Hetero-tandem bicyclic peptide complex

Abstract

The present invention relates to an iso-tandem bicyclic peptide complex, which comprises a first peptide ligand that binds to adhesion protein-4, wherein the first peptide ligand is coupled to two second peptide ligands that bind to CD137 through a linker. The present invention also relates to the use of the iso-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 nectin-4, wherein the first peptide ligand is coupled 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] A combinatorial approach based on phage display has 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 combinatorial library of linear peptides containing three cysteine ​​residues and two regions of six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the invention

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

[0007] (a) A first peptide ligand that binds to adhesion protein 4, having a sequence C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1; BCY8116); which was coupled to the following via an N-(acid-PEG3)-N-bis(PEG3-azide) linker:

[0008] (b) Two second peptide ligands that bind to CD137, both of which 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, 1Nal represents 1-naphthylalanine, tBuAla represents tert-butylalanine, PYA represents 4-pentenoic acid, and Nle represents norleucine.

[0011] According to a further 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 : (A) The nectin-4 / CD137 heterotandem bicyclic peptide complex was analyzed in the Promega CD137 luciferase reporter assay in the presence of H292 cells expressing nectin-4. BCY11617 is a heterotandem bicyclic peptide complex that binds to nectin-4 with the same affinity as BCY11863, but does not bind to CD137. (B) Summary of the EC50 (nM) of BCY11863 in the Promega CD137 luciferase reporter assay co-cultured with different cell lines that endogenously express nectin-4 or are engineered to overexpress nectin-4.

[0014] Figure 2 : In a PBMC-4T1 co-culture assay, the adhesion protein-4 / CD137 heterotandem bicyclic peptide complex induces IFN-γ ( Figure 2 A) and IL-2 ( Figure 2 B) Cytokine secretion. 4T1 cells were engineered to express fibronectin-4. BCY11617 is a hetero-tandem bicyclic peptide complex that binds to fibronectin-4 with the same affinity as BCY11863, but does not bind to CD137. Figure 2C shows the summary of the EC50 (nM) of BCY11863 in cytokine secretion assays of multiple human PBMC donors and tumor cell lines.

[0015] Figure 3 : Pharmacokinetics of the heterotandem bicyclic peptide complex BCY11863 in SD rats and cynomolgus monkeys (cyno) administered intravenously at 2 mg / kg (n=3) and 1 mg / kg (n=2), respectively.

[0016] Figure 4 : Antitumor activity of BCY11863 in the MC38 tumor model (MC38#13) overexpressing adhesion protein-4 in syngeneic mice. Tumor volume during and after BCY11863 treatment. The number of complete responder (CR) mice at D69 is indicated in brackets. QD: daily dosing; Q3D: every three days dosing; ip: intraperitoneal administration.

[0017] Figure 5 : BCY11863 treatment leads to immunogenic memory against MC38 tumor cells overexpressing fibronectin-4 (MC38#13). Tumor volumes after inoculation of C57BL / 6J-hCD137 mice or mice with complete response (CR) to BCY11863. Note that at the end of the observation period (22 days), none of the CR mice developed tumors.

[0018] Figure 6 : BCY11863 shows antitumor activity in the mouse syngeneic CT26 tumor model (CT26#7) overexpressing adhesion protein-4. Tumor volume during BCY11863 treatment. Q3D: dosing every three days; ip: intraperitoneal administration.

[0019] Figure 7 : Total T cells and CD8+T cells increased in CT26#7 tumor tissue 1 hour after the last (6th) Q3D administration of BCY11863. (A) Total T cells, CD8+T cells, CD4+T cells, Tregs, and (B) CD8+T cell / Treg ratio in CT26#7 tumor tissue 1 hour after the last Q3D administration of BCY11863 were analyzed.

[0020] Figure 8 : Pharmacokinetic profile of BCY11863 in plasma and tumor tissue of CT26#7 syngeneic tumor-bearing animals after a single intravenous (iv) administration of 5 mg / kg BCY11863.

[0021] Fig. 9: Plot of plasma concentration versus time of BCY11863 and terminal plasma half-life of BCY11863 in CD-1 mice (n=3) dosed intraperitoneally with 15 mg / kg.

[0022] Fig.10 : Surface plasmon resonance (SPR) binding studies of BCY11863 with immobilized (A) nectin-4 and (B) CD137. Dual binding SPR assay (C) CD137 and (D) nectin-4 were first immobilized on the SPR chip and then BCY11863 was captured. The affinity of bound BCY11863 for soluble human nectin-4 (C) or CD137 (D) was measured by flowing different concentrations of soluble receptors through the chip. (E) Binding of BCY13582 (biotinylated BCY11863) to soluble human CD137 immobilized on a streptavidin SPR chip.

[0023] Fig.11 : Cellular microarray technology (Retrogenix) used to explore non-specific off-target interactions of BCY13582 (biotinylated BCY11863). The screening data shown here show that 1 μM BCY13582 was added to a microarray slide expressing 11 different proteins, which only bound to CD137 and adhesion protein-4 (detected using AlexaFluor647-labeled streptavidin). When incubated with BCY11863, the binding signal was displaced.

[0024] Fig.12 : Tumor growth curves of MC38#13 tumors in huCD137C57Bl / 6 mice demonstrate the antitumor activity of BCY11863 after different doses and dosing intervals. The number of complete responder animals (CR; no palpable tumors) at day 15 after the start of treatment is indicated in brackets.

[0025] Fig.13 : Tumor growth curves (mean ± SEM) of MC38#13 tumors (n=6 / cohort) in huCD137C57Bl / 6 mice demonstrate the antitumor activity of BCY11863 at different doses and dosing regimens. The number of complete responder animals (CR; no palpable tumors) at day 52 after the start of treatment is indicated in brackets. (A) Cohorts administered with vehicle or BCY11863 at a total weekly dose of 3 mg / kg. (B) Cohorts administered with vehicle or BCY11863 at a total weekly dose of 10 mg / kg. (C) Cohorts administered with vehicle or BCY11863 at a total weekly dose of 30 mg / kg.

[0026] Fig.14: Pharmacokinetics of the heterotandem bicyclic peptide complex BCY11863 in SD rats intravenously administered at 100 mg / kg (n=3), and concentrations of BCY11863 and potential metabolites BCY15155 and BCY14602 in plasma were measured. DETAILED DESCRIPTION

[0027] According to a first aspect of the present invention, there is provided an iso-tandem bicyclic peptide complex comprising:

[0028] (a) A first peptide ligand that binds to adhesion protein 4, having a sequence C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1; BCY8116); which was coupled to the following via an N-(acid-PEG3)-N-bis(PEG3-azide) linker:

[0029] (b) Two second peptide ligands that bind to CD137, both of which have the sequence Ac-C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii -A (SEQ ID NO: 2; BCY8928);

[0030] 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 ), 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 forms a covalent bond with the reactive cysteine ​​group of the polypeptide, so that two polypeptide rings are formed on the molecular scaffold;

[0031] 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.

[0032] The N-(acid-PEG3)-N-bis(PEG3-azide) mentioned herein includes:

[0033]

[0034] N-(acid-PEG3)-N-bis(PEG3-azide).

[0035] In one embodiment, the heterotandem bicyclic peptide complex is BCY11863:

[0036]

[0037] The complete details of BCY11863 are shown in Table A below:

[0038] Table A: Composition of BCY11863

[0039]

[0040] In this article Figure 1 The data provided in Table 1 show that BCY11863 exhibited strong CD137 activation in a CD137 reporter gene assay. Figure 2 The data presented in Table 2 show that BCY11863 induces robust IL-2 and IFN-γ cytokine secretion in PBMC co-culture assays with multiple tumor cell lines and human PBMC donors. Figure 3 The data provided in Table 5 show that BCY11863 exhibits excellent PK characteristics, with a terminal half-life of 4.1 hours in SD rats and 5.3 hours in cynomolgus monkeys. Fig.10 and Fig.11 Together with the data shown in Methods sections 11 and 12, the binding and exquisite selectivity of BCY11863 for its targets adhesion protein 4 and CD137 are demonstrated. Figure 4 and Figure 5 We demonstrated significant antitumor activity of BCY11863 in MC38#13 syngeneic mice and the formation of immunogenic memory after BCY11863 treatment. Figure 6 and Figure 7 The antitumor activity of BCY11863 in the CT26#7 syngeneic model was shown, with corresponding infiltration of cytotoxic T cells into the tumor. Fig.12 and Fig.13 It was clearly shown that 1.5 mg / kg BIW and 5 mg / kg dosing at 0 and 24 h over a week produced robust antitumor activity, so BCY11863 was not necessary to maintain measurable plasma concentrations.

[0041] Certain analogs (ie modified derivatives) and metabolites of BCY11863 are mentioned herein, each of which forms a further aspect of the invention and are summarised in Table B below:

[0042] Table B: Composition of BCY11863 analogs and metabolites

[0043]

[0044]

[0045] BCY14601 represents a protein with sequence C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii -A (SEQ ID NO: 3) bicyclic peptide ligand, TATA as a molecular scaffold;

[0046] 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) bicyclic peptide ligand, TATA as the molecular scaffold.

[0047] 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.

[0048] the term

[0049] serial number

[0050] When referring to amino acid residue positions within the compounds of the invention, cysteine ​​residues (C i , C ii and C iii ) remains unchanged and is omitted from the numbering, therefore, the numbering of the amino acid residues in SEQ ID NO: 1 is as follows:

[0051] C i -P1-1Nal2-dD3-C ii -M4-HArg5-D6-W7-S8-T9-P 10 -HyP 11 -W 12 -C iii (SEQ ID NO: 1).

[0052] 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.

[0053] Molecular form

[0054] 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:

[0055] βAla-Sar10-A-(SEQ ID NO:X).

[0056] Reverse peptide sequence

[0057] 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.

[0058] Advantages of peptide ligands

[0059] 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:

[0060] - Species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations;

[0061] - 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;

[0062] - 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;

[0063] - Selectivity. Certain heterotandem bicyclic peptide complexes of the present invention exhibit good selectivity over other targets;

[0064] - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop heterotandem bicyclic peptide complexes with short exposure in an acute disease management setting; or to develop heterotandem bicyclic peptide complexes with enhanced retention in the circulation, which are therefore optimal for the management of more chronic disease states. Other factors driving the ideal plasma half-life are the requirement for sustained exposure for maximum therapeutic efficacy versus the toxicity that accompanies sustained exposure to the agent.

[0065] Crucially, data are provided herein in which the heterotandem bicyclic peptide complexes of the invention are not maintained at plasma concentrations above the in vitro EC of the compound. 50 When the frequency of administration is high, anti-tumor efficacy is shown. This is in contrast to the larger recombinant biological (i.e. antibody-based) methods of 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 attributed to the fact that the hetero-tandem bicyclic complex has a relatively low molecular weight (usually <15 kDa), they are fully synthesized, and they are tumor-targeted agonists of 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 with humanized CD13, daily or every 3 days showed antitumor efficacy. In addition, intraperitoneal pharmacokinetic data showed a plasma half-life of <3 hours, which would predict that the circulating concentration of the complex would continue to decline between doses to below the in vitro EC 50 Furthermore, tumor pharmacokinetic data suggest that levels of the heterotandem bicyclic complex may be higher and more sustained in tumor tissue compared with plasma levels.

[0066] 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 an iso-tandem bicyclic peptide complex as defined herein in a manner that does not maintain a plasma concentration of said complex above the in vitro EC of said complex. 50 The dosing frequency is .

[0067] -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. The data presented herein demonstrate that the hetero-tandem bicyclic peptide complexes of the invention not only eradicate tumors, but also that after re-administration of the tumorigenic agent, the inoculated complete responder mice did not develop tumors (see Figure 5 ). This indicates that treatment with the selected heterotandem bicyclic peptide complexes of the present invention has induced immunogenic memory in complete responder mice. This has significant clinical advantages to prevent the tumor from recurring after it has been initially controlled and eradicated.

[0068] Peptide ligand

[0069] 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 present opposite to 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 the present 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.

[0070] Pharmaceutically acceptable salts

[0071] It will be appreciated that salt forms are within the scope of the present invention and reference to a peptide ligand includes salt forms of the ligand.

[0072] 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.

[0073] 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.

[0074] A particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthylsulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. A particular salt is the hydrochloride. Another particular salt is the acetate.

[0075] 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., NH4 + ) and substituted ammonium ions (e.g. NH3R + NH2R2+ 、NHR3 + and NR4 + ). 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(CH3)4 + .

[0076] 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.

[0077] Modified derivatives

[0078] It will be appreciated 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, and replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with alternative bonds; modification of the length of the peptide backbone; replacement of hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamic acid / aspartic acid and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introduction or substitution of amino acids suitable for functionalization with orthogonal reactivity, such as amino acids with azide or alkynyl groups, which allow functionalization with moieties with alkynyl or azido groups, respectively.

[0079] 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.

[0080] 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 capped with acetic anhydride or other suitable reagents to produce N-terminally acetylated molecules. This embodiment provides the advantage of removing potential recognition points for aminopeptidases and avoids the possibility of degradation of the bicyclic peptide.

[0081] In an alternative embodiment, the N-terminal modification includes the addition of a molecular spacer group that facilitates the coupling of the effector group and maintains the potency of the bicyclic peptide towards its target.

[0082] 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-terminally acetylated 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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, the correct combination and quantity of charged and hydrophobic amino acid residues can reduce the stimulation at the injection site (if the peptide drug has been subcutaneously administered).

[0088] 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).

[0089] 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.

[0090] 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:

[0091] - Incorporation of hydrophobic moieties that exploit hydrophobic interactions and result in lower off-rates, allowing higher affinity to be achieved;

[0092] - incorporation of charged groups that exploit long-range ionic interactions, resulting in faster on-rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0093] - 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.

[0094] (For review, see Gentilucci et al., Curr. Pharmaceutical Design (2010) 16, 3185-203 and Nestor et al. (2009), Curr. Medicinal Chem 16, 4399-418).

[0095] Isotopic variants

[0096] 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 isotopically labeled functional groups.

[0097] 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 64 Cu, 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.

[0098] Certain isotope-labeled peptide ligands of the present 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 adhesion protein-4 target on diseased tissues. The peptide ligands of the present invention can further have valuable diagnostic properties, which 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 14 C, is particularly useful for this purpose due to its ease of incorporation and readily available detection methods.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] synthesis

[0103] 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).

[0104] 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.

[0105] When making a conjugate or complex, amino acid residues in the target polypeptide may be optionally substituted.

[0106] The peptide may also be extended to incorporate, for example, another loop and thereby introduce multiple specificities.

[0107] 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- or C-terminus or within a ring. Standard (bio) coupling techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, addition can be made 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 by enzyme addition, such as using subtiligase, such as (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91 (26): 12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters Vol. 18, No. 22, November 15, 2008, pp. 6000-6003).

[0108] Alternatively, the peptide may be extended or modified by further coupling 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.

[0109] Similar techniques are also used for the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[0110] Furthermore, other functional groups or effector groups can 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.

[0111] Pharmaceutical composition

[0112] 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.

[0113] 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.

[0114] 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).

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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 in similar or slightly lower doses.

[0119] 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 to kill, consume or otherwise effectively remove target cell populations from heterogeneous cell collections in vitro or in vitro. Blood from mammals can be combined in vitro with selected peptide ligands, thereby killing or otherwise removing undesirable cells from the blood for return to mammals according to standard techniques.

[0120] Therapeutic Uses

[0121] 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.

[0122] 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 various types of carcinomas, including adenocarcinomas, squamous 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), gallbladder and biliary system cancer, 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., tongue cancer, buccal cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), 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, and hyperplastic nevus); hematologic malignancies (i.e., leukemias and 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, hematologic malignancies and related conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilia, 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 sarcomas, bone or chondrosarcomas 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 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); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor);or congenital or other syndromes that predispose the patient to malignancy (e.g. xeroderma pigmentosum). ;

[0123] 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), chronic B 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).

[0124] 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.

[0125] 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.

[0126] The present invention is further described below with reference to the following examples.

[0127] Example

[0128] Generally, the hetero-tandem bicyclic peptide complex of the present invention can be prepared according to the following general method:

[0129]

[0130] All solvents were degassed and purged with N2 3 times. A DMF solution of BP-23825 (1.0 eq), HATU (1.2 eq) and DIEA (2.0 eq) was mixed for 5 minutes, and then bicyclic 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.

[0131] A mixture of intermediate 2 (1.0 eq) and bicyclic 2 (2.0 eq) was dissolved in t-BuOH / H2O (1:1), followed by the addition of CuSO4 (1.0 eq), VcNa (4.0 eq) and THPTA (2.0 eq). Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. The reaction mixture was stirred at 40°C for 16 hours under N2 atmosphere. The reaction mixture was purified directly by preparative HPLC.

[0132] The following provides more detailed experiments of the hetero-tandem bicyclic peptide complex of the present invention:

[0133] Example 1: Synthesis of BCY11863

[0134]

[0135] Procedure for preparation of BCY12476

[0136]

[0137] A mixture of N-(acid-PEG3)-N-bis(PEG3-azide) (70.0 mg, 112.2 μmol, 1.0 eq), HATU (51.2 mg, 134.7 μmol, 1.2 eq) and DIEA (29.0 mg, 224.4 μmol, 40 μL, 2.0 eq) was dissolved in DMF (2 mL) and mixed for 5 min. BCY8116 (294.0 mg, 135.3 μmol, 1.2 eq) was then added. The reaction mixture was stirred at 40° C. for 16 hours. LC-MS showed a major peak with the desired m / z. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by preparative HPLC. BCY12476 (194.5 mg, 66.02 μmol, 29% yield, 94% purity) was obtained as a white solid. Calculated MW: 2778.17, observed m / z: 1389.3 ([M+2H] 2+ ),926.7([M+3H] 3+ ).

[0138] Procedure for preparation of BCY11863

[0139]

[0140] First, a mixture of BCY12476 (100.0 mg, 36.0 μmol, 1.0 eq) and BCY8928 (160.0 mg, 72.0 μmol, 2.0 eq) was dissolved in 2 mL t-BuOH / H2O (1:1), and then CuSO4 (0.4 M, 180 μL, 1.0 eq) and VcNa (28.5 mg, 143.8 μmol, 4.0 eq) and THPTA (31.2 mg, 71.8 μmol, 2.0 eq) were added. Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. All solvents were degassed and purged with N2. The reaction mixture was stirred at 40 ° C for 16 hours under N2 atmosphere. LC-MS showed that BCY8928 was retained and the desired m / z was detected. The reaction mixture was purified directly by preparative HPLC. The first purification gave BCY11863 (117.7 mg, 15.22 μmol, 42.29% yield, 93.29% purity) as a TFA salt, while the less pure fraction was purified again by preparative HPLC to yield BCY11863 (33.2 mg, 4.3 μmol, 12% yield, 95% purity) as a TFA salt. Calculated MW: 7213.32, observed m / z: 1444.0 ([M+5H] 5+ ).

[0141] Example 2: Synthesis of BCY13390

[0142]

[0143] Procedure for preparation of BCY13689

[0144]

[0145] A mixture of BCY12476 (47.0 mg, 16.91 μmol, 1.0 eq), BCY8928 (30.0 mg, 13.53 μmol, 0.8 eq) and THPTA (36.7 mg, 84.55 μmol, 5.0 eq) was dissolved in t-BuOH / H2O (1:1, 8 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 21.0 μL, 0.5 eq) and VcNa (67.0 mg, 338.21 μmol, 20.0 eq) were added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned light yellow. The reaction mixture was stirred at 25°C for 1.5 hours under N2 atmosphere. LC-MS showed that some BCY12476 was retained, BCY8928 was completely consumed, and a peak of the desired m / z was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The crude product was purified by preparative HPLC to obtain BCY13689 (25.3 mg, 4.56 μmol, 27% yield, 90% purity) as a white solid. Calculated MW: 4995.74, observed m / z: 1249.4 ([M+4H] 4+ ),999.9([M+5H] 5+ ).

[0146] Procedure for preparation of BCY13390

[0147]

[0148] A mixture of BCY13689 (43.6 mg, 8.73 μmol, 1.0 eq), BCY13389 (20.8 mg, 9.16 μmol, 1.05 eq) and THPTA (3.8 mg, 8.73 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 22.0 μL, 1.0 eq) and VcNa (3.5 mg, 17.45 μmol, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned light yellow. The reaction mixture was stirred at 25°C for 2 hours under N2 atmosphere. LC-MS showed a significant peak corresponding to the desired m / z. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC to obtain BCY13390 (33.8 mg, 4.21 μmol, 48% yield, 90% purity) as a white solid. Calculated MW: 7270.41, observed m / z: 1454.9 ([M+5H] 5+),1213.2([M+6H] 6+ ).

[0149] Example 3: Synthesis of BCY13582

[0150]

[0151] Procedure for preparation of BCY13582

[0152]

[0153] A mixture of BCY13390 (5.0 mg, 0.6 μmol, 1.0 eq) and biotin-PEG12-NHS ester (CAS 365441-71-0, 0.7 mg, 0.72 μmol, 1.1 eq) was dissolved in MeCN / H2O (1:1, 2 mL). The pH of the solution was adjusted to 8 by the dropwise addition of 1.0 M NaHCO3. The reaction mixture was stirred at 25°C for 0.5 h. LC-MS showed that BCY13390 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 BCY13582 (2.5 mg, 0.30 μmol, 43% yield, 96% purity) as a white solid. Calculated MW: 8096.43, observed m / z: 1351.1 ([M+6H] 6+ ),1158.5([M+7H] 7+ ).

[0154] Example 4: Synthesis of BCY13583

[0155]

[0156] Procedure for preparation of BCY13583

[0157]

[0158] BCY13390 (15.0 mg, 2.06 μmol, 1.0 eq) and Alexa A mixture of 488NHS esters (2.5 mg, 4.12 μmol, 2.0 eq) was dissolved in DMF (0.5 mL). DIEA (2.6 mg, 20.63 μmol, 3.6 μL, 10 eq) was then added dropwise. The reaction mixture was stirred at 25 °C for 1 hour. LC-MS showed that BCY13390 was retained and a major peak with the desired m / z was detected. Additional Alexa Fluor 488NHS ester (2.0 mg, 3.09 μmol, 1.5 eq) was added to the reaction mixture and the reaction mixture was stirred at 25 °C for another hour. HPLC showed complete consumption of BCY13390. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The crude product was purified by preparative HPLC to obtain BCY13583 (5 mg, 0.61 μmol, 29% yield, 95% purity) as a red solid. Calculated MW: 7787.9, observed m / z: 1948.8 ([M+4H+H2O] 4+ ),1558.6([M+5H+H2O] 5+ ),1299.1([M+7H+H2O] 7+ ).

[0159] Example 5: Synthesis of BCY13628

[0160]

[0161] Procedure for preparation of BCY13628

[0162]

[0163] A mixture of BCY13390 (5.6 mg, 0.77 μmol, 1.0 eq) and cyanine 5NHS ester (0.5 mg, 0.85 μmol, 1.1 eq) was dissolved in MeCN / H2O (1:1, 2 mL). The pH of the solution was adjusted to 8 by the dropwise addition of 1.0 M NaHCO3. The reaction mixture was stirred at 25°C for 0.5 h. LC-MS showed that BCY13390 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 BCY13628 (2.9 mg, 0.36 μmol, 46% yield, 95% purity) as a blue solid. Calculated MW: 7736.06, observed m / z: 1289.9 ([M+6H] 6+ ),1105.5([M+7H] 7+ ).

[0164] Example 6: Synthesis of BCY15155

[0165]

[0166] Procedure for preparation of BCY15155

[0167]

[0168] A mixture of BCY13689 (25.0 mg, 5.00 μmol, 1.0 eq), BCY14601 (13.0 mg, 6.01 μmol, 1.2 eq) and THPTA (2.0 mg, 5.00 μmol, 1.0 eq) was dissolved in t-BuOH / 0.2 M NH4HCO3 (1:1, 0.5 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 12.5 μL, 1.0 eq) and Vc (3.5 mg, 20.02 μmol, 4.0 eq) were added under N2. The pH of the solution was adjusted to 8 and the solution turned light yellow. The reaction mixture was stirred at 25°C under N2 atmosphere for 2 hours. LC-MS showed that BCY13689 was completely consumed, some 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 BCY15155 (19.7 mg, 2.41 μmol, 36% yield, 97% purity) as a white solid. Calculated MW: 7171.3, observed m / z: 1434.7 ([M+5H] 5+ ),1196.2([M+6H] 6+ ).

[0169] Example 7: Synthesis of BCY14602

[0170]

[0171] Procedure for preparing BCY14602

[0172]

[0173] A mixture of BCY12476 (100.0 mg, 36.00 μmol, 1.0 eq), BCY14601 (158.0 mg, 72.63 μmol, 2.04 eq) and THPTA (15.6 mg, 36.00 μmol, 1.0 eq) was dissolved in t-BuOH / 0.2 M NH4HCO3 (1:1, 2 mL, pre-degassed and purged with N2), and then CuSO4 (0.4 M, 89.0 μL, 1.0 eq) and VcNa (28.5 mg, 143.98 μmol, 4.0 eq) were added under N2. The pH of the solution was adjusted to 8 and the solution turned light yellow. THPTA and VcNa were supplemented twice and the solution was stirred for a total of 48 hours at 25°C under N2 atmosphere. LC-MS showed that BCY12476 was completely consumed, BCY14601 was retained, and a major peak with the desired m / z was detected. Some by-products were also 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 BCY14602 (45.2 mg, 5.51 μmol, 15% yield, 86% purity) as a white solid. Calculated MW: 7129.2, observed m / z: 1426.6 ([M+5H] 5+ ),1189.1([M+6H] 6+ ).

[0174] Analyze the data

[0175] The following hetero-tandem bicyclic peptide complexes of the present invention were analyzed using mass spectrometry and HPLC. The HPLC settings are as follows:

[0176] Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN

[0177] Flow rate: 1.0ml / min

[0178] Column: Gemini-NX C18 5um 110A 150*4.6mm

[0179] Instrument: Agilent 1200HPLC-BE(1-614)

[0180] The gradient used was 30-60% B in 20 min and the data generated were as follows:

[0181] Complex ID Analytical Data - Mass Spectrometry HPLC retention time (min) BCY11863 MW: 7213.32, observed m / z: 1444.0 ([M / 5+H]+) 10.649

[0182] Biological data

[0183] 1. CD137 reporter gene assay in co-culture with tumor cells

[0184] Prepare 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 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 the cells and test article at 37°C, 5% CO2 for 6 hours. At the end of 6 hours, add 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).

[0185] The tumor cell types used in the co-culture were NCI-H292, CT26#7, MC38#13, HT1376, NCI-H322, and T47D, which have been shown to express fibronectin-4.

[0186] Figure 1 The data presented in A show that in a CD137 reporter gene assay, the fibronectin-4 / CD137 heterotandem (BCY11863) induced strong CD137 activation and that the activation was dependent on the binding of the heterotandem to CD137. The CD137 bicyclic peptide in the BCY11617 molecule is composed entirely of D-amino acids to abrogate binding and does not induce CD137 agonism.

[0187] Table 1 below reports the ECs induced by the heterotandem bicyclic peptide complex BCY11863 and close analogs in a CD137 reporter assay co-cultured with tumor cell lines expressing fibronectin-4. 50 (nM) and in Figure 1 Visualized in B. The data demonstrate the potential of BCY11863 to induce CD137 agonism when co-cultured with a range of cell lines expressing fibronectin-4.

[0188] Table 1: EC50 (nM) of fold induction relative to background induced by fibronectin-4 / CD137 heterotandem bicyclic peptide complexes in CD137 reporter gene assay

[0189] Complex ID Tumor cell line species Cell lines used in co-culture <![CDATA[EC 50 Arithmetic mean (nM)]]> BCY11863 Mouse CT26#7 0.14±0.07 BCY11863 Mouse MC38#13 0.31±0.26 BCY11863 people NCI-H292 0.28±0.20 BCY11863 people HT1376 0.52±0.30 BCY11863 people NCI-H322 0.33±0.21 BCY11863 people T47D 0.42±0.24 BCY11863 people MDA-MB-468 0.23±0.01 BCY13582 people HT1376 0.58±0.27 BCY13582 people MDA-MB-468 0.34±0.02 BCY13583 people HT1376 1.7±0.9 BCY13583 people MDA-MB-468 0.84±0.07

[0190] 2. Human PBMC Co-culture (Cytokine Release) Assay

[0191] Human and mouse tumor cell lines were cultured according to the supplier's recommendations. Frozen PBMCs from healthy human donors were thawed and washed once in room temperature PBS and then resuspended in R10 medium. 100 μl PBMCs (1,000,000 PBMCs / ml) and 100 μl tumor cells (100,000 tumor cells / ml) (effector cell: target cell ratio (E:T) 10:1) were plated in each well of a 96-well flat-bottom plate for co-culture assays. 100 ng / ml soluble anti-CD3 mAb (clone OKT3) was added to the culture on day 0 to stimulate human PBMCs. Test, control compounds or vehicle controls were diluted in R10 medium and 50 μL was added to each well to a final volume of 250 μL per well. The plate was covered with a breathable film and incubated in a humidified chamber at 37°C, 5% CO2 for three days. Supernatants were collected 48 hours after stimulation and human IL-2 and IFN-γ were detected using 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, the plate was washed 3 times. The biotin mixture was then added to the plate and shaken for 1 hour at RT. Using a magnetic stand, the plate was washed 3 times. The streptavidin mixture was added to the plate and shaken for 30 minutes at RT. Using a magnetic stand, the plate was washed 3 times, resuspended in 100 μL wash buffer, shaken at RT for 2 minutes, and then read using a Luminex 2000. Raw data was analyzed using the built-in Luminex software to generate a standard curve and interpolate protein concentrations, and all other data analysis and plotting was performed using Excel and Prism software. The data represent a study testing 3-5 independent donor PBMCs in triplicate technical repeats.

[0192] Figure 2 A and Figure 2 The data presented in B show that the fibronectin-4 / CD137 heterotandem (BCY11863) induces robust IL-2 and IFN-γ cytokine secretion in a PBMC-4T1 co-culture assay. BCY11617 is a negative control that binds fibronectin-4 but not CD137.

[0193] Table 2 below reports the ECs induced by selected fibronectin-4 / CD137 heterotandem bicyclic peptide complexes in a human PBMC co-culture (cytokine release) assay. 50 (nM) and maximum IFN-γ cytokine secretion (pg / ml) and are summarized in Figure 2 Visualized in C. This demonstrates the potential of BCY11863 to induce cytokine secretion in the presence of a number of different tumor cell lines expressing cohesin-4.

[0194] Table 2: Selected ECs that secrete IFN-γ cytokines induced by fibronectin-4 / CD137 heterotandem bicyclic peptide complexes in human PBMC-4T1 co-culture (cytokine release) assay 50

[0195]

[0196] 3. Pharmacokinetics of the fibronectin-4 / CD137 heterotandem BCY11863 in SD rats

[0197] The adhesion protein-4 / CD137 hetero-tandem BCY11863 formulated in 25mM histidine HCl, 10% sucrose pH 7 was administered to male SD rats by IV push, IV infusion (15 minutes) or subcutaneously. Serial bleeding was performed through the submandibular vein or saphenous vein at each time point (approximately 80μL blood / 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 using Orbitrap Q Exactive 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 non-compartmental methods using Phoenix WinNonlin6.3 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 the experiment are shown in Table 3:

[0198] Table 3: Pharmacokinetic parameters in SD rats

[0199]

[0200] Table 3 and Figure 5The data in suggest that BCY11863 is a low clearance molecule with a volume of distribution greater than the plasma volume. In addition, SC-administered BCY11863 has high bioavailability in rats.

[0201] Table 4: Pharmacokinetic parameters of BCY11863 and potential metabolites in SD rat PK study following 100 mg / kg IV administration

[0202]

[0203] Table 4 and Fig.14 The data in show that following IV administration of BCY11863 to SD rats, <1% of BCY11863 was metabolized to BCY15155. No significant conversion to BCY14602 was noted within the first 24 hours of the study.

[0204] 4. Pharmacokinetics of the fibronectin-4 / CD137 heterotandem BCY11863 in cynomolgus monkeys

[0205] 1 mg / kg of the adhesion protein-4 / CD137 heterotandem BCY11863 formulated in 25 mM histidine HCl, 10% sucrose pH 7 was administered to non-naive cynomolgus monkeys by intravenous infusion (15 or 30 min) into the cephalic vein. Serial bleeding (approximately 1.2 ml blood / time point) was performed through a peripheral blood vessel of restrained, non-sedated animals at each time point, and the blood was placed in a commercially available tube containing potassium (K2)-EDTA*2H2O (0.85-1.15 mg), placed on wet ice and processed to obtain plasma. The samples were centrifuged immediately after collection (3,000×g, 10 minutes at 2 to 8°C). 0.1 mL of plasma was transferred to a labeled polypropylene microcentrifuge tube. Immediately add 5 times of precipitant (MeOH solution 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) to plasma, mix thoroughly and centrifuge at 2 to 8°C, 12,000rpm for 10 minutes. Transfer supernatant samples to pre-labeled polypropylene microcentrifuge tubes and freeze on dry ice. Samples are stored at -60°C or below until LC-MS / MS analysis. Aliquots of 40 μL calibration standards, quality control, single blank and double blank samples are added to 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 supernatant was injected using Orbitrap Q Exactive 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 the two bispecific compounds are shown in Table 5.

[0206] Table 5: Pharmacokinetic parameters in cynomolgus monkeys

[0207]

[0208]

[0209] Figure 3Plasma concentration versus time curves for 2 mg / kg IV dosing in SD rats (n=3) and 1 mg / kg IV infusion of BCY11863 in cynomolgus monkeys (n=2) are shown. BCY11863 has a steady-state distribution volume (Vdss) of 1.6 L / kg in rats and a clearance of 7.7 mL / min / kg, resulting in a terminal half-life of 4.1 h. BCY11863 has a steady-state distribution volume (Vdss) of 0.62 L / kg in cynomolgus monkeys and a clearance of 3.3 mL / min / kg, resulting in a terminal half-life of 5.3 h. Subsequent studies are consistent with these results. The PK parameters of the IV study in cynomolgus monkeys indicate that it is a low plasma clearance molecule with a distribution volume similar to total body water.

[0210] 5. Pharmacokinetics of the fibronectin-4 / CD137 heterotandem BCY11863 in CD1 mice

[0211] 15 mg / kg of the adhesion protein-4 / CD137 hetero-tandem BCY11863 formulated in 25 mM histidine HCl, 10% sucrose pH 7 was administered to 6 CD-1 mice by intraperitoneal or IV administration. Serial bleeding was performed through the submandibular vein or saphenous vein at each time point (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 to 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. 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.

[0212] Fig. 9 Shown are graphs of plasma concentration versus time of BCY11863 and the terminal plasma half-life of BCY11863 in CD1 mice (n=3) dosed IP at 15 mg / kg.

[0213] Table 6: Pharmacokinetic parameters in CD-1 mice

[0214]

[0215] Fig. 9 The data in Table 6 above indicate that BCY11863 can be administered to mice by IV bolus and IP. IP-administered BCY11863 has high bioavailability in mice. The PK parameters of the IV study indicate that it is a low clearance molecule with a distribution volume greater than the plasma volume.

[0216] 6. Antitumor activity of BCY11863 in the MC38 tumor model (MC38#13) with syngeneic overexpression of adhesion protein 4 sex

[0217] 1 × 10 cells were inoculated into the flank of 6-8 week old C57BL / 6J-hCD137 female mice. 6 MC38 cells (MC38#13) that overexpress fibronectin-4. When the tumors reached an average of 72 mm 3 Mice were randomized to receive vehicle or BCY11863 (administered intraperitoneally) at 1 mg / kg or 10 mg / kg daily (QD) or every three days (Q3D) (n=6 mice / treatment cohort). Mice dosed QD received 16 doses of BCY11863, and mice dosed Q3D received 10 doses of BCY11863. Tumor growth was monitored by caliper measurements until day 69 after treatment initiation. The results of this experiment can be seen in Figure 4 , where by day 7, a significant reduction in tumor growth was observed in both treatment cohorts (p<0.05, 2-way ANOVA with Dunnett's test for multiple comparisons), with all treatment groups significantly different from the vehicle group by day 14. By day 48, 22 of 24 BCY11863-treated animals had a complete response to treatment and no residual palpable tumors remained.

[0218] Based on the circulating plasma half-life of BCY11863 after IP injection in mice (2.5h), plasma trough levels would be close to 0 after BCY11863 doses (1 and 10 mg / kg) and dosing intervals (QD and Q3D), demonstrating that intermittent dosing of BCY11863 with less than continuous plasma exposure is sufficient to result in significant antitumor activity leading to durable complete responses.

[0219] 7. BCY11863 treatment leads to immunogenic memory in the MC38 tumor model overexpressing fibronectin-4

[0220] On day 69, five animals that fully responded to BCY11863 treatment were re-inoculated with 1 × 10 6 MC38#13 cells. A cohort of 5 naive C57BL / 6J-hCD137 female mice were inoculated with 1×10 6 MC38#13 cells were used as controls. The results of this experiment can be seen in Figure 5By day 13 after vaccination, all five naive C57BL / 6J-hCD137 female mice developed tumors, whereas none of the fully responding mice developed tumors, suggesting that animals that achieved a complete anti-tumor response due to BCY11863 treatment had developed immunogenic memory.

[0221] 8. BCY11863 showed anti-tumor activity in a syngeneic CT26 tumor model (CT26#7) that overexpressed adhesion protein 4. Tumor activity

[0222] 3 × 10 cells were inoculated into the flank of 6-8 week old BALB / c-hCD137 female mice. 5 CT26 cells that overexpress fibronectin-4 (CT26#7) were syngeneically grown. When the tumors reached an average of about 70 mm 3 At 4 dpi, mice were randomized to receive vehicle or 5 mg / kg BCY11863 intraperitoneally every three days (for a total of 6 doses). Tumor growth was monitored by caliper measurement until day 14 after the start of treatment. The results of this experiment can be seen in Figure 6 , where BCY11863 treatment significantly (p<0.0001, Student's t-test) reduced tumor growth from day 7 onwards.

[0223] Based on the circulating plasma half-life of BCY11863 after IP injection in mice (2.5 h), plasma exposure would be discontinuous throughout the dosing period, demonstrating that less than continuous plasma exposure of BCY11863 is sufficient to result in significant antitumor activity.

[0224] 9. One hour after the last (sixth) Q3D administration of BCY11863, the total number of T cells and Increased CD8+ T cells

[0225] One hour after the last administration of vehicle or BCY11863, CD26#7 tumor-bearing mice were sacrificed and tumors were harvested and processed to obtain single-cell suspensions, which were stained for flow cytometric analysis of total T cells (CD45+CD3+), CD8+ T cells (CD45+CD3+CD8+), CD4+ T cells (CD45+CD3+CD4+), and regulatory T cells (Treg; CD45+CD3+CD4+Foxp3+). The results of this experiment can be seen in Figure 7 , where it can be seen that BCY11863 treatment resulted in a significant increase in the total number of T cells (p<0.0001, Student's t-test) and CD8+T cells (p<0.0001, Student's t-test), as well as a significant increase in the ratio of CD8+T cells / Treg (p<0.05, Student's t-test).

[0226] This suggests that after intermittent dosing, BCY11863 treatment can lead to increased local levels of T cells in tumor tissue.

[0227] 10. After a single intravenous (iv) administration of 5 mg / kg BCY11863, BCY11863 was Pharmacokinetic characteristics of the drug in plasma and tumor tissue

[0228] 3 × 10 cells were inoculated into the flank of 6-8 week old female BALB / c mice. 5 CT26 cells that overexpress fibronectin-4 are syngeneically expressed (CT26#7). When the tumors reached an average of 400 mm 3 At 400 μL of age, mice were randomized to receive a single intravenous dose of vehicle or 5 mg / kg BCY11863. A cohort of mice (n=3 / time point) was sacrificed at 0.25, 0.5, 1, 2, 4, 8, and 24 h time points, and plasma and tumor tissues were harvested for BCY11863 analysis. For tumor BCY11863 content analysis, tumor homogenates were prepared by homogenizing tumor tissue with 10 volumes (w:v) of homogenization solution (MeOH / 15 mM PBS (1:2, v:v)). 40 μL of sample was quenched with 200 μL of IS1, and the mixture was vortexed at 800 rpm for 10 min and centrifuged at 4°C, 3220 g for 15 min. The supernatant was transferred to another clean 96-well plate, centrifuged at 4°C, 3220 g for 5 min, and 10.0 μL of supernatant was injected for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte. For analysis of plasma BCY11863 content, blood samples were collected in K2-EDTA tubes and immediately processed to plasma by centrifugation at approximately 4°C, 3000g. 40 μL of plasma samples were quenched with 200 μL of IS1, and the mixture was vortexed at 800 rpm for 10 min and centrifuged at 4°C, 3220g for 15 min. The supernatant was transferred to another clean 96-well plate, centrifuged at 4°C, 3220g for 5 min, and then 10.0 μL of the supernatant was injected for LC-MS / MS analysis using Orbitrap Q Exactive in positive ion mode to determine the concentration of the analyte.

[0229] The results of this experiment are shown in Figure 8 In BCY11863 plasma T 1 / 2 (1.65h) and tumor T 1 / 2 As shown by the difference in (13.4 h), it can be seen that BCY11863 is retained in tumor tissue after plasma BCY11863 is eliminated from the circulation.

[0230] 11. BCY11863 binds to fibronectin-4 and CD137 in four preclinical species

[0231] The binding of BCY11863 to its major targets, adhesion protein 4 and CD137, was characterized using surface plasmon resonance (SPR).

[0232] (a) Adhesion protein-4

[0233] BCY11863 bound to cynomolgus monkey, rat, mouse, and human fibronectin-4 as measured by direct binding to the extracellular domain that had been biotinylated and captured on the surface of a streptavidin sensor chip with a K D Between 5-27nM.

[0234] Table 7: Binding affinity of BCY11863 to biotinylated fibronectin-4 extracellular domain: SPR data

[0235]

[0236]

[0237] In order to understand whether the binding of BCY11863 to fibronectin-4 is altered in the context of a ternary complex (i.e., when also bound to CD137), a multicomponent SPR binding assay was developed. BCY11863 was first captured with human CD137 immobilized on the surface of an SPR chip, and then fibronectin-4 from different species was passed over the chip to determine their affinity for the captured BCY11863 (see Fig.10 C). Affinity for fibronectin-4 is generally maintained in the presence of CD137 binding as shown below:

[0238] Table 8: Binding affinity of BCY11863 to the extracellular domain of fibronectin-4 using biotinylated human CD137 as capture reagent

[0239]

[0240] (b) CD137

[0241] Direct binding of BCY11863 to surface-bound CD137 cannot be accurately measured by SPR because the avidity generated by the two CD137-binding double loops in BCY11863 results in an extremely slow k off (See Fig.10 B). In addition, biotinylation of cynomolgus CD137 abolished the binding of BCY11863, likely due to modification of a lysine on the cynomolgus protein that is important for BCY11863 binding. Therefore, a BCY11863 analog (BCY13582) containing a C-terminal biotinylated lysine was tested in SPR to determine the cross-species specificity of BCY11863. BCY13582 was captured onto a sensor chip using a reversible biotin capture kit and the affinity for adhesion protein-4 from different species was determined. Both strategies showed that these BCY11863 analogs bound to human and cynomolgus CD137 with K D <10 nM, with negligible binding to mouse and rat CD137.

[0242] Table 9: Binding affinity of biotinylated BCY11863 analogs to the CD137 extracellular domain: SPR data

[0243]

[0244] To understand whether the binding of BCY11863 to CD137 is altered in the context of a ternary complex (i.e., when also bound to fibronectin-4), a dual binding SPR binding assay was developed. BCY11863 was first captured with human fibronectin-4 immobilized on the surface of an SPR chip, and then soluble CD137 from different species was passed over the chip to determine their affinity to the captured BCY11863 (see Fig.10 D). Affinity for CD137 is generally maintained in the presence of fibronectin-4 binding as shown below:

[0245] Table 10: Binding affinity of BCY11863 to CD137 ECD using biotinylated human fibronectin-4 as capture reagent

[0246]

[0247] Fig.10 A shows an exemplary sensorgram demonstrating that BCY11863 binds to adhesion protein 4 (human) with an affinity of 4.1 nM. Fig.10 B shows the sensorgram of BCY11863 binding to CD137 (human) with high affinity. Due to the presence of two CD137 binding double loops in BCY11863, the dissociation rate from the immobilized CD137 protein is very slow, and the reported K D May be overestimated ( Fig.10 B). Fig.10 C shows that BCY11863 binds to cohesin-4, while the CD137 arm binds to the CD137 protein immobilized on the chip to form a ternary complex. Fig.10 D shows that BCY11863 binds to CD137, while the fibronectin-4 binding arm binds to the fibronectin-4 protein immobilized on the chip to form a ternary complex. Fig.10 E shows the ability of BCY13582 immobilized on the SPR chip to bind to human CD137.

[0248] 12. Selectivity of BCY11863 for adhesion protein-4 and CD137

[0249] Screening of nectin-4 paralogs: BCY11863 was evaluated for binding to nectin-1 (2880-N1, R&D Systems), nectin-2 (2229-N2, R&D Systems), nectin-3 (3064-N3, R&D Systems), nectin-like-1 (3678-S4-050, R&D Systems), nectin-2 (3519-S4-050, R&D Systems), nectin-3 (4290-S4-050, R&D Systems), nectin-4 (4164-S4, R&D Systems) and nectin-5 (2530-CD-050, R&D Systems) using SPR by labeling these proteins with biotin and immobilizing them on a streptavidin surface. BCY11863 did not show any binding to these targets at concentrations up to 5000 nM.

[0250] Screening for CD137 paralogs: Streptavidin-captured BCY13582 (biotinylated BCY11863) was evaluated for binding to soluble TNF family receptors OX40 and CD40 using SPR. BCY13582 did not bind to these targets at concentrations up to 100 nM.

[0251] Retrogenix Microarray Screening: Cellular microarray technology (Retrogenix) was used to screen for specific off-target binding interactions of biotinylated BCY11863 (referred to as BCY13582).

[0252] The binding level of the test peptides to fixed, non-transfected HEK293 cells and cells overexpressing fibronectin-4 and CD137 (TNFRSF9) was investigated, indicating that 1 μM of the test peptide was an appropriate screening concentration. Under these conditions, the test peptides were screened for binding to human HEK293 cells expressing 5484 full-length human plasma membrane proteins and secreted proteins, respectively. This revealed 9 major hits, including fibronectin-4 and CD137.

[0253] Each lead hit was re-expressed along with two control receptors (TGFBR2 and EGFR) and re-tested with: 1 μM BCY13582 test peptide, 1 μM BCY13582 test peptide in the presence of 100 μM BCY11863, and other positive and negative control treatments ( Figure 4). After removing non-specific, irreproducible and non-significant hits, three specific interactions remained for the tested peptides. These were the primary targets fibronectin-4 and CD137 in both untethered and tethered forms.

[0254] No specific off-target interactions of BCY13582 were found, indicating a high degree of specificity for the primary target.

[0255] 13. When 5 mg / kg was given at 0 and 24 h and 10 mg / kg was given at 0 h twice a week, BCY11863 showed no significant difference in the expression of the same gene. Antitumor activity of MC38 tumor model (MC38#13) expressing fibronectin-4

[0256] 1×10 TNFRSF9 cells were subcutaneously transplanted into 6-8 week old female C57BL / 6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen]. 6 MC38#13 (MC38 cells engineered to overexpress murine adhesion protein-4). When the average tumor volume reached approximately 95 mm 3 At 14:00, mice were randomly divided into treatment groups (n=6 / cohort) and treated weekly with vehicle (25 mM histidine, 10% sucrose, pH 7) or 10 mg / kg BCY11863 for two cycles at two different dosing schedules (5 mg / kg BCY11863 at 0h and 24h on D0 and D7, or 10 mg / kg BCY11863 at 0h on D0 and D7). All treatments were administered intravenously (IV). Tumor growth was monitored from the start of treatment to day 15.

[0257] BCY11863 resulted in significant antitumor activity at both dosing regimens, but when analyzing complete responses on day 15 after the start of treatment, the 5 mg / kg regimen at 0 h and 24 h was superior to the 10 mg / kg regimen at 0 h ( Fig.12 ). Administration of 5 mg / kg BCY11863 at 0h and 24h on D0 and D7 resulted in 4 out of 6 complete tumor responses, while administration of 10 mg / kg BCY11863 at 0h on D0 and D7 resulted in 1 out of 6 complete tumor responses. These data, together with the BCY11863 mouse plasma PK data, indicate that maintaining BCY11863 plasma exposure at the levels produced by administration of 5 mg / kg at 0h and 24h in weekly cycles produces a near complete anti-tumor response in the MC38#13 tumor model.

[0258] 14. Antitumor effect of BCY11863 in the MC38 tumor model with syngeneic overexpression of adhesion protein-4 (MC38#13) active

[0259] 1×10 cells were subcutaneously implanted into 6- to 8-week-old female C57BL / 6J-hCD137 mice [B-hTNFRSF9(CD137) mice; Biocytogen] at three weekly doses of 3, 10, and 30 mg / kg, with weekly, biweekly, and daily dosing. 6 MC38#13 cells (MC38 cells engineered to overexpress mouse adhesion protein-4). When the average tumor volume reached approximately 107 mm 3 At , mice were randomized into treatment groups (n=6 / cohort) and treated with 21 daily doses of vehicle (25mM histidine, 10% sucrose, pH7). BCY11863 treatment was performed in three different schedules (QD: daily; BIW: twice a week or QW: weekly) at three different total dose levels (3, 10 and 30 mg / kg total weekly dose). Different BCY11863 treatment cohorts received 21 daily doses (0.43, 1.4 or 4.3 mg / kg), 6 twice-weekly doses (1.5, 5 or 15 mg / kg) or 3 weekly doses (3, 10 or 30 mg / kg). All treatments were administered intravenously (IV). Tumor growth was monitored until the tumor reached more than 2000 mm 3 The volume of 100 mg / kg was maintained at 400 mg / kg, or until 31 days after the start of treatment. Complete responders (animals without palpable tumors) were followed until D52.

[0260] BCY11863 resulted in significant antitumor activity at many dosing regimens, with the BIW dosing regimen being the most effective, particularly 5 mg / kg BIW dosing. This was demonstrated by the number of complete responder animals at day 52. ​​At day 52 after the start of treatment, 15 / 18 BCY11863 BIW-treated mice were complete responders, 12 / 18 BCY11863 QD-treated mice were complete responders, and 6 / 18 BCY11863 QW-treated mice were complete responders. 5 mg / kg BIW dosing resulted in a 100% complete response rate, with 6 / 6 CRs ( Fig.13 ). These data, together with the BCY11863 mouse plasma PK data, suggest that the anti-tumor response to BCY11863 in the MC38#13 tumor model does not require sustained BCY11863 plasma exposure. Sequence Listing <110> Baisco Technology Development Co., Ltd. <120> Hetero-tandem bicyclic peptide complex <130> BIC-C-P2832PCT <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> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> XA <222> (3) <223> Xaa is 1Nal <220> <221> XA <222> (7) <223> Xaa is HArg <220> <221> XA <222> (13) <223> Xaa is HyP <400> 1 Cys Pro Xaa Asp Cys Met Xaa Asp Trp Ser Thr Pro Xaa Trp Cys 1 5 10 15 <210> 2 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <220> <221> XA <222> (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) <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> Yeah <222> (2)…(2) <223> Stone BuAla <220> <221> Yeah <222> (5)…(5) <223> Stone D-Lys(PYA) <220> <221> Yeah <222> (14)..(14) <223> Stone <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 binding to adhesion protein 4, comprising a polypeptide and a molecular scaffold, wherein the polypeptide has a sequence C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1), and wherein 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 coupled to the following via an N-(acid-PEG3)-N-bis(PEG3-azide) linker (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), and wherein 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 BCY11863: or a pharmaceutically acceptable salt thereof.

3. The hetero-tandem bicyclic peptide complex as defined in claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from a free acid or a 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, or the pharmaceutical composition according to claim 4, in the manufacture of a drug for preventing, inhibiting or treating adhesion protein-4 related cancers; The fibronectin-4-related cancer is selected from colon cancer, lung cancer, prostate cancer and breast cancer.

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 adhesion protein-4-related cancer, wherein the treatment comprises maintaining the 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 fibronectin-4-related cancer is selected from colon cancer, lung cancer, prostate cancer and breast cancer.

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