Hetero-tandem bicyclic peptide complex

Abstract

The present invention relates to a hetero-tandem bicyclic peptide complex, which comprises a first peptide ligand binding to EphA2, wherein the first peptide ligand is conjugated to two second peptide ligands binding to CD137 via a linker. The present invention also relates to the use of the hetero-tandem bicyclic peptide complex in preventing, inhibiting or treating cancer.

Description

Technical Field

[0001] The present invention relates to a heterotandem bicyclic peptide complex, which comprises a first peptide ligand binding to EphA2, wherein the first peptide ligand is conjugated to two second peptide ligands binding to CD137 via a linker. The present invention also relates to the use of the heterotandem bicyclic peptide complex in preventing, inhibiting or treating cancer. Background Art

[0002] Cyclic peptides can bind to protein targets with high affinity and target specificity, and are therefore an attractive class of molecules for therapeutic agent development. In fact, several cyclic peptides have been successfully used clinically, such as 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 good binding properties are due to the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of hundreds of square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 ( Wu et al. (2007), Science 330, 1066-71), has a binding affinity to integrin αVb3 The cyclic peptides with Arg-Gly-Asp motifs (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1 ( Zhao et al. (2007), J Struct Biol 160(1), 1-10).

[0003] Due to its cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss after binding to the target and resulting in higher binding affinity. Compared with linear peptides, the reduced flexibility also leads to locking of target-specific conformations, increasing binding specificity. This effect has been exemplified by an effective and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses selectivity relative to other MMPs when the ring is opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties obtained by macrocyclization are more pronounced in polycyclic peptides with more than one peptide ring, such as in vancomycin, nisin and actinomycin.

[0004] Different research groups have previously tethered peptides with cysteine ​​residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers have used tri(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings to synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). WO 2019 / 122860 and WO 2019 / 122863 disclose methods for generating candidate drug compounds, wherein the compounds are generated by attaching cysteine-containing peptides to molecular scaffolds, such as 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

[0005] Combinatorial methods based on phage display have been developed to generate and screen large libraries of bicyclic peptides against target targets (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). Briefly, a region containing three cysteine ​​residues and two six random amino acids (Cys-(Xaa) 6 -Cys-(Xaa) 6 -Cys) and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the invention

[0006] According to a first aspect of the present invention, there is provided an iso-tandem bicyclic peptide complex comprising:

[0007] (a) The first peptide ligand that binds to EphA2 has the sequence A-[HArg]-DC i [HyP]LVNP LC ii LEP[d1Nal]WTC iii (SEQ ID NO: 1; BCY13118); which is ligated by N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker conjugated to,

[0008] (b) Two second peptide ligands that bind CD137 and have the sequence Ac-C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii-A(SEQ ID NO: 2; BCY8928);

[0009] Each of the peptide ligands comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises three reactive cysteine ​​groups (C i , C ii and C iii ), the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the molecular scaffold forms a covalent bond with the reactive cysteine ​​group of the polypeptide, so that two polypeptide rings are formed on the molecular scaffold;

[0010] Wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, d1Nal represents D-1-naphthylalanine, tBuAla represents tert-butylalanine, PYA represents 4-pentenoic acid, and Nle represents norleucine.

[0011] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising the hetero-tandem bicyclic peptide complex as defined herein, in combination with one or more pharmaceutically acceptable excipients.

[0012] According to another aspect of the present invention, there is provided a hetero-tandem bicyclic peptide complex as defined herein for use in preventing, inhibiting or treating cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 : The EphA2 / CD137 heterotandem bicyclic peptide complex BCY13272 was analyzed in the 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.

[0014] Figure 2 : Plasma concentration versus time curve of BCY13272 in CD1 mice (n=3) dosed at 5.5 mg / kg IV, SD rats (n=3) at 3.6 mg / kg IV infusion (15 min), and cynomolgus monkeys (n=2) at 8.9 mg / kg IV infusion (15 min). The pharmacokinetic profile of BCY13272 has the following terminal half-life: 2.9 hours in CD-1 mice, 2.5 hours in SD rats, and 8.9 hours in cynomolgus monkeys.

[0015] Figure 3: Antitumor activity of BCY13272 in the syngeneic MC38 tumor model. (A) MC38 tumor volume during and after BCY13272 treatment. The number of complete responder (CR) mice at D28 (and maintaining CR at D62) is indicated in parentheses. BIW: twice weekly dosing; IV: intravenous administration. (B) Complete responder animals to BCY13272 and naive animals after transplantation of MC38 tumor cells. Tumor growth curves of age-matched control animals. CR: complete responder.

[0016] Figure 4 : BCY13272 induces IFN-γ cytokine secretion in (A) PBMC / MC38 and (B) PBMC / HT29 co-culture assays. BCY12762 is an iso-tandem bicyclic peptide complex that binds to EphA2 but not CD137. BCY13692 is an iso-tandem bicyclic peptide complex that binds to CD137 but not EphA2. (C) Graph of EC50 (nM) values ​​for IL-2 and IFN-γ secretion induced by BCY13272 in PBMC co-culture assays with MC38 (mouse) cell line and 5 PBMC donors, and HT1080 (human) cell line and 4 PBMC donors.

[0017] Figure 5 : Surface plasmon resonance (SPR) of BCY13272 binding to immobilized (A) EphA2 and (B) CD137. DETAILED DESCRIPTION

[0018] According to a first aspect of the present invention, there is provided an iso-tandem bicyclic peptide complex comprising:

[0019] (a) The first peptide ligand that binds to EphA2 has the sequence A-[HArg]-DC i [HyP]LVNPL C ii LEP[d1Nal]WTC iii (SEQ ID NO: 1; BCY13118); which is ligated by N-(acid-PEG 3 )-N-bis(PEG 3 -azide) linker conjugated to,

[0020] (b) Two second peptide ligands that bind CD137 and have the sequence Ac-C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii -A(SEQ ID NO: 2; BCY8928);

[0021] Each of the peptide ligands comprises a polypeptide and a molecular scaffold, wherein the polypeptide comprises three reactive cysteine ​​groups (C i , C ii and C iii ), the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the molecular scaffold forms a covalent bond with the reactive cysteine ​​group of the polypeptide, so that two polypeptide rings are formed on the molecular scaffold;

[0022] Wherein Ac represents acetyl, HArg represents homoarginine, HyP represents trans-4-hydroxy-L-proline, d1Nal represents D-1-naphthylalanine, tBuAla represents tert-butylalanine, PYA represents 4-pentenoic acid, and Nle represents norleucine.

[0023] The N-(acid-PEG 3 )-N-bis(PEG 3 -azide) connectors include:

[0024]

[0025] In one embodiment, the heterotandem bicyclic peptide complex is BCY13272:

[0026]

[0027] The complete details of BCY13272 are shown in Table A below:

[0028] Table A: Composition of BCY13272

[0029]

[0030] Data shown in Figure 3 In this study, we demonstrated that BCY13272 induced significant antitumor effects in the MC38 tumor model in mice.

[0031] Certain analogs (ie modified derivatives) and metabolites of BCY13272 are mentioned herein, each of which forms a further aspect of the invention and are summarised in Table B below:

[0032] Table B: Composition of labeled analogs and potential metabolites of BCY13272

[0033]

[0034] BCY14601 represents a protein with sequence C i [tBuAla]PE[D-Lys(PYA)]PYCii FADPY[Nle]C iii -A (SEQ ID NO: 3), wherein TATA serves as a molecular scaffold;

[0035] and wherein BCY13389 represents a compound having the sequence [Ac]C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii -K (SEQ ID NO: 4), wherein TATA serves as a molecular scaffold.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), 4th edition, John Wiley & Sons, Inc.), which are incorporated herein by reference.

[0037] the term

[0038] serial number

[0039] When referring to amino acid residue positions within the compounds of the invention, cysteine ​​residues (C i , C ii and C iii ) is unchanged and omitted from the numbering, therefore, the numbering of the amino acid residues in SEQ ID NO: 1 is referred to as follows:

[0040] 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).

[0041] For the purposes of this description, the bicyclic peptide is cyclized with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) to produce a trisubstituted structure. The cyclization with TATA occurs at C i , C ii and C iii superior.

[0042] Molecular form

[0043] The N- or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence, separated by a hyphen. For example, the N-terminal βAla-Sar10-Ala tail would be represented as:

[0044] βAla-Sar10-A-(SEQ ID NO:X).

[0045] Reverse peptide sequence

[0046] According to Nair et al. (2003), J Immunol 170 (3), 1362-1373, it is envisioned that the peptide sequences disclosed herein will also be used in their reverse-inverse form. For example, the sequence is reversed (i.e., the N-terminus becomes the C-terminus, and vice versa), and its stereochemistry is also reversed (i.e., the D-amino acid becomes the L-amino acid, and vice versa). For the avoidance of doubt, unless otherwise indicated, referring to an amino acid by its full name or by its amino acid single letter or three letter code herein is intended to represent an L-amino acid. If such an amino acid is intended to represent a D-amino acid, the amino acid will begin with a lowercase d in square brackets, such as [dA], [dD], [dE], [dK], [d1Nal], [dNle], etc.

[0047] Advantages of peptide ligands

[0048] Certain hetero-tandem bicyclic peptide complexes of the present invention have many favorable properties that make them considered as drug-like molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. Such favorable properties include:

[0049] - Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations;

[0050] - Protease stability. Heterotandem bicyclic peptide complexes should ideally exhibit stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across species so that heterotandem bicyclic peptide lead candidates can be developed in animal models and can be confidently administered to humans;

[0051] - Ideal solubility profile. This is a function of the ratio of charged and hydrophilic residues to hydrophobic residues and intra / inter-molecular hydrogen bonding, which is important for formulation and absorption purposes;

[0052] - Selectivity. Certain heterotandem bicyclic peptide complexes of the present invention exhibit good selectivity over other targets;

[0053] - 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 with short exposure in acute disease management settings; or to develop heterotandem bicyclic peptide complexes with enhanced retention in the circulation, which are therefore optimal for managing more chronic disease states. Other factors that contribute to the ideal plasma half-life are the requirement for sustained exposure to achieve maximum therapeutic efficiency (relative to the toxicity associated with sustained exposure to the agent).

[0054] Crucially, data are provided herein in which the heterotandem bicyclic peptide complexes of the invention are unable to maintain plasma concentrations above the in vitro EC of the compound. 50 When the frequency of administration is , anti-tumor efficacy is shown. This is in contrast to the larger recombinant biological (i.e., antibody-based) methods for 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 being bound by theory, it is believed that the reason for this observation is the fact that the hetero-tandem bicyclic complexes have a relatively low molecular weight (usually <15 kDa), they are fully synthetic, and they are tumor-targeted agonists for CD137. Therefore, they have a relatively short plasma half-life, but have good tumor penetration and retention. The data provided herein fully support these advantages. For example, in a mouse syngeneic rodent model, daily or 3-day administration of humanized CD137 demonstrated antitumor efficacy. In addition, intraperitoneal pharmacokinetic data showed a plasma half-life of <3 hours, which would indicate that the circulating concentration of the complex would continue to decline to below the in vitro EC between doses. 50Furthermore, tumor pharmacokinetic data suggest that levels of the heterotandem bicyclic complex may be higher and more sustained in tumor tissue compared with plasma levels.

[0055] 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 the patient a concentration of the complex which is not capable of maintaining a plasma concentration of the complex above the in vitro EC of the complex. 50 The hetero-tandem bicyclic peptide complex as defined herein is administered at a dosing frequency of .

[0056] -Immune memory. Coupling a bicyclic peptide ligand that binds cancer cells with a bicyclic peptide ligand that binds immune cells provides a synergistic advantage of immune memory. It is believed that the hetero-tandem bicyclic peptide complexes of the present invention not only eradicate tumors, but also that after re-administration of the tumorigenic agent, the inoculated complete responder mice did not develop tumors. This indicates that treatment with the hetero-tandem bicyclic peptide complexes of the present invention has induced immunogenic memory in complete responder mice. This has a significant clinical advantage to prevent the tumor from recurring after it has been initially controlled and eradicated.

[0057] Peptide ligand

[0058] As referred to herein, a peptide ligand refers to a peptide covalently bound to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) capable of forming a covalent bond with the scaffold, and a sequence subtended between the reactive groups, the sequence being referred to as a loop sequence because a loop is formed when the peptide is bound 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.

[0059] Pharmaceutically acceptable salts

[0060] It will be understood that salt forms are within the scope of the present invention and reference to a peptide ligand includes salt forms of the ligand.

[0061] The salts of the present invention can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a suitable base or acid in water or in an organic solvent, or in a mixture of the two.

[0062] Acid addition salts (mono or di) can be formed with a wide variety of inorganic and organic acids. Examples of acid addition salts include mono or di salts formed with an acid selected from 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, butyric acid, (+) camphor, 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, galactaric 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 acid (e.g., hydrobromic acid, hydrochloric acid, hydroiodic acid), hydroxyethanesulfonic 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, 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.

[0063] A particular group of salts consists of salts formed by acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalenesulfonic, valeric, propionic, butyric, malonic, glucuronic and lactobionic acids. One particular salt is the hydrochloride. Another particular salt is the acetate.

[0064] If the compound is anionic, or has a functional group that can be anionic (e.g., -COOH can be -COO - ), then it can form a salt with an organic or inorganic base to generate 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 Mg 2+ , and other cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 +) and substituted ammonium ions (e.g. NH 3 R + NH 2 R 2 + 、NHR 3 + and NR 4 + ). Some examples of suitable substituted ammonium ions are those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4 + .

[0065] When the compound of the present invention contains an amine functional group, it can be reacted with an alkylating agent to form a quaternary ammonium salt, for example, according to methods well known to the skilled person. Such quaternary ammonium compounds are within the scope of the present invention.

[0066] Modified derivatives

[0067] It should 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 one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues (such as replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of spacer groups; replacement of one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues; replacement of one or more amino acid residues with alanine, replacement of one or more D-amino acids with succinimide; replacement of one or more amino acid residues ... The invention also provides methods for replacing one or more L-amino acid residues with a residue; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; modification of the length of the peptide backbone; replacing the hydrogen on the α-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 amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introducing or replacing amino acids with orthogonal reactivity suitable for functionalization, such as amino acids with azide or alkynyl groups, which allow functionalization with moieties with alkynyl or azido groups, respectively.

[0068] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using a suitable amino-reactive chemistry and / or a C-terminal modification using a suitable carboxyl-reactive 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 radiation chelator, or a chromophore.

[0069] 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, during peptide synthesis, the N-terminal cysteine ​​group (referred to herein as C i The group) is blocked by acetic anhydride or other suitable reagents, resulting in the molecule being acetylated at the N-terminus. This embodiment provides the advantage of removing potential recognition points of aminopeptidases and avoids the possibility of degradation of the bicyclic peptide.

[0070] In an alternative embodiment, the N-terminal modification includes the addition of a molecular spacer group that facilitates the conjugation of an effector group and maintains the potency of the bicyclic peptide towards its target.

[0071] 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, during peptide synthesis, the C-terminal cysteine ​​group (referred to herein as C iii The group) is synthesized as an amide, resulting in a C-terminal amidation of the molecule. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidases and reduces the likelihood of proteolytic degradation of the bicyclic peptide.

[0072] In one embodiment, the modified derivatives include replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids with isosteric / isoelectronic side chains can be selected that are neither recognized by degradative proteases nor have any adverse effects on target efficacy.

[0073] Alternatively, unnatural amino acids with constrained amino acid side chains can be used so that proteolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, this involves proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid (Aib)), and cyclic amino acids, a simple derivative being amino-cyclopropylcarboxylic acid.

[0074] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises an N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii ) to add a spacer group.

[0075] In one embodiment, the modified derivatives include replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues. In a further embodiment, the modified derivatives include replacing tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the drug stability characteristics of the resulting bicyclic peptide ligands.

[0076] In one embodiment, the modified derivatives include replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an optional embodiment, the modified derivatives include replacing 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 the bicyclic peptide ligand. For example, hydrophobic amino acid residues affect the degree of plasma protein binding, thereby affecting the concentration of the free available part in the plasma, and charged amino acid residues (particularly arginine) can affect the interaction of the peptide with the cell surface phospholipid membrane. The two are combined to affect the half-life, distribution volume and exposure of the peptide drug, and can be adjusted according to the clinical endpoint. In addition, (if the peptide drug has been subcutaneously administered) the correct combination and quantity of charged and hydrophobic amino acid residues can reduce the stimulation at the injection site.

[0077] In one embodiment, the modified derivative comprises replacing one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005), PNAS, 102 (2), 413-418).

[0078] In one embodiment, the modified derivatives include removal of any amino acid residues and substitution with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

[0079] It should be noted that each of the above modifications is used to intentionally improve the potency or stability of the peptide. Through modification, the potency can be further improved by the following mechanisms:

[0080] - Incorporation of hydrophobic moieties that exploit hydrophobic interactions and result in lower off-rates, allowing higher affinity to be achieved;

[0081] - incorporation of charged groups that exploit long-range ionic interactions, resulting in faster association rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0082] - Incorporating additional constraints into the peptide, for example by properly constraining the amino acid side chains to minimize entropy loss upon target binding, by restricting the torsion angles of the backbone to minimize entropy loss upon target binding, and introducing additional cyclization into the molecule for the same reason.

[0083] (For review, see Gentilucci et al., Curr. Pharmaceutical Design (2010) 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009) 16, 4399-418).

[0084] Isotopic variants

[0085] The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present 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 usually occurring in nature, and peptide ligands of the present invention, in which a metal chelating group (referred to as "effector") is attached, which is capable of holding the relevant (radio)isotope, and peptide ligands of the present invention, in which certain functional groups are covalently substituted by relevant (radio)isotopes or isotope-labeled functional groups.

[0086] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include hydrogen isotopes, such as 2 H(D) and 3 H(T); Carbon isotopes such as 11 C. 13 C and 14 C; chlorine isotopes such as 36 Cl; fluorine isotopes such as 18 F; iodine isotopes such as 123 I. 125 I and 131 I; Nitrogen isotopes such as 13 N and 15 N; oxygen isotopes such as 15 O. 17 O and 18 O; phosphorus isotopes such as 32 P; sulfur isotopes such as 35 S; copper isotopes such as 64Cu; Gallium isotopes such as 67 Ga or 68 Ga; yttrium isotopes such as 90 Y; and lutetium isotopes such as 177 Lu; and bismuth isotopes such as 213 Bi.

[0087] Certain isotope-labeled peptide ligands of the invention, such as those incorporating radioactive isotopes, can be used for tissue distribution studies of drugs and / or substrates, and for clinically evaluating the presence and / or absence of the nectin-4 target on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties that can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with labeling agents, such as radioactive isotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, fluorescein, aequorin and luciferase), etc. The radioactive isotope tritium (i.e. 3 H(T)) and carbon-14 (i.e. 14 C), are particularly useful for this purpose due to their ease of incorporation and readily available detection methods.

[0088] Using heavier isotopes such as deuterium 2 H(D) substitution may offer certain therapeutic advantages due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0089] Positron emitting isotopes such as 11 C. 18 F. 15 O and 13 N substitution can be used in positron emission tomography (PET) studies to examine target occupancy.

[0090] Isotopically-labeled compounds of the peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0091] synthesis

[0092] The peptides of the present invention can be synthesized by standard techniques and then reacted in vitro with the molecular scaffold. When doing this, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or verification. Such methods can be accomplished using conventional chemical methods as disclosed in Timmerman et al. (supra).

[0093] Therefore, the present invention also relates to the manufacture of a polypeptide or conjugate selected as described herein, wherein said manufacture comprises optional further steps as described below. In one embodiment, these steps are performed on a final product polypeptide / conjugate prepared by chemical synthesis.

[0094] When making conjugates or complexes, amino acid residues in the target polypeptide may be optionally substituted.

[0095] The peptide may also be extended to incorporate, for example, another loop and thereby introduce multiple specificities.

[0096] In order to extend the peptide, conventional solid phase or solution phase chemistry can be used, using orthogonally protected lysine (and analogs) to simply chemically extend at its N-terminus or C-terminus or in a ring. Standard (biological) conjugation techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, it can be added by fragment condensation or native chemical connection, such as described in (Dawson et al., 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266: 776-779), or added by enzymes, such as using (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91 (26): 12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, 6000-6003 pages) described in the mutase (subtiligase).

[0097] Alternatively, the peptide may be extended or modified by further conjugation of disulfide bonds. This has the additional advantage of allowing the first and second peptides to dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TATA) may be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; further cysteine ​​or thiol may then be attached to the N- or C-terminus of the first peptide so that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0098] Similar techniques are also used for the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[0099] Furthermore, other functional groups or effector groups may be added in the same manner, at the N- or C-terminus or via side chain coupling using appropriate chemistry. In one embodiment, the coupling is performed in a manner that does not block the activity of either entity.

[0100] Pharmaceutical composition

[0101] 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 pharmaceutically acceptable excipients.

[0102] Generally, the peptide ligands of the present invention will be used in purified form with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / water solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride and lactated Ringer's solution. If it is necessary to keep the polypeptide complex suspended, a suitable physiologically acceptable adjuvant can be selected from thickening agents such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin and alginate.

[0103] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982), Remington's Pharmaceutical Sciences, 16th edition).

[0104] The peptide ligands of the invention can be used as a composition for administration alone or in combination with other agents. They can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in combination with the protein ligands of the invention, or even in combination with polypeptides selected according to the invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are combined before administration.

[0105] The route of administration of the pharmaceutical composition according to the present invention can be any route commonly known to those of ordinary skill in the art. For treatment, the peptide ligand of the present invention can be applied to any patient according to standard techniques. The application can be carried out in any suitable manner, including parenteral, intravenous, intramuscular, intraperitoneal, percutaneous, via pulmonary approach or suitably by direct infusion with a catheter. Preferably, the pharmaceutical composition according to the present invention will be applied by inhalation. The dosage and frequency of application will depend on the patient's age, sex and condition, the simultaneous application of other drugs, contraindications and other parameters that the clinician will consider.

[0106] The peptide ligands of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and lyophilization and reconstitution techniques known in the art may be employed. Those skilled in the art will recognize that lyophilization and reconstitution may result in varying degrees of loss of activity and that levels may have to be adjusted upwards to compensate.

[0107] Compositions comprising the peptide ligands of the present invention or mixtures thereof can be administered for preventive and / or therapeutic treatment. In certain therapeutic applications, an amount sufficient to complete at least partial inhibition (inhibition), inhibition (suppression), regulation, killing or some other measurable parameters of a selected cell population is defined as a "therapeutically effective dose". The amount required to reach the dose will depend on the severity of the disease and the general state of the patient's own immune system, but is generally 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, and a more commonly used dose is 0.05 to 2.0 mg / kg / agent. For preventive applications, compositions comprising the peptide ligands of the present invention or mixtures thereof can also be administered with similar or slightly lower doses.

[0108] Compositions comprising peptide ligands according to the present invention can be used in preventive and therapeutic settings to assist in the alteration, inactivation, killing or removal of selected target cell populations in mammals. In addition, peptide ligands as described herein can be selectively used in vitro (extracorporeally) or in vitro (in vitro) to kill, consume or otherwise effectively remove target cell populations from heterogeneous cell collections. Blood from mammals can be combined in vitro with selected peptide ligands, thereby killing or otherwise removing undesirable cells from blood for return to mammals according to standard techniques.

[0109] Therapeutic Uses

[0110] According to another aspect of the present invention, there is provided a hetero-tandem bicyclic peptide complex as defined herein for use in preventing, inhibiting or treating cancer.

[0111] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) 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 others), such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including cancers of the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), cancer of the gallbladder and biliary system, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancer (e.g., cancer of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinus), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, penis cancer, cervical cancer, uterine cancer, Myometrial cancer, endometrial cancer, thyroid cancer (e.g., follicular thyroid cancer), adrenal cancer, prostate cancer, skin and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, hyperplastic nevus); hematologic malignancies (i.e., leukemias, lymphomas) and hematologic precancerous conditions and borderline malignancies, including hematologic malignancies and related conditions of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, leukemia, unexplained monoclonal immunoglobulinemia, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematologic malignancies and related conditions of the myeloid lineage (e.g., 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, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as soft tissue, bone, or chondrosarcomas such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, cardiomyopathy, Posey 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., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and, pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor);or congenital or other forms of syndromes that predispose the patient to malignancy (e.g. xeroderma pigmentosum). ;

[0112] In a further embodiment, the cancer is selected from hematopoietic malignancies such as selected from: non-Hodgkin lymphoma (NHL), Burkitt 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 lymphoma (HL) and chronic myeloid leukemia (CML).

[0113] The term "prevention" as referred to herein involves administration of a protective composition prior to induction of a disease. "Suppression" refers to administration of a composition after an induction event but prior to clinical manifestation of the disease. "Treatment" involves administration of a protective composition after disease symptoms become apparent.

[0114] Animal model systems exist that can be used to screen peptide ligands for effectiveness in preventing or treating disease. The present invention facilitates the use of animal model systems, which allow the development of polypeptide ligands that can cross-react with both human and animal targets, thereby allowing the use of animal models.

[0115] The present invention is further described below with reference to the following examples.

[0116] Example

[0117] Generally, the hetero-tandem bicyclic peptide complex of the present invention can be prepared according to the following general method:

[0118]

[0119] All solvents were degassed and washed with N 2 Purge 3 times. Mix BP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) in DMF for 5 minutes, then add bicyclic 1 (1.2 eq.). Stir the reaction mixture at 40 ° C for 16 hours. Then concentrate the reaction mixture under reduced pressure to remove the solvent and purify by preparative HPLC to obtain intermediate 2.

[0120] A mixture of intermediate 2 (1.0 eq) and bicyclic 2 (2.0 eq) was dissolved in t-BuOH / H 2 O (1:1), then add CuSO 4 (1.0 eq), VcNa (4.0 eq) and THPTA (2.0 eq). Finally, 0.2 M NH 4 HCO 3 To adjust the pH to 8.2 The reaction mixture was stirred at 40° C. for 16 h under atmospheric pressure. The reaction mixture was directly purified by preparative HPLC.

[0121] The following provides more detailed experiments of the hetero-tandem bicyclic peptide complex of the present invention:

[0122] Example 1: Synthesis of BCY13272

[0123]

[0124] Procedure for preparing BCY14964

[0125]

[0126] 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), and the pH of the solution was adjusted to 8 by the dropwise addition of DIEA (64.6 mg, 499.83 μmol, 87.0 μL, 2.4 eq), and 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, and the pH of the resulting solution was adjusted to 8 by the dropwise addition of DIEA. The reaction mixture was stirred at 25° C. for 45 min. LC-MS showed that BCY13118 was completely consumed, and a major peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC to afford 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+ ).

[0127] Procedure for preparation of BCY13272

[0128]

[0129] BCY8928 (644.0 mg, 290.55 μmol, 2.5 eq), THPTA (50.5 mg, 116.22 μmol, 1.0 eq), CuSO 4 A mixture of sodium ascorbate (0.4 M, 145.0 μL, 0.5 eq) and sodium ascorbate (82.0 mg, 464.89 μmol, 4.0 eq) was dissolved in t-BuOH / 0.2 M NH4 HCO 3 (1:1, 6.0 mL). 0.2 M NH 4 HCO 3 (in 1:1 t-BuOH / 0.2 M NH 4 HCO 3 The pH of the solution was adjusted to 7.5, and then the solution was stirred at 25°C for 3 min. BCY14964 (350.0 mg, 116.22 μmol, 1.0 eq) was dissolved in t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 11.0 mL) and then added dropwise to the stirred solution. All solvents were pre-degassed and heated with N 2 Purge. By adding 0.2M NH 4 HCO 3 (in 1:1t-BuOH / 0.2MNH 4 HCO 3 The pH of the solution was adjusted to 7.5 and the solution turned light yellow. 2 The reaction mixture was stirred at 25 °C for 6 hours under atmosphere. LC-MS showed that a major product peak with the desired m / z 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 obtain 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+ ).

[0130] Example 2: Synthesis of BCY14414

[0131]

[0132] Procedure for preparation of BCY14798

[0133]

[0134] 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 NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed and N 2 purge) and then in N 2Add CuSO 4 (0.4 M, 23.0 μL, 0.5 eq) and sodium ascorbate (72.0 mg, 365.27 μmol, 20.0 eq). 0.2 M NH 4 HCO 3 (in 1:1 t-BuOH / 0.2 M NH 4 HCO 3 The pH of the solution was adjusted to 7.5 and the solution turned light yellow. 2 The reaction mixture was stirred at 25 °C for 1.5 hours under atmosphere. LC-MS showed that BCY14964 was retained, compound BCY8928 was completely consumed, and a major peak with the desired m / z 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 obtain 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+ ).

[0135] Procedure for preparation of BCY14414

[0136]

[0137] 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, pre-degassed and N 2 purge) and then in N 2 Add CuSO 4 (0.4M, 5.0μL, 0.5eq) and sodium ascorbate (2.8mg, 16.06μmol, 4.0eq). 0.2M NH 4 HCO 3 (in 1:1 t-BuOH / 0.2 M NH 4 HCO 3 The pH of the solution was adjusted to 7.5 and the solution turned light yellow. 2The reaction mixture was stirred at 25 °C for 2 hours under atmosphere. LC-MS showed that BCY14798 was completely consumed, some BCY13389 was retained, and a major peak with the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative method to obtain 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+ ).

[0138] Example 3: Synthesis of BCY14417

[0139]

[0140] Procedure for preparation of BCY14417

[0141]

[0142] A mixture of BCY14414 (13.0 mg, 1.73 μmol, 1.0 eq) and biotin-PEG12-NHS ester (CAS365441-71-0, 4.2 mg, 4.50 μmol, 2.6 eq) was dissolved in DMF (0.5 mL). The pH of the solution was adjusted to 8 by the 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 a major peak with the desired m / z 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 obtain 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+ ).

[0143] Example 4: Synthesis of BCY14418

[0144]

[0145] Procedure for preparation of BCY14418

[0146]

[0147] BCY14414 (5.6 mg, 0.75 μmol, 1.0 eq) and Alexa Fluor A mixture of 488 (0.9 mg, 1.49 μmol, 2.0 eq) was dissolved in DMF (0.3 mL). The pH of the solution was then adjusted to 8 by the dropwise addition of DIEA. The reaction mixture was stirred at 25 °C for 1.0 h. LC-MS showed complete consumption of BCY14414 and a major peak with the desired m / z 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 obtain 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+ ).

[0148] Example 5: Synthesis of BCY15217

[0149]

[0150] Procedure for preparation of BCY15217

[0151]

[0152] 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 t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed and N 2 purge) and then in N 2 Add CuSO 4 (0.4M, 16.6μL, 1.0eq) and sodium ascorbate (4.7mg, 26.56μmol, 4.0eq). The pH of the solution was adjusted to 8 and the solution turned light yellow. 2 The reaction mixture was stirred at 25 °C for 2 hours under atmosphere. LC-MS showed that BCY14964 was retained and a major peak with the desired m / z 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 obtain 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+ ).

[0153] Example 6: Synthesis of BCY15218

[0154]

[0155] Procedure for preparation of BCY15218

[0156]

[0157] 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 t-BuOH / 0.2 M NH 4 HCO 3 (1:1, 0.5 mL, pre-degassed and N 2 purge) and then in N 2 Add CuSO 4 (0.4 M, 14.0 μL, 1.0 eq) and sodium ascorbate (4.0 mg, 22.95 μmol, 4.0 eq). 0.2 M NH 4 HCO 3 (in 1:1 t-BuOH / 0.2 M NH 4 HCO 3 The pH of the solution was adjusted to 7.5 and the solution turned light yellow. 2 The reaction mixture was stirred at 25 °C for 2 hours under atmosphere. LC-MS showed that BCY14798 was completely consumed, BCY14601 was retained, and a major peak with the desired m / z 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 obtain 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+ ).

[0158] Analyze the data

[0159] The hetero-tandem bicyclic peptide complex of the present invention was analyzed using mass spectrometry and HPLC. The HPLC settings are as follows:

[0160] Mobile phase: A:H 2 0.1% TFA in O B: 0.1% TFA in ACN

[0161] Flow rate: 1.0ml / min

[0162] Column: Kintex 1.7μm C18 100A 2.1mm*150mm

[0163] Instrument: Agilent UPLC 1290

[0164] The gradient used was 30-60% B in 10 min and the data generated were as follows:

[0165]

[0166] Biological data

[0167] 1. CD137 reporter gene assay in co-culture with tumor cells

[0168] Prepare culture medium, called R1 medium, by adding 1% FBS to RPMI-1640 (component of Promega kit CS196005). Prepare serial dilutions of the test article in R1 in a sterile 96-well plate. Add 25 μL per well of the test article or R1 (as background control) to designated wells of a white cell culture plate. Harvest tumor cells * , and resuspended in R1 medium at a concentration of 400,000 cells / mL. Add 25 μL / well of tumor cells to the white cell culture plate. Thaw Jurkat cells (Promega kit CS196005, 0.5 mL) in a water bath and add 5 ml of pre-warmed R1 medium. Then add 25 μL / well of Jurkat cells to the white cell culture plate. Incubate at 37°C, 5% CO 2 The cells and test substances were incubated for 6 h. At the end of 6 h, 75 μL / well of Bio-Glo TM Reagent (Promega) and incubate for 10 min before reading luminescence in a plate reader (Clariostar, BMG). The fold change relative to cells alone (Jurkat cells + cell line used in co-culture) was calculated and plotted as log(agonist) vs. response in GraphPad Prism to determine the EC 50 (nM) and fold induction relative to background (maximum).

[0169] For EphA2, the tumor cell types used for co-culture were A549, PC-3, and HT29.

[0170] Figure 1 The data presented show that in a CD137 reporter assay, in the presence of an EphA2 expressing cell line (A549), the EphA2 / CD137 heterotandem BCY13272 induced robust CD137 activation, whereas a non-binding control molecule (BCY13626) showed no CD137 activation.

[0171] Figure 1 The data shown in Table 1 below show that BCY13272 induces robust CD137 activation in a CD137 reporter assay. The activation is dependent on the binding of the heterotandem to both CD137 and EphA2, as shown by the lack of activity of a non-binding control (BCY13626) that does not associate with EphA2 or CD137.

[0172] Table 1 below reports the ECs induced by BCY13272 in a CD137 reporter assay co-cultured with tumor cell lines expressing EphA2. 50 Summary of (nM) and fold induction:

[0173] Table 1: Activity of EphA2 / CD137 heterotandem bicyclic peptide complexes in CD137 reporter gene assay

[0174]

[0175] 2. Pharmacokinetics of the heterotandem complex BCY13272 in SD rats

[0176] The heterotandem complex BCY13272 formulated in 25 mM histidine HCl, 10% sucrose pH 7 was administered to male SD rats by IV bolus or IV infusion (15 minutes). Serial bleeding was performed at each time point through the submandibular vein or the great saphenous vein (approximately 80 μL blood / time point). All blood samples were immediately transferred to pre-cooled microcentrifuge tubes containing 2 μL K2-EDTA (0.5M) as an anticoagulant and placed on wet ice. The blood samples were immediately processed into plasma by centrifugation at approximately 4°C and 3000g. The precipitant containing the internal standard was immediately added to the plasma, mixed thoroughly and centrifuged at 12,000rpm and 4°C for 10 minutes. The supernatant was transferred to a pre-labeled polypropylene microcentrifuge tube and then quickly frozen on dry ice. The samples were stored at 70°C or below until analysis as needed. 7.5 μL of the supernatant sample was directly injected for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte. The data of plasma concentration versus time were analyzed by non-compartmental method using Phoenix WinNonlin6.3 software program. The curves of C0, Cl, Vdss, T1 / 2, AUC (0-last), AUC (0-infinity), MRT (0-last), MRT (0-infinity) and plasma concentration versus time were reported. The pharmacokinetic parameters of the experiment are shown in Table 2:

[0177] Table 2: Pharmacokinetic parameters in SD rats

[0178] Compound Route of administration T1 / 2(h) Vdss(L / kg) Clp(ml / min / kg) BCY13272 IV Inf 2.5 1.0 7.4

[0179] 3. Pharmacokinetics of the heterotandem complex BCY13272 in cynomolgus monkeys

[0180] Non-naive cynomolgus monkeys were administered 1 mg / kg of the heterotandem complex BCY13272 formulated in 25 mM histidine HCl, 10% sucrose pH 7 by intravenous infusion (15 or 30 min) into the cephalic vein. Serial bleeding (approximately 1.2 ml blood / time point) was performed at each time point through a peripheral blood vessel of restrained non-sedated animals and the blood was placed in a commercially available 2H-EDTA containing potassium (K2). 2 O (0.85-1.15mg) tube, placed on wet ice and processed to obtain plasma. Immediately after collection, the sample was centrifuged (3,000×g, centrifuged at 2 to 8°C for 10 minutes). 0.1mL plasma was transferred to a labeled polypropylene microcentrifuge tube. Immediately, 5 times of precipitant (containing internal standard 100ng / mL labetalol and 100ng / mL dexamethasone and 100ng / mL tolbutamide and 100ng / mL verapamil and 100ng / mL glibenclamide and 100ng / mL celecoxib methanol solution) was added to the plasma, fully mixed and centrifuged at 2 to 8°C, 12,000rpm for 10 minutes. The supernatant sample was transferred to a pre-labeled polypropylene microcentrifuge tube and frozen on dry ice. The sample was stored at -60°C or below until LC-MS / MS analysis. 40μL calibration standards, quality control, single blank and double blank samples were added to aliquots in 1.5mL tubes. Each sample was quenched with 200 μL IS1 (except for the double blank, which was quenched with 200 μL MeOH and 0.5% tritonX-100), and the mixture was thoroughly vortexed (at least 15 s) with a vortexer and centrifuged at 4°C and 12000g for 15 min. 10 μL of the supernatant was injected using Orbitrap QExactive in positive ion mode for LC-MS / MS analysis to determine the concentration of the analyte. The plasma concentration versus time data were analyzed by a non-compartmental method using the Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T1 / 2, AUC (0-end point), AUC (0-infinity), MRT (0-end point), MRT (0-infinity) and plasma concentration versus time curves are reported. The pharmacokinetic parameters of BCY13272 are shown in Table 3.

[0181] Table 3: Pharmacokinetic parameters in cynomolgus monkeys

[0182] Compound way <![CDATA[T 1 / 2 (h)]]> Clp(ml / min / kg) Vdss(L / kg) BCY13272 IV infusion (15 minutes) 8.9 4.1 0.82

[0183] Figure 2Plasma concentration versus time curves of BCY13272 in SD rats (n=3) with 3.6 mg / kg IV infusion (15 min) and in cynomolgus monkeys (n=3) with 9.2 mg / kg IV infusion (15 min) are shown. 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 h. 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 h.

[0184] 4. Pharmacokinetics of the heterotandem complex BCY13272 in CD1 mice

[0185] 15mg / kg of the heterotandem complex BCY13272 formulated in 25mM histidine HCl, 10% sucrose pH 7 was administered to 6 male CD-1 mice by intraperitoneal or intravenous administration. Continuous bleeding (approximately 80μL blood / time point) was performed through the submandibular vein or the great saphenous vein at each time point. All blood samples were immediately transferred to pre-cooled microcentrifuge tubes containing 2μL K2-EDTA (0.5M) as an anticoagulant and placed on wet ice. The blood samples were immediately processed into plasma by centrifugation at approximately 4°C and 3000g. The precipitant containing the internal standard was immediately added to the plasma, mixed thoroughly and centrifuged at 12,000rpm and 4°C for 10 minutes. The supernatant was transferred to a pre-labeled polypropylene microcentrifuge tube and then quickly frozen on dry ice. The samples were stored at 70°C or below until analysis as needed. 7.5 μL of supernatant samples were 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 non-compartmental approach using Phoenix WinNonlin 6.3 software program. C0, Cl, Vdss, T1 / 2, AUC (0-end point), AUC (0-infinity), MRT (0-end point), MRT (0-infinity) and plasma concentration versus time plots are reported.

[0186] Figure 2 Shown is the plasma concentration versus time curve of BCY13272 in CD1 mice (n=3) dosed IV at 5.5 mg / kg; the volume of distribution (Vdss) of BCY13272 was 1.1 L / kg and the clearance was 7.5 mL / min / kg, resulting in a terminal plasma half-life of 2.9 h.

[0187] 5. EphA2 / CD137 heterotandem bicyclic peptide complex BCY13272 induces IFN-γ cells in MC38 co-culture assay Cytokine secretion

[0188] MC38 and HT1080 cell lines were cultured according to the recommended protocol. Frozen PBMCs from healthy human donors were thawed and washed once with benzonase enzyme in room temperature PBS, then resuspended in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1× penicillin / streptomycin, 10mM HEPES and 2mM L-glutamine (referred to herein as R10 medium). 100 μl PBMCs (1,000,000 PBMCs / ml) and 100 μl tumor cells (100,000 tumor cells / ml) (effector cells: target cell ratio (E:T) 10:1) were spread in each well of a 96-well flat-bottom plate for co-culture assay. 100 ng / ml soluble anti-CD3 mAb (clone OKT3) was added to the culture at day 0 to stimulate human PBMCs. Dilute the test compound, control compound, or vehicle control in R10 medium and add 50 μL to each well to a final volume of 250 μL per well. Cover the plate with a gas permeable membrane and incubate at 37°C, 5% CO 2 Incubate in a humidified chamber for two days. Supernatants were collected 24 and 48 hours after stimulation and human IFN-γ was detected with Luminex. 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 at room temperature for 2 hours. Using a magnetic stand, wash the plate 3 times. The biotin mixture was then added to the plate and shaken for 1 hour at RT. Using a magnetic stand, wash the plate 3 times. The streptavidin mixture was added to the plate and shaken for 30 minutes at RT. Using a magnetic stand, wash the plate 3 times, resuspend the cells in 100 μL of wash buffer, shake at RT for 2 minutes, and then read using Luminex 2000. The raw data was analyzed using built-in Luminex software to generate a standard curve and insert protein concentrations, and all other data analysis and drawing were performed using Excel and Prism software. The data represent a study in which experimental replicates of three independent donor PBMCs were tested.

[0189] Figure 2 The data shown in Table 4 below show that BCY13272 induces strong CD137 activation as evidenced by IFN-γ and IL-2 secretion following CD3 stimulation. The activation is dependent on the binding of the heterotandem to both CD137 and EphA2, as evidenced by the lack of activity of the non-binding controls BCY12762 and BCY13692 (where the CD137 and EphA2 binders, respectively, contain all D-amino acids, resulting in non-binding analogs).

[0190] Table 4: EC50 of IL-2 cytokine secretion induced by the EphA2 / CD137 heterotandem bicyclic complex BCY13272 in a human PBMC-MC38 / HT-1080 co-culture assay

[0191] Complex ID Cell lines <![CDATA[EC 50 (nM)]]> N＝ BCY13272 MC38 0.79±0.24 5 BCY13272 HT-1080 0.55±0.47 4

[0192] 6. Antitumor activity of BCY13272 in the syngeneic MC38 tumor model

[0193] 1×10 TNFRSF9 cells were subcutaneously transplanted into 6-8 week old female C57BL / 6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen]. 6 MC38 cells. When the average tumor volume reached about 80 mm 3 At 4 pm, mice were randomized into treatment groups (n=6 / cohort) and treated intravenously (IV) with vehicle (25 mM histidine, 10% sucrose, pH 7), 8 mg / kg BCY13272, 0.9 mg / kg BCY13272, and 0.1 mg / kg BCY13272. All treatments were given twice weekly (BIW) for a total of 6 doses. Tumor growth was monitored from the start of treatment to day 28. Complete responder animals (n=7) were followed until day 62 after the start of treatment and were transplanted with 2×10 6 In parallel, 2×10 MC38 tumor cells were transplanted into naive age-matched control huCD137 C57Bl / 6 mice (n=5). 6 MC38 tumor cells were monitored for 28 days.

[0194] The results of this experiment can be seen Figure 3 , where it can be seen that BCY13272 resulted in significant antitumor activity, with complete responses observed at dose levels of 0.9 (2 complete responders out of 6) and 8 mg / kg (5 complete responders out of 6) Figure 3 Unlike in the naive age-matched control huCD137 C57Bl / 6 mice (100% tumor yield), no tumor regrowth was observed in the BCY13272 complete responder animals ( Figure 3 B). These data indicate that BCY13272 has significant antitumor activity and that BCY13272 treatment can induce immunogenic memory in complete responder animals.

[0195] 7. BCY13272 binding to EphA2 and CD137 as measured by SPR

[0196] (a) CD137

[0197] Biacore experiments were performed to determine the k of heterotandem peptides binding to human CD137 protein. a (M -1 s -1 ), k d (s -1 ) and K D Recombinant human CD137 (R&D systems) was resuspended in PBS and quantified using EZ-Link according to the manufacturer's recommended protocol. TM Biotinylation was performed using Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher). The protein was desalted using a spin column to remove unconjugated biotin and transferred into PBS.

[0198] For analysis of peptide binding, a Biacore T200 or Biacore 3000 instrument was used using a XanTec CMD500D chip. Streptavidin was immobilized on the chip using standard amine coupling chemistry at 25°C, using HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as running buffer. Briefly, a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) was injected at a flow rate of 10 μL / min for 7 min to activate the carboxymethyl dextran surface. For capture of streptavidin, 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. The residual activation groups were blocked by injection of 1M ethanolamine (pH 8.5) for 7min, and biotinylated CD137 was captured at a level of 270-1500RU. The buffer was changed to PBS / 0.05% Tween 20, and the peptides were prepared in series dilution in the buffer to make a final DMSO concentration of 0.5%. The highest peptide concentration was 500nM, and 6 further 2-fold or 3-fold dilutions were performed. SPR analysis was run at a flow rate of 90μl / min at 25°C, binding for 60 seconds and dissociation for 900 seconds. A regeneration step (10μl of 10mM glycine pH 2) was used after each cycle. The data were corrected for the DMSO exclusion volume effect as required. All data were double-referenced for blank injection and reference surface using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software version 2.0c (BioLogicSoftware). Data were fitted using a simple 1:1 binding model, and the influence of mass transport was considered where appropriate.

[0199] (b) EphA2

[0200] Biacore experiments were performed to determine the k of BCY13272 binding to human EphA2 protein. a (M -1 s -1 ), k d (s -1 ) and K D (nM) value.

[0201] EZ-Link TM EphA2 was biotinylated with Sulfo-NHS-LC-Biotin for 1 hour with a 3-fold molar excess of biotin relative to the protein. The reaction mixture was dialyzed against PBS and the extent of labeling was determined using a fluorescent biotin quantification kit (Thermo). For analysis of peptide binding, 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, using HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) was injected at a flow rate of 10 μL / min for 7 min to activate the carboxymethyl dextran surface. For capture of streptavidin, 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 injecting 1 M ethanolamine (pH 8.5):HBS-N (1:1) for 7 min. The buffer was changed to PBS / 0.05% Tween 20 and the protein was diluted to 0.2 μM in buffer to capture biotinylated EphA2 at levels of 500-1500 RU. A dilution series of peptides was prepared in this buffer with a final DMSO concentration of 0.5% and a maximum peptide concentration of 50 or 100 nM, with six further 2-fold dilutions. SPR analysis was run at 25°C with a flow rate of 90 μl / min, 60 sec association and 900-1200 sec dissociation. Data were corrected for DMSO exclusion volume effects. All data were double-referenced to blank injections and reference surfaces 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, and mass transfer effects were considered where appropriate.

[0202] Figure 5The sensorgram shown in A shows that BCY13272 binds to EphA2 (human) with an affinity of 2.0 nM. Figure 5 B shows a sensorgram of BCY13272 binding to CD137 (human) with high affinity. Due to the presence of two CD137 binding double loops in BCY13272, the dissociation rate from the immobilized CD137 protein is very slow, and the reported K D May be overestimated ( Figure 4 B). Sequence Listing <110> Baisco Technology Development Co., Ltd. <120> Hetero-tandem bicyclic peptide complex <130> BIC-C-P2831PCT <150> 62 / 880,191 <151> 2019-07-30 <150> 62 / 910,088 <151> 2019-10-03 <150> 62 / 931,442 <151> 2019-11-06 <150> 63 / 022,667 <151> 2020-05-11 <150> 63 / 024,715 <151> 2020-05-14 <160> 4 <170> PatentIn version 3.5 <210> 1 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> XA <222> (2)..(2) <223> Xaa is HArg <220> <221> XA <222> (5) <223> Xaa is HyP <220> <221> XA <222> (15) <223> Xaa is d1Nal <400> 1 Ala Xaa Asp Cys Xaa Leu Val Asn Pro Leu Cys Leu Glu Pro Xaa Trp 1 5 10 15 Thr Cys <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> XA <222> (2)..(2) <223> Xaa is tBuAla <220> <221> XA <222> (5) <223> Xaa is D-Lys(PYA) <220> <221> XA <222> (14)..(14) <223> Xaa is Nle <400> 2 Cys Xaa Pro Glu Xaa Pro Tyr Cys Phe Ala Asp Pro Tyr Xaa Cys Ala 1 5 10 15 <210> 3 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> XA <222> (2)..(2) <223> Xaa is tBuAla <220> <221> XA <222> (5) <223> Xaa is D-Lys(PYA) <220> <221> XA <222> (14)..(14) <223> Xaa is Nle <400> 3 Cys Xaa Pro Glu Xaa Pro Tyr Cys Phe Ala Asp Pro Tyr Xaa Cys Ala 1 5 10 15 <210> 4 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> XA <222> (2)..(2) <223> Xaa is tBuAla <220> <221> XA <222> (5) <223> Xaa is D-Lys(PYA) <220> <221> XA <222> (14)..(14) <223> Xaa is Nle <400> 4 Cys Xaa Pro Glu Xaa Pro Tyr Cys Phe Ala Asp Pro Tyr Xaa Cys Lys 1 5 10 15

Claims

1. An iso-tandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein the iso-tandem bicyclic peptide complex consists of: (a) A first peptide ligand that binds to EphA2, comprising a polypeptide and a molecular scaffold, wherein the polypeptide consists of the sequence A-[HArg]-DC i [HyP]LVNPL C ii LEP[d1Nal]WTC iii (SEQ ID NO: 1; BCY13118), and the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the molecular scaffold is compatible with the reactive cysteine ​​group (C i , C ii and C iii ) to form a covalent bond, so that two polypeptide rings are formed on the molecular scaffold; It was conjugated via an N-(acid-PEG3)-N-bis(PEG3-azide) linker to (b) two second peptide ligands that bind to CD137, each comprising a polypeptide and a molecular scaffold, wherein the polypeptide consists of the sequence Ac-C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii -A (SEQ ID NO: 2; BCY8928), and the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the molecular scaffold is compatible with the reactive cysteine ​​group (C i , C ii and C iii ) to form a covalent bond, so that two polypeptide rings 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 tert-butylalanine, PYA represents 4-pentenoic acid, and Nle represents norleucine.

2. The hetero-tandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof according to claim 1, which is BCY13272: or a pharmaceutically acceptable salt thereof.

3. The hetero-tandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof as defined in claim 1 or 2, wherein the pharmaceutically acceptable salt is selected from free acid or sodium, potassium, calcium or ammonium salt.

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

5. Use of the hetero-tandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the manufacture of a medicament for preventing, inhibiting or treating EphA2-related cancers; The EphA2-related cancer is selected from the group consisting of prostate cancer, lung cancer, colon cancer and fibroids.

6. Use of the hetero-tandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 in the manufacture of a medicament for treating EphA2-related cancer, wherein the treatment comprises administering the complex in a manner that does not maintain a plasma concentration of the complex above the in vitro EC of the complex. 50 Administering the heterotandem bicyclic peptide complex or a pharmaceutically acceptable salt thereof at a dosage frequency of The EphA2-related cancer is selected from the group consisting of prostate cancer, lung cancer, colon cancer and fibroids.

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