MT1-MMP-specific bicyclic peptide ligand

Abstract

The present invention relates to polypeptides covalently bound to a non-aromatic molecular scaffold such that two or more peptide rings are present oppositely between the attachment points of the scaffold. In particular, the present invention describes peptides that are high affinity binders of membrane type 1 metalloproteinases (MT1-MMPs), such as the collagen binding site of MT1-MMP. The present invention also describes drug conjugates comprising the peptides coupled to one or more effectors and / or functional groups having utility in imaging and targeted cancer therapy.

Description

Technical Field

[0001] The present invention relates to polypeptides covalently bound to a non-aromatic molecular scaffold such that two or more peptide rings are subtended between the attachment points of the scaffold. In particular, the present invention describes peptides that are high affinity binders of membrane type 1 metalloproteinases (MT1-MMPs), such as the collagen binding site of MT1-MMPs. The present invention also describes drug conjugates comprising the peptides coupled to one or more effectors and / or functional groups having utility in imaging and targeted cancer therapy. 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 containing cysteine ​​residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers 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). A method of generating candidate drug compounds wherein the compounds are generated by attaching cysteine-containing peptides to a molecular scaffold such as TATA (1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one) (Heinis et al. (2014), Angew Chem, Int Ed. 53:1602–1606).

[0005] Combinatorial methods based on phage display have been developed to generate and screen large libraries of bicyclic peptides against target targets (Heinis et al. (2009), Nat Chem Biol 5(7), 502-7 and WO2009 / 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 the first aspect of the present invention, a peptide ligand specific for the collagen binding site of MT1-MMP is provided, which comprises a polypeptide and a non-aromatic molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two ring sequences, and the non-aromatic molecular scaffold forms a covalent bond with the cysteine ​​residues of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold.

[0007] According to a further aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein coupled to one or more effectors and / or functional groups.

[0008] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide ligand or a drug conjugate as defined herein, in combination with one or more pharmaceutically acceptable excipients.

[0009] According to a further aspect of the present invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, inhibiting or treating a disease or condition mediated by MT1-MMP. DETAILED DESCRIPTION

[0010] In one embodiment, the loop sequence comprises 6 amino acids.

[0011] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, each consisting of 6 amino acids.

[0012] In one embodiment, the peptide ligand comprises an amino acid sequence selected from the group consisting of:

[0013] C i -PF / I / YD / SWHTC ii -LFGD / EYT / SC iii (SEQ ID NO:1)

[0014] Among them C i , C ii and C iii represents the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

[0015] In one embodiment, the C i -PF / I / YD / SWHTC ii -LFGD / EYT / SC iii The peptide ligand of (SEQ ID NO: 1) is selected from:

[0016] CPYSWETCLFGDYRC(SEQ ID NO:2);

[0017] CPFDWHTCLFGDYTC(SEQ ID NO:3);

[0018] CPFDWHTCLFGEYSC(SEQ ID NO:4);

[0019] CPIDWHTCLFGDYTC(SEQ ID NO:5);

[0020] CPFSWHTCLFGEYSC(SEQ ID NO:6);

[0021] CPFSWHTCLFGDYTC(SEQ ID NO:7);

[0022] CPISWHTCLFGDYSC (SEQ ID NO:8); and

[0023] CPYSWHTCLFGDYSC (SEQ ID NO:9).

[0024] In a further embodiment, the C i -PF / I / YD / SWHTC ii -LFGD / EYT / SC iii The peptide ligand of (SEQ ID NO: 1) is selected from:

[0025] A-(SEQ ID NO:2)-A(BCY1026);

[0026] A-(SEQ ID NO:3)-A(BCY1057);

[0027] A-(SEQ ID NO:4)-A(BCY1065);

[0028] A-(SEQ ID NO:5)-A(BCY1067);

[0029] A-(SEQ ID NO:6)-A(BCY1073);

[0030] A-(SEQ ID NO:7)-A(BCY1074);

[0031] A-(SEQ ID NO:8)-A(BCY1075); and

[0032] A-(SEQ ID NO:9)-A(BCY1076).

[0033] In one embodiment, the molecular scaffold is TATA, the C i -PF / I / YD / SWHTC ii -LFGD / EYT / SC iii The peptide ligand of (SEQ ID NO: 1) is selected from:

[0034] A-(SEQ ID NO:2)-A(BCY1026);

[0035] A-(SEQ ID NO:3)-A(BCY1057);

[0036] A-(SEQ ID NO:4)-A(BCY1065);

[0037] A-(SEQ ID NO:5)-A(BCY1067);

[0038] A-(SEQ ID NO:6)-A(BCY1073);

[0039] A-(SEQ ID NO:7)-A(BCY1074);

[0040] A-(SEQ ID NO:8)-A(BCY1075); and

[0041] A-(SEQ ID NO:9)-A(BCY1076).

[0042] 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.

[0043] the term

[0044] serial number

[0045] When referring to amino acid residue positions within the peptides of the invention, cysteine ​​residues (C i , C ii and C iii ), therefore, the numbering of the amino acid residues in the peptides of the present invention is as follows:

[0046] C i -P1-Y2-S3-W4-E5-T6-C ii -L7-F8-G9-D 10 -Y 11 -R 12 -C iii (SEQ ID NO:2).

[0047] For the purpose of this description, it is assumed that all bicyclic peptides cyclize with 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)tripropan-1-one (TATA) to produce a trisubstituted structure. Cyclization with TATA occurs at C i , Cii and C iii superior.

[0048] Molecular form

[0049] 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:

[0050] βAla-Sar10-A-(SEQ ID NO:X).

[0051] Reverse peptide sequence

[0052] According to the disclosure in Nair et al. (2003), J Immunol 170(3), 1362-1373, it is contemplated that the peptide sequences disclosed herein will also be used in their retro-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., D-amino acids become L-amino acids, and vice versa).

[0053] Peptide ligand

[0054] As referred to herein, a peptide ligand refers to a peptide that is covalently bound to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) that are capable of forming a covalent bond with the scaffold, and a sequence that is present opposite the reactive groups, which is referred to as a loop sequence because a loop is formed when the peptide is bound to the scaffold. In this case, the peptide comprises at least three cysteine ​​residues (referred to herein as C i , C ii and C iii ), and forming at least two rings on the support.

[0055] Advantages of peptide ligands

[0056] Certain bicyclic peptides 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:

[0057] - Species cross-reactivity. Certain ligands exhibit cross-reactivity between PBPs from different bacterial species, thus being able to treat infections caused by multiple bacterial species. Other ligands may be highly specific for PBPs from certain bacterial species, which may be beneficial in treating infections without causing collateral damage to the patient's beneficial flora;

[0058] - Protease stability. Bicyclic peptide ligands 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 bicyclic lead candidates can be developed in animal models and administered to humans with confidence;

[0059] - 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;

[0060] - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides with short exposure in the acute disease management setting; or to develop bicyclic peptides with enhanced retention in the circulation, which are therefore optimal for treating more chronic disease states. Other factors contributing to the ideal plasma half-life are the requirement for sustained exposure to achieve maximum therapeutic efficacy, versus the toxicology that accompanies sustained exposure to the agent; and

[0061] - Selectivity. Certain peptide ligands of the present invention show selectivity for MT1-MMP but do not cross-react with MMP isoforms such as MMP-1, MMP-2, MMP-15 and MMP-16.

[0062] Pharmaceutically acceptable salts

[0063] 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.

[0064] The salts of the present invention can be synthesized from parent compounds containing a basic or acidic moiety by conventional chemical methods such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Hein-rich 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.

[0065] 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, dodecyl sulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactic 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), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, 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.

[0066] A particular class 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, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One particular salt is the hydrochloride. Another particular salt is the acetate.

[0067] 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 + .

[0068] When the peptide of the present invention comprises 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 peptide of the present invention.

[0069] Modified derivatives

[0070] 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 azide groups, respectively.

[0071] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification.

[0072] In a further embodiment, wherein the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry and / or a C-terminal modification using suitable carboxyl-reactive chemistry.

[0073] 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 radiochelator, or a chromophore.

[0074] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, during peptide synthesis, the N-terminal cysteine ​​group (referred to herein as C i The group) is blocked by acetic anhydride or other suitable reagents, resulting in the molecule being acetylated at the N-terminus. This embodiment provides the advantage of removing potential recognition points of aminopeptidases and avoids the possibility of degradation of the bicyclic peptide.

[0075] 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.

[0076] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, during peptide synthesis, the C-terminal cysteine ​​group (referred to herein as C iii The group) is synthesized as an amide, resulting in a C-terminal amidation of the molecule. This embodiment provides the advantage of removing a potential recognition point for carboxypeptidases and reduces the likelihood of proteolytic degradation of the bicyclic peptide.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] In one embodiment, the modified derivative comprises replacing one or more oxidation-sensitive amino acid residues with one or more antioxidant amino acid residues.

[0081] 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).

[0082] 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).

[0083] 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.

[0084] 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:

[0085] - Incorporation of hydrophobic moieties that exploit hydrophobic interactions and result in lower off-rates, allowing higher affinity to be achieved;

[0086] - 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 as-sociation of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0087] - 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.

[0088] (For review, see Gentilucci et al. (2010), Curr. Pharmaceutical Design 16, 3185-203 and Nestor et al. (2009), Curr. Medicinal Chem 16, 4399-418).

[0089] Isotopic variants

[0090] 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.

[0091] 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.

[0092] 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. 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.

[0093] 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.

[0094] Positron emitting isotopes such as 11 C. 18 F, 15O and 13 N substitution can be used in positron emission tomography (PET) studies to examine target occupancy.

[0095] 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.

[0096] Non-aromatic molecular scaffolds

[0097] The term "non-aromatic molecular scaffold" referred to herein refers to any molecular scaffold as defined herein that does not contain an aromatic (ie, unsaturated) carbocyclic or heterocyclic ring system.

[0098] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014), Angewandte Chemie, International Edition 53(6), 1602-1606.

[0099] As mentioned in the above document, the molecular scaffold may be a small molecule, such as an organic small molecule.

[0100] In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0101] In one embodiment, the molecular scaffold comprises reactive groups capable of reacting with functional groups of a polypeptide to form covalent bonds.

[0102] The molecular scaffold may comprise chemical groups that form linkages with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0103] An example of a compound containing an αβ unsaturated carbonyl group is 1,1′,1″-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie International Edition (2014), 53(6), 1602-1606).

[0104] Effectors and functional groups

[0105] According to a further aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein coupled to one or more effectors and / or functional groups.

[0106] The effector and / or functional group can be attached to, for example, the N- and / or C-terminus of a polypeptide, to an amino acid within a polypeptide, or to a molecular scaffold.

[0107] Suitable effector groups include antibodies and parts or fragments thereof. For example, the effector group may include, in addition to one or more constant region domains, an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof. The effector group may also include the hinge region of an antibody (usually a region existing between the CH1 and CH2 domains of an IgG molecule).

[0108] In a further embodiment of this aspect of the invention, the effector group according to the invention is the Fc region of an IgG molecule. Advantageously, the peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of one day or more, two days or more, three days or more, four days or more, five days or more, six days or more or seven days or more. Most advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of one day or more.

[0109] Functional groups typically include binding groups, drugs, reactive groups for attachment to other entities, functional groups that assist in the uptake of the macrocyclic peptide into cells, etc.

[0110] The ability of peptides to penetrate into cells will allow the peptides to effectively target intracellular targets. Targets that peptides with the ability to penetrate into cells can contact include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in apoptotic pathways. Functional groups that enable penetration of cells include peptides or chemical groups that have been added to peptides or molecular scaffolds. Peptides such as those derived from homeobox proteins such as VP22, HIV-Tat, and Drosophila (Antennapedia), for example, are described in Chen and Harrison (2007), Biochemical Society Transactions Volume 35, part 4, p821; Gupta et al. (2004), Advanced Drug Discovery Reviews Volume 57, 9637. The example of the short peptide effectively translocated by plasma membrane includes the penetratin (penetratin) (Derossi et al. (1994), J Biol.Chem.Volume 269p10444) of 16 amino acids from Drosophila antennapedia protein, "model amphipathic peptide" (Oehlke et al. (1998), Biochim Biophys Acts Volume 1414, p127) and the arginine-rich region of HIV TAT protein of 18 amino acids. Non-peptide methods include using small molecule mimics or SMOC, which can be easily connected to biomolecules (Okuyama et al. (2007), Nature Methods Volume 4, p153). Other chemical strategies by which guanidino groups are added to molecules also enhance cell penetration (Elson-Scwab et al. (2007), J Biol Chem Volume 282, p13585). Small molecular weight molecules such as steroids can be added to molecular scaffolds to enhance the intake of cells.

[0111] One type of functional group that can be attached to the peptide ligand includes antibodies and binding fragments thereof, such as Fab, Fv or single domain fragments. In particular, antibodies that bind to proteins that can increase the in vivo half-life of the peptide ligand can be used.

[0112] In one embodiment, the peptide ligand-effector group according to the invention has a tβ half-life selected from the group consisting of 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more. Advantageously, the peptide ligand-effector group or composition according to the invention will have a tβ half-life in the range of 12 to 60 hours. In a further embodiment, it will have a tβ half-life of one day or more. In another further embodiment, it will be in the range of 12 to 26 hours.

[0113] In a particular embodiment of the invention, the functional group is selected from metal chelators, which are suitable for complexing drug-related metal radioisotopes.

[0114] Possible effector groups also include enzymes such as carboxypeptidase G2 for enzyme / prodrug therapy, where a peptide ligand replaces the antibody in ADEPT.

[0115] In a particular embodiment of the invention, the functional group is selected from drugs, such as cytotoxic agents for cancer treatment. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, dichloromethyl diethylamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs azathiopurine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, formerly known as paclitaxel (Taxol); topoisomerase inhibitors, including camptothecins: irinotecan and topotecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate and teniposide. Further agents may include antitumor antibiotics, which include the immunosuppressant actinomycin (used in kidney transplants), doxorubicin, epirubicin, bleomycin, calicheamycins, and others.

[0116] In a further particular embodiment of the present invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE).

[0117] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure:

[0118]

[0119] Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent with the following structure:

[0120]

[0121] In still further particular embodiments of the invention, the cytotoxic agent is selected from monomethyl auristatin E (MMAE).Data are presented herein in Figure 1 and Tables 3 and 4 demonstrating the effects of peptide ligands coupled to toxins comprising MMAE.

[0122] In one embodiment, the cytotoxic agent is linked to the bicyclic peptide via a cleavable bond, such as a disulfide bond or a protease-sensitive bond. In a further embodiment, the groups adjacent to the disulfide bond are modified to control the hindrance of the disulfide bond and thereby control the cleavage rate and concomitant release of the cytotoxic agent.

[0123] Published work has established the potential to modify the sensitivity of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011), Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance would reduce the rate of reduction by intracellular glutathione as well as extracellular (systemic) reducing agents, thereby reducing the ease of release of the toxin both intracellularly and extracellularly. Thus, the optimization of disulfide stability in circulation (which minimizes the undesirable side effects of the toxin) relative to efficient release in the intracellular environment (which maximizes the therapeutic effect) can be selected by carefully selecting the degree of hindrance on either side of the disulfide bond.

[0124] The hindrance on either side of the disulfide bond can be modulated by introducing one or more methyl groups on the targeting entity (here the bicyclic peptide) or toxin side of the molecular construct.

[0125] In one embodiment, the cytotoxic agent and linker are selected from any combination of those described in WO 2016 / 067035 (which cytotoxic agents and linkers are incorporated herein by reference).

[0126] synthesis

[0127] 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).

[0128] Therefore, the present invention also relates to the production of a polypeptide selected as described herein, wherein said production comprises optional further steps as described below. In one embodiment, these steps are performed on a final product polypeptide prepared by chemical synthesis.

[0129] The peptide may also be extended to incorporate, for example, another loop and thereby introduce multiple specificities.

[0130] In order to extend the peptide, conventional solid phase or solution phase chemistry can be used, using orthogonally protected lysine (and analogs) to simply chemically extend at its N-terminus or C-terminus or in a ring. Standard (biological) coupling techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, it can be added by fragment condensation or native chemical connection, such as described in (Dawson et al. (1994), Synthesis of Proteins by Native Chemical Ligation, Science 266: 776-779), or added by enzymes, such as using 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 Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0131] 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.

[0132] Similar techniques are also used for the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[0133] Furthermore, other functional groups or effector groups may be added in the same manner, at the N- or C-terminus or via side chain coupling using appropriate chemistry. In one embodiment, the coupling is performed in a manner that does not block the activity of either entity.

[0134] Pharmaceutical composition

[0135] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide ligand as defined herein, together with one or more pharmaceutically acceptable excipients.

[0136] 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.

[0137] 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).

[0138] The compounds of the invention can be used alone or in combination with another agent or agents. The other agent used in combination can be, for example, another antibiotic, or an antibiotic "adjuvant", such as an agent for increasing penetration into gram-negative bacteria, a resistance determinant inhibitor, or a virulence mechanism inhibitor.

[0139] Suitable antibiotics for use in combination with the compounds of the present invention include, but are not limited to:

[0140] β-lactams, such as penicillins, cephalosporins, carbapenems or monobactams. Suitable penicillins include oxacillin, methicillin, ampicillin, cloxacillin, carbenicillin, piperacillin, ticarcillin, flucloxacillin and nafcillin; suitable cephalosporins include cefazolin, cephalexin, cephalothin, ceftazidime, cefepime, ceftobiprole, ceftaroline, ceftolozane and cefiderocol; suitable carbapenems include meropenem, doripenem, imipenem, ertapenem, biapenem and tebipenem; suitable monobactams include aztreonam;

[0141] Lincosamides, such as clindamycin and lincomycin;

[0142] Macrolides, such as azithromycin, clarithromycin, erythromycin, telithromycin, and solithromycin;

[0143] Tetracyclines, such as tigecycline, omadacycline, eravacycline, doxycycline, and minocycline;

[0144] Quinolones, such as ciprofloxacin, levofloxacin, moxifloxacin, and delafloxacin;

[0145] rifamycins, such as rifampin, rifabutin, rifalazil, rifapentine, and rifaximin;

[0146] Aminoglycosides, such as gentamicin, streptomycin, tobramycin, amikacin, and plazomicin;

[0147] Glycopeptides, such as vancomycin, teichoplanin, telavancin, dalbavancin, and oritavancin,

[0148] pleuromutilins, such as lefamulin;

[0149] Oxazolidinones, such as linezolid or tedizolid;

[0150] Polymyxins, such as polymyxin B or colistin;

[0151] trimethoprim, ilaprim, sulfamethoxazole;

[0152] Metronidazole;

[0153] Fidaxomicin:

[0154] Mupirocin;

[0155] Fusidic acid;

[0156] Daptomycin;

[0157] murepavidin;

[0158] Fosfomycin; and

[0159] Nitrofurantoin.

[0160] Suitable antibiotic "adjuvants" include, but are not limited to:

[0161] Drugs known to improve bacterial uptake, such as outer membrane permeabilizing agents or efflux pump inhibitors; outer membrane permeabilizing agents may include polymyxin B nonapeptide or other polymyxin analogs, or edetate sodium;

[0162] Inhibitors of resistance mechanisms, such as beta-lactamase inhibitors; suitable beta-lactamase inhibitors include clavulanic acid, tazobactam, sulbactam, avibactam, relebactam, and nacubactam; and

[0163] Inhibitors of virulence mechanisms such as toxins and secretion systems, including antibodies.

[0164] The compounds of the invention may also be used in combination with biological therapies, such as nucleic acid-based therapies, antibodies, bacteriophages or bacteriophage lytic enzymes.

[0165] The route of administration of the pharmaceutical composition according to the present invention can be any route generally 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 route of administration includes, but is not limited to: oral (e.g., by ingestion); buccal; sublingual; transdermal (including, for example, by patches, plasters, etc.); transmucosal (including, for example, by patches, plasters, etc.); intranasal (e.g., by nasal spray); ocular (e.g., by eye drops); pulmonary (e.g., by inhalation or insufflation therapy, such as by using an aerosol, such as by mouth or nose); rectal (e.g., suppositories or enemas); vaginal (e.g., by vaginal plugs); parenteral, such as by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcutaneous, intraarticular, subarachnoid and substernal; by, for example, subcutaneous or intramuscular implantation of a depot or reservoir. Preferably, the pharmaceutical composition according to the present invention is administered parenterally. The dosage and frequency of administration will depend on the age, sex and condition of the patient, concurrent administration of other medications, contraindications and other parameters to be considered by the clinician.

[0166] 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.

[0167] Compositions comprising the peptide ligands of the present invention or mixtures thereof may be administered for therapeutic treatment. In certain therapeutic applications, an amount sufficient to accomplish at least partial inhibition, suppression, regulation, killing or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease and the general state of the patient's own immune system, but is generally 10 μg to 250 mg of the selected peptide ligand per kilogram of body weight, with a more commonly used dose of 100 μg to 25 mg / kg / dose.

[0168] Compositions comprising peptide ligands according to the present invention can be used in therapeutic settings to treat microbial infections or to provide prevention for subjects at risk of infection, such as those undergoing surgery, chemotherapy, artificial ventilation or other conditions or planned interventions. 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 (extracorporeally) or in vitro (invitro). 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.

[0169] Therapeutic Uses

[0170] The bicyclic peptides of the present invention have a special use as high affinity binders of membrane type 1 metalloproteinases (MT1-MMP, also known as MMP14). More specifically, they have a high affinity for the collagen binding region of the hemopexin domain (Arkadash et al. (2017), J. Biol. Chem. 292 (8), 3481-3495). MT1-MMP is a transmembrane metalloproteinase that plays a major role in extracellular matrix remodeling directly by degrading several of its components and indirectly by activating pro-MMP2. MT1-MMP is essential for tumor angiogenesis (Sounni et al. (2002), FASEB J. 16 (6), 555-564) and is overexpressed on a variety of solid tumors, so drug conjugates comprising the bicyclic peptides of the present invention that bind to MT1-MMP have a particular utility in the targeted treatment of cancer, particularly solid tumors such as non-small cell lung cancer. In one embodiment, the bicyclic peptides of the present invention are specific for human MT1-MMP. In a further embodiment, the bicyclic peptides of the invention are specific for mouse MT1-MMP. In a still further embodiment, the bicyclic peptides of the invention are specific for human and mouse MT1-MMP. In a still further embodiment, the bicyclic peptides of the invention are specific for human, mouse and dog MT1-MMP.

[0171] The polypeptide ligands of the present invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, etc. Ligands with selected specificity levels can be used in applications involving testing in non-human animals where cross-reactivity is desired, or in diagnostic applications where cross-reactivity with homologs or paralogs needs to be carefully controlled. In certain applications such as vaccine applications, the ability to elicit an immune response to a predetermined range of antigens can be used to tailor vaccines to specific diseases and pathogens.

[0172] Substantially pure peptide ligands having at least 90% to 95% homogeneity are preferred for administration to mammals, and for pharmaceutical use, particularly when the mammal is a human, 98% to 99% or higher homogeneity is most preferred. The selected polypeptide, once partially purified or purified to the desired homogeneity, can be used for diagnosis or therapy (including in vitro) or for developing and conducting experimental procedures, immunofluorescence staining, etc. (Lefkovite and Pernis (1979 and 1981), Immunological Methods, Volumes I and II, Academic Press, NY).

[0173] The peptide ligand conjugates of the present invention will generally be useful for preventing, inhibiting or treating cancer, particularly solid tumors such as non-small cell lung cancer.

[0174] Therefore, according to a further aspect of the present invention, there is provided a drug conjugate of a peptide ligand as defined herein for use in preventing, inhibiting or treating cancer, particularly solid tumors such as non-small cell lung cancer.

[0175] According to a further aspect of the present invention, there is provided a method for preventing, inhibiting or treating cancer, particularly solid tumors such as non-small cell lung cancer, comprising administering a peptide ligand-drug conjugate as defined herein to a patient in need thereof.

[0176] 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 cell carcinomas, transitional cell carcinomas, and others) such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including cancers of the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), 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 marginal 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, unexplained monoclonal immunoglobulinemia, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematologic malignancies and related conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], 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, 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). ;

[0177] 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 symptoms of the disease become apparent.

[0178] Animal model systems exist that can be used to screen drug conjugates for effectiveness in preventing or treating disease. The present invention facilitates the use of animal model systems, which allow for the development of polypeptide ligands that can cross-react with both human and animal targets, thereby allowing the use of animal models.

[0179] The present invention is further described below with reference to the following examples.

[0180] Example

[0181] Materials and Methods

[0182] Peptide synthesis

[0183] The synthesis of peptides was based on Fmoc chemistry using a Symphony peptide synthesizer produced by Peptide Instruments and a Syro II synthesizer produced by MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) were used with appropriate side chain protecting groups: standard coupling conditions were used in each case, followed by deprotection using standard methods.

[0184] Alternatively, the peptide is purified using HPLC and modified with 1,3,5-triacryloyl hexahydro-1,3,5-triazine (TATA, Sigma) after separation. To this end, the linear peptide is diluted to about 35 mL with 50:50 MeCN:H2O, about 500 μL of 100 mM TATA in acetonitrile is added, and then the reaction is initiated with 5 mL of 1M NH4HCO3 in H2O solution. The reaction is allowed to proceed at room temperature for about 30 to 60 minutes, and once the reaction is complete, lyophilized (judged by MALDI). After completion, 1 mL of 1M L-cysteine ​​hydrochloride monohydrate (Sigma) in H2O solution is added to the reaction for about 60 minutes at room temperature to quench any excess TATA.

[0185] After lyophilization, the modified peptide was purified as above, while using a Gemini C18 column (Phenomenex) instead of Luna C8 and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA modified material were pooled, lyophilized and stored at -20°C.

[0186] Unless otherwise stated, all amino acids were used in the L-configuration.

[0187] In some cases, the peptide was converted to an activated disulfide prior to coupling to the free thiol group of the toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol equiv) was added to a solution of the peptide (20 mM) in dry DMSO (1 mol equiv). The reaction was mixed well and DIPEA (20 mol equiv) was added. The reaction was monitored by LC / MS until completion.

[0188] Biological data

[0189] Human Fluorescence Polarization Competition Binding Assay

[0190] Due to its high affinity for the hemopexin domain (PEX) of MT1-MMP, the fluorescein derivative of 17-88-N006 (SEQ ID NO: 10) can be used in competition assays (detected using FP). Here, a preformed complex of PEX and a fluorescent tracer bound to PEX is titrated with a free non-fluoresceinized bicyclic peptide. Since all 17-69-based peptides are expected to bind at the same site, the titrant will displace the fluorescent tracer from PEX. The dissociation of the complex can be quantitatively measured, and the Kd of the competitor (titrant) for the target protein can be determined. The advantage of the competition method is that the affinity of the non-fluoresceinized bicyclic peptide can be accurately and quickly determined.

[0191] The concentration of the tracer is usually at or below the Kd (here 1 nM), and the binding protein (here the hemobindin of MT1-MMP) is in 15-fold excess, so that >90% of the tracer is bound. Subsequently, a non-fluorescent competing bicyclic peptide (usually just the bicyclic core sequence) is titrated so that it displaces the fluorescent tracer from the target protein. The displacement of the tracer is measured, which is associated with a decrease in fluorescence polarization. The decrease in fluorescence polarization is proportional to the proportion of target protein bound to the non-fluorescent titrant and is therefore a measure of the affinity of the titrant to the target protein.

[0192] The raw data are fitted to the analytical solution of the cubic equation describing the balance between the fluorescent tracer, the titrant and the bound protein. The fitting requires the affinity value of the fluorescent tracer to the target protein, which can be determined separately by directly combining the FP experiment (see next section). Sigmaplot 12.0 is used to carry out the curve fitting, and is used as an adapted version of the equation described by Zhi-Xin Wang (FEBS Letters (1995) 360, 111-114).

[0193] Selected peptide ligands of the invention were tested in the above human fluorescence polarization competition binding assay, the results of which are shown in Table 1:

[0194] Table 1: Human MT1-MMP fluorescence polarization competition binding

[0195] Sequence Listing <110> Baisco Technology Development Co., Ltd. <120> MT1-MMP-specific bicyclic peptide ligand <130> BIC-C-P2492PCT <150> GB1820288.7 <151> 2018-12-13 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <220> <221> MISC_FEATURE <222> (3) <223> Xaa is selected from F, I or Y <220> <221> MISC_FEATURE <222> (4) <223> Xaa is selected from D or S <220> <221> MISC_FEATURE <222> (12)..(12) <223> Xaa is selected from D or E <220> <221> MISC_FEATURE <222> (14)..(14) <223> Xaa is selected from T or S <400> 1 Cys Pro Xaa Xaa Trp His Thr Cys Leu Phe Gly Xaa Tyr Xaa Cys 1 5 10 15 <210> 2 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 2 Cys Pro Tyr Ser Trp Glu Thr Cys Leu Phe Gly Asp Tyr Arg Cys 1 5 10 15 <210> 3 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 3 Cys Pro Phe Asp Trp His Thr Cys Leu Phe Gly Asp Tyr Thr Cys 1 5 10 15 <210> 4 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 4 Cys Pro Phe Asp Trp His Thr Cys Leu Phe Gly Glu Tyr Ser Cys 1 5 10 15 <210> 5 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 5 Cys Pro Ile Asp Trp His Thr Cys Leu Phe Gly Asp Tyr Thr Cys 1 5 10 15 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 6 Cys Pro Phe Ser Trp His Thr Cys Leu Phe Gly Glu Tyr Ser Cys 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 7 Cys Pro Phe Ser Trp His Thr Cys Leu Phe Gly Asp Tyr Thr Cys 1 5 10 15 <210> 8 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 8 Cys Pro Ile Ser Trp His Thr Cys Leu Phe Gly Asp Tyr Ser Cys 1 5 10 15 <210> 9 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic <400> 9 Cys Pro Tyr Ser Trp His Thr Cys Leu Phe Gly Asp Tyr Ser Cys 1 5 10 15 <210> 10 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic <220> <221> MISC_FEATURE <222> (18) <223> Xaa represents Sar6 <220> <221> MISC_FEATURE <222> (19)..(19) <223> Xaa represents K-Fl <400> 10 Ala Cys Pro Tyr Ser Trp Glu Thr Cys Leu Phe Gly Asp Tyr Arg Cys 1 5 10 15 Alas!

Claims

1. A collagen binding site-specific peptide ligand of MT1-MMP, comprising a polypeptide and a non-aromatic molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two ring sequences, and the non-aromatic molecular scaffold forms a covalent bond with the cysteine ​​residues of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold, wherein the peptide ligand comprises an amino acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, Among them C i , C ii and C iii represents the first, second and third cysteine ​​residues or pharmaceutically acceptable salts thereof, respectively.

2. A peptide ligand as defined in claim 1, wherein said SEQ ID NO: 1 is selected from the group consisting of SEQ ID NOs: 3-9.

3. A peptide ligand as defined in claim 1, wherein the peptide ligand comprises an amino acid selected from the group consisting of: A-(SEQ ID NO:2)-A; A-(SEQ ID NO:3)-A; A-(SEQ ID NO:4)-A; A-(SEQ ID NO:5)-A; A-(SEQ ID NO:6)-A; A-(SEQ ID NO:7)-A; A-(SEQ ID NO:8)-A; and A-(SEQ ID NO:9)-A.

4. A peptide ligand as defined in claim 1, wherein the molecular scaffold is TATA.

5. A peptide ligand as defined in claim 4, wherein the molecular scaffold is TATA and the peptide ligand comprises an amino acid selected from the group consisting of: A-(SEQ ID NO:2)-A; A-(SEQ ID NO:3)-A; A-(SEQ ID NO:4)-A; A-(SEQ ID NO:5)-A; A-(SEQ ID NO:6)-A; A-(SEQ ID NO:7)-A; A-(SEQ ID NO:8)-A; and A-(SEQ ID NO:9)-A.

6. A peptide ligand as defined in claim 1, wherein the pharmaceutically acceptable salt is selected from sodium, potassium, calcium or ammonium salts.

7. A peptide ligand as defined in claim 1, wherein the MT1-MMP is human MT1-MMP.

8. A drug conjugate comprising a peptide ligand as defined in any one of claims 1 to 7 coupled to one or more effectors and / or functional groups.

9. A drug conjugate as defined in claim 8, conjugated to one or more cytotoxic agents.

10. The drug conjugate as defined in claim 9, wherein the cytotoxic agent is selected from MMAE or DM1.

11. A pharmaceutical composition comprising the peptide ligand of any one of claims 1 to 7 or the drug conjugate of any one of claims 8 to 10, in combination with one or more pharmaceutically acceptable excipients.

12. A drug conjugate as defined in claim 11, additionally comprising one or more therapeutic agents.

13. Use of a drug conjugate as defined in any one of claims 8 to 10 for the preparation of a medicament for preventing, inhibiting or treating solid tumor cancer.

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