EA044591B1 - PEPTIDE LIGANDS FOR BINDING TO MT1-MMP - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to peptide ligands that exhibit high binding affinity to MT1-MMP. In particular, the invention relates to peptide ligands of this type having novel chemical structures to form two or more bonds between the peptide and the framework molecule.

Prerequisites for creating the invention

Various teams of researchers have previously linked peptides to scaffold moieties by forming two or more thioester bonds between the cysteine residues of the peptide and suitable functional groups of the scaffold molecule. For example, methods for preparing potential drug compounds by coupling cysteine-containing peptides to a molecular scaffold, such as tris(bromomethyl)benzene, are disclosed in WO 2004/077062 and WO 2006/078161.

The advantage of using cysteine thiols to form covalent thioester bonds to achieve cyclization of residues is their selective and biorthogonal reactivity. Thiol-containing linear peptides can be cyclized with a thiol-reactive backbone such as 1,3,5-tris-bromomethylbenzene (TBMB) to form Bicyclic Peptides, and the resulting product contains three thioesters at the benzyl sites. The overall reaction of a linear peptide with TBMB to form a looped bicyclic peptide with thioether linkages is shown in FIG. 1.

There is a need for alternative chemical technologies to link peptides to scaffold moieties to form peptide loop structures using suitable substitutions of the thioester moiety, thereby achieving compatibility with different peptides, changes in physicochemical properties such as improved solubility, changes in biodistribution and other benefits.

WO 2011/018227 describes a method of altering the conformation of a first peptide ligand or group of peptide ligands, each peptide ligand comprising at least two reactive groups separated by a loop sequence covalently linked to a molecular framework that forms covalent bonds with said reactive groups, to produce a second a peptide ligand or group of peptide ligands, comprising combining said second derivative or group of derivatives from the peptide(s) and the backbone of said first derivative or group of derivatives, including one of the following: (a) changing at least one reactive group; or (b) changing the type of molecular framework; or (c) changing the bond between at least one reactive group and the molecular framework; or any combination of (a), (b) or (c).

The present inventors' previously published application WO 2016/067035 and pending application GB 1607827.1, filed May ⁴ , 2016, describe bicyclic peptide ligands having high binding affinity for MT1-MMP. These applications also describe conjugates of peptide ligands with therapeutic agents, in particular cytotoxic agents. The full disclosure of these applications is expressly incorporated herein.

The essence of the invention

The present inventors have discovered that replacement of thioester linkages in loop peptides having affinity for MT1-MMP with alkylamino linkages results in loop peptide conjugates that exhibit similar affinities for MT1-MMP as the corresponding conjugates prepared with all thioester linkages. It is expected that replacement of thioether linkages with alkylamino linkages will result in improved solubility and/or improved oxidative stability of the conjugates of the present invention.

Accordingly, in a first aspect, the present invention provides an MT1-MMP-specific peptide ligand comprising a polypeptide comprising three residues selected from cysteine, L-2,3-diaminopropionic acid (Dap), N-beta-alkyl-L-2 ,3-diaminopropionic acid (N-AlkDap) and N-beta-haloalkyl-L-2,3-diaminopropionic acid (N-HAlkDap), wherein said three residues are separated by at least two loop sequences, and a molecular framework, wherein the peptide linked to the framework by covalent alkylamino bonds with Dap or N-AlkDap or N-HAlkDap residues of the polypeptide and thioester bonds with cysteine residues of the polypeptide when these three residues include cysteine, thus forming two polypeptide loops on the molecular framework, where the peptide ligand includes the amino acid sequence formulas (II):

-Ai-Xi-U/O ₂ -X ₃ -X4-G ₅ -A ₂ -E6-D ₇ -F ₈ -Y ₉ -Xio-Xn-A ₃ - (SEQ ID NO: 1) (II) or a pharmaceutically acceptable salt thereof;

Where:

A1, A2, and A ₃ are independently cysteine, L-2,3-diaminopropionic acid (Dap), N-beta-alkyl-L-2,3-diaminopropionic acid (N-AlkDap) or N-beta-haloalkyl- L-2,3diaminopropionic acid (N-HAlkDap), provided that at least one of A1, A2, and A ₃ is Dap, N-AlkDap or N-HAlkDap;

X is any amino acid residue;

- 1 044591

U is a polar, uncharged amino acid residue selected from N, C, Q,

M,S and T;i

O is a non-polar aliphatic amino acid residue selected from G, A, I,

L, P and V.

As can be seen, the derivatives of the present invention include a peptide loop linked to the backbone by at least one alkylamino linkage to Dap or N-AlkDap N-HAlkDap residues and up to two thioether linkages to cysteine. Accordingly, A _b A2, and A ₃ consist of one cysteine and two residues selected from Dap, N-AlkDap or N-HAlkDap. The alkyl prefix in N-AlkDap and N-HAlkDap refers to an alkyl group containing from one to four carbon atoms, preferably methyl. The prefix halogen is used in this context in its usual sense to designate alkyl groups having one or more, preferably one, fluorine, chlorine, bromo or iodo substituents.

When cysteine is present, the thioester bond(s) provide an anchor during the formation of cyclic peptides, as explained below. In these embodiments, the thioether linkage is preferably the central linkage of the bicyclic peptide conjugate, that is, in the peptide sequence, the two alkylamino linkage-forming residues in the peptide are spaced on either side of the cysteine residue forming the thioether linkage. The loop structure of the peptide is therefore a bicyclic peptide conjugate having a central thioether linkage and two peripheral alkylamino linkages. In alternative embodiments, the thioether linkage is at the N-terminus or C-terminus of the peptides, with the central linkage and the other end linkage selected from Dap, N-AlkDap or N-HAlkDap.

In embodiments of the present invention, all three of A ₁ , A 2 , and A ₃ may preferably be Dap or N-AlkDap or N-HAlkDap. In these embodiments, the peptide ligands of the present invention are preferably bicyclic conjugates having a central alkylamino bond and two peripheral alkylamino bonds, wherein the peptide forms two loops that share a central alkylamino bond. In these embodiments, Ai, A2, and A ₃ are all preferably selected from N-AlkDap or N-HAlkDap, most preferably NAlkDap, due to favorable reaction kinetics with alkylated Daps.

Suitably, X ₁ is selected from any one of the following amino acids: Y, M, F or V, such as Y, M or F, in particular Y or M, more particularly Y.

Suitably, U/O2 is selected from U such as N or O such as G.

Suitably, 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, in particular, U at position 3 is selected from Q, or Z at position 3 is selected from E.

Suitably, X ₄ is selected from J, where J is a non-polar aromatic amino acid residue selected from F, W and Y.

Suitably, X ₁₀ is selected from Z, where Z is a polar, negatively charged amino acid residue selected from D or E, such as D.

Suitably, X _p is selected from O, where O is a non-polar aliphatic amino acid residue selected from G, A, I, L, P and V, such as I.

Suitably, the bicyclic compound of formula (II) is a compound of formula (IIa):

- A ₁ -Y/M/F/VU/OU/ZJGA ₂ -EDFYZOA ₃ - (SEQ ID NO: 6 (IIa) where U, O, J and Z are as defined above; or a compound of formula (IIb) :

- A ₁ -Y/M/F/VN/GE/QFGA ₂ -EDFYDIA ₃ - (SEQ ID NO: 7) (IIb); or a compound of formula (IIc):

- A ₁ -Y/M/FN/GE/QFG-A2-EDFYDIA ₃ - (SEQ ID NO: 8) (IIc); or a compound of formula (IId):

- A ₁ -Y/MNE/QFGA ₂ -EDFYDIA ₃ - (SEQ ID NO: 9) (IId); or a compound of formula (IIe):

- A ₁ -YNEFGA ₂ -EDFYDIA ₃ - (17-69-07) (SEQ ID NO: 2) (IIe).

Suitably, the bicyclic compound of formula (II) includes a sequence selected from:

- A ₁ -YNEFGA ₂ -EDFYDIA ₃ (17-69-07) (SEQ ID NO: 2);

- A ₁ -MNQFG- A ₂ -EDFYDIA ₃ (17-69-12) (SEQ ID NO: 10);

- A ₁ -FGEFG- A2-EDFYDIA ₃ (17-69-02) (SEQ ID NO: 11);

- A ₁ -VNEFG- A ₂ -EDFYDIA ₃ (17-69-03) (SEQ ID NO: 12);

- A ₁ -FNEFG- A2-EDFYDI-A3 (17-69-04) (SEQ ID NO: 13);

- A ₁ -YNEYG- A2-EDFYDI-A3 (SEQ ID NO: 14); And

- A ₁ -YNEWG- A2-EDFYDI-A3 (SEQ ID NO: 15), such as:

- A ₁ -YNEFG- A2-EDFYDI- A ₃ (17-69-07) (SEQ ID NO: 2); And

- A ₁ -MNQFG- A2-EDFYDI- A ₃ (17-69-12) (SEQ ID NO: 10),

- 2 044591 in particular:

- A ₁ -YNEFG- A2-EDFYDI- A ₃ (17-69-07) (SEQ ID NO: 2), more specifically:

Dap homologs 17-69-07-N241 designated as SEQ ID 16: ((bAla)-Sar10-AA1 (D-Ala)NE(1Nal) (DAla) A2EDFYD (tBuGly)A ₃ ;

and Dap homologs 17-69-07-N268, designated SEQ ID 17: AA1 (D-Ala) NE (1Nal) (D-Ala) A2EDFYD (tBuGly) A ₃ .

In all of the above sequences, A1, A2, and _A3 are as defined above. Suitable and preferred types and positions of A1, _A2 , and _A3 are as defined above.

In embodiments, the peptide ligand of the present invention further includes one or more modifications selected from: N-terminal and/or C-terminal modifications; replacing one or more amino acid residues with one or more unnatural amino acid residues (eg, replacing one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacing one or more hydrophobic amino acid residues with other unnatural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacing one or more amino acid residues with alanine; replacing one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; modification of the length of the peptide backbone; replacing hydrogen on the α-carbon of one or more amino acid residues with another chemical group, and postsynthetic bioorthogonal modification of amino acids such as cysteine, lysine, glutamate and tyrosine with suitable reagents reactive with an amine, thiol, carboxylic acid and phenol.

Suitably, these embodiments may include N-terminal modification using suitable amino-reactive chemistry and/or C-terminal modification using suitable carboxy-reactive chemistry. For example, an N-terminal modification may involve the addition of a molecular spacer group that facilitates conjugation of effector groups and retention of activity of the bicyclic peptide to its target. The spacer group is preferably an oligopeptide group containing from about 5 to about 30 amino acids, such as an Ala, G-Sar10-A group or bAla-Sar10-A group. Alternatively or additionally, the N-terminal and/or C-terminal modification includes the addition of a cytotoxic agent.

Other possible peptide modifications include modification at amino acid positions 1 and/or 9.

In embodiments, the peptide modification includes replacing one or more amino acid residues with one or more unnatural amino acid residues. For example, where the substitution with an unnatural amino acid residue occurs at position 4, and the residue is selected from: 1 naphthylalanine; 2-naphthylalanine; 3,4-dichlorophenylalanine; and homophenylalanine such as 1-naphthylalanine; 2-naphthylalanine; and 3,4-dichlorophenylalanine, in particular 1-naphthylalanine. Alternatively or additionally, the substitution with an unnatural amino acid residue occurs at position 9 and/or 11, and the residue is selected from: 4-bromophenylalanine or pentafluorophenylalanine for position 9 and/or tert-butylglycine for position 11. In these embodiments, the unnatural amino acid residues, such as those present at position 9, may be selected from: 4-bromophenylalanine, and/or unnatural amino acid residues, such as those present at position 11, selected from: tert-butylglycine.

In embodiments, the amino acid residue at position 1 is replaced with a D-amino acid, such as D-alanine. In other embodiments, the amino acid residue at position 5 is replaced with a D-amino acid, such as D-alanine or D-arginine.

Suitably, the peptide ligand may include a plurality of the above modifications, for example, 2, 3, 4 or 5 or more of the following modifications, for example all of the following 5 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.

In all of the peptide sequences defined in the present invention, one or more tyrosine residues may be replaced by phenylalanine. This has been found to improve the yield of the bicyclic peptide product during the base-catalyzed coupling reaction of the peptide with the scaffold molecule.

Suitably, the peptide ligand of the present invention is a high affinity binder of the hemopexin domain of human, mouse and canine MT1-MMP. Preferably, the binding affinity ki is less than about 100 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM.

Accordingly, the peptide ligand of the present invention is selective for MT1-MMP, but does not cross-react with MMP-1, MMP-2, MMP-15 and MMP-16. Accordingly, the binding affinity of ki for each of these ligands is greater than about 500 nM, greater than about 1000 nM, or greater than about 10,000 nM.

Suitably, the framework includes a (hetero)aromatic or (hetero)alicyclic moiety. Suitably, the framework includes a tris-substituted (hetero)aromatic or (hetero)alicyclic moiety, for example a tris-methylene-substituted (hetero)aromatic or (hetero)alicyclic moiety. The (hetero)aromatic or (hetero)alicyclic moiety is preferably a six-membered ring structure, preferably tris-substituted, such that the framework has a 3-fold symmetry axis. Thus, in some preferred embodiments, the framework is 1,3,5-tris-methylbenzene. In other preferred embodiments, the scaffold is a 1,3,5-tris-(acetamido)benzene group, which can be formed by coupling the peptide with 1,3,5-tris-(bromoacetamido)benzene (TBAB) as described above details below.

In a second aspect, the present invention provides a peptide comprising the amino acid sequence of formula (II) as defined above with respect to the first aspect of the present invention. Accordingly, the peptide is suitable for preparing the peptide ligand of the present invention by coupling to a suitable scaffold molecule, as described below. Suitably, the peptide is a linear peptide.

In a further aspect, the present invention provides a method for producing a peptide ligand in accordance with the first aspect of the present invention, the method comprising: providing a peptide in accordance with the second aspect of the present invention; providing a framework molecule having at least three reactive sites for the formation of alkylamino bonds with the amino groups of the side chains of said cysteine residues and diaminopropionic acid or β-Nalkyldiaminopropionic acid; and forming said alkylamino bonds between the peptide and the framework molecule.

The reactive sites are also suitable for the formation of thioester bonds with -SH groups of cysteine in embodiments where the third residue is a cysteine. The -SH group of cysteine is highly nucleophilic and in these embodiments is expected to first react with the electrophilic centers of the scaffold molecule to anchor the peptide to the scaffold molecule, after which the amino groups react with the remaining electrophilic centers of the scaffold molecule to form a loop peptide ligand.

In embodiments, the peptide contains protecting groups on nucleophilic groups other than amino groups and -SH groups (when present) to form alkylamino bonds.

Suitably, the method of the present invention involves reacting, in a nucleophilic substitution reaction, a peptide as defined herein with a framework molecule having three or more leaving groups.

In alternative methods, the compounds of the present invention can be prepared by converting two or more side chain groups of a peptide into leaving groups and then reacting the peptide, in a nucleophilic substitution reaction, with a backbone molecule having two or more amino groups.

Nucleophilic substitution reactions can be carried out in the presence of a base when the leaving group is a conventional anionic leaving group. The present inventors have discovered that the yields of cyclized peptide ligands can be significantly increased by suitable choice of solvent and base for the nucleophilic substitution reaction, and further that the preferred solvent and base differs from known solvent and base combinations that only result in the formation of thioester bonds. In particular, the present inventors have discovered that improved yields are achieved when using a trialkylamine base, that is, a base of the formula NR1R2R3, wherein R1, R2 and _R3 are independently C1-C5 alkyl groups, preferably C2-C4 alkyl groups, in particular C2- C3 alkyl groups. Particularly suitable bases are triethylamine and diisopropylethylamine (DIPEA). These bases have the property of being only weakly nucleophilic, and this property is thought to account for the fewer side reactions and higher yields observed with these bases. The present inventors have also discovered that the preferred solvents for the nucleophilic substitution reaction are polar and protic solvents, in particular MeCN/H ₂ O (50:50).

In a further aspect, the present invention provides a drug conjugate comprising a peptide ligand of the present invention conjugated to one or more effector and/or functional groups, such as a cytotoxic agent or a metal chelator.

Suitably, the conjugate contains a cytotoxic agent linked to the peptide ligand by a cleavable bond, such as a disulfide bond. Suitably, the cytotoxic agent is selected from DM1 or MMAE.

In embodiments, the drug conjugate has the following structure:

- 4 044591

where R1, R2, R3 and R4 represent hydrogen or C1-C6 alkyl groups;

Toxin refers to any suitable cytotoxic agent;

The bicyclic compound is a loop peptide structure;

n is an integer selected from 1-10; and m is an integer selected from 0-10.

Accordingly, either: R1, R2, R3 and R4 are all H; or R1, R2, R3 are all H and I4=methyl; or R1, R ₂ =methyl and R3, R ₄ =H; or R1, R ₃ =methyl and R2, R4=H; or R1, R2=H and R3, R ₄ =C1-C6 alkyl.

The linker between the toxin and the bicyclic peptide may include a triazole group formed by a click reaction between the azide-functionalized toxin and the alkyne-functionalized bicyclic peptide structure (or vice versa). In other embodiments, the bicyclic peptide may contain an amide bond formed by the interaction between the carboxylate-functionalized toxin and the N-terminal amino group of the bicyclic peptide.

The linker between the toxin and the bicyclic peptide may include a cathepsin cleavable group to provide selective release of the toxin in target cells. A suitable cathepsin cleavable group is valine-citrulline.

The linker between the toxin and the bicyclic peptide may include one or more spacer groups to provide the desired functionality, for example, binding affinity or cathepsin cleavability, of the conjugate. A suitable spacer group is para-aminobenzylcarbamate (PABC), which may be located between the valine-citrulline group and the toxin moiety.

Thus, in embodiments, the bicyclic peptide-drug conjugate may have the following structure consisting of Toxin-PABC-cit-val-triazole-Bicyl:

In other embodiments, the bicyclic peptide-drug conjugate may have the following structure consisting of Toxin-PABC-cit-val-dicarboxylate-Bicyl:

O

Where (alk) is an alkylene group of the formula C _n H _2n where n is from 1 to 10 and may be straight or branched, preferably (alk) is n-propylene or n-butylene.

In another aspect, the present invention also provides a kit comprising at least a peptide ligand or conjugate of the present invention.

In yet another aspect, the present invention provides a composition comprising a peptide

- 5 044591 ligand or conjugate of the present invention and a pharmaceutically acceptable carrier, diluent or excipient.

In addition, the present invention provides a method of treating a disease using a peptide ligand, conjugate or composition of the present invention. Suitably, the disease is a neoplastic disease such as cancer.

In a further aspect, the present invention provides a diagnostic method comprising diagnosing a disease using a peptide ligand or composition of the present invention. Thus, in general, the binding of an analyte to a peptide ligand can be used to displace the agent, resulting in the generation of a signal upon displacement. For example, binding of an analyte (second target) can displace an enzyme (first target) bound to a peptide ligand, providing the basis for binding assays, especially if the enzyme is retained by the peptide ligand through its active site.

Brief description of drawings

Fig. 1 shows a reaction scheme for the preparation of thioether-linked bicyclic peptide ligands in accordance with the prior art.

Fig. 2 shows a thioether-linked bicyclic peptide ligand in accordance with the prior art, designated 17-69-07-N241.

Fig. 3 shows the first secondary amino-linked bicyclic peptide ligand of the present invention.

Fig. 4 shows a tertiary N-methyl amino-linked bicyclic peptide ligand of the present invention.

Fig. 5 shows a third secondary amino-linked bicyclic peptide ligand of the present invention, which is a Dap analogue of 17-69-07-N241.

Fig. 6 shows a fourth secondary amino-linked bicyclic peptide ligand of the present invention cyclized with a TBAB scaffold.

Fig. 7 shows competitive affinity binding assay data for MT1MMP for the derivative of FIG. 3.

Fig. 8 shows competitive affinity binding assay data for MT1MMP for the derivative of FIG. 4.

Fig. 9 shows MT1MMP competitive affinity binding assay data for another derivative of the present invention.

Fig. 10 shows schematic structures of certain bicyclic peptide-TBMB derivatives in accordance with the present invention.

Fig. 11 shows schematic structures of other bicyclic peptide-TBMB derivatives in accordance with the present invention.

Fig. 12 shows schematic structures of other bicyclic peptide-TBMB derivatives in accordance with the present invention.

Fig. 13 shows schematic structures of other bicyclic peptide-TBMB derivatives in accordance with the present invention.

Fig. 14 shows a reaction scheme for preparing the bicyclic peptide-drug conjugate of the present invention by click reaction to form a triazole bond.

Fig. 15 shows a reaction scheme for preparing a bicyclic peptide-drug conjugate of the present invention in the presence of an amido linkage.

Fig. 16 shows tumor volume and body weight over time for Balb/c nude mice bearing tumors with HT1020 tumor cells after treatment with the bicyclic peptide-drug conjugate of the present invention.

Fig. 17 shows tumor volume and body weight over time for Balb/c nude mice bearing tumors with HT1020 tumor cells after treatment with another bicyclic peptide-drug conjugate of the present invention.

Fig. 18 shows tumor volume and body weight over time for Balb/c nude mice bearing tumors with HT1020 tumor cells after treatment with another bicyclic peptide-drug conjugate of the present invention.

Fig. 19 shows tumor volume and body weight over time for Balb/c nude mice bearing tumors with HT1020 tumor cells after treatment with another bicyclic peptide-drug conjugate of the present invention;

Fig. 20 shows tumor volume and body weight over time for Balb/c nude mice bearing tumors with HT1020 tumor cells after treatment with another bicyclic peptide-drug conjugate of the present invention.

Detailed Description of the Invention

Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the art, e.g., peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd 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.

The present invention provides a loop peptide structure as defined in claim 1, comprising two peptide loops sandwiched between three bonds on a molecular scaffold, with a central bond being common to the two loops. The central bond is preferably a thioether bond formed with a cysteine residue of the peptide, or it is an alkylamino bond formed with a Dap or N-AlkDap residue of the peptide. The two outer bonds are preferably alkylamino bonds formed with Dap or N-AlkDap residues of the peptide, or one of the outer bonds can be a thioether bond formed with a cysteine residue of the peptide.

One skilled in the art will appreciate that X at positions 1, 3, 4, 10 and 11 of formula (II) can be any amino acid according to alanine scanning and selection results that allow well-tolerated substitutions at these positions.

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

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

In one embodiment, X at position 3 of formula (II) 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 an amino acid residue selected from D or E. In a further embodiment, the U at position 3 of formula (II) is selected from Q. In an alternative embodiment, the Z at position 3 of formula (II) is selected from E.

In one embodiment, X at position 4 of formula (II) is selected from J, where J is a non-polar aromatic amino acid residue selected from F, W and Y. In a further embodiment, J at position 4 of formula (II) is selected from F. B in an alternative embodiment, the J in position 4 of formula (II) is selected from Y. In an alternative embodiment, the J in position 4 of formula (II) is selected from W.

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

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

In one embodiment, the compound of formula (II) is a compound of formula (IIa):

- A1-Y/M/F/VU/OU/ZJG- A2-EDFYZO- A ₃ - (SEQ ID NO: 6) (IIa);

where U, O, J and Z are as defined above.

In one embodiment, the compound of formula (II) is a compound of formula (IIb):

- A1-Y/M/F/VN/GE/QFGA ₂ -EDFYDIA ₃ - (SEQ ID NO: 7) (IIb).

In one embodiment, the compound of formula (II) is a compound of formula (IIc):

- A1-Y/M/F-N/G-E/Q-F-G-A2-E-D-F-Y-D-I-A3- (SEQ ID NO: 8) (IIc).

In one embodiment, the compound of formula (II) is a compound of formula (IId):

- A1-Y/M-N-E/Q-F-G-A2-E-D-F-Y-D-I-A3- (SEQ ID NO: 9) (IId).

In one embodiment, the compound of formula (II) is a compound of formula (IIe):

- A1-Y-N-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-07) (SEQ ID NO: 2) (IIe).

In yet another embodiment, the peptide of formula (II) includes a sequence selected from:

- A1-Y-N-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-07) (SEQ ID NO: 2);

- A1-M-N-Q-F-G-A2-E-D-F-Y-D-I-A3- (17-69-12) (SEQ ID NO: 10);

- A1-F-G-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-02) (SEQ ID NO: 11);

- A1-V-N-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-03) (SEQ ID NO: 12);

- A1-F-N-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-04) (SEQ ID NO: 13);

- A1-Y-N-E-Y-G-A2-E-D-F-Y-D-I-A3- (17-69-07-N057) (SEQ ID NO: 14); And

- 7 044591

- A ₁ -YNEWG-A2-EDFYDIA ₃ - (17-69-44-N002) (SEQ ID NO: 15).

The peptides of this embodiment were identified as strong candidates following affinity maturation for the hemopexin domain of MT1-MMP.

In yet another embodiment, the peptide of formula (II) includes a sequence selected from:

- A1-Y-N-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-07) (SEQ ID NO: 2); And

- A1-M-N-Q-F-G-A2-E-D-F-Y-D-I-A3- (17-69-12) (SEQ ID NO: 10).

The peptides of this embodiment were identified as candidates with the highest affinity after affinity maturation against the hemopexin domain of MT1-MMP, synthesis of the bicyclic core sequences, and quantitative affinity measurements using competition experiments.

In a further embodiment, the peptide of formula (II) includes a sequence selected from - A1-Y-N-E-F-G-A2-E-D-F-Y-D-I-A3- (17-69-07) (SEQ ID NO: 2). The peptide of this embodiment has been identified as the most potent and stable member of the family of peptide ligands of formula (II).

In yet another embodiment, the peptide of formula (II) includes a sequence selected from:

(bAla)-Sar10-AA1 (D-Ala)NE(1Nal) (D-Ala) A ₂ EDFYD(tBuGly)A ₃ ,); or

AA1 (D-Ala)NE(1Nal)(D-Ala)A ₂ EDFYD (tBuGly) A ₃ ), respectively, where the N-terminus is preferably present as a free amino group and the C-terminus is preferably amidated.

In all of the above sequences, A1, A2, and _A3 are as defined above. Suitable and preferred types and positions of A1, A2, and A ₃ are as defined above.

In one embodiment, certain peptides of formula (II) are fully cross-reactive with mouse, dog, cynomolgus and human MT1-MMP. In a further embodiment, specific exemplary peptide ligands of the present invention are fully cross-reactive with mouse, dog, cynomolgus and human MT1-MMP. For example, both the unstabilized and stabilized derivatives of 17-69-07 (i.e., 17-69-07-N219, 17-69-07-N241, and 17-69-07-N268) are fully cross-reactive.

In yet another embodiment, the peptide of formula (II) is selective for MT1-MMP, but does not cross-react with MMP-1, MMP-2, MMP-15 and MMP-16. The core sequence 17-69-07 and the stabilized variant 17-69-07-N258 are uniquely selective for MT1-MMP. Preferably, the binding affinity of ki for MT1-MMP is less than about 100 nM, less than about 50 nM, less than about 25 nM, or less than about 10 nM. Accordingly, the binding affinity of ki for MMP-1, MMP-2, MMP-15 and MMP-16 is greater than about 500 nM, greater than about 1000 nM, or greater than about 10,000 nM.

It should be understood that modified derivatives of the peptide ligands defined in the present invention are included within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from: N-terminal and/or C-terminal modifications; replacing one or more amino acid residues with one or more unnatural amino acid residues (such as replacing one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacing one or more non-polar amino acid residues with other unnatural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacing one or more amino acid residues with alanine; replacing one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; modification of the length of the peptide backbone; substituting a hydrogen on the alpha carbon of one or more amino acid residues with another chemical group, modifying amino acids such as cysteine, lysine, glutamate/aspartate and tyrosine with suitable 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 an azide or alkyne group, which allow functionalization with alkyne- or azide-containing moieties, respectively.

In one embodiment, the modified derivative includes modification at amino acid positions 1 and/or 9. These positions, especially where tyrosine is present, are most susceptible to proteolytic degradation.

In one embodiment, the modified derivative includes an N-terminal and/or C-terminal modification. In a further embodiment, the modified derivative includes an N-terminal modification using suitable amino-reactive chemistry and/or a C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, said N-terminal or C-terminal modification includes the addition of an effector group including, but not limited to, a cytotoxic agent, a radiochelator, or a chromophore.

In a further embodiment, the modified derivative includes an N-terminal modification. In a further embodiment, the N-terminal modification includes an N-terminal acetyl group. In this embodiment, the N-terminal cysteine group (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 avoids potential degradation of the bicyclic peptide.

In an alternative embodiment, the N-terminal modification includes the addition of a molecular spacer group that facilitates conjugation of effector groups and maintains the activity of the bicyclic peptide towards its target. The spacer group is preferably an oligopeptide group containing from about 5 to about 30 amino acids, such as an Ala, G-Sar10-A or bAla-Sar10-A group. In one embodiment, the spacer group is selected from bAlaSar10-A (ie 17-69-07-N241). The addition of these spacer groups to the bicyclic peptide 17-69-07 does not change the activity against the target protein.

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

In one embodiment, the modified derivative includes 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 degradation proteases and do not have any adverse effect on the target activity.

Alternatively, unnatural amino acids may be used that have limited amino acid side chains such that proteolytic hydrolysis of an adjacent peptide bond is conformationally and sterically hindered. This particularly applies to proline analogues, bulky side chains, C-disubstituted derivatives (eg aminoisobutyric acid, Aib) and cycloamino acids, a simple aminocyclopropylcarboxylic acid derivative.

In one embodiment, the substitution with a non-natural amino acid residue occurs at position 4. Various non-natural amino acid residues are well tolerated at this position. In a further embodiment, the 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 yet another embodiment, unnatural amino acid residues, such as those present at position 4, are selected from: 1-naphthylalanine; 2-naphthylalanine and 3,4-dichlorophenylalanine. These substitutions increase affinity compared to the unmodified wild-type sequence.

In yet another embodiment, the unnatural amino acid residues, such as those present at position 4, are selected from: 1-naphthylalanine. This substitution provided the highest level of affinity enhancement (more than 7-fold) compared to the wild type.

In one embodiment, the unnatural amino acid residue is introduced at position 9 and/or 11. A number of unnatural amino acid residues are well tolerated at these positions.

In a further embodiment, the unnatural amino acid residues, such as those present at position 9, are selected from: 4-bromophenylalanine, pentafluorophenylalanine, for example 4-bromophenylalanine.

In yet another embodiment, unnatural amino acid residues, such as those present at position 11, are selected from: tert-butylglycine. Increased activity and strong protection of the vicinal amino acid backbone from proteolytic hydrolysis is achieved through steric hindrance.

In one embodiment, the modified derivative includes a plurality of the above modifications, such as 2, 3, 4 or 5 or more modifications. In a further embodiment, the modified derivative includes 2, 3, 4 or 5 or more of the following modifications, for example, 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. This multi-substitution is tolerated in combination with potency that is superior to the wild type. In yet another embodiment, the modified derivative includes the following modifications: D-alanine at positions 1 and 5, 1-naphthylalanine at position 4, and tert-butylglycine at position 11. This multi-substitution is tolerated in combination with potency that is superior to the wild type.

- 9 044591

In one embodiment, the modified derivative includes the addition of a spacer group.

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

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

In all peptide sequences defined in the present invention, one or more tyrosine residues may be replaced by phenylalanine. This has been found to improve the yield of the bicyclic peptide product in the base-catalyzed coupling reaction of the peptide with the scaffold molecule.

In a further embodiment, the amino acid residue at position 1 is replaced with a D-amino acid such as D-alanine. This replacement achieves preservation of effectiveness without subsequent decomposition.

In a further embodiment, the amino acid residue at position 5 is replaced with a D-amino acid such as D-alanine or D-arginine. This replacement achieves preservation of effectiveness without subsequent decomposition.

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

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

inclusion of hydrophobic moieties, which exploit the hydrophobic effect and lead to lower dissociation rates, thus achieving higher affinities;

inclusion of charged groups that utilize a wide range of ionic interactions leading to faster association rates and higher affinities (see, for example, Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427- 31); and incorporating additional hindrance into the peptide, for example by properly hindering amino acid side chains so that entropy loss is minimal upon target binding, limiting backbone torsion angles so that entropy loss is minimal upon target binding, and introducing additional cyclizations into the molecule for the same reasons. (See Gentilucci et al, Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al, Curr. Medicinal Chem (2009), 16, 4399-418.)

The present invention includes all pharmaceutically acceptable (radio)isotopically labeled compounds of the present invention, that is, compounds of formula (II) 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 commonly found in nature, and compounds of formula (II) in which metal chelate groups (called effectors) are attached which are capable of retaining the corresponding (radio)isotopes, and compounds of formula (1) wherein certain functional groups are covalently substituted corresponding (radio)isotopes or isotopically labeled functional groups.

Examples of isotopes suitable for inclusion in the compounds of the present invention include isotopes of hydrogen such as ^2H (D) and ^3H (T), carbon such as ^PS , ^13C and ^14C , chlorine such 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 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 (II), for example those that include a radioactive isotope, are useful in tissue distribution studies of drug and/or substrate, as well as for clinical assessment of the presence and/or absence of the MT1-MMP target in affected tissues. such as tumors, and other places. The compounds of formula (II) may also have valuable diagnostic properties because they can be used to detect or identify the formation of a complex between the labeled compound and other molecules, peptides, proteins, enzymes or receptors. Detection or identification methods may use compounds that are labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (eg, luminol, luminol derivatives, luciferin, aequorin and luciferase), etc. The radioactive isotopes tritium, i.e. ^3H (T), and carbon-14, i.e. ^14C , are particularly useful for this purpose given their ease of inclusion and detection.

Substitution with heavier isotopes such as deuterium, i.e. ^2H (D), may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some cases. circumstances.

Substitution with positron-emitting isotopes such as ^PS , ¹⁸ F, ¹⁵ O and ¹³ N can be useful in positron emission tomography (PET) studies to study target occupancy.

Incorporation of isotopes into metal-chelate effector groups such as ⁶⁴ Cu, ⁶⁷ Ga, ⁶⁸ Ga, and ¹⁷⁷ Lu may be useful for imaging tumor-specific antigens using PET or SPECT imaging.

Incorporation of isotopes into metal-chelate effector groups, such as, but not limited to, ⁹⁰ Y, ¹⁷⁷ Lu, and ²¹³ Bi, may provide the opportunity for targeted radiotherapy in which metal chelator-containing compounds of formula (II) transfer a therapeutic radionuclide to a protein target and location.

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

Specificity as used herein refers to the ability of a ligand to bind or otherwise interact with its cognate target to the exclusion of entities 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 other species. Using the approach described in the present invention, specificity can be modulated, which increases or decreases to make ligands more or less capable of interacting with homologs or paralogs of the intended target. Specificity should not be considered synonymous with activity, affinity or avidity, and the potency of a ligand on its target (such as, for example, binding affinity or level of inhibition) is not necessarily related to its specificity.

Binding activity, in the context of the present application, refers to quantitative indicators of binding taken from binding assays, for example, as described in the present invention. Therefore, binding activity refers to the amount of peptide ligand that binds at a given target concentration.

Multispecificity is the ability to bind to two or more targets. Typically, binding peptides are able to bind 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; antibodies with dual specificity, for example, as is known in the field, as stated above. In the present invention, peptide ligands may be capable of binding to two or more targets and are therefore multispecific. Accordingly, they bind to two targets and have dual specificity. The binding may be independent, which would mean that the binding sites for the targets on the peptide are not structurally hindered by the binding of one or the other of the targets. In this case, both targets can be coupled independently. More generally, binding to one target is expected to at least partially interfere with binding to another.

There is a fundamental difference between a ligand with dual specificity and a ligand with a specificity that spans two related targets. In the first case, the ligand is specific for both targets individually and interacts with each in a specific manner. For example, the first loop in a ligand may bind to a first target, and the second loop to a second target. In the second case, the ligand is nonspecific because it does not distinguish between the two targets,

- 11 044591 for example, interacting with an epitope of targets that is common to both.

In the context of the present invention, it is possible that the ligand which has activity towards, for example, a target and an orthologue, may be a bispecific ligand. However, in one embodiment, the ligand is not bispecific, but has a less precise specificity, for example, it binds both the target and one or more orthologues. In general, a ligand that is not selected against both the target and its orthologue is less likely to be bispecific due to the lack of selective pressure for bispecificity. The length of the loop in a bicyclic peptide may be critical to providing a suitable binding surface so that good cross-reactivity of target and ortholog can be achieved while maintaining high selectivity for less closely related homologues.

If the ligands are truly bispecific, in one embodiment, at least one of the target ligand specificities will be common among the selected ligands, and the level of this specificity can be modulated by the methods disclosed in the present invention. The second or additional specificities need not be general, and should not be considered in the procedures described in the present invention.

A molecular scaffold is any molecule that is capable of binding a peptide at multiple points to impart one or more structural features to the peptide. Preferably, the molecular scaffold includes at least three peptide binding sites, called scaffold reactive groups. These groups are capable of reacting with Dap or N-AlkDap or cysteine residues (when present) on the peptide to form stable covalent alkylamino and thioether bonds. Preferred structures for molecular scaffolds are described below.

Thus, the compounds of the invention include, consist essentially of, or consist of a peptide covalently linked to a molecular framework. The term scaffold or molecular framework in the present invention refers to the chemical moiety that is linked to the peptide by alkylamino bonds and thioether bonds (when the third residue is a cysteine) in the compounds of the present invention. The term scaffold molecule or molecular scaffold molecule as used herein refers to a molecule that is capable of reacting with a peptide or peptide ligand to form derivatives of the present invention having alkylamino and, in some embodiments, also thioether linkages. Thus, the framework molecule has the same structure as the framework fragment, except that the corresponding reactive groups (such as leaving groups) of the molecule are replaced by alkylamino and thioether bonds with the peptide in the framework fragment.

A molecular scaffold molecule is any molecule that is capable of binding to a peptide at multiple sites to form thioester and alkylamino bonds to the peptide. It is not a cross-linker because it does not typically link two peptides; instead, it provides two or more attachment points for a single peptide. A molecular scaffold molecule includes at least three attachment points for the peptide, called scaffold reactive groups. These groups are capable of reacting with -SH and amino groups on the peptide to form thioether and alkylamino bonds. Thus, the molecular framework is the framework fragment up to, but not including, the thioether and alkylamino linkages in the conjugates of the present invention. The framework molecule has the structure of a framework, but with reactive groups at the locations of the thioether and alkylamino bonds in the conjugate of the present invention.

Suitably, the framework includes, consists essentially of, or consists of a (hetero)aromatic or (hetero)alicyclic moiety.

As used herein, (hetero)aryl includes aromatic rings, for example aromatic rings having from 4 to 12 members, such as phenyl rings. These aromatic rings may optionally contain one or more heteroatoms (eg, one or more of N, O, S, and P), such as thienyl rings, pyridyl rings, and furanyl rings. The aromatic rings may optionally be substituted. (Hetero)aryl also includes aromatic rings to which one or more other aryl rings or non-aryl rings are fused. For example, naphthyl groups, indole groups, thienothienyl groups, dithienothienyl and 5,6,7,8-tetrahydro-2naphthyl groups (each of which may be optionally substituted) are aryl groups for purposes of this application. As stated above, the aryl rings may be optionally substituted. Suitable substituents include alkyl groups (which may be optionally substituted), other aryl groups (which may themselves be substituted), heterocyclic rings (saturated or unsaturated), alkoxy groups (which include aryloxy groups (e.g. phenoxy groups)), hydroxy groups, aldehyde groups, nitro groups, amine groups (e.g. unsubstituted or mono- or disubstituted with aryl or alkyl groups), carboxylic acid groups, carboxylic acid derivatives (e.g. esters, carboxylic acid amides, etc.), halogen atoms (e.g. Cl, Br and I ) and so on.

As used herein, (hetero)alicyclic refers to a homocyclic or heterocyclic saturated ring. The ring may be unsubstituted or it may be substituted with one or more substituents. Substituents may be saturated or unsaturated, aromatic or non-aromatic, and examples of suitable substituents include those mentioned above in the discussion regarding substituents on alkyl and aryl groups. In addition, two or more ring substituents can combine to form another ring, thus the term ring as used herein includes fused ring systems.

Preferably, the framework includes a tris-substituted (hetero)aromatic or (hetero)alicyclic moiety, for example a tris-methylene-substituted (hetero)aromatic or (hetero)alicyclic moiety. The (hetero)aromatic or (hetero)alicyclic moiety is preferably a six-membered ring structure, preferably tris-substituted, such that the framework has a 3-fold symmetry axis.

In embodiments, the framework is a tris-methylene (hetero)aryl moiety, such as a 1,3,5-tris-methylenebenzene moiety. In these embodiments, the corresponding framework molecule preferably has a leaving group on the methylene carbon atoms. The methylene group then forms an R ₁ alkylamino linkage moiety as defined herein. In these methylene-substituted (hetero)aromatic compounds, electrons from the aromatic ring can stabilize the transition state during nucleophilic substitution. For example, benzyl halides are 100-1000 times more reactive to nucleophilic substitution than alkyl halides that are not associated with a (hetero)aromatic group.

In these embodiments, the framework and framework molecule have the general formula:

,LG

LG

LG

Wherein LG represents a leaving group as described below for the framework molecule, or LG (including the adjacent methylene group forming the R ₁ moiety of the alkylamino group) represents the alkylamino bond to the peptide in the conjugates of the present invention.

In embodiments, the above LG group may be a halogen, such as, but not limited to, a bromine atom, in which case the framework molecule is 1,3,5 Tris(bromomethyl)benzene (TBMB). Another suitable molecular scaffold molecule is 2,4,6-tris(bromomethyl)mesitylene. It is similar to 1,3,5-tris(bromomethyl)benzene, but contains an additional three methyl groups attached to the benzene ring. In the case of this scaffold, additional methyl groups can form additional contacts with the peptide and therefore add additional structural hindrance. A different range of diversity is thus achieved than with 1,3,5-tris(bromomethyl)benzene.

Another preferred molecule to form a scaffold for reaction with a peptide by nucleophilic substitution is 1,3,5-tris(bromoacetamido)benzene (TBAB):

In other embodiments, the molecular scaffold may have a tetrahedral geometry such that the reaction of four functional groups of the encoded peptide with the molecular scaffold results in no more than two product isomers. Other geometries are also possible; indeed, an almost infinite number of scaffold geometries are possible, leading to great potential for peptide ligand diversification.

Peptides used to form the ligands of the present invention include Dap or N-AlkDap or N-HAlkDap residues to form alkylamino bonds to the framework. The structure of diaminopropionic acid is similar and isosteric to the structure of cysteine, which was used to form thioester bonds with the framework in the prior art, with the replacement of the terminal -SH group of cysteine with nα-NH ₂ :

- 13 044591

The term alkylamino is used in the present invention in its ordinary chemical sense to denote a bond consisting of NH or N(R ₃ ) bonded to two carbon atoms, where the carbon atoms are independently selected from alkyl, alkylene or aryl carbon atoms, and R ₃ represents alkyl group. Accordingly, the alkylamino bonds of the present invention include an NH group bonded to two saturated carbon atoms, most preferably methylene carbon atoms (-CH2-). The alkylamino bonds of the present invention have the general formula:

S - R1 - N(R ₃ ) - R2 - P where:

S represents a framework core, for example, a (hetero)aromatic or (hetero)alicyclic ring, as explained below;

R1 represents C1-C3 alkylene groups, preferably methylene or ethylene groups, and most preferably methylene ( _CH2 );

R2 represents a side chain methylene group of Dap or N-AlkDap;

R ₃ represents C1-4 alkyl, including branched alkyl and cycloalkyl, for example methyl, or H; And

P represents a peptide backbone, that is, the R2 group of the above bond is bonded to a carbon atom in the peptide backbone adjacent to the carboxyl carbon of the Dap or N-AlkDap residue.

Certain bicyclic peptides of formula (II) have a number of advantages that make them suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such benefits include: Interspecies cross-reactivity. This is a typical requirement for preclinical pharmacodynamics and pharmacokinetic evaluation.

Protease resistance.

Bicyclic peptide ligands should ideally demonstrate resistance to plasma proteases, epithelial (membrane-bound) proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, and the like. Protease resistance must be maintained between different species so that the bicyclic lead candidate can be developed in animal models and also confidently administered to humans.

Desired solubility profile.

It depends on the ratio of charged and hydrophilic versus hydrophobic residues and intra/intermolecular H-bonding, which is important for formulation and absorption purposes.

Optimal half-life in blood plasma.

Depending on the clinical indication and treatment regimen, it may be necessary to develop a short-acting bicyclic peptide for the acute treatment of acute diseases, or to develop a bicyclic peptide with longer retention in the bloodstream and therefore optimal for the treatment of a wider range of chronic diseases. Other factors that determine the desired plasma half-life are the requirements of prolonged exposure for maximum therapeutic efficacy as opposed to associated toxicology due to prolonged exposure to the agent.

It should be understood that salt forms are included within the scope of this invention, and references to bicyclic peptide compounds of formula (II) include salt forms of these compounds.

The salts of the present invention can be synthesized from a starting compound that contains a basic or acidic group 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. In general, such salts can be prepared by reacting these compounds in free acid or base form with a suitable base or acid in water or in an organic solvent or in mixtures of them.

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, butane, (+)camphor, camphorsulfonic, (+)-(1S)-camphor-10sulfonic, capric, capronic, caprylic, cinnamon, citric, cyclamine, dodecylsulfur

- 14 044591 noic, ethane-1,2-disulfonic, ethanesulfonic, 2-hydroxyethanesulfonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (for example, D-glucuronic), glutamic (for example, L-glutamic), α -oxoglutaric, glycolic, hippuric, hydrohalic acids (for example, hydrobromic, hydrochloric, hydroiodic), isethionic, lactic (for example, (+)-L-lactic, (±)-DL-lactic), lactobionic, maleic, malic, (- )-L-malic, malonic, (±)-DL-mandal, methanesulfonic, naphthalene-2-sulfonic, naphthalene-1,5-disulfonic, 1-hydroxy-2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamic, phosphoric, propionic, pyruvic, L-pyroglutamic, salicylic, 4-aminosalicylic, sebacic, stearic, succinic, sulfuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulfonic, undecylenic and valeric acids, and also acylated amino acids and cation exchange resins.

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

If the compound is anionic or has a functional group that can be anionic (for example, COOH can be ^-COO- ), then a salt can be formed with an organic or inorganic base to form a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li ⁺ , Na ⁺ and K + , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ³⁺ or Zn ⁺ . Examples of suitable organic cations include, but are not limited to, ammonium ion (ie, NH4 ⁺ ) and substituted ammonium ions (eg, NH3R ⁺ , NH2R2+, NHR3 ⁺ , NR4 ⁺ ). Examples of some suitable substituted ammonium ions include those formed 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(CH ₃ ) ₄ ⁺ .

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

Multiple conjugated peptides may be included together in the same molecule according to the present invention. For example, two such peptide conjugates with the same specificity can be linked to each other through a molecular scaffold, increasing the avidity of the derivative for its targets. Alternatively, in another embodiment, multiple peptide conjugates are combined to form a multimer. For example, two different peptide conjugates are combined to create a multispecific molecule. Alternatively, three or more peptide conjugates, which may be the same or different, can be combined to form multispecific derivatives. In one embodiment, multivalent complexes can be constructed by linking molecular scaffolds, which may be the same or different.

In a further aspect, the present invention provides a method for producing a peptide ligand of the present invention, which includes: providing a peptide of the present invention and a framework molecule; and the formation of thioether (when the third residue is a cysteine) and alkylamino bonds between the peptide and the framework molecule.

The details of the framework molecule and peptide are preferably as described above in connection with the first aspect of the present invention.

Peptides for use in the methods of the present invention can be prepared using conventional solid phase synthesis from amino acid starting materials, which may include suitable protecting groups as described in the present invention. These methods for producing peptides are well known in the art.

Preferably, the peptide has protecting groups on nucleophilic groups other than -SH and amino groups intended to form alkylamino bonds.

The nucleophilicity of amino acid side chains has been the subject of several studies, and they are listed in descending order: thiolate in cysteines, amines in lysine, secondary amine in histidine and tryptophan, guanidamines in arginine, hydroxyls in serine/threonine, and finally carboxylates in aspartate and glutamate. Accordingly, in some cases it may be necessary to apply protecting groups to the more nucleophilic groups on the peptide to prevent unwanted side reactions with these groups.

In embodiments, the method of the invention includes: synthesizing a peptide having protective

- 15 044591 groups on nucleophilic groups other than amino groups intended to form alkylamino bonds, and second protecting groups on amino groups intended to form alkylamino bonds, where the protecting groups on amino groups intended to form alkylamino bonds can be removed under conditions different from those used for protecting groups on other nucleophilic groups, followed by treating the peptide under conditions selected to deprotect amino groups intended to form alkylamino bonds without deprotecting other nucleophilic groups. The scaffold coupling reaction is then carried out followed by removal of the remaining protecting groups to obtain a peptide conjugate.

Preferably, the method of the present invention involves reacting, in a nucleophilic displacement reaction, a peptide having reactive -SH and side chain amino groups with a backbone molecule having three or more leaving groups.

The term leaving group in the present invention is used in its usual chemical sense to designate a moiety capable of nucleophilic substitution with an amino group. Any such leaving group can be used in the present invention, provided that it can be easily removed by nucleophilic substitution with an amine. Suitable leaving groups are conjugate acid bases having a pKa of less than about 5. Non-limiting examples of leaving groups useful in the invention include halogen such as bromine, chlorine, iodine, O-tosylate (OTos), O-mesylate (OM), O -triflate (OTf) or O-trimethylsilyl (OTMS).

Nucleophilic substitution reactions can be carried out in the presence of a base, for example when the leaving group is a conventional anionic leaving group. The present inventors have discovered that the yield of cyclized peptide ligands can be significantly increased by suitable selection of solvent and base (and pH) for the nucleophilic substitution reaction, and further that the preferred solvent and base differs from prior art solvent and base combinations that lead to the formation of only thioester bonds. In particular, the present inventors have discovered that improved yields are achieved when using a trialkylamine base, that is, a base of the formula NR ₁ R2R ₃ wherein R _b R2 and R ₃ independently represent C1-C5 alkyl groups, preferably C2-C4 alkyl groups, in particularly C2-C3 alkyl groups. Particularly suitable bases are triethylamine and diisopropylethylamine (DIPEA). These bases have the property of being only weakly nucleophilic, and this property is thought to account for the fewer side reactions and higher yields observed with these bases. The present inventors have also discovered that the preferred solvents for the nucleophilic substitution reaction are polar and protic solvents, in particular MeCN/ _H2O containing MeCN and H2O in volume ratios of 1:10 to 10:1, preferably 2:10 to 10: 2, and more preferably from 3:10 to 10:3, in particular from 4:10 to 10:4.

Additional linkages or functional activities may be added to the N or C terminus of the peptide covalently linked to the molecular scaffold. The functional group is, for example, selected from the group consisting of: a group capable of binding to a molecule that prolongs the half-life of the peptide ligand in vivo, and a molecule that prolongs the half-life of the peptide ligand in vivo. Such a molecule may be, for example, HSA or a cell matrix protein, and the moiety capable of binding to the molecule that prolongs the half-life of the peptide ligand in vivo is an antibody or antibody fragment specific for HSA or a cell matrix protein. Such a molecule can also be a conjugate with high molecular weight PEGs.

In one embodiment, the functional group is a binding molecule selected from the group consisting of a second peptide ligand including a peptide covalently linked to the molecular scaffold and an antibody or antibody fragment. 2, 3, 4, 5 or more peptide ligands can be combined together. The specificities of any two or more of these derivatives may be the same or different; if they are the same, a multivalent binding structure is formed that has increased avidity for the target compared to monovalent binding molecules. Moreover, the molecular scaffolds may be the same or different and may contain the same or different numbers of loops.

In addition, the functional group may be an effector group, such as the Fc region of an antibody.

Attachments to the N or C terminus can be made before or after binding of the peptide to the molecular scaffold. Thus, the peptide can be produced (synthetically or using expression systems of biological origin) with a pre-existing N- or C-terminal peptide group. Preferably, however, the attachment to the N- or C-terminus occurs after the peptide has been combined with the molecular backbone to form a conjugate. For example, fluorenylmethyloxycarbonyl chloride can be used to introduce an Fmoc protecting group at the N-terminus of the peptide. Fmoc binds to serum albumins, including HSA, with high affinity, and Fmoc-Trp or Fmoc-Lys bind with increased affinity. The peptide can be synthesized with the remaining Fmoc protecting group and then linked to the framework via alkylamino groups. An alternative is palmitoyl

- 16 044591 fragment, which also binds to HSA and, for example, is used in liraglutide to extend the half-life of this GLP-1 analog.

Alternatively, the peptide can be conjugate to the scaffold and then modified at the N terminus, for example with the amine and sulfhydryl reactive linker N-e-maleimidocaproyloxy succinimide ester (EMCS). Through this linker, the peptide conjugate can be linked to other peptides, for example the Fc fragment of an antibody.

The binding function may be another peptide linked to the molecular scaffold to form a multimer; other binding protein, including an antibody or antibody fragment; or any other desired compound, including serum albumin or an effector group, for example the F region of an antibody.

In addition, additional binding or functional activities may be associated directly with the molecular scaffold.

In embodiments, the framework may further include a reactive group to which additional activities may be associated. Preferably, this group is orthogonal to other reactive groups on the molecular framework to avoid interaction with the peptide. In one embodiment, the reactive group may be protected and deprotected as needed to conjugate additional activities.

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

Effector and/or functional groups can be attached, for example, to the N or C termini of the polypeptide or to the molecular framework.

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

In a further embodiment of this aspect of the present invention, the effector group of the present invention is the Fc region of an IgG molecule. Preferably, the effector group of the peptide ligand of the present invention includes or consists of an Fc fusion with a peptide ligand having a half-life te of one 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 includes or consists of an Fc fusion with a peptide ligand having a half-life of one day or more.

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

The ability of peptides to penetrate cells will allow the peptides to be effective against intracellular targets. Targets that can be accessed by cell-penetrating peptides include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in the apoptosis pathway. Functional groups that provide cell penetration include peptides or chemical groups that have been added to either the peptide or the molecular scaffold. Peptides such as those derived from VP22, HIV-Tat, Drosophila homeobox protein (Antennapedia) are described, for example, in 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 translocation across plasma membranes include the 16 amino acid peptide penetratin from the Drosophila Antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), a model 18 amino acid amphipathic peptide (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 pl27) and arginine-rich regions of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimetics or SMOCs, which can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies to add guanidinium groups to molecules also improve cell penetration (Elson-Schwab et al (2007) J Biol Chem Volume 282 p13585). Small molecular weight molecules, such as steroids, can be added to the molecular scaffold to enhance uptake into cells.

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

- 17 044591

RGD peptides that bind to integrins present in many cells may also be included.

In one embodiment, the peptide effector ligand group of the present invention has a half-life te 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 group or composition of the present invention will have a half-life ίβ in the range of 12 to 60 hours. In a further embodiment, the half-life ίβ will be one day or more. In a further embodiment, the half-life will be in the range of 12 to 26 hours.

In one particular embodiment of the present invention, the functional group conjugated to the loop peptide is selected from a metal chelator that is suitable for complexing metal radioisotopes of medical importance. Such effectors, when complexed with these radioisotopes, may provide useful agents for cancer therapy. Suitable examples include DOTA, NOTA, EDTA, DTPA, HEHA, SarAr and others (Targeted Radionuclide therapy, Tod Speer, Wolters/Kluver Lippincott Williams & Wilkins, 2011).

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

In one specific embodiment of this aspect of the present invention, the functional group is selected from a drug, such as a cytotoxic agent for cancer therapy. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors, including the camptothecins: irinotecan and topotecan, and type II inhibitors, including amsacrine, etoposide, etoposide phosphate and teniposide. Other agents may include antitumor antibiotics, which include the immunosuppressant dactinomycin (which is used in kidney transplants), doxorubicin, epirubicin, bleomycin, and others.

In yet another specific embodiment of the present invention in accordance with this aspect, the cytotoxic agent is selected from DM1 or MMAE.

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

he n^,0

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

N t ^H OH

In one embodiment, the cytotoxic agent is linked to a bicyclic peptide

- 18 044591 using a cleavable bond such as a disulfide bond. In a further embodiment, the groups adjacent to the disulfide bond are modified to control hindrance of the disulfide bond and thereby the rate of cleavage and concomitant release of the cytotoxic agent.

Published work has established the potential for modifying the susceptibility of a disulfide bond to reduction by introducing steric hindrance on both sides of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione as well as extracellular (systemic) reducing agents, therefore reducing the ease with which the toxin is released both inside and outside the cell. Thus, the choice of optimal disulfide stability in the bloodstream (which minimizes unwanted side effects of the toxin) versus efficient release in the intracellular environment (which maximizes therapeutic effect) can be achieved by carefully selecting the degree of hindrance on both sides of the disulfide bond.

The hindrances on each side of the disulfide bond are modulated by introducing one or more methyl groups either on the targeting moiety side (here a bicyclic peptide) or on the toxin side of the molecular construct.

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

where n is an integer selected from 1-10; And

R ₁ and R 2 independently represent hydrogen or methyl groups.

In one embodiment of a compound of the above formula, n is 1 and R ₁ and R 2 are both hydrogen (ie, a maytansine derivative DM1).

In an alternative embodiment of a compound of the above formula, n is 2, R1 is hydrogen, and R2 is a methyl group (ie, a maytansine DM3 derivative).

In one embodiment of the compound, n is 2 and R1 and R2 are both methyl groups (ie, a maytansine derivative of DM4).

It will be appreciated that a cytotoxic agent can form a disulfide bond, and in a bicyclic peptide conjugate structure, a disulfide bond is introduced between the toxin thiol and the bicyclic peptide thiol through several possible synthetic routes.

In one embodiment, the bicyclic peptide component of the conjugate has the following structure:

Bicycle where m is an integer selected from 0-10.

The bicycle is any suitable loop peptide structure as described in the present invention; And

R3 and _R4 are independently hydrogen or methyl.

Compounds of the above formula wherein R3 and R4 are both hydrogen are considered unhindered, and compounds of the above formula where one or all of R3 and R4 are methyl are considered hindered.

It will be understood that a bicyclic peptide of the above formula can form a disulfide bond, and in the cytotoxic agent conjugate structure described above, a disulfide bond between the thiol-toxin and the thiol-bicyclic peptide is introduced through several possible synthetic routes.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide using the following linker:

where R1, R2, R3 and R4 represent hydrogen or C1-C6 alkyl groups;

Toxin refers to any suitable cytotoxic agent as defined herein.

The bicyclic compound is any suitable loop peptide structure as described in the present invention;

n is an integer selected from 1-10; and m is an integer selected from 0-10.

When R ₁ , R 2 , R 3 and R ₄ are each hydrogen, the disulfide bond is the least hindered and most susceptible to reduction. When R1, R2, R3 and R4 are each alkyl, the disulfide bond is most hindered and least susceptible to reduction. Partial substitutions of hydrogen and alkyl lead to a gradual increase in resistance to reduction, as well as concomitant degradation and release of the toxin. Preferred embodiments include: R1, R2, R3 and R4 are all H; R1, R2, R3 are all H and R ₄ =methyl; R1, R ₂ =methyl and R3, R4=H; R _b R ₃ =methyl and R2, R4=H; and R1, R ₂ =H, R ₃ , R4 = C1-C6 alkyl.

In one embodiment, the toxin of the compound is maytansine, and the conjugate includes a compound of the following formula:

where R1, R2, R3 and R4 are as defined above;

A bicyclic compound is any suitable loop peptide structure as defined herein;

n is an integer selected from 1-10; and m is an integer selected from 0-10.

Additional details and methods for preparing the above-described bicyclic peptide ligand toxin conjugates are described in detail in the authors' published patent application WO 2016/067035 and pending application GB 1607827.1, filed May 4, 2016. The full disclosure of these applications is expressly incorporated herein by links.

The linker between the toxin and the bicyclic peptide may include a triazole group formed by a click reaction between the azide-functionalized toxin and the alkyne-functionalized bicyclic peptide structure (or vice versa). In other embodiments, the bicyclic peptide may contain an amide bond formed by the interaction between the carboxylate-functionalized toxin and the N-terminal amino group of the bicyclic peptide.

The linker between the toxin and the bicyclic peptide may include a cathepsin cleavable

- 20 044591 group to ensure selective release of the toxin in target cells. A suitable cathepsin cleavable group is valine-citrulline.

The linker between the toxin and the bicyclic peptide may include one or more spacer groups to provide the desired functionality, for example, binding affinity or cathepsin cleavability, of the conjugate. A suitable spacer group is para-aminobenzylcarbamate (PABC), which may be located between the valine-citrulline group and the toxin moiety.

Thus, in embodiments, the bicyclic peptide-drug conjugate may have the following structure consisting of Toxin-PABC-cit-val-triazole-Bicycl:

In other embodiments, the bicyclic peptide-drug conjugate may have the following structure consisting of Toxin-PABC-cit-val-dicarboxylate-Bicycl:

where (alk) is an alkylene group of the formula C _n H _2n where n is from 1 to 10 and may be straight or branched, preferably (alk) is n-propylene or nbutylene.

Preferably, the bicyclic peptide-drug conjugate is selected from the group consisting of BT17BDC53, BT17BDC59, BT17BDC61, BT17BDC62 and BT17BDC68, as defined below.

The peptide ligands of the present invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, and the like.

Typically, the use of a peptide ligand can replace the use of an antibody. The derivatives selected in accordance with the invention are used for diagnostic purposes in Western analysis and for protein detection in situ using standard immunohistochemical methods; for use in these applications, derivatives of the selected spectrum can be labeled according to methods known in the art. In addition, such peptide ligands can be used preparatively in affinity chromatography procedures when they are complexed with a chromatographic support such as a resin. All such methods are well known to those skilled in the art. The peptide ligands of the present invention have binding abilities similar to those of antibodies and can replace antibodies in such assays.

Diagnostic applications include any application where antibodies are typically used, including dipstick assays, laboratory assays, and immunodiagnostic assays.

Therapeutic and prophylactic uses of the peptide ligands prepared in accordance with the invention include the administration of derivatives selected in accordance with the invention to a mammalian recipient, such as a human. Substantially pure peptide ligands with at least 90 to 95% homogeneity are preferred for administration to a mammal, and homogeneity of 98 to 99% or more is most preferred for pharmaceutical use, especially when the mammal is a human. Once purified, partially or to homogeneity, as desired, the selected peptides can be used diagnostically or therapeutically (including extracorporeally) or in the development and implementation of assay procedures, immunofluorescent stains, etc. (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

Typically, the peptide ligands of the present invention will be used in purified form together with pharmacologically suitable carriers. Typically these carriers include aqueous

- 21 044591 or alcohol/aqueous solutions, emulsions or suspensions, any of which include saturated saline and/or buffered medium. Parenteral vehicles include sodium chloride solution, dextrose Ringer's solution, dextrose-sodium chloride solutions, and lactated Ringer's solution. Suitable physiologically acceptable adjuvants, if it is necessary to maintain the peptide complex in suspension, can be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

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

The peptide ligands of the present invention can be used as separately administered compositions or in combination with other agents. These may include antibodies, antibody fragments and various immunotherapy drugs such as cyclosporine, methotrexate, adriamycin or cisplatin, and immunotoxins. Pharmaceutical compositions may include cocktails of various cytotoxic or other agents in combination with selected antibodies, their receptors or binding proteins of the present invention, or even combinations of selected peptides of the present invention having different specificities, such as peptides selected using different target derivatives, regardless of whether they were combined before introduction or not.

The route of administration of the pharmaceutical compositions of the invention may be any of those generally known to those skilled in the art. For therapy, including without limitation immunotherapy, selected antibodies, receptors or their binding proteins of the present invention can be administered to any patient in accordance with standard methods. Administration can be by any suitable route, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary or also, where appropriate, by direct infusion via a catheter. The dosage and frequency of administration will depend on the age, gender and condition of the patient, concomitant use of other medications, contraindications and other parameters that the physician must take into account.

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

Compositions containing the peptide ligands of the invention, or a mixture 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, killing, or some other measurable parameter, of a population of selected cells is defined as a therapeutically effective dose. The amounts required to achieve this dosage will depend on the severity of the disease and the general condition of the patient's own immune system, but typically range from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses ranging from 0.05 to 2.0 mg/kg/dose are more widely used. For prophylactic applications, compositions containing the peptide ligands of the invention or a mixture thereof can also be administered at similar or slightly lower dosages.

A composition containing a peptide ligand of the present invention can be used in prophylactic and therapeutic settings to promote the modification, inactivation, destruction or removal of a selected population of target cells in a mammal. In addition, a selected repertoire of peptides described in the present invention can be used extracorporeally or selectively in vitro to kill, deplete or otherwise effectively remove a population of target cells from a heterogeneous group of cells. Blood from a mammal can be combined extracorporeally with selected peptide ligands, whereby unwanted cells are killed or otherwise removed from the blood returned to the mammal according to standard techniques.

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

Examples

Materials and methods.

Protein expression.

MT1-MMP hemopexin-like repeats (also known as the MT1MMP hemopexin domain), Cys319-Gly511 residues from the human gene, were transiently expressed in HEK293 cells as an N-terminally secreted His6-tagged soluble protein using the expression vector pEXPR-IBA42 ( IBA). After expression, the protein was purified using Nickel-NTA affinity chromatography followed by gel filtration and purity was checked using SDS-PAGE. Batch-to-batch variability was also monitored using fluorescence thermal shift experiments

- 22 044591 in the presence/absence of a bicycle binding to the hemopexin domain.

Peptide synthesis.

Peptide synthesis is based on Fmoc chemistry using the Symphony peptide synthesizer from Peptide Instruments and the Syro II synthesizer from MultiSynTech. Standard Fmocamino 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), the base was diisopropylethylamine (DIPEA, Sigma), and deprotection was achieved with 20% piperidine in DMF (AGTC). Syntheses were carried out using 0.37 mmol/g Fmoc-Rink amide AM resin (AGTC), Fmoc amino acids were used in fourfold excess and the base was used in fourfold excess relative to the amino acids. Amino acids were dissolved at 0.2 M in DMSO, HCTU at 0.4 M in DMF, and DIPEA at 1.6 M in N-methylpyrrolidone (Alfa Aesar). Conditions were such that the coupling reactions contained 20 to 50% DMSO in DMF, which reduced aggregation and deletions during solid-phase synthesis and increased yields. Bonding time was typically 30 minutes and deprotection time was 2x5 minutes. Fmoc-N-methylglycine (Fmoc-Sar-OH, Merck) was coupled for 1 hour, and the deprotection and coupling times for the next residue were 20 minutes and 1 hour, respectively. After synthesis, the resin was washed with dichloromethane and dried. Cleavage of side chain and carrier protecting groups was carried out using 10 ml of 95:2.5:2.5:2.5 v/v/v/w TFA/H ₂ O/iPr ₃ SiH/dithiothreutol for 3 hours. After cleavage, the spent resin was removed by filtration and the filtrate was added to 35 ml of diethyl ether, which was cooled at -80°C. The peptide precipitate was centrifuged, the ether supernatant was discarded, and the peptide precipitate 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 to analyze the purity of the crude product using mass spectrometry (MALDI-TOF, Voyager DE from Applied Biosystems). After lyophilization, the peptide powders were placed in 10 ml of a 6 M solution of guanidinium 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 a 0.1% solution of heptafluorobutyric acid. A gradient of 30-70% acetonitrile was used over 15 minutes at a flow rate of 15-20 ml/min using a Gilson preparative HPLC system. Fractions containing pure linear peptide (as determined by MALDI) were used to prepare bicyclic derivatives by coupling to the scaffold molecule as described below.

All amino acids, unless otherwise stated, were used in the L configuration. Unnatural amino acids were incorporated into the peptide sequence using the general methods described above. The list of unnatural amino acid precursors used is given in the table below.

Provider Short name Full chemical name AGTC D-Asp Fmoc-D-Asp(tBu)-OH Iris Biotech HPhe Fmoc-L-Homophenylalanine Alfa Aesar 5FPhe Fmoc-pentafluoro-N-phenylalanine PolyPeptide Group 4BrPhe Fmoc-4-bromo-L-phenylalanine

- 23 044591

Iris Biotech bAla Gtos-beta-A1a-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-Nur(tBu)-OH Merck Novabiochem D-Leu Fmoc-D-Leu-OH Merck Novabiochem HArg Fmoc-L-HArg(Boc)2-OH Peptech Corporation 4,4-BPA1 Fmoc-L-4,4'-biphenylalanine Peptech Corporation 3,3-DPA Fmoc-L-3,3-diphenylalanine Peptech Corporation Dpg Fmoc-dipropylglycine Peptech Corporation INal Fmoc-L-1-Naphthylalanine Peptech Corporation 2 NAI 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-pyrrolidine2-carboxylic acid AGTC D-Asp Fmoc-D-Asp(tBu)-OH Merck tBuGly Fmoc-α-tert-butylglycine Iris Biotech Chg Fmo c-L-cyclo g e xylglycine 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-N-phenylalanine Polypeptide Group Cha Fmoc-beta-cyclohexyl-L-alanine

In addition, the following unnatural amino acid precursors were used to obtain the modified DAP and N-MeDAP peptides:

Compound CAS Mol. weight Provider Fmoc-L-Dap(Boc,Me)-OH 44684780-9 440.49 Iris Biotech GMBH Fmoc-Dap(Vos)-OH 162558- 25-0 426, 46 Sigma Aldrich

Binding affinity for MT1-MMP.

Binding affinity was measured using fluorescence polarization (anisotropy) competition assays.

The fluorescent tags referred to in the present invention are bicyclic peptides that have been labeled using 5,6-carboxyfluorescein.

Fluorescent labeling can be performed on the N-terminal amino group of the peptide, which is separated from the core sequence of the bicycle by a sarcosine spacer (usually Sar5). This can be done 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. Fluorescent labeling can also be carried out at the C-terminus, usually at a lysine introduced as the first C-terminal residue, which is then separated from the core sequence of the bicycle by a sarcosine spacer (usually Sar6). Thus, the N-terminal tags may have the molecular format described as Fluo-Gly-Sar5A(BiCyclCoreSequence), and (BiCycLCoreSequence)-A-Sar6-K(Fluo) for a construct tagged with fluorescein at the C-terminus. The fluorescent labels 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). Due to the acidic nature of fluorescent peptides 17-69, they were usually prepared as concentrated stock solutions in DMSO, from which dilutions were made in 100 mM Tris buffer pH 8.

Due to their high affinity for the hemopexin domain of MT1-MMP (PEX), fluorescein-labeled derivatives can be used for competition experiments (using FP for detection). In this case, a preformed complex of PEX with fluorescent PEX

- 24 044591 binding label was titrated with free, non-fluorescein labeled bicyclic peptide. Since all 17-69 based peptides are expected to bind at the same site, the titrant will displace the fluorescent tag from the PEX. The dissociation of the complex can be measured quantitatively and the Kd of the competitor (titrant) relative to the target protein can be determined. The advantage of the competitive method is that the affinities of non-fluorescein-labeled bicyclic peptides can be determined accurately and quickly.

Label concentrations are typically at or below the Kd (here 1 nM) and the binding protein (hemopexin MT1-MMP here) is in 15-fold excess so that >90% of the label is bound. A non-fluorescent competing bicyclic peptide (usually just the core sequence of the bicyclic) is then titrated so that it displaces the fluorescent tag from the target protein. The displacement of the labeled substance is measured and related to the drop 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 thus an indication of the affinity of the titrant for the target protein.

The raw data is used to fit an analytical solution to a cubic equation that describes the equilibrium between the fluorescent label, the titrant, and the binding protein. Fitting requires the affinity value of the fluorescent tag for the target protein, which can be determined separately using direct FP binding experiments (see previous section). 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.

Reference example 1.

The bicyclic peptide chosen for the thioether and alkylamino backbone comparison was designated 17-69-07-N241. It is a bicyclic conjugate of a thioether-forming peptide with a trimethylenebenzene backbone. The structure of this bicyclic derivative is shown schematically in FIG. 2. The linear peptide before conjugation has the sequence:

H-(β-Ala)-Sar10-Ala-Cys-(D-Ala)-Asn-Glu-(1Nal)-(D-Ala)-Cys-Glu-Asp-Phe-Tyr-Asp-(tBuGly)- Cys- _NH2

Conjugation with 1,3,5-tris(bromomethyl)benzene (TBMB, Sigma) was carried out as follows. 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 after completion of the reaction (as measured by MALDI). After lyophilization, the modified peptide was purified as above, replacing the Luna C8 with a Gemini C18 column (Phenomenex) and replacing the acid with 0.1% trifluoroacetic acid. Pure fractions containing the desired TMB-modified substance were pooled, lyophilized and maintained at -20°C for storage.

The resulting bicyclic derivative, designated 17-69-07-N241, showed high affinity for MT1-MMP. The measured affinity (Kd) of this derivative for MT1-MMP was 0.23 nM. Therefore, the derivative is considered as a promising candidate for targeting tumor cells that express the metalloproteinase MT1-MMP on the cell surface.

Example 1.

A bicyclic peptide designated 17-69-07-N385 was prepared, corresponding to the bicyclic region of the peptide ligand of Reference Example 1, minus the b-Ala-Sar10 tail, and replacing the first and third cysteine residues of DAP with residues forming alkylamino bonds to the TBMB backbone. The structure of this derivative is shown schematically in FIG. 3.

The linear peptide used to form this bicycle was as follows:

Ac-A(Dap)(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)(Dap)

The linear peptide and bicyclic peptide had the following LCMS characteristics:

Retention time present m/z Linear peptide 4.17 min. 886.1, 1771.7 Cyclized peptide 4.39 min. 942.7

Various reagents for the cyclization step were tested as follows. The reagents were prepared to the concentrations indicated in the table below in the selected solvent. Half of this volume of TBMB solution was added to the volume of the peptide solution, the mixture, which was thoroughly mixed, then made up half the volume of the main solution. The reaction mixture was stirred periodically and samples were taken for LCMS analysis.

Reagent Initial solution concentration Volume equivalents added to reaction Final solution concentration Peptide 1.0 mM 10 0.5 mM TVMV 2.6 mM 0.5 0.65 mM Base 200 mM 0.5 50 mM

- 25 044591

Example: 25 μL of TBMV solution was added to 50 μl of peptide solution. The solution was thoroughly mixed, then 25 μl of base solution was added.

Where the solvent used was DMF, all reagents were prepared in DMF. Where the solvent used was DMSO, all reagents were prepared in DMSO. When the solvent used was MeCN/H ₂ O, peptide solutions were prepared in 50% MeCN/H ₂ O, TBMB solutions were prepared in MeCN, and base solutions were prepared in H ₂ O, except when the base was DIPEA , in this case, a solution of the base was prepared in MeCN. All cyclizations were carried out at room temperature. The results were as follows (the spectrum range was set at 3.5-5.5 min. The spectrum at 220 nm was integrated and the sum of the main peaks was taken):

Solvent Base Total Integration Product Integration % product Time MeCN/ _H2O NEt ₃ 4641.0 2857.1 62% 5 o'clock Na ₂ CO ₃ 5470.8 546, 1 10% 5 o'clock _NaHCO3 9956.5 3530.4 35% 4 hours NH ₄ HCO ₃ 6948.9 0 0% 5 o'clock tetramethyl guanidine 12130.5 211, 9 2% 5 o'clock DIPEA 9081, 1 5951.6 66% 16 hours

It can be seen that the purity after cyclization is highly dependent on the choice of base. Product purity ranges from 2 to 66%, the latter comprising acetonitrile/water in the presence of DIPEA. In contrast to the cyclization of Reference Example 1, the yield is relatively low when using normal NaHCO ₃ as the base. The best yields are achieved using trialkylamines, namely triethylamine and diisopropylethylamine (DIPEA).

Comparative binding data to MT1-MMP are shown in FIG. 7. The Kd value was measured to be 0.45 nM, which demonstrates that the change to alkylamino linkages in this example resulted in a surprisingly small change in binding affinity compared to the thioether-linked derivative of Reference Example 1.

Example 2.

A bicyclic peptide designated 17-69-07-N426, corresponding to the bicyclic peptide of Example 1, was prepared by replacing the DAP residues with N-MeDAP residues. The structure of this derivative is shown schematically in FIG. 4. The linear peptide used to form this bicycle was as follows:

Ac-A(Dap(Me))(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)(Dap(Me))

The linear peptide and bicyclic peptide had the following LCMS characteristics:

Retention time present m/z Linear peptide 4.19 min. 900.1, 1799.1 Cyclized peptide 4.38 min. 956.0, 1913.7

Various reaction conditions, solvents and bases were used for the cyclization step as described in Example 1, with the following results (all cyclizations were performed at room temperature):

Solvent Base Total Integration Product Integration % Product Time MeCN/ _H2O Na ₂ CO ₃ 21659.8 12714.5 59% 1 hour MeCN/ _H2O DIPEA 10547.7 9841.3 93% 16 hours

Purity after cyclization again depends on the nature of the base. As expected, the purity with Na ₂ CO ₃ as the base is low (see example 1). Using the optimal condition of acetonitrile/water in the presence of DIPEA, the purity after cyclization is very high (93%), indicating that N-methylation of Dap reduces the level of side reactions.

Comparative binding data to MT1-MMP is shown in FIG. 8. The measured Kd value is 0.36 nM, which is essentially unchanged compared to the thioether-linked derivative of Reference Example 1. This activity is retained despite two N-methylations on the bond and thus the derivative of the present example is of particular interest.

Example 3.

A bicyclic peptide designated 17-69-07-N428 was obtained, corresponding to the bicyclic peptide of Example 1 with Tyr9 replaced by Phe9 (removal of the Tyr hydroxyl). The linear peptide used to form this bicycle was as follows:

Ac-A(Dap) (D-Ala)NE (1Nal) (D-Ala)CEDFF ₉ D (tBuGly) (Dap) The linear peptide and bicyclic peptide had the following LCMS characteristics:

Retention time present m/z Linear peptide 4.51 min. 876.5, 1755.4 Cyclized peptide 4.80 min. 935, 3

- 26 044591

Various reaction conditions, solvents and bases were used for the cyclization step as described in Example 1, with the following results (LCMS spectrum range set at 4-6 min.

The spectrum at 220 nm was integrated and the sum of the main peaks was taken):

Solvent Base Pace. Total Integration Product Integration Product Time MeCN/ _H2O DIPEA room pace. 5962.8 4209.3 71% 6 hours MeCN/ _H2O DIPEA 50°C 5936.4 2898.3 49% 1 hour DMF DIPEA room pace. 4804.6 84.7 2% 1 hour DMF DIPEA 50°С 4384.8 309, 7 7% 1 hour MeCN/ _H2O TMG room temp. 5050.8 2023.8 40% 1 hour MeCN/ _{H2O K2CO3 _} room temp. 6366.7 4109.2 65% 4 hours MeCN/ _{H2O K2CO3 _} 50°С 5314.5 2306.7 43% 1 hour

It can be seen that the purity of the product ranges from 2 to 71%, the latter comprising acetonitrile/water in the presence of DIPEA. Removal of Tyr-OH (Tyr >Phe9) significantly increases the yield of the product compared to the same reaction with a tyrosine-containing peptide under MeCN/H ₂ O/TMG/room temperature, or McCN/H ₂ O/K ₂ CO ₃ / comh. pace. Using DMSO as a solvent produced very dirty chromatograms with several peaks that could not be easily analyzed.

Comparative binding data of this 17-69-07-N428 derivative to MT1-MMP are shown in FIG. 9. The measured Kd value is 3.5 nM. The slight decrease in binding affinity compared to the thioether-linked derivative of Reference Example 1 appears to be primarily due to the substitution of Tyr9 for Phe9.

Example 4.

A bicyclic peptide designated 17-69-07-N434, corresponding to the bicyclic peptide of Example 1, was prepared with a Sar10 N-terminal spacer similar to that used in Reference Example 1 and a PYA (4-pentynic acid, for toxin click-derivatization) conjugating group. The structure of this derivative is shown schematically in FIG. 5. The linear peptide used to form this bicycle was as follows:

(PYA)-(B-Ala)-Sar10-A(Dap)(D-Ala)NE(1Nal)(D-Ala)CEDFYD(tBuGly)(Dap)

The linear peptide and bicyclic peptide had the following LCMS characteristics:

Retention time present m/z Linear peptide 4.18 863.7, 1294.8 Cyclized peptide 4.37 902.6, 1353.0

Cyclization was carried out as follows:

Solvent Base Pace. Total Integration Product Integration Product Time MeCN/ _H2O DIPEA room temp. 4445.6 2666.5 60% 4 hours

The resulting derivative 17-69-07-N434 is the Dap1/3 equivalent of N241 (Reference Example 1) with the N-terminal alkyne required for derivatization with effectors, i.e. toxins. This peptide can be cyclized with TBMB at 60% purity. The kd value with MT1-MMP was measured to be 1.52 nM, making this bicyclic peptide very suitable for targeting MT1-MMP.

Example 5.

Replacing the framework molecule TBMB used in examples 1-3 with TBAB was carried out as follows.

The linear peptides used to form 17-69-07-N385, 17-69-07-N426 and 17-69-07-N428 in Examples 1-3 were cyclized with TBAB at the same concentrations and equivalents as those used used for TVMV. The structure of the TBAB derivative with the N385 peptide is shown schematically in FIG. 6.

DIPEA was used as the base of choice with a solvent mixture of MeCN/H2O at room temperature. The following results were obtained.

Peptide Product retention time Total Integration Product Integration % Product Time 17-69- 07-N385 4.28 min. 9399.8 8830.0 94% 16 hours 17-69- 07-N426 4.47 min. 11941.6 11485.1 96% 16 hours 17-69- 07-N428 4.70 min. 8321.8 7941.5 95% 16 hours

These results indicate that TBAV (haloacetyl) chemistry offers higher rates

- 27 044591 cyclization and higher selectivity than TBMV, as can be seen from the % product column.

Example 6.

A bicyclic peptide designated 17-69-07-N474 was obtained, corresponding to the bicyclic peptide of Example 1, with Cys6 replaced by Dap(Me). The linear peptide used to form this bicycle was as follows:

Ac-A(Dap(Me))(D-Ala)NE(1Nal)(D-Ala)(Dap(Me))EDFYD(tBuGly)(Dap(Me))

The structure of the TBMB derivative with the N385 peptide is shown schematically in FIG. 10.

The linear peptide and bicyclic peptide had the following LCMS characteristics:

Retention time present m/z Linear peptide 3.61 min. 599, 64, 898.96, 1795, 90 Cyclized peptide 3.98 min. 637.68, 956.03, 1910.04

Cyclization was carried out according to the following procedure: 50 μl of a 1 mM solution of the peptide in MeCN/H2O (1:1) was mixed with 25 μl of 2.6 mM TBMB in MeCN, then 25 μl of a 200 mM solution of DIPEA in MeCN/H2O was added (pH adjusted to 10 using acetic acid) and the solution was stirred. (1.3 equivalents of TBMB and 100 equivalents of base relative to the peptide present in the reaction). Samples for LCMS were taken after 4 hours and overnight, and the reaction proceeded as shown in the table below.

Total Integration Product Integration % Product Time 3120 1248 40 4 hours 3784 3632 96 18 hours (at during the night)

Binding to MT1-MMP was assessed in the same way as in other examples. The measured Kd value is 8.0 nM, which is less than that of the thioether-linked derivative of Reference Example 1. This compound still binds with high affinity despite three N-methyl Daps on the linkage, and thus the derivative of the present Example represents special interest.

Example 7.

The following additional bicyclic peptides of the present invention were prepared and tested for binding affinity to MT1-MMP using the methods described above. Schematic structures of these bicyclic peptide compounds are shown in FIG. 10-13.

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Connection ID code Subsequence Binding affinity 17-69-07-N428 Ac-A(Dap) (D-Ala) NE (INal) (DAla)CEDFFD (tBuGly)(Dap) no data 17-69-07-N438 PYA-A(Dap) (D-Ala) NE (INal) (DAla)CEDFYD (tBuGly)(Dap) Ki (h) =3.1 nM 17-69-07-N455 Ac-(B-Ala)-SarlO-A(Dap)(DAla)NE(INal)(D-Ala) CEDFYD(tBuGly)(Dap) Ki (h)=4 nM 17-69-07-N470 Ac-A(Dap(Me))(D- Ala)NE(INal)(D- Ala)(Dap(Me))EDFYD (tBuGly)C Ki (h) =8.3 nM 17-69-07-N473 (PYA)-(D-Asp)3-Sar2A(Dap(Me)) (D-Ala)NE (INal) (DAla) (Dap(Me))EDFYD(tBuGly)C Ki (h) =7.3 nM 17-69-07-N443 PYA-A(Dap(Me))(D- Ala)NE(lNal)(D-Ala)CEDFYD (tBuGly) (Dap(Me)) Ki (h)=3.2 nM 17-69-07-N471 (PYA)-(D-Asp)3-Sar2A(Dap(Me)) (D-Ala)NE (INal) (DAla)CEDFYD(tBuGly) (Dap(Me)) Ki (h)=4, b nM 17-69-07-N472 (PYA)-(В-Ala)-Sar5A(Dap(Me)) (D-Ala)NE (INal) (DAla)CEDFYD(tBuGly) (Dap(Me)) Ki (h)=3.4 nM 17-69-07-N454 Ac-(B-Ala)-SarlO- A(Dap(Me)) (D-Ala)NE (INal) (D- Ala)CEDFYD (tBuGly) (Dap(Me) ) Ki (h)=3.0 nM 17-69-07-N452 A(Dap(Me)) (D-Ala)NE (INal) (DAla)CEDFYD (tBuGly) (Dap(Me) ) Ki (h) =4.0 nM 17-69-07-N450 (PYA)-(B-Ala)-SarlOA(Dap(Me)) (D-Ala)NE (INal) (DAla) CEDFYD(tBuGly)(Dap(Me)) Ki=3.7 nM; Kd=l.91 (SPR) 17-69-07-N451 (B-Ala)-SarlO-A(Dap(Me))(DAla)NE(INal)(D-Ala) CEDFYD(tBuGly)(Dap(Me)) Ki (h)=3.7 nM 17-69-07-N461 Ac (Dap(Me)) (D-Ala)NE (INal) (D- Ki (h)=6.0 nM Ala)CEDFYD (tBuGly) (Dap(Me)) 17-69-07-N479 Ac-A(Dap(Me)) (D- Ala)NE(INal)(D- Ala)CEDFYD(tBuGly)(Dap(Me)) Ki(h)=15.6 nM 17-69-07-N474 Ac-A(Dap(Me)) (D- Ala)NE(INal)(D- Ala)(Dap(Me))EDFYD(tBuGly) (Da P (Me) ) Ki (h)=7.5 nM

It can be seen that high affinity for MT1-MMP is achieved with these alkylamino-linked bicyclic compounds of the present invention. Further studies showed complete cross-reactivity of the bicyclic peptides of the present invention with canine, mouse/rat and human MT1-MMP. Further studies showed the high specificity of the bicyclic peptides of the present invention, without significant cross-reactivity with the MMP1 ectodomain, MMP2 ectodomain, MMP15 ectodomain (hemopexin domain) or MMP16 hemopexin domain. It has also been determined that the pharmacokinetics of the bicyclic peptides of the present invention are similar to the corresponding bicyclic peptides having three thioester linkages to the backbone, but with a slightly longer half-life when measured in serum for the bicyclic peptides of the present invention.

Example 8.

Bicyclic peptide-drug conjugates (BCDs) in which the bicyclic peptides of the present invention are coupled to monomethyl auristatin E (MMAE) via a triazole cyclization reaction were prepared according to the reaction scheme shown in FIG. 14. In addition to the triazole group, linker groups in the conjugates include valine-citrulline (a cathepsin cleavable group) and para-aminobenzylcarbamate (PABC), a spacer group. The steps of the reaction scheme were carried out as follows.

General procedure for obtaining connection 3.

- 29 044591

T i ° _{l l} T c O f OH ^V »%\%A _OH A ^B0C 'N AMCH-M ^N έ LH _? N ^J -·^ Η 0 I Η

Ί EEDQ,DCM,MeOH Ί ^ΝΗ ⁴ ΝΗ

Ο^ΝΗ ₂ ο^νη ₂

3

To a solution of compound 2 (30 g, 80 mmol) in DCM (300 ml) and MeOH (150 ml) was added 4-aminophenylmethanol (11 g, 88 mmol) and EEDQ (40 g, 160 mmol) in the dark. The mixture was stirred at 30°C for 16 hours. TLC (DCM:MeOH=10/1, Rf=0.43) showed that compound 2 was completely consumed and many new spots formed. According to TLC, the reaction mixture was pure. The resulting reaction mixture was concentrated to give a residue, which was purified by silica gel flash chromatography (ISCO®; 330 g x 3 SepaFlash® silica gel flash chromatography column, eluent 0~20% MeOH/Dichloromethane @ 100 ml/min). Compound 3 (20 g, 52% yield) was obtained as a white solid.

General procedure for obtaining connection 4.

To a solution of compound 3 (5.0 g, 10.4 mmol) in DMF (40 ml) was added DIEA (5.4 g, 7.26 ml, 41.7 mmol) and bis(4-nitrophenyl) carbonate (12, 7 g, 41.7 mmol). The mixture was stirred at 0°C and under nitrogen atmosphere for 1 hour. TLC (DCM:MeOH=10/1, Rf=0.66) showed that compound 3 was completely consumed and one new spot formed. The reaction mixture was clear by TLC and LCMS (ES8241-10-P1A, product: RT=1.15 min) indicated the formation of the desired product. The resulting reaction mixture was purified directly using preparative HPLC under neutral conditions. Compound 4 (12 g, 60% yield) was obtained as a white solid.

General procedure for obtaining connection 5.

One batch reaction was carried out as follows: to a solution of compound 4 (1.2 g, 1.68 mmol) in DMF (10 ml) was added DIEA (1.22 ml, 6.98 mmol) under nitrogen atmosphere, the solution was stirred at 0 °C for 10 min, then HOBt (22 6 mg, 1.68 mmol) and MMAE (1.00 g, 1.40 mmol) were added to this mixture, the mixture was degassed and purged with N ₂ 3 times and stirred at 35°C for 16 hours. LC-MS (ES8396-1-P1A1, product: RT=1.19 min) showed that compound 4 was completely consumed and one major peak was detected with the required mass. The resulting reaction mixture from five batches was combined in a 1 L beaker and 500 ml of water was added, then the resulting precipitate was filtered for collection. The precipitate was triturated with EtOAc overnight. Compound 5 (5 g, 59% yield) was obtained as a white solid.

General procedure for obtaining connection 6.

Compound 5 (3.3 g, 2.7 mmol) was dissolved in DCM (18 ml) in the presence of TFA (44 mmol, 3.5 ml), then the solution was stirred at 25°C for 3 hours. The reaction mixture was then concentrated under reduced pressure to remove DCM and TFA to obtain a residue. The residue was dissolved in THF (20 ml), treated with K ₂ CO ₃ (1.8 g, 13 mmol) and the mixture was further stirred at 25°C for a further 12 hours. LC-MS (ES8396-2-P1B1, product: RT=l.04 min) showed that one major peak with the required mass was detected. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was dissolved in 10 ml DMF and purified using preparative HPLC (neutral conditions). Compound 6 (1.6 g, 53% yield) was obtained as a white solid.

General procedure for obtaining compound 7-1.

Compound 6 (1.2 g, 1.1 mmol) and 2-azidoacetic acid (162 mg, 1.6 mmol) were dissolved in DMF (10 ml). TEA (4 50 μl, 3.2 mmol), HOBt (217 mg, 1.6 mmol) and EDCI (307 mg, 1.6 mmol) were added to the solution under nitrogen atmosphere and the mixture was stirred at 0°C for 30 min , then the mixture was slowly heated to 25°C, followed by stirring for 15.5 hours. LC-MS (ES8396-3-P1A, product: RT=1.04 min) showed that compound 6 was completely consumed and one major peak was detected with the required mass. 2 ml of water was added to the reaction mixture to form a clear solution. The solution was then purified directly using preparative HPLC under neutral conditions. Compound 7-1 (0.9 g, 70% yield) was obtained as a white solid.

General procedure for obtaining BT17BDC-53.

To a mixture of (2-azidoacetic acid)-Val-Cit-PABC-MMAE (16 mg, 13.26 µmol, 1 eq.) and BICYCLE alkyne (17-69-07-N434, 30 mg, 11.09 µmol, 0 .8 eq.) in DMF (3 ml) and H ₂ O (2 ml) was added CuI (1.26 mg, 6.63 µmol, 0.5 eq.). The mixture was stirred at 25°C under N2 atmosphere for 20 hours. LC-MS showed that (2-azidoacetic acid)-Val-Cit-PABC-MMAE was completely consumed and one major peak was detected with the desired mass. The resulting reaction mixture was purified using preparative HPLC (TFA conditions). Compound BT17BDC-53 (23.7 mg, 6.06 µmol, 54.64% yield) was obtained as a white solid.

General procedure for obtaining BT17BDC-59.

UN UN (2-end&acetic acid IVaJ-Cit-F ABC-MM AE k BHTBDC-W 1

To a mixture of (2-azidoacetic acid)-Val-Cit-PABC-MMAE (31 mg, 25.69 µmol, 1.2 eq.) and (17-6907-N438, 40 mg, 20.8 µmol, 1 eq. ) in DMF (3 ml) was added a solution of CuSO ₄ (10.25 mg, 64.24 µmol, 3 eq.) in water (0.4 ml) and a solution of ascorbic acid (37.71 mg, 214.12 µmol, 10 eq.) in water (0.4 ml) under nitrogen atmosphere. The mixture was then stirred at 25°C for 1 hour. LC-MS showed that (2azidoacetic acid)-Val-Cit-PABC-MMAE was completely consumed and one major peak with the desired mass was detected. The resulting reaction mixture was purified using preparative HPLC (TFA condition). BT17BDC-59 (26.7 mg, 8.53 μmol, 41.02% yield) was obtained as a white solid.

General procedure for obtaining BT17BDC61.

Compound 7-1 (250 mg, 207 µmol) and BICY-ALKYNE 17-69-07-N450 (515 mg, 188 µmol) were added to a 50 ml round bottom flask, DMF (5 ml) was added and then an aqueous solution of ascorbic acid ( 1 M, 1.88 ml) and an aqueous solution of CuSO ₄ (1 M, 570 μl) under nitrogen atmosphere, then the mixture was stirred at 25°C for 1 hour. LC-MS (ES8396-8-P1A, product: RT=1.03 min) showed that BICYALKYNE was completely consumed and one major peak with the required mass was detected. The reaction mixture was filtered to remove undissolved material, and the filtrate was purified directly using preparative HPLC (TFA conditions). BT17BDC61 (262 mg, 35% yield) was obtained as a white solid.

General procedure for obtaining BT17BDC62.

- 31 044591

Compound 7-1 (250 mg, 207 µmol) and BICY-ALKYNE 17-69-07-N443 (368 mg, 188 µmol) were added to a 50 ml round bottom flask. DMF (5 mL) was added, followed by the addition of aqueous ascorbic acid (1 M, 1.88 mL) and aqueous CuSO ₄ (1 M, 570 μL). The mixture was then stirred at 25°C for 1 hour. LC-MS (ES8396-9-P1A, product: RT=1.07 min) showed that BICY-ALKYNE was completely consumed and one major peak with the required mass was detected. The reaction mixture was filtered to remove undissolved material. The resulting filtrate was purified directly using preparative HPLC (TFA conditions). BT17BDC62 (253 mg, 42% yield) was obtained as a white solid.

Example 9.

Bicyclic-drug conjugates (BCDs) in which the bicyclic peptides of the present invention are linked to monomethyl auristatin E (MMAE) by amide formation between the glutaryl terminal group of the linker and the amino terminal group of the peptide were prepared according to the reaction scheme shown in FIG. 15. The steps of the reaction scheme were carried out as follows.

General procedure for obtaining connection 3.

(4-Aminophenyl)methanol (2.53 g, 20.56 mmol, 1.10 eq.) and EEDQ (9.25 g, 37.39 mmol, 2.00 eq.) in the dark, and the mixture was stirred at 25°C for 8 hours. LC-MS showed that compound 2 was completely consumed and one major peak was detected with the desired mass. The resulting reaction mixture was concentrated under reduced pressure to remove the solvent to obtain a residue.

The residue was purified by silica gel flash chromatography (ISCO®; 120 g SepaFlash® silica gel flash chromatography column, eluent 0~10% MeOH/DCM @ 85 ml/min). Compound 3 (7.00 g, 14.60 mmol, 78.06% yield) was obtained as a white solid.

General procedure for obtaining connection 4.

To a solution of compound 3 (4.00 g, 8.34 mmol, 1.00 eq) and 4-nitrophenylchloroformate (6.72 g, 33.36 mmol, 4.00 eq) in THF (20.00 ml) and DCM (10.00 ml), pyridine (2.64 g, 33.36 mmol, 2.69 ml, 4.00 eq.) was added and the reaction mixture was stirred at 25°C for 5 hours. LC-MS showed that compound 3 was completely consumed and one major peak was detected with the desired mass. The reaction mixture was concentrated under reduced pressure to obtain a residue, which was purified by silica gel flash chromatography (ISCO®; 120 g Silica gel flash column

SepaFlash®, Eluent 0~20% DM/MeOH @ 85 ml/min). Compound 4 (2.20 g, 3.41 mmol, 40.92% yield) was obtained as a white solid.

General procedure for obtaining connection 5.

A mixture of compound 4 (500.00 mg, 775.59 µmol, 1.00 eq) and DIEA (1.00 g, 7.76 mmol, 1.35 ml, 10.00 eq) in DMF (10.00 ml) was stirred under nitrogen atmosphere at 0°C for 30 min and MMAE (445.49 mg, 620.47 µmol, 0.80 eq.) and HOBt (104.80 mg, 775.59 µmol, 1 .00 eq.). The reaction mixture was stirred under a nitrogen atmosphere at 0°C for 10 minutes and at 30°C for another 18 hours. LC-MS showed that compound 4 was completely consumed and one major peak was detected with the desired mass. The resulting reaction mixture was purified directly using flash chromatography on silica gel C18 (ISCO®; 330 g SepaFlash® Flash column C18, Eluent 0~50%

MeCN/H ₂ O @ 85 ml/min). Compound 5 (400.00 mg, 326.92 µmol, 42.15% yield) was obtained as a white solid.

- 32 044591

General procedure for obtaining connection 6.

To a solution of compound 5 (430.00 mg, 351.44 µmol, 1.00 eq.) in DCM (36.00 ml) was added TFA (6.16 g, 54.03 mmol, 4.00 ml, 153.73 eq.) and the mixture was stirred at 25°C for 2 hours. The mixture was then concentrated under reduced pressure to obtain a residue, which was dissolved in THF (10.00 mL), and K2CO3 (1.21 g, 8.79 mmol, 25.00 eq.) was added to the mixture. The reaction mixture was stirred at 25°C for 12 hours. LC-MS showed that compound 5 was completely consumed and one major peak was detected with the desired mass. The resulting reaction mixture was filtered and the filtrate was concentrated under reduced pressure to obtain a residue, which was purified using C18 silica gel flash chromatography (ISCO®; 120 g SepaFlash® C18 Flash Column, Eluent 0~50% MeCN/H2O @ 85 ml/min). Compound 6 (290.00 mg, 258.14 µmol, 73.45% yield) was obtained as a white solid.

General procedure for obtaining connection 7.

The vessel containing (400 mg, 356 µmol) was purged using a nitrogen cylinder. Anhydrous DMA (5 ml) was added with stirring and the solution was cooled to 0°C in an ice water bath. DIEA (130 μl, 712 μmol) was then added and the reaction mixture was stirred at 0°C for 10 minutes. Tetrahydropyran-2,6-dione (81 mg, 712 µmol) was added and then the ice bath was removed. The reaction mixture was stirred at 25°C for 1 hour. LC-MS (ES8396-4-P1A, product: RT=1.08 min) showed that compound 6 was completely consumed and one major peak was detected with the required mass. The mixture was diluted with 5 ml of water and then purified using preparative HPLC (neutral conditions). Compound 7-2 (330 mg, 75% yield) was obtained as a white solid.

General procedure for obtaining connection 8.

To compound 7-2 (330 mg, 267 µmol) in anhydrous DMA (4.5 ml) and DCM (1.5 ml) was added HOSu (92 mg, 800 µmol) under a nitrogen atmosphere with stirring for 10 min at 0° C using an ice bath. EDCI (154 mg, 800 μmol) was then added to the mixture, followed by further stirring at 25°C for 16 hours. LC-MS (ES8396-5-P1A, product: RT=1.15 min) showed that compound 7-2 was completely consumed and one major peak was detected with the required mass. The resulting reaction mixture was diluted with 5 ml of water and then purified by preparative HPLC (neutral conditions). Compound 8 (250 mg, 70% yield) was obtained as a white solid.

General procedure for obtaining BT17BDC68.

A 50 mL round bottom flask that contained BICY-NH ₂ 17-69-07-N451 (80.0 mg, 30 μmol) in DMA (4 mL) was purged using a nitrogen bottle. DIEA (20 μL, 114 μmol) was then added with stirring at 25°C for 10 min. Compound 8 (40 mg, 30 μmol) was then added and the reaction mixture was stirred under positive nitrogen atmosphere for 18 hours at 25°C. LC-MS (ES6635-127-P1A1, product: RT=1.06 min) showed that compound 8 was completely consumed and one major peak was detected with the desired mass. The resulting reaction mixture was purified using preparative HPLC (TFA conditions). BT17BDC68 (33.9 mg, 29% yield) was obtained as a white solid.

Example 10.

The in vitro binding affinity of the bicyclic peptide-drug conjugates obtained above for MT1-MMP was measured as described above. The results were as follows.

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Claims (40)

Connection ID code Identification code of the original bike Linker Binding affinity (Ki in nM) BT17BDC-53 17-69-07-N434 Triazole-2azidoacetyl -ValCitPABC Person ki=3.6, 2.5; rat/mouse ki=l.8, 1.6 BT17BDC-59 17-69-07-N438 Triazole-2azidoacetyl -ValCitPABC Person ki=2.8, 2.7; rat/mouse ki=l.8, 1.7 BT17BDC-61 17-69-07-N450 Triazole-2azidoacetyl -ValCitPABC Human ki=3.2, 2.9; rat/mouse ki=l.8, 1.6 BT17BDC-62 17-69-07-N443 Triazol-2azidoacetyl -ValCitPABC-CO2- Person ki=3.9, 3.3; rat/mouse ki=2,l, 1,9 BT17BDC-68 17-69-07-N451 glutaryl- Val-Cit RAV S Person ki=2.9, 3.3; rat/mouse ki=l.8, 1.6 It can be seen that in all cases the binding affinity of the bicyclic peptides is maintained after conjugation with MMAE. Example 11. The plasma stability of BT17BDC-53 in mouse and human sera was studied. The conjugate was found to be stable (T1/2 greater than 50 hours at a concentration of 4 μM) in both mouse and human sera. The stability appears to be slightly higher than that of the corresponding conjugates in which the peptide is linked to the scaffold by three thioester bonds. Example 12. The in vivo antitumor efficacy of the bicyclic peptide-drug conjugates obtained above was assessed as follows. HT1080 tumor cells were maintained in vitro as a monolayer culture in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere of 5% CO ₂ and air. Tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. Cells growing in the exponential growth phase were collected and counted for tumor inoculation. BALB/c nude mice were inoculated subcutaneously on the right flank with HT1080 tumor cells (%x10 ⁶ ) in 0.2 ml PBS for tumor development. 39 animals were randomized when the average tumor volume reached 134 mm ² . BDC compounds were formulated at a concentration of 0.03 mg/ml in a carrier buffer containing 25 mM histidine and 10% sucrose. The compositions were administered twice weekly (biw) at concentrations of 0.3, 1, 3 and 10 mg/kg. Tumor volume and body weight were measured for 14 days after the first dose. The results are shown in Fig. 16-20. The results show that all five conjugates tested exhibit potent dose-dependent tumor inhibition. At doses of 3 mg/kg and 10 mg/kg, tumors were completely eliminated. BT17BDC53, 61 and 68 were well tolerated up to 10 mg/kg. BT17BDC62 was tolerable up to about 5 mg/kg. BT17BDC59 was tolerable up to 3 mg/kg. This suggests that the presence of the N-terminal spacer Sar10 in BT17BDC53, 61 and 68 reduces the systemic toxicity of the conjugates. All publications identified in the above description are incorporated herein by reference. Various modifications and variations of the described aspects and embodiments of the present invention will be apparent to those skilled in the art without departing from the scope of the present invention. Although the present invention has been described in connection with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described methods of carrying out the invention, which are obvious to those skilled in the art, are intended to be within the scope of the following claims. CLAIM 1. A peptide ligand specific for MT1-MMP, comprising a polypeptide comprising three residues selected from cysteine, L-2,3-diaminopropionic acid (Dap), N-beta-alkyl-L-2,3diaminopropionic acid (N- AlkDap) and N-beta-haloalkyl-L-2,3-diaminopropionic acid (N - 34 044591 HAlkDap), wherein said three residues are separated by at least two loop sequences, and a molecular framework, wherein the peptide is linked to the framework by covalent alkylamino bonds to Dap or N-AlkDap or N-HAlkDap residues of the polypeptide and thioether bonds to cysteine residues of the polypeptide, when these three residues include cysteine, thus forming two polypeptide loops on the molecular framework, where the peptide ligand includes the amino acid sequence of formula (II): - A ₁ -X ₁ -U/O ₂ -X ₃ -X ₄ -G ₅ -A ₂ -E ₆ -D ₇ -F ₈ -Y ₉ -X ₁₀ -X ₁₁ -A ₃ - (SEQ ID NO: 1) (II) or a pharmaceutically acceptable salt thereof; Where: A1, A2, and A ₃ are independently cysteine, L-2,3-diaminopropionic acid (Dap), Nbeta-alkyl-k-2,3-diaminopropionic acid (N-AlkDap) or N-beta-haloalkyl-L- 2,3-diaminopropionic acid (N-HAlkDap), provided that at least one of A1, _A2 , and _A3 is Dap, N-AlkDap or N-HAlkDap; X is 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. 2. The peptide ligand according to claim 1, wherein X1 is selected from any one of the following amino acids: Y, M, F or V, such as Y, M or F, in particular Y or M, more particularly Y. 3. The peptide ligand according to claim 1 or 2, wherein U/O ₂ is selected from U such as N or O such as G. 4. A peptide ligand as claimed in any one of the preceding claims, wherein X ₃ 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 an amino acid residue selected from D or E, in particular, U at position 3 is selected from Q, or Z at position 3 is selected from E. 5. The peptide ligand as claimed in any one of the preceding claims, wherein X ₄ is selected from J, wherein J is a non-polar aromatic amino acid residue selected from F, W and Y. 6. The peptide ligand as claimed in any one of the preceding claims, wherein X ₁₀ is selected from Z, wherein Z is a polar negatively charged amino acid residue selected from D or E, such as D. 7. The peptide ligand as claimed in any one of the preceding claims, wherein X is selected from O, wherein O is a non-polar aliphatic amino acid residue selected from G, A, I, L, P and V, such as I. 8. A peptide ligand according to any one of the preceding claims, wherein the peptide ligand of formula (II) is a compound of formula (IIa): - A ₁ -Y/M/F/VU/OU/ZJGA ₂ -EDFYZOA ₃ - (SEQ ID NO: 6) (IIa) where U, O, J and Z are as defined above; or a compound of formula (IIb): - A ₁ -Y/M/F/VN/GE/QFGA ₂ -EDFYDIA ₃ - (SEQ ID NO: 7) (IIb); or a compound of formula (IIc): - A ₁ -Y/M/FN/GE/QFGA ₂ -EDFYDIA ₃ - (SEQ ID NO: 8) (IIc); or a compound of formula (IId): - A ₁ -Y/MNE/QFGA ₂ -EDFYDIA ₃ - (SEQ ID NO: 9) (IId); or a compound of formula (IIe): - A ₁ -YNEFGA ₂ -EDFYDIA ₃ - (17-69-07) (SEQ ID NO: 2) (IIe). 9. The peptide ligand according to any one of the preceding claims, wherein the peptide ligand of formula (II) includes a sequence selected from: - A ₁ -YNEFGA ₂ -EDFYDIA ₃ - (17-69-07) (SEQ ID NO: 2); - A1-M-N-Q-F-G-A2-E-D-F-Y-D-I-A3- (17-69-12) (SEQ ID NO: 10); - A ₁ -FGEFGA ₂ -EDFYDIA ₃ - (17-69-02) (SEQ ID NO: 11); - A ₁ -VNEFGA ₂ -EDFYDIA ₃ - (17-69-03) (SEQ ID NO: 12); - A ₁ -FNEFGA ₂ -EDFYDIA ₃ - (17-69-04) (SEQ ID NO: 13); - A ₁ -YNEYGA ₂ -EDFYDIA ₃ - (17-69-07-N057) (SEQ ID NO: 14); And - A ₁ -YNEWGA ₂ -EDFYDIA ₃ - (17-69-44-N002) (SEQ ID NO: 15), such as: - A ₁ -YNEFGA ₂ -EDFYDIA ₃ - (17-69-07) (SEQ ID NO: 2); And - A ₁ -MNQFGA ₂ -EDFYDIA ₃ - (17-69-12) (SEQ ID NO: 10). 10. The peptide ligand according to claim 9, wherein the peptide ligand of formula (II) includes the sequence A ₁ -YNEFGA ₂ -EDFYDIA ₃ -(17-69-07) (SEQ ID NO: 2). 11. The peptide ligand according to claim 9, wherein the peptide ligand of formula (II) includes a sequence selected from the Dap homologue designated as SEQ ID 16 ( (bAla)-Sar10-AA ₁ (D-Ala)NE(1Nal) (D- Ala) A2EDFYD (tBuGly)A3; and a Dap homolog designated SEQ ID 17 AA ₁ (D-Ala) NE (1Nal) (D-Ala) A2EDFYD (tBuGly)A ₃ . 12. A peptide ligand as claimed in any one of the preceding claims, wherein two of A ₁ , A ₂ and A ₃ are selected from - 35 044591 Dap, N-AlkDap or N-HAlkDap, and the third of A ₁ , A 2 and A ₃ is cysteine. 13. The peptide ligand according to claim 12, where A ₂ represents cysteine. 14. The peptide ligand according to any one of claims 1 to 9, wherein A _, A ₂ and A ₃ are each N-AlkDap or N-HalkDap. 15. The peptide ligand according to any of the preceding claims, wherein one or more tyrosine residues are replaced by a phenylalanine residue. 16. The peptide ligand according to any of the preceding paragraphs, which further includes one or more modifications selected from: N-terminal and/or C-terminal modifications; replacing one or more amino acid residues with one or more unnatural amino acid residues (such as replacing one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacing one or more hydrophobic amino acid residues with other unnatural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacing one or more amino acid residues with alanine; replacing one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the peptide ligand; replacing one or more peptide bonds with a surrogate bond; modifications to the length of the peptide backbone; substituting a hydrogen on the α-carbon of one or more amino acid residues with another chemical group, and postsynthetic bioorthogonal modification of amino acids such as cysteine, lysine, glutamate and tyrosine with suitable amine, thiol, carboxylic acid and phenol-reactive reagents. 17. The peptide ligand of claim 16, wherein the N-terminal modification includes the addition of a molecular spacer group that facilitates the conjugation of effector groups and maintains the activity of the peptide ligand to its target, such as an Ala group, a G-Sar10-A group, or a bAla-Sar10- group. A. 18. The peptide ligand according to any one of claims 16, 17, which includes a modification at amino acid position 1 and/or 9, and it is selected from: D-alanine at position 1 and/or 4-bromophenylalanine at position 9. 19. The peptide ligand according to any one of claims 16 to 18, which includes replacing one or more amino acid residues with one or more unnatural amino acid residues. 20. The peptide ligand according to claim 19, where the replacement with an unnatural amino acid residue occurs at position 4, and it is selected from: 1-naphthylalanine; 2-naphthylalanine; 3,4-dichlorophenylalanine; and homophenylalanine such as 1-naphthylalanine; 2-naphthylalanine; and 3,4-dichlorophenylalanine. 21. The peptide ligand according to claim 20, wherein the unnatural amino acid residue is 1-naphthylalanine. 22. The peptide ligand according to claim 19 or claim 20, where the replacement with a non-natural amino acid residue occurs at position 9 and/or 11, and it is selected from: 4-bromophenylalanine or pentafluorophenylalanine for position 9 and/or tert-butylglycine for position eleven. 23. The peptide ligand of claim 22, wherein the non-natural amino acid residue, such as those present at position 9, is 4-bromophenylalanine. 24. The peptide ligand of claim 22, wherein the non-natural amino acid residue, such as those present at position 11, is tert-butylglycine. 25. The peptide ligand of claim 16, wherein the amino acid residue at position 1 is replaced with a D-amino acid such as D-alanine. 26. The peptide ligand of claim 16, wherein the amino acid residue at position 5 is replaced with a D-amino acid such as D-alanine or D-arginine. 27. The peptide ligand of claim 19, which includes modifications such as 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/or 5, 1-naphthylalanine at position 4, 4-bromophenylalanine at position 9 and tert-butylglycine at position 11. 28. The peptide ligand according to any one of claims 1 to 27, wherein the peptide ligand of formula (II) is a high-affinity binder of the hemopexin domain of human, mouse and canine MT1-MMP. 29. The peptide ligand according to any one of claims 1 to 28, wherein the peptide ligand of formula (II) is selective for MT1-MMP, but does not cross-react with MMP-1, MMP-2, MMP15 and MMP-16. 30. Linear peptide comprising the amino acid sequence of formula (II) according to any one of claims 1 to 27. 31. A method for producing a peptide ligand according to any one of claims 1 to 29, including: providing the peptide according to claim 30; providing a framework molecule having at least three reactive sites for the formation of thioester and alkylamino bonds with -SH and side chain amino groups of cysteine residues and diaminopropionic acid or e-N-alkyldiaminopropionic acid residues of the peptide; and forming said thioether and alkylamino bonds between the peptide and the framework molecule. 32. A drug conjugate comprising the peptide ligand of any one of claims 129 conjugated to one or more cytotoxic agents. - 36 044591 33. An MT1-MMP-specific conjugate comprising a peptide ligand according to any one of claims 1 to 29 conjugated to one or more functional groups. 34. The conjugate according to claim 32, wherein the functional groups include a metal chelator. 35. The drug conjugate of claim 32, wherein the cytotoxic agent is linked to the peptide ligand by a cleavable bond, such as a disulfide bond. 36. The drug conjugate of claim 32 or claim 35, wherein the cytotoxic agent is selected from DM1 or MMAE. 37. The drug conjugate according to any one of claims 32, 35, 36, where the drug conjugate has the following structure: where R ₁ , R ₂ , R ₃ and R4 represent hydrogen or C1-C6 alkyl groups; toxin refers to any suitable cytotoxic agent; The bicycle is any suitable peptide ligand according to any one of claims 1-29; n is an integer selected from 1-10; and m is an integer selected from 0-10. 38. The drug conjugate of claim 37, wherein either: R _b R ₂ , R ₃ and R ₄ are all H; or R _b R ₂ , R ₃ are all H and R ₄ =methyl; or R _b R ₂ =methyl and R ₃ , R ₄ =H; or R _b R ₃ =methyl and R ₂ , R4 = H; or R _b R ₂ =H and R ₃ , R4 = C ₁ -C ₆ alkyl. 39. The drug conjugate according to any one of claims 32, 35, 36, where the drug conjugate has the following structure: 40. The drug conjugate according to any one of claims 32, 35, 36, where the drug conjugate has the following structure: where (alk) represents a linear or branched alkylene group of the formula _CnH2n , where n has a value _from 1 to 10. -