阅读说明：本技术 特异于epha2的双环肽配体 (Bicyclic peptide ligands specific for EPHA2 ) 是由 L·陈 S·帕万 C·斯泰斯 D·托伊费尔 K·泛里特肖特 于 2018-12-19 设计创作，主要内容包括：本发明涉及与芳族分子支架共价结合的多肽,使得两个或更多个肽环在与支架的连接点之间相对。具体而言,本发明描述了作为Eph受体酪氨酸激酶A2(EphA2)的高亲和力结合物的肽。本发明还包括药物缀合物,其包含缀合至一个或多个效应物和/或官能团的所述肽的、涉及包含所述肽配体和药物缀合物的药物组合物、以及涉及所述肽配体和药物缀合物在预防、抑制或治疗特征在于患病组织(例如肿瘤)中EphA2过度表达的疾病或病症中的用途。(The present invention relates to polypeptides covalently bound to an aromatic molecule scaffold such that two or more peptide loops are opposed between points of attachment to the scaffold. In particular, the invention describes peptides that are high affinity conjugates of Eph receptor tyrosine kinase A2(EphA 2). The invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and/or functional groups, to pharmaceutical compositions comprising the peptide ligands and drug conjugates, and to the use of the peptide ligands and drug conjugates in the prevention, inhibition, or treatment of diseases or disorders characterized by over-expression of EphA2 in diseased tissues (e.g., tumors).)

1. A peptide ligand specific for EphA2 comprising a polypeptide comprising at least three cysteine residues separated by at least two loop sequences and an aromatic molecule scaffold forming a covalent bond with the cysteine residues of the polypeptide thereby forming at least two polypeptide loops on the molecule scaffold.

2. The peptide ligand as defined in claim 1, wherein the loop sequence comprises 4,5,6 or 7 amino acids.

3. The peptide ligand as defined in claim 1 or 2, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, each consisting of 5 amino acids (such as those listed in table 3, table 4 and table 94).

4. The peptide ligand as defined in claim 1 or 2, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, each consisting of 6 amino acids (such as those listed in tables 3-10).

5. The peptide ligand as defined in claim 1 or 2, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, one of which consists of 6 amino acids and the other of which consists of 5 amino acids (such as those listed in table 4).

6. The peptide ligand as defined in claim 1 or 2, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, one of which consists of 6 amino acids and the other of which consists of 4 amino acids (such as those listed in table 4).

7. The peptide ligand as defined in claim 1 or 2, wherein the loop sequence comprises three cysteine residues separated by two loop sequences, one of which consists of 6 amino acids and the other of which consists of 7 amino acids (such as those listed in table 8).

8. The peptide ligand as defined in any one of claims 1 to 7, wherein the peptide ligand comprises an amino acid sequence selected from the group consisting of:

C_i-X₁-C_ii-X₂-C_ii，

wherein X₁And X₂Represents the amino acid residues between the cysteine residues listed in tables 3 to 10, C_i、C_iiAnd C_iiiRespectively represent a first, a second and a third cysteine residue, or a pharmaceutically acceptable salt thereofAn acceptable salt.

9. The peptide ligand as defined in any one of claims 1 to 8, wherein the peptide ligand comprises an amino acid sequence of one or more peptide ligands selected from one or more of those listed in tables 3 to 10.

10. The peptide ligand as defined in any one of claims 1 to 9, wherein the molecular scaffold is selected from (1,3, 5-tris (bromomethyl) benzene) (TBMB) and the peptide ligand is selected from any one of the peptide ligands listed in tables 3 to 10.

11. The peptide ligand as defined in any one of claims 1 to 10, which is selected from any one of compounds 1-286, 289, 292-293 and 296-297, or a pharmaceutically acceptable salt thereof.

12. The peptide ligand as defined in any one of claims 1 to 11, wherein the pharmaceutically acceptable salt is selected from the group consisting of the free acid or the sodium, potassium, calcium, ammonium salts.

13. The peptide ligand of any one of claims 1-12, wherein the EphA2 is human EphA 2.

14. A drug conjugate comprising the peptide ligand of any one of claims 1 to 13 conjugated to one or more effectors and/or functional groups.

15. The drug conjugate of claim 14, wherein the cytotoxic agent is selected from DM 1.

16. A drug conjugate according to claim 14 or 15, selected from any one of BDC-1 to BDC-5, for example from any one of BDC-1 to BDC-4.

17. A pharmaceutical composition comprising the peptide ligand of any one of claims 1 to 13 or the drug conjugate of any one of claims 14 to 16, and one or more pharmaceutically acceptable excipients.

18. The drug conjugate of any one of claims 14 to 16, for use in preventing, inhibiting or treating a disease or disorder characterized by overexpression of EphA2 in diseased tissue.

19. A drug conjugate according to any one of claims 14 to 16 for use in the prevention, inhibition or treatment of cancer.

20. A drug conjugate according to any one of claims 14 to 16 for use in the prevention, inhibition or treatment of lung cancer.

21. The drug conjugate according to any one of claims 14 to 16, for use in the prevention, inhibition or treatment of non-small cell lung cancer.

Technical Field

The present invention relates to polypeptides covalently bound to an aromatic molecule scaffold such that two or more peptide loops are opposed between points of attachment to the scaffold (protected). In particular, the invention describes peptides that are high affinity conjugates of Eph receptor tyrosine kinase A2(EphA 2). The invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and/or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and uses of the peptide ligands and drug conjugates in preventing, inhibiting, or treating diseases or disorders characterized by over-expression of EphA2 in diseased tissues (e.g., tumors).

Background

Cyclic peptides are capable of binding to protein targets with high affinity and target specificity and are therefore an attractive class of molecules for therapeutic development. In fact, several cyclic peptides have been used successfully clinically, such as the antibacterial peptide vancomycin, the immunosuppressant Drug cyclosporin or the anticancer Drug octreotide (draggers et al (2008), Nat Rev Drug Discov 7(7), 608-24). Good binding properties result from the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are bound to surfaces of several hundred square angstroms, for example the cyclic peptide CXCR4 antagonist CVX15(Wu et al (2007), Science330,1066-71), cyclic peptides with an Arg-Gly-Asp motif that bind to integrin α Vb3

(Xiong et al (2002), Science 296(5565),151-5), or cyclic peptide inhibin in combination with urokinase-type plasminogen activatorPreparation upain-1(Zhao et al (2007), J Structure Biol 160(1), 1-10).

Because of its cyclic configuration, the peptidic macrocycle is less flexible than a linear peptide, resulting in less loss of entropy upon binding to the target and higher binding affinity. The reduced flexibility also results in locking in the target-specific conformation, increasing the binding specificity compared to the linear peptide. This effect has been exemplified by a potent, selective matrix metalloproteinase 8(MMP-8) inhibitor which loses selectivity for other MMPs when its ring is opened (Cherney et al (1998), J Med Chem 41(11), 1749-51). The advantageous binding properties obtained by macrocyclization are more pronounced in polycyclic peptides having more than one peptide ring, for example in vancomycin, nisin and actinomycin.

Different research groups have previously attached polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J.Org.Chem; Timmerman et al (2005), ChemBioChem). Meloen and colleagues have used tris (bromomethyl) benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds for structural simulation of protein surfaces (Timmerman et al (2005), ChemBiochem). Methods of producing drug candidate compounds are disclosed in WO 2004/077062 and WO2006/078161, wherein the compounds are produced by linking cysteine-containing polypeptides to a molecular scaffold, such as tris (bromomethyl) benzene.

Combinatorial methods based on phage display have been developed to generate and screen large libraries of bicyclic peptides against a target of interest (Heinis et al (2009), Nat Chem Biol 5(7),502-7 and WO 2009/098450). Briefly, two regions containing three cysteine residues and six random amino acids (Cys- (Xaa) are displayed on phage₆-Cys-(Xaa)₆-Cys) and cyclized by covalent attachment of the cysteine side chain to a small molecule (tris (bromomethyl) benzene).

Disclosure of Invention

According to a first aspect of the present invention there is provided a peptide ligand specific for EphA2, comprising a polypeptide comprising at least three cysteine residues separated by at least two loop sequences and an aromatic molecule scaffold forming a covalent bond with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

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

According to another aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein and one or more pharmaceutically acceptable excipients.

According to another aspect of the present invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or condition characterised by overexpression of EphA2 in a diseased tissue (e.g. a tumour).

Detailed Description

In one embodiment, the loop sequence comprises 4,5,6 or 7 amino acids.

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, each consisting of 5 amino acids (e.g., those listed in table 3, table 4, and table 9).

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, both of which consist of 6 amino acids (such as those listed in tables 3-10).

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, wherein one loop sequence consists of 6 amino acids and the other loop sequence consists of 5 amino acids (such as those listed in table 4).

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, wherein one loop sequence consists of 6 amino acids and the other loop sequence consists of 4 amino acids (such as those listed in table 4).

In another embodiment, the loop sequence comprises three cysteine residues separated by two loop sequences, wherein one loop sequence consists of 6 amino acids and the other loop sequence consists of 7 amino acids (such as those listed in table 8).

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

C_i-X₁-C_ii-X₂-C_iii

wherein X₁And X₂Represents the amino acid residues between the cysteine residues listed in tables 3 to 10, C_i、C_iiAnd C_iiiRespectively, represents a first, second and third cysteine residue, or a pharmaceutically acceptable salt thereof.

In another embodiment, the peptide ligand comprises an amino acid sequence of one or more peptide ligands selected from one or more of the list in tables 3 to 10.

In one embodiment, the molecular scaffold is selected from (1,3, 5-tris (bromomethyl) benzene) (TBMB) and the peptide ligand is selected from any one of the peptide ligands listed in tables 3 to 10.

In one embodiment, the peptide ligand is selected from any one of compounds 1-308, or a pharmaceutically acceptable salt thereof.

In another embodiment, the peptide ligand is selected from any one of compounds 1-286, 289, 292-.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, e.g., in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for Molecular biological, genetic and biochemical methods (see Sambrook et al, Molecular cloning: A Laboratory Manual, third edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al, Short Protocols in Molecular Biology (1999) fourth edition, John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides of the invention, cysteine residues are omitted from the numbering (C) since they are invariant_i、C_iiAnd C_iii) Thus, the numbering of amino acid residues within the peptides of the invention is referenced as follows:

-C_i-M₁-N₂-D₃-W₄-L₅-C_ii-S₆-L₇-G₈-W₉-T₁₀-C_iii-(SEQ ID NO:1)。

for the purposes of this description, it is assumed that all bicyclic peptides are cyclized by TBMB (1,3, 5-tris (bromomethyl) benzene) to yield trisubstituted 1,3, 5-trimethylbenzene structures. Cyclization with TBMB takes place at C_i、C_iiAnd C_iiiThe above.

Molecular form

N-or C-terminal extensions of the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen, e.g., N-terminal (β -Ala) -Sar₁₀the-Ala tail will be represented as:

(β-Ala)-Sar₁₀-A-(SEQ ID NO:X)。

reverse peptide sequence

It is envisaged that the peptide sequences disclosed herein will also be used in their reverse form as disclosed in Nair et al (2003) J Immunol 170(3), 1362-1373. For example, the order is reversed (i.e., N-terminal to C-terminal, and vice versa), and their stereochemistry is reversed (i.e., D-amino acid to L-amino acid, and vice versa).

Peptide ligands

Peptide ligands as referred to herein refer to peptides, peptides or peptidomimetics covalently bound to a molecular scaffold. In general, such peptides, peptides or peptidomimetics include peptides having natural or unnatural amino acids, two or more reactive groups capable of forming covalent bonds with the scaffold (i.e., cysteine residues), and sequences that are opposite between the reactive groups, which are referred to as loop sequences because when the peptide, peptide or peptidomimetic is attached to the scaffoldWhen combined, it forms a ring. In the context of the present application, a peptide, peptide or peptidomimetic comprises at least three cysteine residues (referred to herein as C)_i、C_iiAnd C_iii) Which forms at least two rings on the stent.

Advantages of peptide ligands

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

species cross-reactivity. This is a typical requirement for preclinical pharmacodynamic and pharmacokinetic assessments.

-protease stability. Bicyclic peptide ligands should in most cases exhibit stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained between species so that bicyclic peptide lead candidates can be developed in animal models and reliably administered to humans;

-ideal solubility curve. This is a function of the ratio of charged hydrophilic residues to hydrophobic residues and intramolecular/intermolecular H bonds, which is important for formulation and absorption purposes;

optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides with short or prolonged in vivo exposure times to manage chronic or acute disease states. The optimal exposure time depends on the requirement of a sustained exposure (to achieve maximum therapeutic efficiency) and the requirement of a short exposure time (to minimize the toxicological effects due to sustained exposure to the agent); and

-selectivity. Certain peptide ligands of the invention exhibit good selectivity for other Eph receptor tyrosine kinases such as EphA1, EphA3, EphA4, EphA6, EphA7 and EphB1 and factor XIIA, carbonic anhydrase 9 and CD38 (see table 12 for selectivity data for selected peptide ligands of the invention). It should also be noted that selected peptide ligands of the invention exhibit cross-reactivity with other species (e.g., mice) to allow testing in animal models (table 3, table 5, table 7, table 8 and table 11).

Pharmaceutically acceptable salts

It will be understood that salt forms are within the scope of the invention and reference to peptide ligands includes salt forms of the ligands.

Salts of the invention may be synthesized from the parent compound containing a base or acid moiety by conventional chemical methods, for example as described in Pharmaceutical Salts: Properties, Selection, and Use, P.Heinrich Stahl (ed.), Camile G.Wermuth (ed.), ISBN:3-90639-026-8, Hardcover, page 388, 8.2002. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent or in a mixture of the two.

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

One particular group of salts includes salts formed from: acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (methanesulfonate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One specific salt is the hydrochloride salt. Another specific salt is acetate.

If the compound is anionic or has a functional group which may be anionic (for example, -COOH may be-COO^-) Salts may be formed with organic or inorganic bases which generate the appropriate cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li⁺、Na⁺And K⁺Alkaline earth metal cations such as Ca²⁺And Mg²⁺And other cations such as Al³⁺Or Zn⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)⁴⁺) And substituted ammonium ions (e.g., NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺). Some examples of suitable substituted ammonium ions are those derived from: methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N (CH)₃)₄ ⁺。

When the peptides of the invention contain amine functional groups, they may form quaternary ammonium salts, for example by reaction with alkylating agents according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

Modified derivatives

It is to be understood that modified derivatives of the peptide ligands as defined herein are within the scope of the invention. Examples of such suitable modified derivatives comprise one or more modifications selected from: n-terminal and/or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-tolerant amino acid residues; replacement of one or more amino acid residues with one or more replacement amino acids (e.g., alanine), replacement of one or more L-amino acid residues with one or more D-amino acid residues; n-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; peptide backbone (peptide backbone) length modification; substitution of one or more amino acid residues with another chemical group for a hydrogen on the alpha-carbon, modification of amino acids such as cysteine, lysine, glutamic/aspartic acid and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introduction or substitution of amino acids to introduce orthogonal reactivity suitable for functionalization, e.g., amino acids bearing an azide or alkyne group allow functionalization with an alkyne or azide bearing moiety, respectively.

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

In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, during peptide synthesis, the N-terminal residue is capped with acetic anhydride or other suitable reagent, resulting in an N-terminally acetylated molecule. This embodiment provides the advantage of removing potential recognition sites for aminopeptidases and avoiding the possibility of degradation of bicyclic peptides.

In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates conjugation of the effector group and maintains the potency of the bicyclic peptide on its target.

In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In this embodiment, the C-terminal residue is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment provides the advantage of removing potential recognition sites for carboxypeptidases and reducing the potential for proteolytic degradation of bicyclic peptides.

In one embodiment, the modified derivative comprises the replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, unnatural amino acids can be selected that have isosteric/isoelectronic side chains that are not recognized by degradative proteases, nor do they have any adverse effects on target potency.

Alternatively, unnatural amino acids with constrained amino acid side chains can be used such that proteolysis of adjacent peptide bonds is conformationally and sterically hindered. In particular, these relate to proline analogues, bulky side chains, C α -disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cyclic amino acids, simple derivatives that are amino-cyclopropyl carboxylic acids.

In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises a cysteine (C) towards the N-terminus_i) And/or a C-terminal cysteine (C)_iii) Spacer groups are added.

In one embodiment, the modified derivative comprises the replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-tolerant amino acid residues. In a further embodiment, the modified derivative comprises replacing a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the advantage of improving the drug stability profile of the resulting bicyclic peptide ligands.

In one embodiment, the modified derivative comprises the replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In alternative embodiments, the modified derivative comprises the replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged amino acid residues and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, whereas charged amino acid residues (in particular arginine) can affect the interaction of peptides with phospholipid membranes on cell surfaces. The combination of the two can affect the half-life, volume of distribution and exposure of the peptide drug and can be adjusted according to the clinical endpoint. In addition, the correct combination and number of charged amino acid residues and hydrophobic amino acid residues may reduce stimulation at the injection site (if the peptide drug has been administered subcutaneously).

In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and to stabilize the conformation of the beta-turn by the propensity of the D-amino acid (Tugyi et al (2005) PNAS,102(2), 413-418).

In one embodiment, the modified derivative includes removing any amino acid residues and replacing with alanine, such as D-alanine. This embodiment provides the advantage of identifying key binding residues and removing potential proteolytic attack sites. The results for such alanines, e.g., D-alanine substitutions (also known as alanine scans) can be found in Table 9.

It should be noted that each of the above mentioned modifications is used to intentionally improve the efficacy or stability of the peptide. Further efficacy improvement based on modification can be achieved by the following mechanisms:

incorporation of hydrophobic moieties that exhibit hydrophobic effects and lead to reduced dissociation rates, thereby achieving higher affinities;

incorporation of charged groups that exhibit extensive ionic interactions, leading to faster rates of binding and higher affinities (see, e.g., Schreiber et al, Rapid, electronically associated association of proteins (1996), Nature struct. biol.3, 427-31); and

incorporation of additional constraints in the peptide, for example by correctly constraining the side chains of the amino acids, so as to minimize the entropy loss after target binding; constraining the torsion angle of the scaffold, thereby minimizing entropy loss after target binding; and for the same reason introducing additional cyclization in the molecule.

(for review see Gentilucci et al, Current pharmaceutical Design, (2010),16, 3185-.

Isotopic variation

The present invention includes all pharmaceutically acceptable (radio) isotopically-labelled peptide ligands of the present invention in which one or more atoms are replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature, and in which metal-chelating groups capable of accommodating the relevant (radio) isotope(s) (referred to as "effectors") are attached, and in which certain functional groups in the peptide ligands of the present invention are covalently replaced by the relevant (radio) isotope(s) or isotopically-labelled functional group(s).

Examples of isotopes suitable for inclusion in a peptide ligand of the invention include isotopes of hydrogen, such as²H, (D) and³h (T), isotopes of carbon, e.g.¹¹C、¹³C and¹⁴isotopes of C, chlorine, e.g.³⁶Cl, isotopes of fluorine, e.g.¹⁸F, isotopes of iodine, e.g.¹²³I、¹²⁵I and¹³¹i, isotopes of nitrogen, e.g.¹³N and¹⁵isotopes of N, oxygen, e.g.¹⁵O、¹⁷O and¹⁸o, isotopes of phosphorus, e.g.³²P, isotopes of sulfur, e.g.³⁵S, isotopes of copper, e.g.⁶⁴Isotopes of Cu and gallium, e.g.⁶⁷Ga or⁶⁸Isotopes of Ga, Y, e.g.⁹⁰Y, and isotopes of lutetium, e.g.¹⁷⁷Lu, and bismuthIsotopes of (e.g. of²¹³Bi。

Certain isotopically-labeled peptide ligands of the present invention, for example those incorporating a radioisotope, are useful in drug and/or substrate tissue distribution studies and in clinical assessment of the presence and/or absence of EphA2 targets in diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties as they may be used to detect or identify the formation of complexes between the marker compounds and other molecules, peptides, proteins, enzymes or receptors. The detection or identification method may use a compound labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance (e.g., luminol, a luminol derivative, luciferin, a luminescent protein, luciferase), or the like. Radioisotope tritium, i.e.³H (T) and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and convenient detection means.

With heavier isotopes such as deuterium (i.e.²H (d)) substitution may provide certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferred in some circumstances.

Using positron emitting isotopes (e.g. of the type¹¹C、¹⁸F、¹⁵O and¹³n) substitution can be used in Positron Emission Tomography (PET) studies to examine target occupancy.

Isotopically-labelled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples using appropriate isotopically-labelled reagents in place of the non-labelled reagents previously used.

Aromatic molecule scaffold

The term "aromatic molecular scaffold" as referred to herein refers to any molecular scaffold defined herein comprising an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

It is to be understood that the aromatic molecule scaffold may comprise aromatic moieties. Examples of suitable aromatic moieties within the aromatic scaffold include biphenylene (biphenylene), terphenyl (terphenylene), naphthalene, or anthracene.

It is also understood that the aromatic molecular scaffold may comprise a heteroaromatic moiety. Examples of suitable heteroaromatic moieties within the aromatic scaffold include pyridine, pyrimidine, pyrrole, furan and thiophene.

It is also understood that the aromatic molecule scaffold may comprise a halomethyl arene moiety, such as bis (bromomethyl) benzene, tris (bromomethyl) benzene, tetrakis (bromomethyl) benzene, or derivatives thereof. Non-limiting examples of aromatic molecule scaffolds include: bis, tris, or tetrakis (halomethyl) benzene; bis, tris or tetrakis (halomethyl) pyridines; bis, tris or tetrakis (halomethyl) pyridazine; bis, tris or tetrakis (halomethyl) pyrimidine; bis, tris or tetrakis (halomethyl) pyrazines; bis, tris or tetrakis (halomethyl) -1,2, 3-triazine; bis, tris or tetrakis (halomethyl) -1,2, 4-triazine; bis, tris or tetrakis (halomethyl) pyrroles, furans, thiophenes; bis, tris or tetrakis (halomethyl) imidazoles, oxazoles, thiazoles; bis, tris or tetrakis (halomethyl) -3H-pyrazole, -isoxazole, -isothiazole; bis, tris or tetrakis (halomethyl) biphenylene; bis, tris or tetrakis (halomethyl) terphenyl; 1, 8-bis (halomethyl) naphthalene; bis, tris or tetrakis (halomethyl) anthracenes; and bis, tris or tetrakis (2-halomethylphenyl) methane.

More specific examples of aromatic molecule scaffolds include: 1, 2-bis (halomethyl) benzene; 3, 4-bis (halomethyl) pyridine; 3, 4-bis (halomethyl) pyridazine; 4, 5-bis (halomethyl) pyrimidine; 4, 5-bis (halomethyl) pyrazines; 4, 5-bis (halomethyl) -1,2, 3-triazine; 5, 6-bis (halomethyl) -1,2, 4-triazine; 3, 4-bis (halomethyl) pyrroles, furans, thiophenes and other positional isomers; 4, 5-bis (halomethyl) imidazole, oxazole, thiazole; 4, 5-bis (halomethyl) -3H-pyrazole, -isoxazole, -isothiazole; 2,2' -bis (halomethyl) biphenylene; 2,2 "-bis (halomethyl) terphenyl; 1, 8-bis (halomethyl) naphthalene; 1, 10-bis (halomethyl) anthracene; bis (2-halomethylphenyl) methane; 1,2, 3-tris (halomethyl) benzene; 2,3, 4-tris (halomethyl) pyridine; 2,3, 4-tris (halomethyl) pyridazine; 3,4, 5-tris (halomethyl) pyrimidine; 4,5, 6-tris (halomethyl) -1,2, 3-triazine; 2,3, 4-tris (halomethyl) pyrrole, furan, thiophene; 2,4, 5-bis (halomethyl) imidazole, oxazole, thiazole; 3,4, 5-bis (halomethyl) -1H-pyrazole, -isoxazole, -isothiazole; 2,4,2' -tris (halomethyl) biphenylene; 2,3',2 "-tris (halomethyl) terphenyl; 1,3, 8-tris (halomethyl) naphthalene; 1,3, 10-tris (halomethyl) anthracene; bis (2-halomethylphenyl) methane; 1,2,4, 5-tetrakis (halomethyl) benzene; 1,2,4, 5-tetrakis (halomethyl) pyridine; 2,4,5, 6-tetrakis (halomethyl) pyrimidine; 2,3,4, 5-tetrakis (halomethyl) pyrrole, furan, thiophene; 2,2',6,6' -tetrakis (halomethyl) biphenylene; 2,2 ", 6, 6" -tetrakis (halomethyl) terphenyl; 2,3,5, 6-tetrakis (halomethyl) naphthalene and 2,3,7, 8-tetrakis (halomethyl) anthracene; and bis (2, 4-bis (halomethyl) phenyl) methane.

As described in the previous document, the molecular scaffold may be a small molecule, such as an organic small molecule.

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

In one embodiment, the molecular scaffold comprises a reactive group capable of reacting with a functional group of the polypeptide to form a covalent bond.

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

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

In one embodiment, the molecular scaffold is 2,4, 6-tris (bromomethyl) mesitylene. The molecule is similar to 1,3, 5-tris (bromomethyl) benzene, but contains three additional methyl groups attached to the benzene ring. This has the following advantages: additional methyl groups may form further contacts with the polypeptide and thus add additional structural constraints.

The molecular scaffold of the invention contains chemical groups that allow the functional groups of the polypeptides of the encoded library of the invention to form covalent bonds with the molecular scaffold. The chemical group is selected from a variety of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimides, maleimides, azides, alkyl halides, and acyl halides.

Scaffold reactive groups that can be used to react with the thiol group of cysteine on a molecular scaffold are alkyl halides (or also known as halocarbons or haloalkanes).

Examples include bromomethylbenzene (a scaffold reactive group such as TBMB) or iodoacetamide. Other scaffold reactive groups for selectively coupling compounds to cysteines in proteins are maleimides, compounds containing α β unsaturated carbonyl groups and compounds containing α halomethylcarbonyl groups. Examples of maleimides that can be used as molecular scaffolds of the present invention include: tris- (2-maleimidoethyl) amine, tris- (2-maleimidoethyl) benzene, tris- (maleimido) benzene. An example of an alpha halomethylcarbonyl-containing compound is N, N' - (benzene-1, 3, 5-triyl) tris (2-bromoacetamide). Selenocysteine is also a natural amino acid with similar reactivity to cysteine and can be used in the same reaction. Thus, whenever cysteine is mentioned, substitution with selenocysteine may be accepted generally, unless the context indicates otherwise.

Effectors and functional groups

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

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

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

In a further embodiment of this aspect of the invention, the effector group according to the invention is the Fc region of an IgG molecule. Advantageously, the peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion having a t β half-life 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 advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion having a t β half-life of one day or more.

Functional groups typically include binding groups, drugs, reactive groups for attachment of other entities, functional groups that facilitate uptake of the macrocyclic peptide into a cell, and the like.

The ability of the peptide to penetrate cells will allow the peptide to be effectively directed against the target within the cell. Targets that can be accessed by peptides with the ability to penetrate cells include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in apoptotic pathways. Functional groups capable of penetrating cells include peptides or chemical groups that have been added to peptide or molecular scaffolds. Peptides, such as those derived from homeobox proteins such as VP22, HIV-Tat, Drosophila (Antennapedia), e.g., such as Chen and Harrison, Biochemical Society Transactions (2007) Vol.35, part 4, page 821; gupta et al, Advanced Drug Discovery Reviews (2004) 57, volume 9637. Examples of short peptides that have been shown to be effective in translocation across the plasma membrane include the 16 amino acid penetrating peptide from drosophila antennapedia protein (desossi et al (1994) J biol. chem. 269, p. 10444), the 18 amino acid "model amphipathic peptide" (Oehlke et al (1998) Biochim biophysis Acts, p. 1414, p. 127) and the arginine-rich region of the HIV TAT protein. Non-peptide Methods include the use of small molecule mimetics or SMOCs that can be readily attached to biomolecules (Okuyama et al (2007) Nature Methods, vol.4, p.153). Other chemical strategies that add guanidino groups to the molecule also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem, Vol.282, p.13585). Small molecular weight molecules such as steroids may be added to the molecular scaffold to enhance cellular uptake.

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

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

In a particular embodiment of the invention, the functional group is selected from metal chelators, which are suitable for complexing pharmaceutically relevant metal radioisotopes.

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

In a particular embodiment of the invention, the functional group is selected from drugs, e.g. cytotoxic agents for cancer therapy. Suitable examples include: alkylating agents such as cisplatin and carboplatin, and oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites including the purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids, such as vincristine, vinblastine, vinorelbine, and vindesine; etoposide and teniposide, which are derivatives of podophyllotoxin; taxanes, including paclitaxel (paclitaxel), originally referred to as paclitaxel (Taxol); topoisomerase inhibitors include camptothecin: irinotecan and topotecan, and type II inhibitors, including ambridine, etoposide phosphate, and teniposide. Other agents may include antitumor antibiotics including the immunosuppressive agents actinomycin D (for kidney transplantation), doxorubicin, epirubicin, bleomycin, calicheamicin and the like.

In a further specific embodiment of the invention, the cytotoxic agent is selected from a maytansinoid (such as DM1) or a monomethyl auristatin (e.g. MMAE).

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

monomethyl auristatin e (mmae) is a synthetic antineoplastic agent and has the following structure:

in yet another embodiment of the invention, the cytotoxic agent is selected from maytansinoids (e.g., DM 1). The data, shown in table 11 herein, demonstrates the effect of peptide ligands conjugated to DM 1-containing toxin.

In one embodiment, the cytotoxic agent is linked to the bicyclic peptide through a cleavable bond (e.g., a disulfide bond or a protease-sensitive bond). In a further embodiment, groups adjacent to the disulfide bonds are modified to control the blockage of the disulfide bonds and thereby control the rate of cleavage and concomitant release of the cytotoxic agent.

Published work has identified the potential to alter the susceptibility of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al (2011) Bioconjugate Chemistry,22,717). Greater steric hindrance reduces the rate of reduction of intracellular glutathione and extracellular (systemic) reducing agents, thereby reducing the ease of toxin release both intracellularly and extracellularly. Thus, the optimal choice of disulfide stability in circulation (which minimizes the adverse side effects of the toxin) and efficient release in the intracellular environment (which maximizes the therapeutic effect) can be achieved by carefully selecting the degree of hindrance on either side of the disulfide bond.

The hindrance on either side of the disulfide bond is modulated by the introduction of one or more methyl groups on the targeting entity (here the bicyclic peptide) or toxin side of the molecular construct.

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

In one embodiment, the cytotoxic agent is DM1, and the drug conjugate comprises a compound of formula (a):

wherein said bicyclic ring is selected from any one of compounds 72 as defined herein.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B):

wherein said bicyclic ring is selected from any one of compounds 72, 226, 227, and 303 as defined herein.

In one embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (a), wherein the bicyclic ring is selected from compound 72 as defined herein. This BDC is referred to herein as BDC-1. The data provided herein demonstrate that BDC-1 competes for excellent binding in the EphA2 competitive binding assay, as shown in table 11.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein the bicyclic ring is selected from compound 72 as defined herein. This BDC is referred to herein as BDC-2. The data provided herein demonstrate that BDC-2 competes for excellent competitive binding in the EphA2 competitive binding assay, as shown in table 11.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein the bicyclic ring is selected from compound 226 as defined herein. This BDC is referred to herein as BDC-3. The data provided herein demonstrate that BDC-3 competes for excellent competitive binding in the EphA2 competitive binding assay, as shown in table 11.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein the bicyclic ring is selected from compound 227 as defined herein. This BDC is referred to herein as BDC-4. The data provided herein demonstrate that BDC-4 competes for excellent binding in the EphA2 competitive binding assay, as shown in table 11.

In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate comprises a compound of formula (B), wherein the bicyclic ring is selected from compound 303 as defined herein. This BDC is referred to herein as BDC-5. The data provided herein demonstrate that BDC-5 competes for excellent binding in the EphA2 competitive binding assay, as shown in table 11.

In one embodiment, the drug conjugate is selected from BDC-1 to BDC-5. In another embodiment, the drug conjugate is selected from BDC-1 to BDC-4.

Synthesis of

The peptides of the invention may be prepared synthetically by standard techniques and then reacted with the molecular scaffold in vitro. When doing so, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such a process can be accomplished using conventional chemistry such as that disclosed in Timmerman et al, supra.

Thus, the present invention also relates to the preparation of a polypeptide or conjugate selected as described herein, wherein the preparation comprises optional further steps as illustrated below. In one embodiment, these steps are performed on the final product polypeptide/conjugate prepared by chemical synthesis.

When preparing the conjugate or complex, amino acid residues in the polypeptide of interest may optionally be substituted.

The peptide may also be extended to incorporate, for example, another loop, thus introducing multispecific properties.

For extension of the peptide, it can be chemically extended using standard solid or solution phase chemistry, using orthogonally protected lysines (and the like), simply at its N-or C-terminus or within a loop. The activated or activatable N-or C-terminus can be introduced using standard (bio) conjugation techniques. Alternatively, the addition may be by fragment condensation or Native chemical ligation (e.g., as described in (Dawson et al 1994.Synthesis of Proteins by Natural chemical ligation. science 266: 776-.

Alternatively, the peptide may be extended or modified by further conjugation via a disulfide bond. This has the additional advantage of allowing the first and second peptides to be separated from one another once in the cell reducing environment. In this case, a molecular scaffold (e.g., TBMB) may be added during the chemical synthesis of the first peptide to allow reaction with the three cysteine groups; an additional cysteine or thiol may then be added to the N-or C-terminus of the first peptide such that the cysteine or thiol reacts only with the free cysteine or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

Similar techniques are equally applicable to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially leading to tetraspecific molecules.

Furthermore, the addition of other functional or effector groups can be accomplished in the same manner, using appropriate chemistry, at the N-or C-terminus or by coupling of side chains. In one embodiment, the coupling is performed in a manner that does not block the activity of either entity.

Pharmaceutical composition

According to a further aspect of the invention, there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein and one or more pharmaceutically acceptable excipients.

Generally, the peptide ligands of the invention will be used in purified form together with a pharmacologically appropriate excipient or carrier. Typically, such excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including physiological saline and/or buffered media. Parenteral vehicles include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride and lactated ringer's agents. Suitable physiologically acceptable adjuvants may be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates, if necessary to maintain the polypeptide complex in suspension.

Intravenous carriers include liquid and nutritional supplements and electrolyte supplements such as those based on ringer's dextrose. Preservatives and other additives may also be present, such as antimicrobials, oxidation resistance agents, chelating agents and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16 th edition).

The peptide ligands of the invention may be used as compositions administered alone or in combination with other agents. These may include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Pharmaceutical compositions may include "cocktail mixtures" of various cytotoxins or other agents in combination with the protein ligands of the invention, or even combinations of polypeptides selected according to the invention with different specificities, e.g., polypeptides selected using different target ligands, whether or not combined prior to administration.

The route of administration of the pharmaceutical composition according to the present invention may be those generally known to those of ordinary skill in the art. For therapeutic methods, the peptide ligands of the invention may be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary routes, or, where appropriate, by direct catheter infusion. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dose and frequency of administration depends on the age, sex and condition of the patient, concurrent administration of other drugs, contraindications and other parameters to be considered by the clinician.

The peptide ligands of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective and lyophilization and reconstitution techniques known in the art can be employed. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of loss of activity, and that levels may have to be adjusted upward to compensate.

Compositions containing the peptide ligands of the invention or cocktail thereof can be administered for prophylactic and/or therapeutic treatment. In certain therapeutic applications, a sufficient amount to effect at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease and the general state of the patient's own immune system, but will generally be in the range of from 0.005 to 5.0mg of the selected peptide ligand per kilogram of body weight, more usually in the range of from 0.05 to 2.0 mg/kg/dose. For prophylactic use, compositions containing the peptide ligands of the invention or cocktail thereof can also be administered at similar or slightly lower doses.

Compositions comprising peptide ligands according to the invention may be used to help alter, inactivate, kill or eliminate selective target cell populations in mammals in prophylactic and therapeutic settings. In addition, the peptide ligands described herein can be selectively used to kill, deplete, or otherwise effectively remove a target cell population from a heterogeneous collection of cells in vitro (extracorporeally) or in vitro (in vitro). Blood from the mammal may be combined in vitro with selected peptide ligands, whereby undesirable cells are killed or otherwise removed from the blood and returned to the mammal according to standard techniques.

Therapeutic uses

The bicyclic peptides of the invention have particular utility as EphA2 binding agents. Eph receptor tyrosine kinases (Eph) belong to a large group of Receptor Tyrosine Kinases (RTKs) that phosphorylate proteins on tyrosine residues. Eph and its membrane-bound ephrin ligands (ephrins) control the localization of cells and organization of tissues (tissue) (Poliakov et al (2004) Dev Cell 7, 465-80). Functional biochemical Eph reactions occur at higher ligand oligomerization states (Stein et al (1998) Genes Dev 12, 667-Across 678).

Among other modes of function, various ephs and ephrins are shown to play a role in vascular development. Knockdown of EphB4 and ephrin-B2 resulted in a lack of ability to remodel the capillary bed into blood vessels (Poliakov et al, supra) and resulted in embryonic lethality. Sustained expression of some Eph receptors and ephrins was also observed in newly formed adult microvasculature (Brantley-Sieders et al (2004) Curr Pharm Des 10,3431-42; Adams (2003) J Ant 202,105-12).

It has also been observed that unregulated regeneration of some ephrins and their receptors in adults promotes tumor invasion, metastasis and neovascularization (Nakamoto et al (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al, supra). In addition, some Eph family members have been found to be overexpressed on tumor cells from a variety of human tumors (Brantley-Sieders et al, supra); marme (2002) Ann hemtool 81 Suppl 2, S66; booth et al (2002) Nat Med 8,1360-1).

The EPH receptor a2 (ephrin receptor type a2) is a protein encoded by the EPHA2 gene in humans.

EphA2 is upregulated in a variety of human cancers, and is commonly associated with disease progression, metastasis, and poor prognosis, for example: breast Cancer (Zelinski et al (2001) Cancer Res.61, 2301-2306; Zhuang et al (2010) Cancer Res.70, 299-308; Brantley-Sieders et al (2011) PLoS One 6, e24426), lung Cancer (Brannan et al (2009) Cancer Prev Res (Phila)2, 1039-.

The overall role of EphA2 in cancer progression has not been established, although evidence suggests interactions at many stages of cancer progression, including tumor cell growth, survival, invasion, and angiogenesis. Down-regulation of EphA2 expression inhibited tumor Cancer Cell proliferation (Binda et al (2012) Cancer Cell 22,765-780), while blockade of EphA2 inhibited VEGF-induced Cell migration (Hess et al (2001) Cancer Res.61, 3250-3255), sprouting, and angiogenesis (Cheng et al (2002) Mol Cancer Res.1, 2-11; Lin et al (2007) Cancer 109,332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB J.19, 1884-1886).

Antibody drug conjugates to EphA2 have been shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68,9367-9374), and similar approaches have been tried in humans, although treatment has been forced to be terminated due to treatment-related adverse events (Annunziata et al (2013) InvestNew drugs 31, 77-84).

Polypeptide ligands selected according to the methods of the invention are useful for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, and the like. Ligands with selected levels of specificity are useful in applications involving testing in non-human animals where cross-reactivity is desired, or in diagnostic applications where careful control of cross-reactivity with homologues or paralogs is desired. In some applications, such as vaccine applications, the ability to elicit an immune response to a predetermined range of antigens can be exploited to tailor vaccines against specific diseases and pathogens.

Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for mammalian administration, and 98 to 99% or more homogeneity are most preferred for pharmaceutical use, particularly when the mammal is a human. Once purified as desired (partially purified or purified to homogeneity), the selected polypeptide may be used in diagnosis or therapy (including in vitro) or in developing and performing assay procedures, immunofluorescent staining and the like (Lefkovite and Pernis, (1979 and 1981) immunologicals methods, volumes I and II, Academic Press, NY).

According to another aspect of the present invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or condition characterised by overexpression of EphA2 in a diseased tissue (e.g. a tumour).

According to another aspect of the present invention there is provided a method of preventing, inhibiting or treating a disease or condition characterized by overexpression of EphA2 in a diseased tissue (e.g., a tumor) comprising administering to a patient in need thereof an effector group of a peptide ligand as defined herein and a drug conjugate.

In one embodiment, EphA2 is mammalian EphA 2. In another embodiment, the mammalian EphA2 is human EphA 2.

In one embodiment, the disease or disorder characterized by overexpression of EphA2 in diseased tissue is selected from cancer.

Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and various types of cancers including adenocarcinomas, squamous carcinomas, transitional cell carcinomas, and other cancers), such as bladder and urinary tract cancers, breast cancers, gastrointestinal cancers (including esophageal cancers, gastric (stomach) cancers, small intestine cancers, colon cancers, rectal cancers, and anal cancers), liver cancers (hepatocellular carcinomas), carcinomas of the gallbladder and biliary tract, exocrine pancreatic cancers, kidney cancers, lung cancers (such as adenocarcinomas, small cell lung cancers, non-small cell lung cancers, bronchoalveolar carcinomas, and mesotheliomas), head and neck cancers (such as tongue cancers, buccal cavity cancers, laryngeal cancers, pharyngeal cancers, nasopharyngeal cancers, tonsillar cancers, salivary gland cancers, nasal cavity cancers, and paranasal sinus cancers), ovarian cancers, fallopian tube cancers, peritoneal cancers, vaginal cancers, vulval cancers, penile cancers, cervical cancers, uterine muscle cancers, endometrial cancers, Thyroid cancer (e.g., follicular thyroid cancer), renal cancer, prostate cancer, skin and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematologic malignancies (i.e., leukemia, lymphoma) and premalignant hematologic disorders as well as peripheral malignancies, including hematologic malignancies and disorders associated with lymphoid lineage (e.g., acute lymphocytic leukemia [ ALL ], chronic lymphocytic leukemia [ CLL ], B-cell lymphomas such as diffuse large B-cell lymphoma [ DLBCL ], follicular lymphoma, burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [ NK ] cell lymphoma, hodgkin's lymphoma, hairy cell leukemia, univocal monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and disorders associated with hematologic malignancies and myeloid lineage (e.g., acute myelogenous leukemia [ AML ], chronic myelogenous leukemia [ CML ], chronic myelomonocytic leukemia [ ml ], eosinophilic syndrome, myeloproliferative disorders, such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, and promyelocytic leukemia); tumors of mesenchymal origin, such as soft tissue sarcomas, bone or cartilage sarcomas, e.g. osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, kaposi's sarcoma, ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant tissue sarcomas and dermatofibrosarcoma eminensis; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid tumors); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, wilms' tumor, and primitive neuroectodermal tumors); or congenital or other forms of syndrome, predisposes patients to malignancy (e.g., xeroderma pigmentosum).

In further embodiments, the cancer is selected from: breast cancer, lung cancer, stomach cancer, pancreatic cancer, prostate cancer, liver cancer, glioblastoma and angiogenesis.

In another embodiment, the cancer is selected from lung cancer, e.g., non-small cell lung cancer. The data presented herein demonstrate that BDC (BDC-8) of the present invention completely eradicates non-small cell lung cancer from day 32, with no tumor regrowth occurring at day 28 after the suspended dose. This data clearly demonstrates the clinical utility of BDC of the invention in cancer such as lung cancer, particularly non-small cell lung cancer.

The term "prevention" as referred to herein relates to the administration of a protective composition prior to induction of disease. By "inhibit" is meant administration of the composition after an induction event but prior to clinical manifestation of the disease. "treatment" refers to the administration of a protective composition after symptoms of the disease become apparent.

Animal model systems for screening peptide ligands for effectiveness in preventing or treating disease are available. The present invention allows the development of polypeptide ligands that can cross-react with human and animal targets, thereby allowing the use of animal models, which facilitates the use of animal model systems.

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