RU2824959C2 - Bicyclic peptide ligands specific for mt1-mmp - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to polypeptides which are covalently bonded to molecular scaffolds so that two or more peptide loops are closed between points of attachment to the scaffold. In particular, the invention describes peptides which bind with high affinity to type 1 membrane metalloprotease (MT1-MMP). Invention also describes drug conjugates containing said peptides conjugated with one or more effector and/or functional groups, which are used in imaging and targeted therapy of cancer.

EFFECT: bicyclic peptide ligands specific for MT1-MMP are disclosed.

30 cl, 13 dwg, 17 tbl, 5 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to polypeptides that are covalently linked to molecular scaffolds such that two or more peptide loops are stacked between the attachment points on the scaffold. In particular, the invention describes peptides that are high-affinity ligands for membrane metalloproteinase type 1 (MT1-MMP). The invention also describes drug conjugates comprising said peptides conjugated to one or more effector and/or functional groups that find use in imaging and targeted cancer therapy.

LEVEL OF TECHNOLOGY

Cyclic peptides are able to bind with high affinity and specificity to protein targets and are therefore an attractive class of molecules for the development of therapeutic agents. Indeed, several cyclic peptides have already been successfully used in the clinic, such as the antibacterial peptide vancomycin, the immunosuppressant drug cyclosporine or the antitumor drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The good binding properties result from the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of cyclic structures. Typically, macrocycles bind to surfaces of several hundred square angstroms, such as the cyclic peptide CVX15, a CXCR4 antagonist (400 ; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVb3 (355 ) (Xiong et al. (2002), Science 296 (5565), 151-5) or the cyclic peptide inhibitor upain-1, which binds to urokinase-type plasminogen activator (603 ; Zhao et al. (2007), J Struct Biol 160 (1), 1-10).

Because of their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less loss of entropy upon binding to targets and leading to higher binding affinity. The reduced flexibility also results in locking of target-specific conformations, increasing binding specificity compared to linear peptides. This effect can be illustrated by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity for other MMPs when its ring is opened (Cherney et al. (1998) J Med Chem 41 (11), 1749–51). The favorable binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides having more than one peptide ring, such as vancomycin, nisin, and actinomycin.

Various research groups have previously linked polypeptides with cysteine residues to a synthetic molecular framework (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and co-workers used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops on synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for creating drug candidates, wherein said compounds are formed by linking cysteine-containing polypeptides to a molecular framework such as, for example, tris(bromomethyl)benzene, are disclosed in WO 2004/077062 and WO 2006/078161.

To generate and screen large libraries of bicyclic peptides against targets of interest, a phage display based on combinatorial approaches was developed (Heinis et al. (2009), Nat Chem Biol 5 (7), 502-7 and patent WO2009/098450). Briefly, combinatorial libraries of linear peptides containing three cysteine residues and two regions of six random amino acids (Cys-(Xaa) ₆ -Cys-(Xaa) ₆ -Cys) were displayed on phage and cyclized by covalently linking the cysteine side chains to a small molecule (tris-(bromomethyl)benzene).

ESSENCE OF THE INVENTION

According to a first aspect of the present invention, there is provided a peptide ligand specific for MT1-MMP, comprising a polypeptide comprising at least three cysteine residues separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises an amino acid sequence of formula (I):

-C _i -XU/OXXGC _ii -EDFYXXC _iii - (SEQ ID NO: 1) (I)

or a modified derivative or a pharmaceutically acceptable salt thereof;

Where:

C _i , C _ii and C _iii represent the first, second and third cysteine residues, respectively;

X represents any amino acid residue;

U is a polar, uncharged amino acid residue selected from N, C, Q, M, S and T; and O is a non-polar aliphatic amino acid residue selected from G, A, I, L, P and V.

According to another aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups, such as cytotoxic agents, in particular DM1 and MMAE.

According to another aspect of the present invention there is provided a conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups, such as a chelator group, bearing a radionuclide, in particular DOTA.

According to another 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 pharmaceutically acceptable excipients.

According to another aspect of the present invention, there is provided a peptide ligand as defined herein for use in the prevention, suppression or treatment of cancer, in particular solid tumors such as non-small cell lung cancer.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Stability of 17-69-07-N219 in mouse plasma. Several ions were monitored as indicated in the legend, as well as two transitions in MRM mode. Excellent correlation between the ions is observed. The half-life of the peptide in mouse plasma at 37°C is 6 hours.

Figure 2: PK profile of the bicyclic peptide 17-69-07-N004 in mouse. 2 animals per time point.

Figure 3: Stability in mouse (A) and human (B) plasma of two stabilized 17-69-07 molecules (with 4-bromophenylalanine at position 9: 17-69-07-N244, without 4-bromophenylalanine at position 9: 17-69-07-N231) compared to unstabilized 17-69-07-N219. Several MRI transitions for this analyte were observed and correlated well with each other. In accordance with the purpose of this plot, only one transition is shown.

Figure 4: Biodistribution of ¹⁷⁷ Lu 17-69-07-N144 in HT-1080 xenograft bearing mice.

Figure 5: Biodistribution of ¹⁷⁷ Lu 17-69-07-N246 in HT-1080 xenograft bearing mice.

Figure 6: Biodistribution of ¹⁷⁷ Lu 17-69-07-N248 in HT-1080 xenograft bearing mice.

Figure 7: (A): Plot of mean tumor volume versus time for BT17BDC-1 and 9. Doses were administered on days 0, 2, 4, 7, 9, and 11. (B): Body weight during treatment, which is an indicator of drug-related toxicity and an indicator of the overall health of the animal.

Figure 8: List of sequence outputs from affinity maturation using libraries with fixed loop 2 residues 17-69. The sequence ligation plot on the right indicates a general preference for residues in loop 1 over residues 1, 2, 3, 4, and 5.

Figure 9: Top: Plot of mean tumor volume versus time for BT17BDC-17 in EBC-1 xenograft bearing mice. Doses were administered on days 0, 2, 4, 7, 9, 11, and 14. Bottom: Body weight during treatment, which is an indicator of drug-related toxicity and an indicator of the overall health of the animal.

Figure 10: Top: Plot of mean tumor volume versus time for BT17BDC-18 in EBC-1 xenograft bearing mice. Doses were administered on days 0, 2, 4, 7, 9, 11, and 14. Bottom: Body weight during treatment, which is an indicator of drug-related toxicity and an indicator of the overall health of the animal.

Figure 11: Top: Plot of mean tumor volume versus time for BT17BDC-19 in EBC-1 xenograft bearing mice. Doses were administered on days 0, 2, 4, 7, 9, 11, and 14. Bottom: Body weight during treatment, which is an indicator of drug-related toxicity and an indicator of the overall health of the animal.

Figure 12: Top: Plot of mean tumor volume versus time for BT17BDC-20 in EBC-1 xenograft bearing mice. Doses were administered on days 0, 2, 4, 7, 9, 11, and 14. Bottom: Body weight during treatment, which is an indicator of drug-related toxicity and an indicator of the overall health of the animal.

Figure 13: Plot of area under the curve (AUC) of tumor volume over time associated with a particular BDC by dose group. Curve fitting was performed using all available data points normalized to tumor volume at time zero using standard IC50 equations.

DETAILED DESCRIPTION OF THE INVENTION

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

Nomenclature

Numbering

When referring to the positions of amino acid residues in the compounds of formula (I), the cysteine residues (C _i , C _ii and C _iii ) are not included in the numbering since they are invariant, therefore the numbering of the amino acid residues in the compound of formula (I) is denoted as shown below:

-C _i -X ₁ -U/O ₂ -X ₃ -X ₄ -G ₅ -C _ii -E ₆ -D ₇ -F ₈ -Y ₉ -X ₁₀ -X ₁₁ -C _iii - (SEQ ID NO: 1).

For the purposes of this description, all bicyclic peptides are assumed to be cyclized with TBMB (1,3,5-tris(bromomethyl)benzene) to give the trisubstituted 1,3,5-trismethylbenzene structure. Cyclization with TBMB occurs at C _i , C _ii , and C _iii .

Sequence of the bicyclic peptide backbone

Each bicyclic peptide disclosed herein has been assigned a unique framework sequence number, which is defined as the amino acid sequence between the first N-terminal cysteine (C _i ) and the last C-terminal cysteine (C _iii ). For example, for the identification number 17-69-07, the framework sequence is _Ci YNEFGC _ii EDFYDIC _iii (SEQ ID NO: 2) and is designated as "17-69-07" or "(17-69-07)".

Peptide code

Certain bicyclic peptides described herein are also assigned a unique identification number using a peptide code, such as 17-69-07-N241, where N241 denotes a particular derivative of the bicyclic scaffold sequence 17-69-07. Different derivatives of 17-69-07 have different N numbers, i.e., N001, N002, Nxxx.

Molecular format

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

βAla-Sar10-A-(17-69-07)

and have the complete sequence βAla-Sar10-A-CYNEFGCEDFYDIC (SEQ ID NO: 3).

Modifications

Unnatural amino acid substitutions within the bicyclic framework sequence are listed following the molecular format description. For example, if tyrosine 1 in 17-69-07 is replaced by D-alanine, the description is (17-69-07) D-Ala1, and the complete sequence would be described as C(D-Ala1)NEFGCEDFYDIC (SEQ ID NO: 4).

If the N-terminal or C-terminal tail is attached to a bicyclic peptide that also contains modifications in the framework sequence, then using 17-69-07-N241 as an example, the molecular description of the Format is:

βAla-Sar10-A-(17-69-07) DAla1 1NAl4 DAla5 tBuGly11.

The complete amino acid sequence of 17-69-07-N241 is therefore:

βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 5).

Peptide ligands

A peptide ligand, as referred to herein, refers to a peptide covalently linked to a molecular scaffold. Typically, such peptides include two or more reactive groups (i.e., cysteine residues) that are capable of forming covalent bonds with the scaffold, and a sequence formed between said reactive groups, which is referred to as a loop sequence because it forms a loop when the peptide is linked to the scaffold. In this case, the peptides include at least three cysteine residues (referred to herein as C _i , C _{ii ,} and C _iii ), and form at least two loops on the scaffold.

It will be appreciated by one skilled in the art that X at positions 1, 3, 4, 10 and 11 of formula (I) can be any amino acid, given the alanine scan results (see Table 5) and the selection of yields (Figure 8), which yields well tolerated substitutions at these positions.

In one embodiment, X at position 1 of formula (I) is selected from any of the following amino acids: Y, M, F, or V. In another embodiment, X at position 1 of formula (I) is selected from Y, M, or F. In yet another embodiment, X at position 1 of formula (I) is selected from Y or M. In yet another embodiment, X at position 1 of formula (I) is selected from Y.

In one embodiment, U/O at position 2 of formula (I) is selected from U, such as N. In an alternative embodiment, U/O at position 2 of formula (I) is selected from O, such as G.

In one embodiment, X at position 3 of formula (I) is selected from U or Z, wherein U is a polar, uncharged amino acid residue selected from N, C, Q, M, S and T, and Z is a polar, negatively charged amino acid residue selected from D or E. In a further embodiment, U at position 3 of formula (I) is selected from Q. In an alternative embodiment, Z at position 3 of formula (I) is selected from E.

In one embodiment, X at position 4 of formula (I) is selected from J, wherein J is a non-polar residue of an aromatic amino acid selected from F, W and Y. In another embodiment, J at position 4 of formula (I) is selected from F. In an alternative embodiment, J at position 4 of formula (I) is selected from Y. In an alternative embodiment, J at position 4 of formula (I) is selected from W.

In one embodiment, X at position 10 of formula (I) is selected from Z, wherein Z is a polar, negatively charged amino acid residue selected from D or E. In one embodiment of the invention, Z at position 10 of formula (I) is selected from D.

In one embodiment, X at position 11 of formula (I) is selected from O, where O is a non-polar aliphatic amino acid residue selected from G, A, I, L, P and V. In one embodiment, O at position 11 of formula (I) is selected from I.

In one embodiment, the compound of formula (I) is a compound of formula (Ia):

-C _i -Y/M/F/VU/OU/ZJGC _ii -EDFYZOC _iii - (SEQ ID NO: 6)

(Ia);

where U, O, J and Z represent the above defined.

In one embodiment, the compound of formula (I) is a compound of formula (Ib):

-C _i -Y/M/F/VN/GE/QFGC _ii -EDFYDIC _iii - (SEQ ID NO: 7)

(Ib).

In one embodiment, the compound of formula (I) is a compound of formula (Ic):

-C _i -Y/M/FN/GE/QFGC _ii -EDFYDIC _iii - (SEQ ID NO: 8)

(Ic).

In one embodiment, the compound of formula (I) is a compound of formula (Id):

-C _i -Y/MNE/QFGC _ii -EDFYDIC _iii - (SEQ ID NO: 9)

(Id).

In one embodiment, the compound of formula (I) is a compound of formula (Ie):

-C _i -YNEFGC _ii -EDFYDIC _iii - (17-69-07) (SEQ ID NO: 2)

(Ie).

In another embodiment, the peptide of formula (I) comprises a sequence selected from:

-C _i -YNEFGC _ii -EDFYDIC _iii - (17-69-07) (SEQ ID NO: 2);

-C _i -MNQFGC _ii -EDFYDIC _iii - (17-69-12) (SEQ ID NO: 10);

-C _i -FGEFGC _ii -EDFYDIC _iii - (17-69-02) (SEQ ID NO: 11);

-C _i -VNEFGC _ii -EDFYDIC _iii - (17-69-03) (SEQ ID NO: 12);

-C _i -FNEFGC _ii -EDFYDIC _iii - (17-69-04) (SEQ ID NO: 13);

-C _i -YNEYGC _ii -EDFYDIC _iii - (17-69-07-N057) (SEQ ID NO: 14); And

-C _i -YNEWGC _ii -EDFYDIC _iii - (17-69-44-N002) (SEQ ID NO: 15).

Peptides according to this embodiment were identified as being strong candidates for subsequent affinity maturation towards the hemopexin domain of MT1-MMP (see Example 1 and Tables 1 and 8).

In another embodiment, the peptide of formula (I) comprises a sequence selected from:

-C _i -YNEFGC _ii -EDFYDIC _iii - (17-69-07) (SEQ ID NO: 2); And

-C _i -MNQFGC _ii -EDFYDIC _iii - (17-69-12) (SEQ ID NO: 10).

The peptides of this embodiment were identified as being the highest affinity candidates for subsequent affinity maturation towards the hemopexin MT1-MMP domain, for synthesis of bicyclic scaffold sequences, and quantitative measurement of affinity using competition binding experiments (see Example 1 and Tables 1-3).

In another embodiment, the peptide of formula (I) comprises a sequence selected from -C _i -YNEFGC _ii -EDFYDIC _iii - (17-69-07) (SEQ ID NO: 2). The peptide according to this embodiment has been identified as being the most potent and stable member of the family of peptide ligands of formula (I) (see Examples 1 to 4).

In one embodiment, certain peptide ligands of the invention are fully cross-reactive with mouse, dog, cynomolgus monkey, and human MT1-MMP. In another embodiment, particularly illustrated peptide ligands of the invention are fully cross-reactive with mouse, dog, cynomolgus monkey, and human MT1-MMP. For example, provided herein are data that demonstrate that both unstabilized and stabilized derivatives of 17-69-07 (i.e., 17-69-07-N219 and 17-69-07-N241) are fully cross-reactive (see Table 13).

In another embodiment, the peptide ligand of the invention is selective for MT1-MMP, but does not cross-react with MMP-1, MMP-2, MMP-15, and MMP-16. Data are provided herein that show that the scaffold sequence 17-69-07 and the stabilized variant 17-69-07-N258 are exceptionally selective for MT1-MMP (see Table 14).

Advantages of peptide ligands

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

Cross-reactivity with other species. This is a typical requirement for preclinical assessment of pharmacodynamics and pharmacokinetics;

Protease stability. Bicyclic peptide ligands should exhibit ideal stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across species so that the bicyclic lead candidate can be developed in animal models and also administered to humans with confidence;

Desirable solubility profile. This depends on the proportion of charged and hydrophilic relative to hydrophobic residues and intra-/intermolecular H-bonds, which are important for formulation and absorption purposes; and

Optimal circulating half-life. Depending on the clinical indication and treatment regimen, it may be desirable to design a bicyclic peptide for short exposure in the setting of acute disease or to design a bicyclic peptide with increased circulating retention and therefore optimal for the treatment of more chronic disease. Other factors governing the desired plasma half-life are subordinate to the requirement for sustained exposure to achieve maximum therapeutic efficacy versus the attendant toxicity of prolonged exposure to the agent.

Pharmaceutically acceptable salts

It should be understood that salt forms are within the scope of the present invention and references to compounds of formula (I) include salt forms of said compounds.

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.

Acid addition salts (mono- or di-salts) can be formed with a wide range of acids, both inorganic and organic. Examples of acid addition salts include mono- or di-salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g., L-ascorbic), L-aspartic, benzenesulfonic, benzoic, 4-acetamidobenzoic, butanoic, (+)-camphoric acids, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulfuric acids, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), α-oxoglutaric, glycolic, hippuric, hydrohalic acids (e.g. hydrobromic, hydrochloric, hydroiodic), isethionic, lactic (e.g. (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic acids, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-aminosalicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic acids, p-toluenesulfonic acid, undecylenic and valeric acids, as well as acylated amino acids and cation exchange resins.

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

If the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO-), then a salt can be formed with an organic or inorganic base to generate a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li ⁺ , Na ⁺ , and K ⁺ , alkaline earth metal cations such as ^Ca2+ and ^Mg2+ , and other cations such as ^Al3+ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., _NH4 ⁺ ) and substituted ammonium ions (e.g., _NH3R ⁺ , _{NH2R2 +} , _NHR3 ⁺ , _NR4 ^{+ )} . Examples of some suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N( _CH3 ) ₄₊ ^.

If the compounds of formula (I) contain an amino group, they can form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of formula (I).

Modified derivatives

It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of suitable such modified derivatives include one or more modifications selected from N-terminal and/or C-terminal modifications; substitution of one or more amino acid residues with one or more unnatural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other unnatural isosteric or isoelectronic amino acids); addition of a spacer group; 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 acid residues with alanine, substitution of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacing one or more peptide bonds with a substituent bond; modifying the length of the peptide backbone; replacing a hydrogen on the alpha carbon of one or more amino acid residues with another chemical group, modifying amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with the corresponding amine, thiol, carboxylic acid and phenol-reactive reagents so as to functionalize said amino acids, and introducing or replacing amino acids that introduce orthogonal reactivity that are suitable for functionalization, for example, amino acids bearing azide or alkyne groups that allow functionalization of the alkyne or azide bearing moieties, respectively.

In one embodiment of the invention, the modified derivative comprises an amino acid modification at position 1 and/or 9. Data are presented herein that show that these positions, particularly where tyrosine is present, are most susceptible to proteolytic degradation.

In one embodiment of the invention, the modified derivative comprises an N-terminal and/or a C-terminal modification. In a further embodiment, the modified derivative comprises a modification of the N-terminus using suitable amine-reactive reagents, and/or a modification of the C-terminus using suitable carboxyl-reactive reagents. In another embodiment, said modification of the N-terminus or C-terminus comprises the addition of an effector group, including, but not limited to, a cytotoxic agent, a radiochelator, or a chromophore.

In a further embodiment of the invention, the modified derivative comprises a modification of the N-terminus. In another embodiment, the modification of the N-terminus comprises an N-terminal acetyl group, such as in 17-69-07-N004 disclosed herein. In this embodiment, the N-terminal group of the cysteine (a group referred to herein as _Ci ) is capped with acetic anhydride or other suitable reagents during peptide synthesis, resulting in a molecule that is acetylated at the N-terminus. This embodiment provides the advantage of removing a potential recognition point for aminopeptidases and eliminates the possibility of degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of effector groups and maintains the activity of the bicyclic peptide towards its target, such as an Ala, G-Sar10-A or bAla-Sar10-A group. The data presented herein show that the addition of these groups to the bicyclic peptide 17-69-07 does not alter the activity towards the target protein (see Tables 11-12).

In another embodiment of the invention, the modified derivative comprises a C-terminal modification. In yet another embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal group of the cysteine (the group referred to herein as C _iii ) is synthesized as an amide during peptide synthesis, leading to a molecule that is amidated at the C-terminus. This embodiment provides the advantage of removing a potential carboxypeptidase recognition point and reduces the possibility of proteolytic degradation of the bicyclic peptide.

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

Alternatively, unnatural amino acids may be used as those having limited amino acid side chains, so that proteolytic hydrolysis of the nearby peptide bond is conformationally and sterically hindered. This applies in particular to proline analogues, bulky side chains, disubstituted Cα- derivatives (e.g., aminoisobutyric acid, Aib) and cycloamino acids, of which aminocyclopropylcarboxylic acid is a simple derivative.

In one embodiment, an unnatural amino acid residue is substituted at position 4. Data are provided herein that demonstrate that a number of unnatural amino acid residues are well tolerated at this position (see Table 8). In another embodiment, unnatural amino acid residues, such as those present at position 4, are selected from: 1-naphthylalanine; 2-naphthylalanine; cyclohexylglycine, phenylglycine; tert-butylglycine; 3,4-dichlorophenylalanine; cyclohexylalanine; and homophenylalanine.

In another embodiment, the non-natural amino acid residues, such as those present at position 4, are selected from: 1-naphthylalanine; 2-naphthylalanine; and 3,4-dichlorophenylalanine. Data are provided herein that show that these substitutions increase affinity compared to the unmodified wild-type sequence (see Table 8).

In another embodiment, the non-natural amino acid residues, such as those present at position 4, are selected from: 1-naphthylalanine. Data are provided herein that show that this substitution provides the greatest amount of affinity enhancement (greater than 7-fold) compared to the wild type (see Table 8).

In one embodiment of the invention, an unnatural amino acid residue is introduced at position 9 and/or 11. Data are provided herein that show that a number of unnatural amino acid residues are well tolerated at these positions (see Table 9).

In another embodiment, the non-natural amino acid residues, such as those present at position 9, are selected from: 4-bromophenylalanine, pentafluorophenylalanine.

In another embodiment, the non-natural amino acid residues, such as those present at position 11, are selected from: tert-butylglycine.

In another embodiment, the non-natural amino acid residues, such as those present at position 9, are selected from: 4-bromophenylalanine. The present disclosure provides data that demonstrate a change in the Tyr9 recognition point by proteolytic enzymes (see Table 9).

In another embodiment, the non-natural amino acid residues, such as those present at position 11, are selected from: tert-butylglycine. The present disclosure provides data that demonstrate increased activity and strong protection of the adjacent amino acid scaffold from proteolytic hydrolysis due to steric hindrance (see Table 9).

In one embodiment, the modified derivative comprises a plurality of the above modifications, such as 2, 3, 4, or 5 or more modifications. In another embodiment, the modified derivative comprises 2, 3, 4, or 5 or more of the following modifications, such as all of the following 5 modifications: D-alanine at position 1 and 5, 1-naphthylalanine at position 4, 4-bromophenylalanine at position 9, and tert-butylglycine at position 11. Data are provided herein that demonstrate that this multiple substitution (17-69-07-N252; 17-69-07-N244 and 17-69-07-N255) is consistently tolerated with activity that is superior to the wild type (see Tables 10-12). In another embodiment of the invention, the modified derivative comprises the following modifications: D-alanine at position 1 and 5, 1-naphthylalanine at position 4, and tert-butylglycine at position 11. Data are provided herein that demonstrate that this multi-substitution (17-69-07-N239) is consistently tolerated with activity that is superior to the wild type (see Table 11).

In one embodiment of the invention, the modified derivative comprises the addition of a spacer group. In a further embodiment of the invention, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine (C _i ) and/or to the C-terminal cysteine (C _iii ).

In one embodiment of the invention, the modified derivative comprises replacing one or more amino acid residues susceptible to oxidation with one or more amino acid residues resistant to oxidation. In another embodiment of the invention, the modified derivative comprises replacing a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

In one embodiment of the invention, the modified derivative comprises replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment of the invention, the modified derivative comprises replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged residues relative to hydrophobic amino acid residues is an important characteristic of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the extent of plasma protein binding and hence the concentration of the free available fraction in plasma, while charged amino acid residues (particularly arginine) can affect the interaction of the peptide with phospholipid membranes on cell surfaces. Both of these in combination can affect the half-life, volume of distribution and exposure of the peptide drug, all of which can be tailored to the final clinical requirements. In addition, the correct combination and amount of charged residues relative to hydrophobic amino acid residues can reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

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

In a further embodiment, the amino acid residue at position 1 is replaced with a D-amino acid, such as D-alanine. Data are provided herein that demonstrate retention of activity without subsequent degradation (see Table 6).

In another embodiment of the invention, the amino acid residue at position 5 is replaced with a D-amino acid, such as D-alanine or D-arginine. Data are provided herein that demonstrate retention of activity without subsequent degradation (see Table 7).

In one embodiment of the invention, the modified derivative comprises removal of any amino acid residues and substitution with alanines. This embodiment provides the advantage of removing potential site(s) of proteolytic attack.

It should be noted that each of the above modifications serves to intentionally improve the activity or stability of the peptide. Further improvement in activity based on modifications can be achieved through the following mechanisms:

- inclusions of hydrophobic moieties that utilize the hydrophobic effect and result in a decrease in dissociation rates, so that higher affinity is achieved;

- inclusion of charged groups that utilize long-range ionic interactions, leading to higher association rates and higher affinities (see, e.g., Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

- incorporating additional constraint into the peptide, such as by correctly constraining the amino acid side chains such that the loss of entropy is minimal upon target binding, constraining the torsion angles of the backbone such that the loss of entropy is minimal upon target binding, and introducing additional cycles into the molecule for identical reasons.

(For reviews, see Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185–203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399–418).

Isotopic variants

The present invention includes all pharmaceutically acceptable (radio)isotope-labeled compounds of the present invention, i.e. compounds of formula (I) wherein one or more atoms are replaced by atoms having the same atomic number but an atomic mass or mass number different from the atomic mass or mass number normally found in nature, and compounds of formula (I) to which metal chelating groups (called "effectors") are attached which are capable of retaining the corresponding (radio)isotopes, and compounds of formula (I) in which certain functional groups are covalently replaced by the corresponding (radio)isotopes or isotope-labeled functional groups.

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

Certain isotopically labeled compounds of formula (I), such as those comprising a radioactive isotope, are useful for medicinal and/or substrate tissue distribution studies, as well as for clinical evaluation of the presence and/or absence of the MT1-MMP target in pathological tissues such as tumors and elsewhere. The compounds of formula (I) may additionally have valuable diagnostic properties such that they can be used to detect or identify complex formation between the labeled compound and other molecules, peptides, proteins, enzymes or receptors. The detection or identification methods may employ compounds that are labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g. luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. Radioactive isotopes tritium, i.e. ³ H (T), and carbon-14, i.e. ¹⁴ C, are particularly useful for this purpose because of their ease of incorporation and ready means of detection.

Substitution with heavier isotopes such as deuterium, i.e. ² H (D), may provide certain therapeutic advantages as a result of higher metabolic stability, such as increased half-life in vivo or reduced drug dosage requirements, and may therefore be preferable in some circumstances.

Substitution with positron-emitting isotopes such as ¹¹ C, ¹⁸ F, ¹⁵ O, and ¹³ N may be useful in positron emission tomography (PET) studies to verify distribution in targets.

Incorporation of isotopes into the chelating effector groups of metals, such as ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, and ¹⁷⁷ Lu, may be useful for imaging tumor-specific antigens using PET or SPECT imaging. In particular, such biodistribution data are presented herein in Example 3.

The incorporation of isotopes into the chelating effector groups of metals, such as, but not limited to, ⁹⁰ Y, ¹⁷⁷ Lu and ²¹³ Bi, may enable targeted radiotherapy, whereby the compounds of formula (I) bearing the metal chelator carry the therapeutic radionuclide towards the target protein and site of action.

Isotopically labeled compounds of formula (I) can generally be prepared by conventional methods known to those skilled in the art or by methods analogous to those described in the accompanying examples using a suitable isotopically labeled reagent in place of the unlabeled reagent previously used.

Binding activity

Specificity as used herein refers to the ability of a ligand to bind or otherwise interact with a recognizable target excluding portions that are similar to the target. For example, specificity may refer to the ability of a ligand to inhibit the interaction of a human enzyme but not a homologous enzyme from another species. Using the approach described herein, specificity can be modulated, i.e., increased or decreased, so as to make ligands more or less able to interact with homologs or paralogs of the intended target. Specificity is not intended to be synonymous with potency, affinity, or avidity, and the potency of a ligand for its target (such as, for example, binding affinity or level of inhibition) is not necessarily related to its specificity.

Binding activity, as used herein, refers to quantitative measurements of binding obtained from a binding assay, such as those described herein. Thus, binding activity refers to the amount of peptide ligand that is bound at a given target concentration.

Multispecificity is the ability to bind to two or more targets. Typically, binding peptides are capable of binding to a single target, such as an epitope in the case of an antibody, due to their conformational properties. However, peptides can be designed that can bind to two or more targets; for example, dual-specificity antibodies, as known in the art, as noted above. In the present invention, the peptide ligands can have the ability to bind to two or more targets, and therefore be multispecific. Accordingly, they bind to two targets, and have dual specificity. The binding can be independent, which would mean that the target binding sites on the peptide do not structurally interfere with binding to one or the other target. In this case, both targets can be bound independently of each other. More generally, it is expected that binding to one target should at least partially interfere with binding to the other.

There is a fundamental difference between a ligand with dual specificity and a ligand with specificity that spans two related targets. In the former case, the ligand is specific for both targets separately and interacts with each in a specific manner. For example, the first loop in the ligand may bind to the first target and the second loop to the second target. In the latter case, the ligand is non-specific because it does not discriminate between the two targets, for example by interacting with an epitope of the targets that is common to both.

In the context of the present invention, it is contemplated that a ligand that has activity against, for example, a target and an ortholog may be a bispecific ligand. However, in one embodiment of the invention, the ligand is not bispecific, but has a less precise specificity, such that it binds both the target and one or more orthologs. In general, a ligand that has not been selected against both the target and its ortholog is less likely to be bispecific due to the lack of selective pressure towards bispecificity. The length of the loop in the bicyclic peptide may be critical in providing an optimized binding surface such that good cross-activity against the target and the ortholog can be obtained while maintaining high selectivity for less related homologs.

If the ligands are indeed bispecific, in one embodiment, at least one of the ligands' target specificities should be common among the selected ligands, and the level of this specificity can be modulated using the methods disclosed herein. The second or additional specificity need not be compatible and should not be the subject of the methods described herein.

The target is the molecule or portion thereof with which the peptide ligands bind or otherwise interact. Although binding is considered a prerequisite for activity in most classes, and may be an activity in itself, other activities are envisaged. Thus, the present invention does not require direct or indirect measurement of binding.

A molecular scaffold is any molecule that is capable of attaching a peptide at multiple points to impart one or more structural characteristics to the peptide. Preferably, the molecular scaffold includes at least three points of attachment of the peptide, referred to as scaffold reactive groups. These groups are capable of reacting with cysteine residues ( _Ci , _Cii, and _Ciii ) on the peptide to form a covalent bond. They not only form a disulfide bond, which is involved in reductive cleavage and concomitant degradation of the molecule, but also form stable, covalent thioether bonds. Preferred molecular scaffold structures are described below.

Molecular frameworks

Molecular scaffolds are described, for example, in WO 2009/098450 and the references cited therein, in particular in WO 2004/077062 and WO 2006/078161.

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

In one embodiment, the molecular scaffold may be or may be based on natural monomers such as nucleosides, sugars or steroids. For example, the molecular scaffold may include a short polymer of such units, such as a dimer or trimer.

In one embodiment, the molecular scaffold is a compound with known toxicity, such as low toxicity. Examples of suitable compounds include cholesterols, nucleotides, steroids, or existing drugs such as tamazepam.

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

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

The molecular scaffold may include chemical groups that form a bond with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

In one embodiment, the molecular scaffold may comprise or may consist of tris(bromomethyl)benzene, especially 1,3,5-tris(bromomethyl)benzene (“TBMB”) or a derivative thereof.

In one 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 provides an advantage in that the additional methyl groups can form additional contacts with the polypeptide and therefore add additional structural constraint.

The molecular scaffold of the present invention comprises chemical groups that allow functional groups of the polypeptide encoded by the library of the invention to form covalent bonds with the molecular scaffold. These chemical groups are 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.

Reactive scaffold groups that can be used on the molecular scaffold to react with the thiol groups of cysteines are alkyl halides (or also referred to as haloalkanes or haloalkanes). Examples include bromomethylbenzene (a reactive scaffold group exemplified by TBMB) or iodoacetamide. Other reactive scaffold groups that are used to selectively attach compounds to cysteines in proteins are maleimides. Examples of maleimides that can be used as molecular scaffolds according to the invention include: tris-(2-maleimidoethyl)amine, tris(2-maleimidoethyl)benzene, tris(maleimide)benzene. Selenocysteine is also a natural amino acid that has a reactivity similar to cysteine and can be used for the same reactions. Thus, where cysteine is mentioned, it is generally acceptable to substitute selenocysteine unless the context suggests otherwise.

Effector and functional groups

According to another aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effector and/or functional groups.

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

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

In another embodiment of this aspect of the invention, the effector moiety of the present invention is the Fc region of an IgG molecule. Preferably, the peptide ligand-effector moiety conjugate of the present invention comprises or consists of a peptide ligand coupled to an Fc having a tβ half-life of the order of a day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Most preferably, the peptide ligand of the present invention comprises or consists of a peptide ligand coupled to an Fc having a tβ half-life of the order of a day or more.

Functional groups generally include linking groups, drugs, reactive groups for attaching other moieties, functional groups that help to uptake macrocyclic peptides into cells, and the like.

The ability of peptides to penetrate cells must allow the peptides to act effectively against intracellular targets. Targets that may be accessible to peptides through their ability to penetrate cells include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in the apoptotic pathway. Functional groups that enable cell penetration include peptide or chemical groups that are added either to the peptide or to the molecular scaffold. Peptides are peptides such as those derived from, for example, VP22, HIV-Tat, the homeobox protein of Drosophila (Antennapedia), for example, as described by Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637. Examples of short peptides that have been shown to be effective in translocating across plasma membranes include the 16-amino acid peptide penetratin of the Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18-amino acid "model amphipathic peptide" (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127), and the arginine-rich regions of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimetics or SMOCs, which can be readily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies adding guanidinium groups to the molecules also enhance cellular penetration (Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585). Low molecular weight molecules such as steroids can be added to the molecular scaffold to improve cellular uptake.

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

RGD peptides, which bind to integrins present on many cells, may also be included.

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

In one particular embodiment of the present invention, the functional group is selected from a metal chelator that is suitable for complexing metal radioisotopes that are sought after as a drug. Such effectors, when complexed with said radioisotopes, can be useful agents for the treatment of cancer. Suitable examples include DOTA, NOTA, EDTA, DTPA, HEHA, SarAr, and others (Targeted Radionuclide therapy, Tod Speer, Wolters/Kluver Lippincott Williams & Wilkins, 2011).

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

In one specific embodiment of the present invention, the functional group is selected from a drug, such as a cytotoxic agent for the treatment of cancer. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites including purine analogues, azathioprine and mercaptopurine or pyrimidine analogues; plant alkaloids and terpenoids including 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 and topotecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Other agents may include antineoplastic antibiotics, which include the immunosuppressant dactinomycin (which is used in kidney transplants), doxorubicin, epirubicin, bleomycin, calicheamicins, and others.

In one further specific embodiment of the present invention, the cytotoxic agent is selected from maytansinoids (e.g., DM1) or monomethyl substituted auristatins (e.g., MMAE).

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

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

Data are presented herein in Examples 4 and 5, which demonstrate the effects of peptide ligands conjugated to DM1 or MMAE containing toxins.

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

Published work has demonstrated the potential to modify the susceptibility of the disulfide bond to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). Higher degrees of steric hindrance reduce the rate of reduction by intracellular glutathione as well as extracellular (systemic) reducing agents, thereby reducing the ease with which the toxin is released both intra- and extracellularly. Thus, selection of optimal disulfide stability in circulation (which minimizes unwanted side effects of the toxin) versus efficient release in the intracellular environment (which maximizes therapeutic effect) can be achieved by carefully tailoring the degree of steric hindrance on either side of the disulfide bond.

The hindrance on either side of the disulfide bond is modulated by introducing one or more methyl groups either in the targeting portion (herein in the bicyclic peptide) or in the toxin portion of the molecular construct.

Thus, in one embodiment, the cytotoxic agent is a maytansinoid selected from a compound of formula (II):

in which n is an integer selected from 1 to 10; and

R ₁ and R ₂ independently of each other represent hydrogen, C _1-6 alkyl, or a carbocyclic or heterocyclic group.

The term C _1-6 -alkyl as used herein refers to a linear or branched saturated hydrocarbon group containing from 1 to 6 carbon atoms, respectively. Examples of such groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl or hexyl and the like.

The terms "heterocyclyl" and "carbocyclyl" as used herein, unless the context otherwise requires, include both aromatic and non-aromatic ring systems. Thus, for example, the terms "heterocyclic group" and "carbocyclic group" include within their scope aromatic, non-aromatic, unsaturated, partially saturated, fully saturated carbocyclic or heterocyclic ring systems. In general, unless the context otherwise requires, such groups may be monocyclic or bicyclic (including fused bicyclic groups and bridged bicyclic groups) and may contain, for example, from 3 to 12 ring members, more typically from 5 to 10 ring members.

In one embodiment of the compound of formula (II), R ₁ and R ₂ are independently hydrogen or methyl.

In one embodiment of the compound of formula (II), n is 1 and R ₁ and R ₂ are independently hydrogen (i.e., the maytansine derivative DM1).

In an alternative embodiment of the compound of formula (II), n is 2 and R ₁ is hydrogen and R ₂ is a methyl group (i.e., the maytansine derivative DM3).

In one embodiment of the compound of formula (II), n is 1 and R ₁ and R ₂ are both a methyl group (i.e., the maytansine derivative DM4).

It should be borne in mind that the cytotoxic agent of formula (II) can form a disulfide bond, and in the conjugated structure with the bicyclic peptide of formula (I), the disulfide bond between the thiol-toxin (II) and the thiol-bicyclic peptide (III) is introduced using several possible synthesis schemes, two of which are described in Scheme II or Scheme III.

In one embodiment, the bicyclic peptide component of the conjugate has the structure shown in formula (III):

where M is an integer selected from 0 to 10, and

R ₃ and R ₄ independently represent hydrogen, C _1-6 alkyl, or a carbocyclic or heterocyclic group.

In one embodiment of the compound of formula (III), R ₃ and R ₄ are independently hydrogen or methyl.

Compounds of formula (III) wherein R ₃ and R ₄ are both hydrogen are considered to be unhindered, and compounds of formula (III) wherein one or all of R ₃ and R ₄ are methyl are considered to be hindered.

It should be borne in mind that the bicyclic peptide of formula (III) can form a disulfide bond, and in the conjugated structure with the cytotoxic agent of formula (II), the disulfide bond between the thiol-toxin (II) and the thiol-bicyclic peptide (III) is introduced using several possible synthesis schemes, one of which is described in scheme II.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide via a linker defined in formula (IV):

where R ₁ , R ₂ , R ₃ and R ₄ represent hydrogen, C _1-6 -alkyl or a carbocyclic or heterocyclic group;

toxin refers to any suitable cytotoxic agent as defined herein;

the bicycle is any suitable bicyclic peptide as defined herein;

n is an integer chosen from 1 to 10; and

m is an integer chosen from 0 to 10.

In one embodiment, R ₁ , R ₂ , R ₃ and R ₄ are hydrogen or methyl.

When R ₁ , R ₂ , R ₃ , and R ₄ are each hydrogen, the disulfide bond is the least protected and most sensitive to reduction. When R ₁ , R ₂ , R ₃ , and R ₄ are each methyl, the disulfide bond is the most protected and least sensitive to reduction. Partial hydrogen and methyl substitutions produce progressively increased resistance to reduction and concomitant cleavage and release of the toxin.

In one embodiment, the toxin of compound (IV) is maytansine and the conjugate comprises a compound of formula (V):

where R ₁ , R ₂ , R ₃ and R ₄ represent hydrogen, C _1-6 -alkyl or a carbocyclic or heterocyclic group;

the bicycle is any suitable bicyclic peptide as defined herein;

n is an integer chosen from 1 to 10; and

m is an integer chosen from 0 to 10.

In another embodiment of the compound of formula (V), n is 1 and R ₁ , R ₂ , R ₃ and R ₄ are hydrogen, i.e., the compound of formula (V) ^a :

The BDC of formula (V) ^a is known as BT17BDC-17. The exposed disulfide bond in BDC BT17BDC-17 is equivalent to BT17BDC-9, resulting in a difference in the bicyclic peptide portion: BT17BDC-9 uses an unstabilized sequence (17-69-07-N219), while BT17BDC-17 uses a stabilized bicyclic peptide analogue (17-69-07-N241), which is an amide-linked construct to the toxin disulfide. This exposed maytansine derivative with n=1 is called DM1.

In another embodiment of the compound of formula (V), n is 1, R ₁ is methyl, and R ₂ , R ₃ and R ₄ are each hydrogen, i.e., the compound of formula (V) ^b :

The BDC of formula (V) ^b is known as BT17BDC-18 and contains one protecting methyl group on the bicyclic peptide side and in the context of an antibody drug conjugate gives a 7-fold reduction in its sensitivity to a reducing agent such as dithiothreitol (compared to the unprotected disulfide) (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). The reduced sensitivity to reduction correlates with a slower rate of toxin release. This unprotected maytansine derivative with n=1 is called DM1. BT17BDC-18 uses a stabilized bicyclic peptide analogue (17-69-07-N241) which is an amide-linked construct to the toxin-disulfide.

In another embodiment of the compound of formula (V), n is 2, R ₁ and R ₂ are both hydrogen, and R ₃ and R ₄ are both methyl, i.e., the compound of formula (V) ^is :

The BDC of formula (V) ^c is known as BT17BDC-19 and contains two protecting methyl groups on the maytansine side and in the context of an antibody drug conjugate gives a 14-fold decrease in its sensitivity to a reducing agent such as dithiothreitol. The decreased sensitivity to reduction correlates with a slower rate of toxin release. This protected derivative of maytansine with n=2 is called DM4. BT17BDC-19 uses a stabilized bicyclic peptide analogue (17-69-07-N241) which is a construct linked via an amide to the toxin-disulfide.

In another embodiment of the compound of formula (V), n is 2, R ₁ and R ₃ are both methyl, and R ₂ and R ₄ are both hydrogen, i.e., the compound of formula (V) ^d :

The BDC of formula (V) ^d is known as BT17BDC-20 and contains one protecting methyl group on the maytansine side and one protecting methyl group on the bicyclic peptide side and in the context of an antibody drug conjugate gives a 170-fold decrease in its sensitivity to a reducing agent such as dithiothreitol. The decreased sensitivity to reduction correlates with a slower rate of toxin release. This protected maytansine derivative with n=2 is called DM3. BT17BDC-20 uses a stabilized bicyclic peptide analog (17-69-07-N241) which is a construct linked via an amide to the toxin-disulfide.

Indeed, in the context of the antibody-drug conjugate, the balance of efficacy versus tolerability in an animal model has shown that its optimum is associated with a certain level of protection, i.e. with the protection present in DM4 (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717), which is also present as such in BT17BDC-19.

In one embodiment of the invention, the conjugate is selected from BT17BDC-9, BT17BDC-17 (compounds of formula (V) ^a ), BT17BDC-18 (compounds of formula (V) ^b ), BT17BDC-19 (compounds of formula (V) ^c ) and BT17BDC-20 (compounds of formula (V) ^d ). The data are presented in Example 5 and in Tables 16 and 17, which demonstrate the useful properties of BT17BDC-9, BT17BDC-17, BT17BDC-18, BT17BDC-19 and BT17BDC-20.

In another embodiment of the invention, the conjugate is selected from BT17BDC-9, BT17BDC-17 (compounds of formula (V) ^a ), BT17BDC-18 (compounds of formula (V) ^b ) and BT17BDC-19 (compounds of formula (V) ^c ). The data are presented in Example 5 and in Tables 16 and 17, they demonstrate that these conjugates are considered as suitable molecules for use in targeted cancer therapy.

In another embodiment of the invention, the conjugate is selected from BT17BDC-17 (compound of formula (V) ^a ), BT17BDC-18 (compound of formula (V) ^b ) and BT17BDC-19 (compound of formula (V) ^c ). The data are presented in Example 5 and in Tables 16 and 17, which show that these conjugates are considered suitable molecules for use in targeted cancer therapy and are well tolerated at effective doses.

Synthesis

The peptides of the present invention can be prepared synthetically using standard methods, followed by in vitro reaction with a molecular scaffold. Once this has been accomplished, standard chemistry can be used. This allows for rapid, large-scale production of a soluble substance for further downstream experimentation or validation. Such methods can be accomplished using conventional chemistry, such as that disclosed in Timmerman et al (supra).

Thus, the invention also relates to the production of polypeptides or conjugates selected as described herein, wherein the production comprises optional additional steps as described below. In one embodiment, these steps are carried out with the final polypeptide/conjugate product obtained by chemical synthesis.

Optionally, amino acid residues in the polypeptide of interest may be replaced in producing the conjugate or complex.

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

To extend a peptide, it can be simply chemically extended at its N-terminus or C-terminus or within its loops using orthogonally protected lysines (and analogues) using standard solid-phase chemistry or solution synthesis methods. Standard (bio)conjugation methods can be used to introduce an activated or activatable N- or C-terminus. Alternatively, additions can be made by fragment condensation or native chemical ligation, such as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779), or by enzymes, such as using subtiligase, as described in (Chang et al. Proc Natl Acad Sci U S A. 1994 Dec 20; 91(26):12544-8 or in Hikari et al. Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

Alternatively, the peptides may be extended or modified by additional conjugation via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other upon exposure to the reducing environment of the cell. In this case, a molecular scaffold (e.g., TBMB) may be added during the chemical synthesis of the first peptide in such a way as to react with three cysteine groups; an additional cysteine or thiol may then be attached to the N- or C-terminus of the first peptide such that this cysteine or thiol reacts only with the free cysteine or thiol of the second peptide, forming a disulfide-linked bicyclic peptide-peptide conjugate.

Similar methods are equally applicable to the synthesis/addition of two bicyclic and bispecific macrocycles, potentially creating a tetraspecific molecule.

In addition, addition of other functional groups or effector groups can be accomplished in the same manner, using appropriate chemistry, by coupling at the N- or C-termini or via side chains. In one embodiment, the attachment is accomplished in such a way that it does not block the activity of any of the parts.

According to another aspect of the present invention, there is provided a method for preparing a drug conjugate as defined herein, which comprises the synthetic route described in any of Schemes I, II or III.

Pharmaceutical compositions

According to another 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 pharmaceutically acceptable excipients.

Typically, the present peptide ligands should be used in purified form together with pharmacologically acceptable excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including physiological saline and/or buffered media. Parenteral carriers include sodium chloride solution, Ringer's solution with dextrose, dextrose and sodium chloride, and Ringer's solution with lactate. Suitable physiologically acceptable adjuvants, if it is necessary to maintain the polypeptide complex in suspension, can be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

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

The peptide ligands of the present invention may be used as single-administered compositions or in combination with other agents. They may include antibodies, antibody fragments, and various immunotherapeutic drugs such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins. Pharmaceutical compositions may include "cocktails" of various cytotoxic or other agents in combination with the protein ligands of the present invention, or even combinations of selected polypeptides of the present invention having different specificities, such as polypeptides selected using different target ligands, whether or not they are combined prior to administration.

The route of administration of the pharmaceutical compositions according to the invention may be any of those generally known to those skilled in the art. For therapy, including but not limited to immunotherapy, the peptide ligands of the invention may be administered to any patient according to standard methods. The administration may be carried out by any suitable route, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary routes, or also, respectively, by direct infusion using a catheter. The dosage and frequency of administration should depend on the age, sex and condition of the patient, the simultaneous administration of other drugs, contraindications and other parameters that should be taken into account by the physician.

The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This method has been shown to be effective, and art-known lyophilization and reconstitution methods can be used. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of loss of activity, and that levels should be adjusted upward to compensate.

Compositions comprising the present peptide ligands or mixtures thereof can be administered for prophylactic and/or therapeutic treatment. In some therapeutic applications, an adequate amount to achieve at least partial inhibition, suppression, modulation, destruction, or some other measurable parameter of a population of selected cells is defined as a "therapeutically effective dose." Amounts necessary to achieve this dose will depend on the severity of the disease and the general condition of the patient's own immune system, but are typically in the range of 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg/kg/dose most commonly used. For prophylactic purposes, compositions comprising the present peptide ligands or mixtures thereof can also be administered in similar or slightly lower doses.

A composition comprising a peptide ligand according to the present invention can be used for prophylactic and therapeutic purposes to promote the alteration, inactivation, destruction or removal of a selected population of target cells in the body of a mammal. In addition, the peptide ligands described herein can be used extracorporeally or in vitro to selectively destroy, reduce or otherwise effectively remove a population of target cells from a heterogeneous population of cells. Blood from a mammal can be combined extracorporeally with selected peptide ligands, resulting in unwanted cells being destroyed or otherwise removed from the blood for return to the mammal in accordance with standard methods.

Therapeutic use

The bicyclic peptides of the invention have particular utility as high-affinity binding agents to membrane metalloproteinase type 1 (MT1-MMP, also known as MMP14). MT1-MMP is a transmembrane metalloproteinase that plays a major role in extracellular matrix remodeling, directly by stimulating the degradation of several of its components and indirectly by activating pro-MMP2. MT1-MMP is critical for tumor angiogenesis (Sounni et al (2002) FASEB J. 16(6), 555-564) and is overexpressed on a variety of solid tumors, so the bicyclic MT1-MMP binding peptides of the present invention have particular utility in targeted cancer therapy, particularly solid tumors such as non-small cell lung cancer. In one embodiment, the bicyclic peptide of the invention is specific for human MT1-MMP. In another embodiment, the bicyclic peptide of the invention is specific for mouse MT1-MMP. In another embodiment, the bicyclic peptide of the invention is specific for human and mouse MT1-MMP. In another embodiment, the bicyclic peptide of the invention is specific for human, mouse and dog MT1-MMP.

The polypeptide ligands selected according to the method of the present invention can be used for therapeutic and prophylactic purposes in vivo, in vitro and in vivo diagnostic applications, in vitro testing and use as reagents, and the like. Ligands having selected levels of specificity are useful for applications that involve testing in non-human animals where cross-reactivity is desirable, or for diagnostic applications where cross-reactivity with homologues or paralogues must be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response to predetermined ranges of antigens can be used to tailor a vaccine to specific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to a mammal, and homogeneity of 98 to 99% or more is most preferred for pharmaceutical applications, particularly when the mammal is a human. Once purified, either partially or to homogeneity, as required, the selected polypeptides can be used diagnostically or therapeutically (including in vitro use) or for developing and performing assays, immunofluorescence staining, and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

The effector group and peptide ligand conjugates of the present invention generally find use in the prevention, suppression or treatment of cancer, in particular solid tumors such as non-small cell lung cancer.

Thus, according to a further aspect of the present invention, there are provided effector groups and drug conjugates with a peptide ligand as defined herein for use in the prevention, suppression or treatment of cancer, in particular solid tumors such as non-small cell lung cancer.

According to another aspect of the present invention, there is provided a method for the prevention, suppression or treatment of cancer, in particular solid tumors such as non-small cell lung cancer, which comprises administering to a patient in need thereof an effector moiety and a drug-peptide ligand conjugate as defined herein.

Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and carcinomas of various types, including adenocarcinomas, squamous cell carcinomas, transitional cell carcinomas, and other carcinomas) such as carcinomas of the bladder and urinary tract, breast, gastrointestinal tract (including carcinomas of the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinomas, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinomas, and mesotheliomas), head and neck carcinomas (e.g., cancer of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity and paranasal sinuses), ovaries, fallopian tubes, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid gland (eg, follicular thyroid carcinoma), adrenal glands, prostate gland, skin and adjacent organs (eg, melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevus); hematologic malignancies (e.g., leukemias, lymphomas) and premalignant hematologic disorders and disorders with borderline malignancy, including hematologic malignancies and related lymphoid cell conditions (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, T-cell lymphomas and leukemias, natural killer [NK] cell lymphomas, Hodgkin lymphomas, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and hematologic malignancies and related myeloid cell conditions (e.g., acute myelogenous leukemia [AML], chronic myelogenous leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndrome, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplasia syndrome, and promyelocytic leukemia); tumors of mesenchymal origin such as sarcomas of soft tissue, bone, or cartilage such as osteosarcomas, fibrosarcomas, chondrosarcomas, rhabdomyosarcomas, leiomyosarcomas, liposarcomas, angiosarcomas, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcomas, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberance; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblast tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinoma); and pediatric and embryonal tumors (e.g., medulloblastomas, neuroblastomas, Wilms tumor, and primitive neuroectodermal tumors); or syndromes, congenital or otherwise, that render the patient susceptible to malignancy (e.g., xeroderma pigmentosum).

References herein to the term "prevention" include administration of a prophylactic composition prior to the onset of disease induction. "Suppression" refers to administration of a composition after an event has been induced but prior to clinical manifestation of the disease. "Treatment" includes administration of a protective composition after the onset of disease symptoms.

Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease. The use of animal model systems facilitates the present invention by allowing the development of polypeptide ligands that can cross-react with human and animal targets for use in animal models.

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

EXAMPLES

Materials and methods

Selection of phages

6×6 phage libraries of bicyclic peptides were generated as described by Heinis et al (2009), Nat Chem Biol 5(7), 502-507, in patents WO 2009/098450, WO 2013/050615 and WO 2013/050616. Phage display selection was performed using the indicated 6×6 phage library against the biotinylated domain of human hemopexin MT1-MMP.

Protein expression

Hemopexin-like MT1-MMP repeats (also known as the hemopexin domain of MT1-MMP), residues Cys319-Gly511 of the human gene, were transiently expressed in HEK293 cells as a secreted soluble protein with an N-terminal His6 tag using the PExpr-IBA42 (IBA) expression vector. After expression, the protein was purified by Nickel-NTA affinity chromatography followed by gel filtration, and purity was verified by SDS-PAGE. Batch-to-batch variability was also monitored by fluorescence temperature shift experiments in the presence/absence of the bicyclic hemopexin domain-binding peptide.

Peptide synthesis

The peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer from Peptide Instruments and a Syro II synthesizer from MultiSynTech. Standard Fmoc amino acids (Sigma, Merck) with the following side chain protecting groups were used: Arg(Pbf); Asn(Trt); Asp(OtBu); Cys(Trt); GIu(OtBu); Gln(Trt); His(Trt); Lys(Boc); Ser(tBu); Thr(tBu); Trp(Boc); and Tyr(tBu) (Sigma). The coupling reagent was HCTU (Pepceuticals), diisopropylethylamine (DIPEA, Sigma) was used as the base, and deprotection was achieved with 20% piperidine in DMF (AGTC). Syntheses were performed using 0.37 mmol/g Fmoc-Rink amide resin AM (AGTC), Fmoc-amino acids were used in a fourfold excess, and base was used in a fourfold excess relative to amino acids. Amino acids were dissolved to 0.2 M in DMSO, HCTU to 0.4 M in DMF, and DIPEA to 1.6 M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that coupling reaction mixtures included 20 to 50% DMSO in DMF, which reduced aggregation and deletions during solid-phase synthesis and increased yield. Coupling times were typically 30 min and deprotection times were 2 x 5 min. Fmoc-N-methylglycine (Fmoc-Cap-OH, Merck) was coupled for 1 h and the deprotection and coupling times for the subsequent residue were 20 min and 1 h, respectively. After synthesis, the resin was washed with dichloromethane and dried. Side chain deprotection and support formation were accomplished using 10 mL of 95:2.5:2.5:2.5 v/v/v/w TFA/ _H2O / _iPr3SiH /dithiothreitol for 3 h. After cleavage, the spent resin was removed by filtration and the filtrate was added to 35 mL of diethyl ether that had been cooled to -80°C. The peptide pellet was centrifuged, the ether supernatant was discarded and the peptide pellet was washed with cold ether two more times. The peptides were then redissolved in 5-10 mL of acetonitrile-water and lyophilized. A small sample was taken for analysis of the crude product purity by mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). After lyophilization, the peptide powders were transferred to 10 mL of 6 M guanidine hydrochloride in H ₂ O supplemented with 0.5 mL of 1 M dithiothreitol and loaded onto a C8 Luna preparative HPLC column (Phenomenex). Solvents (H ₂ O, acetonitrile) were acidified with 0.1% heptafluorobutyric acid. The gradient range was 30-70% acetonitrile over 15 min at a flow rate of 15-20 mL/min using a Gilson preparative HPLC system. Fractions containing pure linear peptide material (as determined by MALDI) were pooled and modified with 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma). For this, the linear peptide was diluted with _H2O to ~35 mL, ~500 µL of 100 mM TBMB in acetonitrile was added, and the reaction was initiated with 5 mL of 1 M _NH4HCO3 in _{H2O .} The reaction was allowed to proceed for ~30-60 min at room temperature and lyophilized immediately after completion of the reaction (judged by MALDI). After lyophilization, the modified peptide was purified as described above, replacing the Luna C8 column with a Gemini C18 column (Phenomenex), and replacing the acid with 0.1% trifluoroacetic acid. Pure fractions containing the correct TMB-modified material were pooled, lyophilized, and maintained at -20°C for storage.

All amino acids, unless otherwise noted, were used in L-configurations.

DOTA was attached to the peptide chain during solid-phase peptide synthesis using the protected precursor DOTA(tBu) ₃ (TCI, CAS 137076-54-1).

Unnatural amino acids were incorporated into the peptide sequence using the general methods described above.

The list of unnatural amino acid precursors used in this description is summarized in the table below:

Supplier Short name Full chemical name AGTC D-Asp Fmoc-D-Asp(tBu)-OH Iris Biotech HPhe Fmoc-L-homophenylalanine Alfa Aesar 5FPhe Fmoc-pentafluoro-L-phenylalanine Polypeptide Group 4BrPhe Fmoc-4-bromo-L-phenylalanine Iris Biotech B-Ala Fmoc-beta-Ala-OH Iris Biotech 3Pal Fmoc-L-3Pal-OH Iris Biotech 4Pal Fmoc-L-4Pal-OH Iris Biotech D-Pro Fmoc-D-Pro-OH Merck Novabiochem Aib Fmoc-Aib-OH Merck Novabiochem D-Ala Fmoc-D-Ala-OH Merck Novabiochem D-Arg Fmoc-D-Arg(Pbf)-OH Merck Novabiochem D-Gln Fmoc-D-Gln(Trt)-OH Merck Novabiochem D-His Fmoc-D-His(Trt)-OH Merck Novabiochem Hyp Fmoc-Hyp(tBu)-OH Merck Novabiochem D-Leu Fmoc-D-Leu-OH Merck Novabiochem HArg Fmoc-L-HArg(Boc)2-OH Peptech Corporation 4,4-BPAl Fmoc-L-4, 4'-biphenylalanine Peptech Corporation 3,3-DPA Fmoc-L-3,3-diphenylalanine Peptech Corporation Dpg Fmoc-dipropylglycine Peptech Corporation 1NAl Fmoc-L-1-naphthylalanine Peptech Corporation 2NAl Fmoc-L-2-naphthylalanine Peptech Corporation Pip Fmoc-L-pipecolic acid Polypeptide group Aze Fmoc-L-azetidine-2-carboxylic acid Polypeptide group Cha Fmoc-beta-cyclohexyl-L-alanine Polypeptide group 4FluoPro (2S,4R)-Fmoc-4-fluoro-pyrrolidine-2-carboxylic acid AGTC D-Asp Fmoc-D-Asp(tBu)-OH Merck tBuGly Fmoc-α-tert-butylserine Iris Biotech Chg Fmoc-L-cyclohexylglycine Fluorochem Phg Fmoc-phenylglycine-OH Iris Biotech 3Pal Fmoc-L-3Pal-OH Iris Biotech 4Pal Fmoc-L-4Pal-OH Merck Novabiochem D-Leu Fmoc-D-Leu-OH Merck Novabiochem HArg Fmoc-L-HArg(Boc)2-OH Polypeptide group 3.4 DCPhe Fmoc-3,4-dichloro-L-phenylalanine Polypeptide group Cha Fmoc-beta-cyclohexyl-L-alanine

Peptides used for pharmacokinetic studies were lyophilized from 0.1% TFA in water to yield TFA salts or free acids of the compounds.

Synthesis of BDCs using 17-69-07-N219 as a bicyclic peptide precursor

Two bicyclic peptide-drug conjugates (BDCs) were synthesized using 17-69-07-N219 as a precursor peptide. Activated vcMMAE or disulfide-DM1 constructs (dissolved in DMSO) were directly conjugated in 1.4× excess to 17-69-07-N219 under aqueous conditions (100 sodium phosphate, pH 8) (see Schemes I and II). Peptide concentrations were 9 mg/mL or higher. The reaction was monitored by LC/MS and considered complete after 3.5 h. This was followed by standard reversed-phase purification using a C18 semi-preparative column. Fractions with purity greater than 95% were isolated and lyophilized. The materials did not contain measurable amounts of free toxin. For in vitro and in vivo studies, lyophilized powders were converted into concentrated DMSO stock solutions (100 mg/ml) and diluted in the appropriate buffer for further use.

Synthesis of BDCs using 17-69-07-N241 as a bicyclic peptide precursor

The following N-hydroxysuccinimide esters (NHS esters) of maytansinoid disulfides were used for the synthesis of BT17BDC-17, BT17BDC-18, BT17BDC-19 and BT17BDC-20:

where R ₁ , R ₂ , R ₃ , R ₄ , m and n are as described above for compounds of formulas (V) ^a , (V) ^b , (V) ^c , (V) ^d for BT17BDC-17, BT17BDC-18, BT17BDC-19 and BT17BDC-20, respectively.

BT17BDC-18 was further synthesized via an alternative route as described below and in Scheme III. Here, 17-69-07-N277 was synthesized by reacting 17-69-07-N241 with SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate) in DMSO. Concentrations of 17-69-07-N241 were 10 mM or higher in 1.3-fold excess SPP and 20-fold excess diisopropylethylamine at room temperature. The reaction was considered complete after 1 h as judged by LC/MS. Purification was performed by reverse phase as described above. The appropriate fractions were lyophilized.

17-69-07-N277 was disulfide exchanged with 1.15 equivalents of DM1 (as free thiol) under semiaqueous conditions (50% dimethylacetamide and 50% 100 mM sodium acetate, pH 5.0, supplemented with 2 mM EDTA) for 21 h at room temperature under a nitrogen gas blanket. Concentrations of 17-69-07-N277 in the reaction mixture were 10 mM or greater. This was followed by standard reversed-phase purification using a C18 semi-preparative column. Fractions greater than 95% pure were isolated and lyophilized. The materials did not contain measurable amounts of free toxin. For in vitro and in vivo studies, the lyophilized powders were solubilized in an aqueous formulation as described above or added directly to the appropriate buffer.

Determination of the dissociation rate constant of a bicyclic peptide binding to MT1-MMP

Direct binding detection methods using fluorescence polarization (anisotropy)

Direct binding assays using fluorescence polarization or anisotropy were performed by titrating a constant concentration of a fluorescent label (here a fluorescent bicyclic peptide) with its binding partner (here the hemopexin domain of MT1-MMP). As the concentration of the binding partner increases during the titration, the polarization signal changes proportionally to the fraction of bound and unbound material. This allows the dissociation rates (Kd) to be determined quantitatively. Assay data can be fitted using standard ligand binding equations.

Typically, label concentrations are ideally well below the Kd of the label:titrant pair, and the concentrations chosen are typically 1 nM or less. The titrant (binding partner) concentration ranges from 0.1 nM typically to 5 μM. The range is chosen such that the maximum change in fluorescence polarization can be observed. The buffers used are phosphate buffered saline in the presence of 0.01% Tween. Experiments were performed in black 384-well low binding/low volume plates (Corning 3820), and the fluorescence polarization signal was measured using a BMG Pherastar FS plate reader.

The fluorescent labels mentioned in the text are bicyclic peptides rendered fluorescent by the use of 5,6-carboxyfluorescein. Attachment of the fluorescent label can be performed at the N-terminal amino group of the peptide, which is separated from the bicyclic backbone sequence by a sarcosine spacer (usually Sar5). This can be accomplished during Fmoc solid phase synthesis or post-synthesis (after cyclization with TBMB and purification) if the N-terminal amino group is unique to the peptide. Attachment of the fluorescent label can also be performed at the C-terminus, typically at a lysine introduced as the first C-terminal residue, which is then separated from the bicyclic backbone sequence by a sarcosine spacer (usually Sar6). Thus, the N-terminal tags may have the molecular format described as Fluo-Gly-Sar5-A (bicyclic scaffold sequence), and (bicyclic scaffold sequence)-A-Sar6-K (Fluo) for the construct with a C-terminal fluorescent tag. The fluorescent tags used in the examples are A-(17-69)-A-Sar6-K (Fluo), A-(17-69-07)-A-Sar6-K (Fluo), and A-(17-69-12)-A-Sar6-K (Fluo). Because of the acidic nature of the fluorescent peptides 17-69, they are typically prepared as concentrated stock solutions in DMSO, from which dilutions are made in 100 mM Tris buffer, pH 8.

Competitive assay using fluorescence polarization (anisotropy)

Due to their high affinity for the hemopexin (PEX) domain of MT1-MMP, fluorescently labeled derivatives of 17-69-07 and 17-69-12 (designated 17-69-07-N040 and 17-69-12-N005, respectively) can be used for competition experiments (using FP for detection). Here, a pre-formed complex of PEX with a fluorescently labeled PEX-binding peptide is titrated with a free, non-fluorescent bicyclic peptide. Since all 17-69-based peptides are expected to bind to the same site, the titrating compound will displace the fluorescent label from PEX. Dissociation of the complex can be quantified and the Kd of the competitor (titrating compound) from the target protein is determined. The advantage of the competitive assay is that the affinity of non-fluorescent bicyclic peptides can be determined accurately and quickly.

Label concentrations are typically at or below the Kd level (herein 1 nM) and the binding protein (herein hemopexin MT1-MMP) is in 15-fold excess such that >90% of the label is bound. This is followed by titration with a non-fluorescent competitive bicyclic peptide (usually only the bicyclic scaffold sequence) such that it displaces the fluorescent label from the target protein. The label displacement is measured and related to the decrease in fluorescence polarization. The decrease in fluorescence polarization is proportional to the fraction of the target protein bound to the non-fluorescent titrant and is therefore a measure of the affinity of the titrant for the target protein.

The raw data are fitted to an analytical solution of a cubic equation describing the equilibrium between the fluorescent label, titrant, and binding protein. The fit requires knowledge of the affinity of the fluorescent label for the target protein, which can be determined separately by direct FP binding experiments (see previous section). The curve fitting was performed using Sigmaplot 12.0 and an adapted version of the equation described by Zhi-Xin Wang (FEBS Letters 360 (1995) 111–114) was used.

Plasma stability profile

Method #1:

A rapid assessment of the plasma stability profile was developed using mass spectrometric detection (MALDI-TOF, Voyager DE, Applied Biosystems) of the parent mass as well as protease-induced fragments in plasma. By assessing the nature of the fragments, preferred cleavage sites can be identified. Here, 1-1.5 mM peptide stock solution (in DMSO) was directly diluted in mouse/rat/human plasma (Sera labs, using citrate as anticoagulant) to give a final concentration of 50 μM peptide and incubated for 48 h at 37°C. 5 μL samples were removed at appropriate time points and frozen at -80°C. For analysis, samples were thawed, mixed with 25 μl of 3:3:1 acetonitrile:methanol:water, and centrifuged at 13k for 5 min. 5 μl of the peptide-containing supernatant was aspirated and mixed with 30 mM ammonium bicarbonate in 1:1 acetonitrile: _H2O . 1 μl of this solution was then spotted on a MALDI plate, dried, and 1 μl of Matrix (alpha-cyanocinnamic acid, Sigma, prepared as a saturated solution in 1:1 acetonitrile:water containing 0.1% trifluoroacetic acid) was layered on top of the sample, dried, and analyzed by MALDI TOF. It should be noted that this is a qualitative assay and serves to identify comparative changes in plasma stability between different bicyclic peptide sequences and functions as an excellent tool for identifying preferred cleavage sites.

Method #2

To quantify the stability of bicyclic peptides in plasma, peptide stock solutions (200 μM in DMSO) were mixed with plasma (human or mouse) to give final concentrations of 10 μM. Samples of 40 μL were withdrawn periodically for up to 8 h and frozen at -80°C. Prior to LC/MS analysis, samples were thawed and mixed with 3 volumes (here 120 μL) of 1:1 acetonitrile/aqueous MeOH. The milky suspension was centrifuged for 30 min at 13,000 rpm and the peptide-containing supernatants were quantified for doubly/triple-charged species and their MS/MS fragments using a Waters Xevo TQ-D instrument using a standard curve of plasma-extracted same peptides as reference compounds. The plasma degradation half-life was used to assess the relative stability of the molecules.

Pharmacokinetics of 17-69-07 in mice, identification of metabolites

Pharmacokinetics 17-69-07-N004

Pharmacokinetics in mice were studied using the bicyclic peptide 17-69-04-N004, which was dosed to one group of 12 male CD1 mice as a single intravenous dose of 5.925 mg/kg as a 5 mL/kg bolus of a 1.19 mg/mL solution. Formulation solutions were prepared from 100 μL of a 23.7 mg/mL DMSO stock solution that was diluted with 1.9 mL phosphate-buffered saline immediately prior to dosing, resulting in a vehicle consisting of 5% DMSO in PBS at pH 7.4. Blood samples were collected from two animals per time point by cardiac puncture under terminal anesthesia at 0.08, 0.5, 1, 2, and 4 hours post-dose and transferred to EDTA tubes to obtain plasma. Plasma samples were immediately frozen at -20°C. For analysis, samples were rapidly thawed and 50 μl aliquots were treated with 3 volumes of extraction solvent (a 2:9:9 mixture of 10 mM ammonium bicarbonate, pH 8, acetonitrile and methanol containing the analytical internal standard). Precipitated proteins were removed by centrifugation and the supernatant was analyzed by LC-MS/MS. Samples were quantified by calculation against a calibration curve generated in control mouse plasma. Pharmacokinetic parameters were determined by noncompartmental analysis using the PK Solutions 2.0 software package from Summit Research Services.

Definition of terms:

Cmax: Maximum measured concentration;

Tmax: Time at which the maximum concentration is measured;

AUC 0-t: Area under the plasma drug concentration/time curve from 0 minutes to the last quantitative data point; and

AUC 0-∞: Area under the plasma drug concentration/time curve from 0 minutes with extrapolation of data beyond the endpoint based on terminal half-life.

Identification of bicyclic peptide metabolites in mouse plasma

Three plasma samples were available for use (0.5, 1 and 2 h) in the in vivo analysis of potential metabolites of 17-69-07-N004 in mice. Analysis was performed by LC/MS and LC/MS using an LTQ Orbitrap XL mass spectrometer. The approach to search for peptide metabolites in circulation was to calculate the exact mass (10 ppm window) of putative metabolites (addition of water 1 or 2 (+18, +36) to cleave loop 1 and/or loop 2, respectively; then loss of individual amino acids or loss of amino acid stretches from loop 1 and/or loop 2). This was followed by manual searching by comparing total ion chromatograms with control mouse plasma.

Efficacy of BT17BDC-1 and BT17BDC-9 in HT-1080 xenograft-bearing mice.

Nude BALB/C mice bearing subcutaneous HT-1080 tumor xenografts were treated with BDCs or vehicle (PBS). BDCs were administered 3 times per week for 2 weeks with dosing initiated when tumors measured approximately 150-200 ^mm3 . Mice were monitored and tumor volume and body weight measurements were recorded 3 times per week.

Example 1: Identification of high-affinity bicyclic peptides using the hemopexin MT1-MMP domain

Using previously established methods for generating bicyclic peptide phage libraries, peptides were selected against the human MT1-MMP hemopexin domain. After three rounds of selection from the intact library using successively lower concentrations of target, the output was sequenced. Bicyclic peptide 17-69 (CKNRGFGCEDFYDIC) (SEQ ID NO: 16) was identified as one of the peptides with the most enriched sequence yield, and good target binding was confirmed using AlphaScreen.

Three small phage libraries were constructed to provide complete sequence coverage of each of the 3 parts of the 17-69 sequence. These three libraries were subjected to two rounds of selection against the hemopexin protein. Interestingly, the most promising sequencing yields were 5x6 bicyclic peptides, while the starting libraries were in the 6x6 format. It is likely that the shorter loop length was selected due to the higher affinity of the bicyclic peptide for the target protein. The shorter loop lengths result from incorrectly synthesized primers that are included during the construction of the phage libraries.

The main sequencing yield was peptide 17-69-07, which has the sequence CYNEFGCEDFYDIC (SEQ ID NO: 2).

Based on the observation that the 5x6 format was apparently the most successful and that discrimination between 5x6 binders was required, two further libraries were generated testing deliberate truncations of the first loop, yielding bicyclic peptides 17-69-02, 17-69-03, 17-69-04 and 17-69-12. These are contained within the sequences disclosed in Figure 8.

A trend was visible for some residues, although the binding assay was unable to distinguish the best binders as they reached the maximum limit of the assay.

The most abundant sequences were analyzed for hemopexin binding using alpha screening, where all signals were high compared to the original sequence 17-69 (see Table 1):

Table 1: Hemopexin binding assay using bicyclic peptides of the invention

Since all affinity matured 17-69 based clones produced signals that were near the maximal limit of the assay, the plot presented in Table 1 does not allow for unambiguous identification of the best clones. Therefore, some of the peptides were synthesized as fluorescent derivatives (17-69, 17-69-07 and 17-69-12) and used for direct binding assay experiments using fluorescence polarization (FP). Here, fluorescein is separated from the bicyclic sequence or from the N- or C-terminus relative to the scaffold sequence by a linker (usually Sar6) (Table 2).

Table 2: Results of direct binding assay experiments using fluorescence polarization (FP)

Peptide code Molecular format FP Method Label Kd (nM) 17-69-N004 A-(17-69)-A-Sar6-K(Fl) Direct binding A-(17-69)-A-Sar6-K(fluorescein) 692 17-69-02-N001 A-(17-69-02)-A Competition A-(17-69-07)-A-Sar6-K(fluorescein) 191 17-69-03-N001 A-(17-69-03)-A Competition A-(17-69-07)-A-Sar6-K(fluorescein) 16.7 17-69-12-N005 A-(17-69-12)-A-Sar6-K(Fl) Direct binding A-(17-69-07)-A-Sar6-K(fluorescein) 3.1 17-69-07-N040 A-(17-69-07)-A-Sar6-K(Fl) Direct binding A-(17-69-07)-A-Sar6-K(fluorescein) 0.52

Fluorescent derivatives of 17-69-07 and 17-69-12 (designated 17-69-07-N040 and 17-69-12-N005) showed strong binding with off-rates of 0.52 and 3.1 nM, respectively. These are markedly improved over the original, immature 17-69 sequence (17-69-N004). Sequences that contain the 5x6 format appear to induce high affinities in the context of an affinity maturation library.

Due to their high affinity for the hemopexin (PEX) domain of MT1-MMP, fluorescent derivatives 17-69-07 and 17-69-12 (designated 17-69-07-N040 and 17-69-12-N005, respectively) were used for subsequent competition experiments (using FP for detection). Here, a preformed PEX complex with a PEX-binding fluorescent label was titrated with a free, non-fluorescent bicyclic peptide. Since all 17-69-based peptides (containing the conserved second loop motif CEDFYDIC; SEQ ID NO: 17) are expected to bind to the same site, the titrating agent will displace the fluorescent label from PEX. The dissociation of the complex can be measured quantitatively and the Kd of the competitor (titrating agent) determined. The advantage of the competitive assay is that the affinity of non-fluorescent bicyclic peptides can be determined accurately and rapidly.

In this context, it is important to ensure that the scaffold sequences 17-69-07 and 17-69-12 are responsible only for the high affinity for PEX. Therefore, the peptides were synthesized as variants with N- and C-terminal alanines, thus closely mimicking the sequence expressed on the phage particle, but without the Sar5/6 molecular spacer that was used in the fluorescent constructs.

The affinity determined by competition experiments was nearly identical to that obtained with the fluorescent derivatives, demonstrating unambiguously that the bicyclic scaffold sequences are responsible for the high binding affinity to PEX (Table 3). In addition, the fluorescent constructs show that C-terminal binding of molecular spacers and effector groups (herein as -A-Sar6-K(fluorescein)) is allowed.

Table 3: Results of direct binding experiments using fluorescence polarization (FP)

Peptide code Molecular format Subsequence FP Method Label Kd (nM) 17-69-07-N001 A-(17-69-07)-A ACYNEFGCEDFYDICA (SEQ ID NO: 18) Competition A-(17-69-12)-A-Sar6-K(fluorescein) 1.65±0.29 17-69-12-N003 A-(17-69-12)-A ACMNQFGCEDFYDICA (SEQ ID NO: 19) Competition A-(17-69-07)-A-Sar6-K(fluorescein) 3.7±2.52

Example 2: Proteolytic stability of the mouse 17-69-07 scaffold sequence

Stability in plasma 17-69-07

For therapeutic use in humans, as well as for preclinical evaluation in animal species, it is appropriate for the lead bicyclic peptide to be sufficiently stable in the circulation following intravenous administration. Adequate stability is required to allow sufficient levels of the bicyclic peptide to bind to its target and exert its biological function.

Common species used in preclinical models include mouse, rat, rabbit, and macaque. In the former, the bicyclic peptide 17-69-07-N219 was tested for stability in the presence of mouse plasma using the methods described previously (method #2). The bicyclic scaffold sequence retains the original natural proteinogenic amino acid sequence of 17-69-07 and additionally contains an N-terminal molecular spacer, which is used for conjugation of effector groups (sequence: G-Sar10-ACYNEFGCEDFYDIC; SEQ ID NO: 20). The affinity of 17-69-07-N219 for PEX was maintained despite the presence of the molecular spacer (Kd=0.82 nM).

This compound exhibited moderate stability in mouse plasma ex vivo with a half-life of 6 hours (Figure 1).

Identification of proteolytic cleavage sites 17-69-07 ex/in vivo

To gain insight into the chemical nature of 17-69-07-N219 degradation in mouse plasma, samples were analyzed by MALDI-TOF for any potential degradation products. Mass spectra showed possible loss of tyrosine, implying loop opening (hydrolysis) and removal of Tyr1 in loop 1 and/or Tyr9 in loop 2.

A pharmacokinetic (PK) study in mice using the minimal bicyclic peptide 17-69-07-N004 (Ac-CYNEFGCEDFYDIC (SEQ ID NO: 2), which consists of the minimal bicyclic peptide scaffold 17-69-07) was performed to determine clearance and elimination rate from circulation in vivo. Resulting blood samples were analyzed further and plasma samples were analyzed for any potential proteolytic degradation products of 17-69-07-N004.

The PK profile is shown in Figure 2.

Table 4: Pharmacokinetic parameters 17-69-07-N004

PK parameter Average Cmax (ng/ml) 15789 Tmax (min) 5.0 Distribution half-life (min) NC Elimination half-life (min) 14 AUC 0-t (mcg/ml.min) 286 AUC 0-∞ (µg/ml.min) 286 Volume of distribution based on AUC 0-∞ (L/kg) 0.4 Clearance based on AUC 0-∞ (ml/min/kg) 20.7 Time points for elimination half-life regression (min) 5-60

NC: not calculated due to limited data

Table 4 shows that the peptide has a half-life of 14 min and a clearance of 20.7 ml/min/kg. The clearance rate is higher than the glomerular filtration rate observed in mice (summarized in Qi et al, American Journal of Physiology - Renal Physiology (2004) Vol. 286 no. 3, F590-F596), suggesting that the peptide is cleared by additional pathways, such as plasma-mediated proteolysis and endothelial proteases.

To address whether in vivo proteolysis significantly contributes to clearance, plasma samples collected at t=0.5, 1 and 2 h were subjected to targeted analysis for bicyclic peptide fragments using LC-MS/MS methods.

A variety of proteolytic metabolites could be identified, and the fragments with the most intense signals are listed below in descending order:

Ac-C _i YNEFGC _ii EDFYDIC _iii (SEQ ID NO: 2):

elimination of YNE in loop 1;

exclusion of YNEF in loop 1 (SEQ ID NO: 21);

deletion of YNEFG (SEQ ID NO: 22) in loop 1 (complete loop is removed);

elimination of Y in loop 1 and/or 2;

elimination of YD in loop 2;

FYD elimination in loop 2;

YDI exception in loop 2; and

exclusion of EDFYDI (SEQ ID NO: 23), in loop 2 (the entire loop is deleted).

The top three metabolites were present at moderate signal levels, while the remaining fragments were detected only in trace amounts.

Taken together, both ex vivo and in vivo metabolites appear to concentrate with an initial cleavage at or near Tyr 1/9, followed by sequential removal of residues in close proximity. This ultimately results in the removal of one or both loops in their entirety.

Increased proteolytic stability and activity of sequence 17-69-07

Approaches to stabilize peptide sequences against proteolytic degradation are numerous (see reviews by Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185–203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399–418) and patents WO 2009/098450, WO 2013/050615, and WO 2013/050616). Briefly, they include substitution of the amino acid that provides the recognition point for the protease(s), alteration of the amino acid backbone at the cleavage site (i.e., N-methylation, pseudopeptide bonds, etc.), steric hindrance to adjacent bonds (i.e., β-substituted amino acids), and incorporation of D-enantiomeric amino acids. Some of these modifications (i.e. N-methylation, D-amino acids) can protect/shield the proteolytic cleavage site from hydrolysis, even if they are located up to two residues away from the cleavage site. Although this is a relatively direct way of protecting a sequence from proteolytic action, it is much more difficult to incorporate stabilizing changes that do not significantly alter activity (and specificity) for the target protein.

From the ex/in vivo proteolytic degradation data of 17-69-07 presented in the previous section, it is clear that Tyr1/9 and residues in its immediate vicinity are potential sites for stabilization of the bicyclic peptide. Quantitative measures of successful peptide stabilization include increases in its half-life in mouse and human plasma.

First, eleven derivatives of 17-69-07 were created in which each position was replaced with alanine (referred to as "alanine scanning"). This type of information provides information about the energetic contribution and role of certain residues and potentially removes proteolytic recognition points.

Table 5: Alanine scan results

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) C _i YNEFGC _ii EDFYDIC _iii
(SEQ ID NO: 2) 2.47±0.25 17-69-07-N014 Ac-(17-69-07) Ala1 3.8 17-69-07-N015 Ac-(17-69-07) Ala2 >5000 17-69-07-N016 Ac-(17-69-07) Ala3 5.1 17-69-07-N017 Ac-(17-69-07) Ala4 34 17-69-07-N018 Ac-(17-69-07) Ala5 >5000 17-69-07-N019 Ac-(17-69-07) Ala6 >5000 17-69-07-N020 Ac-(17-69-07) Ala7 >5000 17-69-07-N021 Ac-(17-69-07) Ala8 >5000 17-69-07-N022 Ac-(17-69-07) Ala9 >5000 17-69-07-N023 Ac-(17-69-07) Ala10 11.1 17-69-07-N024 Ac-(17-69-07) Ala11 8

17-69-07-N004 is an unmodified wild-type peptide containing a capped N-terminus (N-terminal acetylation, referred to as "Ac"). Several of the Ala substitutions are well tolerated, particularly at positions 1, 3, 4, 10, and 11 within the sequence. Since the side chains of these residues are not required for high affinity binding to the target, the compounds of formula (I) are broadly defined at these particular sequence positions.

This makes the substitution at residue 1 (Tyr 1) a very attractive possibility, as it could remove one of the proteolytic recognition points. The substitution of Gly5 by Ala5 is of interest, as the chemical modification is minor (addition of a methyl group) but results in a dramatic decrease in activity. It is possible that Gly5 adopts unusual phi/psi angles outside the general Ramachendran diagram, and these angles could potentially be induced by D-amino acids. An additional benefit would be the stabilization of peptide bonds adjacent to this site.

In this regard, a partial D-alanine scan was performed to assess whether they are stable in terms of maintaining activity: the inclusion of D-amino acids in the sequence is highly desirable due to their stabilizing effect against proteolytic cleavage.

Table 6: Effect of substitution of residues in the first loop with D-alanines

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) 2.47±0.25 17-69-07-N061 Ac-(17-69-07) D-Ala1 23.6 17-69-07-N062 Ac-(17-69-07) D-Ala2 >5000 17-69-07-N063 Ac-(17-69-07) D-Ala3 105 n=1 17-69-07-N056 Ac-(17-69-07) D-Ala4 >5000 17-69-07-N058 Ac-(17-69-07) D-Ala5 2.37±0.86 17-69-07-N059 Ac-(17-69-07) D-Pro5 >5000

D-Ala1 instead of Tyr1 binds with an affinity 10-fold lower than wild type. However, it is of interest because the Tyr1 proteolytic recognition point is removed and replaced by a stabilizing amino acid without inducing a significant loss of activity.

Notably, the substitution of Gly5 with D-Ala5 is well tolerated, as the affinity compared to wild type remains unchanged.

In this context, D-Arg5 is also transferred (and similarly, therefore, all D-amino acids except D-Pro, see Table 6), which may be of interest if the physicochemical properties need to be modified during further design of the molecule (Table 7).

Table 7: Effect of substitution of residues at position 5

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) 2.47±0.25 17-69-07-N018 Ac-(17-69-07) Ala5 >5000 17-69-07-N058 Ac-(17-69-07) D-Ala5 2.37±0.86 17-69-07-N120 Ac-(17-69-07) D-Arg5 4.24±1.48

Furthermore, in view of the phage selection yields indicating a definite preference for hydrophobic and aromatic amino acids at position 4 of the sequence, a number of derivatives were synthesized containing selected aromatic amino acids, including natural tyrosine and tryptophan (Table 8).

Table 8: Effect of substitution of residues at position 4

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) 2.47±0.25 17-69-07-N057 Ac-(17-69-07) Tyr4 1.78±0.45 17-69-44-N002 Ac-(17-69-07) Trp4 0.75±0.23 17-69-07-N067 Ac-(17-69-07)1NAl4 0.36±0.12 17-69-07-N068 Ac-(17-69-07)2NAl4 0.7±0.19 17-69-07-N065 Ac-(17-69-07) Chg4 >5000 17-69-07-N071 Ac-(17-69-07) Phg4 628 17-69-07-N072 Ac-(17-69-07) tBuGly4 >5000 17-69-07-N137 Ac-(17-69-07)3,4-DCPhe4 1.85±2.45 17-69-07-N139 Ac-(17-69-07) Cha4 362 17-69-07-N140 Ac-(17-69-07) HPhe4 43.3

1Nal: 1-naphthylalanine; 2Nal: 2-naphthylalanine; Chg: cyclohexylglycine, Phg: phenylglycine; tBuGly: tert-butylglycine; 3,4-DCPhe4: 3,4-dichlorophenylalanine; Cha: cyclohexylalanine; and HPhe: homophenylalalnine.

Several substitutions at position 4 increase affinity compared to the wild type, including tyrosine, tryptophan, 1- and 2-naphthylalanine (1/2 Nal), and 3,4-dichlorophenylalanine (3,4-DCPhe). The most potent substitution is 1-naphthylalanine, which increases affinity 7-fold.

Several residues in loop 2 of 17-69-07 were examined to improve the stability of the molecule. These include residue 9, which includes a potential Tyr9 recognition site. Residue 11 is also attractive because it allows substitutions (Table 5) and is close to the Tyr9 recognition site.

Table 9: Summary of tolerated amino acid substitutions

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) 2.47±0.25 17-69-07-N130 Ac-(17-69-07)4BrPhe9 2.45±1.08 17-69-07-N182 Ac-(17-69-07) 5FPhe9 13.1 17-69-07-N100 Ac-(17-69-07) tBuGly11 1.56±1.14

Of interest are 4-bromophenylalanine (4BrPhe), which alters the proteolytic recognition site of Tyr9, and tert-butylglycine (tBuGly), which slightly increases affinity and, importantly, significantly protects adjacent scaffold amino acids from proteolytic hydrolysis via steric hindrance.

Multiple site substitutions to achieve global proteolytic protection 17-69-07

The previous section described multiple positions within the 17-69-07 sequence that permit the inclusion of amino acids that enhance proteolytic stability and/or affinity.

In order to combine these modifications into one molecule, a molecule was synthesized that included Tyr1 substitutions. D-Ala1, Phe4 1-naphthylalanine4, Gly5 D-Ala5, Tyr9 4BrPhe9 and Ile11 tBuGly11. Notably, all modifications are cotransported and the activity is higher than that of the wild-type molecule (Tables 10 and 11).

Table 10: Results of specific multiple substitution

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) 2.47±0.25 17-69-07-N252 Ac-(17-69-07) D-Ala1 1Nal4 D-Ala5 4BrPhe9 tBuGly11 0.8±0.39

In anticipation of the attachment of effector groups to such a bicyclic peptide, further molecular design generated variants using an N-terminal sarcosine molecular spacer (10 consecutive Sar, designated Sar10) starting with an N-terminal glycine and ending with a C-terminal alanine. For the purpose of this experiment, the N-terminal Gly was capped with an acetyl group to remove the positive charge.

Table 11: Comparative data after addition of G-Sarl0-A

Peptide code Molecular description Kd (nM) 17-69-07-N004 Ac-(17-69-07) (wild type) 2.47±0.25 17-69-07-N225 Ac-G-Sar10-A-(17-69-07) 0.95±0.1 17-69-07-N239 Ac-G-Sar10-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 tBuGly11 0.94±0.08 17-69-07-N255 Ac-G-Sar10-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 4BrPhe9 tBuGly11 0.3

These data indicate that both molecular spacers (attached to the N-terminus as shown) and amino acid substitutions within the bicyclic sequence backbone are well tolerated, while activity is maintained or improved.

Table 12 presents non-acetylated, (un)stabilized derivatives of the molecules shown in Table 11. Their stability was quantitatively assessed in mouse and human plasma to demonstrate improvement over unstabilized 17-69-07-N219 (Figure 1).

Table 12: Molecules selected for activity-related plasma stability assessment

Peptide code Molecular description Kd (nM) 17-69-07-N219 G-Sar10-A-(17-69-07) (not stabilized by spacer) 0.82±0.09 17-69-07-N244 G-Sar10-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 4BrPhe9 tBuGly11 0.45±0.19 17-69-07-N231 G-Sar10-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 tBuGly11 1.07±0.28 17-69-07-N241 bAla-Sar10-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 tBuGly11 1.21±0.24

Figure 3A shows the stability in mouse plasma of the penta- and tetra-substituted molecules 17-69-07-N244 and 17-69-07-N231, respectively, compared to the parent unstabilized wild-type molecule 17-69-07-N219. The half-life of the peptide in mouse plasma at 37°C is >>20 hours compared to 6 hours for the wild-type molecule without enhanced stabilization.

Figure 3B shows the stability in human plasma of the penta- and tetra-substituted molecules 17-69-07-N244 and 17-69-07-N231, respectively, compared to the parent unstabilized wild-type molecule 17-69-07-N219. The half-life of the peptide in mouse plasma at 37°C is >>20 hours compared to 6 hours for the wild-type molecule without enhanced stabilization.

Thus, targeted substitutions of up to 5 positions in the bicyclic framework sequence (Tyr1 D-Ala1, Phe4 1Naphthylalanine4, Gly5 D-Ala5, Tyr9 4BrPhe9 and Ile11 tBuGly11) yield superior molecules with increased potency and significantly improved plasma stability.

Selectivity of the stabilized derivative 17-69-07 (17-69-07-N241)

The stabilized, molecular spacer-containing derivative 17-69-07-N241 was tested in a competitive FP assay for affinity to the hemopexin domain of MT1-MMP derived from other species. The data are presented in Table 13:

Table 13: Species cross-reactivity of 17-69-07 and its derivatives

Human/macaque (Kd, nM) Dog (Kd, nM) Mouse (Kd, nM) 17-69-07-N219 0.82 0.9 Not defined. 17-69-07-N241 1.21 1.4 0.63

Both unstabilized and stabilized derivatives of 17-69-07 exhibit complete cross-reactivity.

A derivative of 17-69-07-N241 with an N-terminal fluorescein (designated 17-69-07-N258, with the sequence: fluorescein-(bAla)-Sar10-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 tBuGly11) was tested against related human metalloproteinases. The data obtained show that the 17-69-07 scaffold sequence and this stabilized variant are uniquely selective for MT1-MMP (Table 14).

Table 14: Selectivity of 17-69-07 and its derivatives towards related metalloproteinases

MT1-MMP
(=MMP-14)
(Kd, in nM) MMP-1
(Kd, in nM) MMP-2
(Kd, in nM) MMP-15
(Kd, in nM) MMP-16
(Kd, in nM) 17-69-07-N258 2,2 >1000 >1000 >1000 >1000

Example 3: In vivo analysis of proteolytically stabilized chelator-conjugated 17-69-07 variants

Peptides linked to metal chelators have many applications in diagnostics and therapy. Certain imaging agents or therapeutic radioisotopes can be “loaded” onto the chelator, while the peptide carries said isotopes to the target. In this way, tumor-specific antigens can be visualized using, for example, PET and SPECT scanners. For targeted radiotherapy, therapeutic radionuclides (e.g., ⁹⁰ Y and ¹⁷⁷ Lu) are loaded onto a chelator peptide and selectively delivered to the tumor by interaction with the tumor-associated antigen. There, they exert their antitumor activity as a result of the high energy radiation they emit.

Synthesis of chelator-linked bicyclic peptides 17-69-07

Five derivatives were synthesized in which the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-ethylenediaminetetraacetic acid) was linked to the N-terminal alanine of the bicyclic peptide 17-69-07.

Table 15: Brief description of molecules and structures

Peptide code Molecular description Kd (nM) 17-69-07-N144 DOTA-A-(17-69-07) 0.5 17-69-07-N147 DOTA[Lu]-A-(17-69-07) 0.95±0.5 17-69-07-N248 DOTA-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 4BrPhe9 tBuGly11 0.5 17-69-07-N246 DOTA-A-(17-69-07) All D amino acids >5000

17-69-07-N144 is the wild-type sequence of 17-69-07 where DOTA is directly linked to the N-terminal alanine, which serves as a single amino acid spacer. This molecule retains full activity against MT1-MMP. To demonstrate that activity is retained upon loading with a therapeutic/imaging radionuclide, the chelator was loaded with natural (cold) ¹⁷⁵ lutetium as a safe substitute. Activity was retained.

A fully stabilized variant, 17-69-07-N248, also retained activity in the context of a conjugate with DOTA.

A control molecule, 17-69-07-N246, was generated which is the complete D-amino acid equivalent of 17-69-07-N144. This molecule is completely resistant to proteolytic degradation, as the amide bonds adjacent to the D-amino acids are protected from proteases, and does not have any activity towards MT1-MMP. Importantly, this peptide retains exactly the same sequence and chemical composition.

Biodistribution of 17-69-07-N144 loaded with ¹⁷⁷ Lu in HT1080 xenograft bearing mice

HT-1080 (cells known to overexpress MT1-MMP) was inoculated subcutaneously into the right side of the trunk of 6-week-old male BALB/c nu/nu mice. Tumors were allowed to grow for approximately 1 week.

150 pmol of peptide 17-69-07-N144 were loaded with 1 MBq of the γ- and β-emitter ¹⁷⁷ Lu using standard complexation methods. Then, 150 pmol of loaded 17-69-07-N144 was administered per tumor-bearing mouse (equivalent to 17 μg/kg). Three animals were euthanized at each time point, the various organs and tumors were removed and weighed, and the gamma radiation level per gram of tumor/organ tissue was determined. The resulting graph provides a quantitative representation of the localization of the peptide in vivo in the mouse. In particular, the selective accumulation of HT1080 in the tumor indicates MT1-MMP binding in vivo.

Figure 4 summarizes the biodistribution data. Notably, the bicyclic peptide is tumor specific and appears to persist for up to 24 hours despite an estimated circulating half-life of 14 min. This likely indicates uptake by tumor cells. Significant localization is seen in the kidney, possibly due to renal amino acid transporters transiently binding the peptide. No significant uptake of the molecule is seen in any other organ. It should be noted that the mouse hemopexin domain shares complete homology with the human hemopexin domain, suggesting that if mouse PEX were expressed in other tissues, corresponding signals would be observed.

To assess whether tumor uptake in the xenograft model was selective and mediated by PEX binding, an additional study was conducted using the peptide 17-69-07-N246, which is a D-amino acid analogue of 17-69-07-N144 (Figure 5).

When comparing the biological distribution pattern obtained with the active bicyclic peptide 17-69-07-N144, it is clear that the non-MT1-MMP-binding bicyclic peptide 17-69-07-N246 does not accumulate in the tumor, confirming that the tumor signal of 17-69-07-N144 is driven by the targeted binding of MT1-MMP in vivo.

Finally, to assess the effect of proteolytic stabilization of the 17-69-07 sequence on biodistribution, the bicyclic peptide 17-69-07-N248 (DOTA-A-(17-69-07) D-Ala1 1Nal4 D-Ala5 4BrPhe9 tBuGly11) was used in an identical biodistribution study (Figure 6).

Strikingly, the increased proteolytic stability of the peptide results in an overall doubling of tumor uptake at any given time point compared to 17-69-07-N144, illustrating the superior properties of the molecule compared to the parent, unstabilized 17-69-07 sequence.

It is expected that this effect may result in a favourable therapeutic index, both in the case of targeted radionuclide therapy using the conjugates described in this example and in the context of toxin-conjugated bicyclic peptides (Example 4).

Example 4: Conjugation of unstabilized bicyclic peptides with cytotoxic agents

For targeted cancer therapy, highly potent cytotoxic drugs are attached via a cleavable linker to a targeting moiety (herein, a bicyclic peptide) that binds to tumor-associated proteins expressed on the cell surface. The overexpressed tumor-associated cell surface protein target is selected for its ability to be internalized into the cell. Upon binding of the cytotoxic agent-targeting moiety-tumor-associated cell surface protein compound, the entire molecular complex is internalized into the cell. After transferring from the systemic circulation to a separate intracellular environment, the cytotoxic drug is cleaved from the targeting moiety as a result of intracellular environmental conditions, and the cleaved drug then exerts its antitumor effect by inducing targeted cell death by cell cycle arrest followed by apoptosis.

The bicyclic peptide 17-69-07-N219 (see Examples 2 and 3) consists of the wild-type bicyclic scaffold sequence linked at the N-terminal end to a Gly-Sar10-Ala molecular spacer (G-Sar10-A-(17-69-07), where the complete sequence description is G-Sar10-A-CYNEFGCEDFYDIC; SEQ ID NO: 20). This derivative (17-69-07) retains full activity (Table 12) against MT1-MMP with a Kd of 0.8 nM. The unique free amino group at the N-terminal end of the molecular spacer is ideal for conjugation with effector groups such as potent cytotoxic agents. The long intramolecular distance imparted by the Sar10 linker between the conjugation site at the N-terminal Gly and the bicyclic scaffold sequence is used to ensure that target binding activity is fully retained after conjugation to effector groups of significant molecular size. Shorter molecular spacers can be chosen as desired as long as the activity of the bicyclic scaffold sequence towards the target protein is retained.

In order to obtain an experimental proof of concept of a bicyclic peptide drug conjugate (designated BDC) targeting MT1-MMP, two conjugates 17-69-07-N219 were prepared that utilize either the microtubule polymerization inhibiting toxins DM1 (maytansine, also known as N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) or MMAE (monomethyl auristatin E, also known as (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)butanamido)butanamide).

The MMAE conjugate is designated BT17BDC-1 and is separated from the precursor 17-69-07-N219 by a valine-citrulline (Val-Cit) linker, including a self-destructing para-aminobenzylcarbonyl group (PABC). The Val-Cit linker is selectively cleaved (hydrolyzed) by the cathepsin B-rich environment found in intracellular lysosomes, and after hydrolysis, PABC is destroyed releasing MMAE as the active toxic component. The Val-Cit-PABC linker is stable in the bloodstream, and thus releases the toxin only after cellular internalization.

The structure of the conjugate and the synthesis scheme for obtaining BT17BDC-1 are shown in Scheme I:

Scheme I

Synthetic strategy for the preparation of BDC17BDC-1: fully purified cyclized TMB bicyclic precursor 17-69-07-N219 is reacted with the succinimide ester of glutaryl-valine-citrulline-p-aminobenzylcarbonyl-MMAE (commonly abbreviated as VC-MMAE or Val-Cit-PABC-MMAE) to afford the conjugate BDC17BDC-1.

The DM1 conjugate 17-69-07-N219 is designated BT17BDC-9 and is separated from the precursor 17-69-07-N219 by a disulfide bond that can be cleaved in the reducing environment encountered in the intracellular environment. The reduction is thought to occur via intracellular glutathione, which is present intracellularly at a concentration of ~10 mM. In contrast, the concentration of glutathione and free reducing agents in the circulating blood is much lower (<10 μM); thus, the toxin is released preferentially into the intracellular environment where it can exert its cell killing activity.

The structure of the conjugate and the synthesis scheme for obtaining BT17BDC-9 are shown in Scheme II:

Scheme II:

Synthetic strategy for the preparation of BDC17BDC-9: fully purified cyclized TMB bicyclic precursor 17-69-07-N219 containing a free N-terminal amine is reacted with succinimide ester disulfide-DM1 (structure as shown) to afford the conjugate BDC17BDC-9.

The release of toxins in BDC17BDC-1 and BDC17BDC-9 is therefore mechanistically different, i.e. in the former case due to intracellular proteolytic conditions and in the latter due to intracellular reducing conditions.

Two BDCs were used in in vitro cytotoxicity studies and showed low nanomolar/picomolar activity on cell cultures such as HT1080 (data not shown).

In an in vivo mouse xenograft model (bearing HT1080 tumor), both BDCs induced a significant reduction in tumor volume 9 days post-injection compared to vehicle control. The dosing schedule is shown in Figure 7 (arrows). Tumors were completely cleared by day 14 for both BDCs as assessed by palpation (Figure 7). It should be noted that animal weights were largely stable for both BDC17BDC-1 and BDC17BDC-9, indicating a therapeutic window that may be sufficient for therapeutic purposes.

Example 5: Conjugation of proteolytically stabilized bicyclic peptides with cytotoxic agents

The bicyclic framework sequence 17-69-07, containing the modifications: D-alanine at position 1, 1-naphthylalanine at position 4, D-alanine at position 5, α-tert-butylglycine at position 11 with or without 4-bromophenylalanine at position 9, had increased proteolytic stability in plasma ex vivo and in vivo (examples 2, 3).

In the context of bicyclic peptide-drug conjugates, it is likely that a more stable bicyclic scaffold sequence may result in greater tumor exposure due to decreased systemic clearance and higher stability in the proteolytically aggressive tumor microenvironment. In both cases, this may result in increased MT1-MMP-driven intracellular uptake of BDC, leading to increased therapeutic efficacy.

The stabilized bicyclic peptide 17-69-07-N241 was designed with a molecular scheme similar to the unstabilized 17-69-07-N219 (described in Examples 3 and 4). It has the following sequence:

(B-Ala)-Sar10-AC(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 5)

which is cyclized by TBMB as described above.

The N-terminal beta-alanine was chosen over the previously used glycine as in 17-69-07-N219 to minimize the formation of diketopiperazine by-products during the addition of N-hydroxysuccinimide (NHS) esters (Purdie et al (1973), J. Chem. Soc., Perkin Trans. 2, 1845), in this example specifically NHS esters of cytotoxic agents (Scheme I, II).

The sarcosine decameric spacer is retained as in 17-69-07-N219 to ensure complete retention of the binding of the bicyclic peptide to the hemopexin domain of MT1-MMP.

The class of maytansine toxins was selected for conjugation to 17-69-07-N241 based on efficacy and tolerability in the mouse xenograft model described in Example 4, and a disulfide cleavable linker was again chosen.

Four BDCs (referred to herein above as BT17BDC-17, BT17BDC-18, BT17BDC-19, and BT17BDC-20) were generated whereby both the toxin and 17-69-07-N241 were invariant and the sensitivity of the disulfide bond to reduction/cleavage was altered by modulating the degree of hindrance adjacent to the sulfur atoms.

The scheme of synthesis of conjugates corresponds to that described in scheme II, where 17-69-07-N219 is replaced by 17-69-07-N241.

For BT17BDC-18, an additional synthetic route involved the creation of a pyridyl disulfide bicyclic precursor (designated 17-69-07-N277) that reacts with DM1 to form the complete conjugate. The synthetic route is described in Scheme III.

Scheme III

Synthetic strategy for the preparation of BDC17BDC-18: fully purified cyclized TMB bicyclic precursor 17-69-07-N219 containing a free N-terminal amine is reacted with SPP (N-succinimidyl 4-(2-pyridyldithio)pentanoate) to afford intermediate 17-69-07-N277. This is then reacted with DM1 as the free thiol to afford the desired conjugate BT17BDC-18.

In vitro characterization of BT17BDC-17, BT17BDC-18, BT17BDC-19 and BT17BDC-20

The four BDCs were assessed for several in vitro parameters such as retention of activity against the human hemopexin MT1-MMP domain, ex vivo stability in mouse, rat and human plasma, and stability against reducing agents such as dithiothreitol.

The data are presented in Table 16 below:

Table 16: Properties of BT17BDC-17, BT17BDC-18, BT17BDC-19 and BT17BDC-20 in vitro

Bicyclic peptide drug conjugate Kd (nM)
(hemopexin domain) ^a t _1/2 (hour)
(human plasma) ^b t _1/2 (hour)
(mouse plasma) ^b t _1/2 (hour)
(rat plasma) ^b Relative stability to DTT
(ADC) ^c Relative stability to DTT
(BDC) ^d 17-69-07-N219 0.82±0.09
(n=3) ^e 30.3±4.7
(n=1) 3.9±0.3
(n=1) 3.7±0
(n=1) n/a n/a 17-69-07-N241 1.4±0.3
(n=10) ^e >36
(n=2) >36
(n=2) >36
(n=1) n/a n/a BT17BDC-17 0.7
(n=1) ^e 4.2±0
(n=2) 5.5±0.7
(n=1) 1.2±0.1
(n=1) 1 1 BT17BDC-18 0.99±0.17
(n=6) ^e 12.7±2.2
(n=2) 14.3±1
(n=2) 4.3±0.6
(n=2) 7 5 BT17BDC-19 0.5
(n=1) ^e 23.5±4.1
(n=2) >36
(n=1) >36
(n=1) 14 30 BT17BDC-20 2.95±1.3
(n=4) ^f 32±1
(n=1) >36
(n=1) 34±4
(n=1) 170 93

where n/a: not used, and where n=number of repetitions

^a : determined by fluorescence polarization competition experiments using 17-69-07-N040 as a label

^b : determined using quantitative LC-MS. Incubation time in plasma containing 4 μM BDC up to 24 h.

^c : from Kellogg et al (2011) Bioconjugate Chemistry, 22, 717. Note that these values are for antibody-drug conjugates containing the disulfide linker described in the text.

^d : determined by quantitative LC-MS. Note that these values apply to bicyclic drug conjugates containing the disulfide linker described in the text. Methods were adapted from Kellogg et al (2011) Bioconjugate Chemistry, 22, 717.

^e : used 17-69-07-N004 as a tag in the FP competition assay

^f : used 17-69-07-N040 as a tag in FP competition assay

All molecular constructs retain their affinity for the hemopexin target (second column).

The data indicate that plasma stability is determined by the nature of the disulfide bond (associated with modulation of sensitivity to reduction) rather than the nature of the bicyclic peptide, as all BDCs contain the same bicyclic peptide (17-69-07-N241), which is stable in plasma of the three species tested.

Furthermore, BT17BDC-18, BT17BDC-19 and BT17BDC-20 exhibit stability in human plasma adequate for therapeutic use, as renal filtration-driven clearance of peptides of this molecular size in humans is expected to have an estimated half-life of 2 to 4 hours, which is several-fold faster than the degradation half-life of BDCs in human plasma (>14 hours for BT17BDC-18/19/20, see Table 16). Thus, the major portion of BDCs is expected to be excreted via the kidneys and only a fraction is degraded in the bloodstream, making these BDCs potentially suitable for therapeutic purposes.

In vivo efficacy of BT17BDC-17, BT17BDC-18, BT17BDC-19 and BT17BDC-20

All BDCs containing a stabilized bicyclic scaffold sequence were tested for their efficacy in in vivo mouse xenograft models using the human squamous cell lung cancer line EBC-1.

BT17BDC-17, BT17BDC-18, and BT17BDC-19 were effective and produced tumor freedom at 9 days (Figures 9, 10, and 11). BT17BDC-17 showed good efficacy but some weight loss at high doses. BT17BDC-20 was ineffective and produced little tumor shrinkage based on constant weight tolerance (Figure 12).

The area under the curve (AUC) of the tumor volume versus time and BDC plot was taken and plotted against the corresponding dose group (Figure 13). From this, the effective dose to achieve a 50% reduction in tumor AUC (ED50) could be determined, which is summarized in Table 17.

Table 17: Effective dose to achieve 50% tumor AUC reduction for BT17BDC-9, BT17BDC-17, BT17BDC-18, BT17BDC-19, and BT17BDC-20

BDC BT17BDC-9 BT17BDC-17 BT17BDC-18 BT17BDC-19 BT17BDC-20 ED50 ^a 2.1±1.1 ^b 1.3±0.6 ^b 2.1±0.8 ^b 3.1±1.6 ^b 22±13 ^b

^a ED50±95% confidence interval

^b units in mg/kg, in mice bearing EBC-1 tumors

Thus, BT17BDC-9, BT17BDC-17, BT17BDC-18 and BT17BDC-19 are suitable molecules for use in targeted cancer therapy based on their efficacy, and BT17BDC-17, BT17BDC-18 and BT17BDC-19 are well tolerated at effective doses.

--->

LIST OF SEQUENCES

<110> Bicycle Therapeutics Limited

<120> Bicyclic peptide ligands specific for MT1-MMP

<130> BIC-C-P1803PCT

<150>GB1419237.1

<151> 2014-10-29

<150>GB1515245.7

<151> 2015-08-27

<160> 23

<170> Patent version 3.5

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<221> Xaa

<222> (4)..(4)

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<---

Claims (125)

1. A peptide ligand specific for MT1-MMP, comprising a polypeptide comprising three cysteine residues separated by two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine residues of the polypeptide such that two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises an amino acid sequence of formula (I): -C _i -X ₁ -U ₂ /O ₂ -X ₃ -X ₄ -G ₅ -C _ii -E ₆ D ₇ -F ₈ -Y ₉ -X ₁₀ -X ₁₁ -C _iii - (SEQ ID NO: 1) (I) or its pharmaceutically acceptable salt, Where: C _i , C _ii and C _iii represent the first, second and third cysteine residues, respectively; X ₁ is selected from any of the following amino acids: Y, M, F or V; U ₂ /O ₂ is selected from N or G; X ₃ is selected from U or Z, where U is a polar, uncharged amino acid residue selected from N, C, Q, M, S and T, and Z is a polar, negatively charged amino acid residue selected from D or E; X ₄ is selected from J, where J is a non-polar aromatic amino acid residue selected from F, W and Y; X ₁₀ is selected from Z, where Z is a polar, negatively charged amino acid residue selected from D or E; and X ₁₁ is selected from O, where O is a non-polar aliphatic amino acid residue selected from G, A, I, L, P and V; optionally, the peptide ligand comprises one or more modifications selected from: (i) an N-terminal modification which comprises the addition of a molecular spacer group selected from Ala, a G-Sar10-A group and a bAla-Sar10-A group; (ii) an N-terminal and/or C-terminal modification that includes the addition of a cytotoxic agent; (iii) substitution of the amino acid at position 1 with D-alanine; (iv) replacing the amino acid residue at position 4 with a non-natural amino acid residue selected from 1-naphthylalanine; 2-naphthylalanine; 3,4-dichlorophenylalanine; and homophenylalanine; (v) substitution of the amino acid residue at position 9 with 4-bromophenylalanine; (vi) substitution of the amino acid residue at position 11 with tert-butylglycine; and (vii) 2, 3, 4 or 5 of the following modifications: D-alanine at position 1 and/or 5, 1-naphthylalanine at position 4, 4-bromophenylalanine at position 9 and tert-butylglycine at position 11. 2. The peptide ligand of claim 1, wherein the compound of formula (I) is a compound of formula (Ia): -C _i -Y/M/F/VU/OU/ZJGC _ii -EDFYZOC _iii - (SEQ ID NO: 6), (Ia) where U, O, J and Z are as defined above; or compound of formula (Ib): -C _i -Y/M/F/VN/GE/QFGC _ii -EDFYDIC _iii - (SEQ ID NO: 7) (Ib); or compound of formula (Ic): -C _i -Y/M/FN/GE/QFGC _ii -EDFYDIC _iii - (SEQ ID NO: 8) (Ic); or compound of formula (Id): -C _i -Y/MNE/QFGC _ii -EDFYDIC _iii - (SEQ ID NO: 9) (Id); or compound of formula (Ie): -C _i -YNEFGC _ii -EDFYDIC _iii - (17-69-07) (SEQ ID NO: 2) (Ie). 3. A peptide ligand according to claim 1 or 2, wherein the peptide of formula (I) comprises a sequence selected from: -C _i -YNEFGC _ii -EDFYDIC _iii - (17-69-07) (SEQ ID NO: 2); -C _i -MNQFGC _ii -EDFYDIC _iii - (17-69-12) (SEQ ID NO: 10); -C _i -FGEFGC _ii -EDFYDIC _iii - (17-69-02) (SEQ ID NO: 11); -C _i -VNEFGC _ii -EDFYDIC _iii - (17-69-03) (SEQ ID NO: 12); -C _i -FNEFGC _ii -EDFYDIC _iii - (17-69-04) (SEQ ID NO: 13); -C _i -YNEYGC _ii -EDFYDIC _iii - (17-69-07-N057) (SEQ ID NO: 14); And -C _i -YNEWGC _ii -EDFYDIC _iii - (17-69-44-N002) (SEQ ID NO: 15). 4. A peptide ligand according to any one of claims 1-3, which comprises one or more modifications selected from: (i) an N-terminal modification which comprises the addition of a molecular spacer group selected from Ala, a G-Sar10-A group or a bAla-Sar10-A group; (ii) an N-terminal and/or C-terminal modification that includes the addition of a cytotoxic agent; (iii) substitution of the amino acid at position 1 with D-alanine; (iv) replacing the amino acid residue at position 4 with a non-natural amino acid residue selected from: 1-naphthylalanine; 2-naphthylalanine; 3,4-dichlorophenylalanine; and homophenylalanine; (v) substitution of the amino acid residue at position 9 with 4-bromophenylalanine; (vi) substitution of the amino acid residue at position 11 with tert-butylglycine; and (vii) 2, 3, 4 or 5 of the following modifications: D-alanine at position 1 and/or 5, 1-naphthylalanine at position 4, 4-bromophenylalanine at position 9 and tert-butylglycine at position 11. 5. A peptide ligand according to any one of claims 1-4, wherein the pharmaceutically acceptable salt is selected from a free acid or a salt of sodium, potassium, calcium, ammonium. 6. A peptide ligand according to any one of claims 1-5, which is a ligand with high affinity binding to the hemopexin domain of MT1-MMP of humans, mice and dogs. 7. A peptide ligand according to any one of claims 1-6, which is selective for MT1-MMP but does not cross-react with MMP-1, MMP-2, MMP-15 and MMP-16. 8. A drug conjugate comprising a peptide ligand according to any one of claims 1-7 conjugated to one or more effector and/or functional groups, wherein the effector and/or functional group comprises a cytotoxic agent, a chromophore or a metal chelator. 9. A drug conjugate according to claim 8, wherein the effector and/or functional group comprises a cytotoxic agent or a metal chelator. 10. The drug conjugate of claim 9, wherein the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulfide bond, or a protease-sensitive bond. 11. A drug conjugate according to claim 9 or 10, wherein the cytotoxic agent is selected from DM1, which has the following structure: or MMAE, which has the following structure: 12. A drug conjugate according to any one of claims 8 to 11, which is a compound that has the following structure: wherein 17-69-07-N219 is G-Sar10-ACYNEFGCEDFYDIC (SEQ ID NO: 20). 13. A drug conjugate according to any one of claims 8 to 11, which is a compound that has the following structure: wherein 17-69-07-N219 is G-Sar10-ACYNEFGCEDFYDIC (SEQ ID NO: 20). 14. A drug conjugate according to claim 9 or 10, wherein the cytotoxic agent is a maytansinoid and is selected from a compound of formula (II): (II) where n is an integer chosen from 1 to 10; and R ₁ and R ₂ independently of each other represent hydrogen, C _1-6 alkyl, or a carbocyclic or heterocyclic group such as hydrogen or methyl. 15. A drug conjugate according to claim 14, wherein: n is 1 and R ₁ and R ₂ are both hydrogen (i.e. the maytansine derivative DM1); or n is 2 and R ₁ is hydrogen and R ₂ is a methyl group (i.e. the maytansine derivative of DM3); or n is 2, and R ₁ and R ₂ are both methyl groups (i.e., the maytansine derivative of DM4). 16. A drug conjugate according to any one of claims 8 to 15, wherein the bicyclic peptide component of the conjugate has a structure shown in formula (III): (III) where m is an integer chosen from 0 to 10, and R ₃ and R ₄ independently of one another represent hydrogen, C _1-6 alkyl, or a carbocyclic or heterocyclic group such as hydrogen or methyl. 17. A drug conjugate according to any one of claims 9 to 16, wherein the cytotoxic agent is linked to the bicyclic peptide via a linker defined in formula (IV) (IV) where R ₁ , R ₂ , R ₃ and R ₄ represent hydrogen, C _1-6 -alkyl or a carbocyclic or heterocyclic group such as hydrogen or methyl; Toxin refers to any suitable cytotoxic agent; The bicycle is a peptide ligand according to any one of claims 1-6; n is an integer chosen from 1 to 10; and m is an integer chosen from 0 to 10. 18. A drug conjugate according to claim 17, wherein the toxin of compound (IV) is maytansine and the conjugate comprises a compound of formula (V): (V) where R ₁ , R ₂ , R ₃ and R ₄ represent hydrogen, C _1-6 -alkyl or a carbocyclic or heterocyclic group; The bicycle is a peptide ligand according to any one of claims 1-6; n is an integer chosen from 1 to 10; and m is an integer chosen from 0 to 10. 19. A drug conjugate according to claim 18, wherein: n is 1, and R ₁ , R ₂ , R ₃ , and R ₄ are each hydrogen, i.e., a compound of formula (V) ^a : (V) ^a or n is 1, and R ₁ is methyl and R ₂ , R ₃ and R ₄ are each hydrogen, i.e. the compound is of formula (V) ^b : or n is 2, R ₁ and R ₂ are both hydrogen, R ₃ and R ₄ are both methyl, i.e. the compound has formula (V) ^c : (V) ^c or n is 2, R ₁ and R ₂ are both methyl, R ₃ and R ₄ are both hydrogen, i.e. the compound has formula (V) ^d : (V) ^d . 20. A drug conjugate according to claim 8 or 9, which is selected from BT17BDC-9: wherein 17-69-07-N219 is G-Sar10-ACYNEFGCEDFYDIC (SEQ ID NO: 20); BT17BDC-17: wherein the bicycle is βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 5; 17-69-07-N241); BT17BDC-19: wherein the bicycle is βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 5; 17-69-07-N241); and BT17BDC-20: wherein the bicycle is βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 5; 17-69-07-N241). 21. A drug conjugate according to claim 8 or 9, which is BT17BDC-18: wherein the bicycle is βAla-Sar10-A-C(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)C (SEQ ID NO: 5; 17-69-07-N241). 22. A drug conjugate according to claim 8, wherein the effector and/or functional group comprises a metal chelator. 23. A drug conjugate according to claim 22, wherein the metal chelator is suitable for complexing metal radioisotopes that are in demand as a drug. 24. A drug conjugate according to claim 22 or 23, wherein the metal chelator is complexed with a metal radioisotope. 25. A drug conjugate according to claim 23 or 24, wherein the metal radioisotope is selected from 64Cu, 67Ga, 68Ga, 177Lu, 90Y and 213Bi. 26. A drug conjugate according to any one of claims 22-25, wherein the metal chelator is selected from DOTA, NOTA, EDTA, DTPA, HEHA and SarAr. 27. A method for producing a drug conjugate according to claim 20, which includes a synthetic route described in the scheme 28. A pharmaceutical composition for treating cancer characterized by overexpression of MT1-MMP, which comprises a therapeutically effective amount of a drug conjugate according to any one of claims 8-26, in combination with one or more pharmaceutically acceptable excipients. 29. A drug conjugate according to any one of claims 8 to 26 for use in the treatment of cancer characterized by overexpression of MT1-MMP. 30. A method for diagnosing or imaging cancer in a patient, the method comprising administering to said patient a therapeutically effective dose of a drug conjugate according to any one of claims 22-26.