EA046810B1 - BICYCLIC PEPTIDE LIGANDS SPECIFIC TO MT1-MMP - Google Patents

Abstract

The invention relates to polypeptides covalently linked to non-aromatic molecular scaffolds such that two or more peptide loops are located between the attachment points to the scaffold. Specifically, the invention describes peptides capable of binding with high affinity to membrane metalloprotease type 1 (MT1-MMP), for example, to the collagen-binding region of MT1-MMP. The invention also describes drug conjugates comprising said peptides conjugated to one or more effector and/or functional groups, which can be used for imaging and targeted cancer therapy.

Description

Field of technology to which the invention relates

The present invention relates to polypeptides covalently linked to non-aromatic molecular scaffolds such that two or more peptide loops are located between the points of attachment to the scaffold. In particular, the invention describes peptides capable of binding with high affinity to membrane metalloprotease type 1 (MT1-MMP), for example, to the collagen-binding site of MT1-MMP. The invention also describes drug conjugates containing these peptides conjugated to one or more effector and/or functional groups that can be used for imaging and targeted cancer therapy.

State of the art

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

Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in reduced entropy loss upon binding to targets and higher binding affinities. Reduced flexibility also leads to fixation of target-specific conformations, increasing binding specificity compared to linear peptides. This effect can be illustrated by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses selectivity over other MMPs after ring opening (Cherney et al. (1998), J. Med. Chem. 41(11), 1749-51). The beneficial binding properties achieved through macrocyclization are even more pronounced for polycyclic peptides containing more than one peptide ring, such as, for example, vancomycin, nisin and actinomycin.

Various research groups have previously attached polypeptides containing cysteine residues to a synthetic molecular structure (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen et al. used tris-(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops on synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the preparation of candidate drug compounds, wherein said compounds are prepared by attaching cysteine-containing polypeptides to a molecular scaffold such as, for example, TATA (1,1',1-(1,3,5-triazinan-1,3,5-triyl )triprop-2-en-1-one, Heinis et al. Angew Chem., Int. Ed., 53:1602-1606).

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

The essence of the invention

According to a first aspect, the invention provides a peptide ligand specific for the collagen binding site of MT1-MMP, which comprises a polypeptide containing at least three cysteine residues separated by at least two loop sequences, and a non-aromatic molecular scaffold that forms covalent bonds with cysteine residues of the polypeptide to form at least two polypeptide loops on a molecular scaffold.

In accordance with another aspect, the invention relates to a drug conjugate containing a peptide ligand described herein conjugated to one or more effector and/or functional groups.

In accordance with a further aspect, the invention relates to a pharmaceutical composition containing a peptide ligand or drug conjugate described herein, in combination with one or more pharmaceutically acceptable excipients.

In accordance with another aspect, the invention relates to a peptide ligand or drug conjugate described herein for the prevention, suppression or treatment of a disease or disorder mediated by MT1-MMP.

Detailed Description of the Invention

In one embodiment, said loop sequences comprise six

- 1,046,810 amino acids.

In another embodiment, the loop sequences comprise three cysteine residues separated by two loop sequences each consisting of six amino acids.

In one embodiment, the peptide ligand contains an amino acid sequence selected from:

C _i -PF/I/YD/SWHTC _ii -LFGD/EYT/SC _iii (SEQ ID NO: 1), where Ci, Cii and C _iii represent the first, second and third cysteine residues, respectively, or pharmaceutically acceptable salts thereof.

In one embodiment, the peptide ligand Ci-P-F/I/Y-D/S-W-H-T-Cii-L-F-G-D/E-Y-T/S-Ciii (SEQ ID NO: 1) is selected from:

CPYSWETCLFGDYRC(SEQ ID NO: 2);

CPFDWHTCLFGDYTC(SEQ ID NO: 3);

CPFDWHTCLFGEYSC (SEQ ID NO: 4);

CPIDWHTCLFGDYTC(SEQ ID NO: 5);

CPFSWHTCLFGEYSC (SEQ ID NO: 6):

CPFSWHTCLFGDYTC(SEQ ID NO: 7);

CPISWHTCLFGDYSC(SEQ ID NO: 8);

CPYSWHTCLFGDYSC (SEQ ID NO: 9).

In another embodiment, the peptide ligand Ci-P-F/I/Y-D/S-W-H-T-Cii-L-F-G-D/E-Y-T/S-Ciii (SEQ ID NO: 1) is selected from:

A-(SEQ ID NO: 2)-A (BCY1026);

A-(SEQ ID NO: 3)-A (BCY1057);

A-(SEQ ID NO: 4)-A (BCY1065);

A-(SEQ ID NO: 5)-A (BCY1067);

A-(SEQ ID NO: 6)-A (BCY1073);

A-(SEQ ID NO: 7)-A (BCY1074);

A-(SEQ ID NO: 8)-A (BCY1075);

A-(SEQ ID NO: 9)-A (BCY1076).

In one embodiment, the molecular scaffold is TATA and the peptide ligand Ci-P-F/I/Y-D/S-W-H-T-Cii-L-F-G-D/E-Y-T/S-Ciii (SEQ ID NO: 1) is selected from:

A-(SEQ ID NO: 2)-A (BCY1026);

A-(SEQ ID NO: 3)-A (BCY1057);

A-(SEQ ID NO: 4)-A (BCY1065);

A-(SEQ ID NO: 5)-A (BCY1067);

A-(SEQ ID NO: 6)-A (BCY1073);

A-(SEQ ID NO: 7)-A (BCY1074);

A-(SEQ ID NO: 8)-A (BCY1075);

A-(SEQ ID NO: 9)-A (BCY1076).

Unless otherwise noted, all technical and scientific terms used herein have their usual meanings known to those skilled in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Use standard methods of molecular biology, genetics and biochemistry (see Sambrook et al., Molecular Cloning: A Laboratory Manual, ^3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999), ^4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature.

Numbering.

When mentioning the positions of amino acid residues in the peptides of the present invention, cysteine residues (Ci, Cii and Ciii) are not included in the numbering since they are invariant, therefore the numbering of amino acid residues in the peptides of the present invention is carried out as follows:

C ₁ -P ₁ -Y2-S3-W4-E5-T6-C ₁₁ -L ₇ -F8-G9-D ₁ 0-Y11-R12-C ₁₁₁ (SEQ ID NO: 2).

For the purposes of the present invention, all bicyclic peptides are assumed to be cyclized with 1,1',1''-(1,3,5-triazinan-1,3,5-triyl)tripropan-1-one (TATA) to give a trisubstituted structure. Cyclization involving TATA is carried out at Ci, Cii and Ciii.

Molecular format.

N- or C-terminal extensions of the bicyclic core sequence are added to the left or right side of the sequence and separated by a hyphen. For example, the N-terminal tail of βAla-Sar10-Ala is designated as follows:

βAla-Sar10-A-(SEQ ID NO: X).

- 2 046810

Inverted peptide sequences.

Information provided in Nair et al. (2003), J. Immunol. 170(3), 1362-1373, suggests that the peptide sequences described here will also find use in their retroinverted form. For example, a reverse sequence is used (ie, the N-terminus becomes the C-terminus, and vice versa), the stereochemistry of which is also reversed (ie, D-amino acids become L-amino acids, and vice versa).

Peptide ligands.

A peptide ligand, as used herein, refers to a peptide covalently linked to a molecular framework. Typically, such peptides contain two or more reactive groups (such as cysteine residues) that are capable of forming covalent bonds with the scaffold, and a sequence between these reactive groups, which is called a loop sequence because it forms a loop when the peptide binds to the scaffold. In this case, the peptides contain at least three cysteine residues (denoted here as Ci, Cii and Cin) and form at least two loops on the framework.

Advantages of peptide ligands.

Some of the bicyclic peptides of the present invention have a number of useful properties that make them drug molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. Such beneficial properties include:

Interspecies cross-reactivity. Some ligands show cross-reactivity against PBPs of different bacterial species and can therefore be used to treat infections caused by multiple bacterial species. Other ligands may be highly specific for PBPs of certain bacterial species, which may be useful in treating the infection without collateral damage to the patient's beneficial flora;

Protease stability. Bicyclic peptide ligands should ideally exhibit resistance to plasma proteases, epithelial (membrane-anchored) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, and the like. Protease stability must be maintained across species so that a leading bicyclic candidate can be developed in animal models and also confidently administered to humans;

Desired solubility profile. Solubility depends on the ratio of charged and hydrophilic residues to hydrophobic residues and the number of intra/intermolecular hydrogen bonds, it is a parameter important for preparing compositions and achieving absorption;

Optimal half-life in bloodstream plasma. Depending on the clinical indication and treatment regimen, it may be necessary to develop a bicyclic peptide for short-term action in the treatment of acute disease, or to develop a bicyclic peptide with an increased retention time in the bloodstream that is optimal for the treatment of chronic disease states. Other factors that determine the desired plasma half-life include the requirement of prolonged exposure to ensure maximum therapeutic efficacy compared to associated toxicology due to prolonged exposure to the drug; And

Selectivity. Certain peptide ligands of the present invention exhibit selectivity for MT1-MMP but are unable to cross-react with MMP isoforms such as MMP-1, MMP-2, MMP-15 and MMP-16.

Pharmaceutically acceptable salts.

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

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

Acid addition salts (mono- or di-salts) can be prepared using a wide range of acids, both inorganic and organic. Examples of acid addition salts include mono or di salts prepared using an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (eg, L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4 -acetamidobenzoic, butane, (+)-camphor, camphorosulfonic, (+)-(^)-camphor-10-sulfonic, capric, capronic, caprylic, cinnamic, citric, cyclamine, dodecylsulfuric, ethane-1,2-disulfonic, ethanesulfonic , 2-hydroxyethanesulfonic, formic, fumaric, galactic, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic acid), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acid (e.g. , hydrobromic, hydrochloric, hydroiodic), isethionic, lactic (for example, (+)-b-lactic,

- 3 046810 (±)-DL-lactic), lactobionic, maleic, apple, (-)-b-apple, malonic, (±)-DL-almond, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5- disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-aminosalicylic sebacic, stearic, succinic, sulfuric, tannic, ( +)-b-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

One particular group of salts consists of salts derived from acetic, hydrochloric, hydroiodic, phosphoric, nitric, sulfuric, citric, lactic, succinic, maleic, malic, isethionic, fumaric, benzenesulfonic, toluenesulfonic, sulfuric, methanesulfonic (mesylate), ethanesulfonic, naphthalene sulfonic , valeric, propane, butanoic, malonic, glucuronic and lactobionic acids. The specific salt is a hydrochloride. Another specific salt is acetate.

If the compound is anionic or contains a functional group that can be anionic (for example, -COOH can be converted to ^-COO- ), then a salt formed by an organic or inorganic base generating a suitable cation can be obtained. 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 ion (ie, NH4 ⁺ ) and substituted ammonium ions (eg, NH3R ⁺ , NH2R2+, _NHR3 ⁺ , NR4+). Examples of some 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⁺ .

If the peptides of the present invention contain an amino group, they can form quaternary ammonium salts, for example, by reaction with an alkylating agent according to methods well known to one skilled in the art. Such quaternary ammonium compounds are included within the scope of the peptides of the present invention.

Modified derivatives.

It should be understood that the modified peptide ligand derivatives defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from N-terminal and/or C-terminal modifications; replacing one or more amino acid residues with one or more unnatural amino acid residues (for example, replacing one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacing one or more non-polar amino acid residues with other unnatural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-stable amino acid residues; replacing one or more amino acid residues with alanine; replacing one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; changes in the length of the peptide backbone; substituting a hydrogen on the alpha carbon of one or more amino acid residues with another chemical group, modifying amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable amines, thiols, carboxylic acid and phenol-reactive reagents to functionalize said amino acids, as well as the introduction or replacement of amino acids to achieve orthogonal reaction properties that provide functionalization, for example, amino acids bearing azide or alkyne groups, which allow functionalization with alkyne or azide-containing moieties, respectively.

In one embodiment, the modified derivative contains a modification at the N-terminus and/or the C-terminus. In another embodiment, the modified derivative is prepared by N-terminal modification using suitable amino reactants and/or C-terminal modification using suitable carboxy reactants. In another embodiment, said N-terminal or C-terminal modification includes the addition of an effector group, non-limiting examples of which include a cytotoxic agent, a radiochelator, or a chromophore.

In another embodiment, the modified derivative contains an N-terminal modification. In another embodiment, the N-terminal modification includes the introduction of an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (referred to here as Ci) is capped with acetic anhydride or other suitable reagents during peptide synthesis to produce an N-terminally acetylated molecule. The advantages of this embodiment are the removal of a potential aminopeptidase recognition site and avoidance

- 4 046810 prevention of possible degradation of the bicyclic peptide.

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

In another embodiment, the modified derivative contains a C-terminal modification. In another embodiment, the C-terminal modification includes the introduction of an amide group. In this embodiment, a C-terminal cysteine group (herein referred to as C _iii ) is added as an amide during peptide synthesis to produce a C-terminally amidated molecule. This embodiment has the advantage of removing a potential carboxypeptidase recognition site and reducing the potential for proteolytic degradation of the bicyclic peptide.

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

Alternatively, unnatural amino acids may be used that contain spatially restricted side chains that provide conformational and steric hindrance to proteolytic hydrolysis of the adjacent peptide bond. In particular, such amino acids can be proline analogues, amino acids with bulky side chains, C-disubstituted derivatives (such as aminoisobutyric acid, Aib) and cycloamino acids, a simple derivative being aminocyclopropylcarboxylic acid.

In one embodiment, the modified derivative is prepared by adding a spacer group. In another embodiment, the modified derivative is prepared by adding a spacer group to the N-terminal cysteine (Ci) and/or the C-terminal cysteine ( _Ciii ).

In one embodiment, the modified derivative includes replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-stable amino acid residues.

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

In one embodiment, the modified derivative includes replacing one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability through steric hindrance and through the ability of D-amino acids to stabilize β-bend conformations (Tugyi et al. (2005), PNAS, 102(2), 413-418).

In one embodiment, the modified derivative is prepared by removing any amino acid residues and replacing them with alanine residues. This embodiment has the advantage of removing potential proteolytic attack site(s).

It should be noted that each of the above modifications are intentionally carried out to increase the activity or stability of the peptide. Further modification-induced increases in activity can be achieved through the following mechanisms:

introduction of hydrophobic fragments, the hydrophobicity of which leads to a decrease in dissociation rates and, as a consequence, to an increase in affinity;

introduction of charged groups that are characterized by long-range ionic interactions leading to increased rates of association and hence affinity (see, for example, Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and introducing additional spatial constraint into the peptide, for example, by appropriately spacing the amino acid side chains so that entropy loss is minimal upon binding to the target, spatially constraining the torsion angles of the main chain so that entropy loss is minimal upon binding to the target, and introducing additional

- 5,046,810 cyclizations per molecule for identical reasons, (reviews can be found in Gentilucci et al., Curr. Pharmaceutical Design (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

Isotopic options.

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

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

Some isotopically labeled peptide ligands of the present invention, for example containing a radioactive isotope, can be used to study the distribution of drugs and/or substrates in tissues. The peptide ligands of the present invention may additionally have valuable diagnostic properties such that they can be used to detect or identify complex formation between a labeled compound and other molecules such as peptides, proteins, enzymes or receptors. Detection or identification methods can use compounds labeled with such means as radioisotopes, enzymes, fluorescent substances, luminescent substances (for example, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. Radioactive isotopes such as tritium, those. ³ H (T), and carbon-14, i.e. ¹⁴ C, due to the ease of their administration and detection.

Substitution with heavier isotopes such as deuterium, such as ^2H (D), may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or the ability to use lower doses, and may therefore be preferred in some circumstances .

Substitution with positron-emitting isotopes such as ¹¹ C, ¹⁸ F, ¹⁵ O and ¹³ N can be used to study target occupancy using positron emission tomography (PET).

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

Non-aromatic molecular framework.

References herein to the term non-aromatic molecular framework refer to any molecular framework as defined herein that does not contain an aromatic (ie, unsaturated) carbocyclic or heterocyclic moiety.

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

As noted in the above documents, the molecular framework may be a small molecule, such as a small organic molecule.

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.

In one embodiment, the molecular framework contains reactive groups capable of reacting with the functional group(s) of the polypeptide to form covalent bonds.

The molecular framework may contain chemical groups capable of forming bonds with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1-(1,3,5-triazinan1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014) , 53(6), 16021606).

Effector and functional groups.

In another aspect, the invention provides a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups.

- 6 046810

Effector and/or functional groups may be attached, for example, to the N- and/or C-terminus of the polypeptide, to an amino acid within the polypeptide, or to a molecular scaffold.

Suitable effector groups include antibodies and parts or fragments thereof. For example, an effector group may comprise an antibody light chain (CL) constant region, an antibody heavy chain CH1 domain, an antibody heavy chain CH2 domain, an antibody heavy chain CH3 domain, or any combination thereof in addition to one or more constant region domains. The effector group may also contain an antibody hinge region (this region is typically located between the CH1 and CH2 domains of an IgG molecule).

In a further embodiment of this aspect of the invention, the effector group of the present invention is the Fc region of an IgG molecule. Preferably, the peptide ligand effector moiety of the present invention contains or includes a peptide ligand-Fc hybrid having a tβ half-life of 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more. Most preferably, the peptide ligand of the present invention includes or contains a peptide ligand-Fc hybrid having a half-life tβ of 1 day or more.

Functional groups typically include linking groups, drugs, reactive groups for attaching other moieties, functional groups that promote the uptake of macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate cells allows peptides to act against intracellular targets. Targets that can be targeted by cell-penetrating peptides include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in the apoptosis pathway. Functional groups that provide cell penetration include peptides or chemical groups attached to either a peptide or a molecular scaffold. Peptides such as derivatives of, for example, VP22, HIV-Tat, the Drosophila homeobox protein (Antennapedia) can be used, for example as described in Chen and Harrison, Biochemical Society Transactions (2007), Volume 35, part 4, p. 821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57, 9637. Examples of short peptides that have been shown to be efficiently transported across plasma membranes include the peptide penetratin from the Drosophila Antennapedia protein (Derossi et al. (1994), J. Biol. Chem . Volume 269, p. 10444), containing 16 amino acids, a model of an amphipathic peptide (Oehlke et al. (1998), Biochim. Biophys Acts, Volume 1414, p. 127), containing 18 amino acids, and arginine-rich regions of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimics or SMOCs that can be easily attached to biomolecules (Okuyama et al. (2007), Nature Methods, Volume 4, p. 153). Other chemical strategies of adding guanidinium groups to molecules also increase cell penetration (Elson-Schwab et al. (2007), J. Biol. Chem. Volume 282, p. 13585). Low molecular weight molecules, such as steroids, can be attached to a molecular scaffold to increase cellular uptake.

One class of functional groups that can be attached to peptide ligands includes antibodies and their binding fragments, such as Fab, Fv or single domain fragments. In particular, antibodies that bind to proteins capable of increasing the half-life of the peptide ligand in vivo can be used.

In one embodiment, the peptide effector moiety ligand of 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 more or 20 days or more. Preferably, the peptide ligand effector moiety or composition of the invention has a tβ half-life ranging from 12 to 60 hours. In another embodiment, its tβ half-life is one day or more. In a further embodiment, the half-life is in the range of 12 to 26 hours.

In one particular embodiment of the invention, the functional group is selected from a metal chelator capable of complexing metal radioisotopes used in medicine.

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

In one particular embodiment of the invention, the functional group is selected from a drug, such as a cytotoxic agent for the treatment of cancer. Suitable examples include alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites such as purine analogs, azathioprine 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 such as paclitaxel, initially

- 7 046810 known as Taxol; topoisomerase inhibitors such as the camptothecins: irinotecan and topotecan, and type II inhibitors such as amsacrine, etoposide, etoposide phosphate and teniposide. Other agents may include antitumor antibiotics such as the immunosuppressant dactinomycin (which is used in kidney transplants), doxorubicin, epirubicin, bleomycin, calichemycins, and others.

In another specific embodiment of the invention, the cytotoxic agent is selected from maytansinoids (such as DM1) or monomethyl auristatins (such as MMAE).

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

Monomethyl auristatin E (MMAE) is a synthetic anticancer agent and has the following structure:

In the following specific embodiment of the invention, the cytotoxic agent is selected from monomethyl auristatin E (MMAE). Data demonstrate the effects of peptide ligands conjugated to MMAE-containing toxin.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by 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 blocking of the disulfide bond and thus the rate of cleavage and concomitant release of the cytotoxic agent.

Published work identifies the possibility of changing the sensitivity of a disulfide bond to reduction by introducing steric hindrance on both sides of the disulfide bond (Kellogg et al. (2011), Bioconjugate Chemistry, 22, 717). A higher degree of steric hindrance reduces the rate of reduction by intracellular glutathione as well as by extracellular (systemic) reducing agents, resulting in reduced ease of release of the toxin both inside and outside the cell. Thus, the optimal degree of disulfide stability in the bloodstream (which minimizes unwanted side effects of the toxin) versus efficient release into the intracellular environment (which maximizes therapeutic effect) can be achieved by carefully selecting the degree of steric hindrance on both sides of the disulfide bond.

The hindrance on both sides of the disulfide bond is modulated by introducing one or more methyl groups on either the targeting moiety (here the bicyclic peptide) or the toxin

- 8 046810 side of the molecular design.

In one embodiment, the cytotoxic agent and linker are selected from any combinations described in WO 2016/067035 (cytotoxic agents and linkers are incorporated herein by reference).

Synthesis.

The peptides of the present invention can be obtained synthetically using standard methods followed by interaction with a molecular scaffold in vitro. standard chemical methods can then be used. This allows for rapid large-scale production of soluble material for subsequent experiments or testing. Such methods can be carried out using conventional chemical methods such as those described in Timmerman et al. (higher).

Thus, the invention also relates to the production of polypeptides selected in accordance with the present invention, where the production includes optional additional steps described below. In one embodiment, these steps are carried out on the final polypeptide product obtained by chemical synthesis.

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

The peptide can be extended by conventional chemical methods at the N-terminus or C-terminus or within loops using orthogonally protected lysines (and their analogues) using standard solid-phase chemistry or liquid-phase chemistry methods. Standard (bio)conjugation techniques can be used to introduce an activated or activated N- or C-terminus. Alternatively, additions can be introduced by condensation of fragments or by native chemical ligation, for example as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779), or using enzymes such as subtiligases as described in (Chang et al. Proc. Natl. Acad. Sci. USA. 1994 Dec 20; 91(26):12544-8 or Hikari et al Bioorganic & Medicinal Chemistry Letters, Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

Alternatively, the peptides can be extended or modified by further conjugation via disulfide bonds. An additional advantage of such conjugation is that it allows the first and second peptides to be separated from each other in the reducing environment of the cell. In this case, a molecular scaffold (eg TATA) can be added during chemical synthesis of the first peptide so that it reacts with the three cysteine groups; an additional cysteine or thiol residue can then be added to the N- or C-terminus of the first peptide such 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.

Similar methods are equally applicable to the synthesis/coupling of two bicyclic and bispecific macrocycles, possibly resulting in a tetraspecific molecule.

In addition, the addition of other functional or effector groups can be accomplished in a similar manner by attaching them to the N- or C-terminus or to the side chains using appropriate chemical methods. In one embodiment, the coupling is carried out in such a way that it does not block the activity of either moiety.

Pharmaceutical compositions.

In accordance with another aspect, the invention relates to a pharmaceutical composition containing a peptide ligand described herein in combination with one or more pharmaceutically acceptable excipients.

Typically, the peptide ligands of the present invention are used in purified form together with pharmacologically acceptable excipients or carriers. Typically, such excipients or carriers include aqueous or alcohol/aqueous solutions, emulsions or suspensions such as saline and/or buffered media. Parenteral media include sodium chloride solution, Ringer's solution containing dextrose, Ringer's solution containing dextrose and sodium chloride, and lactate. If it is necessary to maintain the polypeptide complex in suspension, suitable physiologically acceptable excipients selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates are added.

Intravenous media include liquid and nutrient replenishment agents, as well as electrolyte-containing replenishment agents, such as dextrose-containing Ringer's solution media. Media may also contain preservatives and other additives such as antimicrobial agents, antioxidants, chelating agents and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th edition).

The compounds of the invention may be used alone or in combination with another agent or agents. Another agent for use in combination may be, for example, another antibiotic or an antibiotic adjuvant, such as a Gram-negative bacterial penetration enhancer, an inhibitor of resistance determinants, or an inhibitor of virulence mechanisms.

- 9 046810

Antibiotics suitable for use in combination with the compounds of the present invention include, but are not limited to:

beta-lactams such as penicillins, cephalosporins, carbapenems or monobactams. Suitable penicillins include oxacillin, methicillin, ampicillin, cloxacillin, carbenicillin, piperacillin, tricarcillin, 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;

lincosamides such as clindamycin and lincomycin;

macrolides such as azithromycin, clarithromycin, erythromycin, telithromycin and solithromycin; tetracyclines such as tigecycline, omadacycline, eravacycline, doxycycline and minocycline;

quinolones such as ciprofloxacin, levofloxacin, moxifloxacin and delafloxacin; rifamycins such as rifampicin, rifabutin, rifalazil, rifapentine and rifaximin; aminoglycosides such as gentamicin, streptomycin, tobramycin, amikacin and plazomycin; glycopeptides such as vancomycin, teichoplanin, telavancin, dalbavancin and oritavancin, pleuromutilins such as lefamulin, oxazolidinones such as linezolid or tedizolid, polymyxins such as polymyxin B or colistin; trimethoprim, iclaprim, sulfamethoxazole;

metronidazole;

fidaxomicin;

mupirocin;

fusidic acid;

daptomycin;

murepavidin;

fosfomycin and nitrofurantoin.

Suitable antibiotic adjuvants include, but are not limited to:

agents known to improve bacterial penetration, such as outer membrane permeability enhancers or efflux pump inhibitors; outer membrane permeabilizers may include polymyxin B nonapeptide or other polymyxin analogues, or sodium edetate;

inhibitors of resistance mechanisms such as beta-lactamase inhibitors;

suitable beta-lactamase inhibitors include clavulanic acid, tazobactam, sulbactam, avibactam, relebactam and nacubactam;

inhibitors of virulence mechanisms such as toxins and secretion systems including antibodies.

The compounds of the present invention can also be used in combination with biological therapies, such as nucleic acid, antibody, bacteriophage or phage lysine therapies.

The method of administration of the pharmaceutical compositions according to the invention may be any of the conventional methods known to those skilled in the art. For the purpose of treatment, the peptide ligands of the invention can be administered to any patient in accordance with standard methods. Routes of administration include, but are not limited to, oral (eg, by oral administration); buccal; sublingual; transdermal (for example, using a patch, plaster, etc.); transmucosal (for example, using a patch, plaster, etc.); intranasal (for example, using a nasal spray); ophthalmic (for example, using eye drops); pulmonary (for example, by inhalation or insufflation therapy using, for example, an aerosol, for example, through the mouth or nose); rectal (for example, using a suppository or enema); vaginal (for example, using a pessary); parenteral, for example, by injection, such as subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardial, intrathecal, intraspinal, intracapsular, subcapsular, intraocular, intraperitoneal, intratracheal, subcutaneous, intraarticular, subarachnoid and intrasternal; by implanting a depot or reservoir, for example, subcutaneously or intramuscularly. Preferably, the pharmaceutical compositions according to the invention are administered parenterally. The dosage and frequency of administration depend on the age, gender and condition of the patient, concomitant use of other medications, contraindications and other parameters that must be taken into account by the clinician.

The peptide ligands of the present invention can be lyophilized for storage and redissolved in a suitable carrier before use. This method has been shown to be effective and can be carried out using lyophilization and redissolution methods known in the art. Those skilled in the art will recognize that lyophilization and redissolution can result in varying degrees of loss of activity, which may need to be compensated for by the use of

- 10 046810 dose adjustments are being made upward.

Compositions containing the peptide ligands of the present invention, or a mixture thereof, can be administered for the purpose of therapeutic treatment. For certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or other measurable parameter of the population of selected cells is defined as a therapeutically effective dose. The amount required to achieve this dose depends on the severity of the disease and the general condition of the patient's own immune system, but usually varies from 10 μg to 250 mg of the selected peptide ligand per 1 kg of body weight, with a dose of 100 μg to 25 mg/kg typically used /dose.

A composition containing a peptide ligand of the present invention can be used therapeutically for the treatment of a microbial infection or for prophylaxis in an individual at risk of infection, for example, undergoing surgery, chemotherapy, mechanical ventilation or other condition or planned intervention. In addition, the peptide ligands described herein can be used in vitro or in vitro to selectively kill, deplete, or otherwise effectively reduce a population of target cells in a heterogeneous mixture of cells. The mammal's blood can be combined extracorporeally with selected peptide ligands to kill or otherwise remove unwanted cells from the blood, followed by the blood being returned to the mammal according to standard techniques.

Therapeutic use.

The bicyclic peptides of the present invention have a specific use as high-affinity membrane metalloproteinase type 1 (MT1-MMP, also known as MMP14) binding agents, more specifically the collagen-binding region of the hemopexin domain (Arkadash et al. J. Biol. Chem. 2017 , 292(8), 3481-3495). MT1-MMP is a transmembrane metalloproteinase that plays an important role in the remodeling of the extracellular matrix, directly by degrading some of its components and indirectly by activating pro-MMP2. MT1-MMP also plays a key role in tumor angiogenesis (Sounni et al. (2002), FASEB J. 16(6), 555-564) and is expressed at elevated levels in a number of solid tumors, so drug conjugates containing MT1-MMP- The bicyclic peptide binding peptides of the present invention may be specifically used for targeted treatment of cancer, in particular solid tumors such as non-small cell lung carcinomas. In one embodiment, the bicyclic peptide of the present invention is specific for human MT1-MMP. In a further embodiment, the bicyclic peptide of the present invention is specific for murine MT1-MMP. In another embodiment, the bicyclic peptide of the present invention is specific for human and murine MT1-MMP. In a further embodiment, the bicyclic peptide of the present invention is specific for human, murine and canine MT1-MMP.

The polypeptide ligands of the present invention can be used for in vivo therapeutic and prophylactic purposes, in vitro and in vivo diagnostic purposes, in vitro assays, as reagents, and the like. Ligands having a certain level of specificity can be used in applications that involve testing non-human animals where cross-reactivity is desired, or in diagnostic applications where cross-reactivity with homologs or paralogs must be carefully monitored. In some applications, such as vaccines, the ability to induce an immune response to predefined ranges of antigens can be used to tailor the vaccine to specific diseases and pathogens.

Substantially pure peptide ligands with at least 90 to 95% homogeneity are preferred for administration to a mammal, and homogeneity of 98 to 99% or greater is most preferred for pharmaceutical use, particularly when the mammal is a human. Once purified, partially or to the desired degree of homogeneity, the selected polypeptides can be used for diagnostic or therapeutic purposes (including in vitro) or for the development and implementation of analytical procedures, immunofluorescence staining, etc. (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

The peptide ligand conjugates of the present invention generally find use in methods of preventing, suppressing or treating cancer, in particular solid tumors such as non-small cell lung carcinomas.

Thus, in another aspect, the invention provides drug conjugates containing a peptide ligand described herein for use in methods of preventing, suppressing or treating cancer, particularly solid tumors such as non-small cell lung carcinomas.

In accordance with another aspect, the invention relates to a method of preventing, suppressing or treating cancer, in particular solid tumors such as non-small cell lung carcinomas, which includes administering to a patient in need thereof a drug conjugate containing a peptide ligand described herein.

Examples of cancer (and its benign counterparts) that can be treated (or suppressed) include

- 11 046810 includes, without limitation, tumors of epithelial origin (adenomas and carcinomas of various types, including adenocarcinomas, squamous cell, transitional cell and other carcinomas), such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including yourself esophagus, stomach, small intestine, colon, rectum and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (eg, adenocarcinomas, small cell lung carcinomas, non-small cell lung carcinomas, bronchoalveolar carcinomas and mesotheliomas), head and neck (eg, cancer of the tongue, mouth, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity and paranasal sinuses), ovary, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (eg, follicular thyroid carcinoma), adrenal gland, prostate, skin and adnexa (eg, melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (such as leukemias, lymphomas) and precancerous hematologic disorders and borderline malignancies such as hematologic malignancies and related conditions of the lymphoid lineage (eg, acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, such such as diffuse large cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer cell [NK] lymphomas, Hodgkin lymphomas, hairy cell leukemia, monoclonal gammopathy of unknown origin, plasmacytoma, multiple myeloma and post transplantation lymphoproliferative disorders), hematologic malignancies, and related conditions of myeloid origin (eg, acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome and promyelocytic leukemia); tumors of mesenchymal origin, such as sarcomas of soft tissue, bone or cartilage, such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcomas, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histiocytomas and elevated dermatofibrosarcomas; tumors of the central or peripheral nervous system (eg, astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and schwannomas); endocrine tumors (eg, pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary thyroid carcinoma); tumors of the eye and appendages (for example, retinoblastoma); germ cell and trophoblast tumors (eg, teratomas, seminomas, dysgerminomas, benign gestational trophoblastic disease and choriocarcinomas); as well as pediatric and embryonal tumors (eg, medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, that make the patient susceptible to malignancy (eg, xeroderma pigmentosum).

References herein to the term prophylaxis include administration of the protective composition prior to induction of disease. The term suppression refers to the administration of the composition after an induction event, but before the clinical manifestation of the disease. The term treatment includes administration of the protective composition after the onset of symptoms of the disease.

Animal model systems exist that can be used to screen for the effectiveness of drug conjugates in preventing or treating disease. The present invention facilitates the use of animal model systems because the polypeptide ligands of the invention can cross-react with human and animal targets.

The invention is further described with reference to the following examples.

Examples

Materials and methods.

Peptide synthesis.

Peptide synthesis was carried out using the Fmoc strategy on a Symphony peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer manufactured by MultiSynTech. Standard Fmoc amino acids (Sigma, Merck) with appropriate side chain protecting groups are used: in each case, standard condensation conditions are used whenever possible, followed by deprotection using standard methods.

Alternatively, the peptides are purified by HPLC and, after isolation, modified with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma). To do this, the linear peptide is diluted 50:50 MeCN:H2O to ~35 ml, add ~500 µl of 100 mM TATA in acetonitrile and initiate the reaction with 5 ml of 1 M NH ₄ HCO ₃ in H2O. The reaction is allowed to proceed for ~30-60 min at room temperature and after completion of the reaction (determined by MALDI) is lyophilized. After completion of the reaction to

- 12 046810 to the reaction mixture, add 1 ml of a 1 M solution of L-cysteine hydrochloride monohydrate (Sigma) in H2O and hold for ~60 min at room temperature to quench any excess TATA.

After lyophilization, the modified peptide is purified as described above, replacing the Luna C8 column with a Gemini C18 column (Phenomenex) and the acid with 0.1% trifluoroacetic acid. Pure fractions containing the target TATA-modified substance are pooled, lyophilized and stored at -20°C.

Unless otherwise noted, all amino acids used are of the L configuration.

In some cases, before binding to the free thiol group of the toxin, the peptides are converted to activated disulfides using the following method: a solution of 4-methyl(succinimidyl-4(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol-eq. ) is added to a solution of the peptide (20 mM) in dry DMSO (1 mol equiv.). The reaction mixture is thoroughly stirred and DIPEA (20 mol-eq.) is added. The reaction is monitored by LC/MS until completion.

Results of biological tests.

Fluorescence polarization competitive binding assay of human proteins.

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

The concentration of the label is typically at or below the Kd (here 1 nM) and the binding protein (here hemopexin MT1-MMP) is in 15-fold excess so that >90% of the label is bound. A non-fluorescent competing bicyclic peptide (usually just the bicyclic core sequence) is then titrated so that it displaces the fluorescent tag from the target protein. Label displacement is measured and correlated with a decrease in fluorescence polarization. The decrease in fluorescence polarization is proportional to the fraction of the target protein bound to the non-fluorescent titrant and is therefore a measure of the affinity of the titrant for the target protein.

The raw data is fitted to an analytical solution to a cubic equation that describes the equilibrium between the fluorescent label, the titrant, and the binding protein. Fitting requires an affinity value of the fluorescent tag for the target protein, which can be determined separately using direct FP binding experiments. Curve fitting is performed using Sigmaplot 12.0 and is used as an adaptation of the equation described by Zhi-Xin Wang (FEBS Letters (1995), 360, 111-114).

Selected peptides of the present invention are tested using the above fluorescence polarization human protein competitive binding assay, the results are presented in the table.

Competitive binding of human MT1-MMP with fluorescence polarization

Bicyclic peptide Molecular framework Label Ki n BCY1026 TATA ACPYSWETCLFGDYRCA[Sar] ₆ [KFl] (17-88-N006) (SEQ ID NO: 10) 2801.67±1040.54 3 BCY1057 TATA ACPYSWETCLFGDYRCA[Sar]6[KFl] (17-88-N006) (SEQ ID NO: 10) 436.5±81.34 2 BCY1065 TATA ACPYSWETCLFGDYRCA[Sar]6[KFl] (17-88-N006) (SEQ ID NO: 10) 567±137.2 2 BCY1067 TATA ACPYSWETCLFGDYRCA[Sar]6[KFl] (17-88-N006) (SEQ ID NO: 10) 429.5±44.1 2 BCY1073 TATA ACPYSWETCLFGDYRCA[Sar]6[KFl] (17-88-N006) (SEQ ID NO: 10) 554.5±238.14 2 BCY1074 TATA ACPYSWETCLFGDYRCA[Sar]6[KFl] (17-88-N006) (SEQ ID NO: 10) 356±45.08 2 BCY1075 TATA ACPYSWETCLFGDYRCA[Sar]6[KFl] (17-88-N006) (SEQ ID NO: 10) 441±113.68 2 BCY1076 TATA ACPYSWETCLFGDYRCA[Sar] ₆ [KFl] (17-88-N006) (SEQ ID NO: 10) 903.5±12.74 2

Claims (13)

1. A peptide ligand specific for the collagen-binding site of MT1-MMP, containing a polypeptide containing at least three cysteine residues separated by at least two loop-like sequences, and a non-aromatic molecular framework that forms covalent bonds with the cysteine residues of the polypeptide to obtain at least two polypeptide loops on a molecular scaffold, wherein the peptide ligand contains an amino acid sequence selected from: Ci-PF/I/YD/SWHT-Cii-LFGD/EYT/S-Ciii (SEQ ID NO: 1), where Ci, Gy and C _iii represent the first, second and third cysteine residues, respectively, or pharmaceutically acceptable salts thereof. 2. The peptide ligand according to claim 1, wherein the peptide ligand C ₁ -P-F7I/YD/SWHTC ₁₁ -LFGD/EYT/SC ₁₁₁ (SEQ ID NO: 1) is selected from: CPYSWETCLFGDYRC(SEQ ID NO: 2); CPFDWHTCLFGDYTC(SEQ ID NO: 3); CPFDWHTCLFGEYSC (SEQ ID NO: 4); CPIDWHTCLFGDYTC(SEQ ID NO: 5); CPFSWHTCLFGEYSC (SEQ ID NO: 6): CPFSWHTCLFGDYTC(SEQ ID NO: 7); CPISWHTCLFGDYSC(SEQ ID NO: 8); CPYSWHTCLFGDYSC (SEQ ID NO: 9). 3. The peptide ligand according to claim 1 or 2, wherein the peptide ligand Ci-P-F/I/Y-D/S-W-H-T-Cii-L-F-G-D/E-Y-T/S-Ciii (SEQ ID NO: 1) is selected from: A-(SEQ ID NO: 2)-A (BCY1026); A-(SEQ ID NO: 3)-A (BCY1057); A-(SEQ ID NO: 4)-A (BCY1065); A-(SEQ ID NO: 5)-A (BCY1067); A-(SEQ ID NO: 6)-A (BCY1073); A-(SEQ ID NO: 7)-A (BCY1074); A-(SEQ ID NO: 8)-A (BCY1075); A-(SEQ ID NO: 9)-A (BCY1076). 4. Peptide ligand according to any one of claims 1 to 3, where the molecular framework is TATA. 5. The peptide ligand according to claim 4, wherein the molecular framework is TATA and the peptide ligand C ₁ -PF/I/YD/SWHTC ₁₁ -LFGD/EYT/SC ₁₁₁ (SEQ ID NO: 1) is selected from: A-(SEQ ID NO: 2)-A (BCY1026); A-(SEQ ID NO: 3)-A (BCY1057); A-(SEQ ID NO: 4)-A (BCY1065); A-(SEQ ID NO: 5)-A (BCY1067); A-(SEQ ID NO: 6)-A (BCY1073); A-(SEQ ID NO: 7)-A (BCY1074); A-(SEQ ID NO: 8)-A (BCY1075); A-(SEQ ID NO: 9)-A (BCY1076). 6. The peptide ligand according to any one of claims 1 to 5, where the pharmaceutically acceptable salt is selected from a free acid salt or a sodium, potassium, calcium, ammonium salt. 7. The peptide ligand according to any one of claims 1 to 6, wherein MT1-MMP is human MT1-MMP. 8. A drug conjugate containing a peptide ligand according to any one of claims 1 to 7, conjugated to one or more effector and/or functional groups. 9. The drug conjugate according to claim 8, containing one or more cytotoxic agents. 10. The drug conjugate according to claim 9, wherein said cytotoxic agent is selected from MMAE or DM1. 11. A pharmaceutical composition which contains a peptide ligand according to any one of claims 1 to 7 or a drug conjugate according to any one of claims 8 to 10 in combination with one or more pharmaceutically acceptable excipients. 12. The pharmaceutical composition according to claim 11, which further contains one or more therapeutic agents. 13. Use of a drug conjugate according to any one of claims 8 to 10 in the prevention, suppression or treatment of a disease or disorder mediated by MT1-MMP.