Bicyclic peptide ligand drug conjugates

Abstract

The present invention relates to a drug conjugate comprising two cytotoxic agents bound to a peptide ligand.The present invention also relates to a pharmaceutical composition comprising said drug conjugate and a method of using said drug conjugate in the prevention, suppression or treatment of diseases.Examples of such diseases include diseases that can be alleviated by cell death, particularly diseases characterized by defective cells, cell proliferative diseases such as cancer, and autoimmune diseases such as rheumatoid arthritis.

Description

[Technical field]

[0001] The present invention relates to a drug conjugate comprising two cytotoxic agents bound to a peptide ligand.The present invention also relates to a pharmaceutical composition comprising said drug conjugate and a method of using said drug conjugate in the prevention, suppression or treatment of diseases that can be alleviated by cell death, in particular diseases characterized by defective cells, cell proliferative diseases such as cancer and autoimmune diseases such as rheumatoid arthritis. [Background technology]

[0002] Cyclic peptides can bind to target proteins with high affinity and target specificity, which makes them an attractive group of molecules for therapeutic drug development. Indeed, several cyclic peptides have already been successfully used in the clinic, e.g., 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. In general, cyclic peptides are well-known, e.g., the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), the cyclic peptide of the Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å2; 2 ) (Xiong et al. (2002), Science 296 (5565), 151-5), and upein-1 (603 Å), a cyclic peptide inhibitor that binds to urokinase-type plasminogen activator. 2 ; Zhao et al. (2007), J Struct Biol 160 (1), 1-10), macrocyclic peptides bind to surfaces measuring hundreds of square angstroms.

[0003] Due to their ring structure, peptide macrocycles are less flexible than linear peptides, leading to a smaller entropy loss upon binding to the target, and thus a higher binding affinity. The reduced flexibility also leads to a more rigid target-specific conformation, which increases the binding specificity compared to linear peptides. This effect is illustrated by the example of a potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity compared to other MMPs upon ring opening (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved by macrocyclization are even more pronounced for polycyclic peptides with two or more peptide rings, e.g. vancomycin, nisin, and actinomycin.

[0004] Previously, various research teams have attached polypeptides with cysteine ​​residues to synthetic molecular structures (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 onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds, generated by linking cysteine-containing polypeptides to molecular scaffolds such as tris(bromomethyl)benzene, are disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] A combinatorial approach based on phage display has been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009 / 098450). Briefly, three cysteine ​​residues and two regions of six random amino acids (Cys-(Xaa) 6-Cys-(Xaa) 6 A combinatorial library of linear peptides containing the cysteine ​​side chain (Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chain to a small molecule (tris-(bromomethyl)benzene). Summary of the Invention

[0006] According to a first aspect of the present invention, (i) a peptide ligand comprising at least three reactive groups separated by at least two loop sequences, and an aromatic molecular scaffold that forms covalent bonds with the reactive groups of the peptide ligand such that at least two polypeptide loops are formed on the molecular scaffold; and (ii) two cytotoxic agents conjugated to said peptide ligand; A drug conjugate comprising:

[0007] According to a second aspect of the present invention, there is provided a pharmaceutical composition comprising a drug conjugate as defined herein, in combination with one or more pharma- ceutically acceptable excipients.

[0008] According to a further aspect of the invention there is provided a drug conjugate as defined herein for use in the prevention, suppression or treatment of diseases which can be alleviated by cell death, in particular diseases characterised by defective cells, cell proliferative diseases such as cancer and autoimmune diseases such as rheumatoid arthritis. [Brief description of the drawings]

[0009] [Figure 1] Tumor volume tracings after administration of BT17BDC23 to female BALB / c nude mice bearing HT1080 xenografts. Error bars represent standard error of the mean (SEM). [Diagram 2] Tumor volume tracings after administration of BT17BDC69 to female BALB / c nude mice bearing HT1080 xenografts. Error bars represent standard error of the mean (SEM). [Diagram 3]Tumor volume tracings after administration of BT17BDC70 to female BALB / c nude mice bearing HT1080 xenografts. Error bars represent standard error of the mean (SEM). [Figure 4] Tumor volume tracings following administration of BT17BDC61 to female BALB / c nude mice bearing HT1080 xenografts. Error bars represent standard error of the mean (SEM). [Diagram 5] Tumor volume tracings after administration of BT17BDC75 to female BALB / c nude mice bearing HT1080 xenografts. Error bars represent standard error of the mean (SEM). [Figure 6] Tumor volume tracings after administration of BCY16278 to female BALB / c nude mice bearing HT1080 xenografts. Error bars represent standard error of the mean (SEM). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention According to a first aspect of the present invention, (i) a peptide ligand comprising at least three reactive groups separated by at least two loop sequences, and an aromatic molecular scaffold that forms covalent bonds with the reactive groups of the peptide ligand such that at least two polypeptide loops are formed on the molecular scaffold; and (ii) two cytotoxic agents conjugated to said peptide ligand; A drug conjugate comprising:

[0011] Surprisingly, data presented in Figures 1-5 and Tables 1-8 herein show that bicyclic peptide drug conjugates (BDCs) containing two cytotoxic agents exhibited the greatest antitumor efficacy compared to corresponding BDCs containing one and four cytotoxic agents at comparable doses.

[0012] In one embodiment, the peptide ligand is specific for MT1-MMP.

[0013] MT1-MMP is a transmembrane metalloprotease that plays a major role in the remodeling of the extracellular matrix, directly by degrading some components of the matrix and indirectly by activating pro-MMP2. Because MT1-MMP is important for tumor angiogenesis (Sounni et al (2002) FASEB J. 16(6), 555-564) and is overexpressed in various solid tumors, the MT1-MMP-binding bicyclic peptides of the invention are particularly useful for targeted therapy of cancer, particularly solid tumors such as non-small cell lung cancer. In one embodiment, at least one of the bicyclic peptides of the invention is specific for human MT1-MMP. In a further embodiment, at least one of the bicyclic peptides of the invention is specific for mouse MT1-MMP. In a further embodiment, at least one of the bicyclic peptides of the invention is specific for human and mouse MT1-MMP. In still further embodiments, at least one of said bicyclic peptides of the invention is specific for human, mouse and canine MT1-MMP.

[0014] Examples of suitable MT1-MMP-specific peptide ligands are described in WO 2016 / 067035 and WO 2017 / 191460, which bicyclic peptide ligands are incorporated herein by reference.

[0015] In one embodiment, the peptide ligand is specific for MT1-MMP and the loop sequence comprises 5 or 6 amino acids.

[0016] In a further embodiment, the peptide ligand is specific for MT1-MMP and the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of five amino acids and the other of which consists of six amino acids. Thus, in one embodiment, the peptide ligand is specific for MT1-MMP and C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 1) (Here, 1Nal represents 1-naphthylalanine, and tBuGly represents t-butylglycine.) It has a core sequence of

[0017] In a further embodiment, the peptide ligand is specific for MT1-MMP; βAla-Sar 10 -A- (SEQ ID NO: 1) (described as 17-69-07-N241 and SEQ ID NO: 5 in WO2016 / 067035).

[0018] In another embodiment, the peptide ligand is specific for MT1-MMP and the loop sequence comprises two (S)-2-amino-3-(methylamino)propanoic acid (Dap(Me)) residues and one cysteine ​​residue separated by two loop sequences, one of five amino acids and the other of six amino acids. Thus, in one embodiment, the peptide ligand is specific for MT1-MMP and [Dap(Me)](D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)[Dap(Me)] (SEQ ID NO: 2) (Here, Dap(Me) represents (S)-2-amino-3-(methylamino)propanoic acid, 1Nal represents 1-naphthylalanine, and tBuGly represents t-butylglycine.) It has a core sequence of

[0019] In a further embodiment, the peptide ligand is specific for MT1-MMP; βAla-Sar 10 -A- (SEQ ID NO: 2) It has the full length sequence.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry.Standard procedures are used for molecular biology, genetics and biochemistry methods, which are incorporated herein by reference (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.).

[0021] nomenclature Numbering When referring to amino acid residue positions within the bicyclic peptides of the present invention, a cysteine ​​residue (C i , C ii and C iii ) are invariant and therefore omitted from the numbering, and therefore the numbering of amino acid residues within selected bicyclic peptides of the invention is -C i -dA 1 -N 2 -E 3 -1Nal 4 -dA 5 -C ii -E 6 -D 7 -F 8 -Y 9 -D 10 -tBuGly 11 -C iii - (SEQ ID NO:1) It is written as follows.

[0022] For the purposes of this description, it is assumed that all bicyclic peptides are cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) to yield trisubstituted 1,3,5-trismethylbenzene structures. Cyclization with TBMB yields the C i , C ii , and C iii It is held.

[0023] molecular format N- or C-terminal extensions of the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, N-terminal βAla-Sar 10 -Ala tail is βAla-Sar 10 -A-(sequence number X) It is expressed as follows.

[0024] Inverted peptide sequence In light of the disclosure of Nair et al (2003) J Immunol 170(3), 1362-1373, it is anticipated that the peptide sequences disclosed herein will also be useful in retro-inverso forms, for example where the sequence is reversed (i.e., N-terminus becomes C-terminus and vice versa) and the stereochemistry is also reversed (i.e., D-amino acids become L-amino acids and vice versa).

[0025] Peptide Ligands As used herein, a peptide ligand refers to a peptide, peptides, or peptidomimetic that is covalently attached to a molecular scaffold. Typically, such peptides, peptides, or peptidomimetics contain a peptide having a natural or unnatural amino acid, two or more reactive groups (i.e., cysteine ​​residues) that can form covalent bonds with the scaffold, and a sequence sandwiched between the reactive groups, referred to as a loop sequence because the peptide, peptides, or peptidomimetic forms a loop when bound to the scaffold. In the present case, the peptide, peptide, or peptidomimetic contains at least three cysteine ​​residues (herein referred to as C i , C ii and C iii) which forms at least two loops on the scaffold.

[0026] Advantages of peptide ligands Certain bicyclic peptides of the invention have many advantageous properties that allow them to be considered as drug-like molecules suitable for injection, inhalation, nasal, ocular, oral, or topical administration. Such advantageous properties include: -Species cross-reactivity, which is a common requirement for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability. Bicyclic peptide ligands 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 different species so that bicyclic lead candidates can not only be developed in animal models but also confidently administered to humans; - a desirable solubility profile, which is a function of charged residues, the ratio of hydrophilic to hydrophobic residues, and intra- / inter-molecular hydrogen bonding, which is important for formulation and absorption purposes; - Optimal plasma half-life in the systemic circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides for short exposure periods in order to develop bicyclic peptides that are optimal for managing more chronic disease states by improving circulation retention. Other factors that lead to a desirable plasma half-life are the need for sustained exposure to maximize therapeutic efficacy and the associated toxicity of sustained exposure of the drug; and -Selectivity. Certain peptide ligands of the present invention exhibit better selectivity over other receptor subtypes. For example, if a bicyclic peptide is specific for MT1-MMP, the bicyclic peptide is ideally selective for MT1-MMP over other metalloproteases.

[0027] Pharmaceutically acceptable salts It will be understood that salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt forms of said ligand.

[0028] The salts of the present invention can be synthesized from a parent compound that contains a basic or acidic moiety by 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. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of both.

[0029] Acid addition salts (monosalts or disalts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include 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, butanoic acid, (+)camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, and cyclamic acid. , dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g. D-glucuronic acid), glutamic acid (e.g. L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g. hydrobromic acid, hydrochloric acid, iodine acid, etc.) 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 Included are mono- or di-salts formed with acids selected from the group consisting of 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, valeric acid, acylated amino acids, and cation exchange resins.

[0030] One particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0031] Functional groups in which the compound is anionic or can exist in an anionic form (e.g., COOH is COO - In the case of cations having a structure such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, + , Na + and K. + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH 4+ ) and substituted ammonium ions (e.g., NH 3 R + , N.H. 2 R 2+ , N.H.R. 3+ , N.R. 4+ ) are included, but are not limited to. Some examples of suitable substituted ammonium ions include methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH 3 ) 4+ It is.

[0032] When the compounds of the present invention contain amine functions, they may form quaternary ammonium salts, for example, by reaction with alkylating agents by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the compounds of the present invention.

[0033] Modified Derivatives It is understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (such as 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 non-natural isosteric or isoelectronic amino acids); addition of spacer groups; substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more amino acids with one or more amino acids (e.g., replacement with alanine); substitution of one or more L-amino acid residues with one or more D-amino acid residues; bicyclic peptides. modification of the length of the peptide backbone; replacement of the hydrogen on the alpha carbon of one or more amino acid residues with an alternative chemical group; modification of amino acids such as cysteine, lysine, glutamic / aspartic acid, tyrosine, etc. with appropriate amine-, thiol-, carboxylic acid-, and phenol-reactive reagents to functionalize said amino acids; introduction or substitution of amino acids with orthogonal reactivity suitable for functionalization (e.g., amino acids bearing an azide or alkyne group to allow functionalization at alkyne- or azide-bearing sites, respectively).

[0034] In certain embodiments, the modified derivatives include an N-terminal and / or a C-terminal modification. In further embodiments, the modified derivatives include an N-terminal modification using appropriate amino-reactive chemistry and / or a C-terminal modification using appropriate carboxy-reactive chemistry. In further embodiments, the N-terminal or C-terminal modification includes the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator, or a chromophore.

[0035] 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, the N-terminal residue is capped with acetic anhydride or other suitable reagent during peptide synthesis, resulting in an N-terminally acetylated molecule. This embodiment provides the advantage of removing potential recognition sites for aminopeptidases, avoiding possible degradation of the bicyclic peptide.

[0036] In another embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates the attachment of an effector group and retention of potency of the bicyclic peptide against its target.

[0037] 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, the C-terminal residue is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment provides the advantage of removing potential recognition sites for carboxypeptidases, reducing the likelihood of proteolysis of the bicyclic peptide.

[0038] In one embodiment, the modified derivatives include the substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids can be selected that have isocoordinate / isoelectronic side chains that are not recognized by degradative proteases and do not have any adverse effect on targeting efficacy.

[0039] Alternatively, unnatural amino acids with constrained amino acid side chains can be used such that proteolytic hydrolysis of nearby peptide bonds is structurally and sterically hindered. In particular, these concern proline analogues, bulky side chains, Cα-disubstituted derivatives (such as aminoisobutyric acid, Aib), and cycloamino acids (a simple derivative is amino-cyclopropyl carboxylic acid).

[0040] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of an N-terminal cysteine ​​(Ci ) and / or a C-terminal cysteine ​​(C iii ) including the addition of a spacer group to

[0041] In some embodiments, the modified derivatives also include substitutions of one or more oxidation-resistant amino acid residues for one or more oxidation-sensitive amino acid residues. In further embodiments, the modified derivatives include substitutions of tryptophan residues with naphthylalanine or alanine residues. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0042] In one embodiment, the modified derivative comprises a substitution of one or more hydrophobic amino acid residues for one or more charged amino acid residues. In another embodiment, the modified derivative comprises a substitution of one or more hydrophobic amino acid residues for one or more charged amino acid residues. The appropriate balance of charged and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and therefore the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. The two can combine to affect the half-life, volume of distribution, and exposure of the peptide drug, which can be tailored depending on the clinical endpoint. Additionally, the appropriate combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0043] In one embodiment, the modified derivatives include the substitution of one or more D-amino acid residues for one or more L-amino acid residues, which is believed to enhance proteolytic stability due to steric hindrance and the propensity of D-amino acids to stabilize β-turn structures (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0044] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine, such as D-alanine. This embodiment provides the advantage of identifying critical binding residues and removing potential attack sites for proteolysis.

[0045] It should be noted that each of the above modifications serves to purposefully improve the potency or stability of the peptide. Further potency improvements based on modifications can be achieved through the following mechanisms: -Utilizing the hydrophobic effect, decreasing off-rates and achieving higher affinity by incorporating hydrophobic moieties; - the incorporation of charged groups that exploit long-range ionic interactions, resulting in faster on-rates and higher affinities (see, e.g., Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - Incorporating additional constraints into the peptide, for example by appropriately constraining the amino acid side chains so that the entropic loss upon target binding is minimized, constraining the backbone torsion angles so that the entropic loss upon target binding is minimized, or introducing additional cyclizations into the molecule for the same reasons (for reviews see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203 or Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418).

[0046] Isotope variants The present invention includes all pharma- ceutically acceptable (radio)isotope-labeled peptide ligands of the invention in which one or more atoms have been replaced with an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, peptide ligands of the invention to which metal chelating groups (referred to as "effectors") have been attached and which may bear relevant (radio)isotopes, and peptide ligands of the invention in which certain functional groups have been covalently replaced with relevant (radio)isotopes or isotopically labeled functional groups.

[0047] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention are: 2 H(D) and 3 Hydrogen, such as H(T), 11 C. 13 C and 14 Carbon, such as C 36 Chlorine such as Cl, 18 Fluorine such as F 123 I, 125 I and 131 Iodine, such as I 13 N and 15 Nitrogen such as N 15 O. 17 O and 18 Oxygen, such as O 32 Phosphorus such as P 35 Sulfur such as S 64 Copper, such as Cu 67 Ga and 68 Gallium such as Ga, 90 Yttrium, such as Y 177 Lu and other lutetium 213 Includes isotopes of bismuth such as Bi.

[0048] Certain isotopically labeled peptide ligands of the invention, such as those incorporating radioisotopes, are useful for drug and / or substrate tissue distribution studies, as well as for clinically assessing the presence and / or absence of EphA2 targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they may be used to detect or identify conjugate formation between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may employ compounds labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, luciferase), and the like. The radioisotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14 C is particularly useful for this purpose in view of its ease of uptake and rapid detection.

[0049] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), may be preferable in some circumstances because they may offer therapeutic advantages due to greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements.

[0050] 11 C. 18 F, 15 O. 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0051] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the Examples below, substituting appropriate isotopically labeled reagents for the non-labeled reagents previously used.

[0052] Reactive Groups The molecular scaffolds of the invention can be attached to polypeptides via functional or reactive groups on the polypeptide, which are usually formed from the side chains of certain amino acids found in the polypeptide polymer.

[0053] A reactive group is a group that can form a covalent bond with a molecular scaffold. Typically, reactive groups are present on the amino acid side chains of peptides. Examples are sulfur-containing groups such as lysine, arginine, histidine and analogs such as cysteine, methionine and selenocysteine.

[0054] In one embodiment, the reactive groups include cysteine ​​and / or (S)-2-amino-3-(methylamino)propanoic acid (Dap(Me)). In a further embodiment, all three reactive groups include cysteine. In another embodiment, two reactive groups include (S)-2-amino-3-(methylamino)propanoic acid (Dap(Me)) and one reactive group includes cysteine.

[0055] Examples of reactive groups of natural amino acids are the thiol group of cysteine, the amino group of lysine, the carboxyl group of aspartic acid or glutamic acid, the guanidinium group of arginine, the phenol group of tyrosine, or the hydroxyl group of serine. Non-natural amino acids can provide a wide range of reactive groups, including azide, ketocarbonyl, alkyne, vinyl, and aryl halide groups. The amino and carboxyl groups at the termini of polypeptides can also serve as reactive groups for forming covalent bonds with molecular scaffolds / cores.

[0056] The polypeptides of the invention contain at least three reactive groups. They can also contain four or more reactive groups. The more reactive groups used, the more loops can be formed within the molecular scaffold.

[0057] In a preferred embodiment, a polypeptide having three reactive groups is generated. The reaction of said polypeptide with a molecular scaffold / core having three-fold rotational symmetry generates a single product isomer. The generation of a single product isomer is advantageous for several reasons. The nucleic acid of the compound library only codes for the primary sequence of the polypeptide, and does not code for the isomeric state of the molecule formed upon reaction of the polypeptide with the molecular core. If a single product isomer is generated, the assignment of the nucleic acid to the product isomer is unambiguously defined. If multiple product isomers are formed, the nucleic acid cannot give information about the nature of the product isomer isolated in the screening or selection process. The formation of a single product isomer is also advantageous when synthesizing a specific compound in the library of the invention. In this case, the chemical reaction of the polypeptide with the molecular scaffold results in a single product isomer, not a mixture of isomers. In another embodiment of the invention, a polypeptide having four reactive groups is generated. The reaction of said polypeptide with a molecular scaffold / core having tetrahedral symmetry generates two product isomers. Even if two different product isomers are encoded by one and the same nucleic acid, the isomeric nature of the isolated isomers can be determined by chemically synthesizing both isomers, separating the two isomers, and testing both isomers for binding to the target ligand.

[0058] In one embodiment of the present invention, at least one of the reactive groups of the polypeptide is orthogonal to the remaining reactive groups. The use of orthogonal reactive groups allows the orthogonal reactive groups to be directed to a specific site of the molecular core. To limit the number of isomers produced, a linking strategy involving orthogonal reactive groups can be used. In other words, by selecting a reactive group for one or more of at least three bonds that is different from that selected for the remaining portion of at least three bonds, a specific order of binding or derivatization of a specific reactive group of the polypeptide to a specific position on the molecular scaffold can be usefully achieved. In another embodiment, the reactive group of the polypeptide of the present invention is reacted with a molecular linker, where the linker can react with the molecular scaffold, such that the linker is ultimately interposed between the molecular scaffold and the polypeptide in a bound state.

[0059] Alternative methods to thiol-mediated conjugation can be used to attach the molecular scaffold to the peptide via a covalent bond. Alternatively, these techniques can be used to modify or attach additional moieties (e.g., small molecules of interest different from the molecular scaffold) to the polypeptide after it has been selected or isolated according to the invention - although in this embodiment, obviously the attachment does not have to be covalent and can include non-covalent attachment. These methods can be used instead of (or in combination with) the thiol-mediated method by generating phage expressing proteins and peptides with unnatural amino acids bearing the required chemically reactive groups, by combining small molecules bearing complementary reactive groups, or by incorporating unnatural amino acids into chemically or recombinantly synthesized polypeptides when generating the molecules after the selection / isolation step. Further details can be found in WO 2009 / 098450 or in Heinis, et al., Nat Chem Biol 2009, 5 (7), 502-7.

[0060] It is understood that the looped bicyclic peptide structure is further linked to the molecular scaffold via at least one thioether bond. The thioether bond provides an anchor during the formation of the bicyclic peptide. In some embodiments, there is only one such thioether bond. In further embodiments, there is one such thioether bond and two amino bonds. In further embodiments, there is one such thioether bond and two alkylamino bonds. Suitably, the thioether bond is the central bond of the bicyclic or polycyclic peptide conjugate, i.e., in the peptide sequence, the two residues forming the amino bonds in the peptide (e.g., diaminopropionic acid residues) are spaced apart on either side of the amino acid residues forming the thioether bond (e.g., lysine). Suitably, the looped peptide structure is thus a bicyclic peptide conjugate with a central thioether bond and two peripheral amino bonds. In some embodiments, the location of the thioether bond can be N-terminal or C-terminal to the two N-alkylamino bonds.

[0061] In one embodiment, the reactive groups are one cysteine ​​residue and two (S)-2-amino-3-(methylamino)propanoic acid (Dap(Me)) or N-β-C 1-4 It contains alkyl-L-2,3-diaminopropionic acid (N-AlkDap) residues.

[0062] Aromatic molecular scaffolds As used herein, the term "aromatic molecular scaffold" refers to any molecular scaffold as defined herein that contains an aromatic carbocyclic or heterocyclic ring system.

[0063] It will be appreciated that the aromatic molecular scaffold may comprise an aromatic moiety. Examples of suitable aromatic moieties within the aromatic scaffold include biphenylene, terphenylene, naphthalene or anthracene.

[0064] It is also understood that the aromatic molecular scaffold may contain heteroaromatic moieties. Examples of suitable heteroaromatic moieties within the aromatic scaffold include pyridine, pyrimidine, pyrrole, furan and thiophene.

[0065] It is also understood that the aromatic molecular scaffold may contain a halomethylarene moiety. Examples of halomethylarene moieties include bis(bromomethyl)benzene, tris(bromomethyl)benzene, tetra(bromomethyl)benzene or derivatives thereof. Non-limiting examples of aromatic molecular scaffolds include bis-, tris- or tetra(halomethyl)benzene; bis-, tris- or tetra(halomethyl)pyridine; bis-, tris- or tetra(halomethyl)pyridazine; bis-, tris- or tetra(halomethyl)pyrimidine; bis-, tris- or tetra(halomethyl)pyrazine; bis-, tris- or tetra(halomethyl)-1,2,3-triazine; bis-, tris- or tetra-(halomethyl)-1,2,4-triazine; bis-, tris- or tetra(halomethyl)pyrrole; These include -furans, -thiophenes; bis-, tris- or tetra(halomethyl)imidazoles, -oxazoles, -thiazoles; bis-, tris- or tetra(halomethyl)-3H-pyrazoles, -isoxazoles, -isothiazoles; bis-, tris- or tetra(halomethyl)biphenylenes; bis-, tris- or tetra(halomethyl)terphenylenes; 1,8-bis(halomethyl)naphthalenes; bis-, tris- or tetra(halomethyl)anthracenes; bis-, tris- or tetra(2-halomethylphenyl)methanes.

[0066] More specific examples of aromatic molecular scaffolds include 1,2-bis(halomethyl)benzenes; 3,4-bis(halomethyl)pyridines; 3,4-bis(halomethyl)pyridazines; 4,5-bis(halomethyl)pyrimidines; 4,5-bis(halomethyl)pyrazines; 4,5-bis(halomethyl)-1,2,3-triazines; 5,6-bis(halomethyl)-1,2,4-triazines; 3,4-bis(halomethyl)pyrroles, -furans, -thiophenes and other positional isomers; 4,5-bis(halomethyl)imidazoles, -oxazoles, -thiazoles; 4,5-bis(halomethyl)-3H-pyrazoles, -isoxazoles, -isothiazoles; 2,2'-bis(halomethyl)biphenylenes; 2,2'' -Bis(halomethyl)terphenylenes;1,8-Bis(halomethyl)naphthalenes;1,10-Bis(halomethyl)anthracenes;Bis(2-halomethylphenyl)methanes;1,2,3-Tris(halomethyl)benzenes;2,3,4-Tris(halomethyl)pyridines;2,3,4-Tris(halomethyl)pyridazines;3,4,5-Tris(halomethyl)pyrimidines;4,5,6-Tris(halomethyl)-1,2,3-triazines;2,3,4-Tris(halomethyl)pyrroles, -furans, -thiophenes;2,4,5-Bis(halomethyl)imidazoles, -oxazoles, -thiazoles;3,4,5-Bis(halomethyl)-1H-pyrazoles, -isoxazoles, -isothiazoles; 2,4,2'-Tris(halomethyl)biphenylenes;2,3',2''-Tris(halomethyl)terphenylenes;1,3,8-Tris(halomethyl)naphthalenes;1,3,10-Tris(halomethyl)anthracenes;Bis(2-halomethylphenyl)methanes;1,2,4,5-Tetra(halomethyl)benzenes;1,2,4,5-Tetra(halomethyl)pyridines;2,4,5,6-Tetra(halomethyl)pyridines )pyrimidines; 2,3,4,5-tetra(halomethyl)pyrroles, -furans, -thiophenes; 2,2',6,6'-tetra(halomethyl)biphenylenes; 2,2'',6,6''-tetra(halomethyl)terphenylenes; 2,3,5,6-tetra(halomethyl)naphthalenes and 2,3,7,8-tetra(halomethyl)anthracenes; bis(2,4-bis(halomethyl)phenyl)methanes.

[0067] As described in the aforementioned publications, the molecular scaffold can also be a small molecule, for example a small organic molecule.

[0068] In some embodiments, the molecular scaffold can also be a macromolecule, hi some embodiments, the molecular scaffold is a polymer composed of amino acids, nucleotides, or carbohydrates.

[0069] In some embodiments, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0070] The molecular scaffold may contain chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0071] In certain embodiments, the molecular scaffold may comprise or consist of tris(bromomethyl)benzene, in particular 1,3,5-tris(bromomethyl)benzene ("TBMB"), or a derivative thereof.

[0072] In an embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene, but contains three additional methyl groups attached to the benzene ring. This has the advantage that the additional methyl groups can form additional contacts with the polypeptide, resulting in additional structural constraints.

[0073] The molecular scaffolds of the invention comprise chemical groups that enable functional groups of the polypeptides of the encoded libraries of the invention to form covalent bonds with the molecular scaffold, said chemical groups being selected from a wide range of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides and acyl halides.

[0074] Scaffold reactive groups that can be used on a molecular scaffold to react with the thiol group of cysteine ​​are alkyl halides (also called halogenoalkanes or haloalkanes).

[0075] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups used to selectively attach compounds to cysteines in proteins are maleimides, αβ-unsaturated carbonyl-containing compounds and α-halomethylcarbonyl-containing compounds. Examples of maleimides that may be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. An example of an α-halomethylcarbonyl-containing compound is N,N',N''-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a natural amino acid with similar reactivity to cysteine ​​and can be used in the same reactions. Thus, where cysteine ​​is mentioned, it is generally acceptable to substitute selenocysteine ​​unless the context suggests otherwise.

[0076] Cytotoxic Agents Suitable examples of cytotoxic agents include alkylating agents such as cisplatin, carboplatin, oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites such as the purine analogues azathioprine, mercaptopurine, and pyrimidine analogues; plant alkaloids and terpenoids, including the vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel (originally known as taxol); topoisomerase inhibitors, including camptothecins (irinotecan, topotecan), and type II inhibitors, such as amsacrine, etoposide, etoposide phosphate, and teniposide. Additional drugs may include the immunosuppressants dactinomycin (used in kidney transplants), antitumor antibiotics including doxorubicin, epirubicin, bleomycin, calicheamicin, and the like.

[0077] In one embodiment of the invention, the cytotoxic agent is selected from a maytansinoid (such as DM1) or a monomethylauristatin (such as MMAE).

[0078] DM1 is a thiol-containing derivative of maytansine, a cytotoxic agent with the following structure: [ka]

[0079] (S)-N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide) (monomethylauristatin E; MMAE) is a synthetic antineoplastic agent with the following structure: [ka]

[0080] In a more particular embodiment of the invention, the cytotoxic agent is MMAE.

[0081] In certain embodiments, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulfide bond or a protease-sensitive bond, hi further embodiments, the groups adjacent to the disulfide bond are modified to control inhibition of the disulfide bond, thereby controlling cleavage and subsequent release of the cytotoxic agent.

[0082] Published studies have demonstrated that it may be possible to tune the susceptibility of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). Greater steric hindrance reduces the rate of reduction by intracellular glutathione and extracellular (systemic) reducing agents, resulting in increased susceptibility to toxin release both intracellularly and extracellularly. Thus, optimal selection of disulfide stability in circulation (minimizing undesirable side effects of the toxin) and efficient release in the intracellular environment (maximizing therapeutic efficacy) can be achieved by carefully selecting the degree of hindrance on either side of the disulfide bond.

[0083] The disorder on either side of the disulfide bond is modulated by the introduction of one or more methyl groups on either the target (here the bicyclic peptide) or toxin side of the molecular construct.

[0084] In one embodiment, the cytotoxic agents and linkers are selected from any combination of those described in WO2016 / 067035 (which cytotoxic agents and linkers are incorporated herein by reference).

[0085] In some embodiments, the linker between the cytotoxic agent and the bicyclic peptide comprises one or more amino acid residues. Examples of amino acid residues suitable for suitable linkers include Ala, Cit, Lys, Trp and Val. In further embodiments, the linker between the cytotoxic agent and the bicyclic peptide comprises a Val-Cit moiety. In further embodiments, the linker between the cytotoxic agent and the bicyclic peptide comprises a β-Ala moiety.

[0086] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises p-aminobenzylcarbamate (PABC).

[0087] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises a triazolyl moiety.

[0088] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises a propanol group.

[0089] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises an acetyl group.

[0090] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises N-(bisaminopropyl)glycine (BAPG).

[0091] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises one or more (eg, 10) sarcosine (Sar) residues.

[0092] In a further embodiment, the linker between the cytotoxic agent and the bicyclic peptide is -Sar 10 -βAla-BAPG-(Propanoyl-triazolyl-acetyl-Val-Cit-PABC) 2 linker (i.e., the resulting bicyclic peptide drug conjugate is 10-βAla-BAPG-(Propanoyl-triazolyl-acetyl-Val-Cit-PABC-MMAE) 2 (including parts).

[0093] In one embodiment, the bicyclic peptide ligand is specific for MT1-MMP, the cytotoxic agent is MMAE, the number of MMAE moieties is two, and the drug conjugate comprises the compound BT17BDC69: [ka] (where R is βAla-Sar 10 -A- (SEQ ID NO: 1), where R is linked via the N-terminus of the peptide (i.e., the βAla residue).

[0094] Data showing that BT17BDC69 demonstrates potent antitumor activity at 0.3 mg / kg, biw are presented in Figure 2 and Tables 3 and 7 herein.

[0095] In another embodiment, the bicyclic peptide ligand is specific for MT1-MMP, the cytotoxic agent is MMAE, the number of MMAE moieties is two, and the drug conjugate comprises the compound BT17BDC75: [ka] (where R is βAla-Sar 10 -A- (SEQ ID NO: 2), where R is linked via the N-terminus of the peptide (i.e., the βAla residue).

[0096] Data showing that BT17BDC75 dramatically shrank tumors, eradicating 3 / 3 and 1 / 3 tumors at 0.3 mg / kg, biw and 1 mg / kg, biw, respectively, are presented in Figure 5 and Tables 4 and 8 herein.

[0097] In another embodiment, the bicyclic peptide ligand is specific for MT1-MMP, the cytotoxic agent is MMAE, the number of MMAE moieties is two, and the drug conjugate comprises the compound BCY16278: [ka] (where R is βAla-Sar 10 -A- (SEQ ID NO: 2), where R is linked via the N-terminus of the peptide (i.e., the βAla residue).

[0098] Data showing that BCY16278 exhibits potent antitumor activity at 0.3 mg / kg, qw are presented in Figure 6 and Tables 9 and 10 herein.

[0099] synthesis The peptides of the present invention can be synthetically produced by standard techniques followed by reaction with molecular scaffolds in vitro. Standard chemical reactions can be used to carry this out. This allows for rapid and large-scale preparation of soluble material for further downstream experiments or validation. Such methods can be achieved using conventional chemistry, such as that disclosed in Timmerman et al (supra).

[0100] The present invention therefore also relates to the production of a polypeptide or conjugate selected as defined herein, which production comprises optional further steps as explained below, which in one embodiment are carried out on the final product polypeptide / conjugate produced by chemical synthesis.

[0101] In producing the conjugate or complex, amino acid residues in the polypeptide of interest can be optionally substituted.

[0102] The peptides can also be extended, for example to incorporate additional loops, thereby introducing multiple specificities.

[0103] To extend a peptide, it can simply be chemically extended at the N- or C-terminus or in a loop using standard solid- or solution-phase chemistry with orthogonally protected lysines (and analogues). Standard (bio)conjugation techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, they can be added by fragment condensation or native chemical ligation (e.g. as shown in Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or enzymatically (e.g. using subtiligase as described in 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).

[0104] Alternatively, the peptide may be extended or modified by further bonding via a disulfide bond. This also has the advantage of allowing the first and second peptides to dissociate from each other once in the reductive environment of the cell. In this case, a molecular scaffold (e.g., TBMB) can be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups. Then, a further cysteine ​​or thiol can be added to the N-terminus or C-terminus of the first peptide, which can only react with the free cysteine ​​or thiol of the second peptide to form a disulfide-bonded bicyclic peptide-peptide conjugate.

[0105] Similar techniques could equally be applied to the synthesis / coupling of two bicyclic and bispecific macrocycles to create tetraspecific molecules.

[0106] Furthermore, the addition of other functional or effector groups can be achieved similarly using appropriate chemical methods, coupling at the N-terminus or C-terminus or coupling via a side chain, in some embodiments, coupling is performed in such a way as not to interfere with the activity of either entity.

[0107] Pharmaceutical Compositions According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0108] Generally, the peptide ligands of the present invention are utilized in purified form together with pharmacologically appropriate excipients or carriers.Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media.Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and the like.If necessary to keep the polypeptide conjugate in suspension, suitable physiologically acceptable adjuvants can be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0109] Intravenous vehicles include fluid, nutrient and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0110] The peptide ligands of the invention can be used as a separately administered component or together with other agents. These can include various immunotherapeutic agents and immunotoxins, such as antibodies, antibody fragments, cyclosporine, methotrexate, adriamycin or cisplatinum. Pharmaceutical compositions can include "cocktails" of various cytotoxic or other agents in combination with the protein ligands of the invention, or even combinations of polypeptides with different specificities selected according to the invention (e.g., polypeptides selected with different targeting ligands, etc.), whether or not pooled prior to administration.

[0111] The route of administration of the pharmaceutical composition according to the invention may be any of those generally known to those skilled in the art. For treatment, the peptide ligand of the invention may be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, via the pulmonary route, or by direct injection by catheter, as appropriate. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, the co-administration of other drugs, contraindications and other parameters that should be taken into account by the clinician.

[0112] The peptide ligand of the present invention can be freeze-dried for storage and reconstituted in a suitable carrier before use.This technique has been shown to be effective, and can use freeze-drying and reconstitution techniques known in the art.Those skilled in the art will understand that freeze-drying and reconstitution may result in varying degrees of activity loss, and may have to adjust levels upward to compensate.

[0113] Compositions containing the peptide ligands or cocktails thereof of the present invention can be administered for prophylactic and / or therapeutic treatments. In a particular therapeutic application, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or other measurable parameters of a population of selected cells is defined as a "therapeutic effective amount". The amount required to achieve this dosage will vary depending on the severity of the disease and the general state of the patient's own immune system, but typically within 0.005-5.0 mg of the selected peptide ligand per kg of body weight is used, with 0.05-2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof of the present invention can also be administered at similar or slightly lower doses.

[0114] Compositions containing peptide ligands according to the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of selective target cell populations in mammals. In addition, the peptide ligands described herein can be selectively used ex vivo or in vitro to kill, deplete or otherwise effectively remove target cell populations from heterogeneous cell populations. Blood from a mammal can be combined ex vivo with the selected peptide ligand, whereby undesirable cells are killed or otherwise removed from the blood for return to the mammal according to standard techniques.

[0115] Therapeutic Use Due to the presence of a cytotoxic agent, the drug conjugates of the present invention are particularly useful in the treatment of diseases that can be alleviated by cell death. Examples of suitable diseases include diseases characterized by defective cells, cell proliferative diseases such as cancer, and autoimmune diseases such as rheumatoid arthritis.

[0116] The presence of a cytotoxic agent attached to the cancer cell-binding bicyclic peptide gives the bicyclic peptide of the invention specific utility in the treatment of cancer. Thus, according to a further aspect of the invention there is provided a drug conjugate as defined herein for use in the prevention, inhibition or treatment of cancer (such as a tumor).

[0117] According to a further aspect of the present invention, there is provided a method of preventing, inhibiting or treating cancer (such as a tumor), comprising administering a drug conjugate as defined herein to a patient in need thereof.

[0118] Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other cancers), such as tumours of the bladder, urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric portion), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (such as adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (such as cancer of the tongue, buccal cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, Cancers of the vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal glands, prostate, skin and adnexa (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); borderline malignancies and related diseases, including hematological malignancies (leukemia, lymphoma) and premalignant hematological disorders and hematological malignancies (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, leukemia, ... cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, post-transplant lymphoproliferative disorder), as well as hematological malignancies and related diseases of the myeloid system (acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], myeloproliferative disorders such as eosinophilic syndrome, polycythemia vera, essential thrombocythemia, primary myelofibrosis, myeloproliferative disorders, myelodysplastic syndromes, promyelocytic leukemia, etc.); Mesenchymal tumors, for example sarcomas of soft tissue, bone or cartilage such as osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytoma, dermatofibrosarcoma; tumors of the central or peripheral nervous system (such as astrocytoma, glioma and glioblastoma, meningioma, ependymoma, pineal tumor and neurinoma); endocrine tumors (such as pituitary tumor, adrenal tumor, pancreatic islet cell tumor, parathyroid tumor, carcinoid tumor, medullary thyroid carcinoma); tumors of the eye and adnexa (such as retinoblastoma);These include, but are not limited to, germ cell and trophoblastic tumors (such as teratomas, seminomas, ectodermal tumors, hydatidiform moles, and choriocarcinomas); pediatric and embryonal tumors (such as medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors); congenital or other syndromes that predispose patients to malignancies (such as xeroderma pigmentosum);

[0119] In further embodiments, the cancer is selected from breast cancer, lung cancer, gastric cancer, pancreatic cancer, prostate cancer, liver cancer, glioblastoma and angiogenesis.

[0120] As used herein, the term "prevention" includes administration of the protective composition prior to the induction of the disease. "Suppression" refers to administration of the composition after an inducing event, prior to the appearance of clinical symptoms of the disease. "Treatment" includes administration of the protective composition after symptoms of the disease are manifest.

[0121] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease, and their use is facilitated by the present invention, which allows for the development of polypeptide ligands that can cross-react with human and animal targets.

[0122] The present invention will now be further described with reference to examples. EXAMPLES

[0123] Abbreviation 1NAI 1-Naphthylalanine HyP Hydroxyproline HArg Homoarginine β-Ala β-alanine Sar Sarcosine x represents x Sar residues)

[0124] material and method Peptide synthesis Peptides were synthesized by solid phase synthesis. Rink Amide MBHA Resin was used. DMF was added to a mixture containing Rink Amide MBHA (0.4-0.45 mmol / g) and Fmoc-Cys(Trt)-OH (3.0 equiv.), followed by addition of DIC (3 equiv.) and HOAt (3 equiv.) and mixing for 1 h. 20% piperidine in DMF solvent was used for deblocking. Each subsequent amino acid was coupled in 3 equiv. using the activation reagents DIC (3.0 equiv.) and HOAT (3.0 equiv.) in DMF. The reaction was monitored by ninhydrin or tetrachloride color reaction. After synthesis, the peptide resin was washed with DMF x 3 and MeOH x 3 and N 2 The peptide resin was then dried overnight under bubbling. 2 The peptide was precipitated with cold isopropyl ether and centrifuged (3000 rpm for 3 min). The pellet was washed twice with isopropyl ether and the crude peptide was dried under vacuum for 2 h and lyophilized. The lyophilized powder was diluted with ACN / H2O and purified by HPLC. 2 HO (50:50) and add 100 mM TBMB solution in ACN solvent, followed by HO. 2 Ammonium bicarbonate (1M) in 0 solvent was added and the solution was stirred for 1 h. It was quenched with cysteine ​​hydrochloride (10 equivalents relative to TBMB), mixed and left for 1 h. The solution was lyophilized to give the crude product. The crude peptide was purified by preparative HPLC and lyophilized to give the product.

[0125] Unless otherwise noted, all amino acids were used in the L-configuration.

[0126] Preparation of Drug Conjugates of the Invention General conditions Separation conditions: 1) Phase A: 0.075% TFA aqueous solution, Phase B: MeCN 2) Phase A: 0.08% NH 4 HCO 3 Aqueous solution, B phase: MeCN Separation method: 18-48-55 minutes, RT=53.5 minutes Separation columns: Luna 200*25 mm 10 μm, C18, 110 A and Gemin150*30 mm, C18, 5 μm, 110 A, connected, 50℃. Dissolution method:DMF Separation purity: 95%

[0127] Scheme 1: Preparation of triazole-Val-Cit-PABC-MMAE-containing drug conjugates [ka]

[0128] Preparation of compound 3 [ka] To a solution of compound 2 (30 g, 80 mmol) in DCM (300 mL) and MeOH (150 mL) solvent, 4-aminophenylmethanol (11 g, 88 mmol) and EEDQ (40 g, 160 mmol) were added in the dark. The mixture was stirred at 30 °C for 16 h. TLC (DCM:MeOH = 10 / 1, R f =0.43), compound 2 was completely consumed and many new spots formed. The reaction was clean by TLC. The resulting reaction mixture was concentrated to give a residue which was purified by flash silica gel chromatography (ISCO®; 330 g x 3 SepaFlash® silica flash column, eluent of 0-20% MeOH / dichloromethane at 100 mL / min). Compound 3 (20 g, 52% yield) was obtained as a white solid.

[0129] Preparation of compound 4 [ka] To a solution of compound 3 (5.0 g, 10.4 mmol) in DMF (40 mL) solvent, DIEA (5.4 g, 7.26 mL, 41.7 mmol) and bis(4-nitrophenyl)carbonate (12.7 g, 41.7 mmol) were added. The mixture was stirred at 0 °C under nitrogen for 1 h. TLC (DCM:MeOH = 10 / 1, Rf = 0.66) showed that compound 3 was completely consumed and one new spot was formed. The reaction was clean by TLC and LCMS (:RT = 1.15 min) showed that the desired product was formed. The resulting reaction mixture was directly purified by preparative HPLC under neutral conditions. Compound 4 (12 g, 60% yield) was obtained as a white solid.

[0130] Preparation of compound 5 [ka] To a solution of compound 4 (1.2 g, 1.68 mmol) in DMF (10 mL) solvent, DIEA (1.22 mL, 6.98 mmol) was added under nitrogen atmosphere, and the solution was stirred at 0 °C for 10 min, after which HOBt (226 mg, 1.68 mmol) and MMAE (1.00 g, 1.40 mmol) were added thereto, and the mixture was degassed and purified with N 2 The mixture was purged with 0.5 mL of ethyl acetate three times and stirred at 35° C. for 16 h. LC-MS (product: RT = 1.19 min) showed that compound 4 was completely consumed and one main peak with the desired mass was detected. The resulting five batches of reaction mixture were combined in a 1 L beaker and 500 mL of water was added, after which a precipitate formed and was collected by filtration. The precipitate was triturated with EtOAc overnight. Compound 5 (5 g, 59% yield) was obtained as a white solid.

[0131] Preparation of compound 6 [ka] Compound 5 (3.3 g, 2.7 mmol) was dissolved in DCM (18 mL) in the presence of TFA (44 mmol, 3.5 mL) and the solution was stirred at 25° C. for 3 h. The reaction mixture was then concentrated under reduced pressure to remove DCM and TFA to give a residue. The residue was dissolved in THF (20 mL) and K 2 CO 3 (1.8 g, 13 mmol) and the mixture was stirred at 25° C. for an additional 12 h. LC-MS (product: RT = 1.04 min) showed that one main peak with the desired mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to give a residue. The residue was dissolved in 10 mL of DMF and purified by preparative HPLC (neutral conditions). Compound 6 (1.6 g, 53% yield) was obtained as a white solid.

[0132] Preparation of compound 7-1 [ka] Compound 6 (1.2 g, 1.1 mmol) and 2-azidoacetic acid (162 mg, 1.6 mmol) were dissolved in DMF (10 mL). TEA (450 μL, 3.2 mmol), HOBt (217 mg, 1.6 mmol) and EDCI (307 mg, 1.6 mmol) were added to the solution under nitrogen, and the mixture was stirred at 0 °C for 30 min, after which the mixture was slowly warmed to 25 °C with stirring for another 15.5 h. LC-MS (product: RT = 1.04 min) showed that compound 6 was completely consumed and one main peak with the desired mass was detected. 2 mL of water was added to the reaction mixture to form a clear solution. The solution was then directly purified by preparative HPLC under neutral conditions. Compound 7-1 (0.9 g, 70% yield) was obtained as a white solid.

[0133] Scheme 2: Preparation of drug conjugates containing Glutaryl-Val-Cit-PABC-MMAE [ka]

[0134] Preparation of compound 7-2 [ka] A vial containing 6-dimethylformamide (400 mg, 356 μmol) was purged with a nitrogen balloon. Anhydrous DMA (5 mL) was added with stirring and the solution was cooled to 0 °C in an ice-water bath. DIEA (130 μL, 712 μmol) was then added and the reaction was stirred at 0 °C for 10 min. Tetrahydropyran-2,6-dione (81 mg, 712 μmol) was added and the ice bath was removed. The reaction was stirred at 25 °C for 1 h. LC-MS (product: RT = 1.08 min) showed that compound 6 was completely consumed and one main peak with the desired mass was detected. The mixture was diluted with 5 mL of water and purified by preparative HPLC (neutral conditions). Compound 7-2 (330 mg, 75% yield) was obtained as a white solid.

[0135] Preparation of compound 8 [ka] To compound 7-2 (330 mg, 267 μmol) in anhydrous DMA (4.5 mL) and DCM (1.5 mL) was added HOSu (92 mg, 800 μmol) with stirring for 10 min at 0 °C with ice bath under nitrogen. Then, EDCI (154 mg, 800 μmol) was added to the mixture and further stirred at 25 °C for 16 h. LC-MS (product: RT = 1.15 min) showed that compound 7-2 was completely consumed and one main peak with the desired mass was detected. The resulting reaction mixture was diluted with 5 mL of water and purified by preparative HPLC (neutral conditions). Compound 8 (250 mg, 70% yield) was obtained as a white solid.

[0136] Preparation of the reference bicyclic peptide drug conjugate BT17BDC61 (mono-MMAE) BT17BDC61 was prepared as described in WO 2018 / 115204.

[0137] The alkyne side chain derivatized peptides used to prepare the following compounds were synthesized on resin according to the methods described in the Materials and Methods section and have the following general structure: [ka] (17-69-07-N475 and 17-69-07-N466) [ka] 17-69-07-N468

[0138] Preparation of bicyclic peptide drug conjugate BT17BDC75 (bis-MMAE) [ka] (where BICY is βAla-Sar 10 -A- (including SEQ ID NO: 2) Compound 7-1 (350 mg, 290 μmol) and bisBICY-alkyne 17-69-07-N475 (289 mg, 96.7 μmol) were taken in a 50 mL round flask, DMSO (5 mL) was added, and then aqueous ascorbic acid (1 M, 2.90 mL) and CuSO4 (1 M, 870 μL) were added under nitrogen atmosphere. The mixture was then stirred at 25 °C for 30 min. LC-MS (product: RT = 1.11 min) showed that the BICY-alkyne was completely consumed and one main peak with the desired mass was detected. The DMSO solution was simply diluted with 10% DMF / H 2 The mixture was diluted to approximately 10 ml with 200 mL of 1H2O and directly purified by preparative HPLC under TFA conditions. BT17BDC75 (233 mg, 44% yield) was obtained as a white solid. m / z was 1350 [M+H]. 4+ HPLC, RT = 16.84 min.

[0139] Preparation of the reference bicyclic peptide drug conjugate BT17BDC23 (mono-MMAE) BT17BDC23 was prepared as described in WO 2018 / 127699.

[0140] Preparation of the reference bicyclic peptide drug conjugate BT17BDC69 (bis-MMAE) [ka] (Here, BICY is βAla-Sar 10 -A- (including SEQ ID NO: 1) Compound 7-1 (60.0 mg, 49.7 μmol) and bis BICY-alkyne 17-69-07-N466 (40.0 mg, 13.4 μmol) were dissolved in DMF (3 mL). After adding an aqueous solution of ascorbic acid (0.8 M, 620 μL), the mixture was stirred under a nitrogen atmosphere with CuSO 4 Aqueous solution (0.8 M, 190 μL) was added and the mixture was stirred at 25 °C for 14 h. LC-MS showed that the bisBICY-alkyne was completely consumed. The resulting reaction mixture was filtered and the filtrate was purified by prep-HPLC (TFA conditions). BT17BDC69 (26 mg, 36% yield) was obtained as a white solid. m / z showed 1081 [M+H]5+. HPLC, RT = 20.18 min.

[0141] Preparation of the reference bicyclic peptide drug conjugate BT17BDC70 (Tet-MMAE) [ka] (where BICY is βAla-Sar 10 -A- (including SEQ ID NO: 1) Compound 7-1 (60.0 mg, 50.0 μmol) and tetra BICY-alkyne 17-69-07-N468 (20.0 mg, 5.72 μmol) were dissolved in DMF (5 mL). Aqueous ascorbic acid (0.8 M, 620 μL) was added, followed by aqueous CuSO4 (0.8 M, 190 μL) under nitrogen atmosphere, and the mixture was stirred at 25 °C for 14 h. LCMS (ES6635-184-P1A1) showed that tetra BICY-alkyne was consumed, and HPLC (ES6635-184-P1H1) showed that a new substance was formed according to compound 7-1 (HPLC: ES6635-SM-1206). The resulting reaction mixture was filtered, and the filtrate was purified by preparative HPLC (TFA conditions). BT17BDC70 (8 mg, 17% yield) was obtained as a white solid. The target substance was confirmed by HRMS (TOF). m / z was 2773.7 [M+H] 3+ HPLC RT = 23.04 min.

[0142] Preparation of the reference bicyclic peptide drug conjugate BCY16278 (bis-MMAE) Preparation of Compound 10 [ka] Compound 9 can be prepared in a similar manner to compound 2 described above. For example, compound 9 was prepared by solid-phase peptide synthesis using chlorotrityl resin and standard methods for coupling and deprotection. To a solution of compound 9 (150 mg, 167 μmol, 1 equiv.) in pyridine (4 mL), EDCI (224 mg, 1.17 mmol, 7 equiv.) and (4-aminophenyl)methanol (41.23 mg, 334 μmol, 2 equiv.) were added. The mixture was stirred at 25° C. for 2 h. LC-MS showed that compound 9 was completely consumed and one main peak of the desired m / z was detected. The residue was purified by preparative HPLC (TFA buffer) to give compound 10 (135 mg, 134 μmol, 80% yield) as a white solid. Calculated MW: 1001.1, observed m / z: 1001.5 [M+H] + .

[0143] Preparation of compound 11 [ka] To a solution of compound 10 (129 mg, 128 μmol, 1 equiv.) in DMF (2 mL), DIEA (66.6 mg, 515 μmol, 89.8 μL, 4 equiv.) and bis(4-nitrophenyl)carbonate (117 mg, 386 μmol, 3 equiv.) were added. The mixture was stirred at 30° C. under nitrogen atmosphere for 16 h. LC-MS showed one main peak of the desired m / z. The residue was purified by prep-HPLC (neutral conditions). Compound 11 (85 mg, 72 μmol, 56% yield) was obtained as a white solid. Calculated MW: 1166.2, observed m / z: 1166.3 [M+H] + .

[0144] Preparation of compound 12 [ka] To a solution of compound 11 (80 mg, 68 μmol, 1 equiv.) in DMF (1.5 mL), HOBt (11.1 mg, 82 μmol, 1.2 equiv.), DIEA (35.46 mg, 274 μmol, 47 μL, 4 equiv.), and MMAE (49.2 mg, 68 μmol, 1 equiv.) were added. The mixture was stirred at 30° C. under nitrogen atmosphere for 16 h. LC-MS showed that compound 11 was completely consumed and one main peak of the desired m / z was detected. The residue was purified by preparative HPLC (TFA condition). Compound 12 (81 mg, 46 μmol, 67% yield) was obtained as a white solid. Calculated MW: 1746.1, observed m / z: 873.4 [M / 2+H] + .

[0145] Preparation of the reference bicyclic peptide drug conjugate BCY16278 (bis-MMAE) [ka] (where BICY is βAla-Sar 10-A- (SEQ ID NO: 2). Compound 12 (30.7 mg, 1769 μmol, 2.1 equiv.), 17-69-07-N475 (25.0 mg, 8.4 μmol, 1.0 equiv.) and tris(3-hydroxyprotriazolylmethyl)amine (8.0 mg, 18.4 μmol, 2.2 equiv.) in t-BuOH (0.5 mL) and H 2 Degas the mixture in O (0.5 mL) and add N 2 After purging three times with CuSO 4 (0.4 M, 20.9 μL, 1.0 equiv.), and VcNa (3.3 mg, 16.75 μmol, 2.0 equiv.) were added to the mixture under a nitrogen atmosphere. Then, 0.2 M NH 4 HCO 3 The pH was adjusted to 8 by dropwise addition. The mixture was stirred under nitrogen at 25° C. for 1 h. LC-MS showed that compound 13 was the major component in the mixture (calculated MW: 6474.5, observed m / z: 1081.0 [M / 6+H] + The reaction mixture was used in the next step without further purification.

[0146] The reaction mixture was diluted with NH 2 NH 2 .H 2 O (20 μL) was added. The mixture was stirred at 25 °C for 0.5 h. LC-MS showed that compound 13 was completely consumed and one main peak of the desired m / z was detected. The residue was purified by preparative HPLC (TFA condition) to give BCY16278 (15.5 mg, 2.55 μmol, 33% yield, 96.1% purity) as a white solid. Calculated MW: 5851.6, observed m / z: 1463.7 [M / 4+H] + , 1171.2 [M / 5+H] + .

[0147] Biological Data Example 1: In vivo efficacy testing of BT17BDC23, BT17BDC61, BT17BDC69, BT17BDC70 and BT17BDC75 in treating HT1080 xenografts in BALB / c nude mice

[0148] 1. Research purpose The aim of the study was to evaluate the in vivo antitumor efficacy of BT17BDC23, BT17BDC61, BT17BDC69, BT17BDC70 and BT17BDC75 in treating HT1080 xenografts in BALB / c nude mice. 2. Experimental Design [Table 1]

[0149] 3.Material 3.1 Animals and housing conditions 3.1.1 Animals Species: Mus Musculus Lineage: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18-22g Number of mice: 39 + extra mice Animal donor: Shanghai LC Laboratory Animal Co., LTD. 3.1.2 Rearing conditions The mice were housed in individually ventilated cages at constant temperature and humidity, with three mice per cage. ·Temperature: 20-26℃. ·Humidity: 40-70%. Cage: Made of polycarbonate, size 300mm x 180mm x 150mm, bedding material was corncob, changed twice weekly. Diet: Animals were fed radiation-sterilized dry granular food ad libitum throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: The identification label on each cage contained the following information: number of animals, sex, strain, date received, treatment, study number, group number, and start date of treatment. Animal Identification: Animals were ear tagged.

[0150] 3.2 Experiments and positive controls Product identification: BT17BDC23 Manufacturer: Bicycle Therapeutics Lot Number: N / A Physical properties: Freeze-dried powder Molecular weight: 3878.5 Packaging and storage conditions: Store at -80℃ Product Identification: BT17BDC61 Manufacturer: Bicycle Therapeutics Lot Number: 1 Physical properties: Freeze-dried powder Molecular weight: 3939.46 Purity: 95.11% Packaging and storage conditions: Store at -80℃ Product Identification: BT17BDC69 Manufacturer: Bicycle Therapeutics Lot Number: 1 Physical properties: Freeze-dried powder Molecular weight: 5401.78 Purity: 95.20% Packaging and storage conditions: Store at -80℃ Product Identification: BT17BDC70 Manufacturer: Bicycle Therapeutics Lot Number: 1 Physical properties: Freeze-dried powder Molecular weight: 8315.87 Purity: 97.40% Packaging and storage conditions: Store at -80℃ Product Identification: BT17BDC75 Manufacturer: Bicycle Therapeutics Lot Number: 1 Physical properties: Freeze-dried powder Molecular weight: 5397.25 Purity: 97.60% Packaging and storage conditions: Store at -80℃

[0151] 4. Experimental methods and procedures 4.1 Cell culture HT1080 tumor cells were grown as monolayers in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in 5% CO in air. 2 The tumor cells were maintained in vitro at 25°C for 1 h at 4°C. Tumor cells were periodically recultured twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor implantation.

[0152] 4.2 Tumor transplantation Each mouse was treated with HT1080 tumor cells (5 × 10 6 ) were inoculated subcutaneously in 0.2 ml of PBS into the right flank to allow tumors to develop. The mean tumor volume was 170 mm 3 When the number of animals reached 100, 39 animals were randomized. The administration of the test substance and the number of animals in each group are shown in the experimental design table. 4.3 Preparation of test substance formulations [Table 2]

[0153] 4.4 Observations All procedures regarding the handling, care and treatment of animals in this study were performed according to the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec, in accordance with the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). During regular observations, animals were checked daily for any effects of tumor growth or treatment on normal behavior, including motor ability, food and water consumption (visual observation only), weight gain / loss, eye / hair mattness, and any other abnormal effects as described in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0154] 4.5 Tumor Measurements and Endpoints The primary endpoint was whether tumor growth could be slowed or whether mice could be cured. Tumor volumes were measured bidimensionally three times weekly using calipers, and volume was calculated in mm using the following formula: 3 Expressed in units: V = 0.5 axb 2 where a and b are the long and short diameters of the tumor, respectively. The tumor size was then used to calculate the T / C value. The T / C value (%) is an index of antitumor effect, where T and C are the average volumes of the treatment and control groups on a given day, respectively. The TGI was calculated for each group using the following formula: TGI(%)=[1-(T i -T 0 ) / (V i -V 0 )]×100;T i is the mean tumor volume of the treatment group on a given day, T 0 is the mean tumor volume of the treatment group on the day treatment started, V i is T i and mean tumor volume of the vehicle control group on the same day, V 0 is the mean tumor volume of the vehicle control group on the day treatment began.

[0155] 4.6 Sampling At the end of the study, mice were re-challenged and plasma was collected for PK analysis at 5, 15, 30, 60, and 120 minutes after dosing.

[0156] 4.7 Statistical analysis Summary statistics including mean and standard error of the mean (SEM) are provided for tumor volumes in each group at each time point. Statistical analysis of between-group differences in tumor volumes was performed using data obtained at the therapeutic best time point after the last dose. One-way ANOVA was performed to compare tumor volumes between groups, and when a significant F-statistic (ratio of treatment variance to error variance) was obtained, the Games-Howell test was used to compare between groups. All data were analyzed using Prism. P < 0.05 was considered statistically significant.

[0157] 5.Results 5.1 Tumor growth curves Tumor growth curves are shown in Figures 1 to 5.

[0158] 5.2 Tumor volume tracing The mean tumor volumes over time in female Balb / c nude mice bearing HT1080 xenografts are shown in Tables 1 to 4. [Table 3] [Table 4] [Table 5] [Table 6]

[0159] 5.3 Tumor growth inhibition analysis The tumor growth inhibition rate of BT17BDCs in the HT1080 xenograft model was calculated based on the tumor volume measurement value 14 days after the start of administration, and is shown in Tables 5 to 8. [Table 7] [Table 8] a Mean ± SEM. b Tumor growth inhibition rate is calculated by dividing the group mean tumor volume of the treatment group by the group mean tumor volume of the control group (T / C). [Table 9] a Mean ± SEM. b Tumor growth inhibition rate is calculated by dividing the group mean tumor volume of the treatment group by the group mean tumor volume of the control group (T / C). [Table 10]

[0160] 6. Summary and discussion of results In this study, we evaluated the therapeutic efficacy of BT17BDCs in an HT1080 xenograft model. Tumor volume measurements for all treatment groups at each time point are shown in Figures 1 to 5 and Tables 1 to 8.

[0161] The mean tumor size in mice in the vehicle control group was 1012 mm on day 14. 3 reached.

[0162] BT17BDC23 (Mono-MMAE) - Reference BT17BDC23 showed dose-dependent antitumor activity at 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg, with tumor measurements of 1639 mm 3 (TGI=-2.7%, p>0.05), 1081mm 3 (TGI=36.1%, p>0.05), 12mm 3 (TGI=110.7%, p<0.001), 0mm 3 (TGI=111.8%, p<0.001).

[0163] BT17BDC69 (Bis-MMAE) BT17BDC69 at 0.3mg / kg biw (TV=6mm 3 , TGI=119.6%, p<0.001) showed potent antitumor activity. 3 , TGI=12.2%, p>0.05) did not show significant antitumor activity.

[0164] BT17BDC70 (Tetra-MMAE) - Reference BT17BDC70 at 0.1mg / kg biw (TV=1315mm 3 , TGI=-36.3%, p>0.05) and 0.3 mg / kg biw BT17BDC70 (TV=903 mm 3 , TGI=12.8%, p>0.05) did not show significant antitumor activity.

[0165] BT17BDC61 (Mono-MMAE) - Reference The mean tumor size in vehicle control mice was 797 mm on day 14. 3 BT17BDC61 reached 0.3 mg / kg (TV=606 mm 3 , TGI=28.5%, p>0.05) and 1 mg / kg (TV=435 mm 3 , TGI=54.2%, p<0.05) and 3 mg / kg (TV=0 mm 3 , TGI=119.7%, p<0.001) and 10 mg / kg (TV=0 mm 3 , TGI=120.1%, p<0.001), demonstrating dose-dependent antitumor activity. Among them, BT17BDC61 at 3mg / kg and 10mg / kg completely eliminated the tumor.

[0166] BT17BDC75 (Bis-MMAE) BT17BDC75 is 0.1mg / kg biw(TV=1070mm 3 , TGI=33.5%, p>0.05) did not show any clear antitumor activity, but 0.3mg / kg biw (TV=0mm 3 , TGI=113.6%, p<0.001) and 1mg / kg biw (TV=1mm 3 , TGI=113.4%, p<0.001), dramatically regressed tumors, eradicating 3 / 3 and 1 / 3 tumors, respectively.

[0167] Thus, the data presented herein support the present invention that bicyclic peptide drug conjugates containing two cytotoxic agents (i.e., MMAE) provide surprisingly favorable results compared to not only single cytotoxic agents, but also more than two (i.e., four) cytotoxic agents. For example, a BDC containing two MMAE moieties (BT17BDC69) exhibited potent antitumor activity at low concentrations (0.3 mg / kg biw). In contrast, the corresponding BDC with a single MMAE moiety (BT17BDC23) only exhibited dose-dependent antitumor activity, and the corresponding BDC with four MMAE moieties (BT17BDC70) did not produce significant antitumor activity.

[0168] Furthermore, another BDC containing two MMAE moieties (BT17BDC75) dramatically regressed tumors at low concentrations (0.3 mg / kg biw and 1 mg / kg biw), eradicating 3 / 3 and 1 / 3 tumors, respectively. In contrast, the corresponding BDC with a single MMAE moiety (BT17BDC61) only showed dose-dependent antitumor activity, eradicating tumors only at the highest concentrations tested (3 mg / kg and 10 mg / kg).

[0169] Thus, the data surprisingly show that the best results were obtained with BDCs containing two cytotoxic agents.

[0170] Example 2: In vivo efficacy study of BCY16278 in treating HT1080 xenografts in BALB / c nude mice

[0171] 1. Research purpose The aim of the study was to evaluate the in vivo antitumor efficacy of BT17BDC23, BT17BDC61, BT17BDC69, BT17BDC70 and BT17BDC75 in treating HT1080 xenografts in BALB / c nude mice. 2. Experimental Design [Table 11]

[0172] 3.Material 3.1 Animals and housing conditions 3.1.1 Animals Species: Mus Musculus Lineage: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18-22g Number of mice: 45 + extra mice Animal provider: Beijing Vital River Laboratory Animal Technology Co., Ltd. 3.1.2 Rearing conditions The mice were housed in individually ventilated cages with constant temperature and humidity, five mice per cage. ·Temperature: 20-26℃. ·Humidity: 40-70%. Cage: Made of polycarbonate, size 300mm x 180mm x 150mm, bedding material was corncob, changed twice weekly. Diet: Animals were fed radiation-sterilized dry granular food ad libitum throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: The identification label on each cage contained the following information: number of animals, sex, strain, date received, treatment, study number, group number, and start date of treatment. Animal Identification: Animals were ear tagged.

[0173] 3.2 Experiments and positive controls Product identification: BCY16278 Manufacturer: Bicycle Therapeutics Lot Number: 1 Physical properties: Freeze-dried powder Molecular weight: 5851.68 Purity: 96.10% Packaging and storage conditions: Store at -80℃

[0174] 4. Experimental methods and procedures 4.1 Cell culture HT1080 tumor cells were grown as monolayers in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in 5% CO in air. 2 The tumor cells were maintained in vitro at 25°C for 1 h at 4°C. Tumor cells were periodically recultured twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were harvested and counted for tumor implantation.

[0175] 4.2 Tumor transplantation To generate tumors, each mouse was inoculated with HT1080 tumor cells (5*10 6 The mean tumor volume was 253 mm3 The animals were randomized when the dose of the test substance and the number of animals in each group were shown in the experimental design table. 4.3 Preparation of test substance formulations [Table 12]

[0176] 4.4 Observations Observations were made as described in Example 1 above.

[0177] 4.5 Tumor Measurements and Endpoints Measurements were performed as described in Example 1 above.

[0178] 4.7 Statistical analysis Analysis was performed as described in Example 1 above.

[0179] 5.Results 5.1 Tumor growth curves Tumor growth curves are shown in FIG.

[0180] 5.2 Tumor volume tracing The mean tumor volumes over time in female Balb / c nude mice bearing HT1080 xenografts are shown in Table 9. [Table 13]

[0181] 5.3 Tumor growth inhibition analysis The tumor growth inhibition rate of BCY16278 in the HT1080 xenograft model was calculated based on the tumor volume measured 14 days after the start of administration, and is shown in Table 10. [Table 14] a Mean ± SEM. b Tumor growth inhibition rate is calculated by dividing the group mean tumor volume of the treatment group by the group mean tumor volume of the control group (T / C).

[0182] 6. Summary and discussion of results In this study, we evaluated the therapeutic efficacy of BT17BDCs in an HT1080 xenograft model. Tumor volume measurements for all treatment groups at each time point are shown in Figure 6 and Tables 9 to 10.

[0183] The mean tumor size in mice in the vehicle control group was 2062 mm on day 18. 3 reached. BCY16278 was administered at 0.1 mg / kg, qw (TV=1200 mm 3 , TGI=47.6%, p<0.001) and 0.3 mg / kg, qw (TV=440 mm 3 , TGI=89.7%, p<0.001), demonstrating significant antitumor activity.

[0184] Thus, the data surprisingly demonstrate significant antitumor activity for a bicyclic peptide drug conjugate of the present invention containing two cytotoxic agents.

Claims

1. (i) a peptide ligand comprising at least three reactive groups separated by at least two loop sequences, and an aromatic molecular scaffold that forms covalent bonds with the reactive groups of the peptide ligand such that at least two polypeptide loops are formed on the molecular scaffold; and (ii) two cytotoxic agents conjugated to said peptide ligand; A drug conjugate comprising:

2. The drug conjugate of claim 1 , wherein the peptide ligand is specific for MT1-MMP.

3. The drug conjugate of claim 1 or claim 2, wherein the loop sequence comprises 5 or 6 amino acids.

4. The drug conjugate of claim 1 or claim 2, wherein the reactive group comprises cysteine ​​and / or (S)-2-amino-3-(methylamino)propanoic acid (Dap(Me)).

5. The drug conjugate of claim 2, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of which consists of five amino acids and the other of which consists of six amino acids.

6. the peptide ligand C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 1) (wherein 1Nal represents 1-naphthylalanine and tBuGly represents t-butylglycine) The drug conjugate of claim 5 having a core sequence of:

7. the peptide ligand βAla-Sar 10 -A-(SEQ ID NO: 1) The drug conjugate of claim 5 or claim 6, having a full-length sequence of:

8. The drug conjugate of claim 2, wherein the loop sequence comprises two (S)-2-amino-3-(methylamino)propanoic acid (Dap(Me)) residues and one cysteine ​​residue separated by two loop sequences (one consisting of five amino acids and the other consisting of six amino acids).

9. the peptide ligand [Dap(Me)](D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)[Dap(Me)] (SEQ ID NO: 2) (wherein Dap(Me) represents (S)-2-amino-3-(methylamino)propanoic acid, 1Nal represents 1-naphthylalanine, and tBuGly represents t-butylglycine.) The drug conjugate of claim 8 having a core sequence of:

10. the peptide ligand βAla-Sar 10 -A-(SEQ ID NO: 2) The drug conjugate of claim 8 or claim 9, having a full-length sequence of:

11. The drug conjugate of any one of claims 1, 2, 5, 6, 8 and 9, wherein the aromatic molecular scaffold is selected from TBMB (1,3,5-tris(bromomethyl)benzene).

12. 5. The cytotoxic agent has the structure: 【Chemical 1】 10. The drug conjugate of claim 1, wherein the compound is (S)—N-((3R,4S,5S)-1-((S)-2-((1R,2R)-3-(((1S,2R)-1-hydroxy-1-phenylpropan-2-yl)amino)-1-methoxy-2-methyl-3-oxopropyl)pyrrolidin-1-yl)-3-methoxy-5-methyl-1-oxoheptan-4-yl)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamide)butanamide) (monomethylauristatin E; MMAE), having the formula:

13. The drug conjugate of any one of claims 1, 2, 5, 6, 8 and 9, further comprising a linker between the peptide ligand and each of the cytotoxic agents.

14. The drug conjugate of claim 13, wherein the linker is selected from one or more of Val-Cit, β-Ala, p-aminobenzylcarbamate (PABC), triazolyl, propanoyl, acetyl, N-(bisaminopropyl)glycine (BAPG) and one or more (e.g., 10) sarcosine (Sar) residues, such as -Sar 10 -βAla-BAPG-(propanoyl-triazolyl-acetyl-Val-Cit-PABC) 2 linker.

15. 2. The drug conjugate of claim 1, selected from BT17BDC69, BT17BDC75 and BCY16278.

16. 20. A pharmaceutical composition comprising the drug conjugate of any one of claims 1, 2, 5, 6, 8, 9 and 15 in combination with one or more pharmaceutically acceptable excipients.

17. 17. The pharmaceutical composition of claim 16 for use in the prevention, suppression or treatment of a disease.

18. 18. The pharmaceutical composition of claim 17, wherein the disease is a disease that can be alleviated by cell death.

19. 19. The pharmaceutical composition of claim 18, wherein the disease is selected from diseases characterized by defective cells, cell proliferative diseases such as cancer, and autoimmune diseases such as rheumatoid arthritis.

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