WO2021220011A1

WO2021220011A1 - Anti-infective bicyclic peptide conjugates

Info

Publication number: WO2021220011A1
Application number: PCT/GB2021/051047
Authority: WO
Inventors: Matthew BALMFORTH; Paul Beswick; Mike Dawson; Nick Lewis; Michael Skynner; Katerine VAN RIETSCHOTEN
Original assignee: Bicycletx Limited
Priority date: 2020-05-01
Filing date: 2021-04-30
Publication date: 2021-11-04

Abstract

The present invention relates to polypeptides which are covalently bound to molecular scaffolds such that two or more peptide loops are subtended between attachment points to the scaffold. In particular, the bicyclic peptides of the invention are conjugated to a carrier peptide in order to greatly enhance the bacterial cell killing activity. More particularly, the invention describes peptides which are high affinity binders of Lipid II. The invention also includes pharmaceutical compositions comprising said conjugates and to the use of said conjugates in suppressing or treating a disease or disorder mediated by bacterial infection or for providing prophylaxis to a subject at risk of infection.

Description

ANTI-INFECTIVE BICYCLIC PEPTIDE CONJUGATES

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Lipid ll is a precursor molecule in the synthesis of the cell wall of bacteria, it is a peptidoglycan, which is amphipathic and named for its bactoprenol hydrocarbon chain, which acts as a lipid anchor, embedding itself in the bacterial cell membrane. Lipid II must translocate across the cell membrane to deliver and incorporate its disaccharide-pentapeptide "building block" into the peptidoglycan mesh. Lipid II is the target of several antibiotics, however, with the advent of increasing species of bacteria developing antibiotic resistance, there is a need to provide alternative binding agents directed to this target in order to provide efficacious anti-infective medicaments. Hart et a! (2017) Chem Sci 8, 7991-7997 describes a series of Lipid II binding lipopeptides having antibacterial activity against vancomycin-resistant bacteria. The invention therefore is therefore directed to providing alternative peptides having improved antibacterial properties.

SUMMARY OF THE INVENTION

According to a first aspect of the invention, there is provided an anti-infective peptide conjugate which comprises:

(i) a bicyclic peptide ligand capable of binding to Lipid II comprising a polypeptide which comprises 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; and

(ii) a carrier peptide. According to a further aspect of the invention, there is provided a pharmaceutical composition comprising the conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

According to a further aspect of the invention, there is provided the conjugate as defined herein for use in suppressing or treating a disease or disorder mediated by bacterial infection or for providing prophylaxis to a subject at risk of infection.

DETAILED DESCRIPTION OF THE INVENTION

(i) a bicyclic peptide ligand capable of binding to Lipid II comprising a polypeptide which comprises 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; and (ii) a carrier peptide.

Bicyclic Peptide Ligands

In one embodiment, said loop sequences comprise 6 amino acids.

In a further embodiment, said loop sequences comprise three reactive groups separated by two loop sequences both of which consist of 6 amino acids.

In an alternative embodiment, said loop sequences comprise two [Dap(Me)] residues and one cysteine residue separated by two loop sequences both of which consist of 6 amino acids.

References herein to Lipid II include any form or isoform of Lipid II which may be present in any bacterial species. In one embodiment, the Lipid II is present within one or more pathogenic bacterial species. In a further embodiment, the one or more pathogenic bacterial species is selected from any of: Acinetobacter baumannii, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostrium tetani, Corynebacterium diphtheriae, Echinococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (such as Enterotoxigenic E. coli, Enteropathogenic E. coli, Enterohemorragic E. coli or Enteroaggregative E. coli), Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pneumococcus, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella such as, Salmonella bongori, Salmonella enterica, Salmonella subterranean, Salmonella typhi or Salmonella typhimurium, Shigella (such as Shigella sonnei or Shigella dysenteriae), Staphylococcus aureus (such as MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae or Yersinia pestis.

In one embodiment, the Lipid II is present within E. coli.

In an alternative embodiment, the Lipid II is present within A. baumannii.

In an alternative embodiment, the Lipid II is present within P. aeruginosa.

In one embodiment, the bicyclic peptide is selected from any peptides described in Hart etai (2017) Chem Sci 8, 7991-7997, the peptides of which are herein incorporated by reference.

In a further embodiment, the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

C_iDWPDWQC_iiYGWSSHC_iii (SEQ ID NO: 1);

C_iKHQAQEC_iiVAI REGC_iii (SEQ ID NO: 2);

C_iWAMPMWC_iiDSWSQNC_iii (SEQ ID NO: 3);

C_iRPQKFNC_iilSANIRC_iii (SEQ ID NO: 4);

C_iKAMIGAC_iiVAMQFAC_iii (SEQ ID NO: 5);

C_iYPVDWYC_iiLFQTVDC_iii (SEQ ID NO: 6);

C_iRYVSGDCYYAQAHC_iii (SEQ ID NO: 7);

C_iLLQSLLC_iiPYSTHRC_iii (SEQ ID NO: 8);

C_iLLQSLLC_iiPYSTHAC_iii (SEQ ID NO: 9);

C_iLLQSLLC_iiPYSTRAC_iii (SEQ ID NO: 10);

C_iLLQSLLC_iiRYSTHAC_iii (SEQ ID NO: 11);

C_iLLQRLLC_iiPYSTHAC_iii (SEQ ID NO: 12); [Dap(Me)]_iLLQRLL[Dap(Me)]_iiPYSTHAC_iii (SEQ ID NO: 13); [Dap(Me)]_iLLQRLLC_iiPYSTHA[Dap(Me)]_iii (SEQ ID NO: 14); and C_iLLQRLL[Dap(Me)]_iiPYSTHA[Dap(Me)]_iii (SEQ ID NO: 15); wherein i, ii and iii represent first, second and third reactive groups, respectively, such that C_i, C_ii and C_iii represent first, second and third cysteine residues, respectively, and [Dap(Me)]_i, [Dap(Me)]_ii and [Dap(Me)]_iii represent first, second and third [Dap(Me)] residues, respectively, wherein Dap represents diaminopropionic acid, or a pharmaceutically acceptable salt thereof.

In a yet further embodiment, the bicyclic peptide ligand comprises an amino acid sequence which is selected from:

In a still yet further embodiment, the bicyclic peptide ligand comprises an amino acid sequence which is:

C_iLLQRLLC_iiPYSTHAC_iii (SEQ ID NO: 12); wherein C_i, C_ii and C_iii represent first, second and third cysteine residues, respectively, or a pharmaceutically acceptable salt thereof.

In a further embodiment, the bicyclic peptide ligand additionally comprises N- and/or C- terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 1)-G (referred to in Hart et al (2017) as P1);

A-(SEQ ID NO: 2)-G (referred to in Hart et al (2017) as P2);

A-(SEQ ID NO: 3)-G (referred to in Hart et al (2017) as P3);

A-(SEQ ID NO: 4)-G (referred to in Hart et al (2017) as P4);

A-(SEQ ID NO: 5)-G (referred to in Hart et al (2017) as P5);

A-(SEQ ID NO: 6)-G (referred to in Hart et al (2017) as P6);

A-(SEQ ID NO: 7)-G (referred to in Hart et al (2017) as P7);

A-(SEQ ID NO: 8)-G (referred to in Hart et al (2017) as P8);

A-(SEQ ID NO: 9)-G (herein referred to in Hart et al (2017) as P8-D-(R15A)); A-(SEQ ID NO: 10)-G (herein referred to in Hart et al (2017) as P8-D-(H14R,

R15A)); A-(SEQ ID NO: 11)-G (herein referred to in Hart et al (2017) as P8-D-P10R,

R15A));

A-(SEQ ID NO: 12)-G (herein referred to herein as BCY11898 and in Hart et al (2017) as P8-D-S6R, R15A));

A-(SEQ ID NO: 13)-G;

A-(SEQ ID NO: 14)-G; and A-(SEQ ID NO: 15)-G.

In a yet further embodiment, the bicyclic peptide ligand additionally comprises N- and/or C- terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 13)-G;

A-(SEQ ID NO: 14)-G; and A-(SEQ ID NO: 15)-G.

In a still yet further embodiment, the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is:

A-(SEQ ID NO: 12)-G (herein referred to herein as BCY11898 and in Hart et al (2017) as P8-D-S6R, R15A)).

In one embodiment, the carrier peptide comprises a linear peptide.

In one embodiment, the carrier peptide comprises between 3 and 15 amino acids. In a further embodiment, the carrier peptide comprises between 4 and 12 amino acids. In a yet further embodiment, the carrier peptide is either 4, 7, 8, 10, 11 or 12 amino acids in length. In a still yet further embodiment, the carrier peptide is 11 amino acids in length.

In one embodiment, the carrier peptide is selected from one of the following peptides: KSLRRVWRSWR (SEQ ID NO: 16; herein referred to as DRAMP); KSL[HArg][HArg]VW[HArg]SW[HArg] (SEQ ID NO: 17);

[dK] [dS] [dL] [dR] [dR] [dV][dW] [dR] [dS] [dW] [dR] (SEQ ID NO: 18; herein referred to as D form DRAMP);

[dR][dW][dS][dR][dW][dV][dR][dR][dL][dS][dK] (SEQ ID NO: 19; herein referred to as retroinverso DRAMP); VKLFPVKLFP (SEQ ID NO: 20);

SLLSLIRKLIT (SEQ ID NO: 21);

FFFLSRIFGK (SEQ ID NO: 22);

PLILLRLLRGQF (SEQ ID NO: 23);

NAGSLLSGWG (SEQ ID NO: 24);

NGVQPKY (SEQ ID NO: 25);

DKYLPRPRPV (SEQ ID NO: 26);

KFFKFFK (SEQ ID NO: 27);

KFFK (SEQ ID NO: 28);

KFFKFFKFFK (SEQ ID NO: 29); and RLWVLWRR (SEQ ID NO: 30).

In a further embodiment, the carrier peptide is selected from one of the following peptides: KSLRRVWRSWR (SEQ ID NO: 16; herein referred to as DRAMP);

[dK] [dS] [dL] [dR] [dR] [dV][dW] [dR] [dS] [dW] [dR] (SEQ ID NO: 18; herein referred to as D-form DRAMP); and

[dR][dW][dS][dR][dW][dV][dR][dR][dL][dS][dK] (SEQ ID NO: 19; herein referred to as retroinverso DRAMP).

In one embodiment, the carrier peptide additionally comprises a moiety for facilitating conjugation to the bicyclic peptide. Such conjugation facilitating moieties include: an azidoalanine (Aza) residue; an azidolysine (K(N₃)) residue; or a (PYA-N₃[K] residue, wherein PYA represents propargyl-acid. In a further embodiment, the moiety for facilitating conjugation to the bicyclic peptide is a (PYA-N₃[K]) residue.

In one embodiment, said Aza, (K(N₃)) or (PYA-N₃[K]) residue is present at either the N- or C- terminal of said carrier peptide.

In one particular embodiment, the moiety for facilitating conjugation of the carrier peptide to the bicyclic peptide comprises a moiety of formula (I):

wherein R represents the bicyclic peptide and R₁ represents the carrier peptide. Anti-Infective Conjugates TBMB Conjugates

In one specific embodiment, the bicyclic peptide ligand is attached to a TBMB scaffold and conjugated to a carrier peptide and comprises an anti-infective conjugate which is a compound of formula (I):

and is selected from:

TV SB Conjugates

In an alternative specific embodiment, the bicyclic peptide ligand is attached to a TVSB scaffold and conjugated to a carrier peptide and comprises an anti-infective conjugate which is a compound of formula (I):

(I) and is selected from:

TBAB Conjugates

In an alternative specific embodiment, the bicyclic peptide ligand is attached to a TBAB scaffold scaffold and conjugated to a carrier peptide and comprises an anti-infective conjugate which is a compound of formula (I):

and is selected from:

Surprisingly, the presence of both the carrier peptide and the bicyclic peptide within the conjugate provide a synergistic arrangement wherein the bicyclic peptide is able to bind with affinity to Lipid II, and the carrier peptide allows for bacterial cell entry in order to provide for more effective microbial cell killing activity as is evidenced in the data presented herein. When tested alone in wild type bacteria the unconjugated bicyclic peptides and carrier peptides have little or no anti-microbial activity, but when tested in bacteria with a compromised outer membrane (hyperporinated cells) the unconjugated bicyclic peptides demonstrate similar antimicrobial activity to that seen with with the conjugated peptides in wild type bacteria. The conjugated bacteria surprisingly show similar levels of activity in both wild type and hyperporinated cells. The data of the present conjugates also compares favourably with existing antibiotics (see Table 1 herein).

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 Sam brook et ai, Molecular Cloning: A Laboratory Manual, 3rd ed., 2001 , Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY; Ausubel etal., Short Protocols in Molecular Biology (1999) 4^th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature Numbering

When referring to amino acid residue positions within peptides of the invention, cysteine residues (C_i, C_ii and C_iii) are omitted from the numbering as they are invariant, therefore, the numbering of amino acid residues within peptides of the invention is referred to as below:

C_i- D₁ - W₂- P₃- D₄- W₅-Q₆-C_ii-Y ₇-G₈- W₉-S₁₀-S₁₁ - H ₁₂-C_iii (SEQ ID NO: 1).

For the purpose of this description, all bicyclic peptides are assumed to be cyclised with TBMB, TVSB or TBAB yielding a tri-substituted structure. Cyclisation with TBMB, TVSB or TBAB occurs on the first, second and third reactive groups (i.e. C_i, C_ii, C_iii, [Dap(Me)],, [Dap(Me)]_ii and [Dap(Me)]_iii).

Molecular Format

N- or C-terminal extensions to the bicycle core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAIa-Sar10-Ala tail would be denoted as: βAIa-Sar10-A-(SEQ ID NO:X). Inversed Peptide Sequences

In light of the disclosure in Nair etal (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein would also find utility in their retro-inverso form. For example, the sequence is reversed (i.e. N-terminus becomes C-terminus and vice versa) and their stereochemistry is likewise also reversed (i.e. D-amino acids become L-amino acids and vice versa).

Peptide Ligands

A peptide ligand, as referred to herein, refers to a peptide covalently bound to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e. cysteine residues) 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 peptides comprise at least three cysteine residues (referred to herein as C_i, C_ii and C_iii), and form at least two loops on the scaffold.

Advantages of the Peptide Ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties which enable them to be considered as suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include:

Species cross-reactivity. Certain ligands demonstrate cross-reactivity across Lipid II from different bacterial species and hence are able to treat infections caused by multiple species of bacteria. Other ligands may be highly specific for the Lipid II of certain bacterial species which may be advantageous for treating an infection without collateral damage to the beneficial flora of the patient;

Protease stability. Bicyclic peptide ligands should ideally demonstrate 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;

Desirable solubility profile. This is a function of the proportion of charged and hydrophilic versus hydrophobic residues and intra/inter-molecular H-bonding, which is important for formulation and absorption purposes;

An optimal plasma half-life in the circulation. Depending upon the clinical indication and treatment regimen, it may be required to develop a bicyclic peptide for short exposure in an acute illness management setting, or develop a bicyclic peptide with enhanced retention in the circulation, and is therefore optimal for the management of more chronic disease states. Other factors driving the desirable plasma half-life are requirements of sustained exposure for maximal therapeutic efficiency versus the accompanying toxicology due to sustained exposure of the agent; and

Selectivity.

Pharmaceutically Acceptable Salts

It will be appreciated that salt forms are within the scope of this invention, and references to peptide ligands include the salt forms of said ligands.

The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

Acid addition salts (mono- or di-salts) may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulfonic, (+)-(1 S)-camphor-10-sulfonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1, 2-disulfonic, ethanesulfonic, 2- hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydriodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1 , 5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L- pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L- tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts formed from acetic, hydrochloric, hydriodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propanoic, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

If the compound is anionic, or has a functional group which may be anionic (e.g., -COOH may be -COO^'), then a salt may be formed with an organic or inorganic base, generating a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li⁺, Na⁺ and K⁺, alkaline earth metal cations such as Ca²⁺ and Mg²⁺, and other cations such as Al³⁺ or Zn⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH₄₊) and substituted ammonium ions (e.g., NH₃R⁺, NH₂R₂ ⁺, NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

Where the peptides of the invention contain an amine function, these may form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Modified Derivatives

It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues; replacement of one or more amino acid residues with an alanine, 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; replacement of one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol- reactive reagents so as to functionalise said amino acids, and 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.

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.

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 (the group referred to herein as C_i) 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.

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 (the group referred to herein as C_iii) 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 carboxy peptidase and reduces the potential for proteolytic degradation of the bicyclic peptide. In one embodiment, the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected having isosteric/isoelectronic side chains which are neither recognised by degradative proteases nor have any adverse effect upon target potency.

Alternatively, non-natural amino acids may be used having 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, Cα- disubstituted derivatives (for example, aminoisobutyric acid, Aib), and cyclo amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (C_i) and/or the C-terminal cysteine (C_iii).

In one embodiment, the modified derivative comprises replacement of one or more oxidation sensitive amino acid residues with one or more oxidation resistant amino acid residues.

In one embodiment, the modified derivative comprises replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged versus hydrophobic amino acid residues is an important characteristic of the bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (in particular arginine) 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 amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

In one embodiment, the modified derivative comprises replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by a propensity of D-amino acids to stabilise b-turn conformations (Tugyi et a/ (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above mentioned modifications serve to deliberately improve the potency or stability of the peptide. Further potency improvements based on modifications may be achieved through the following mechanisms:

Incorporating hydrophobic moieties that exploit the hydrophobic effect and lead to lower off rates, such that higher affinities are achieved;

Incorporating charged groups that exploit long-range ionic interactions, leading to faster on rates and to higher affinities (see for example Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

Incorporating additional constraint into the peptide, by for example constraining side chains of amino acids correctly such that loss in entropy is minimal upon target binding, constraining the torsional angles of the backbone such that loss in entropy is minimal upon target binding and introducing additional cyclisations in the molecule for identical reasons.

(for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

Isotopic Variations

The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the invention, wherein one or more atoms are replaced by atoms having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and peptide ligands of the invention, wherein metal chelating groups are attached (termed “effector”) that are capable of holding relevant (radio)isotopes, and peptide ligands of the invention, wherein certain functional groups are covalently replaced with relevant (radio)isotopes or isotopically labelled functional groups.

Examples of isotopes suitable for inclusion in the peptide ligands of the invention comprise isotopes of hydrogen, such as ²H (D) and ³H (T), carbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³⁶CI, fluorine, such as ¹⁸F, iodine, such as ¹²³l, ¹²⁵l and ¹³¹l, nitrogen, such as ¹³N and ¹⁵N, oxygen, such as ¹⁵O, ^1O0 and ¹⁸O, phosphorus, such as ³²P, sulfur, such as ³⁵S, copper, such as ⁶⁴Cu, gallium, such as ⁶⁷Ga or ⁶⁸Ga, yttrium, such as ⁹⁰Y and lutetium, such as ¹⁷⁷Lu, and Bismuth, such as ²¹³Bi.

Certain isotopically-labelled peptide ligands of the invention, for example, those incorporating a radioactive isotope, are useful in drug and/or substrate tissue distribution studies. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used for detecting or identifying the formation of a complex between a labelled compound and other molecules, peptides, proteins, enzymes or receptors. The detecting or identifying methods can use compounds that are labelled with labelling agents such as radioisotopes, enzymes, fluorescent substances, luminous substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ³H (T), and carbon-14, i.e. ¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ²H (D), may afford certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and hence may be preferred in some circumstances.

Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

Isotopically-labeled compounds of peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying Examples using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

Molecular Scaffold

Molecular scaffolds are described in, for example, WO 2009/098450 and references cited therein, particularly WO 2004/077062 and WO 2006/078161.

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

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 which form the linkage with a peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

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.

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 include bromomethylbenzene or iodoacetamide. Other scaffold reactive groups that are used to selectively couple compounds to cysteines in proteins are maleimides, ab unsaturated carbonyl containing compounds and a-halomethylcarbonyl containing compounds. 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.

In one embodiment, the molecular scaffold of the invention is 1,3,5-tris(bromomethyl)benzene (TBMB):

Thus, following cyclisation with the bicyclic peptides of the invention on the three reactive groups, the molecular scaffold forms a tri-substituted derivative of TBMB having the following structure:

wherein * denotes the point of attachment of the reactive groups. In an alternative embodiment, the molecular scaffold of the invention is other than 1,3,5- tris(bromomethyl)benzene (TBMB).

In one embodiment, the molecular scaffold is selected from 1,3,5-tris(vinylsulfonyl)benzene (TVSB) or N,N',N"-1,3,5-benzenetriyltris[2-bromoacetamide] (TBAB).

In a further embodiment, the molecular scaffold is 1,3,5-tris(vinylsulfonyl)benzene (TVSB):

Thus, following cyclisation with the bicyclic peptides of the invention on the three reactive groups, the molecular scaffold forms a tri-substituted derivative of TVSB having the following structure:

wherein * denotes the point of attachment of the reactive groups.

In an alternative embodiment, the molecular scaffold is N,N',N"-1,3,5-benzenetriyltris[2- bromoacetamide] (TBAB):

Thus, following cyclisation with the bicyclic peptides of the invention on the three reactive groups, the molecular scaffold forms a tri-substituted derivative of TBAB having the following structure:

wherein * denotes the point of attachment of the reactive groups.

Reactive Groups

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 [Dap(Me)] group, a lysine side chain, or an N-terminal amine group or any other suitable reactive group. Details may be found in WO 2009/098450.

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.

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.

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.

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.

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. 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.

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.

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. In one embodiment, an polypeptide with three reactive groups has the sequence (X)_lY(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 3 and 6 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.

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 WO 2009/098450 or Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.

Synthesis

The peptides of the present invention may be manufactured synthetically by standard techniques followed by reaction with a molecular scaffold in vitro. When this is performed, standard chemistry may be used. This enables the rapid large scale preparation of soluble material for further downstream experiments or validation. Such methods could be accomplished using conventional chemistry such as that disclosed in Timmerman et al. (supra).

Thus, the invention also relates to manufacture of polypeptides selected as set out herein, wherein the manufacture comprises optional further steps as explained below. In one embodiment, these steps are carried out on the end product polypeptide made by chemical synthesis.

Peptides can also be extended, to incorporate for example another loop and therefore introduce multiple specificities. To extend the peptide, it may simply be extended chemically at its N-terminus or C-terminus or within the loops using orthogonally protected lysines (and analogues) using standard solid phase or solution phase chemistry. Standard (bio)conjugation techniques may be used to introduce an activated or activatable N- or C-terminus. Alternatively, additions may be made by fragment condensation or native chemical ligation e.g. as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci U S A. 1994 Dec 20; 91 (26): 12544-8 or in Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

Alternatively, the peptides may be extended or modified by further conjugation through disulphide bonds. This has the additional advantage of allowing the first and second peptide to dissociate from each other once within the reducing environment of the cell. In this case, the molecular scaffold (e.g. TBMB, TVSB or TBAB) could be added during the chemical synthesis of the first peptide so as to react with the three cysteine groups; a further cysteine or thiol could then be appended to the N or C-terminus of the first peptide, so that this cysteine or thiol only reacted with a free cysteine or thiol of the second peptide, forming a disulfide - linked bicyclic peptide-peptide conjugate.

Similar techniques apply equally to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

Furthermore, addition of other functional groups or effector groups may be accomplished in the same manner, using appropriate chemistry, coupling at the N- or C-termini or via side chains. In one embodiment, the coupling is conducted in such a manner that it does not block the activity of either entity.

Pharmaceutical Compositions

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharmaceutically acceptable excipients.

Generally, the present peptide ligands will be utilised in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, 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 compounds of the invention can be used alone or in combination with another agent or agents. The other agent for use in combination may be for example another antibiotic, or an antibiotic ‘adjuvant’ such as an agent for improving permeability into Gram-negative bacteria, an inhibitor of resistance determinants or an inhibitor of virulence mechanisms.

Suitable antibiotics for use in combination with the compounds of the invention include but are not limited to:

Beta lactams, such as penicillins, cephalosporins, carbapenems or monobactams. Suitable penicillins include oxacillin, methicillin, ampicillin, cloxacillin, carbenicillin, piperacillin, tricarcillin, flucloxacillin, and nafcillin; suitable cephalosporins include cefazolin, cefalexin, cefalothin, ceftazidime, cefepime, ceftobiprole, ceftaroline, ceftolozane and cefiderocol; suitable carbapenems include meropenem, doripenem, imipenem, ertapenem, biapenem and tebipenem; suitable monobactams include aztreonam;

Lincosamides such as clindamycin and lincomycin;

Macrolides such as azithromycin, clarithromycin, erythromycin, telithromycin and solithromycin;

Tetracyclines such as tigecycline, omadacycline, eravacycline, doxycycline, and minocycline; Quinolones such as ciprofloxacin, levofloxacin, moxifloxacin, and delafloxacin;

Rifamycins such as rifampicin, rifabutin, rifalazil, rifapentine, and rifaximin;

Aminoglycosides such as gentamycin, streptomycin, tobramycin, amikacin and plazomicin; Glycopeptides such as vancomycin, teichoplanin, telavancin, dalbavancin, and oritavancin, Pleuromutilins such as lefamulin Oxazolidinones such as linezolid or tedizolid Polymyxins such as polymyxin B or colistin;

Trimethoprim, iclaprim, sulfamethoxazole;

Metronidazole; Fidaxomicin:

Mupirocin;

Fusidic acid;

Daptomycin;

Murepavidin;

Fosfomycin; and Nitrofurantoin.

Suitable antibiotic ‘adjuvants’ include but are not limited to: agents known to improve uptake into bacteria such as outer membrane permeabilisers or efflux pump inhibitors; outer membrane permeabilisers may include polymyxin B nonapeptide or other polymyxin analogues, or sodium edetate; inhibitors of resistance mechanisms such as beta-lactamase inhibitors; suitable beta- lactamase inhibitors include clavulanic acid, tazobactam, sulbactam, avibactam, relebactam and nacubactam; and inhibitors of virulence mechanisms such as toxins and secretion systems, including antibodies.

The compounds of the invention can also be used in combination with biological therapies such as nucleic acid based therapies, antibodies, bacteriophage or phage lysins.

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. For therapy, the peptide ligands of the invention can be administered to any patient in accordance with standard techniques. Routes of administration include, but are not limited to, oral (e.g., by ingestion); buccal; sublingual; transdermal (including, e.g., by a patch, plaster, etc.); transmucosal (including, e.g., by a patch, plaster, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eyedrops); pulmonary (e.g., by inhalation or insufflation therapy using, e.g., via an aerosol, e.g., through the mouth or nose); rectal (e.g., by suppository or enema); vaginal (e.g., by pessary); parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot or reservoir, for example, subcutaneously or intramuscularly. Preferably, the pharmaceutical compositions according to the invention will be administered parenterally. 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 levels may have to be adjusted upward to compensate.

The compositions containing the present peptide ligands or a cocktail thereof can be administered for therapeutic treatments. In certain therapeutic applications, 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 10 μg to 250 mg of selected peptide ligand per kilogram of body weight, with doses of between 100 μg to 25 mg/kg/dose being more commonly used.

A composition containing a peptide ligand according to the present invention may be utilised in therapeutic settings to treat a microbial infection or to provide prophylaxis to a subject at risk of infection e.g. undergoing surgery, chemotherapy, artificial ventilation or other condition or planned intervention. In addition, the peptide ligands 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.

Therapeutic Uses

The bicyclic peptides of the invention have specific utility as Lipid II binding agents.

Lipid II is an important component for bacterial cell wall synthesis. Bacterial cell wall synthesis is essential to growth, cell division (thus reproduction) and maintaining the cellular structure in bacteria. Inhibition of Lipid II leads to irregularities in cell wall structure such as elongation, lesions, loss of selective permeability, and eventual cell death and lysis.

Thus, without being bound by theory it is believed that the peptide ligands of the present invention will be capable of causing bacterial growth inhibition, cell death and lysis by virtue of binding to Lipid II and inhibiting cell wall synthesis. It will be appreciated that the peptide ligands of the present invention may bind to Lipid II at any site capable of interfering with the mechanism of action of said Lipid II. For example, the peptide ligand may bind to the active sites of said Lipid II and inhibit the transpeptidase or transglycosylase. Alternatively, the peptide ligand may bind elsewhere on Lipid II in order to interfere with its mechanism of action.

Polypeptide ligands selected according to the method of the present invention may be employed in in vivo therapeutic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, and the like. In some applications, such as vaccine applications, 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. Once purified, partially or to homogeneity as desired, the selected polypeptides may be used diagnostically or therapeutically (including extracorporeal ly) 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).

According to a further aspect of the invention, there is provided a conjugate as defined herein, for use in suppressing or treating a disease or disorder mediated by bacterial infection or for providing prophylaxis to a subject at risk of infection.

According to a further aspect of the invention, there is provided a method of suppressing or treating a disease or disorder mediated by bacterial infection or for providing prophylaxis to a subject at risk of infection, which comprises administering to a patient in need thereof the conjugate as defined herein.

In one embodiment, the bacterial infection relates to any one or more of the following bacterial species: Acinetobacter baumannii, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostrium tetani, Corynebacterium diphtheriae, Echinococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (such as Enterotoxigenic E. coli, Enteropathogenic E. coli, Enterohemorragic E. coli or Enteroaggregative E. coli), Francisella tularensis, Haemophilus influenzae , Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pneumococcus, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella such as, Salmonella bongori, Salmonella enterica, Salmonella subterranean, Salmonella typhi or Salmonella typhimurium, Shigella (such as Shigella sonnei or Shigella dysenteriae), Staphylococcus aureus (such as MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae or Yersinia pestis.

The conjugates of the invention or pharmaceutical compositions comprising said conjugates are useful for the treatment of skin and soft tissue infections, gastrointestinal infection, urinary tract infection, pneumonia, sepsis, intra-abdominal infection and obstetrical/gynaecological infections. The infections may be caused by Gram-positive bacteria, such as S. pneumoniae, or Gram-negative bacteria, such as E. coli, P. aeruginosa and A. baumannii, or may be due to more than one species of bacterium.

In one embodiment, the disease or disorder mediated by bacterial infection is selected from: pertussis (which may be caused by Bordetella pertussis ); tetanus (which may be caused by Clostrium tetani); diphtheria (which may be caused by Corynebacterium diphtheriae); echinococcal disease (which may be caused by Echinococcus); diarrhea, hemolytic uremic syndrome or urinary tract infection (which may be caused by Escherichia coli); respiratory infections or meningitis (which may be caused by Haemophilus influenzae ); gastritis, peptic ulcer disease or gastric neoplasms (which may be caused by Helicobacter pylori); tuberculosis (which may be caused by Mycobacterium tuberculosis)·, meningitis, pneumonia, bacteremia or otitis media (which may be caused by

Pneumococcus); food poisoning (which may be caused by Salmonella); shigellosis or gastroenteritis (which may be caused by Shigella); and cholera (which may be caused by Vibrio cholerae). References herein to the term "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 invention is further described below with reference to the following examples.

EXAMPLES

Materials and Methods Peptide Synthesis

Peptide synthesis was based on Fmoc chemistry, using a Symphony peptide synthesiser manufactured by Peptide Instruments and a Syro II synthesiser by MultiSynTech. Standard Fmoc-amino acids were employed (Sigma, Merck), with appropriate side chain protecting groups: where applicable standard coupling conditions were used in each case, followed by deprotection using standard methodology.

Alternatively, peptides were purified using HPLC and following isolation they were modified with the required molecular scaffold (namely, TBMB, TVSB or TBAB). For this, linear peptide was diluted with 50:50 MeCN:H₂O up to ~35 mL, ~500 μL of 100 mM scaffold in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M NH₄HCO₃ in H₂O. The reaction was allowed to proceed for -30 -60 min at RT, and lyophilised once the reaction had completed (judged by MALDI). Once completed, 1ml of 1M L-cysteine hydrochloride monohydrate (Sigma) in H2O was added to the reaction for -60 min at RT to quench any excess TBMB, TVSB or TBAB.

Following lyophilisation, the modified peptide was purified as above, while replacing the Luna C8 with a Gemini C18 column (Phenomenex), and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct scaffold-modified material were pooled, lyophilised and kept at -20°C for storage.

All amino acids, unless noted otherwise, were used in the L- configurations. In some cases peptides are converted to activated disulfides prior to coupling with the free thiol group of a toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2- pyridylthio)pentanoate) (100mM) in dry DMSO (1.25 mol equiv) was added to a solution of peptide (20mM) in dry DMSO (1 mol equiv). The reaction was well mixed and DIPEA (20 mol equiv) was added. The reaction was monitored by LC/MS until complete.

The conjugates of the invention were generally produced as described below in Scheme I by reacting the corresponding Aza, (K(N₃)) and (PYA-N₃[K]) derivatives in the preence of copper to produce a conjugate in which the Bicycle is linked to the carrier peptide by a triazole grouping:

BIOLOGICAL DATA Minimum Inhibition Concentration (MIC) Assay

Minimum inhibitory concentration (MIC) assays were carried out using E. coli strains: GKCW101, GKCW102, ATCC25922; A. baumannii strain JWW19; and P. aeruginosa strain PA01 using the method described by Antimicrobial Agents and Chemotherapy December 2016 Volume 60 Number 12 pages 7372-7381 and CLSI, 2020. Performance standards for antimicrobial susceptibility testing. Clinical Lab Standards Institute. The results are shown in Table 1: ble 1 : MIC Data for Selected Peptide Ligands of the Invention and Control Antibiotics Meropenem and Erythromycin

Claims

1. An anti-infective peptide conjugate which comprises:

(ii) a carrier peptide.

2. The anti-infective peptide conjugate according to claim 1 , wherein said loop sequences comprise 6 amino acids.

3. The anti-infective peptide conjugate according to claim 1 or claim 2, wherein said loop sequences comprise: three cysteine residues separated by two loop sequences both of which consist of 6 amino acids; or two [Dap(Me)] residues and one cysteine residue separated by two loop sequences both of which consist of 6 amino acids.

4. The anti-infective peptide conjugate according to any of claims 1 to 3, wherein the Lipid II is present within one or more pathogenic bacterial species.

5. The anti-infective peptide conjugate according to claim 4, wherein the one or more pathogenic bacterial species is selected from any of: Acinetobacter baumannii, Bacillus anthracis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostrium tetani, Corynebacterium diphtheriae, Echinococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (such as Enterotoxigenic E. coli, Enteropathogenic E. coli, Enterohemorragic E. coli or Enteroaggregative E. coli), Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitides, Pneumococcus, Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella such as, Salmonella bongori, Salmonella enterica, Salmonella subterranean, Salmonella typhi or Salmonella typhimurium, Shigella (such as Shigella sonnei or Shigella dysenteriae), Staphylococcus aureus ('such as MRSA), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae or Yersinia pestis.

6. The anti-infective peptide conjugate according to claim 5, wherein the Lipid II is present within E. coli.

7. The anti-infective peptide conjugate according to claim 5, wherein the Lipid II is present within A. baumannii.

8. The anti-infective peptide conjugate according to claim 5, wherein the Lipid II is present within P. aeruginosa.

9. The anti-infective peptide conjugate according to any one of claims 1 to 8, wherein the bicyclic peptide ligand comprises an amino acid sequence which is selected from: C_iDWPDWQC_iiYGWSSHC_iii (SEQ ID NO: 1); C_iKHQAQEC_iiVAI REGC_iii (SEQ ID NO: 2); C_iWAMPMWC_iiDSWSQNC_iii (SEQ ID NO: 3); C_iRPQKFNC_iilSANIRC_iii (SEQ ID NO: 4); C_iKAMIGAC_iiVAMQFAC_iii (SEQ ID NO: 5); C_iYPVDWYC_iiLFQTVDC_iii (SEQ ID NO: 6); C_iRYVSGDCYYAQAHC_iii (SEQ ID NO: 7); C_iLLQSLLC_iiPYSTHRC_iii (SEQ ID NO: 8); C_iLLQSLLC_iiPYSTHAC_iii (SEQ ID NO: 9); C_iLLQSLLC_iiPYSTRAC_iii (SEQ ID NO: 10); C_iLLQSLLC_iiRYSTHAC_iii (SEQ ID NO: 11); C_iLLQRLLC_iiPYSTHAC_iii (SEQ ID NO: 12); [Dap(Me)]_iLLQRLL[Dap(Me)]_iiPYSTHAC_iii (SEQ ID NO: 13); [Dap(Me)]_iLLQRLLC_iiPYSTHA[Dap(Me)]_iii (SEQ ID NO: 14); and C_iLLQRLL[Dap(Me)]_iiPYSTHA[Dap(Me)]_iii (SEQ ID NO: 15); such as:

C_iLLQRLLC_iiPYSTHAC_iii (SEQ ID NO: 12); [Dap(Me)]_iLLQRLL[Dap(Me)]_iiPYSTHAC_iii (SEQ ID NO: 13); [Dap(Me)]_iLLQRLLC_iiPYSTHA[Dap(Me)]_iii (SEQ ID NO: 14); and C_iLLQRLL[Dap(Me)]_iiPYSTHA[Dap(Me)]_iii (SEQ ID NO: 15); in particular: C_iLLQRLLC_iiPYSTHAC_iii (SEQ ID NO: 12); wherein i, ii and iii represent first, second and third reactive groups, respectively, such that C_i, C_ii and C_iii represent first, second and third cysteine residues, respectively, and [Dap(Me)]_i, [Dap(Me)]_ii and [Dap(Me)]_iii represent first, second and third [Dap(Me)] residues, respectively, wherein Dap represents diaminopropionic acid, or a pharmaceutically acceptable salt thereof.

10. The anti-infective peptide conjugate according to claim 9, wherein the bicyclic peptide ligand additionally comprises N- and/or C-terminal additions and comprises an amino acid sequence which is selected from:

A-(SEQ ID NO: 1)-G (referred to in Hart et al (2017) as P1);

A-(SEQ ID NO: 2)-G (referred to in Hart et al (2017) as P2);

A-(SEQ ID NO: 3)-G (referred to in Hart et al (2017) as P3);

A-(SEQ ID NO: 4)-G (referred to in Hart et al (2017) as P4);

A-(SEQ ID NO: 5)-G (referred to in Hart et al (2017) as P5);

A-(SEQ ID NO: 6)-G (referred to in Hart et al (2017) as P6);

A-(SEQ ID NO: 7)-G (referred to in Hart et al (2017) as P7);

A-(SEQ ID NO: 8)-G (referred to in Hart et al (2017) as P8);

R15A));

A-(SEQ ID NO: 11)-G (herein referred to in Hart et al (2017) as P8-D-P10R,

R15A));

A-(SEQ ID NO: 12)-G (herein referred to herein as BCY11898 and in Hart et al

(2017) as P8-D-S6R, R15A));

A-(SEQ ID NO: 13)-G;

A-(SEQ ID NO: 14)-G; and

A-(SEQ ID NO: 15)-G, such as:

A-(SEQ ID NO: 13)-G;

A-(SEQ ID NO: 14)-G; and A-(SEQ ID NO: 15)-G; in particular: A-(SEQ ID NO: 12)-G (herein referred to herein as BCY11898 and in Hart etal (2017) as P8-D-S6R, R15A)).

11. The anti-infective peptide conjugate according to any one of claims 1 to 10, wherein the carrier peptide comprises a linear peptide, such as between 3 and 15 amino acids, in particular between 4 and 12 amino acids, more particularly either 4, 7, 8, 10, 11 or 12 amino acids in length, most particularly 11 amino acids in length.

12. The anti-infective peptide conjugate according to any one of claims 1 to 11, wherein the carrier peptide is selected from one of the following peptides:

KSLRRVWRSWR (SEQ ID NO: 16; herein referred to as DRAMP); KSL[HArg][HArg]VW[HArg]SW[HArg] (SEQ ID NO: 17);

[dK] [dS] [dL] [dR] [dR] [dV] [dW][dR][dS][dW][dR] (SEQ ID NO: 18; herein referred to as D form DRAMP);

[dR] [dW][dS] [dR] [dW][dV] [dR] [dR][dL] [dS][dK] (SEQ ID NO: 19; herein referred to as retroinverso DRAMP);

VKLFPVKLFP (SEQ ID NO: 20);

SLLSLIRKLIT (SEQ ID NO: 21);

FFFLSRIFGK (SEQ ID NO: 22);

PLILLRLLRGQF (SEQ ID NO: 23);

NAGSLLSGWG (SEQ ID NO: 24);

NGVQPKY (SEQ ID NO: 25);

DKYLPRPRPV (SEQ ID NO: 26);

KFFKFFK (SEQ ID NO: 27);

KFFK (SEQ ID NO: 28);

KFFKFFKFFK (SEQ ID NO: 29); and RLWVLWRR (SEQ ID NO: 30), such as:

KSLRRVWRSWR (SEQ ID NO: 16; herein referred to as DRAMP);

[dK] [dS] [dL] [dR] [dR] [dV] [dW] [dR] [dS] [dW] [dR] (SEQ ID NO: 18; herein referred to as D form DRAMP); and

[dR] [dW][dS] [dR] [dW][dV] [dR] [dR][dL] [dS][dK] (SEQ ID NO: 19; herein referred to as retroinverso DRAMP).

13. The anti-infective peptide conjugate according to any one of claims 1 to 12, wherein the carrier peptide additionally comprises a moiety for facilitating conjugation to the bicyclic peptide, such as an azidoalanine (Aza) residue, an azidolysine (K(N₃)) residue or a (PYA- N₃[K]) residue, wherein PYA represents propargyl-acid, in particular a (PYA-N₃[K]) residue.

14. The anti-infective peptide conjugate according to claim 13, wherein said Aza, (K(N₃)) or (PYA-N₃[K]) residue is present at either the N- or C-terminal of said carrier peptide.

15. The anti-infective peptide conjugate according to claim 13, wherein the moiety for facilitating conjugation of the carrier peptide to the bicyclic peptide comprises a moiety of formula (I):

wherein R represents the bicyclic peptide and R₁ represents the carrier peptide.

16. The anti-infective peptide conjugate according to any one of claims 1 to 15, wherein the molecular scaffold is selected from 1,3,5-tris(bromomethyl)benzene (TBMB), 1,3,5- tris(vinylsulfonyl)benzene (TVSB) or N,N',N"-1,3,5-benzenetriyltris[2-bromoacetamide] (TBAB).

17. The anti-infective peptide conjugate according to any one of claims 1 to 16, wherein the bicyclic peptide ligand is attached to a TBMB scaffold and conjugated to a carrier peptide and comprises an anti-infective conjugate which is a compound of formula (I):

and is selected from:

18. The anti-infective peptide conjugate according to any one of claims 1 to 16, wherein the bicyclic peptide ligand is attached to a TVSB scaffold and conjugated to a carrier peptide and comprises an anti-infective conjugate which is a compound of formula (I):

and is selected from:

19. The anti-infective peptide conjugate according to any one of claims 1 to 16, wherein the bicyclic peptide ligand is attached to a TBAB scaffold scaffold and conjugated to a carrier peptide and comprises an anti-infective conjugate which is a compound of formula (I):

and is selected from:

20. The anti-infective peptide conjugate according to one of claims 1 to 19, wherein the pharmaceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium and ammonium salt.

21. A pharmaceutical composition which comprises the anti-infective peptide conjugate of any one of claims 1 to 20, in combination with one or more pharmaceutically acceptable excipients.

22. The pharmaceutical composition according to claim 21 , which additionally comprises one or more therapeutic agents.

23. The anti-infective peptide conjugate according to any of claims 1 to 20, or the pharmaceutical composition as defined in claim 21 or claim 22, for use in suppressing or treating a disease or disorder mediated by bacterial infection or for providing prophylaxis to a subject at risk of infection.