EA046294B1 - HETEROTANDEM BICYCLIC PEPTIDE COMPLEX - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to a heterotandem bicyclic peptide complex that includes a first peptide ligand that binds EphA2 conjugated through a linker to two second peptide ligands that bind CD137. The invention also relates to the use of said heterotandem bicyclic peptide complexes for the prevention, suppression or treatment of cancer. The invention also relates to the use of said heterotandem bicyclic peptide complexes for the prevention, suppression or treatment of cancer.

State of the art

Cyclic peptides can bind with high affinity and specificity to target proteins and, as a result, they represent a promising class of molecules for the development of therapeutics. In fact, several cyclic peptides are already successfully used in clinical practice, for example the antibacterial peptide vancomycin, the immunosuppressant cyclosporine or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The high binding ability is due to the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of the cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstroms, such as the cyclic peptide antagonist CXCR4-CVX15 (400 A ² ; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly- motif Asp binding to integrin aVb3 (355 A ² ) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 binding to urokinase-type plasminogen activator (603 A ² ; Zhao et al (2007), J Struct Biol 160 (1), 1-10).

Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss upon binding to targets and resulting in higher binding affinities. Reduced flexibility also leads to blocking of target-specific conformations, increasing binding specificity compared to linear peptides. This effect was demonstrated with a highly active and selective inhibitor of matrix metalloproteinase-8 (MMP-8), which lost its selectivity over other MMPs upon ring opening (Cherney et al. (1998), J Med Chem 41 (11), 1749-51 ). The high binding capacity achieved through macrocyclization is even more pronounced in polycyclic peptides having more than one peptide ring, such as vancomycin, nisin and actinomycin.

Previously, various groups of researchers have linked polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops on synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for preparing promising medicinal compounds, in which said compounds are obtained by coupling cysteine-containing polypeptides to a molecular scaffold, such as, for example, 1,1',1''-(1,3,5-triazinan-1,3,5-triyl) triprop-2-en-1-one (TATA), disclosed in patent documents WO 2019/122860 and WO 2019/122863.

Combinatorial approaches based on phage display have been developed to generate and screen large libraries of bicyclic peptides for binding to targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and patent document WO 2009/098450) . Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six randomly selected amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) were presented on phage and subjected to cyclization by covalently linking cysteine side chains to small molecule (tris(bromomethyl)benzene).

The essence of the invention

According to a first aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:

(a) the first peptide ligand that binds to EphA2 and which has the sequence A-[HArg]-DC ₁ [HyP]LVNPLC ₁₁ LEP[d1Nal]WTC ₁₁₁ (SEQ ID NO: 1; BCY13118) conjugated through the N-( linker acid-PEG3)-N-bis(PEG3-az) with (b) second peptide ligands that bind to CD137, both of which have the sequence Ac-C ₁ [tBuAla]PE[D-Lys(PYA)]PYC ₁₁ FADPY [Nle]C ₁₁₁ -A (SEQ ID NO: 2; BCY8928);

wherein each of said peptide ligands comprises a polypeptide containing three reactive cysteine groups (Ci, Cii and Ciii) separated by two loop sequences, and a molecular scaffold that is 1,1',1''-(1,3,5- triazinan-1,3,5-triyl)triprop-2-en-1one (TATA) and which forms covalent bonds with the reactive cysteine groups of the polypeptide, resulting in the formation of two polypeptide loops on the molecular framework, where Ac represents acetyl, HArg represents homoarginine, HyP is trans-4-hydroxyL-proline, d1Nal is D-1-naphthylalanine, tBuAla is tert-butyl-alanine, PYA is 4-pentinoic acid and Nle is norleucine.

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

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a heterotandem bicyclic peptide complex as defined herein for use in the prevention, suppression or treatment of cancer.

Brief description of drawings

Fig. 1. Analysis of the heterotandem EphA2/CD137 bicyclic peptide complex BCY13272 by performing a Promega CD137 luciferase reporter gene 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 which includes D-amino acids and does not bind EphA2 or CD 137.

Fig. 2. Plasma concentration-time curve of BCY13272 following intravenous administration of a dose of 5.5 mg/kg to CD1 mice (n=3), intravenous infusion of a dose of 3.6 mg/kg (15 min) to SD rats (n=3) and with an intravenous infusion of a dose of 8.9 mg/kg (15 min) to cynomolgus monkeys (n=2). The pharmacokinetic profile for 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.

Fig. 3. Antitumor activity of BCY13272 in the MC38 syngeneic tumor model. (A) Volumes of MC38 tumors during and after treatment with BCY13272. The number of mice with a complete response (CR) on day D28 (and how many mice remain with a complete response on day D62) is indicated in parentheses. BIW: dosing twice a week; IV: intravenous administration. (B) Tumor growth curves of complete responders to BCY13272 and naïve age-matched controls after implantation with MC38 tumor cells. CR: complete response animal.

Fig. 4. BCY13272 induces secretion of the cytokine IFN-γ in a coculture assay of (A) PBMC/MC38 and (B) PBMC/HT29. BCY12762 is a heterotandem bicyclic peptide complex that binds to EphA2 but does not bind to CD137. BCY13692 is a heterotandem bicyclic peptide complex that binds to CD137 but does not bind to EphA2. (C) Graphical relationship between EC ₅₀ (nM) values for BCY13272 and induced IL-2 and IFN-γ secretion in a PBMC co-culture assay with the MC38 cell line (mouse) for 5 PBMC donors and with the HT1080 cell line (human) ) in case of 4 PBMC donors.

Fig. 5. Data from surface plasmon resonance (SPR) studies of the binding of BCY13272 to immobilized (A) EphA2 and (B) CD137.

Detailed Description of the Invention

According to a first aspect of the invention, there is provided a heterotandem bicyclic peptide complex comprising:

(a) the first peptide ligand that binds to EphA2 and which has the sequence A-[HArg]-DC ₁ [HyP]LVNPLC ₁₁ LEP[d1Nal]WTC ₁₁₁ (SEQ ID NO: 1; BCY13118) conjugated through the N-( linker acid-PEG3)-N-bis(PEG3-azide) with (b) second peptide ligands that bind to CD137, both of which have the sequence Ac-C ₁ [tBuAla]PE[D-Lys(PYA)]PYC ₁₁ FADPY [Nle]C ₁₁₁ -A (SEQ ID NO: 2; BCY8928), wherein each of said peptide ligands comprises a polypeptide containing three reactive cysteine groups (C ₁ , C ₁₁ and C ₁₁₁ ) separated by two loop sequences, and a molecular framework , which is 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1one (TATA) and which forms covalent bonds with reactive cysteines groups of the polypeptide, resulting in the formation of two polypeptide loops on the molecular framework, where Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxyL-proline, d1Nal represents D-1-naphthylalanine, tBuAla represents tert-butyl-alanine, PYA represents 4- pentinoic acid and Nle represents norleucine.

Mention in the invention of the linker N-(acid-PEG3)-N-bis(PEG3-azig) includes:

K-(acid-PEO3)-N-bis(PEO3-azide).

In one embodiment, the heterotandem bicyclic peptide complex is BCY13272:

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Details for BCY13272 are given in Table. A below.

Table A

Composition BCY13272

Complex No. BCY No. for EphA2 The point is attached to the IA Linker No.BCY For CD137 The point is attached to the IA BCY13272 BCY13118 N-terminus EC acidPEG ₃ )-N6nc(PEG ₃ azide) BCY8928, BCY8928 dLys(PYA)4

In fig. Figure 3 presents data that demonstrates that BCY13272 produces a significant antitumor effect in the MC38 tumor mouse model.

The invention refers to specific analogs (ie, modified derivatives) and metabolites of BCY13272, each of which form additional aspects of the invention, and data for which are shown in table. B below.

Table B

Composition of labeled analogues and possible metabolites of BCY13272

Complex No. BCY No. for EphA2 The point is attached to the IA Linker No.BCY for CD 137 Point joined!! I Modific ator BCY14414 BCY13118 N-terminus N-(xncnoTa-PEG ₃ )-N-6nc(PEG ₃ azide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 N/A BCY14417 BCY13118 N-terminus N-(xncnoTa-PEG ₃ )-N-6nc(PEG ₃ azide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 Peg12- Biotin

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azide) BCY14418 BCY13118 N-terminus N-(KiicnomaPEG ₃ )-N6hc(PEGjazide) BCY8928 BCY13389 dLys(PYA)4 dLys(PYA)4 Alexa Fluor® 488 BCY15217 BCY13118 N-terminus N-(KiicnomaPEG.0-N6iic(PEGjazide) BCY14601 BCY14601 dLys(PYA)4 dLys(PYA)4 N/A BCY15218 BCY13118 N-terminus N-(Kncnoma- PEG.0-N- 6iic(PEGsazide) BCY8928 BCY14601 dLys(PYA)4 dLys(PYA)4 N/A

where BCY14601 is a bicyclic peptide ligand having the sequence C ₁ [tBuAla]PE[D-Lys(PYA)]PYC ₁₁ FADPY[Nle]C ₁₁₁ -A (SEQ ID NO: 3), with TATA as the molecular framework;

and where BCY13389 is a bicyclic peptide ligand having the sequence [Ac]C ₁ [tBuAla]PE[D-Lys(PYA)]PYC ₁₁ FADPY[Nle]C ₁₁₁ -K (SEQ ID NO: 4), with TATA as the molecular frame.

Unless otherwise noted, all technical and scientific terms used in the present invention have the same meanings as commonly understood by those skilled in the art, for example, peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard methods of molecular biology, genetic and biochemical studies are used (see monographs 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.), the contents of which are incorporated herein by reference.

Nomenclature.

Numbering.

When specifying amino acid residue positions in the compounds of the invention, cysteine residues (Ci, C ₁₁ and C _{1i 1} ) are not numbered because they are invariant, so the numbering of amino acid residues in SEQ 1D NO: 1 is as shown below:

Ci-HyP1-L2-V3-N4-P5-L6-Ci _i -L ₇ -E8-P ₉ -d1Nal10-W11-T1 ₂ -Ciii (SEQ ID NO: 1).

For purposes of the present invention, it is assumed that all bicyclic peptides are cyclized with 1,1',1-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1-one (TATA) with the formation of a trisubstituted structure. Cyclization with TATA occurs at _C1 , _C11 and _C111 .

Molecular format.

N- or C-terminal extensions for the bicycle core sequence are added to the left or right side of the sequence and separated by a hyphen. For example, the N-terminal eAla-Sar10-Ala tail would be designated as:

eAla-Sar10-A-(SEQ ID NO: X).

Reverse peptide sequences.

In light of Nair et al (2003) J Immunol 170(3), 1362-1373, it is believed that the peptide sequences disclosed herein may also find use in their retro-inverse form. For example, the order of a sequence is reversed (that is, the N-terminus becomes the C-terminus and vice versa), and its stereochemistry is likewise reversed (that is, D-amino acids become L-amino acids and vice versa). To avoid misinterpretation, when amino acids are specified, either by their full name or by their one- or three-letter letter code, they are intended to be L-amino acids in the invention unless otherwise stated. If it is intended that such an amino acid should be represented in the invention as a D-amino acid, then, in this case, the amino acid designation will be preceded by a lowercase letter d and the entire designation will be placed in square brackets, for example, [dA], [dD], [ dE], [dK], [d1Nal], [dNle], and so on.

Advantages of peptide ligands.

The particular heterotandem bicyclic peptide complexes of the present invention have a number of advantageous properties that make them suitable drug-like molecules for injection, inhalation, nasal,

- 4 046294 ophthalmic, oral or topical administration. Such preferred properties include:

interspecies cross-reactivity. It is a common requirement for preclinical pharmacodynamic and pharmacokinetic studies;

resistance to proteases. Heterotandem bicyclic peptide complexes should ideally exhibit resistance to plasma proteases, epithelial (membrane-anchored) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases and other similar proteases. Resistance to proteases should be maintained between different species, so that a promising heterotandem bicyclic peptide can be developed in animal models, which can then be used in humans with a high degree of confidence;

required solubility profile. It depends on the ratio of charged and hydrophilic residues to hydrophobic residues and on external/internal molecular hydrogen bonds, which are important from the point of view of drug preparation and absorption;

selectivity. Specific heterotandem bicyclic peptide complexes of the invention exhibit high selectivity in the presence of other targets;

optimal half-life from plasma in the bloodstream. Depending on the clinical indications and treatment regimen, it may be necessary to develop a heterotandem bicyclic peptide complex with a short-term effect in the case of treatment of an acute disease, or the development of a heterotandem bicyclic peptide complex with an increased residence time in the bloodstream, which is therefore optimal for the treatment of chronic diseases . Other factors that determine the required plasma half-life include the requirement for prolonged exposure to achieve maximum therapeutic efficacy, taking into account the accompanying toxicity associated with prolonged exposure to the drug.

Importantly, the invention provides evidence that selected heterotandem bicyclic peptide complexes demonstrate antitumor activity when dosed at a frequency that does not maintain plasma concentrations of the compound above its in vitro EC50 value. This is in contrast to larger biologic (i.e. antibody) approaches for CD137 agonism or bispecific CD137 agonism (Segal et al., Clin Cancer Res., 23(8):1929-1936 (2017), Claus et al. al., Sci Trans Med., 11(496): eaav5989, 1-12 (2019), Hinner et al., Clin Cancer Res., 25(19):5878-5889 (2019)). Without being bound by any theory, it can be assumed that the observed phenomenon is due to the fact that heterotandem bicyclic complexes have a relatively low molecular weight (typically <15 kDa), they are completely synthetic products, and they are targeted CD137 agonists on tumors. As such, they are characterized by relatively short plasma half-lives but high tumor permeability and long tumor residence time. The invention provides data that fully supports these advantages. For example, antitumor activity in syngeneic tumor mouse models with humanized CD137 occurs either daily or every third day. In addition, pharmacokinetic data obtained with intraperitoneal administration indicate a plasma half-life of <3 hours, suggesting a steady decline in circulating concentrations of the complex below the in vitro EC ₅₀ value between dosing administrations. In addition, tumor pharmacokinetic data indicate that levels of the heterotandem bicyclic complex in tumor tissue may be higher and more sustained compared to plasma levels.

It should be noted that this finding forms an important additional aspect of the invention. Thus, according to an additional aspect of the invention, the invention provides a method of treating cancer that includes administering the heterotandem bicyclic peptide complex described herein at a dosing frequency that does not maintain plasma concentrations of said complex above the in vitro EC ₅₀ value for said complex.

Immunological memory.

The combination of a cancer cell-binding bicyclic peptide ligand with an immune cell-binding bicyclic peptide ligand provides a synergistic beneficial effect on immunological memory. The heterotandem bicyclic peptide complex of the invention is believed to not only kill tumors, but upon repeated administration of the tumor-inducing agent, none of the inoculated mice with a complete response developed tumors. This indicates that treatment with the selected heterotandem bicyclic peptide complexes of the invention induced immunological memory in mice with a complete response. This is a significant clinical advantage in terms of preventing the recurrence of the tumor after it has initially been treated with antitumor therapy and destroyed.

Peptide ligands.

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A peptide ligand referred to in the invention refers to a peptide covalently linked to a molecular framework. Typically, such peptides include two or more reactive groups (i.e., cysteine residues) that are capable of forming covalent bonds with the backbone, and a sequence located between these reactive groups, which is called a loop sequence because it forms a loop when binding of the peptide to the scaffold. Here, the peptides include at least three reactive groups selected from cysteine, 3-mercaptopropionic acid and/or cysteamine, and form at least two loops on the framework.

Pharmaceutically acceptable salts.

It should be understood that salt forms are included within the scope of the present invention, and references to peptide ligands include salt forms of said ligands.

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

Acid addition salts (mono- or di-salts) can be formed with a wide range of acids, both inorganic and organic. Examples of salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (eg, L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butane , (+)-camphor, camphorsulfonic, (+)-(U)-camphor-10sulfonic, capric, capronic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic , fumaric, mucus, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydroiodic) , isethionic, lactic (for example, (+)-b-lactic, (±)-DL-lactic), lactobionic, maleic, apple, (-)-apple, malonic, (±)-DL-almond, methanesulfonic, naphthalene- 2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamic, phosphoric, propionic, pyruvic, L-pyroglutamine, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-G-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, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalene sulfonic , valeric, propane, butanoic, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride. Another specific salt is acetate.

If the compound is anionic or has a functional group that can be anionic (for example, -COOH can be in the form of the anion -COO-), then the salt can 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 Ca ²⁺ and Mg2 ⁺ , and other cations such as Al ³⁺ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (ie, NH4 ⁺ ) and substituted ammonium ions (eg, NH3R ⁺ , NH2R2+, NHR3 ⁺ , NR4 ⁺ ). Examples of some suitable substituted ammonium ions are those formed 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 typical quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

When the compounds of the invention contain amine functionality, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent in accordance with methods well known to one skilled in the art. Such quaternary ammonium compounds are within the scope of the invention.

Modified derivatives.

It should be understood that the modified peptide ligand derivatives described 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 modi

- 6 046294 fications; replacing one or more amino acid residues with one or more unnatural amino acid residues (for example, replacing one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacing one or more non-polar amino acid residues with other unnatural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacing one or more amino acid residues with alanine, replacing 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; replacing one or more peptide bonds with a surrogate bond; modification of the length of the peptide main chain; replacing the hydrogen on the alpha carbon of one or more amino acid residues with another chemical group, modifying amino acids such as cysteine, lysine, glutamate/aspartate, and tyrosine with suitable amines, thiols, carboxylic acids, and phenol-reactive reagents to functionalize said amino acids, and introducing or substitutions with amino acids that introduce orthogonal reactive groups that are suitable for functionalization, for example, amino acids bearing azide or alkyne groups, which allow functionalization with alkyne or azide-bearing moieties, respectively.

In one embodiment, the modified derivative includes an N-terminal and/or C-terminal modification. In a further embodiment, wherein the modified derivative comprises N-terminal modification using suitable chemistry with an amino group, and/or C-terminal modification using suitable chemistry with a carboxyl group. In a further embodiment, said N-terminal or C-terminal modification includes the introduction of an effector group, including, but not limited to, a cytotoxic agent, a radiochelating agent, or a chromophore.

In a further embodiment, the modified derivative includes an N-terminal modification. In a further embodiment, the N-terminal modification includes an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (a group referred to herein as Ci) is blocked with acetic anhydride or other suitable reagents during peptide synthesis, resulting in the formation of a molecule with an acetylated N-terminus. This embodiment provides the advantage of removing a potential aminopeptidase recognition site and preventing possible degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification includes the introduction of a molecular spacer group that facilitates the conjugation of effector groups and helps maintain the activity of the bicyclic peptide towards its target.

In a further embodiment, the modified derivative includes a C-terminal modification. In a further embodiment, the C-terminal modification includes an amide group. In this embodiment, the C-terminal cysteine group (referred to in the invention as C _iii ) is synthesized in the amide form during peptide synthesis, resulting in the formation of a molecule with an amidated C-terminus. This embodiment provides the benefit of removing a potential carboxypeptidase recognition site and reducing the likelihood of proteolytic degradation of the bicyclic peptide.

In one embodiment, the modified derivative includes replacing one or more amino acid residues with one or more unnatural amino acid residues. In this embodiment, unnatural amino acids may be selected having isosteric/isoelectronic side chains that are not recognized by degradative proteases and do not have any adverse effect on target efficacy.

Alternatively, unnatural amino acids may be used that have sterically hindered amino acid side chains, resulting in conformationally and sterically hindering proteolytic hydrolysis of the adjacent peptide bond. In particular, they refer to proline analogues, bulky side chains, Ca-disubstituted derivatives (eg aminoisobutyric acid, Aib) and cyclic amino acids, the simple derivative of which is aminocyclopropylcarboxylic acid.

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

In one embodiment, the modified derivative includes replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-stable amino acid residues. In another embodiment, the modified derivative includes replacing the tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the benefit of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

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In one embodiment, the modified derivative includes replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative includes replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. A favorable balance between charged and hydrophobic amino acid residues is an important characteristic of bicyclic peptide ligands. For example, hydrophobic amino acid residues influence the degree of binding to plasma proteins and, thus, the concentration of the available free fraction in plasma, while charged amino acid residues (in particular, arginine) can influence the interaction of the peptide with phospholipid membranes on the cell surface. These two types of residues in combination can influence the half-life, volume of distribution and exposure of the peptide drug, and can be selected according to the desired clinical outcome. In addition, the favorable combination and number of charged amino acid residues relative to hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

In one embodiment, the modified derivative includes replacing one or more L-amino acid residues with one or more D-amino acid residues. In this embodiment, it is believed that such a substitution allows for increased proteolytic stability due to steric hindrance and due to the ability of the D-amino acid to stabilize conformational isomerism (Tugyi et al (2005) PNAS, 102(2), 413-418).

In one embodiment, the modified derivative includes removing any amino acid residues and replacing them with alanines. This embodiment provides the benefit of removing potential site(s) of proteolytic attack.

It should be noted that each of the modifications mentioned above is intended to increase the activity or stability of the peptide in a intended manner. Additional increases in activity through modifications can be achieved based on the following mechanisms:

introduction of hydrophobic moieties, which provide a hydrophobic effect and lead to lower dissociation rates, resulting in higher degrees of affinity;

introduction of charged groups that provide long-range ionic interactions, resulting in higher rates of association and higher degrees of affinity (see, for example, Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3 , 427-31); and introducing additional degrees of freedom restrictions into the peptide by, for example, properly selecting the degrees of freedom restrictions of the amino acid side chains, thereby minimizing the loss of entropy upon binding to the target, limiting the degrees of freedom of the torsion angles of the main chain, thereby minimizing the loss of entropy upon binding to the target, and by introducing additional rings into the molecule for the same reasons (for a review, see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

Isotopic options.

The present invention includes all pharmaceutically acceptable (radio)isotopically labeled peptide ligands of the invention in which 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 typically found in nature, and peptide ligands of the invention to which are attached metal chelating groups (called effectors) that are capable of retaining the corresponding (radioactive) isotopes, and peptide ligands of the invention in which certain functional groups are covalently replaced by the corresponding (radioactive) isotopes or isotopically labeled functional groups .

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

The specific isotopically labeled peptide ligands of the invention, such as those incorporated with a radioactive isotope, may be used in tissue distribution studies of drug compounds and/or substrates, as well as for clinical assessment of the presence and/or absence of the nectin-4 target in diseased tissues . The peptide ligands of the invention may additionally have valuable diagnostic properties whereby they can be used to detect or identify the formation of a complex between a labeled compound and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods may use compounds that are labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (eg, luminol, luminol derivatives, luciferin, aequorin and luciferase), and so on. In particular, for this purpose the following can be used:

- 8 046294 dioactive isotopes of tritium, that is, ³ H (T), and carbon-14, that is, ¹⁴ C, due to the ease of their administration and the availability of means for their detection.

Substitution with heavier isotopes such as deuterium, i.e. ^2H (D), may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dose requirement, and may therefore be preferable in some circumstances.

Substitution with positron-active isotopes such as 11C, ^18F , ^15O and ^13N can be used in positron emission tomography (PET) studies to determine the degree of target occupancy.

The isotope-labeled peptide ligand compounds of the invention can generally be prepared by conventional methods known to those skilled in the art, or by methods similar to those described in the examples herein, by using an appropriate isotope-labeled reagent in place of the previously used unlabeled reagent.

Synthesis.

The peptides of the present invention can be prepared synthetically by standard methods followed by reaction with a molecular scaffold in vitro. Once this process is complete, standard chemistry techniques can be used. This allows you to quickly obtain large quantities of soluble material for subsequent experiments or validation. Such methods can be carried out using traditional chemical techniques, such as those described in the above-mentioned publication by Timmerman et al.

Therefore, the invention also relates to the production of polypeptides or conjugates selected as specified in the invention, where the production includes optional additional steps explained below. In one embodiment, these steps are carried out on the final polypeptide/conjugate product obtained by chemical synthesis.

Amino acid residues in the polypeptide of interest may optionally be substituted to form a conjugate or complex.

Peptides can also be extended, for example to introduce another loop and therefore achieve multiple specificities.

To extend a peptide, it can simply be extended chemically at its N-terminus or C-terminus or within loops using orthogonally protected lysines (and their analogues) using standard solid-phase or solution synthesis methods. Standard (bio)conjugation techniques can be used to introduce an activated or activated N- or C-terminus. Alternatively, additions can be made by fragmentary condensation or native chemical ligation, for example, 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 USA. 1994 Dec 20; 91(26):12544-8 or Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003.

Alternatively, the peptides can be extended or modified by further conjugation through the use of disulfide bonds. This approach has the additional advantage that it allows the first and second peptides to be separated from each other immediately after entering the reducing environment of the cell. In this case, a molecular scaffold (eg TATA) can be added during the chemical synthesis of the first peptide in order to react with the three cysteine groups; an additional cysteine or thiol can then be attached to the N- or C-terminus of the first peptide, whereby this cysteine or thiol reacts only with the free cysteine or thiol of the second peptides, forming a disulfide-linked bicyclic peptide-peptide conjugate.

Similar methods are equally applicable to the synthesis/linking of two bicyclic and bispecific macrocycles, allowing the creation of tetraspecific molecules.

In addition, the addition of other functional groups or effector groups can be accomplished in a similar manner by using appropriate chemistry, addition at the N- or C-termini, or through side chains. In one embodiment, the attachment is carried out in such a way that it does not block the activity of any 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.

In general, the peptide ligands of the present invention will be used in purified form together with pharmacologically acceptable excipients or carriers. Typically, these excipients or carriers include aqueous or alcohol/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Media for parenteral administration include sodium chloride solution, Ringer's dextrose solution,

- 9 046294 dextrose and sodium chloride and lactated Ringer's solution. Suitable physiologically acceptable excipients, if it is necessary to maintain the polypeptide complex in the form of a suspension, can be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

IV media include fluid and nutrient replenishment agents and electrolyte replenishment agents, such as dextrose Ringer's solutions. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

The peptide ligands of the present invention can be used in the form of separately administered compositions or in combination with other drugs. These may include antibodies, antibody fragments and various immunotherapy drugs such as cyclosporine, methotrexate, adriamycin or cisplatin, as well as immunotoxins. Pharmaceutical compositions may include cocktails of various cytotoxic or other drugs in combination with protein ligands of the present invention or even in combination with selected polypeptides of the present invention having different specificities, such as polypeptides selected using different target ligands, regardless of whether they are combined or not combined before administration.

The method of administration of the pharmaceutical compositions of the invention may be any of the methods well known to those skilled in the art. For the purpose of therapy, the peptide ligands of the invention can be administered to any patient by standard methods. Administration may be by any suitable route, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, inhalation, or, if necessary, direct infusion through a catheter. It is preferable to administer the pharmaceutical compositions of the invention by inhalation. The dose and frequency of administration will depend on the age, gender and condition of the patient, simultaneous administration of other drugs, contraindications and other parameters that must be taken into account by the attending physician.

The peptide ligands of the present invention can be lyophilized for storage and then dispersed in a suitable medium before use. This approach has been shown to be effective and lyophilization and dispersion techniques known in the art can be used. It will be apparent to those skilled in the art that lyophilization and dispersion may result in varying degrees of loss of activity, and that levels may need to be adjusted upward to compensate for these losses.

Compositions containing the peptide ligands of the present invention or a cocktail thereof can be administered for the purpose of prophylactic and/or therapeutic treatment. In some therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of the population of selected cells is defined as a therapeutically effective dose. The amounts required to achieve this dose will depend on the severity of the disease and the general condition of the patient's own immune system, and they usually range from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.0. 05 to 2.0 mg/kg/dose. For prophylactic purposes, compositions containing the peptide ligands of the present invention or mixtures thereof may also be administered in similar or slightly lower doses.

A composition containing a peptide ligand of the present invention can be used for prophylactic and therapeutic purposes to promote the modification, inactivation, destruction or removal of a selected population of target cells in a mammal. In addition, the peptide ligands described herein can be used in vitro or selectively in vitro to kill, deplete, or otherwise effectively remove a population of target cells from a heterogeneous set of cells. The mammal's blood can be combined extracorporeally with selected peptide ligands, causing unwanted cells to be killed or otherwise removed from the blood, which is then returned to the mammal using standard methods.

Therapeutic use.

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a heterotandem bicyclic peptide complex as defined herein for use in the prevention, suppression or treatment of cancer.

Examples of cancers (and their benign counterparts) that may be treated (or suppressed) include, but are not limited to, tumors of epithelial origin (adenomas and carcinomas of various types, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas and other carcinomas), such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including carcinomas of the esophagus, stomach (gastric), small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gallbladder and biliary system , exocrine part of the pancreas, by

- 10 046294 check, lung (for example, adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchoalveolar carcinomas and mesotheliomas), head and neck carcinomas (for example, cancer of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, cavity nose and paranasal sinuses), ovaries, fallopian tubes, abdomen, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (eg, follicular thyroid carcinoma), adrenal glands, prostate, skin and adjacent organs (eg, melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (eg, leukemias, lymphomas) and premalignant and borderline hematologic disorders, including hematologic malignancies and associated lymphoid cell conditions (eg, acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T cell lymphomas and leukemias, natural killer cell [NK] lymphomas, Hodgkin lymphomas, hairy cell leukemia, monoclonal gammopathy unknown origin, plasmacytoma, multiple myeloma, and lymphoproliferative disorders after transplantation), as well as hematologic malignancies and related myeloid cell conditions (eg, acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplasia syndrome and promyelocytic leukemia); tumors of mesenchymal origin, for example, sarcomas of soft tissue, bone or cartilage, such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histocytomas and dermatofibrosarcoma protuberance; tumors of the central or peripheral nervous system (eg, astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (eg, pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary thyroid carcinoma); tumors of the eyes and their appendages (for example, retinoblastoma); germ cell and trophoblast tumors (eg, teratomas, seminomas, dysgerminomas, hydatidiform mole and choriocarcinoma); and pediatric and embryonal tumors (eg, medulloblastomas, neuroblastomas, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, that make the patient susceptible to the development of a malignancy (eg, xeroderma pigmentosum).

In a further embodiment, the cancer is selected from hematological malignancies such as non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B-cell chronic lymphocytic leukemia (B-CLL), B-cell and T-cell acute lymphocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL) and chronic myeloid leukemia (CML).

As used herein, the term prevention includes administration of the protective composition before the onset of disease. Suppression refers to the administration of the composition after the event leading to the onset of the disease, but before the clinical manifestation of the disease. Treatment involves administering the protective composition after the onset of symptoms of the disease.

Animal model systems are known and available that can be used to screen for the effectiveness of peptide ligands in preventing or treating disease. The present invention facilitates the use of animal model systems, allowing the development of polypeptide ligands that can cross-react with human and animal targets, thereby enabling the use of animal models.

The invention is further described below using the examples below.

Examples

In most cases, the heterotandem bicyclic peptide complex of the invention can be prepared by the following general method:

- 11 046294

All solvents are degassed and purged with N2 3 times. A solution of BP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) in DMF was mixed for 5 min, then Bicycle 1 (1.2 eq) was added. The reaction mixture was stirred at 40°C for 16 hours. The reaction mixture was then concentrated under reduced pressure to remove the solvent and purified by preparative HPLC to obtain intermediate 2.

A mixture of intermediate 2 (1.0 eq) and bicycle 2 (2.0 eq) was dissolved in t-BuOH/H ₂ O (1:1), and then CuSO ₄ (1.0 eq), VcNa (4, 0 equiv) and THPTA (2.0 equiv). Finally, 0.2 M NH ₄ HCO ₃ is added to adjust the pH to 8. The reaction mixture is stirred at 40°C for 16 hours under N2. The reaction mixture is directly purified by preparative HPLC.

More detailed descriptions of experiments for the preparation of the heterotandem bicyclic peptide complex of the invention are given below.

Example 1. Synthesis of BCY13272.

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Method for obtaining BCY14964.

A mixture of BP-23825 (155.5 mg, 249.40 µmol, 1.2 eq) and HATU (95.0 mg, 249.92 µmol, 1.2 eq) was dissolved in NMP (1.0 ml), then The pH of this solution was adjusted to 8 by dropwise addition of DIEA (64.6 mg, 499.83 µmol, 87.0 µl, 2.4 eq), and then the solution was stirred at 25°C for 5 min. BCY13118 (500.0 mg, 207.83 μmol, 1.0 eq) was dissolved in NMP (5.0 ml), and then added to the reaction solution, the pH of the resulting solution was adjusted to 8 by dropwise addition of DIEA. The reaction mixture was stirred at 25°C for 45 minutes. LC-MS analysis indicated that BCY13118 was completely reacted and one major peak was detected at the desired m/z value. The reaction mixture was concentrated under reduced pressure to remove the solvent to leave 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 value: 3011.53, observed m/z values: 1506.8 ([M+2H]2+), 1005.0 ([M+3H] ³⁺ ).

Method for obtaining BCY13272.

Ν ₃ ^ -O.

Ό'

Ν· •S.

.N

Oh, Ο. L.,BCY13118

Η

CuSO ₄ -6H ₂ O VcNa THPTA

RC Υ892 8 ----------------------t-BuOH/O 2 M NH ₄ HCO ₃ (1:1)

BCY14964

BCY8928 ,O.

Ν-^ _ο ·

-ABOUT.

ABOUT

A..vsU131-1v Ν n θ Ο BCY13272

Mixture of BCY8928 (644.0 mg, 290.55 µmol, 2.5 eq), THPTA (50.5 mg, 116.22 µmol, 1.0 eq), CuSO ₄ (0.4 M, 145.0 µl, 0.5 equiv) and sodium ascorbate (82.0 mg, 464.89 µmol, 4.0 equiv) were dissolved in t-BuOH/0.2 M NH4HCO ₃ (1:1, 6.0 ml). The pH of this solution was adjusted to 7.5 by adding dropwise 0.2 M NH ₄ HCO ₃ (in 1:1 t-BuOH/0.2 M NH4HCO3), and then the solution was stirred at 25°C for 3 minutes. BCY14964 (350.0 mg, 116.22 µmol, 1.0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 11.0 ml), and then added dropwise to the stirred solution. All solvents were previously degassed and purged with N2. The pH of this solution was adjusted to 7.5 by adding dropwise 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3) and the solution turned yellow. The reaction mixture was stirred at 25°C for 6 h under N2 atmosphere. LC-MS analysis indicated that a major product peak at the desired m/z value was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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 value: 7446.64, observed m/z values: 1242.0 ([M+6H]6+), 1491.0 ([M+5H] ⁵⁺ ).

- 13 046294

Example 2. Synthesis of BCY14414.

CuSO ₄ -5H ₂ O VcNa THPTA t-BuOH/O.2 M NH ₄ HCO ₃ (1:1)

Method for obtaining BCY14798.

N ₃

A mixture of BCY14964 (55.0 mg, 18.26 µmol, 1.0 eq), BCY8928 (32.4 mg, 14.61 µmol, 0.8 eq) and THPTA (39.8 mg, 91.32 µmol, 5 .0 eq) was dissolved in t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 ml, previously degassed and purged with N2), and then CuSO ₄ (0.4 M, 23.0 µl) was added , 0.5 eq) and sodium ascorbate (72.0 mg, 365.27 µmol, 20.0 eq) under N ₂ atmosphere. The pH of this solution was adjusted to 7.5 by adding dropwise 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3), and the solution turned light yellow. The reaction mixture was stirred at 25°C for 1.5 h under N2 atmosphere. LC-MS analysis indicated that BCY14964 remained, BCY8928 was fully reacted, and one major peak was detected at the desired m/z value. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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 value: 5229.07, observed m/z values: 1308.3 ([M+4H]4+), 1046.7 ([M+5H] ⁵⁺ ).

- 14 046294

Method for obtaining BCY14414.

A mixture of BCY14798 (21.0 mg, 4.02 µmol, 1.0 eq), BCY13389 (10.0 mg, 4.42 µmol, 1.1 eq) and THPTA (1.8 mg, 4.02 µmol, 1 .0 eq) was dissolved in t-BuOH/0.2 M NH ₄ HCO ₃ (1:1, 0.5 ml, previously degassed and purged with N2), and then CuSO ₄ (0.4 M, 5 .0 µl, 0.5 equiv) and sodium ascorbate (2.8 mg, 16.06 µmol, 4.0 equiv) under N2 atmosphere. The pH of this solution was adjusted to 7.5 by adding dropwise 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3), and the solution turned light yellow. The reaction mixture was stirred at 25°C for 2 hours under N2 atmosphere. LC-MS analysis indicated that BCY14798 was completely reacted, some BCY13389 remained, and one major peak was detected at the desired m/z value. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC 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⁺ ).

Example 3. Synthesis of BCY14417.

- 15 046294

Method for obtaining BCY14417,

A mixture of BCY14414 (13.0 mg, 1.73 µmol, 1.0 eq) and 6uotuh-PEG12-NHS ester (CAS 365441-71-0, 4.2 mg, 4.50 µmol, 2.6 eq) was dissolved in DMF (0.5 ml). The pH of this solution was adjusted to 8 by adding dropwise DIEA. The reaction mixture was stirred at 25°C for 0.5 h. LC-MS analysis indicated that BCY14414 was completely reacted and one major peak was detected at the desired m/z value. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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 value: 8329.74, observed m/z values: 1389.6 ([M+6H] ⁶⁺ ), 1191.9 ([M+7H] ⁷⁺ ).

Example 4. Synthesis of BCY14418.

- 16 046294

Method for obtaining BCY14418.

A mixture of BCY14414 (5.6 mg, 0.75 µmol, 1.0 eq) and Alexa fluor® 488 (0.9 mg, 1.49 µmol, 2.0 eq) was dissolved in DMF (0.3 ml). The pH of this solution was then adjusted to 8 by adding DIEA dropwise. The reaction mixture was stirred at 25°C for 1.0 h. LC-MS analysis indicated that BCY14414 was completely reacted and one major peak was detected at the desired m/z value. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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⁺ ).

Example 5. Synthesis of BCY15217.

- 17 046294

Method for obtaining BCY15217.

A mixture of BCY14964 (20.0 mg, 6.64 µmol, 1.0 eq), BCY14601 (30.5 mg, 13.95 µmol, 2.1 eq) and THPTA (2.9 mg, 6.64 µmol, 1 .0 eq) was dissolved in a mixture of t-BuOH/0.2 M NH4HCO3 (1:1, 0.5 ml, previously degassed and purged with N ₂ ), and then CuSO ₄ (0.4 M, 16.6 µl, 1.0 equiv) and sodium ascorbate (4.7 mg, 26.56 µmol, 4.0 equiv) under N2 atmosphere. The pH value of this solution was adjusted to 8, and the solution acquired a light yellow color. The reaction mixture was stirred at 25°C for 2 hours under N2 atmosphere. LC-MS analysis indicated that BCY14964 remained and one major peak was detected at the desired m/z value. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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 +).

Example 6. Synthesis of BCY15218.

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Method for obtaining BCY15218.

CuSO ₄ -5H ₂ O VcNa ΤΗΡΤΑ

BCY14601 ----------------------t-BuOH/O 2 Μ NH ₄ HCOj(1 1)

A mixture of BCY14798 (30.0 mg, 5.74 µmol, 1.0 eq), BCY14601 (15.0 mg, 6.88 µmol, 1.2 eq) and THPTA (2.5 mg, 5.74 µmol, 1 .0 eq) was dissolved in a mixture of t-BuOH/0.2 M NH ₄ HCO ₃ (1:1, 0.5 ml, previously degassed and purged with N ₂ ), and then CuSO ₄ (0.4 M, 14.0 µl, 1.0 equiv) and sodium ascorbate (4.0 mg, 22.95 µmol, 4.0 equiv) under N2 atmosphere. The pH of this solution was adjusted to 7.5 by adding dropwise 0.2 M NH4HCO3 (in 1:1 t-BuOH/0.2 M NH4HCO3) and the solution turned yellow. The reaction mixture was stirred at 25°C for 2 hours under N2 atmosphere. LC-MS analysis indicated that BCY14798 was completely reacted, leaving BCY14601, and one major peak was detected at the desired m/z value. The reaction mixture was filtered and concentrated under reduced pressure to obtain 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+).

Analysis data

The heterotandem bicyclic peptide complex according to the invention was analyzed by mass spectrometry and high performance liquid chromatography (HPLC). High performance liquid chromatography analyzes were performed under the following conditions:

Mobile phase: A: 0.1%TFA in H ₂ OB: 0.1%TFA in ACN

Flow: 1.0 ml/min

Column: Kintex 1.7 µm C18 100A 2.1 mm x 150 mm

Instrument: Agilent UPLC 1290

A 30-60% B gradient was used over 10 min and the following data were obtained.

Complex No. Mass spectroscopy analysis data Retention time (min) by HPLC BCY13272 Calculated MW: 7102.28, observed m/z: 1776.4 [M+4HJ4+, 1421.3 [M+5H]+ 7.07

Biological activity data

1. Reporter analysis for CD137 in co-culture with tumor cells.

Culture medium, called R1 medium, is prepared by adding 1% FBS to RPMI-1640 (promega kit component CS196005). Serial dilutions of test samples in R1 are prepared in a sterile 96-well plate. Add 25 µl per well of test samples or R1 (as background control) to the indicated wells in a white cell culture plate. Tumor cells* are collected and resuspended in R1 medium at a concentration of 400,000 cells/ml. Twenty-five (25) µl/well of tumor cells is 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 prewarmed R1 medium. Twenty-five (25) µl/well of Jurkat cells is then added to a white cell culture plate. Incubate cells and test samples for 6 hours at 37°C, 5% CO ₂ . After 6 hours, add 75 μl/well of Bio-Glo™ reagent (Promega) and incubate for 10 minutes, then read the luminescence on a plate reader (Clariostar, BMG). Calculate the fold change relative to individual cells (Jurkat cells + cell line used in co-culture) and plot the response versus the logarithm of the concentration (agonist) using GraphPad Prism software to determine the EC ₅₀ value (nM) and the fold induction relative background (Max).

The tumor cells used in co-culture for EphA2 were A549, PC-3 and HT29.

The data presented in Fig. 1 show that the EphA2/CD137 heterotandem BCY13272 induces strong activation of CD137 in a CD137 reporter assay in the presence of EphA2 expression

- 19 046294 binding cell line (A549), while the non-binding control molecule (BCY13626) does not activate CD137.

The data presented in Fig. 1 and in table. 1 below show that BCY13272 induces high CD137 activation in a CD137 reporter assay. Activation is dependent on heterotandem binding to both CD137 and EphA2, as demonstrated by the lack of activation in the case of a nonbinding control (BCY13626), which does not bind EphA2 or CD137.

Data on the EC ₅₀ value (nM) and the fold of induction caused by BCY13272, obtained from a reporter assay for CD137 in a co-culture with an EphA2-expressing tumor cell line, are given in Table. 1 below.

Table 1

Activity of EphA2/CD137 heterotandem bicyclic peptide complexes in CD137 reporter assay

Complex No. Cell line expressing EphA2 ECzo (nM) Etah Geometric mean value EC50/cell line BCY13272 RS-3 0.245 44.5 0.117 0.0805 44.2 0.0898 53 A549 0.1468 25.7 0.127 0.107 23.6 0.132 30.2 NT-29 0.567 36.5 0.279 0.187 26 0.205 36.4

2. Study of the pharmacokinetics of the heterotandem complex BCY13272 in SD rats.

Male SD rats were each administered a heterotandem bicyclic peptide complex prepared in 25 mM histidine HCl, 10% sucrose, pH 7, by intravenous bolus or intravenous drip (15 min). Sequential blood sampling (approximately 80 μl of blood/time point) was 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 of K2-EDTA (0.5 M) as an anticoagulant and placed on natural ice. Blood samples were immediately processed to obtain plasma by centrifugation at approximately 4°C at 3000 g. The precipitant containing the internal standard was quickly added to the plasma, mixed well and centrifuged at 12,000 rpm, 4°C for 10 minutes. The supernatant was transferred to pre-labeled polypropylene microcentrifuge tubes and then quickly frozen on dry ice. Samples were stored at -70°C or below in required quantities until analysis. 7.5 μL supernatant samples were directly injected into an Orbitrap Q Exactive liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument in positive ion mode to determine concentrations in the sample being analyzed. Plasma concentration-time analysis was performed according to non-compartmental methods using Phoenix WinNonlin 6.3 software. C0, Cl, Vdss, T/ ₂ , AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) were recorded and plasma concentration versus time curves were plotted. The pharmacokinetic parameters obtained in this experiment are given in table. 2.

table 2

Pharmacokinetic parameters in SD rats

Connection Dosing method T1/2(h) Vdss (l/kg) С1р (ml/min/kg) BCY13272 intravenously 2.5 1.0 7.4

3. Study of the pharmacokinetics of the heterotandem complex BCY13272 in cynomolgus macaques

Naive cynomolgus monkeys were administered by intravenous infusion (15 or 30 min) into the saphenous vein with 1 mg/kg heterotandem complex BCY13272, prepared in 25 mM histidine HCl, 10% sucrose, pH 7. Serial blood samples were collected (approximately 1.2 ml blood/ moment of time)

- 20 046294 from the peripheral blood vessel of immobilized animals not exposed to sedatives, at each time into commercially produced test tubes containing potassium salt (K2) EDTA*2H2O (0.85-1.15 mg), on natural ice and subjected processing to obtain plasma. Samples were centrifuged (3000 x g for 10 min at 2 to 8°C) immediately after sampling. Plasma in a volume of 0.1 ml was transferred into labeled polypropylene microcentrifuge tubes. A 5-fold amount of precipitant was quickly added to the plasma, including an internal standard of 100 ng/ml labetalol + 100 ng/ml dexamethasone + 100 ng/ml tolbutamide + 100 ng/ml verapamil + 100 ng/ml glyburide + 100 ng/ml celecoxib in solution plasma in MeOH, mixed well and centrifuged at 12,000 rpm for 10 min at a temperature of 2 to 8°C. Supernatant samples were transferred to pre-labeled polypropylene microcentrifuge tubes and frozen on dry ice. Samples were stored at -60°C or lower until LC-MS/MS analysis. A 40 µl aliquot of the calibration standard, quality control, single blank and double blank samples was added to a 1.5 ml tube. Each sample (except the double blank) was quenched with 200 µl IS1, respectively (the double blank was quenched with 200 µl MeOH with 0.5% triton X-100), and the mixture was then mixed well using a Vortex mixer (at least , 15 s) and centrifuged for 15 min at 12000 g at 4°C. 10 μL samples of the supernatant were injected into an Orbitrap Q Exactive liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument in positive ion mode to determine concentrations in the sample being analyzed. Plasma concentration-time analysis was performed according to non-compartmental methods using Phoenix WinNonlin 6.3 software. C0, Cl, Vdss, T/ ₂ , AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) and plasma concentration versus time curves were recorded. Pharmacokinetic parameters for BCY13272 are shown in Table. 3.

Table 3

Pharmacokinetic parameters in the cynomolgus monkey

Compound Dosing method T1/2(4) С1р (ml/min/kg) Vdss (l/c) BCY13272 intravenous infusion (15 min) 8.9 4.1 0.82

In fig. Figure 2 shows the plasma concentration-time curve for BCY13272 following an intravenous infusion of 3.6 mg/kg (15 min) in SD rats (n=3) and an intravenous infusion of 9.2 mg/kg (15 min) in cynomolgus monkeys (n=3). ). BCY13272 has a steady-state volume of distribution (Vdss) of 1.0 L/kg and a clearance of 7.5 mL/min/kg in rats, resulting in a terminal half-life of 2.9 hours. BCY13272 has a steady-state volume of distribution (Vdss ) 0.82 L/kg and a clearance of 4.1 ml/min/kg in cynomolgus monkeys, resulting in a terminal half-life of 8.9 hours.

4. Study of the pharmacokinetics of the BCY13272 heterotandem complex in CD1 mice.

Six male CD-1 mice were each administered intraperitoneally or intravenously with 15 mg/kg heterotandem complex BCY13272 prepared in 25 mM histidine HCl, 10% sucrose, pH 7. Serial blood samples (approximately 80 μl of blood/time point) were collected from the submandibular or subcutaneous veins at any given time. All blood samples were immediately transferred to pre-chilled microcentrifuge tubes containing 2 μl of K2-EDTA (0.5 M) as an anticoagulant and placed on natural ice. Blood samples were immediately processed to obtain plasma by centrifugation at approximately 4°C at 3000 g. The precipitant containing the internal standard was quickly added to the plasma, mixed well and centrifuged at 12,000 rpm, 4°C for 10 min. The supernatant was transferred to pre-labeled polypropylene microcentrifuge tubes and then quickly frozen on dry ice. Samples were stored at -70°C or below in required quantities until analysis. 7.5 μL supernatant samples were directly injected into an Orbitrap Q Exactive liquid chromatography-tandem mass spectrometry (LC-MS/MS) instrument in positive ion mode to determine concentrations in the sample being analyzed. Plasma concentration-time analysis was performed according to non-compartmental methods using Phoenix WinNonlin 6.3 software. C0, Cl, Vdss, T/ ₂ , AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf) were recorded and plasma concentration versus time curves were plotted.

In fig. Figure 2 shows plasma concentration-time curves for BCY13272 when administered intravenously at 5.5 mg/kg to CD1 mice (n=3); the steady-state volume of distribution (Vdss) for BCY13272 is 1.1 L/kg with a clearance of 7.5 mL/min/kg, resulting in a terminal half-life of 2.9 hours.

5. EphA2/CD137 heterotandem bicyclic peptide complex BCY13272 induces sec

- 21 046294 reaction of the cytokine IFN-γ when performing an analysis in a co-culture with MC38.

MC38 and HT1080 cells were cultured according to recommended protocols. Frozen PBMCs obtained from healthy human donors were thawed and washed once at room temperature with PBS with benzonase and then resuspended in RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1x penicillin/streptomycin, 10 mM HEPES, and 2 mM L-glutamine (called R10 medium). Seed 100 μl PBMC (1,000,000 PBMC/ml) and 100 μl tumor cells (100,000 tumor cells/ml) (effector:target cell (E:T) ratio 10:1) in each well of a 96-well flat-bottom plate for analysis in a shared culture. On day 0, 100 ng/ml soluble anti-CD3 monoclonal antibody (mAb) (clone OKT3) was added to the culture to stimulate human PBMCs. Test compounds, control compounds, or placebo media were diluted in R10 medium, and 50 μl was added to the appropriate wells to bring the final well volume to 250 μl. The plates were covered with breathable film and incubated in a humidified chamber at 37°C with 5% CO ₂ for two days. Supernatants were collected at 24 and 48 h post-stimulation and analyzed for IFN-γ on a Luminex analyzer. Briefly, standards and samples were added to a black 96-well plate. A microparticle cocktail (provided in the Luminex kit, R&D Systems) was added and shaken for 2 hours at room temperature. The plate was washed 3 times using a magnetic holder. The biotin cocktail was then added to the plate and shaken for 1 hour at room temperature. The plate was washed 3 times using a magnetic holder. The streptavidin cocktail was added to the plate and shaken for 30 min at room temperature. The plates were washed 3 times using a magnetic holder, resuspended in 100 µl wash buffer, shaken for 2 min at room temperature and read using a Luminex 2000 analyzer. The resulting raw data was analyzed using the built-in Luminex software to generate standard curves and interpolate concentrations protein, all other data analyzes and graphing were performed using Excel and Prism software. Data are from one study with three independent donor PBMCs tested in two parallel experiments.

The data presented in Fig. 2 and in table. 4 below show that BCY13272 induces strong activation of CD137, as evidenced by the secretion of IFN-γ and IL-2 after CD3 stimulation. Activation is dependent on heterotandem binding to both CD137 and EphA2, as evidenced by the lack of activity of the nonbinding controls BCY12762 and BCY13692 when the CD137 and EphA2 binders, respectively, include a D-amino acid that results in a nonbinding analogue.

Table 4

EC ₅₀ values of IL-2 cytokine secretion induced by EphA2/CD137 heterotandem bicyclic complex BCY13272 in human PBMC-MC38/HT-1080 co-culture assay

Complex No. Cell line EC ₅₀ (nM) N= BCY13272 MSZ 8 0.79±0.24 5 BCY13272 NT-1080 0.55±0.47 4

6. Antitumor activity of BCY13272 in the MC38 syngeneic tumor model

Female C57BL/6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] at 6-8 weeks of age were implanted subcutaneously with 1x106 MC38 cells. Mice were randomized to treatment when mean tumor volumes reached approximately 80 mm ³ and treated with IV placebo (25 mM histidine, 10% sucrose, pH 7), IV 8 mg/kg BCY13272, 0.9 mg/kg BCY13272 and 0.1 mg/kg BCY13272. All treatment procedures were carried out twice a week (BIW) for a total of 6 doses. Tumor growth was continuously monitored until day 28 from the start of treatment. Animals with a complete response (n=7) were followed up to day 62 from the start of treatment and were re-implanted with 2x106 MC38 tumor cells, and tumor growth was continuously monitored for 28 days. At the same time, naïve age-matched control huCD137 C57Bl/6 mice (n=5) were implanted with 2x106 MC38 tumor cells and observed for 28 days.

The results of this experiment are presented in Fig. 3, in which it can be seen that BCY13272 leads to significant antitumor activity with complete responses observed at dose levels of 0.9 mg/kg (2 of 6 complete responses) and 8 mg/kg (5 of 6 complete responses) (Fig. 3A). In contrast to naïve age-matched control huCD137 C57Bl/6 mice (100% engraftment rate), there was no tumor regrowth in complete response animals following treatment with BCY13272 (Figure 3B). These data indicate that BCY13272 has significant antitumor activity and that treatment with BCY13272 can induce immunological memory in complete response animals.

7. Binding of BCY13272 to EphA2 and CD137 measured by surface plasmon spectroscopy

- 22 046294 zonans (SPR).

(a) CD137.

Experiments were carried out using a Biacore analyzer to determine the values of ka (M ^-1 s ^-1 ), kd (s ^-1 ), KD (nM) for heterotandem peptides binding to the human CD137 protein. Recombinant human CD137 (R&D systems) was resuspended in PBS and biotinylated using EZ-Link™ Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) according to the manufacturer's protocol. The protein was desalted to remove unbound biotin using spin columns in PBS.

For peptide binding analysis, a Biacore T200 or Biacore 3000 instrument with a XanTec CMD500D chip was used. 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 running buffer. B Briefly, the carboxymethyldextran surface was activated by injection over 7 min of a 1:1 mixture of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)/0.1 M Nhydroxysuccinimide (NHS) at a flow rate of 10 µl/min. For streptavidin capture, the protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μL onto the activated chip surface. Residual activated groups were blocked by injection of 1 M ethanolamine (pH 8.5) for 7 min, and biotinylated CD137 was captured to 270-1500 RU (resonance units). The buffer was replaced with PBS/0.05% Tween 20, and serial dilutions of the peptides were prepared in this buffer with a final DMSO concentration of 0.5%. The highest peptide concentration was 500 nM with 6 additional 2-fold or 3-fold dilutions. Surface plasmon resonance (SPR) analysis was performed at 25°C at a flow rate of 90 μL/min with 60 s association and 900 s dissociation. After each cycle, a regeneration step was used (10 μl of 10 mM glycine pH 2). If necessary, data were adjusted to account for the effects of excluded volume of DMSO. All data were double-checked against blank injections and a reference surface using standard processing techniques, and data processing and kinetic data fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a 1:1 simple binding model allowing for mass transfer effects where appropriate.

(b) EphA2.

Experiments were carried out using a Biacore analyzer to determine the values of ka (M ^-1 s ^-1 ), kd (s ^-1 ), KD (nM) for the binding of BCY13272 to the human EphA2 protein.

EphA2 was biotinylated using EZ-Link™ Sulfo-NHS-LC-Biotin for 1 h in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 at 3x molar excess of biotin to protein. The degree of label introduction was determined using a kit for quantitative determination of biotin using the fluorescent method Fluorescence Biotin Quantification Kit (Thermo) after dialysis of the reaction mixture in PBS. For peptide binding analysis, a Biacore T200 instrument with a XanTec CMD500D chip was used. 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 running buffer. Briefly, the carboxymethyldextran surface was activated by injecting a 1:1 mixture of 0.4 M 1-ethyl-3(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/0.1 M N-hydroxysuccinimide (NHS) over 7 min at a flow rate of 10 μL. /min. For streptavidin capture, the protein was diluted to 0.2 mg/mL in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μL onto the activated chip surface. Residual activated groups were blocked by injection of 1 M ethanolamine (pH 8.5):HBSN (1:1) for 7 min. The buffer was changed to PBS/0.05% Tween 20, and biotinylated EphA2 was captured to 500–1500 RU using protein dilution to 0.2 μM in buffer. Serial dilutions of the peptides were prepared in this buffer with a final DMSO concentration of 0.5%, the highest peptide concentration being 50 or 100 nM with 6 additional 2-fold dilutions. Surface plasmon resonance (SPR) analysis was performed at 25°C at a flow rate of 90 μL/min with association of 60 s and dissociation of 900–1200 s. The data were adjusted to account for the effects of the excluded volume of DMSO. All data were double-checked against blank injections and a reference surface using standard processing techniques, and data processing and kinetic data fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a 1:1 simple binding model allowing for mass transfer effects where appropriate.

In fig. 5A is a sensorgram showing that BCY13272 binds to EphA2 (human) with an affinity of 2.0 nM. In fig. 5B is a sensorgram showing that BCY13272 binds to (human) CD137 with high affinity. Due to the presence of two CD137 binding bicycles in BCY13272, the rate of dissociation from immobilized CD137 protein is very slow and the reported KD value may be overestimated (Fig. 4B).

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Claims (6)

CLAIM 1. Heterotandem bicyclic peptide complex, including: (a) the first peptide ligand that binds to EphA2 and which has the sequence A-[HArg]-DC ₁ [HyP]LVNPLC ₁₁ LEP[d1Nal]WTC ₁₁₁ (SEQ ID NO:1; BCY13118), conjugated through the N-( linker acid-PEG ₃ )-N-bis(PEG ₃ -azide) with (b) second peptide ligands that bind to CD137, both of which have the sequence Ac-Ci[tBuAla]PE[D-Lys(PYA)]PYC _u FADPY[Nle]C _U iA (SEQ ID NO: 2; BCY8928), wherein each of said peptide ligands comprises a polypeptide containing three reactive cysteine groups (Ci, Cii and Ciii) separated by two loop sequences, and a molecular framework that represents is 1,1',1-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1one (TATA) and which forms covalent bonds with the reactive cysteine groups of the polypeptide, resulting two polypeptide loops are formed on the molecular framework; where Ac is acetyl, HArg is homoarginine, HuP is trans-4-hydroxyL-proline, d1Nal is D-1-naphthylalanine, tBuAla is tert-butyl-alanine, PYA is 4-pentinoic acid and Nle is norleucine. 2. Heterotandem bicyclic peptide complex according to claim 1, which is BCY13272: BCY13272 3. Heterotandem bicyclic peptide complex according to claim 1 or 2, where the pharmaceutically acceptable salt is selected from the free acid or sodium, potassium, calcium, ammonium salt. 4. A pharmaceutical composition which comprises a heterotandem bicyclic peptide complex according to any one of claims 1 to 3 in combination with one or more pharmaceutically acceptable excipients. 5. Use of a heterotandem bicyclic peptide complex according to any one of claims 1 to 3 for the prevention, suppression or treatment of cancer. 6. A method of treating cancer, which includes administering a heterotandem bicyclic peptide complex according to any one of claims 1-3 at a dosing frequency that does not maintain plasma concentrations of said complex above the in vitro EC ₅₀ value for said complex. -