

[0139] • protease stability, whether this concerns Bicycle stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and the like. Protease stability should be maintained between different species such that a Bicycle lead candidate can be developed in animal models as well as administered with confidence to humans.

[0140] • replacement of oxidation-sensitive residues, such as tryptophan and methionine with oxidation-resistant analogues in order to improve the pharmaceutical stability profile of the molecule

[0141] • a desirable solubility profile, which is a function of the proportion of charged and hydrophilic versus hydrophobic residues, which is important for formulation and absorption purposes

[0142] • correct balance of charged versus hydrophobic residues, as hydrophobic residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged residues (in particular arginines) may influence the interaction of the peptide with the phospholipid membranes on cell surfaces. The two in combination may influence half-life, volume of distribution and exposure of the peptide drug, and can be tailored according to the clinical endpoint. In addition, the correct combination and number of charged versus hydrophobic residues may reduce irritation at the injection site (were the peptide drug administered subcutaneously).

[0143] • a tailored half-life, depending on the clinical indication and treatment regimen. It may be prudent to develop an unmodified molecule for short exposure in an acute illness management setting, or develop a bicyclic peptide with chemical modifications that enhance the plasma half-life, and hence be optimal for the management of more chronic disease states.

[0144] Approaches to stabilise therapeutic peptide candidates against proteolytic degradation are numerous, and overlap with the peptidomimetics field (for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

[0145] Modifications which may be made to a peptide identified in accordance with the disclosed methods include:

[0146] • Cyclisation of the polypeptide

[0147] • N- and C-terminal capping, usually N-terminal acetylation and C-terminal amidation.

[0148] • Alanine scans, to reveal and potentially remove the proteolytic attack site(s).

[0149] • D-amino acid replacement, to probe the steric requirements of the amino acid side chain, to increase proteolytic stability by steric hindrance and by a propensity of D- amino acids to stabilise p-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413- 418).

[0150] • N-methyl / N-alkyl amino acid replacement, to impart proteolytic protection by direct modification of the scissile amide bond (Fiacco et al, Chembiochem. (2008), 9(14), 2200-3). N-methylation also has strong effect on the torsional angles of the peptide bond, and is believed to aid in cell penetration & oral availability (Biron et al (2008), Angew. Chem. Int. Ed., 47, 2595 -99)

[0151] Incorporation of non-natural amino acids, i.e. by employing Isosteric / isoelectronic side chains that are not recognised by proteases, yet in some embodiments have little or no effect on target potency

[0152] - Constrained amino acid side chains, such that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically impeded. In particular, these concern proline analogues, bulky sidechains, Coc- disubstituted derivatives (where the simplest derivative is Aib, H2N- C(CH3)2-COOH), and cyclo amino acids, a simple derivative being aminocyclopropylcarboxylic acid).

[0153] • Peptide bond surrogates, and examples include

[0154] - N-alkylation (see above, i.e. CO-NR)

[0155] - Reduced peptide bonds (CH2-NH-)

[0156] - Peptoids (N-alkyl amino acids, NR-CH2-CO)

[0157] - Thio-amides (CS-NH)

[0158] - Azapeptides (CO-NH-NR)

[0159] - Trans-alkene (RHC=C-)

[0160] - Retro-inverso (NH-CO)

[0161] - Urea surrogates (NH-CO-NHR)

[0162] • Peptide backbone length modulation

[0163] - i.e. p23- amino acids, (NH-CR-CH2-CO, NH-CH2-CHR-CO),

[0164] • Substitutions on the alpha-carbon on amino acids, which constrains backbone conformations, the simplest derivative being Aminoisobutyric acid (Aib).

[0165] It should be explicitly noted that some of these modifications may also serve to deliberately improve the potency of the peptide against the target, or, for example to identify potent substitutes for the oxidation-sensitive amino acids (Trp and Met).

[0166] Polypeptide modifications may include replacement of one or more amino acid residues with one or more non-natural amino acid residues or vice versa, replacement of one or more amino acids (e.g. one or more natural amino acids) with one or more isosteric and / or isolectronic amino acids, replacement of one or more natural amino acids with one or more isosteric and / or isoelectronic non-natural amino acids, or vice versa, replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; introduction or replacement of amino acids that introduce orthogonal reactivities that are suitable for functionalisation, for example azide or alkyne-group bearing amino acids that allow functionalisation with alkyne or azide- bearing moieties, respectively.

[0167] Amino acid residues may typically be replaced with other amino acid residues of similar chemical structure, similar chemical properties or similar side-chain volume (“conservative substitutions”). The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace.

[0168] Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table A above. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains, which is well-known the person skilled in the art.

[0169] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N- terminal modification using suitable amino-reactive chemistry, and / or C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C- terminal modification comprises addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator or a chromophore.

[0170] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group is capped with acetic anhydride or other appropriate reagents during peptide synthesis leading to a molecule which is N-terminally acetylated. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and avoids the potential for degradation of the bicyclic peptide.

[0171] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group which facilitates the conjugation of effector groups and retention of potency of the bicyclic peptide to its target. In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal cysteine group is synthesized as an amide during peptide synthesis leading to a molecule which is C-terminally amidated. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidase and reduces the potential for proteolytic degradation of the bicyclic peptide.

[0172] Unnatural amino acids may be incorporated into proteins and peptides displayed on phage by transforming E. coli with plasmids or combinations of plasmids bearing: 1) the orthogonal aminoacyl-tRNA synthetase and tRNA that direct the incorporation of the unnatural amino acid in response to a codon, 2) The phage DNA or phagemid plasmid altered to contain the selected codon at the site of unnatural amino acid incorporation (Proc Natl Acad Sci U S A. 2008 Nov 18; 105(46): 17688-93. Protein evolution with an expanded genetic code. Liu CC, Mack AV, Tsao ML, Mills JH, Lee HS, Choe H, Farzan M, Schultz PG, Smider W; A phage display system with unnatural amino acids. Tian F, Tsao ML, Schultz PG. J Am Chem Soc. 2004 Dec 15;126(49):15962-3). The orthogonal aminoacyl-tRNA synthetase and tRNA may be derived from the Methancoccus janaschii tyrosyl pair or a synthetase (Addition of a photocrosslinking amino acid to the genetic code of Escherichiacoli. Chin JW, Martin AB, King DS, Wang L, Schultz PG. Proc Natl Acad Sci U S A. 2002 Aug 20;99(17):11020-4) and tRNA pair that naturally incorporates pyrrolysine (Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for sitespecific protein modification. Yanagisawa T, Ishii R, Fukunaga R, Kobayashi T, Sakamoto K, Yokoyama S. Chem Biol. 2008 Nov 24; 15(11 ): 1187-97; Genetically encoding N(epsilon)- acetyllysine in recombinant proteins. Neumann H, Peak-Chew SY, Chin JW. Nat Chem Biol. 2008 Apr;4(4):232-4. Epub 2008 Feb 17). The codon for incorporation may be the amber codon (UAG) another stop codon (UGA, or UAA), alternatively it may be a four base codon. The aminoacyl-tRNA synthetase and tRNA may be produced from existing vectors, including the pBK series of vectors, pSUP (Efficient incorporation of unnatural amino acids into proteins in Escherichia coli. Ryu Y, Schultz PG. Nat Methods. 2006 Apr;3(4):263-5) vectors and pDULE vectors (Nat Methods. 2005 May;2(5):377-84. Photo-cross-linking interacting proteins with a genetically encoded benzophenone. Farrell IS, Toroney R, Hazen JL, Mehl RA, Chin JW). The E.coli strain used will express the F’ pilus (generally via a tra operon). When amber suppression is used the E. coli strain will not itself contain an active amber suppressor tRNA gene. The amino acid will be added to the growth media, preferably at a final concentration of 1 mM or greater. Efficiency of amino acid incorporation may be enhanced by using an expression construct with an orthogonal ribosome binding site and translating the gene with ribo-X(Evolved orthogonal ribosomes enhance the efficiency of synthetic genetic code expansion. Wang K, Neumann H, Peak-Chew SY, Chin JW. Nat Biotechnol. 2007 Jul;25(7):770-7). This may allow efficient multi-site incorporation of the unnatural amino acid providing multiple sites of attachment to the ligand.

[0173] (B) Repertoires, sets and groups of polypeptide ligands

[0174] (I) Construction of Libraries

[0175] Libraries intended for selection may be constructed using techniques known in the art, for example as set forth in W02004 / 077062, or biological systems, including phage vector systems as described herein. Other vector systems are known in the art, and include other phage (for instance, phage lambda), bacterial plasmid expression vectors, eukaryotic cellbased expression vectors, including yeast vectors, and the like. For example, see W02009098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7. The present invention employs biological libraries which can be screened by genetic display.

[0176] Non-biological systems such as those set forth in W02004 / 077062 are based on conventional chemical screening approaches. They are simple, but lack the power of biological systems since it is impossible, or at least impracticably onerous, to screen large libraries of peptide ligands. Screening by such individual assays, however, may be timeconsuming and the number of unique molecules that can be tested for binding to a specific target generally does not exceed 106chemical entities.

[0177] In contrast, biological screening or selection methods generally allow the sampling of a much larger number of different molecules. Thus biological methods can be used in application of the invention. In biological procedures, molecules are assayed in a single reaction vessel and the ones with favourable properties (i.e. binding) are physically separated from inactive molecules. Selection strategies are available that allow to generate and assay simultaneously more than 1013individual compounds. Examples for powerful affinity selection techniques are phage display, ribosome display, mRNA display, cis display, yeast display, bacterial display, mammalian display or RNA / DNA aptamer methods (see Jaroszewicz et al., FEMS Microbiology Reviews, fuab052, 46, 2022, 1-25, incorporated herein by reference, for a more complete list). These biological in vitro selection methods have in common that ligand repertoires are encoded by DNA or RNA. They allow the propagation and the identification of selected ligands by sequencing. Phage display technology has for example been used for the isolation of antibodies with very high binding affinities to virtually any target. When using a biological system, once a vector system is chosen and one or more nucleic acid sequences encoding polypeptides of interest are cloned into the library vector, one may generate diversity within the cloned molecules by undertaking mutagenesis prior to expression; alternatively, the encoded proteins may be expressed and selected before mutagenesis and additional rounds of selection are performed.

[0178] Thus, for example, a library of sequences encoding polypeptides according to the invention, said sequences comprising areas of random or otherwise variant sequence differing between individual members of the repertoire, can be cloned into the nucleic acid encoding a phage, for use in a phage display. Therefore, in one embodiment, displaying the polypeptides on a genetic display system comprises encoding said repertoire of nucleic acids into a phage vector such that the polypeptide is expressed as part of a phage coat protein, and expressing the phage in a bacterial host to display the phage coat protein bearing the polypeptide at the bacterial surface.

[0179] The displayed polypeptide is a polypeptide as described herein, that is a polypeptide which comprises the sequence (X)IY(X)mY(X)nY(X)o, wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 1 and 20 defining the length of intervening polypeptide segments and I and o are numbers between 0 and 20 defining the length of the flanking polypeptide segments.

[0180] Preferably, I and o are independently between 0 and 15. In embodiments, m and n are independently between 2 and 10, preferably between 4 and 8, for example 4, 5, 6, 7 or 8.

[0181] In embodiments, the polypeptide comprises a sequence Y(X)mY(X)nY, wherein m is 4, 5, 6, 7, or 8 and n is 4, 5, 6, 7, 8; preferably wherein m is 5, 6 or 7, and n is 5, 6, or 7. Y is a reactive group, and is preferably Cysteine (C). X is any amino acid.

[0182] In some embodiments, the peptide ligand of the invention may comprise a polypeptide with the sequence C(X)6C(X)6C, wherein C is Cysteine and X represents any amino acid.

[0183] Said phage nucleic acid may be further mutated by subjecting the nucleic acid to mutagenesis, before the polypeptides are expressed as part of the phage and displayed on the phage coat.

[0184] In different genetic display systems, such as mRNA display, nucleic acids encoding the repertoire of polypeptides are cloned into the display system such that the nucleic acid is expressed in the appropriate manner for the display system to operate (in the case of mRNA display, in a manner such that the nascent polypeptide remains bound to the encoding mRNA).

[0185] Mutagenesis of nucleic acid sequences encoding structurally optimised polypeptides is carried out by standard molecular methods. Of particular use is the polymerase chain reaction, or PCR, (Mullis and Faloona (1987) Methods Enzymol., 155: 335, herein incorporated by reference). PCR, which uses multiple cycles of DNA replication catalysed by a thermostable, DNA-dependent DNA polymerase to amplify the target sequence of interest, is well known in the art. The construction of various antibody libraries has been discussed in Winter et al. (1994) Ann. Rev. Immunology 12, 433-55, and references cited therein.

[0186] Alternatively, polypeptide variants can be introduced by introducing using synthetic DNA of a specific sequence or mixture of sequences (e.g. degenerate oligonucleotides or mixes of oligonucleotides) to code for the polypeptides and these can be inserted into library or expression vectors using standard molecular biology techniques.

[0187] (ii) Genetically encoded diversity

[0188] In one embodiment, the polypeptides of interest are genetically encoded. This offers the advantage of enhanced diversity together with ease of handling. An example of a genetically polypeptide library is a mRNA display library. Another example is a replicable genetic display package (rgdp) library such as a phage display library. In one embodiment, the polypeptides of interest are genetically encoded as a phage display library. Thus, in one embodiment the genetic display system of the invention comprises a replicable genetic display package (rgdp) such as a phage particle. In these embodiments, the nucleic acid can be comprised by the phage genome. In these embodiments, the polypeptide can be comprised by the phage coat.

[0189] In some embodiments, the invention may be used to produce a genetically encoded combinatorial library of polypeptides which are generated by translating a number of nucleic acids into corresponding polypeptides and linking molecules of said molecular scaffold to said polypeptides.

[0190] The genetically encoded combinatorial library of polypeptides may be generated by phage display, yeast display, ribosome display, bacterial display or mRNA display.

[0191] Techniques and methodology for performing phage display can be found in W02009098450. In one embodiment, screening may be performed by contacting a library, set or group of polypeptide ligands with a target and isolating one or more member(s) that bind to said target.

[0192] In another embodiment, individual members of said library, set or group are contacted with a target in a screen and members of said library that bind to said target are identified.

[0193] In another embodiment, members of said library, set or group are simultaneously contacted with a target and members that bind to said target are selected.

[0194] The target(s) may be a peptide, a protein, a polysaccharide, a lipid, a DNA or a RNA.

[0195] The target may be a receptor, a receptor ligand, an enzyme, a hormone or a cytokine.

[0196] In some embodiments the target is a target expressed on a cancer cell. In some embodiments the target is a protein expressed on the surface of a cancer cell. In some embodiments the target is an epitope on the surface of a protein expressed on the surface of a cancer cell. In some embodiments the epitope is a continuous epitope comprising from about 3 to about 30 amino acids, e.g. from about 5 to about 20 amino acids.

[0197] The target maybe intracellular, secreted, membrane spanning or membrane tethered or associated.

[0198] The target may be a protein involved in mobility, adhesion, structure, signalling, storage, transport, catalytic activity or immunity.

[0199] The target may be a prokaryotic protein, a eukaryotic protein, or an archeal protein. More specifically the target ligand may be a mammalian protein or an insect protein or a bacterial protein or a fungal protein or a viral protein.

[0200] The target ligand may be an enzyme, such as a protease, kinase, hydrolase, transferase etc.

[0201] It should be noted that the invention also embraces polypeptide ligands isolated from a screen according to the invention. In one embodiment the screening method(s) of the invention further comprise the step of: manufacturing a quantity of the polypeptide isolated as capable of binding to said targets.

[0202] The invention also relates to peptide ligands having more than two loops. For example, tricyclic polypeptides joined to a molecular scaffold can be created by joining the N- and C- termini of a bicyclic polypeptide joined to a molecular scaffold according to the present invention. In this manner, the joined N and C termini create a third loop, making a tricyclic polypeptide. This embodiment need not be carried out on phage, but can be carried out on a polypeptide-molecular scaffold conjugate as described herein. Joining the N- and C- termini is a matter of routine peptide chemistry. In case any guidance is needed, the C- terminus may be activated and / or the N- and C- termini may be extended for example to add a cysteine to each end and then join them by disulphide bonding. Alternatively the joining may be accomplished by use of a linker region incorporated into the N / C termini. Alternatively the N and C termini may be joined by a conventional peptide bond. Alternatively any other suitable means for joining the N and C termini may be employed, for example N-C-cyclization could be done by standard techniques, for example as disclosed in Linde et al. Peptide Science 90, 671-682 (2008) "Structure-activity relationship and metabolic stability studies of backbone cyclization and N-methylation of melanocortin peptides", or as in Hess et al. J. Med. Chem. 51 , 1026-1034 (2008) "backbone cyclic peptidomimetic melanocortin-4 receptor agonist as a novel orally administered drug lead for treating obesity". One advantage of such tricyclic molecules is the avoidance of proteolytic degradation of the free ends, in particular by exoprotease action. Another advantage of a tricyclic polypeptide of this nature is that the third loop may be utilised for generally applicable functions such as BSA binding, cell entry or transportation effects, tagging or any other such use. It will be noted that this third loop will not typically be available for selection (because it is not produced on the phage but only on the polypeptide-molecular scaffold conjugate) and so its use for other such biological functions still advantageously leaves both loops 1 and 2 for selection / creation of specificity.

[0203] (iii) Phage purification

[0204] In accordance with the present invention, phage purification before reaction with the molecular scaffold is optional. In the event that purification is desired, any suitable means for purification of the phage may be used. Standard techniques may be applied in the present invention. For example, phage may be purified by filtration or by precipitation such as PEG precipitation; phage particles may be produced and purified by polyethylene-glycol (PEG) precipitation as described previously. Details can be found in W02009098450.

[0205] In case further guidance is needed, reference is made to Jespers et al (Protein Engineering Design and Selection 2004 17(10):709-713. Selection of optical biosensors from chemisynthetic antibody libraries.) In one embodiment phage may be purified as taught therein. The text of this publication is specifically incorporated herein by reference for the method of phage purification; in particular reference is made to the materials and methods section starting part way down the right-column at page 709 of Jespers et al. Moreover, the phage may be purified as published by Marks et al J. Mol. Biol vol 222 pp581- 597, which is specifically incorporated herein by reference for the particular description of how the phage production / purification is carried out.

[0206] If phage purification is not desired, culture medium including phage can be mixed directly with a purification resin and a reducing agent (such as TCEP), as set forth in the examples described in WO2014 / 140342.

[0207] (C) Expression of Screened Polypeptides

[0208] (i) Cloning of polypeptides

[0209] In order to assay any desired activity, polypeptides should be isolated from the components of the display system used. For example, phage polypeptides should be isolated form phage components such as coat proteins, which they are fused to during the phage display procedure. Yeast display and bacterial display likewise incorporate the polypeptide into the Aga2p gene or the OmpA gene. mRNA display employs a library in which the polypeptide is fused to puromycin and a DNA / RNA duplex encoding the polypeptide.

[0210] Advantageously, polypeptide “genes” can be cloned from library members identified during the genetic display screen and inserted into plasmids for expression of the polypeptide. Suitable cloning techniques include PCR and / or restriction digestion followed by ligation. As an alternative to cloning, or where cloning of specific polypeptide coding sequences is not possible (e.g. desired peptide sequence identified from large mixed population by next generation sequencing or when it is desirable to modify the sequence from a parent such as during affinity maturation), custom DNA sequences encoding for the desired polypeptide(s) can be purchased from commercial vendors and used equivalently.

[0211] Expression vectors for polypeptide expression can be selected from a wide variety known in the art and available commercially. A list of around 1700 bacterial expression plasmids is available on addgene.org. An exemplary expression vector is pET-3a, available from Novagen / Millipore.

[0212] Advantageously, the expression vector comprises tags and features to assist cloning and expression of the sequence encoding the polypeptide. For example, a purification tag, such as a polyhistidine tag, or a Strep tag can be used. Both tag systems facilitate purification of proteins. Any suitable purification tag can be used. For example, the purification tag may comprise or consist of biotin. Biotin is particularly suitable for use in the disclosed methods as it forms a strong non-covalent attachment with streptavidin and related proteins (neutravidin, avidin, etc)

[0213] More often, the purification tag is a peptide purification tags suitable for IMAC (immobilised metal affinity chromatography) chemistry. For example, the purification tag may comprise a poly-His tag (e.g. HHHH, HHHHHH or HHHHHHHH;). Such tags are suitable for binding to a purification support comprising a metal such as nickel or cobalt. Still other purification tags include peptide tags such as Strep (WSHPQFEK), FLAG (DYKDDDDK), Human influenza hemagglutinin (HA) (YPYDVPDYA), Myc (EQKLISEED), and V5 (GKPIPNPLLGLDST), etc.

[0214] Other suitable purification tags include: Biotin-carboxy carrier protein (BCCP); Calmodulin binding peptide (CBP); Chitin binding domain (CBD); Histidine affinity tag (HAT); Polyarginine (Arg-tag); Polyaspartate (Asp-tag); Polylysine (Lys-tag); Polyphenylalanine (Phe-tag); and Streptavadin-binding peptide (SBP).

[0215] Thus, in one embodiment the polypeptide comprises a purification tag and the method comprises purifying the polypeptide. In one embodiment purifying the polypeptide comprises contacting the polypeptide with a complementary purification matrix such as a chromatography column or bead (e.g. magnetic bead).

[0216] An expression partner may also be used, to improve expression of the polypeptide from the vector. In one embodiment the expression partner is selected from an optically-detectable protein (e.g. a fluorescent protein) and a detectable enzyme tag). For instance, DHFR or GFP may be coexpressed with the polypeptide. The coexpression of a partner may be desirable depending on the expression system used, and may be functional in detecting expression or in a downstream screening assay, such as in the case of GFP. A large number of expression or fusion partners is available in the art, for example as described in Costa S, et al., Front Microbiol. 2014 Feb 19;5:63.

[0217] Optionally, a protease cleavage site or “tag” may be incorporated, such as forTEV orthrombin. Use of such cleavage tags permits cleavage of the polypeptide from accompanying sequences by protease digestion.

[0218] Other sequences may be included, such as cell-penetrating peptides or proteins, avi-tags for site specific biotinylation, and the like.

[0219] PCR can be used to recover the polypeptide sequence from the display system and precisely insert the nucleic acid encoding the polypeptide into the cloning site of the vector. Additional elements may be added, such as selection markers and protein tags; these can be added sequentially, or together with the polypeptide nucleic acid, for example by Gibson assembly. Other cloning strategies include Golden Gate cloning, restriction digestion and ligation, TOPO cloning, SLIC, in-fusion cloning and Gateway cloning.

[0220] Exemplary markers and tags include spacer elements, protease recognition sequences (to cleaver markers if desired), streptavidin recognition sequences, and enzyme tags such as DHFR.

[0221] Cloning methods including PCR can be automated using liquid-handling machines and performed on multiwell plates to provide very high throughput analysis of Bicycles®.

[0222] In some embodiments a polypeptide expressed in a method disclosed herein may comprise a polypeptide portion having a sequence corresponding to the polypeptide displayed on the genetic display system. In some embodiments the polypeptide may further comprise a purification tag, such as a purification tag disclosed herein. In some embodiments the polypeptide may further comprise a cleavage site, such as protease cleavage site as disclosed herein. In some embodiments the polypeptide comprises a protease cleavage site between the sequence corresponding to the polypeptide displayed on the genetic display system and a purification tag. In some embodiments the polypeptide comprises one or more spacer groups. In some embodiments the one or more spacer groups each independently comprise from about 1 to about 10, such as from about 2 to about 5 amino acids. In some embodiments the spacer groups comprise amino acids selected from alanine, glycine, proline, and serine. Exemplary spacer groups include AGAA and PAS. In some embodiments a purification tag is flanked by one or more spacer group. In some embodiments a cleavage site is flanked by one or more spacer groups. In some embodiments a polypeptide expressed in a method disclosed herein may comprise or consist of a structure selected from

[0223] B-Sw-C-Sx-P-Sy-E; and

[0224] B-Sw-P-Sx-C-Sy-E wherein B is a polypeptide portion having a sequence corresponding to the polypeptide displayed on the genetic display system; each S is independently a spacer (e.g. AGAA or PAS); w, x and y are each independently 0 or 1 ; C is a cleavage site (e.g. a TEV site); P is a purification tag (e.g. a Strep tag); and E is an expression partner (e.g. GFP or DHFR).

[0225] (ii) Expression of polypeptides Expression advantageously takes place in a cell-free expression system (CFES) or in vitro transcription and translation system (IVTT). A selection of such systems is available commercially. An IVTT system may include, for example, an RNA polymerase, a ribosome, nucleotide phosphates, amino acid-loaded tRNAs, and translation factors, such as initiation and elongation factors. Suitable in vitro transcription / translation reagents are well known in the art (e.g. Isalan, M. et al (2005) PLoS Biol. 3 e64). An example is PURExpress, available from New England Biolabs.

[0226] PureExpress is a cell lysate; other expression systems based on cell lysates include TNT SP6 from Promega, and can potentially be made from any organism used to express proteins.

[0227] Expression systems may also be based on reconstituted pure components such as PUREfrex.

[0228] Alternatively expression can be cell-based, for example using bacteria (e.g. E. coll), mammalian cells (e.g. HEK293), or others including yeast, insect, plant cells such as wheat, and the like.

[0229] The expression reaction is preferably conducted in multiwell plates, such as 96 well plates, and automated using robotics.

[0230] (iii) Polypeptide modification

[0231] Modification of polypeptides is preferably carried out on multiwell plates, for example as described in WO2014 / 140342. This can be combined with use of automated liquid handling robots and / or magnetic particle processing robots.

[0232] (iii) Screening

[0233] Screening for a desired characteristic, such as the binding affinity of the polypeptide for target, is preferably carried out in a high-throughput screening apparatus, such as Clariostar from BMG Labtech. A number of assays can be envisaged, including biochemical assays such as HTRF, FRET-, luminescence- and absorbance-based assays, AlphaScreen, AlphaLISA, ELISA assays, and fluorescence polarization assays; biophysical assays such as SPR, BLI, grating-coupled interferometry (GCI), isothermal titration calorimetry (ITC), mass spectrometry, thermal shift (e.g. DSF), microscale thermophoresis (MST), temperature related intensity change (TRIC) and microfluidic diffusional sizing; imaging assays including microscopy and flow cytometry; other binding assays including radioligand binding and equilibrium dialysis; and other / functional activity assays including cell reporter assays, enzyme inhibition assays, cell uptake / efflux, cell killing or viability or cell cycle assays, in vitro ADME assays such as plasma stability assays and ion flux assays. EXAMPLES

[0234] Assaying Bicycles independent of phage overcomes the drawbacks of uncertain avidity and dosage effects, and allows for easier quantification and quality checks before committing to chemical polypeptide synthesis. It is not practical to use solid-phase peptide synthesis (SPPS) in a high-throughput manner for primary screening.

[0235] Cell-Free Expression Systems (CFES) provide in vitro transcription and translation and are used to biologically express the linear peptides in vitro. This system overcomes issues encountered by more traditional In vivo expression, namely proteolytic degradation (Martemyanov et al., 2001 , Protein Expression and Purification, 21 (3), pp.456-461). This is particularly useful when attempting to express low molecularweight peptides such as Bicycles, which are more susceptible to this (Li, 2011 , Protein Expression and Purification, 80(2), pp.260-267). It can also provide a higher throughput than in vivo expression, due to fewer method steps.

[0236] The peptides are subsequently modified with scaffold to cyclise, before being suitable for any desired assay.

[0237] We have thus developed a high-throughput workflow for the production and cyclisation of Bicycles for use in primary screening assays, using in vitro expression.

[0238] Methodology

[0239] Plasmid Design

[0240] An engineered pET-3a plasmid, with additional inserted regions was produced (Table 1 , Table 2, Table 3). All inserts contained a Bicycle® sequence, followed by one of two generic sequences, termed ‘tag’ regions. One of these, termed ‘non-conjugated’, was a four residue spacer, TEV protease recognition site, proline-alanine-serine (PAS) spacer and then Strep-tag II (Figure 1 A). The other, ‘DHFR’, was a four residue spacer, Strep-tag II, PAS spacer, TEV protease recognition site, PAS spacer and then Dihydrofolate Reductase (DHFR) (Figure 1 B).

[0241] Table 1

[0242] SUBSTITUTE SHEET (RULE 26)

[0243] Table 2

[0244] SUBSTITUTE SHEET (RULE 26)

[0245] Table 3

[0246] Agarose Gels

[0247] Gel loading dye (NewEngland Biolabs®) was added to each sample, at 1X. 5 pL per well of each sample with dye was then loaded into an agarose E-gel EX (ThermoFisher Scientific).

[0248] Either 1 % or 2% were used. For the 1 % gels, 20 pL of 1 kb DNA ladder (diluted 3:200) (New England BioLabs®) was used. For the 2% gel 5 pL 50 bp ladder (New England Biolabs®) was used. The gels were run for 10 minutes and imaged using the E-gel machine.

[0249] Gel Extraction

[0250] SUBSTITUTE SHEET (RULE 26) Desired bands from the agarose gel were excised and DNA purified using the Qiagen QIAquick Gel Extraction kit (Qiagen). The manufacturers protocol was followed, with the DNA being eluted in 30 pL of nuclease free water, after incubating on the column for 2 minutes.

[0251] General PCR All PCRs contained the reagents in Table 4, with the addition of the template DNA, and water to achieve a final volume of 20 pL. The primers (Table 5) and the template DNA used are detailed in the associated experiment section. The parameters of the reactions in a MiniAmp™ Plus Thermal Cycler (ThermoFisher Scientific) shown in Table 6 were used, with differences stated in the specific methods section.

[0252] Table 4: PCR reagents. Template DNA is described in the appropriate text. Table 5: Primer Sequences. Red indicates overhang region

[0253] SUBSTITUTE SHEET (RULE 26)

[0254] Table 6:. Thermocycler parameters for PCR reactions. Experiment specific changes are described in the appropriate text.

[0255] Template DNA vector production

[0256] PCR was performed on an engineered pET-3a plasmid, to linearise it and introduce Ndel restriction endonuclease sites. PCR was performed with primers 1 and 2 (Table 5). These are complementary to the pET-3a plasmid to ensure the inclusion of the tag region (and the rest of the plasmid), and exclusion of the Bicycle® sequence. The final concentration of the template DNA used was 0.62 ng / pL. The reaction was then purified using QIAquick PCR Purification Kit (Qiagen), by adding 30 pL per column, and eluting in 15 pL of water. A restriction digest was performed on all the purified DNA, for one hour at 37°C. The reaction reagents were at the following final concentrations in 25 pL: Ndel 200 units / mL, R3.1 Buffer 1X and 20 ng / pL of DNA. The sample was run on a 1% agarose gel, and the bands excised and purified. The purified DNA was then ligated using T4 ligase (New England BioLabs®). The reaction volumes and final concentrations in 20 pL used were as follows: 1 pL of T4 DNA Ligase (1x105 units / mL), 2 pL T4 DNA Ligase reaction buffer (1X) and 2.5 ng / pL of DNA. The reaction was incubated at 20°C for 90 minutes, before being heat inactivated at 65°C for 10 minutes.

[0257] The samples were transformed and sequenced. The cultures containing the correct sequence were mega prepped (Qiagen) in-house, following the manufacturer’s protocol.

[0258] Extraction of Bicycle® Sequence from Phage

[0259] SUBSTITUTE SHEET (RULE 26) PCR was performed on samples of phage glycerol stocks, using primers 3 and 4 (Table 5). The extension time during the amplification stage of the PCR was reduced to 15 seconds, from 78 seconds.

[0260] Gibson Assembly

[0261] The vector plasmid produced was incubated at room temperature overnight with the following: Ndel restriction endonuclease (1 unit per 100 ng of DNA), 1X R3.1 buffer and rSAP (1 unit per 1500 ng DNA). Following this, the sample was heat inactivated for 20 minutes at 65°C.

[0262] 10 pL of 2X Gibson Assembly Master mix (New England BioLabs®) was added to the DNA fragments (PCR product of Bicycle® from phage and the generated linear vector plasmid), along with 1 unit of Ndel, and enough water to achieve a final volume of 20 pL. The ratio of the DNA fragments was 1 :5 moles of vector: Insert, with 50 ng of template vector being used. The reactions were left at room temperature for 5 minutes, before being incubated at 50°C for 15 minutes, and then returned to room temperature for a further 5 minutes. The reaction was transformed and sequenced.

[0263] Transformation

[0264] 5 ng of plasmid DNA (less than 5% total volume of cells) was added to an aliquot of DH5- alpha competent E.coli (high efficiency) on ice. These were mixed and left on ice for 30 minutes. The cells were heat shocked at 42°C for 30 seconds, before being placed back on ice for 2 minutes. 950 pL of 37°C recovery media was then added to the tubes before being incubated at 37°C for 1 hour, while shaking at 250 rpm. 100 pL of each culture was then spread on warm agar plates (100 pg / mL Ampicillin +LB). The plates were incubated overnight at 37°

[0265] Sequencing

[0266] The following day, colonies were picked and sent for Sanger sequencing. Each colony was picked into a well containing 125 pL of 2xYT media with 100 pg / mL ampicillin. The samples were then incubated overnight at 37°C with 150rpm shaking. The next morning, 62.5 pL of 50% glycerol was mixed into each well, before 10 pL of each sample was transferred to a sequencing plate.

[0267] Sequence analysis was performed in Geneious Prime.

[0268] CFES New England Biolabs (NEB) PURExpress® In Vitro Protein Synthesis Kit (E6800S) was used. The manufacturers protocol was followed. However, the total reaction volume was adjusted, to 27 pL for increased DNA or water addition. The template DNA was added to achieve a final concentration of 18.5 ng / pL. 20 units RNAse Inhibitor (ThermoFisher Scientific - EO0384) was also added per 27 pL total reaction volume. The samples were incubated at 37°C for 2 hours while shaking at 250 rpm.

[0269] SDS-PAGE

[0270] Tricine SDS sample buffer with 0.02 M DTT was mixed with the samples at a 1 :1 ratio, and then incubated for 5 minutes at 95°C. 10 pL was loaded into the wells of a 10-20% tricine, 1 .0 mm gel (note, if a western blot was also being performed, the volume pipetted into wells was 5 pL, due to there being two gels). 5 pL of Novex™ pre-stained protein standard (ThermoFisher Scientific) was used as the ladder. This gel was in a tricine SDS running buffer. The electrophoresis was performed at 125 volts for 75 minutes. The gel was then stained on a rocker for 1 hour with Coomassie protein stain, before being washed for 10 minutes with water. This was then imaged and photographed on an iBright FL1500 device (ThermoFisher Scientific).

[0271] Western Blot

[0272] After electrophoresis the gel was rinsed with cell grade water for 30 minutes on a rocker, with the water being replaced at 10-minute intervals. The gel was transferred to a PVDF membrane (0.2 pm pore size) using an iBIot machine that provided 23 volts for 6 minutes. The following wash and incubation steps were performed. 50 ml of blocking buffer (Phosphate-Buffered Saline (PBS), 0.1% tween and 7% w / v skim milk powder) for 30 minutes, followed by 4 rinses with wash buffer (PBS with 0.1% tween). 25 ml of primary antibody solution was added for 30 minutes. This was 1.55 mg / ml mouse mAB to Strep-tag II, diluted in wash buffer (with 0.5% w / v skim milk powder) at a ratio of 1 :10,000 (antibody:wash buffer). A 30-minute wash was performed, replenishing the buffer at 10-minute intervals. 25 ml of secondary antibody solution was added for 30 minutes. This was 1 mg / ml goat pAB to mouse IgG (HRP), diluted in wash buffer (with 0.5% w / v skim milk powder) at a ratio of 1 :20,000 (antibody:wash buffer). Following this a final 30-minute wash was performed, replenishing the buffer at 10-minute intervals.

[0273] 2 ml of enhanced chemiluminescent (ECL) horseradish peroxidase (HRP) substrate (ThermoFisher Scientific, 34094) was applied to the membrane for five minutes. This was then imaged and photographed on an iBright FL1500 device (ThermoFisher Scientific).

[0274] Purification of peptide For the purification of peptide 121 -83-01 -strep, Strep-Tactin® Sepharose resin (IBA Lifesciences) was used. 60 pL of 50% stock was pipetted into a fresh 1.5 ml Eppendorf tube. The resin was then washed three times by mixing in 1 ml of wash buffer (100 mM tris-HCL pH 8, 150 mM NaCI, 1 mM EDTA) before then centrifuging at 300g for 2 minutes, followed by removing the supernatant, and repeating. After the washes, the resin was resuspended in 30 pL of wash buffer and the expression sample was mixed with resin, and incubated at room temperature for 15 minutes, at 250 rpm. The samples were centrifuged at 300g for 2 minutes, and then the supernatant was taken as the flow through. 1 mL of wash buffer was mixed with the resin, before being centrifuged at 300g for 2 minutes. The supernatant was taken as the first wash, and this process repeated for the second wash. 50 uL of Strep-Tactin elution buffer (1X) was mixed, and centrifuged at 21 ,300g for 2 minutes. The supernatant was then removed as the elution fraction. 45 pL for first elution fraction was taken, which was then centrifuged for a further 2 minutes at 21 ,300g. Following this, 35 pL of the supernatant was removed. This was the second elution fraction.

[0275] Modification with scaffold

[0276] 20 pL of MagStrep "type3" XT beads (IBA Lifesciences), were washed 3 times in PBS, before being added to the CFES sample. Beads were then moved sequentially through 96-well plates containing various solutions (Table 7). 1 M Tris-HCI pH 8 was used to neutralize the elution fractions.

[0277] Table 7: Plate contents for automated modification of peptides.

[0278] Mass Spectrometry Peptides from the cyclisation experiments were analysed by either Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF), or by liquid chromatography mass spectrometry (LC-MS).

[0279] Example 1

[0280] PCR of Bicycle sequences from Phage

[0281] Each 20 pL reaction was composed of the reagents listed in Table 8. Primers 3 and 4 (Table 5), and plasmid pET-3a, were used.

[0282] Table 8. PCR master mix recipe for Bicycle DNA extraction from phage infected TGls

[0283] A clean pipet tip was used to transfer a small amount of phage infected TGI glycerol stock or culture to the PCR tube

[0284] A thermocycler was set up to perform PCR on the samples using the settings in Table 9:

[0285] Table 9. Thermocycler protocol for Bicycle extraction PCR

[0286] The samples were placed on ice after being removed from the Thermocycler. 3.3 pL of SDS free, purple, gel loading dye (6X) was added to each sample. 5pL of each sample with dye was then loaded into a 2% agarose E-gel EX. 5 pL of the 50bp DNA ladder was also loaded. The gel was run for 10 minutes and imaged using the E-gel machine.

[0287] All reactions produced the PCR product at the appropriate number of base pairs. Example 2

[0288] Plasmid assembly

[0289] In a fresh PCR tube, 2 ul of PCR reaction, 0.5 ul of linear plasmid (containing the AA sequences to be conjugated to the Bicycle), 7 ul water, and 10 ul Gibson master mix were combined.

[0290] A 1 : 10 dilution Ndel was made in the supplied buffer and 0.5 ul added to each of the reaction tubes. The reaction was incubated for 1 hour at 50 °C.

[0291] More PCR master mix was assembled as in table 8, using a forward primer with sequence AGTCGTGTCTTACCGGG and a reverse primer with sequence TATCGCCACTGGCAGCAG. 1 ul of Gibson reaction mix was added and incubated in a thermocycler using the setting in Table 10.

[0292] Table 10. Thermocycler protocol amplification of linear Gibson product

[0293] The resulting PCR reaction results in linear vector containing the Bicycle sequence in the center. The concentration is around 0.5 uM.

[0294] Both template vector plasmids showed efficient linearisation through single endonuclease digest (Figure 2). This was indicated by the higher running of the digested sample, compared to the uncut plasmid, which presents a band of supercoiled plasmid, which is only achievable with circular DNA.

[0295] The Gibson assembly generated correct sequences (Figure 3).

[0296] Example 3

[0297] Cell-free polypeptide expression

[0298] In a protein low bind Eppendorf tube, the cell free expression mixture according to table 11 was assembled. Volume (ul) for 1x reaction

[0299] Table 11. Cell free expression recipe for a 100 ul reaction using NEB Purexpress

[0300] The reaction mixture was incubated in a shacking incubator at 37°C 250 rpm for 2 hours.

[0301] The expression mix was then flash frozen or progressed to purification and cyclisation.

[0302] The peptide 121-83-01 was successfully purified using Strep-Tactin Sepharose resin, producing a single band in the second elution fraction (Figure 4). The majority of the CFES proteins were removed in the flow-through, with any remaining being washed out. The higher molecular weight contaminant seen in the first elution fraction was determined to be the Strep- Tactin® monomer. Further steps to reduce pipetting error removed the accidental collection of the resin. The identity of the peptide was confirmed by a western blot (Figure 5).

[0303] Using either purification, so the peptide band was visible by Coomassie stain, or a western blot, the successful expression of peptides containing different Bicycle® sequences in CFES was determined. Use of a DHFR construct significantly increase the efficiency of expression.

[0304] Example 4

[0305] Cyclisation

[0306] For each 100 ul cell free expression reaction, 20 ul of magnetic strep-tactin XT beads were washed 3 times in PBS and resuspended in the original volume.

[0307] The reagents listed in Table 12 were added to deep 96 well plates.

[0308] Table 12. Solutions for purifying and scaffolding the peptides.

[0309] The Strep-tactin beads were added to the cell free expression reactions in plate number 1 and mixed. The beads were then moved sequentially through the other plates. After treatment in the final solution (50mM glycine pH2), the magnetic beads are discarded and the eluted peptides are neutralised by additional of 5 ul of 1 M Tris-HCL pH8.

[0310] Firstly, the gel (Figure 6) showed that the peptide can be successfully purified out of the CFES, and eluted using a low pH buffer, during the automated cyclisation process. The results matched previous purification only studies performed. While the gel is not high enough resolution to confidently determine if scaffold addition has occurred, the data from LC-MS confirms a correct mass shift from linear to cyclised peptide (Figure 7).

[0311] Similar results were seen when the modification process was performed on the same peptides but manually. These peptides were analysed using MALDI-TOF (Table 13). The dimer forming, that is seen as the higher molecular weight band on the gel, is seen in the spectra (Figures 8-16). There was an unexpected mass increase of around 29 Da on all the samples tested.

[0312] Table 13

[0313] Example 5

[0314] Binding of expressed cyclized polypeptides

[0315] Bicycles® specific for TFRIwere selected from previous screens based on synthesized polypeptides. Nucleic acid sequences encoding the polypeptide components of the Bicycles® were cloned from phage and expressed and cyclized, and Kd was tested by SPR . Cell binding was assessed fluorescence of cells bound by fluorescently labelled Bicycles®. Binding was also assessed by ELISA using fluorescently labelled Bicycles®.

[0316] Cloning

[0317] DNA sequences corresponding to Bicycle peptides (scaffolded on TATB) identified by phage display against TFR1 were codon optimised for expression in E.coli B. The sequences were fused to the n-terminal of AvsfGFP{C48A} - superfolder GFP (A heavily mutated version of the green fluorescent protein from Aequorea Victoria) spaced by a poly Pro / Ala / Ser (“PAS”) linker that mimics the linker used in phage display of Bicycles. C-terminally to the AvsfGFP{C48A}, a TEV protease (C4 peptidase from tobacco etch virus) recognition site was engineered followed by a short PAS linker and a polyhistidine tag. Designed sequences were cloned into a pET3a protein expression vector.

[0318] Expression and purification

[0319] 1. Following transformation of the complete plasmids into BL21 (DE3) E.coli, starter cultures were grown in TB (Terrific Broth) supplemented with 2% (w / v) D-Glucose, overnight 37C. Larger cultures were inoculated in autoinduction media (Terrific Broth + 5 g / L D-Glucose, 2 g / L D-Lactose, 10 mM MgS04, 1 g / L ammonium sulfate) and grown at 25C for 24 hours.

[0320] 2. Cultures were harvested by sedimentation via centrifugation (4000 g, 30 mins, 4C).

[0321] 3. E.coli pellets were resuspended in a minimal volume of lysis buffer (25 mM HEPES pH8, 300 mM NaCI, 0.25 mM TCEP, 5 mM Imidazole, 0.1 mg / mL Lysozyme, 100U / mL DNAase) and sonicated at 40% power for 10mins in 10 second intervals on ice.

[0322] 4. Lysates were clarified by sedimentation via centrifugation (60 000 g, 30 min, 4C).

[0323] 5. Lysates were further clarified by sterile vacuum filtration (0.45 urn)

[0324] 6. Lysates incubated with 5 mL of PureCube 100 Ni-INDIGO agarose Resin (Cube Biotech, GmbH) under constant stirring overnight at 4C.

[0325] 7. Resin was collected by sedimentation via centrifugation (400 g, 10 mins, 20C)

[0326] 8. Resin was washed with 10 bed volumes of wash buffer (25 mM HEPES pH8, 150 mM NaCI, 0.25 mM TCEP, 5 mM Imidazole). 9. The protein was eluted with wash buffer supplemented with 1 M imidazole.

[0327] Cyclisation

[0328] 10. The protein was diluted to 5 uM in modification buffer (25 mM HEPES pH8, 150 mM NaCI, 0.25 mM TCEP) and incubated with 50 uM TATB with a final concentration of 20% (v / v) Acetonitrile. The mixture was left for 30 mins and subsequently quenched in a 10 M excess of D-Cysteine to TATB for 10 min, followed by supplementation to a final concentration of 50 mM ammonium bicarbonate.

[0329] 11 . The reaction was diluted in modification buffer to a final concentration of less than 50 mM Imidazole.

[0330] 12. Reaction mixtures were incubated with 5 mL of PureCube 100 Ni-INDIGO agarose Resin (Cube Biotech, GmbH) under constant stirring overnight at 4C.

[0331] 13. Resin was collected by sedimentation via centrifugation (400 g, 10 mins, 20C)

[0332] 14. Resin was washed with 10 bed volumes of wash buffer (25 mM HEPES pH8, 150 mM NaCI, 0.25 mM TCEP, 5 mM Imidazole).

[0333] 15. The protein was eluted with wash buffer supplemented with 1 M imidazole.

[0334] Protein eluates were concentrated via centrifugal concentrators (MWCO <3K) and aggregates were removed via gel filtration (HiLoad Superdex 200 pg, 25 mM HEPES pH 8, 150 mM NaCI)

[0335] ELISA

[0336] To appropriate wells of a Thermo Scientific™, Pierce™ Streptavidin Coated Plate (Clear, 96- Well) was added either 50 pL of 40nM biotinylated TfR1 (AC RO Biosystems TFR-H82E5) or PBS alone, and incubated for 1 h at room temperature. The wells were washed (3x) with PBS + 0.1% Tween (PBST) to remove excess protein. The plate was then blocked with 200 pL PBST-1% BSA for 1 hour. Excess liquid was removed, and 50pL of 200nM of expressed Bicycle was added, and incubated for 1 hr at room temperature. The wells were washed again (3x) with PBST to remove non-bound peptide, and 50pL of 1 :1000 anti-GFP antibody (Abeam, cat ab1218) was added to each well, and incubated at room temperature for 1 hr at room temperature. The wells were washed again (3x) with PBST and 50pL of 1 :10,000 anti-mouse HRP (Abeam, cat ab97023) added to each well, and incubated at room temperature for 1 hr at room temperature. The wells were washed again (6x) with PBST before 50 pL of TMB solution (Sigma, T0440-1 L) was added to each well for ~1-2 mins. The colour change reaction was stopped with 50pL 1% (v / v) HCI. The plate was then read on a on a Pherastar FS / FSX (BMG Labtech)at OD of 450 nM. Data is displayed as signal (TfR1) - background (PBS alone).

[0337] Cell Binding

[0338] The cell binding ability of expressed Bicycles was measured using flow cytometry using standard cell culture techniques. For this, 200 pL U87MG cells were seeded into 96 well U- bottomed plates at a density of 80,000 cells per well. The plate was centrifuged for 5 min at 100 ref, and supernatant removed. 100 pL of expressed Bicycles starting from 1 pM concentration with 1 :2 dilution, 8 dilution points. These were incubated 45 min on ice. Followed by 3x wash with PBS. Cells were resuspending in 150ul of cell staining buffer containing DAPI (3uM solution), and incubated in the dark for 10 min. Run on the Attune Nxt with 100 pL injection with 200 pL / sec speed. Cells were gated as singlets followed by low DAPI and the median green fluorescent value was plotted as data.

[0339] It was found that both the relative Kd and the cell binding data were consistent between the synthesized and expressed polypeptides, confirming that polypeptide synthesis is a viable, faster and lower cost alternative to synthesis in polypeptide selection experiments. Absolute Kd values varied between the expressed polypeptide and synthesized polypeptide experiments, as might be expected between different experiments, but the relative binding performance was maintained between sequences.

[0340] Cell binding data using labelled expressed polypeptides was also in agreement with SPR data from both synthesized and expressed polypeptides.

[0341] The invention is described in the foregoing examples for the purposes of illustration only, and variations of the exemplified embodiments will be apparent to those skilled in the art which fal within the scope of the appended claims. All documents cited herein are incorporated by reference.

Claims

Claims1 . A method for selecting a polypeptide displayed on a genetic display system according to an activity, comprising the steps of: a. displaying the polypeptide on the genetic display system, screening the system for binding to the target, and selecting members which bind to the target; b. isolating nucleic acid encoding the polypeptide selected in (a) directly from the display system members or synthesizing nucleic acid with the sequence encoding the polypeptide selected in (a) and expressing the nucleic acid in an expression system to produce the polypeptide,; c. determining the activity of the polypeptide in an assay.

2. A method according to claim 1 , wherein nucleic acid encoding the polypeptide is cloned from the genetic display assay member and expressed in an expression system.

3. A method according to claim 1 , wherein the genetic display assay member is sequenced and the sequence information is used to design synthetic DNA used to express a polypeptide or a repertoire of polypeptides, the activity of which is determined in step (c).

4. A method according to any preceding claim, wherein the selected polypeptide comprises reactive groups, and said polypeptide is covalently bound to a molecular scaffold by reaction with said reactive groups such that two or more peptide loops are subtended between attachment points to the scaffold.

5. A method according to claim 4, wherein the polypeptide is attached to the scaffold at two attachment points.

6. A method according to claim 4 or claim 5, wherein the scaffold is selected from the group consisting of 1 ,3,5-triacryloyl-1 ,3,5-triazinane (TATA), 1-[3,5-bis(2-bromoacetyl)-1 ,3,5- triazinan-1-yl]-2-bromoethanone (TATB), N,N’,N”-(benzene-1 ,3,5-triyl)- tris(2- bromoacetamide) (TBAB) 1 ,1',1"-(1 ,4,7-triazonane-1 ,4,7-triyl)tris(2-chloroethan-1-one) (TCAZ), 1 ,1 ',1"-(1 ,4,7-triazonane-1 ,4,7-triyl)tris(2-bromothan-1-one) (TBAZ), 2,4,6- tris(bromomethyl)-s-triazine (TBMT), 2,4,6-tris(chloromethyl)-1 ,3,5-triazine (TCTZ), 1 ,T,1"-(1 H,4H-3a,6a-(methaniminomethano)pyrrolo[3,4-c]pyrrole-2,5,8(3H,6H)-triyl)tris[2-chloro- ethanone] (TBCU), 1 ,3,5-tri(ethenesulfonyl)-1 ,3,5-triazinane (TSTA), 2-bromo-1-[7,10-bis(2- bromoacetyl)-3,7,10-triazatricyclo[3.3.3.0A1 ,5]undecan-3-yl]ethenone (TBCU) and [3,5-bis[4- (bromomethyl)benzoyl]-1 ,3,5-triazinan-1-yl]-[4-(bromomethyl)phenyl]methanone (TBPM).

7. A method according to any preceding claim, wherein the activity is selected from binding affinity, cell permeability, internalisation into cells, cell reporter functionality, enzyme inhibition activity, activity in cell killing, cell viability modification, cell cycle inhibition, and activity in ion flux assays.

8. A method according to claim 7, wherein the activity is binding affinity.

9. A method according to claim 1 or claim 2, wherein the expression system is a cell- free expression system (CFES).

10. A method according to any preceding claim, wherein the polypeptide includes one or more polypeptide tags and / or protease recognition sequences.

11. A method according to claim 10, wherein one or more polypeptide tags are not derived from the genetic display system.

12. A method according to any preceding claim, wherein the polypeptide is expressed fused with an expression partner.

13. A method according to claim 12, where the expression partner is a detectable enzyme or a detectable protein label.

14. A method according to claim 13, wherein the expression partner is GFP.

15. A method according to any preceding claim, wherein the genetic display system displays a repertoire of polypeptide variants.

16. A method according to claim 15, wherein the repertoire has a complexity of at least 104, 105, 106, 107, 108, 109, 1010or more polypeptide variants.

17. A method according to any preceding claim, wherein the genetic display system is selected from phage display, ribosome display, mRNA display, cis display, yeast display, bacterial display, mammalian display or RNA / DNA aptamer methods.

18. A method according to any preceding claim, wherein nucleic acid encoding the polypeptide is cloned from the genetic display system substantially free of any genetic display system components.

19. A method according to claim 18, wherein the genetic display system is phage display and the cloned nucleic acid encoding the polypeptide is free of any phage coat protein component.

20. A method according to any preceding claim, wherein the expressed polypeptide does not comprise a component of the genetic display system.21 . The method according to any preceding claim, wherein the activity of the polypeptide is determined by an assay selected from the group consisting of including biochemical assays such as HTRF, FRET-, luminescence- and absorbance-based assays, AlphaScreen, AlphaLISA, ELISA assays and fluorescence polarization assays; biophysical assays such as SPR, BLI, Grating-Coupled Interferometry (GCI), Isothermal Titration Calorimetry (ITC), Mass spectrometry, Thermal shift (e.g. DSF), Microscale Thermophoresis (MST), Temperature Related Intensity Change (TRIC) and Microfluidic diffusional sizing; imaging assays including microscopy and flow cytometry; other binding assays including radioligand binding and equilibrium dialysis; and other / functional activity assays including cell reporter assays, cell internalisation, uptake / efflux assays, enzyme inhibition assays, cell killing or viability or cell cycle assays and ion flux assays.

22. The method according to any preceding claim, further comprising attaching the expressed or selected polypeptide to one or more functional groups.

23. The method of claim 22, wherein the functional group is selected from a binding group, such as a polypeptide, including an antibody domain, an Fc domain or a further structured peptide, a reactive group, an effector group, a drug such as a toxin, a metal chelator, a chromophore, a fluorophore, a label, or a cell penetrating peptide.

24. A method according to any preceding claim, comprising formulating the selected polypeptide into a pharmaceutical composition.

25. A method according to any preceding claim, comprising synthesising the selected polypeptide.

26. A polypeptide produced by the method according to claim 24.

27. A library of expressed polypeptides, each corresponding to a polypeptide displayed on a member of a genetic display system having an activity against a target; wherein each expressed polypeptide comprises a polypeptide having a sequence corresponding to the sequence of the polypeptide displayed on the member; and a purification tag; preferably wherein the expressed polypeptides in the library do not comprise a component of the genetic display system.

28. A system, comprising:(a) a genetic display system for displaying a plurality of polypeptides and selecting one or more displayed polypeptides having activity against a target; and(b) an assay system for determining the activity of one or more isolated polypeptides corresponding to the displayed polypeptides thereby selected.

29. A system according to claim 28, further comprising an expression system for expression isolated polypeptides corresponding to the selected displayed peptides.

30. A system according to claim 28 or claim 29, which is a high-throughput system.

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