Heterotandem bicyclic peptide conjugates

Abstract

The present invention relates to a heterotandem bicyclic peptide complex comprising a first peptide ligand that binds to EphA2 conjugated via linkers to two second peptide ligands that bind to CD137, and the present invention also relates to the use of the heterotandem bicyclic peptide complex in the prevention, suppression, or treatment of cancer.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a heterotandem bicyclic peptide complex comprising a first peptide ligand that binds to EphA2 conjugated via a linker to two second peptide ligands that bind to CD137. The present invention also relates to the use of said heterotandem bicyclic peptide complex in the prevention, inhibition or treatment of cancer. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are able to bind protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutic drugs. Indeed, several cyclic peptides have already been successfully used in the clinic, such as, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The excellent binding properties are due not only to the relatively large interaction surface formed between the peptide and the target, but also to the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are used with the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 (Wu et al., 2007), Science 330, 1066-71), a cyclic peptide (355 Å) with an Arg-Gly-Asp motif that binds to integrin αVb3 2 ) (Xiong et al., 2002, Science 296(5565), 151-5), or upain-1 (603 Å), a cyclic peptide inhibitor that binds to urokinase-type plasminogen activator. 2 ; Zhao et al. (2007), J Struct Biol 160(1), 1-10), which bind to surfaces measuring several hundred square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides and experience less entropy loss when binding to targets, resulting in higher binding affinity. The reduced flexibility also results in a target-specific conformational fixation, increasing binding specificity compared to linear peptides. This effect is exemplified by a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity against other MMPs when its ring is opened (Cherney et al., 1998, J Med Chem 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with multiple peptide rings, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously tethered polypeptides bearing cysteine ​​residues to synthetic molecular structures (Kemp and McNamara, 1985, J. Org. Chem; Timmerman et al., 2005, ChemBioChem). Meloen and coworkers used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al., 2005, ChemBioChem). Methods for producing candidate drug compounds (wherein the compounds are generated by linking cysteine-containing polypeptides to molecular scaffolds such as, for example, 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA)) are disclosed in WO 2019 / 122860 and WO 2019 / 122863.

[0005] A phage display-based combinatorial approach has been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al., 2009, Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a linear peptide containing three cysteine ​​residues and two random six amino acid regions (Cys-(Xaa) 6 -Cys-(Xaa) 6 A combinatorial library of cysteine ​​(Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the Invention

[0006] (Summary of the invention) According to a first aspect of the present invention, (a) a peptide that binds to EphA2 and has the sequence [ka] a first peptide ligand having an N-(acid-PEG) 3 )-N-Bis(PEG 3 -azide) linker, (b) binds to CD137, both of which have the sequence [ka] two second peptide ligands having the following structure: Conjugated to wherein each of the peptide ligands comprises three reactive cysteine ​​groups (C i , C ii , and C iii), and a molecular scaffold which is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and forms a covalent bond with a reactive cysteine ​​group of the polypeptide, such that two polypeptide loops are formed on the molecular scaffold; Here, there is provided a heterotandem bicyclic peptide complex, wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, d1Nal represents D-1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, and Nle represents norleucine.

[0007] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a heterotandem bicyclic peptide complex as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0008] According to a further aspect of the present invention there is provided a heterotandem bicyclic peptide complex as defined herein for use in the prevention, inhibition or treatment of cancer. [Brief description of the drawings]

[0009] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Analysis of the EphA2 / CD137 heterotandem bicyclic peptide complex BCY13272 in a Promega CD137 luciferase reporter assay in the presence of EphA2-expressing A549, PC-3, and HT29 cells (n=3). BCY13626 is a heterotandem bicyclic peptide complex similar to BCY13272 but contains D-amino acids and does not bind to EphA2 or CD137. [Diagram 2]Figure 2: Plasma concentration versus time plots of BCY13272 resulting from a 5.5 mg / kg IV dose in CD1 mice (n=3), a 3.6 mg / kg IV infusion (15 min) in SD rats (n=3), and an 8.9 mg / kg IV infusion (15 min) in cynomolgus monkeys (n=2). The pharmacokinetic profile of BCY13272 has a terminal half-life of 2.9 hours in CD-1 mice, 2.5 hours in SD rats, and 8.9 hours in cynomolgus monkeys. [Diagram 3] Figure 3: Antitumor activity of BCY13272 in a syngeneic MC38 tumor model. (A) MC38 tumor volume at and after BCY13272 treatment. The number of complete responder (CR) mice at D28 (and the number of mice remaining in CR at D62) is indicated in brackets. BIW: twice weekly dosing; IV: intravenous administration. (B) Tumor growth curves of complete responder animals and age-matched untreated control animals to BCY13272 after MC38 tumor cell implantation. CR: complete responder. [Figure 4] Figure 4: BCY13272 induces IFN-γ cytokine secretion in (A) PBMC / MC38 and (B) PBMC / HT29 co-culture assays. BCY12762 is a heterotandem bicyclic peptide complex that binds EphA2 but not CD137. BCY13692 is a heterotandem bicyclic peptide complex that binds CD137 but not EphA2. (C) Plots of EC50 (nM) values ​​for BCY13272-induced IL-2 and IFN-γ secretion in PBMC co-culture assays using the MC38 (mouse) cell line and five PBMC donors and the HT1080 (human) cell line and four PBMC donors. [Diagram 5] FIG. 5: Surface plasmon resonance (SPR) binding of BCY13272 to immobilized (A) EphA2 and (B) CD137. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention According to a first aspect of the present invention, (a) a peptide that binds to EphA2 and has the sequence [ka] a first peptide ligand having an N-(acid-PEG) 3 )-N-Bis(PEG 3 -azide) linker, (b) binds to CD137, both of which have the sequence [ka] two second peptide ligands having the following structure: Conjugated to wherein each of the peptide ligands comprises three reactive cysteine ​​groups (C i , C ii , and C iii ), and a molecular scaffold which is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and forms a covalent bond with a reactive cysteine ​​group of the polypeptide, such that two polypeptide loops are formed on the molecular scaffold; Here, there is provided a heterotandem bicyclic peptide complex, wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, d1Nal represents D-1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, and Nle represents norleucine.

[0011] N-(Acid-PEG 3 )-N-Bis(PEG 3 Reference herein to a -azido) linker is [ka] Contains :.

[0012] In one embodiment, the heterotandem bicyclic peptide complex is BCY13272: [ka] It is.

[0013] Full details of BCY13272 are shown in Table A below: Table A: Composition of BCY13272 [Table 1]

[0014] Data demonstrating that BCY13272 produces significant antitumor effects in the murine MC38 tumor model are presented herein in FIG.

[0015] Reference is made herein to specific analogues (i.e. modified derivatives) and metabolites of BCY13272, each of which forms a further aspect of the invention, and are summarized in Table B below: Table B: Composition of labeled analogs and potential metabolites of BCY13272 [Table 2] Here, BCY14601, together with TATA as a molecular scaffold, [ka]

[0023] The present invention relates to a bicyclic peptide ligand having a sequence of And BCY13389, with TATA as the molecular scaffold, [ka] 5 represents a bicyclic peptide ligand having the sequence:

[0016] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, for example, peptide chemistry, cell culture, and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetics, and biochemistry methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc., incorporated herein by reference).

[0017] (Nomenclature) (Numbering) When referring to amino acid residue positions within the compounds of the invention, a cysteine ​​residue (C i , C ii , and C iii ) are invariant, they are omitted from the numbering and therefore the numbering of amino acid residues in SEQ ID NO:1 is referred to as follows: C i -HyP 1 -L 2 -V 3 -N 4 -P 5 -L 6 -C ii -L 7 -E 8 -P 9 -d1Nal 10 -W 11 -T 12 -C iii (SEQ ID NO: 1) .

[0018] For this illustration, the bicyclic peptide is cyclized with 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) to give a trisubstituted structure. Cyclization with TATA results in the C i , C ii , and C iii This occurs above.

[0019] (Molecular format) N- or C-terminal extensions to the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO: X) This is expressed as:

[0020] (Reverse peptide sequence) In view of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, it is envisaged that the peptide sequences disclosed herein will also find utility in their retro-inverso forms. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus and the C-terminus becomes the N-terminus) and the stereochemistry is likewise reversed (i.e., D-amino acids become L-amino acids and L-amino acids become D-amino acids). For the avoidance of doubt, reference to an amino acid, either as its full name or as its one-letter or three-letter designation, is intended herein to be represented as an L-amino acid, unless otherwise specified. When such an amino acid is intended to be represented as a D-amino acid, the amino acid is preceded by a lowercase d in square brackets, e.g., [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0021] (Advantages of peptide ligands) Certain heterotandem bicyclic peptide conjugates of the present invention have several advantageous properties that make them suitable drug-like molecules for injection, inhalation, nasal, ocular, oral, or topical administration. Such advantageous properties include: -Species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability. Heterotandem bicyclic peptide conjugates should ideally demonstrate stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that heterotandem bicyclic peptide lead candidates can not only be developed in animal models but also be administered with confidence to humans; - a desirable solubility profile, which is a function of charged and hydrophilic to hydrophobic residue ratios and intra / inter-molecular H-bonds, which are important for formulation and absorption purposes; - Selectivity. Certain heterotandem bicyclic peptide complexes of the present invention exhibit good selectivity over other targets; - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop heterotandem bicyclic peptide complexes for short-term exposure in acute disease management settings or to develop heterotandem bicyclic peptide complexes with enhanced retention in the circulation, making them optimal for managing more chronic disease states. Other factors driving the desired plasma half-life are the requirement for sustained exposure for maximum therapeutic efficacy and the associated toxicities of sustained exposure of the drug.

[0022] Importantly, the heterotandem bicyclic peptide complexes of the present invention have the following in vitro EC 50Data are presented herein that show antitumor potency when administered at a frequency that does not maintain plasma concentrations above 100 kDa. This is in contrast to larger recombinant biologic (i.e., antibody-based) approaches to CD137 agonism or bispecific CD137 agonism (Segal et al., Clin Cancer Res., 23(8):1929-1936 (2017); Claus et al., Sci Trans Med., 11(496): eaav5989, 1-12 (2019); Hinner et al., Clin Cancer Res., 25(19):5878-5889 (2019)). Without wishing to be bound by theory, it is believed that the reason for this observation is due to the fact that the heterotandem bicyclic complexes have a relatively low molecular weight (usually <15 kDa), are fully synthetic, and are tumor-targeted agonists of CD137. Thus, it has a relatively short plasma half-life, but good tumor penetration and retention. Data are presented herein that fully support these advantages. For example, antitumor efficacy in a syngeneic rodent model of mice with humanized CD137 has been shown either daily or every 3 days. Furthermore, intraperitoneal pharmacokinetic data show that the plasma half-life is less than 3 hours, which allows the circulating concentration of the complex to be reduced by 100% between doses in vitro EC 50 Furthermore, tumor pharmacokinetic data indicate that the levels of heterotandem bicyclic complexes in tumor tissues can be higher and more sustained compared to plasma levels.

[0023] It will be appreciated that this observation forms an important further aspect of the present invention. Thus, according to a further aspect of the present invention there is provided a method of treating cancer comprising administering to a subject in need thereof an in vitro EC of a heterotandem bicyclic peptide complex as defined herein. 50 In accordance with another aspect of the present invention, methods are provided that include administering the conjugate at a dosage frequency that does not maintain a plasma concentration of the conjugate above 100 mg / kg / day.

[0024] Immune memory. Coupling a bicyclic peptide ligand that binds to cancer cells with a bicyclic peptide ligand that binds to immune cells provides the synergistic advantage of immune memory. Not only do the heterotandem bicyclic peptide complexes of the present invention appear to eradicate tumors, but none of the inoculated full responder mice developed tumors when re-administered with tumorigenic agents. This indicates that treatment with the heterotandem bicyclic peptide complexes of the present invention induces immunogenic memory in the full responder mice. This has important clinical benefits for preventing recurrence of the tumor once it has been initially controlled and eradicated.

[0025] (Peptide Ligand) The peptide ligand referred to herein refers to a peptide covalently bound to a molecular scaffold.Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond with the scaffold, and an internal sequence between the reactive groups, called a loop sequence, because the peptide forms a loop when it binds to the scaffold.In this case, the peptide comprises at least three reactive groups selected from cysteine, 3-mercaptopropionic acid, and / or cysteamine, and forms at least two loops on the scaffold.

[0026] (Pharmaceutical acceptable salts) It will be understood that the salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of that ligand.

[0027] The salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, page 388, August 2002. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0028] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, Cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, Hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, Examples of suitable salts include mono- or di-salts formed with acids selected from the group consisting of acetic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0029] One particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0030] The compound is anionic or has a functional group which may be anionic (e.g., -COOH can be -COO - In the case of cations such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29 + , Na + , and K + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R + , N.H. 2 R 2 + , N.H.R. 3 + , N.R. 4 + ) are included, but are not limited to. Some suitable examples of substituted ammonium ions include those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. Examples of common quaternary ammonium ions are N(CH 3 ) 4 + It is.

[0031] When the compounds of the present invention contain amine functions, they may form quaternary ammonium salts, for example, by reaction with alkylating agents by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0032] (Modified derivative) It will be understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., 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 spacer groups; 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 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 in the bicyclic peptide ligand; replacement of one or more peptide bonds with surrogate bonds; modification of the peptide backbone length; Modifications include one or more selected from: replacement of the hydrogen on the α-carbon of one or more amino acid residues with another chemical group; modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid, and tyrosine with suitable amine-, thiol-, carboxylic acid-, and phenol-reactive reagents to functionalize the amino acids; and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, e.g., amino acids with azide or alkyne groups that allow functionalization with alkyne- or azide-bearing moieties, respectively.

[0033] In one embodiment, the modified derivative comprises an N-terminal and / or a C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using a suitable amino reaction chemistry and / or a C-terminal modification using a suitable carboxy reaction chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator, or a chromophore.

[0034] 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, an N-terminal cysteine ​​group (referred to herein as C iThe N-terminal acetyl group (referred to as "acetyl group") is capped with acetic anhydride or other suitable reagent during peptide synthesis to yield an N-terminally acetylated molecule. This embodiment offers the advantage of eliminating a potential recognition point for aminopeptidases, avoiding possible degradation of the bicyclic peptide.

[0035] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates the conjugation of an effector group and retention of potency of the bicyclic peptide against its target.

[0036] 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 (herein referred to as C iii The C-terminally amidated group, referred to as the C-terminus, is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment offers the advantage of eliminating a potential recognition point for carboxypeptidases, reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0037] In one embodiment, the modified derivatives include the substitution 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 that have isosteric / isoelectronic side chains that are not recognized by degradative proteases or have any adverse effect on targeting efficacy.

[0038] Alternatively, unnatural amino acids with constrained amino acid side chains may be used such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, these concern proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids that are simple derivatives of amino-cyclopropyl carboxylic acids.

[0039] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of an N-terminal cysteine ​​(C i) and / or a C-terminal cysteine ​​(C iii ) including the addition of a spacer group to

[0040] In one embodiment, the modified derivative comprises the substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises the substitution of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0041] In one embodiment, the modified derivative comprises the substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises the substitution of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. The combination of the two can affect the half-life, volume of distribution, and exposure of the peptide drug, which can be adjusted depending on the clinical endpoint. Furthermore, the correct combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

[0042] In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to enhance proteolytic stability due to steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0043] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine. This embodiment has the advantage of removing potential sites of proteolytic attack.

[0044] It should be noted that each of the above mentioned modifications serves to purposefully improve the potency or stability of the peptide. Further potency improvements based on the modifications can be achieved by the following mechanisms: - taking advantage of the hydrophobic effect and incorporating hydrophobic sites resulting in lower dissociation rates so that higher affinities are achieved; - taking advantage of long-range ionic interactions, resulting in faster association rates and incorporating charged groups resulting in higher affinity (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - building further constraints into the peptide, for example by constraining the amino acid side chains correctly so that the entropy loss is minimal upon target binding, constraining the backbone torsion angles so that the entropy loss is minimal upon target binding, and introducing further cyclizations into the molecule for the same 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).

[0045] (Isotopic Variation) The present invention includes all pharma- ceutically acceptable (radio)isotope-labeled peptide ligands of the invention in which one or more atoms have been replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, as well as peptide ligands of the invention to which a metal chelating group capable of bearing a relevant (radio)isotope has been attached (referred to as "effectors"), and peptide ligands of the invention in which a particular functional group has been covalently replaced with a relevant (radio)isotope or an isotopically labeled functional group.

[0046] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, e.g., 2 H(D) and 3 H(T), an isotope of carbon, e.g. 11 C. 13 C and 14 C, an isotope of chlorine, e.g. 36 Cl, isotopes of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I, and 131 I, isotopes of nitrogen, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O, and 18 O, isotopes of phosphorus, e.g. 32 P, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y, as well as isotopes of lutetium, e.g. 177 Lu, as well as isotopes of bismuth, e.g. 213 Contains Bi.

[0047] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating radioisotopes, are useful in drug and / or substrate tissue distribution studies and for clinically assessing the presence and / or absence of Nectin-4 targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may use compounds that are labeled with labeling agents, such as, for example, radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). The radioisotope tritium, i.e., 3 H(T) and carbon-14, i.e. 14 C is particularly useful for this purpose given its ease of incorporation and the means of detection available.

[0048] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), may confer certain therapeutic advantages resulting from greater metabolic stability, for example, increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0049] 11 C. 18 F, 15 O, and 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0050] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the accompanying Examples, substituting appropriate isotopically labeled reagents for previously utilized non-labeled reagents.

[0051] (synthesis) The peptides of the present invention can be synthetically produced by standard techniques and then reacted with molecular scaffolds in vitro. Standard chemistry can be used to carry this out. This allows for rapid large-scale preparation of soluble material for further downstream experimentation or validation. Such methods can be achieved using conventional chemistry such as that disclosed in Timmerman et al. (supra).

[0052] The present invention therefore also relates to the production of a polypeptide or conjugate selected as described herein, which production comprises any further steps as explained below, in one embodiment these steps are carried out on the final polypeptide conjugate made by chemical synthesis.

[0053] Optionally, amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

[0054] The peptide can also be extended, for example, to incorporate additional loops and thus introduce multiple specificities.

[0055] To extend a peptide, it may simply be chemically extended at its N- or C-terminus or within a loop using orthogonally protected lysines (and analogues) using standard solid- or solution-phase chemistries. Activated or activatable N- or C-termini may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be performed enzymatically by fragment condensation or native chemical ligation as described, for example, in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or using subtiligase as described, for example, in (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, 15 Nov. 2008, pp. 6000-6003).

[0056] Alternatively, peptides may be extended or modified by further conjugation via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other in the reducing environment of cells. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; then, an additional cysteine ​​or thiol can be added to the N- or C-terminus of the first peptide, so that this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0057] Similar techniques apply equally to the synthesis / coupling of two bicyclic bispecific macrocycles, potentially giving rise to a tetraspecific molecule.

[0058] Furthermore, the addition of other functional or effector groups may be achieved in the same manner, coupling at the N- or C-terminus or via a side chain using appropriate chemistry, hi one embodiment, the coupling is carried out in such a way as not to block the activity of either entity.

[0059] Pharmaceutical Composition According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0060] Usually, the peptide ligands are utilized 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 the polypeptide complex in suspension, may be selected from viscosity-increasing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0061] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose, and preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack, 1982, Remington's Pharmaceutical Sciences, 16th ed.).

[0062] The peptide ligands of the invention may be used as a separately administered composition or in conjunction with other drugs. These may include antibodies, antibody fragments, and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin, or cisplatin, and immunotoxins. Pharmaceutical compositions may include "cocktails" of various cytotoxic or other drugs in conjunction with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities, such as polypeptides selected with different targeting ligands, whether pooled or not prior to administration.

[0063] The route of administration of the pharmaceutical composition according to the invention may be any one generally known to those skilled in the art. For therapy, the peptide ligand of the invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also suitably by direct injection using a catheter. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dosage and frequency of administration will depend on the age, sex, and condition of the patient, the simultaneous administration of other drugs, contraindications, and other parameters taken into account by the clinician.

[0064] The peptide ligands of the present invention can be lyophilized before storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be utilized. It will be understood by those skilled in the art that lyophilization and reconstitution may result in varying degrees of activity loss, and that levels may need to be adjusted upwards to compensate.

[0065] Compositions containing the peptide ligands or cocktails thereof of the present invention can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a selected population of cells is defined as a "therapeutically effective dose". The amount required to achieve this dosage will depend on the severity of the disease and the general state of the patient's own immune system, but generally ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

[0066] Compositions containing peptide ligands according to the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in a mammal. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove target cell populations from heterogeneous populations of cells. Blood from a mammal can be combined ex vivo with the selected peptide ligand, thereby killing or otherwise removing undesirable cells from the blood for return to the mammal according to standard techniques.

[0067] (therapeutic use) According to a further aspect of the present invention there is provided a heterotandem bicyclic peptide complex as defined herein for use in the prevention, inhibition or treatment of cancer.

[0068] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), such as tumors of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, cancers of the nasal cavity, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and adnexal cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and borderline malignancies including premalignant hematological disorders and hematological malignancies and related disorders of lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [D-cell lymphoma], LBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and related disorders of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndromes, myeloproliferative disorders, e.g., polycythemia vera, essential thrombocytopenia, myeloma, myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia; tumors of mesenchymal origin, e.g., sarcomas of the soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and Schwannoma);These include, but are not limited to, endocrine tumors (e.g., pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas); and pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors); or congenital or other syndromes that predispose a patient to malignancies (e.g., xeroderma pigmentosum);

[0069] In further embodiments, the cancer is selected from hematopoietic malignancies, e.g., selected from: non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myelogenous leukemia (CML).

[0070] References herein to the term "prevention" include administration of the protective composition prior to the induction of disease. "Suppression" refers to administration of the composition after an inductive event but prior to clinical appearance of disease. "Treatment" includes administration of the protective composition after disease symptoms are manifest.

[0071] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease, and the use of animal model systems is facilitated by the present invention, which allows for the development of polypeptide ligands that can cross-react with human and animal targets.

[0072] The invention will now be further described with reference to the following examples. EXAMPLES

[0073] (Example) In general, the heterotandem bicyclic peptide complexes of the invention can be prepared according to the following general method: [ka]

[0074] Degas all solvents and rinse with N 2 The mixture is purged three times with 500 rpm for 10 min. A solution of BP-23825 (1.0 equiv.), HATU (1.2 equiv.), and DIEA (2.0 equiv.) in DMF is mixed for 5 min, after which bicycle 1 (1.2 equiv.) is added. The reaction mixture is stirred at 40° C. for 16 h. The reaction mixture is then concentrated under reduced pressure to remove the solvent and purified by preparative HPLC to give intermediate 2.

[0075] A mixture of intermediate 2 (1.0 equiv.) and bicycle 2 (2.0 equiv.) was dissolved in t-BuOH / H 2 HO (1:1), then CuSO 4 (1.0 equiv.), VcNa (4.0 equiv.), and THPTA (2.0 equiv.). Finally, 0.2 M NH 4 HCO 3 The pH is adjusted to 8 by adding N 2 Stir under atmosphere for 16 hours at 40° C. The reaction mixture was directly purified by preparative HPLC.

[0076] More detailed experimental details of selected heterotandem bicyclic peptide complexes of the present invention are provided herein below:

[0077] Example 1: Synthesis of BCY13272 [ka] Procedure for preparation of BCY14964 [ka] A mixture of BP-23825 (155.5 mg, 249.40 μmol, 1.2 equiv) and HATU (95.0 mg, 249.92 μmol, 1.2 equiv) was dissolved in NMP (1.0 mL), then the pH of the solution was adjusted to 8 by dropwise addition of DIEA (64.6 mg, 499.83 μmol, 87.0 μL, 2.4 equiv), and then the solution was allowed to stir at 25° C. for 5 min. BCY13118 (500.0 mg, 207.83 μmol, 1.0 equiv) was dissolved in NMP (5.0 mL), then added to the reaction solution, and the pH of the resulting solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was allowed to stir at 25° C. for 45 min. LC-MS showed that BCY13118 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. The residue was then purified by preparative HPLC to give BCY14964 (1.35 g, 403.46 μmol, 64.7% yield, 90% purity) as a white solid. Calculated MW: 3011.53, observed m / z: 1506.8 ([M+2H] 2+ ), 1005.0([M+3H] 3+ ).

[0078] (Procedure for preparation of BCY13272) [ka] BCY8928 (644.0mg, 290.55μmol, 2.5eq), THPTA (50.5mg, 116.22μmol, 1.0eq), CuSO 4 A mixture of 1,2-dichlorophenyl ether (0.4 M, 145.0 μL, 0.5 equiv.), and sodium ascorbate (82.0 mg, 464.89 μmol, 4.0 equiv.) was dissolved in t-BuOH / 0.2 M NH4HCO3 (1:1, 6.0 mL), and the pH of the solution was adjusted to 0.2 M NH 4 HCO 3 (1:1 t-BuOH / 0.2M NH 4 HCO 3The pH was adjusted to 7.5 by dropwise addition of t-BuOH / 0.2M NH 4 HCO 3 (1:1, 11.0 mL) and then added dropwise to the stirred solution. All solvents here were pre-degassed and N 2 The pH of this solution was adjusted to 0.2M NH 4 HCO 3 (1:1 t-BuOH / 0.2M NH 4 HCO 3 The temperature was adjusted to 7.5 by dropwise addition of 1,2-dichloroethane, and the solution turned pale yellow. The reaction mixture was then cooled to 100° C. 2 Stirred at 25° C. under ambient atmosphere for 6 h. LC-MS showed one major product peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY13272 (1.75 g, 235.01 μmol, 67.40% yield, 94% purity) as a white solid. Calculated MW: 7446.64, observed m / z: 1242.0 ([M+6H] 6+ ), 1491.0([M+5H] 5+ ).

[0079] Example 2: Synthesis of BCY14414 [ka] (Procedure for preparation of BCY14798) [ka] A mixture of BCY14964 (55.0 mg, 18.26 μmol, 1.0 equiv.), BCY8928 (32.4 mg, 14.61 μmol, 0.8 equiv.), and THPTA (39.8 mg, 91.32 μmol, 5.0 equiv.) was dissolved in t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed, N 2 (purged with CuSO 4(0.4 M, 23.0 μL, 0.5 equiv.) and sodium ascorbate (72.0 mg, 365.27 μmol, 20.0 equiv.) in N 2 The pH of this solution was adjusted to 0.2M NH 4 HCO 3 (1:1 t-BuOH / 0.2M NH 4 HCO 3 The temperature was adjusted to 7.5 by dropwise addition of 1,2-dichloroethane, and the solution turned pale yellow. The reaction mixture was then cooled to 100° C. 2 Stirred at 25° C. under ambient atmosphere for 1.5 h. LC-MS showed that BCY14964 remained and compound BCY8928 was completely consumed with one major peak with the desired m / z detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY14798 (51 mg, 9.17 μmol, 33.37% yield, 94% purity) as a white solid. Calculated MW: 5229.07, observed m / z: 1308.3 ([M+4H] 4+ ), 1046.7([M+5H] 5+ ).

[0080] (Procedure for preparation of BCY14414) [ka] A mixture of BCY14798 (21.0 mg, 4.02 μmol, 1.0 equiv.), BCY13389 (10.0 mg, 4.42 μmol, 1.1 equiv.), and THPTA (1.8 mg, 4.02 μmol, 1.0 equiv.) was dissolved in t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed, N 2 (purged with CuSO 4 (0.4 M, 5.0 μL, 0.5 equiv.) and sodium ascorbate (2.8 mg, 16.06 μmol, 4.0 equiv.) in N 2 The pH of this solution was adjusted to 0.2M NH 4 HCO 3 (1:1 t-BuOH / 0.2M NH 4 HCO 3The temperature was adjusted to 7.5 by dropwise addition of 1,2-dichloroethane, and the solution turned pale yellow. The reaction mixture was then cooled to 100° C. 2 The mixture was stirred at 25° C. under ambient atmosphere for 2 h. LC-MS showed that BCY14798 was completely consumed, some BCY13389 remained, and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative separation to give BCY14414 (20 mg, 2.40 μmol, 59.73% yield, 90.9% purity) as a white solid. Calculated MW: 7503.74, observed m / z: 1251.5 ([M+5H] 5+ ), 1072.9([M+7H] 7+ ).

[0081] Example 3: Synthesis of BCY14417 [ka] (Procedure for preparation of BCY14417) [ka] A mixture of BCY14414 (13.0 mg, 1.73 μmol, 1.0 equiv.) and biotin-PEG12-NHS ester (CAS 365441-71-0, 4.2 mg, 4.50 μmol, 2.6 equiv.) was dissolved in DMF (0.5 mL). The pH of the solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 0.5 h. LC-MS showed that BCY14414 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY14417 (9.0 mg, 1.07 μmol, 80.49% yield, 90.8% purity) as a white solid. Calculated MW: 8329.74, observed m / z: 1389.6 ([M+6H] 6+ ), 1191.9([M+7H] 7+ ).

[0082] Example 4: Synthesis of BCY14418 [ka] (Procedure for preparation of BCY14418) [ka] A mixture of BCY14414 (5.6 mg, 0.75 μmol, 1.0 equiv.) and Alexa fluor® 488 (0.9 mg, 1.49 μmol, 2.0 equiv.) was dissolved in DMF (0.3 mL). The pH of the solution was then adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 1.0 h. LC-MS showed that BCY14414 was completely consumed and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY14418 (2.3 mg, 0.25 μmol, 32.89% yield, 85.6% purity) as a red solid. Calculated MW: 8020.19, observed m / z: 1337.2 ([M+6H] 6+ ).

[0083] Example 5: Synthesis of BCY15217 [ka] (Procedure for preparation of BCY15217) [ka] A mixture of BCY14964 (20.0 mg, 6.64 μmol, 1.0 equiv.), BCY14601 (30.5 mg, 13.95 μmol, 2.1 equiv.), and THPTA (2.9 mg, 6.64 μmol, 1.0 equiv.) was dissolved in t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed, N 2 (purged with CuSO 4 (0.4 M, 16.6 μL, 1.0 equiv.) and sodium ascorbate (4.7 mg, 26.56 μmol, 4.0 equiv.) in N2 The pH of the solution was adjusted to 8, and the solution turned pale yellow. The reaction mixture was diluted with N 2 The mixture was stirred at 25° C. under ambient atmosphere for 2 h. LC-MS showed that BCY14964 remained and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY15217 (19.7 mg, 2.41 μmol, 36.26% yield, 96.2% purity) as a white solid. Calculated MW: 7362.5, observed m / z: 1473.5 ([M+5H] 5+ ), 1228.2([M+6H] 6+ ), 1052.8([M+7H] 7+ ).

[0084] Example 6: Synthesis of BCY15218 [ka] (Procedure for preparation of BCY15218) [ka] A mixture of BCY14798 (30.0 mg, 5.74 μmol, 1.0 equiv.), BCY14601 (15.0 mg, 6.88 μmol, 1.2 equiv.), and THPTA (2.5 mg, 5.74 μmol, 1.0 equiv.) was dissolved in t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed, N 2 (purged with CuSO 4 (0.4 M, 14.0 μL, 1.0 equiv.) and sodium ascorbate (4.0 mg, 22.95 μmol, 4.0 equiv.) in N 2 The pH of this solution was adjusted to 0.2M NH 4 HCO 3 (1:1 t-BuOH / 0.2M NH 4 HCO 3 The temperature was adjusted to 7.5 by dropwise addition of 1,2-dichloroethane, and the solution turned pale yellow. The reaction mixture was then cooled to 100° C. 2Stirred at 25° C. under ambient atmosphere for 2 h. LC-MS showed that BCY14798 was completely consumed, BCY14601 remained, and one major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to give BCY15218 (22 mg, 2.67 μmol, 46.61% yield, 95.0% purity) as a white solid. Calculated MW: 7404.6, observed m / z: 1234.8 ([M+6H] 6+ ).

[0085] (Analysis Data) The heterotandem bicyclic peptide conjugates of the present invention were analyzed using mass spectrometry and HPLC. The HPLC settings were as follows: Mobile phase: A: H 2 B: 0.1% TFA in O. C: 0.1% TFA in ACN. Flow rate: 1.0ml / min Column: Kintex 1.7um C18 100A 2.1mm*150mm Instrument: Agilent UPLC 1290

[0086] The gradient used was 30-60% B over 10 min. Data was generated as follows: [Table 3]

[0087] (Biological Data) (1. CD137 reporter assay co-culture with tumor cells) A culture medium called R1 medium is prepared by adding 1% FBS to RPMI-1640 (a component of Promega kit CS196005). Serial dilutions of test article in R1 are prepared in sterile 96-well plates. 25 μL per well of test article or R1 (as background control) is added to designated wells in white cell culture plates. Tumor cells *are harvested and resuspended in R1 medium at a concentration of 400,000 cells / mL. Twenty-five (25) μL / well of tumor cells are added to a white cell culture plate. Jurkat cells (Promega kit CS196005, 0.5 mL) are thawed in a water bath and then added to 5 ml of pre-warmed R1 medium. Twenty-five (25) μL / well of Jurkat cells are then added to a white cell culture plate. Cells and test articles are incubated at 37° C., 5% CO 2 At the end of the 6 hours, 75 μL / well of Bio-Glo™ reagent (Promega) is added and incubated for 10 minutes before reading luminescence on a plate reader (Clariostar, BMG). Fold changes compared to cells alone (Jurkat cells + cell line used in co-culture) are calculated and plotted as log(agonist) vs. response in GraphPad Prism to determine EC 50 (nM) and fold induction over background (Max) are determined.

[0088] The tumor cell types used in the co-culture for EphA2 were A549, PC-3, and HT29.

[0089] The data presented in Figure 1 show that the EphA2 / CD137 heterotandem BCY13272 induces strong CD137 activation in a CD137 reporter assay in the presence of an EphA2-expressing cell line (A549), whereas a non-binding control molecule (BCY13626) shows no CD137 activation.

[0090] The data presented in Figure 1 and Table 1 below demonstrate that BCY13272 induces robust CD137 activation in a CD137 reporter assay. Activation is dependent on heterotandem binding to both CD137 and EphA2, as shown by the lack of activity of a non-binding control (BCY13626) that does not bind either EphA2 or CD137.

[0091] BCY13272-induced ECs in CD137 reporter assays in coculture with EphA2-expressing tumor cell lines50 A summary of the induction values ​​(nM) and fold induction are reported in Table 1 below: Table 1: Activity of EphA2 / CD137 heterotandem bicyclic peptide complexes in the CD137 reporter assay. [Table 4]

[0092] (2. Pharmacokinetics of the heterotandem complex BCY13272 in SD rats) Male SD rats were administered the heterotandem complex BCY13272 formulated in 25 mM histidine HCl, 10% sucrose pH 7 by IV bolus or IV infusion (15 min). Serial blood draws (approximately 80 μL blood / time point) were taken from the submandibular or saphenous vein at each time point. All blood samples were immediately transferred to pre-chilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5 M) as anticoagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at 3000 g at approximately 4° C. Precipitants containing internal standards were immediately added to the plasma, mixed thoroughly, and centrifuged at 12,000 rpm at 4° C. for 10 min. The supernatants were transferred to pre-labeled polypropylene microcentrifuge tubes and then flash frozen on dry ice. Samples were stored below 70° C. until analysis as required. 7.5 μL of the supernatant sample was directly injected for LC-MS / MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentration of the analytes. Plasma concentration versus time data were analyzed by a noncompartmental approach using the Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2 , AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf), and plasma concentration versus time profile graphs were reported. The pharmacokinetic parameters of the study are shown in Table 2: Table 2: Pharmacokinetic parameters in SD rats [Table 5]

[0093] 3. Pharmacokinetics of the heterotandem complex BCY13272 in cynomolgus monkeys Previously treated cynomolgus monkeys were administered 1 mg / kg of the heterotandem complex BCY13272, formulated in 25 mM histidine HCl, 10% sucrose pH 7, via intravenous infusion (15 or 30 min) into the cephalic vein. Serial blood draws (approximately 1.2 ml blood / time point) were taken from a peripheral vein of restrained, non-sedated animals at each time point and were then transferred to potassium (K2) EDTA buffer on wet ice. * 2H 20 (0.85-1.15 mg) and processed for plasma. Samples were centrifuged immediately after collection (3,000 × g, 2-8 °C for 10 min). 0.1 mL of plasma was transferred to a labeled polypropylene microcentrifuge tube. 5x precipitant containing internal standards 100 ng / mL labetalol, 100 ng / mL dexamethasone, 100 ng / mL tolbutamide, 100 ng / mL verapamil, 100 ng / mL glyburide, and 100 ng / mL celecoxib in MeOH was immediately added to the plasma, mixed thoroughly, and centrifuged at 12,000 rpm, 2-8 °C for 10 min. A sample of the supernatant was transferred to a pre-labeled polypropylene microcentrifuge tube and frozen on dry ice. Samples were stored at or below -60 °C until LC-MS / MS analysis. Aliquots of 40 μL of calibration standards, quality control samples, single blank samples, and double blank samples were added to 1.5 mL tubes. Each sample (except double blank) was quenched with 200 μL IS1 respectively (double blank sample was quenched with 200 μL MeOH containing 0.5% tritonX-100), after which the mixture was thoroughly vortexed (at least 15 seconds) and centrifuged at 12000 g for 15 min at 4°C. 10 μL of the supernatant was injected for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analytes. Plasma concentration versus time data were analyzed by a noncompartmental approach using Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2 , AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf), and plasma concentration versus time profile graphs were reported. The pharmacokinetic parameters of BCY13272 are shown in Table 3. Table 3: Pharmacokinetic parameters in cynomolgus monkeys [Table 6]

[0094] Figure 2 shows the plasma concentration versus time curves of BCY13272 obtained from 3.6 mg / kg IV infusion (15 min) in SD rats (n=3) and 9.2 mg / kg IV infusion (15 min) in cynomolgus monkeys (n=3). BCY13272 has a steady-state volume of distribution (Vdss) of 1.0 L / kg in rats and a clearance of 7.5 mL / min / kg resulting in a terminal half-life of 2.9 hours. BCY13272 has a steady-state volume of distribution (Vdss) of 0.82 L / kg in cynomolgus monkeys and a clearance of 4.1 mL / min / kg resulting in a terminal half-life of 8.9 hours.

[0095] 4. Pharmacokinetics of the Heterotandem Complex BCY13272 in CD1 Mice Six male CD-1 mice were administered 15 mg / kg of the heterotandem complex BCY13272 formulated in 25 mM histidine HCl, 10% sucrose pH 7 by intraperitoneal or intravenous administration. Serial blood draws (approximately 80 μL blood / time point) were taken from the submandibular or saphenous vein at each time point. All blood samples were immediately transferred to pre-chilled microcentrifuge tubes containing 2 μL K2-EDTA (0.5 M) as anticoagulant and placed on wet ice. Blood samples were immediately processed for plasma by centrifugation at 3000 g at approximately 4° C. Precipitants containing internal standards were immediately added to the plasma, mixed thoroughly, and centrifuged at 12,000 rpm at 4° C. for 10 min. The supernatant was transferred to pre-labeled polypropylene microcentrifuge tubes and then flash frozen on dry ice. Samples were stored below 70° C. until analysis as required. 7.5 μL of the supernatant sample was directly injected for LC-MS / MS analysis using an Orbitrap Q Exactive in positive ion mode to determine the concentration of the analytes. Plasma concentration versus time data were analyzed by a noncompartmental approach using the Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T 1 / 2 , AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf), and graphs of plasma concentration versus time profiles were reported.

[0096] FIG. 2 shows the plasma concentration versus time curve of BCY13272 resulting from a 5.5 mg / kg IV dose in CD1 mice (n=3); the volume of distribution (Vdss) of BCY13272 is 1.1 L / kg and the clearance is 7.5 mL / min / kg resulting in a terminal half-life of 2.9 hours.

[0097] 5. The EphA2 / CD137 heterotandem bicyclic peptide complex BCY13272 induces IFN-γ cytokine secretion in MC38 co-culture assays. MC38 and HT1080 cell lines were cultured according to the recommended protocol. Frozen PBMCs from healthy human donors were thawed, washed once in room temperature PBS containing benzonase, and then resuspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× penicillin / streptomycin, 10 mM HEPES, and 2 mM L-glutamine (referred to herein as R10 medium). 100 μl of PBMCs (1,000,000 PBMCs / ml) and 100 μl of tumor cells (100,000 tumor cells / ml) (effector:target cell ratio (E:T) 10:1) were plated into each well of a 96-well flat-bottom plate for co-culture assay. 100 ng / ml of soluble anti-CD3 mAb (clone OKT3) was added to the cultures on day 0 to stimulate the human PBMCs. Test articles, control compounds, or vehicle controls were diluted in R10 medium and 50 μL was added to each well for a final volume of 250 μL per well. Plates were covered with breathable film and incubated in 5% CO 2The plates were incubated for 2 days in a humidified chamber at 37°C containing 0.1% ethanol. Supernatants were harvested 24 and 48 hours after stimulation and human IFN-γ was detected by Luminex. Briefly, standards and samples were added to a black 96-well plate. Microparticle cocktail (provided in the Luminex kit, R&D Systems) was added and shaken for 2 hours at room temperature. The plates were washed 3 times using a magnetic holder. Then, biotin cocktail was added to the plate and shaken for 1 hour at RT. The plates were washed 3 times using a magnetic holder. Streptavidin cocktail was added to the plate and shaken for 30 minutes at RT. The plates were washed 3 times using a magnetic holder, resuspended in 100 μL of wash buffer, shaken for 2 minutes at RT and read using a Luminex 2000. Raw data was analyzed using the built-in Luminex software to generate standard curves and interpolate protein concentrations, and all other data analysis and graphing was performed using Excel and Prism software. Data represent one study with PBMC from three independent donors studied in experimental duplicates.

[0098] The data presented in Figure 2 and Table 4 below show that BCY13272 induces robust CD137 activation as evidenced by IFN-γ and IL-2 secretion upon CD3 stimulation. Activation is dependent on heterotandem binding of the CD137 and EphA2 binder to both CD137 and EphA2 as evidenced by the lack of activity of the non-binding controls BCY12762 and BCY13692, respectively, which contain all D-amino acids resulting in non-binding analogs. Table 4: EC50 of IL-2 cytokine secretion induced by EphA2 / CD137 heterotandem bicyclic complex BCY13272 in human PBMC-MC38 / HT-1080 co-culture assay. [Table 7]

[0099] 6. Antitumor Activity of BCY13272 in a Syngeneic MC38 Tumor Model 1 × 10 6 MC38 cells were implanted subcutaneously. The average tumor volume was approximately 80 mm. 3 Upon reaching a tumor mass of 1000 mg / kg, mice were randomized into treatment groups (n=6 / cohort) and treated with vehicle (25 mM histidine, 10% sucrose, pH 7) intravenously (IV), 8 mg / kg BCY13272, 0.9 mg / kg BCY13272, and 0.1 mg / kg BCY13272 IV. All treatments were administered twice weekly (BIW) for a total of six doses. Tumor growth was monitored from treatment initiation until day 28. Complete responder animals (n=7) were followed up until day 62 after treatment initiation and included 2×10 6 MC38 tumor cells and tumor growth was monitored for 28 days. Concurrently, age-matched untreated control huCD137 C57Bl / 6 mice (n=5) were rechallenged with 2×10 6 MC38 tumor cells were implanted and monitored for 28 days.

[0100] The results of this experiment can be seen in Figure 3. In this experiment, it can be seen that BCY13272 produced significant antitumor activity, with complete responses observed at dose levels of 0.9 mg / kg (2 of 6 complete responders) and 8 mg / kg (5 of 6 complete responders) (Figure 3A). Unlike age-matched untreated control huCD137 C57Bl / 6 mice (100% tumor take rate), no tumor regrowth was observed in BCY13272 complete responder animals (Figure 3B). These data indicate that BCY13272 has significant antitumor activity and that BCY13272 treatment can produce immunogenic memory in complete responder animals.

[0101] 7. Binding of BCY13272 to EphA2 and CD137 as Measured by SPR (a) CD137 Biacore experiments were performed to determine the k of heterotandem peptide binding to human CD137 protein. a (M -1 s -1 ), k d (s -1 ), K D (nM) values ​​were determined. Recombinant human CD137 (R&D systems) was resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin Reagent (Thermo Fisher) as per the protocol suggested by the manufacturer. Proteins were desalted and uncoupled biotin was removed into PBS using spin columns.

[0102] For peptide binding analysis, Biacore T200 or Biacore 3000 instruments were used with XanTec CMD500D chips. Streptavidin was immobilized on the chips using standard amine coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as running buffer. Briefly, carboxymethyl dextran sulfate was activated by a 7 min injection of 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min. For streptavidin capture, proteins were diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl over the activated chip surface. The remaining activated groups were blocked by a 7 min injection of 1 M ethanolamine (pH 8.5) and biotinylated CD137 was captured to levels of 270-1500 RU. The buffer was changed to PBS / 0.05% Tween 20 and a dilution series of the peptide was prepared in this buffer with a final DMSO concentration of 0.5%. The highest peptide concentration was 500 nM and six further 2- or 3-fold dilutions were performed. SPR analysis was performed at 25°C with a flow rate of 90 μl / min for 60 s association and 900 s dissociation. After each cycle, a regeneration step (10 μl of 10 mM glycine pH 2) was utilized. Data were corrected for volume effects with the exception of DMSO, as necessary. All data were double-referenced to blank injections and baselines using standard processing procedures and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a simple 1:1 binding model, taking mass transfer effects into account where appropriate.

[0103] (b) EphA2 Biacore experiments were performed to determine the k a (M -1 s -1 ), kd (s -1 ), K D (nM) values ​​were determined.

[0104] EphA2 was biotinylated with EZ-Link™ Sulfo-NHS-LC-Biotin for 1 h in 4 mM sodium acetate, 100 mM NaCl, pH 5.4, containing a 3× molar excess of biotin relative to protein. The reaction mixture was dialyzed into PBS, after which the degree of labeling was determined using a fluorescent biotin quantification kit (Thermo). For peptide binding analysis, a Biacore T200 instrument was used with a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine coupling chemistry at 25° C. with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, carboxymethyl dextran sulfate was activated by a 7 min injection of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) in a 1:1 ratio at a flow rate of 10 μl / min. For streptavidin capture, protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl over the activated chip surface. Remaining activated groups were blocked by a 7 min injection of 1 M ethanolamine (pH 8.5):HBS-N (1:1). The buffer was changed to PBS / 0.05% Tween 20, and biotinylated EphA2 was captured to a level of 500-1500 RU using dilution of protein to 0.2 μM in buffer. A dilution series of the peptide was prepared in this buffer containing a final DMSO concentration of 0.5%, with a maximum peptide concentration of 50 or 100 nM, and six further two-fold dilutions. SPR analysis was performed at 25°C with a flow rate of 90 μl / min, with association for 60 s and dissociation for 900-1200 s. Data were corrected for volume effects excluding DMSO. All data were double-referenced to blank injections and a reference surface using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a simple 1:1 binding model, taking into account mass transfer effects where appropriate.

[0105] Figure 5A shows a sensorgram indicating that BCY13272 binds to EphA2 (human) with an affinity of 2.0 nM. Figure 5B shows a sensorgram indicating that BCY13272 binds to CD137 (human) with high affinity. Due to the presence of two CD137-binding bicycles in BCY13272, the dissociation rate of immobilized CD137 protein is very slow, faster than the reported K D may be an overestimate (Figure 4B). The present application provides the following aspects of the invention. (Aspect 1) (a) a peptide that binds to EphA2 and has the sequence (chemical 1) TIFF0007620002000031.tif7170 a first peptide ligand having an N-(acid-PEG) 3 )-N-Bis(PEG 3 -azide) linker, (b) binds to CD137, both of which have the sequence (Case 2) TIFF0007620002000032.tif7170 two second peptide ligands having the following structure: Conjugated to wherein each of the peptide ligands comprises three reactive cysteine ​​groups (C i 、C ii , and C iii ), and a molecular scaffold which is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and forms a covalent bond with a reactive cysteine ​​group of the polypeptide, such that two polypeptide loops are formed on the molecular scaffold; wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, 1Nal represents 1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, and Nle represents norleucine. (Aspect 2) BCY13272: (3) TIFF0007620002000033.tif124170 2. The heterotandem bicyclic peptide complex of embodiment 1, wherein (Aspect 3) 3. The heterotandem bicyclic peptide complex according to embodiment 1 or embodiment 2, wherein said pharma- ceutically acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt. (Aspect 4) A pharmaceutical composition comprising a heterotandem bicyclic peptide complex according to any one of aspects 1 to 3 in combination with one or more pharma- ceutically acceptable excipients. (Aspect 5) 4. The heterotandem bicyclic peptide conjugate according to any one of embodiments 1 to 3 for use in the prevention, suppression or treatment of cancer. (Aspect 6) A method for treating cancer comprising administering to a subject a heterotandem bicyclic peptide complex according to any one of claims 1 to 3 in vitro EC. 50 said method comprising administering said conjugate at a dosing frequency that does not maintain a plasma concentration of said conjugate greater than

Claims

1. (a) a polypeptide that binds to EphA2 and has the sequence 【Chemistry 1】 a first peptide ligand having an N-(acid-PEG) 3 )-N-Bis(PEG 3 -azide) linker, (b) binds to CD137, both of which have the sequence 【Chemistry 2】 two second peptide ligands having the following structure: or a pharma- ceutically acceptable salt thereof, Each of the peptide ligands contains three reactive cysteine ​​groups (C i , C ii , and C iii ), and a molecular scaffold which is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and forms a covalent bond with a reactive cysteine ​​group of the polypeptide, such that two polypeptide loops are formed on the molecular scaffold; wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, 1Nal represents 1-naphthylalanine, tBuAla represents t-butyl-alanine, PYA represents 4-pentynoic acid, and Nle represents norleucine, or a pharma- ceutical acceptable salt thereof.

2. BCY13272: 【Chemistry 3】 2. The heterotandem bicyclic peptide complex according to claim 1, wherein:

3. 3. The heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof according to claim 1 or 2, wherein the pharma- ceutical acceptable salt is selected from sodium, potassium, calcium, or ammonium salts.

4. A pharmaceutical composition comprising the heterotandem bicyclic peptide complex or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 3 in combination with one or more pharma- ceutically acceptable excipients.

5. A pharmaceutical composition for use in the prevention, suppression, or treatment of cancer, comprising the heterotandem bicyclic peptide complex or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3.

6. A pharmaceutical composition for treating cancer, comprising the heterotandem bicyclic peptide complex according to any one of claims 1 to 3 or a medicamentously acceptable salt thereof, comprising the in vitro EC of said complex. 50 The pharmaceutical composition, wherein the conjugate is administered at a dosage frequency that does not maintain a plasma concentration of the conjugate greater than

Images