US20140274759A1 - Modification of polypeptides - Google Patents

Modification of polypeptides Download PDF

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US20140274759A1
US20140274759A1 US13/832,526 US201313832526A US2014274759A1 US 20140274759 A1 US20140274759 A1 US 20140274759A1 US 201313832526 A US201313832526 A US 201313832526A US 2014274759 A1 US2014274759 A1 US 2014274759A1
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Prior art keywords
phage
peptide
polypeptide
nahco
molecular scaffold
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Edward Walker
Catherine Stace
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BicycleRD Ltd
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Bicycle Therapeutics PLC
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Priority to US13/832,526 priority Critical patent/US20140274759A1/en
Priority to DK14710558.9T priority patent/DK2970954T3/da
Priority to PL14710558T priority patent/PL2970954T3/pl
Priority to PT14710558T priority patent/PT2970954T/pt
Priority to EP14710558.9A priority patent/EP2970954B1/en
Priority to CN201480014384.0A priority patent/CN105189747B/zh
Priority to PCT/EP2014/055204 priority patent/WO2014140342A1/en
Priority to JP2015562247A priority patent/JP6437468B2/ja
Priority to AU2014229994A priority patent/AU2014229994B2/en
Priority to SG11201506876XA priority patent/SG11201506876XA/en
Priority to ES14710558T priority patent/ES2700999T3/es
Priority to CA2901535A priority patent/CA2901535C/en
Assigned to BICYCLE THERAPEUTICS LIMITED reassignment BICYCLE THERAPEUTICS LIMITED ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WALKER, EDWARD, STACE, Catherine
Publication of US20140274759A1 publication Critical patent/US20140274759A1/en
Priority to US14/849,637 priority patent/US9932367B2/en
Assigned to Bicyclerd Limited reassignment Bicyclerd Limited CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: BICYCLE THERAPEUTICS LIMITED
Assigned to Bicyclerd Limited reassignment Bicyclerd Limited CORRECTIVE ASSIGNMENT TO CORRECT THE ADDRESS OF THE ASSIGNEE PREVIOUSLY RECORDED ON REEL 044893 FRAME 0767. ASSIGNOR(S) HEREBY CONFIRMS THE CHANGE OF NAME. Assignors: Bicyclerd Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/14—Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support

Definitions

  • the present invention concerns methods for production of polypeptide ligands having a desired binding activity.
  • the invention concerns the production of polypeptides which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. Attachment of the molecular scaffold to the polypeptide is performed on a purification resin, which can take the form of magnetic resin beads.
  • 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.
  • 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.
  • macrocycles bind to surfaces of several hundred square angstrom, as for example the cyclic peptide CXCR4 antagonist CVX15 (400 ⁇ 2 ; Wu, B., et al., Science 330 (6007), 1066-71), a cyclic peptide with the Arg-Gly-Asp motif binding to integrin ⁇ Vb3 (355 ⁇ 2 ) (Xiong, J.
  • 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.
  • MMP-8 matrix metalloproteinase 8
  • WO2004/077062 discloses a method of selecting a candidate drug compound.
  • 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.
  • WO2006/078161 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.
  • FIG. 1 of this document shows a schematic representation of the synthesis of various loop peptide constructs.
  • constructs disclosed in this document rely on —SH functionalised peptides, typically comprising cysteine residues, and heteroaromatic groups on the scaffold, typically comprising benzylic halogen substituents such as bis- or tris-bromophenylbenzene. Such groups react to form a 3yclized3s linkage between the peptide and the scaffold.
  • the best inhibitor, PK15 inhibits human PK (Hpk) with a K i 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 (hfXla; 69% sequence identity) or thrombin (36% sequence identity) at the highest concentration tested (10 ⁇ M) (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.
  • the present invention provides a method for conjugating a peptide displayed on a genetic display system to a molecular scaffold, comprising the steps of:
  • the original method by Heinis et al. performed the conjugation of peptide and molecular scaffold (TBMB) in free solution. Phage, bearing peptides which were (or were not) conjugated to the TBMB scaffold were then isolated by centrifugation.
  • the present invention obtained improved results by conjugating the phage to a solid phase purification resin, which can then be used to isolate the phage.
  • the resin can be isolated by centrifugation or retained in columns; in a preferred embodiment, the resin is magnetic and can be isolated by the application of a magnetic field.
  • the genetic display system is selected from phage display, ribosome display, mRNA display, yeast display and bacterial display. In one embodiment, the genetic display system is phage display.
  • step (a) is followed by a washing step before addition of the molecular scaffold. Washing can be performed, for example, with a solution of a reducing agent, for example the reducing agent used in step (a).
  • a reducing agent for example the reducing agent used in step (a).
  • the reducing agent used in the washing step is less powerful or more dilute than the reducing agent used in step (a).
  • the resin-bound polypeptides may be exposed to the reducing agent in purified form, or can be present in culture.
  • Genetic display systems involve replication in cells, such as bacteria or yeast; these cells may be removed by purification, in which case step (a) can comprise a washing step, in which polypeptides bound to resin are washed in buffer and separated from the cell culture contaminants.
  • a suitable reducing agent is TCEP.
  • Other reducing agents, such as DTT, can be used as set forth herein.
  • the reduction and conjugation reactions are preferably conducted at room temperature, such as 25° C.
  • room temperature such as 25° C.
  • reactions are conducted at temperatures above room temperature, for example 42° C.
  • 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.
  • at least two loops are provided. In some embodiments, three, four, five, six or more loops may be present.
  • Reactive groups in the polypeptide are capable of forming covalent linkages with the scaffold. Most commonly, reactive groups comprise cysteine residues.
  • Peptides are combined with a purification resin, which can be any suitable resin which is useful as a solid phase for the purification of protein material.
  • a purification resin which can be any suitable resin which is useful as a solid phase for the purification of protein material.
  • Many resins, such as ion-exchange resins including beads and chromatography materials are known in the art which are useful for this purpose.
  • the resin is a magnetic resin, which allows magnetic separation of the polypeptides bound to the genetic display system.
  • 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.
  • the genetic display systems incorporating the polypeptide ligands are eluted from the resin.
  • the polypeptides can then be displayed on the genetic display system in conjugated form, and selected by known means.
  • the polypeptide ligands are multispecific.
  • the polypeptide loops formed by the interaction of the polypeptide with the molecular scaffold are capable of binding to more than one target.
  • loops may be selected individually for binding to the desired targets, and then combined.
  • the loops are selected together, as part of a single structure, for binding to different desired targets.
  • 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.
  • a functional group may be attached to the molecular scaffold itself.
  • Examples of functional groups are as for the preceding configuration.
  • 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.
  • 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.
  • polypeptide cyclisation is cyclised by N—C crosslinking, using a crosslinking agent such as EDC.
  • 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.
  • the polypeptide is cyclised by enzymatic means.
  • the enzyme is a transglutaminase, for instance a microbial transglutaminase, such as Streptomyces mobaraensis transglutaminase.
  • a transglutaminase for instance a microbial transglutaminase, such as Streptomyces mobaraensis transglutaminase.
  • a microbial transglutaminase such as Streptomyces mobaraensis transglutaminase.
  • the polypeptide ligands according to the invention are specific for human Kallikrein, and comprise a polypeptide comprising at least three reactive groups, separated by at least two loop sequences, and a molecular scaffold which forms covalent bonds with the reactive groups of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the loops of the peptide ligand comprise three, four or five, but less than six, amino acids.
  • peptides comprising less than 6 amino acids in each loop can have a much higher binding affinity for Kallikrein.
  • the loops of the peptide ligand comprise three amino acids and the polypeptide has the consensus sequence G r FxxG r RVxG r , wherein G r is a reactive group.
  • the loops of the peptide ligand comprise five amino acids and a first loop comprises the consensus sequence G r GGxxNG r , wherein G r is a reactive group.
  • two adjacent loops of the polypeptide may comprise the consensus sequence G r GGxxNG r RxxxxG r .
  • the loops of the peptide ligand comprise five amino acids and a first loop comprises the motif G r x w / F Px K / R G r , wherein G r is a reactive group.
  • the reference to a “first” loop does not necessarily denote a particular position of the loop in a sequence. In some embodiments, however, the first loop may be proximal loop in an amino terminus to carboxy terminus peptide sequence.
  • the polypeptide further comprises a second, distal loop which comprises the motif G r T / L H Q / T xLG r .
  • sequences of the first loop include G r xWPARG r , G r xWPSRG r , G r xFPFRG r and G r xFPYRG r .
  • x may be any amino acid, but is for example S or R.
  • the loops of the peptide ligand comprise five amino acids and a first loop comprises the motif G r xHxDLG r , wherein G r is a reactive group.
  • the loops of the peptide ligand comprise five amino acids and a first loop comprises the motif G r THxxLG r , wherein G r is a reactive group.
  • the polypeptide comprises two adjacent loops which comprise the motif G r x w / F Px K / R G r T / L H Q / T DLG r .
  • the reactive group is preferably a reactive amino acid.
  • the reactive amino acid is cysteine.
  • Variants of the polypeptides according to this aspect of the invention can be prepared as described above, by identifying those residues which are available for mutation and preparing libraries which include mutations at those positions.
  • polypeptide ligand according to the preceding aspect of the invention, which comprises one or more non-natural amino acid substituents and is resistant to protease degradation.
  • the present invention found that certain non-natural amino acids permit binding to plasma Kallikrein with nM Ki, whilst increasing residence time in plasma significantly.
  • the non-natural amino acid is selected from N-methyl Arginine, homoarginine and hydroxyproline.
  • N-methyl and homo-derivatives of Arginine are used to replace Arginine, and proline 3 can be preferably replaced by hydroxyproline, azetidine carboxylic acid, or an alpha-substituted amino acid, such as aminoisobutyric acid.
  • arginine may be replaced with guanidyl-phenylalanine.
  • the polypeptide comprises a first loop which comprises the motif G r xWPARG r , wherein P is replaced with azetidine carboxylic acid; and/or R is replaced with N-methyl arginine; and/or R is replaced with homoarginine; and/or R is replaced with guanidyl-phenylalanine.
  • the polypeptide comprises a first loop which comprises the motif G r xFPYRG r , wherein R is replaced with N-methyl arginine; and/or R is replaced with homoarginine, and wherein proline is replaced by azetidine carboxylic acid; and/or R is replaced with guanidyl-phenylalanine.
  • the polypeptide ligand may further comprise a sarcosine polymer, used as a linker to link polypeptide ligands together, or to attach one or more functional groups.
  • the polypeptide ligand may be protease resistant.
  • Protease resistant conjugates can be selected by screening a repertoire of polypeptide ligands for protease resistance.
  • FIG. 1 Assessment of the reaction conditions for linking phage displayed peptides to tris-(bromomethyl)benzene (TBMB).
  • TBMB tris-(bromomethyl)benzene
  • C Titres (transducing units) of phage reduced and treated with various concentrations of TBMB in 20 mM NH 4 HCO 3 , 5 mM EDTA, pH 8, 20% ACN at 30° C. for 1 hour. Titres of phage from fdg3p0ss21 (black) and from library 1 (white) are shown.
  • FIG. 2 Chemical reaction of the tri-functional compound TBMB with peptides containing one or two cysteines.
  • A Plausible reaction mechanism of TBMB with a peptide fusion protein containing two cysteine residues.
  • B Mass spectra of a peptide fusion proteins with two cysteines before and after reaction with TBMB.
  • C Plausible reaction mechanism of TBMB with a peptide fusion protein containing one cysteine residue.
  • D Mass spectra of a peptide fusion proteins with one cysteine before and after reaction with TBMB.
  • FIG. 3 The binding of resin-processed modified polypeptide ligands to kallikrein is illustrated.
  • FIG. 4 The effect of different buffers on the performance of the modification procedure.
  • A the effect of different modification buffers, NaHCO3 and NH4CO3.
  • B The effect of different concentrations of NaCl elution buffer at different pH.
  • C The effect of different concentrations of NaCl elution buffer and pH on elution in first and second steps in a two-step elution procedure.
  • FIG. 5 Target binding assay from the eluates of different samples treated with different buffers and eluted at different pH.
  • FIG. 6 Illustration of quick and long magnetic modification protocols.
  • FIG. 7 Comparison of quick and long protocols for modification of PK15-bearing phage: (A) comparison of phage titre by qPCR, and (B) functional comparison for Kallikrein binding.
  • a (poly)peptide ligand or (poly)peptide conjugate refers to a polypeptide covalently bound to a molecular scaffold.
  • 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.
  • the polypeptides comprise at least three reactive groups, and form at least two loops on the scaffold.
  • the reactive groups are groups capable of forming a covalent bond with the molecular scaffold.
  • the reactive groups are present on amino acid side chains on the peptide. Examples are amino-containing groups such as cysteine, lysine and selenocysteine.
  • 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.
  • 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.
  • 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.
  • Binding activity 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.
  • Multispecificity is the ability to bind to two or more targets.
  • 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.
  • peptides can be developed which can bind to two or more targets; dual specific antibodies, for example.
  • the peptide ligands can be capable of binding to two or more targets and are therefore be multispecific.
  • 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.
  • a target is a molecule or part thereof to which the peptide ligands bind or otherwise interact with.
  • binding is seen as a prerequisite to activity of most kinds, and may be an activity in itself, other activities are envisaged.
  • the present invention does not require the measurement of binding directly or indirectly.
  • 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.
  • 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.
  • Screening for binding activity is conducted according to methods well known in the art, for instance from phage display technology.
  • 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.
  • 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.
  • each individual organism or cell contains only one or a limited number of library members.
  • the nucleic acids are incorporated into expression vectors, in order to allow expression of the polypeptides encoded by the nucleic acids.
  • a library may take the form of a population of host organisms, each organism containing one or more copies of an expression vector containing a single member of the library in nucleic acid form which can be expressed to produce its corresponding polypeptide member.
  • the population of host organisms has the potential to encode a large repertoire of genetically diverse polypeptide variants.
  • 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.
  • 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.
  • 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 10 2 members. Repertoires of 10 11 or more members can be constructed.
  • a set of polypeptide ligands refers to a plurality of polypeptide ligands which can be subjected to selection in the methods described. Potentially, a set can be a repertoire, but it may also be a small collection of polypeptides, from at least 2 up to 10, 20, 50, 100 or more.
  • a group of polypeptide ligands refers to two or more ligands.
  • a group of ligands comprises only ligands which share at least one target specificity.
  • a group will consist of from at least 2, 3, 4, 5, 6, 7, 8, 9 or 10, 20, 50, 100 or more ligands.
  • a group consists of 2 ligands.
  • a method for conjugating a peptide displayed on a genetic display system to a molecular scaffold comprising the steps of:
  • step (a) is followed by a washing step before addition of the molecular scaffold
  • a method for conjugating a peptide displayed on a genetic display system to a molecular scaffold wherein the display system is washed in a dilute solution of reducing agent.
  • the method for conjugating a peptide displayed on a genetic display system to a molecular scaffold wherein the wash solution further comprises a chelating agent.
  • TCEP conjugating a peptide displayed on a genetic display system to a molecular scaffold, wherein the reducing agent is TCEP.
  • a method for conjugating a peptide displayed on a genetic display system to a molecular scaffold wherein the molecular scaffold is added in the presence of aqueous acetonitrile.
  • step (a) is performed for 20 minutes at room temperature.
  • step (b) is performed for 10 minutes at room temperature.
  • Molecular scaffolds are described in, for example, WO2009098450 and references cited therein, particularly WO2004077062 and WO2006078161.
  • the molecular scaffold may be a small molecule, such as a small organic molecule.
  • the molecular scaffold may be, or may be based on, natural monomers such as nucleosides, sugars, or steroids.
  • the molecular scaffold may comprise a short polymer of such entities, such as a dimer or a trimer.
  • the molecular scaffold is a compound of known toxicity, for example of low toxicity.
  • suitable compounds include cholesterols, nucleotides, steroids, or existing drugs such as tamazepam.
  • the molecular scaffold may be a macromolecule. In one embodiment the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.
  • 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 and acyl halides.
  • the molecular scaffold may comprise or may consist of tris(bromomethyl)benzene, especially 1,3,5-Tris(bromomethyl)benzene (‘TBMB’), or a derivative thereof.
  • TBMB 1,3,5-Tris(bromomethyl)benzene
  • 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.
  • 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 and acyl halides.
  • the reactive groups of the polypeptides can be provided by side chains of natural or non-natural 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.
  • each of the reactive groups of the polypeptide for linking to a molecular scaffold are of the same type.
  • each reactive group may be a cysteine residue. Further details are provided in WO2009098450.
  • the reactive groups for linking to a molecular scaffold may comprise two or more different types, or may comprise three or more different types.
  • 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.
  • 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 groups that are used to couple selectively compounds to cysteines in proteins are maleimides.
  • 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.
  • Lysines are also suited as reactive groups to modify peptides on phage by linking to a molecular scaffold.
  • 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.
  • 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.
  • 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.
  • succinimides 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.
  • succinimides for use as molecular scaffold include tris-(succinimidyl aminotriacetate), 1,3,5-Benzenetriacetic acid.
  • aldehydes for use as molecular scaffold include Triformylmethane.
  • 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.
  • the amino acids with reactive groups for linking to a molecular scaffold may be located at any suitable positions within the polypeptide.
  • 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.
  • loop length can be manipulated in accordance with the present teaching.
  • the polypeptide can comprise the sequence AC(X) n C(X) m CG, wherein X stands for a random natural amino acid, A for alanine, C for cysteine and G for glycine and n and m, which may be the same or different, are numbers between 3 and 6.
  • 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 WO2009098450.
  • 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.
  • 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.
  • 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.
  • a single product isomer is also advantageous if a specific member of a library of the invention is synthesized.
  • the chemical reaction of the polypeptide with the molecular scaffold yields a single product isomer rather than a mixture of isomers.
  • 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.
  • 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.
  • 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.
  • 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.
  • amino acids of the members of the libraries or sets of polypeptides can be replaced by any natural or non-natural amino acid.
  • 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.
  • an polypeptide with three reactive groups has the sequence (X) l Y(X) m Y(X) n Y(X) o , wherein Y represents an amino acid with a reactive group, X represents a random amino acid, m and n are numbers between 3 and 6 defining the length of intervening polypeptide segments, which may be the same or different, and l and o are numbers between 0 and 20 defining the length of flanking polypeptide segments.
  • thiol-mediated conjugations can be used to attach the molecular scaffold to the peptide via covalent interactions.
  • 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.
  • thiol mediated 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 WO2009098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.
  • 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.
  • nucleic acids encoding such polypeptides can be synthesised.
  • 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.
  • Effector and/or functional groups can be attached, for example, to the N or C termini of the polypeptide, or to the molecular scaffold.
  • 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).
  • an effector group according to the present invention is an Fc region of an IgG molecule.
  • a peptide ligand-effector group according to the present invention comprises or consists of a peptide ligand Fc fusion having a t ⁇ 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.
  • the peptide ligand according to the present invention comprises or consists of a peptide ligand Fc fusion having a t ⁇ half-life of a day or more.
  • Functional groups include, in general, binding groups, drugs, reactive groups for the attachment of other entities, functional groups which aid uptake of the macrocyclic peptides into cells, and the like.
  • 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.
  • 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 p 153 ‘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 p 13585 “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.
  • 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.
  • antibodies which bind to proteins capable of increasing the half life of the peptide ligand in vivo may be used.
  • RGD peptides which bind to integrins which are present on many cells, may also be incorporated.
  • a peptide ligand-effector group according to the invention has a t ⁇ half-life 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.
  • a peptide ligand-effector group or composition according to the invention will have a t ⁇ half life in the range 12 to 60 hours. In a further embodiment, it will have at half-life of a day or more. In a further embodiment still, it will be in the range 12 to 26 hours.
  • 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
  • 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.
  • Bicycles peptides conjugated to molecular scaffolds
  • a number of properties need considered.
  • Libraries intended for selection may be constructed using techniques known in the art, for example as set forth in WO2004/077062, 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 cell-based expression vectors, including yeast vectors, and the like.
  • phage for instance, phage lambda
  • bacterial plasmid expression vectors for instance, bacterial plasmid expression vectors
  • eukaryotic cell-based expression vectors including yeast vectors, and the like.
  • yeast vectors for example, see WO2009098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.
  • Non-biological systems such as those set forth in WO2004/077062 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. However, they are useful where, for instance, only a small number of peptide ligands needs to be screened. Screening by such individual assays, however, may be time-consuming and the number of unique molecules that can be tested for binding to a specific target generally does not exceed 10 6 chemical entities.
  • biological screening or selection methods generally allow the sampling of a much larger number of different molecules.
  • biological methods can be used in application of the invention.
  • 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 10 13 individual compounds. Examples for powerful affinity selection techniques are phage display, ribosome display, mRNA display, yeast display, bacterial display or RNA/DNA aptamer methods.
  • 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.
  • a vector system 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.
  • PCR polymerase chain reaction
  • the variants are preferably synthesised de novo and inserted into suitable expression vectors.
  • Peptide synthesis can be carried out by standard techniques known in the art, as described above. Automated peptide synthesisers are widely available, such as the Applied Biosystems ABI 433 (Applied Biosystems, Foster City, Calif., USA)
  • 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.
  • the polypeptides of interest are genetically encoded as a phage display library.
  • the complex of the invention comprises a replicable genetic display package (rgdp) such as a phage particle.
  • rgdp replicable genetic display package
  • the nucleic acid can be comprised by the phage genome.
  • the polypeptide can be comprised by the phage coat.
  • 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.
  • the genetically encoded combinatorial library of polypeptides may be generated by phage display, yeast display, ribosome display, bacterial display or mRNA display.
  • 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.
  • 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.
  • members of said library, set or group are simultaneously contacted with a target and members that bind to said target are selected.
  • the target(s) may be a peptide, a protein, a polysaccharide, a lipid, a DNA or a RNA.
  • the target may be a receptor, a receptor ligand, an enzyme, a hormone or a cytokine.
  • 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.
  • the target ligand may be an enzyme, such as a protease.
  • the invention also embraces polypeptide ligands isolated from a screen according to the invention.
  • 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.
  • the invention also relates to peptide ligands having more than two loops.
  • 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.
  • 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.
  • the joining may be accomplished by use of a linker region incorporated into the N/C termini.
  • the N and C termini may be joined by a conventional peptide bond.
  • 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.
  • 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.
  • phage purification before reaction with the molecular scaffold is optional.
  • any suitable means for purification of the phage may be used. Standard techniques may be applied in the present invention.
  • 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 WO2009098450.
  • 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.
  • the phage may be purified as published by Marks et al J. Mol. Biol vol 222 pp 581-597, which is specifically incorporated herein by reference for the particular description of how the phage production/purification is carried out.
  • 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 herein.
  • a reducing agent such as TCEP
  • reaction chemistry used in the present invention provides for a rapid and efficient generation of polypeptide ligands displayed on phage.
  • Reactions conditions used in the present invention preferably comprise the following steps, all preferably conducted at room temperature:
  • the buffer is preferably pH 8.0; it is not necessary to adjust buffer pH in the final solution.
  • Suitable buffers include NaHCO 3 , initially at pH 8.0.
  • Alternative buffers may be used, including buffers with a pH in the physiological range, including NH 4 CO 3 , HEPES and Tris-hydroxymethyl aminoethane, Tris, Tris-Acetate or MOPS.
  • the NaHCO 3 buffer is preferably used at a concentration of 1M, adding 1 ml to a suspension of resin to equilibrate the resin.
  • the resin is preferably an ion exchange resin.
  • Ion exchange resins are known in the art, and include any material suitable for anion exchange chromatography known in the art, such as an agarose based chromatography material, e.g. sepharoses like Fast Flow or Capto, polymeric synthetic material, e.g. a polymethacrylate such as Toyopearls, polystyrene/divinylbenzene, such as Poros, Source, or cellulose, e.g. Cellufine.
  • the anion exchange resin material includes, but is not limited to a resin that carries a primary amine as ligand, e.g.
  • the anion exchange resin material includes, but is not limited to a resin having a positively charged moiety at neutral pH, such as alkylaminoethane, like diethylaminoethane (DEAE), dimethylaminoethane (DMAE), or trimethylaminoethyl (TMAE), polyethyleneimine (PEI), quaternary aminoalkyl, quaternary aminoethane (QAE), quaternary ammonium (Q), and the like.
  • alkylaminoethane like diethylaminoethane (DEAE), dimethylaminoethane (DMAE), or trimethylaminoethyl (TMAE), polyethyleneimine (PEI), quaternary aminoalkyl, quaternary aminoethane (QAE), quaternary ammonium (Q), and the like.
  • DEAE diethylaminoethane
  • DMAE dimethylaminoethane
  • TMAE trimethylaminoethyl
  • step (1) reducing agent is added to a concentration of 1 mM.
  • the dilute reducing agent used in step (2) is preferably at a concentration of 1 ⁇ M. Both concentrations are for TCEP, and other values may apply to other reducing agents.
  • the dilute reducing agent is used to maintain the polypeptide in a reduced state prior to reaction with the molecular scaffold.
  • a chelating agent is included in the washing step. For example, EDTA may be included.
  • Alternative reducing agents may be selected from dithiothreitol, thioglycolic acid, thiolactic acid, 3-mercaptopropionic acid, thiomalic acid, 2,3-dimercaptosuccinic acid, cysteine, N-glycyi-L-cysteine, L-cysteinylglycine and also esters and salts thereof, thioglycerol, cysteamine and C1-C4 acyl derivatives thereof, N-mesylcysteamine, Nacetylcysteine, N-mercaptoalkylamides of sugars such as N-(mercapto-2-ethyl) gluconamide, pantetheine, N-(mercaptoalkyl)-co-hydroxyalkylamides, for example those described in patent application EP-A-354 835, N-mono- or N,N-dialkylmercapto-4-butyramides, for example those described in patent application EP-A-368 763
  • the conjugation of the molecular scaffold, in the case of TBMB and other scaffolds whose reactive groups are thiol-reactive, is preferably conducted in the presence of acetonitrile.
  • the acetonitrile is preferably at a final concentration of about 20%.
  • Unreacted molecular scaffold is removed from the phage by washing. Subsequently, phage can be eluted from the resin, and selected as set forth previously.
  • the phage-containing culture medium can be washed prior to reduction with the reducing agent.
  • the reducing agent itself can be added in two steps; in a concentrated form, to effect reduction, and then in dilute form (step 2 above), to maintain the displayed polypeptide in a reduced state.
  • the timing of the steps can also be varied, without significantly altering the efficiency of the procedure.
  • the present invention found that reduction in TCEP for 20 minutes is as effective as reduction for 30 minutes.
  • reaction with TBMB for 10 minutes does not give a significantly lower level of binding than reaction for 30 minutes.
  • the resin is magnetic.
  • Magnetic resin beads such as magnetic sepharose beads, can be obtained commercially from, for example, Bangs Laboratories, Invitrogen, Origene and GE Healthcare. See also U.S. Pat. No. 2,642,514 and GB 1239978.
  • Application of a magnetic field permits isolation of the beads, which results in purification of the polypeptides bound to the beads from the medium in which they are contained.
  • the magnetic beads are separated from the medium by insertion of a magnetic probe into the medium. Beads are retained on the magnetic probe, and can be transferred to a washing station, or a different medium. Alternatively, beads can be isolated by applying a magnetic field to the vessel in which they are contained, and removing the medium once the beads are immobilised.
  • Magnetic separation provides faster, more efficient processing of resins in the method of the invention.
  • Polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like.
  • Ligands having selected levels of specificity are useful in applications which involve testing in non-human animals, where cross-reactivity is desirable, or in diagnostic applications, where cross-reactivity with homologues or paralogues needs to be carefully controlled.
  • the ability to elicit an immune response to predetermined ranges of antigens can be exploited to tailor a vaccine to specific diseases and pathogens.
  • Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human.
  • the selected polypeptides may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures, immunofluorescent stainings and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).
  • the peptide ligands of the present invention will typically find use in preventing, suppressing or treating inflammatory states, allergic hypersensitivity, cancer, bacterial or viral infection, and autoimmune disorders (which include, but are not limited to, Type I diabetes, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease and myasthenia gravis).
  • prevention involves administration of the protective composition prior to the induction of the disease.
  • suppression refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after disease symptoms become manifest.
  • Animal model systems which can be used to screen the effectiveness of the peptide ligands in protecting against or treating the disease are available.
  • the use of animal model systems is facilitated by the present invention, which allows the development of polypeptide ligands which can cross react with human and animal targets, to allow the use of animal models.
  • the demyelinating disease is induced by administration of myelin basic protein (see Paterson (1986) Textbook of Immunopathology, Mischer et al., eds., Grune and Stratton, New York, pp. 179-213; McFarlin et al. (1973) Science, 179: 478: and Satoh et al. (1987) J. Immunol., 138: 179).
  • the present peptide ligands will be utilised in purified form together with pharmacologically appropriate carriers.
  • these carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, any including saline and/or buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's.
  • Suitable physiologically-acceptable adjuvants, if necessary to keep a polypeptide complex in suspension may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.
  • Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).
  • the peptide ligands of the present invention may be used as separately administered compositions or in conjunction with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cylcosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with the selected antibodies, receptors or binding proteins thereof of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are pooled prior to administration.
  • immunotherapeutic drugs such as cylcosporine, methotrexate, adriamycin or cisplatinum
  • Pharmaceutical compositions can include “cocktails” of various cytotoxic or other agents in conjunction with the selected antibodies, receptors or binding proteins thereof of the present invention, or even combinations of selected polypeptides according to the present invention having different specificities, such as
  • the route of administration of pharmaceutical compositions according to the invention may be any of those commonly known to those of ordinary skill in the art.
  • the selected antibodies, receptors or binding proteins thereof of the invention can be administered to any patient in accordance with standard techniques.
  • the administration can be by any appropriate mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also, appropriately, by direct infusion with a catheter.
  • the dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other drugs, counterindications and other parameters to be taken into account by the clinician.
  • the peptide ligands of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate.
  • compositions containing the present peptide ligands or a cocktail thereof can be administered for prophylactic and/or therapeutic treatments.
  • an adequate amount to accomplish at least partial inhibition, suppression, modulation, killing, or some other measurable parameter, of a population of selected cells is defined as a “therapeutically-effective dose”. Amounts needed to achieve this dosage will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from 0.005 to 5.0 mg of selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose being more commonly used.
  • compositions containing the present peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.
  • a composition containing a peptide ligand according to the present invention may be utilised in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of a select target cell population in a mammal.
  • the selected repertoires of polypeptides described herein may be used extracorporeally or in vitro selectively to kill, deplete or otherwise effectively remove a target cell population from a heterogeneous collection of cells.
  • Blood from a mammal may be combined extracorporeally with the selected peptide ligands whereby the undesired cells are killed or otherwise removed from the blood for return to the mammal in accordance with standard techniques.
  • the desired diversity is typically generated by varying the selected molecule at one or more positions.
  • the positions to be changed are selected, such that libraries are constructed for each individual position in the loop sequences. Where appropriate, one or more positions may be omitted from the selection procedure, for instance if it becomes apparent that those positions are not available for mutation without loss of activity.
  • variation can then be achieved either by randomisation, during which the resident amino acid is replaced by any amino acid or analogue thereof, natural or synthetic, producing a very large number of variants or by replacing the resident amino acid with one or more of a defined subset of amino acids, producing a more limited number of variants.
  • the preferred method is to synthesise mutant polypeptides de novo. Mutagenesis of structured polypeptides is described above, in connection with library construction.
  • the polypeptide in this example is a phage displayed peptide.
  • the nucleic acid is comprised by the phage particle.
  • the molecular scaffold in this example is a small molecule (TBMB).
  • the volume of the reduced phage was adjusted to 32 ml with reaction buffer and 8 ml of 50 ⁇ M TBMB in ACN were added to obtain a final TBMB concentration of 10 ⁇ M.
  • the reaction was incubated at 30° C. for 1 hr before non-reacted TBMB was removed by precipitation of the phage with 1/5 volume of 20% PEG, 2.5 M NaCl on ice and centrifugation at 4000 rpm for 30 minutes.
  • the small organic compound tris-(bromomethyl)benzene (TBMB) was used as a scaffold to anchor peptides containing three cysteine residues (Kemp, D. S, and McNamara, P. E., J. Org. Chem, 1985; FIG. 1B ).
  • Halogen alkanes conjugated to an aromatic scaffold react specifically with thiol groups of cysteines in aqueous solvent at room temperature (Stefanova, H. I., Biochemistry, 1993).
  • Meloen and co-workers had previously used bromomethyl-substituted synthetic scaffolds for the immobilization of peptides with multiple cysteines (Timmerman, P. et al., ChemBioChem, 2005).
  • the reaction conditions for the modification of a peptide on phage were elaborated next. As it appeared difficult to detect the chemically modified peptide on phage with available techniques, the present invention expressed the peptide N G C GSG C GSG C G C as an N-terminal fusion with the two soluble domains D1 and D2 of the minor phage coat protein plll and analyzed the molecular weight of the protein before and after reaction with TBMB by mass spectrometry. Attempts to selectively link the three cysteines in the peptide to the scaffold but spare the three disulfide bridges of the D1 and D2 domains of plll (C7-C36, C46-053, C188-C201) failed.
  • PK15 is a three cysteines containing peptide (H-ACSDRFRNCPADEALCG-NH 2 ), which when coupled with TBMB, is a specific and potent inhibitor of human plasma kallikrein.
  • This peptide can be displayed as a fusion to gene 3 protein of phage and if correctly modified by TBMB will result in a phage that can specifically bind to kallikrein.
  • Non-modification of PK15 on the phage or cross-linking of the phage would not result in a specific binding signal for the phage binding to kallikrein.
  • Anion exchange resin was used to capture the phage, allowing for quick and easy changing of the buffers that the phage were exposed to during the modification process.
  • the phage were also titred for particle number and infectivity to show that the modification process had not made the phage significantly less infectious.
  • the particle and infectious titres for the samples were compared to see if the modification procedure had “damaged” the phage and rendered them less infectious than before the modification.
  • a colony from E. coli containing PK15/WT FdTet which had been freshly streaked on an agar plate was used to inoculate 25 ml of either 2TY/tet or LB/tet, and cultures were incubated overnight at 37 C shaking 250 rpm.
  • the buffers compared were NH 4 CO 3 and NaHCO 3 .
  • Samples to be treated in NaHCO 3 buffer were prepared using 1M NaHCO 3 .
  • the two PK15 cultures were treated as follows:
  • the phage eluates (in citrate buffer) were retained.
  • the eluted phage were screened for Kallikrein binding.
  • a target binding screen was performed on the eluate samples from above, as shown in FIG. 5 . No clear trends were visible, even on repeat assays.
  • pH5 gives the best results, but must be used with high salt.
  • the phage modification process has been optimised from a ‘long’ protocol.
  • the results of the long protocol are compared herein to a shortened protocol.
  • a colony from streaked PK15/WT FdTet plate as in Example 3 was used to inoculate 25 ml of 2TY/tet. The culture incubated overnight at 37° C., shaking at 250 rpm.
  • the quick protocol is as follows
  • the long protocol is as follows:
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