Bicyclic peptide ligands specific for P-selectin

Abstract

Bicyclic peptide ligands specific for P-selectin The present invention relates to a polypeptide covalently bound to a molecular scaffold such that two or more peptide loops are internal between the attachment points to the molecular scaffold. In particular, the present invention describes a peptide that binds to P-selectin. The present invention also relates to a multimeric binding complex of a polypeptide covalently bound to a molecular scaffold such that two or more peptide loops are internal between the attachment points to the molecular scaffold that is a binding agent for P-selectin. The present invention also includes drug conjugates comprising said peptides and complexes conjugated to one or more effector groups and / or functional groups, pharmaceutical compositions comprising said peptide ligands, complexes and drug complexes, and the use of said peptide ligands and drug conjugates in the prevention, inhibition or treatment of diseases or disorders mediated by cell adhesion molecules, such as P-selectin, including vaso-occlusive crisis and sickle cell disease-related conditions, cancer, or COVID-19.

Description

[Technical field]

[0001] The present invention relates to a polypeptide covalently bound to a molecular scaffold such that two or more peptide loops are internal between the attachment points to the molecular scaffold. In particular, the present invention describes a peptide that binds to P-selectin. The present invention also relates to a multimeric binding complex of a polypeptide covalently bound to a molecular scaffold such that two or more peptide loops are internal between the attachment points to the molecular scaffold that is a binding agent for P-selectin. Furthermore, the present invention includes drug conjugates comprising said peptides and complexes conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising said peptide ligands, complexes and drug conjugates, and the use of said peptide ligands and drug conjugates to prevent, inhibit or treat diseases or disorders mediated by cell adhesion molecules such as P-selectin, including vaso-occlusive crisis and sickle cell disease-related conditions, cancer or COVID-19. [Background technology]

[0002] Cyclic peptides are an attractive class of molecules for the development of therapeutic drugs, since they can bind to target proteins with high affinity and specificity. Indeed, several cyclic peptides have already been successfully used clinically, for example, the antibacterial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat. Rev. Drug. Discov. 7(7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target, as well as the low structural flexibility in the cyclic structure. Typically, macrocycles bind surfaces of several hundred square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 (400 Å2; Wu et al. (2007), Science 330, 1066-71), a cyclic peptide with an Arg-Gly-Asp motif that binds to integrin αVb3 (355 Å2) (Xiong et al. (2002), Science 296(5565), 151-5), or the cyclic peptide inhibitor upain-1 that binds to urokinase-type plasminogen activator (603 Å2; Zhao et al. (2007), J. Struct. Biol. 160(1), 1-10).

[0003] Peptide macrocycles, due to their cyclic configuration, are less flexible than linear peptides, which results in less entropy lost upon binding to the target and higher binding affinity. The reduced flexibility also leads to locking of target-specific conformations, which increases binding specificity compared to linear peptides. This effect is exemplified by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), which loses its selectivity against other MMPs when its ring is opened (Cherney et al. (1998), J. Med. Chem. 41(11), 1749-51). The advantageous binding properties achieved by macrocyclization are even more pronounced in polycyclic peptides with multiple peptide rings, such as vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously tethered polypeptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers used tris(bromomethyl)benzene and related molecules for rapid and quantitative cyclization of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for the generation of candidate drug compounds, generated by coupling cysteine-containing polypeptides to molecular scaffolds such as 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606).

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

[0006] According to a first aspect of the present invention, there is provided herein a peptide ligand specific for P-selectin comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

[0007] According to a further aspect of the present invention, there is provided herein a multimeric binding complex comprising at least two peptide ligands, wherein at least one peptide ligand is a peptide ligand specific for P-selectin as defined herein, said peptide ligands may be the same or different, each comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

[0008] According to a further aspect of the present invention, there is provided herein a drug conjugate comprising a peptide ligand or multimeric binding complex as defined herein conjugated to one or more effector groups and / or functional groups.

[0009] According to a further aspect of the present invention, there is provided herein a pharmaceutical composition comprising a peptide ligand, multimeric binding complex or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0010] According to a further aspect of the invention, there is provided herein a peptide ligand, multimeric binding complex, drug conjugate or pharmaceutical composition as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by P-selectin. [Brief description of the drawings]

[0011] [Figure 1] Schematic representation of the P-selectin flow adhesion assay.

[0012] [Diagram 2] Effect of BCY12262 on flow adhesion of isolated leukocytes onto P-selectin. Fold change in AI (adhesion index: cells / mm2) from baseline (no drug control, not shown in graph). Means of triplicate data are shown, error bars indicate standard deviation. Dashed lines indicate the mean of the samples shown.

[0013] Detailed Description of the Invention Peptide Ligands According to a first aspect of the present invention, there is provided herein a peptide ligand specific for P-selectin comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

[0014] In one embodiment, the reactive group comprises a cysteine ​​residue.

[0015] In a further embodiment, said peptide ligand comprises the motif WCDV. WCDV amino acid motif has previously been shown to confer binding of linear peptide ligands to P-selectin. However, the inventors have surprisingly found that the binding affinity of such motifs and their modified derivatives is enhanced when incorporated into the bicyclic peptides described herein. In yet a further embodiment, said peptide ligand comprises the modified derivative of the motif WCDV.

[0016] In a further embodiment, the loop sequence comprises 4 or 6 amino acids.

[0017] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence consisting of four amino acids and a second loop sequence consisting of six amino acids.

[0018] In one embodiment, the peptide ligand is C i -X1-X2-X3-X4-C ii -DV-X5-X6-X7-X 8- C iii (SEQ ID NO:1) [In the sequence, X1 represents D or Y; X2 represents A or M; X3 indicates D or E; X4 represents W, 1Nal or Trp(Me); X5 indicates P or T; X6 indicates S or D; X7 represents L or Y; X8 indicates P or G; (where 1Nal represents 1-naphthylalanine and Trp(Me) represents methyl-tryptophan), and C i , C ii and C iii represent the first cysteine ​​residue, the second cysteine ​​residue, and the third cysteine ​​residue, respectively. or a modified derivative thereof, or a pharma- ceutically acceptable salt thereof.

[0019] In a further embodiment, X4 represents W.

[0020] In still further embodiments, C i -X1-X2-X3-X4-C ii -DV-X5-X6-X7-X 8- C iii The peptide ligand of (SEQ ID NO:1) is C i DAD[1Nal]C ii DVPSLPC iii (SEQ ID NO:2); C i DADWC ii DVPSLPC iii (SEQ ID NO:3); C i YME[1Nal]C ii DVTDYGC iii (SEQ ID NO:4); C i YME[Trp(Me)]C ii DVTDYGC iii (SEQ ID NO:5); and C i YMEWC ii DVTDYGC iii (SEQ ID NO:6); [In the sequence, C i , C ii and C iii represent a first cysteine ​​residue, a second cysteine ​​residue, and a third cysteine ​​residue, respectively, or a modified derivative thereof, or a pharma- ceutically acceptable salt thereof.

[0021] In further embodiments, the peptide ligand comprises an N-terminal and / or C-terminal addition, A-(SEQ ID NO:2)-A (referred to herein as BCY12027); H2N-A-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY12026); A-(SEQ ID NO:3)-A (referred to herein as BCY11648); H2N-A-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY12025); Ac-A-(SEQ ID NO:4)-A (referred to herein as BCY11279); A-(SEQ ID NO:4)-A-[K(PYA)] (referred to herein as BCY11890); Ac-A-(SEQ ID NO:5)-A (referred to herein as BCY11281); Ac-(SEQ ID NO:6) (referred to herein as BCY9717); A-(SEQ ID NO:6)-A (referred to herein as BCY10194); A-(SEQ ID NO:6)-A-[K(PYA)] (referred to herein as BCY18041); [PYA]-A-(SEQ ID NO:6)-A-NH2 (referred to herein as BCY10910); and Ac-A-(SEQ ID NO:6)-[K(PYA)]-NH2 (referred to herein as BCY10911): [In the sequence, PYA represents 4-pentynoic acid] is selected from.

[0022] In still further embodiments, the peptide ligand comprises an N-terminal and / or C-terminal addition, A-(SEQ ID NO:2)-A (referred to herein as BCY12027); H2N-A-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY12026); A-(SEQ ID NO:3)-A (referred to herein as BCY11648); H2N-A-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY12025); Ac-A-(SEQ ID NO:4)-A (referred to herein as BCY11279); Ac-A-(SEQ ID NO:5)-A (referred to herein as BCY11281); Ac-(SEQ ID NO:6) (referred to herein as BCY9717); A-(SEQ ID NO:6)-A (referred to herein as BCY10194); [PYA]-A-(SEQ ID NO:6)-A-NH2 (referred to herein as BCY10910); and Ac-A-(SEQ ID NO:6)-[K(PYA)]-NH2 (referred to herein as BCY10911): [In the sequence, PYA represents 4-pentynoic acid] is selected from.

[0023] The peptide of this embodiment exhibits good binding to P-selectin (see Table 6).

[0024] In certain embodiments, the peptide ligand is selected from BCY11648, BCY12027, BCY12025 and BCY12026. In further embodiments, the peptide ligand is selected from BCY12027 and BCY12026. The peptides of these embodiments show very good binding to P-selectin, for example, BCY12027 has a K D It exhibits high affinity, with a binding affinity of less than 10 nM (see Table 6).

[0025] In a further embodiment, the pharma- ceutically acceptable salt is selected from the free acid or a sodium, potassium, calcium or ammonium salt.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, such as peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry techniques. Standard techniques (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) are used for molecular biology, genetics and biochemistry methods.

[0027] Numbering When referring to the position of an amino acid residue in a peptide of the present invention, a cysteine ​​residue (C i , C ii and C iii ) are invariant and are therefore omitted from the numbering, and the numbering of amino acid residues within the peptides of the invention is therefore referred to as follows: -C i -D1-A2-D3-1Nal4-C ii -D5-V6-P7-S8-L9-P 10 -C- iii -(Sequence number: 2).

[0028] Molecular Format Extensions N- or C-terminal to the bicyclic core sequence are added to the left or right side of the sequence, separated by a hyphen. For example, an N-terminal biotin-G-Sar5 tail is shown as follows: [Biot]-G-[Sar5]-A-(SEQ ID NO:X)

[0029] For the purposes of this description, all bicyclic peptides will be referred to as 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) or 1,1',1''-(1,4,7-triazonane-1,4,7-triyl)tris(2-chloroethan-1-one) (TCAZ) or the bromo derivative 1,1',1''-(1,4,7-triazonane-1,4,7-triyl)tris(2-bromoethan-1-one) (TBAZ): [ka] It has been hypothesized that the cyclization may be carried out either at C or C to result in a three-substituted structure. However, as will be apparent from the description of the invention provided herein, cyclization may be carried out using any suitable molecular scaffold that forms a covalent bond with a reactive group of a polypeptide such that at least two polypeptide loops are formed. i , C ii and C iii Happens on top.

[0030] In one embodiment, the molecular scaffold is TATA and the bicyclic peptide is selected from BCY11279, BCY11281, BCY9717, BCY10194, BCY10910 and BCY10911.

[0031] In an alternative embodiment, the molecular scaffold is TCAZ and the bicyclic peptide is selected from BCY12027, BCY11648, BCY12025 and BCY12026.

[0032] Reverse peptide sequence In light of the disclosure of Nair et al. (2003) J. Immunol. 170(3), 1362-1373, it is contemplated that the peptide sequences disclosed herein will also find utility in their retro-inverso forms, for example where the sequence is reversed (i.e., the N-terminus becomes the C-terminus and vice versa) and the stereochemistry is also reversed (i.e., D-amino acids become L-amino acids and vice versa).

[0033] Definition of Peptide Ligand As referred to herein, a peptide ligand refers to a peptide, peptidic or peptidomimetic that is covalently attached to a molecular scaffold. Typically, such a peptide, peptidic or peptidomimetic comprises a peptide having a natural or unnatural amino acid, two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond with the scaffold, and an internal sequence between the reactive groups, referred to as a loop sequence because the peptide, peptidic or peptidomimetic forms a loop when bound to the scaffold. In this case, the peptide, peptidic or peptidomimetic has at least three cysteine ​​residues (referred to herein as a loop sequence). i , C ii and C iii ), which form at least two loops on the scaffold.

[0034] Advantages of peptide ligands Certain bicyclic peptides of the invention have many advantageous properties that allow them to be considered drug-like molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include: -Species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability. Bicyclic peptide ligands should in most cases demonstrate stability against plasma proteases, epithelial proteases ("membrane-anchored" proteases), gastric proteases, intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic peptide lead candidates can be developed not only in animal models but also safely administered to humans; a desirable solubility profile, which is a function of charged residues, the ratio of hydrophilic to hydrophobic residues, and intra- / inter-molecular H-bonds, which are important for formulation and absorption purposes; and - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides with short or long exposure times in vivo for the management of chronic or acute disease states. The optimal exposure time is determined by the need for sustained exposure (to maximize therapeutic efficacy) and the need for a short exposure time to minimize toxicological effects resulting from sustained exposure to the drug.

[0035] Pharmaceutically acceptable salts Salt forms are within the scope of the present invention, and it is understood that a reference to a peptide ligand includes the salt forms of said ligand.

[0036] The salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water, in an organic solvent, or in a mixture of both.

[0037] Acid addition salts (mono- or di-salts) can be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+)-camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyanosulfonic acid, ethyl ester ... Curamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g., hydrobromic acid, hydrochloric acid, , 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, The mono- or di-salts include those formed with acids selected from the group consisting of carboxylic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, valeric acid, as well as acylated amino acids and cation exchange resins.

[0038] One particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0039] The compound is anionic or has a functional group which may be anionic (e.g., -COOH is -COO - In the case of the cations, which may be Li, the salt may be formed with an organic or inorganic base to generate the appropriate cation. Examples of suitable inorganic cations include Li + , Na + and K. + Alkali metal ions such as Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are included, but are not limited to. Some suitable examples of substituted ammonium ions include methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0040] When the peptides of the invention contain amine functions, they may be reacted, for example, with alkylating agents to form quaternary ammonium salts 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.

[0041] modified derivatives It is understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from the following: modification of the N-terminus and / or C-terminus; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with one or more substituted amino acids, such as 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 the bicyclic peptide ligand; surrogate linkage of one or more peptide bonds. modification of the peptide backbone length; replacement of the hydrogen on the α-carbon of one or more amino acid residues with another chemical group; modification of amino acids such as cysteine, lysine, glutamic / aspartic acid and tyrosine with appropriate amine-, thiol-, carboxylic acid- and phenol-reactive reagents to functionalize said amino acids; and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, such as amino acids with azide or alkyne groups to allow functionalization with alkyne- or azide-bearing moieties, respectively.

[0042] In one embodiment, the modified derivative comprises an N-terminus and / or a C-terminus modification.In a further embodiment, the modified derivative comprises an N-terminus modification with suitable amino-reactive chemistry and / or a C-terminus modification with suitable carboxy-reactive chemistry.In a further embodiment, the N-terminus or C-terminus modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator or a chromophore.

[0043] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, the N-terminal residue is capped with acetic anhydride or other suitable reagent during peptide synthesis, which results in an N-terminal acetylated molecule. This embodiment provides the advantage of removing the potential recognition point of aminopeptidase and avoiding the possibility of decomposition of bicyclic peptides.

[0044] In an alternative embodiment, the N-terminal modification involves the addition of a molecular spacer group that facilitates conjugation of an effector group and retains potency of the bicyclic peptide against its target.

[0045] 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-terminal amidated molecule. This embodiment provides the advantage of removing the potential recognition point of carboxypeptidase, reducing the possibility of proteolysis of the bicyclic peptide.

[0046] In one embodiment, the modified derivatives include the substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, the non-natural amino acids can be selected to have isosteric / isoelectronic side chains that are not recognized by degradative proteases and do not adversely affect targeting potency.

[0047] Alternatively, unnatural amino acids with constrained amino acid side chains can be used such that proteolytic hydrolysis of nearby peptide bonds is structurally and sterically hindered. In particular, these concern proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids (a simple derivative is amino-cyclopropyl carboxylic acid).

[0048] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of an N-terminal cysteine ​​(C i ) and / or a C-terminal cysteine ​​(C iii ) including the addition of a spacer group to

[0049] In one embodiment, the modified derivative comprises one or more oxidation-sensitive amino acid residues substituted with one or more oxidation-resistant amino acid residues.In a further embodiment, the modified derivative comprises tryptophan residues substituted with naphthylalanine residues or alanine residues.This embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting bicyclic peptide ligand.

[0050] In one embodiment, the modified derivative comprises the substitution of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the modified derivative comprises the substitution of 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 feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues can affect the degree of plasma protein binding and thus the concentration of the free available fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membranes of the cell surface. The combination of the two can affect the half-life, distribution volume and exposure of the peptide drug, allowing it to be tailored depending on the clinical endpoint. Furthermore, the appropriate combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0051] 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, an embodiment which is believed to increase proteolytic stability due to steric hindrance and the β-turn conformation stabilizing properties of D-amino acids (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0052] In one embodiment, the modified derivative comprises the removal of an amino acid residue or substitution with an alanine, such as D-alanine. This embodiment offers the advantage of identifying critical binding residues and removing potential proteolytic attack sites.

[0053] It should be noted that each of the above mentioned modifications serves to purposefully improve the potency or stability of the peptide. Further potency improvements based on the modifications can be achieved by the following mechanisms: - taking advantage of the hydrophobic effect and incorporating hydrophobic moieties resulting in a lower dissociation rate so that higher affinity is achieved; - the incorporation of charged groups that take advantage of long-range ionic interactions, leading to faster kinetics and higher affinity (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and -Incorporating additional constraints into the peptide, for example by constraining the amino acid side chains correctly so that entropy loss is minimized upon target binding, constraining the backbone torsion angles so that entropy loss is minimized upon target binding, and introducing additional cyclization into the molecule for the same reasons. (For reviews, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0054] Isotope Variations The present invention includes all pharma- ceutically acceptable (radio)isotope-labeled peptide ligands of the invention in which one or more atoms have been replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, as well as peptide ligands of the invention having attached thereto a metal chelating group capable of bearing a relevant (radio)isotope (termed an "effector"), and peptide ligands of the invention in which a particular functional group has been covalently replaced by a relevant (radio)isotope or an isotopically labeled functional group.

[0055] Examples of isotopes suitable for inclusion in the peptide ligands of the invention include isotopes of hydrogen, e.g. 2 H(D) and 3 H(T), an isotope of carbon, e.g. 11 C. 13 C and 14 C, an isotope of chlorine, e.g. 36 Cl, an isotope of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I and 131 I, an isotope of nitrogen, e.g. 13 N and 15 N, isotopes of oxygen, e.g. 15 O. 17 O and 18 O, isotopes of phosphorus, e.g. 32 P, isotopes of sulfur, e.g. 35 S, isotopes of copper, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90Y and isotopes of lutetium, e.g. 177 Lu and bismuth isotopes, e.g. 213 There is Bi.

[0056] Certain isotopically labeled peptide ligands of the invention, such as peptide ligands incorporating radioisotopes, are useful for drug and / or substrate tissue distribution studies and clinical evaluation of the presence and / or absence of DLL3 targets on diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods can use compounds labeled with labeling agents such as radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase). The radioisotope tritium, i.e. 3 H(T), and carbon-14, i.e. 14 C is particularly useful for this purpose in view of its ease of incorporation and ease of preparation of detection means.

[0057] Deuterium, i.e. 2 Substitution with heavier isotopes, such as H(D), may confer certain therapeutic advantages resulting from greater metabolic stability, for example increased half-life in vivo or reduced dosage requirements and therefore may be preferred in some circumstances.

[0058] 11 C. 18 F, 15 O and 13 Substitution with positron emitting isotopes, such as N, is useful for examining target occupancy in Positron Emission Topography (PET) studies.

[0059] Isotopically labeled compounds of the peptide ligands of the present invention may generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the accompanying Examples, substituting appropriate isotopically labeled reagents for previously employed non-labeled reagents.

[0060] Molecular scaffolds In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. As used herein, "non-aromatic molecular scaffold" refers to any molecular scaffold, as defined herein, that does not contain an aromatic (i.e. unsaturated) carbocyclic or heterocyclic ring system.

[0061] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0062] As described in the aforementioned documents, the molecular scaffold may be a small molecule, for example a small organic molecule.

[0063] In one embodiment, the molecular scaffold may be a polymer, hi one embodiment, the molecular scaffold is a polymer composed of amino acids, nucleotides or carbohydrates.

[0064] In one embodiment, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0065] The molecular scaffold may contain chemical groups that form bonds with peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0066] An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606).

[0067] Multimeric Binding Complex According to one aspect of the invention, there is provided herein a multimeric binding complex comprising at least two peptide ligands, wherein at least one peptide ligand is a peptide ligand specific for P-selectin as defined herein, said peptide ligands may be the same or different, each comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

[0068] The present invention describes a series of multimerized bicyclic peptides with different chemical linkers and hinges of different lengths and rigidities, using different binding sites within the bicyclic peptides to bind and activate targets (such as P-selectin) with a wide range of potency and efficacy.

[0069] It will be appreciated by those skilled in the art that the concept of the present invention is the recognition that multi-sequence (multimeric) bicyclic peptides provide synergistic advantages by virtue of the resulting properties of said multimeric binding complexes compared to corresponding monomeric binding complexes comprising a single bicyclic peptide. For example, multimeric binding complexes of the present invention typically exhibit greater levels of binding potency or functional activity (referred to herein as EC) than their monomeric counterparts. 50 In addition, the multimeric binding complexes of the present invention are designed to be large enough to be cleared by the kidney.

[0070] The complex of the present invention is particularly useful for treating cell adhesion molecule-related diseases and disorders, such as those mediated by cell adhesion molecules. Such diseases and disorders include blood diseases and disorders, such as sickle cell disease or sickle cell anemia and vaso-occlusive crisis. In one embodiment, the disease or disorder is associated with sickle cell disease. In another embodiment, the disease or disorder is cancer. In a further embodiment, the disease or disorder is cancer metastasis. In one embodiment, the cell adhesion molecule is P-selectin. In a further embodiment, the cell adhesion molecule is E-selectin and / or L-selectin.

[0071] In certain embodiments, at least one of said peptide ligands is specific for P-selectin, and at least one of said further peptide ligands binds to P-selectin.Thus, in one embodiment, at least two of said peptide ligands bind to P-selectin.In a further embodiment, said two or more P-selectin molecules are present on different target cells.In another embodiment, said two or more P-selectin molecules are present on the same target cell.

[0072] Thus, in certain embodiments, the multimerizing peptide of the present invention comprises at least two peptide ligands specific for P-selectin. In further embodiments, the multimerizing peptide comprises three peptide ligands specific for P-selectin. In another embodiment, the multimerizing peptide comprises four peptide ligands specific for P-selectin.

[0073] In further embodiments, the cells are selected from blood cells, such as red blood cells. In certain embodiments, the cells are red blood cells affected by sickle cell disease or sickle cell anemia. Thus, in one embodiment, the cells are sickle red blood cells.

[0074] Without being bound by theory, it is believed that the multimerized peptide can block cell surface molecules such as cell adhesion molecules. Thus, in one embodiment, the peptide ligands are specific to the same target. In a further embodiment, the multimeric binding complex comprises at least two identical peptide ligands. By "identical" is meant peptides with the same amino acid sequence, and most crucially, the same amino acid sequence means the binding portion of the peptide (e.g., the sequence may vary depending on the binding position). In this embodiment, each peptide in the multimeric binding complex binds to the exact same epitope on the same target--therefore, the resulting target binding complex forms a homodimer (when the multimeric complex contains two identical peptides), a homotrimer (when the multimeric complex contains three identical peptides), or a homotetramer (when the multimeric complex contains four identical peptides), etc.

[0075] In an alternative embodiment, the multimeric binding complex comprises at least two different peptide ligands. By "different" is meant peptides with different amino acid sequences. In this embodiment, the different peptide ligands in the multimeric binding complex bind to different epitopes on the same target--thus, the resulting target binding complex produces biparatopic (when the multimeric binding complex comprises two different peptides), triparatopic (when the multimeric binding complex comprises three different peptides) or tetraparatopic (when the multimeric binding complex comprises four different peptides), etc.

[0076] It is further understood that the multimerization peptide can block or inhibit cell surface molecules, such as different cell adhesion molecules. Thus, in one embodiment, the peptide ligands are specific to different targets. In this embodiment, it is understood that the multimeric binding complex comprises at least two different peptide ligands (i.e., peptide ligands with different amino acid sequences). In this embodiment, each peptide in the multimeric binding complex binds to a different epitope on a different target--thus, the resulting target binding complex produces a bispecific multimeric binding complex (when the multimeric binding complex comprises two different peptides), a trispecific multimeric binding complex (when the multimeric binding complex comprises three different peptides), a tetraspecific multimeric binding complex (when the multimeric binding complex comprises four different peptides), etc. In a further embodiment, each different epitope and / or target is present on the same cell.

[0077] It is understood that the multimeric binding complex of the present invention can be designed to bind to a variety of different targets, such as receptors. Thus, in the case of the bispecific, trispecific, and tetraspecific multimeric binding complexes described above, it is understood that the peptides can bind to targets on at least two different cells (blood vessel cells, endothelial cells, or other blood cells, such as other red blood cells).

[0078] The peptides in the multimeric binding complexes of the invention can be assembled through a number of different options, for example, a central hinge or branch portion with spacer or arm elements radiating from the hinge or branch point, each containing a peptide, or alternatively, it is envisioned that a circular support member could carry multiple peptides projecting inward or outward.

[0079] In one embodiment, each peptide ligand is attached to the central hinge portion by a spacer group.

[0080] It is understood that the spacer group may be linear and may comprise a single peptide attached to the central hinge region. Thus, in one embodiment, the multimeric binding complex comprises a peptide having the formula (I): [ka] [In the formula, CHM indicates the central hinge part; S1 represents a spacer group; Bicycle refers to a peptide ligand as defined herein; and m is an integer from 2 to 10. This includes compounds of the formula:

[0081] In one embodiment, m represents an integer from 2 to 4. In a further embodiment, m represents an integer selected from 2, 3 or 4.

[0082] It is understood that when m represents 4, the middle hinge moiety requires four attachment points. Thus, in one embodiment, m represents 4 and CHM represents formula (A): [ka] [where "-----" indicates the point of attachment to each S1 group] The motif is referred to herein as Tet.

[0083] It is understood that when m represents 3, the middle hinge moiety requires three attachment points. Thus, in one embodiment, m represents 3 and CHM represents formula (B): [ka] [where "-----" indicates the point of attachment to each S1 group] motif (herein referred to as TCA).

[0084] It is understood that when m represents 2, the middle hinge moiety requires two attachment points. Thus, in one embodiment, m represents 2 and CHM represents formula (C): [ka] [In the formula, [ka] indicates the point of attachment to each S1 group] (herein referred to as GTA).

[0085] It will be readily apparent to one skilled in the art how alternative central hinge portions can be constructed depending on the value of m.

[0086] It is understood that the spacer (S1) may be any suitable structure for attaching the peptide central hinge portion to the peptide. In one embodiment, the spacer (S1) comprises a triazolyl moiety. The advantage of this embodiment is that the triazolyl moiety can be incorporated into the synthesis using commonly available "click" chemistry. Examples of suitable spacer (S1) groups include one or more PEG moieties, peptide sequences, carbohydrates, lipids, and the like.

[0087] In further embodiments, the spacer (S1) comprises one or more PEG moieties. References herein to "PEG" refer to the general structure: (CH2CH2O) n (where n is any number such as 1 to 30) and having a regular repeating unit.

[0088] Thus, in a further embodiment, the spacer (S1) is selected from the group consisting of spacers S1A, S1B, S1C, S1D, S1E, S1F, S1G and S1H: [ka] [where: "-----" indicates the point of attachment to the CHM group; and [ka] indicates the point of attachment to the bicyclic group. is selected from one of the following:

[0089] In a further embodiment, the spacer (S1) is S1A. In a further embodiment, n represents 5, 10 or 23.

[0090] Tetrameric binding complex In one embodiment, the multimeric binding complex comprises a tetrameric binding complex as set forth in Tables 1A and 1B below:

[0091] [Table 1]

[0092] In one embodiment, the multimeric binding complex comprises a tetrameric binding complex other than BCY11903 and / or BCY19238.

[0093] [Table 2]

[0094] Data presented herein (see Table 6) show that the tetrameric binding complexes of Table 1B exhibit good binding to P-selectin with affinity KD values ​​of less than 100 nM or less than 0.02 nM. Data presented herein, particularly in Figure 2, show that BCY12262 significantly inhibits leukocyte adhesion compared to a positive control antibody (crizanlizumab).

[0095] In one embodiment, the multimeric binding complex comprises a tetramer comprising four bicyclic peptides, each BCY10910 as defined herein, linked via an N-terminal Pya moiety to a spacer molecule (S1A) where n represents 10 or 23, where (S1A) is linked to a central hinge moiety, (A) as defined herein. The multimeric binding complexes according to this embodiment are referred to herein as BCY5462 and BCY5463, respectively.

[0096] In a further embodiment, the multimeric binding complex comprises a tetramer comprising four bicyclic peptides, each BCY10911 as defined herein, linked via a C-terminal Lys(Pya) moiety to a spacer molecule (S1A) where n represents 10 or 23, where (S1A) is linked to a central hinge moiety, (A) as defined herein. The multimeric binding complexes according to this embodiment are referred to herein as BCY5464 and BCY5465, respectively.

[0097] In one embodiment, the multimeric binding complex comprises a tetramer comprising four bicyclic peptides, BCY12025 or BCY12026 as defined herein, which are linked via C-terminal Lys(Pya) moieties to a spacer molecule (S1A) [wherein n represents 23], where (S1A) is linked to a central hinge moiety, (A) as defined herein. These multimeric binding complexes are referred to herein as BCY12261 and BCY12262, respectively: [ka] [wherein monomer 1 represents BCY12025 or BCY12026, respectively]

[0098] In the data herein, in Table 6, BCY12261 and BCY12262 have affinities K of 0.019 nM and 0.017 nM, respectively. D It has been shown to bind to P-selectin at .

[0099] In an alternative arrangement, the spacer group may be branched, such that a single spacer group may link multiple peptides at a central hinge region. Thus, in an alternative embodiment, the multimeric binding complex has the formula (II): [ka] [In the formula, CHM indicates the central hinge part; S1 represents a spacer group; Bicyclic refers to a peptide ligand as defined herein; and m is an integer from 2 to 10. The present invention includes compounds represented by the formula:

[0100] It will be appreciated that the peptide ligand can be attached to the spacer through a number of means, in one embodiment, the peptide ligand is conjugated to one half of a binding pair, the other half of the binding pair attaching the respective peptide to the spacer.

[0101] In one embodiment, the binding pair comprises biotin and streptavidin. Thus, each peptide ligand is conjugated to biotin and bound to a spacer via streptavidin.

[0102] trimeric binding complex In one embodiment, the multimeric binding complex comprises a trimeric binding complex as set forth in Tables 2A and 2B below: [Table 3]

[0103] In one embodiment, the multimeric binding complex comprises a trimeric binding complex other than BCY19242 and / or BCY19239.

[0104] [Table 4]

[0105] The data herein show that the tetrameric binding complexes of Table 2B have an affinity K D Values ​​below 500 nM or below 0.5 nM indicate good binding (see Table 6).

[0106] In one embodiment, the multimeric binding complex comprises a trimer comprising three bicyclic peptides, each BCY10910 as defined herein, which are linked via N-terminal Pya moieties to a spacer molecule (S1A) [wherein n represents 10 or 23], where (S1A) is linked to a central hinge moiety, (B) as defined herein. The multimeric binding complexes according to this embodiment are referred to herein as BCY5458 and BCY5459, respectively.

[0107] In a further embodiment, the multimeric binding complex comprises a trimer comprising three bicyclic peptides, each of which is BCY10911 as defined herein, and these bicyclic peptides are linked via a C-terminal Lys(Pya) moiety to a spacer molecule (S1A) [wherein , n represents 10 or 23], where (S1A) is attached to the central hinge portion, which is (B) as defined herein. Multimeric binding complexes according to this embodiment are referred to herein as BCY5460 and BCY5461, respectively.

[0108] In one embodiment, the multimeric binding complex comprises a trimer comprising three bicyclic peptides, BCY12025 or BCY12026 as defined herein, which are linked via C-terminal Lys(Pya) moieties to a spacer molecule (S1A) [wherein n represents 23], where (S1A) is linked to a central hinge moiety, (B) as defined herein. These multimeric binding complexes are referred to herein as BCY12259 and BCY12260, respectively.

[0109] In the data herein, in Table 6, BCY12259 and BCY12260 have affinities K of 0.233 nM and 0.109 nM, respectively. D It has been shown to bind to P-selectin at 100 ng / mL.

[0110] Tandem Binding Complex In one embodiment, the multimeric binding complex comprises a tandem binding complex as set forth in Tables 3A and 3B below:

[0111] [Table 5]

[0112] In one embodiment, the multimeric binding complex comprises a dimeric binding complex other than BCY19243 and / or BCY19240.

[0113] [Table 6]

[0114] The data herein show that the dimeric binding complexes of Table 3B have an affinity for P-selectin, K D Values ​​of about 1200 nM, less than 1000 nM, less than 500 nM or less than 0.5 nM have been shown to indicate good binding (see Table 6).

[0115] In one embodiment, the multimeric binding complex comprises a tandem comprising two bicyclic peptides, each BCY10910 as defined herein, which are linked via their N-terminal PYA moieties to a spacer molecule (S1A) [wherein n represents 10 or 23], where (S1A) is linked to a central hinge moiety, which is (C) as defined herein. The multimeric binding complexes according to this embodiment are referred to herein as BCY5454 and BCY5455, respectively.

[0116] In a further embodiment, the multimeric binding complex comprises a tandem comprising two bicyclic peptides, each BCY10911 as defined herein, which are linked via a C-terminal Lys(Pya) moiety to a spacer molecule (S1A) [wherein n represents 10 or 23], where (S1A) is linked to a central hinge moiety, which is (C) as defined herein. The multimeric binding complexes according to this embodiment are referred to herein as BCY5456 and BCY5457, respectively.

[0117] In one embodiment, the multimeric binding complex comprises a tandem comprising two bicyclic peptides, each of which is BCY12025 or BCY12026 as defined herein, which are linked via a C-terminal Lys(Pya) moiety to a spacer molecule (S1A) [wherein n represents 23], where (S1A) is linked to a central hinge moiety, which is (C) as defined herein. These multimeric binding complexes are referred to herein as BCY12257 and BCY12258, respectively.

[0118] The data herein show that BCY12257 and BCY12258 have affinities K of 11.6 nM and 0.409 nM, respectively, in Table 6. D It has been shown to bind to P-selectin at 100 ng / mL.

[0119] Linker It is understood that the P-selectin peptide ligand may be conjugated to a second peptide ligand via any suitable linker. Typically, the linker is designed in such a way that the two bicyclic peptides can bind unhindered to their respective targets, either alone or simultaneously, while binding to both target receptors. Furthermore, the linker should allow binding to both targets simultaneously while maintaining an appropriate distance between the target cells or receptors that results in the desired functional outcome. The nature of the linker can be adjusted to increase the length, rigidity, or solubility to optimize the desired functional outcome. The linker can also be designed to allow multiple bicyclics to be attached to the same target. Increasing the valency of either binding peptide can help improve the affinity of the complex to the target cell or block one or both of the target cell surface molecules.

[0120] In one embodiment, the linker is selected from the following sequences: -CH2-, -PEG5-, -PEG 10 -,-PEG 12 -,-PEG 23 -,-PEG 24 -,-PEG 15 -Sar5-,-PEG 10 -Sar 10 -,-PEG5-Sar 15 -, -PEG5-Sar5-, -B-Ala-Sar 20 -,-B-Ala-Sar 10 -PEG 10 -,-B-Ala-Sar5-PEG 15 - and -B-Ala-Sar5-PEG5-.

[0121] Structural representations of suitable linkers are detailed below: [ka] [ka] [ka]

[0122] synthesis The peptides of the present invention can be synthetically produced by standard techniques and then reacted in vitro with molecular scaffolds, using standard chemical reactions. This allows for rapid large-scale preparation of soluble material for further downstream experiments or validation. Such methods can be accomplished by conventional chemical methods such as those disclosed in Timmerman et al. (supra).

[0123] The present invention therefore also relates to the production of a polypeptide or conjugate selected as defined herein, which production comprises optional further steps as explained below, which in one embodiment are carried out on a polypeptide / conjugate that is the final product produced by chemical synthesis.

[0124] Amino acid residues in the polypeptide of interest may optionally be substituted in producing the conjugate or complex.

[0125] The peptide can also be extended to introduce multiple specificities, for example by incorporating additional loops.

[0126] To extend a peptide, it can simply be chemically extended at its N- or C-terminus or within a loop using orthogonally protected lysines (and analogues) using standard solid-phase or solution-phase chemistry. Activated or activatable N- or C-termini may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be performed enzymatically, for example by fragment condensation or native chemical ligation as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or using subtiligase as described in (Chang et al. Proc Natl Acad Sci US A. 1994 Dec 20; 91(26):12544-8 or in Hikari et al. Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0127] Alternatively, peptides may be extended or modified by further conjugation via disulfide bonds. This has the added advantage of allowing the first and second peptides to dissociate from each other in the reductive environment of cells. In this case, a molecular scaffold (e.g., TATA or TCAZ) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; thus, an additional cysteine ​​or thiol can be added to the N-terminus or C-terminus of the first peptide, and this cysteine ​​or thiol can only react with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0128] Similar techniques are equally applicable to the synthesis / coupling of two bicyclic bispecific macrocycles, potentially giving rise to tetraspecific molecules.

[0129] Furthermore, the addition of other functional or effector groups may be achieved in the same manner, by appropriate chemical methods, coupling at the N-terminus or C-terminus, or via a side chain. In one embodiment, the coupling is performed in such a way as not to block the activity of either entity.

[0130] Drug Conjugates According to a further aspect of the present invention there is provided a drug conjugate comprising a peptide ligand or multimeric binding complex as defined herein conjugated to one or more effector groups and / or functional groups.

[0131] Effector groups and / or functional groups may be attached, for example, to the N-terminus and / or C-terminus of the polypeptide, to amino acids within the polypeptide, or to a molecular scaffold.

[0132] Suitable effector groups include antibodies and their parts or fragments. For example, effector groups can 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, in addition to one or more constant region domains. Effector groups can also include an antibody hinge region (such a region is usually found between the CH1 and CH2 domains of an IgG molecule).

[0133] In a further embodiment of this aspect of the invention, the effector group according to the invention is an 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 tβ half-life of 1 day or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more or 7 days or more. Most advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion having a half-life of 1 day or more.

[0134] Functional groups generally include linking groups, reactive groups for attaching drugs or other entities, functional groups that aid in cellular uptake of the macrocyclic peptide, and the like.

[0135] The ability of peptides to penetrate cells allows them to exert their effects on intracellular targets. Targets accessible by peptides with cell penetration capabilities include intracellular signaling molecules such as transcription factors, tyrosine kinases, molecules involved in apoptotic pathways, etc. Functional groups that allow cell entry include peptides or chemical groups added to either the peptide or the molecular scaffold. For example, peptides such as peptides derived from VP22, HIV-Tat, Drosophila homeobox protein (antennapedia), etc., as described 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 efficient at crossing plasma membranes include the 16 amino acid penetratin peptide from the Drosophila antennapedia protein (Derossi et al. (1994) J Biol. Chem. Volume 269 p10444), the 18 amino acid "model amphipathic peptide" (Oehlke et al. (1998) Biochim Biophys Acts Volume 1414 p127), and the arginine-rich region of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimetics or SMOCs that can be easily conjugated to biomolecules (Okuyama et al. (2007) Nature Methods Volume 4 p153). Other chemical strategies that add guanidinium groups to molecules also enhance cell penetration (Elson-Scwab et al. (2007) J Biol Chem Volume 282 p13585). Small molecular weight molecules such as steroids can be added to the molecular scaffold to improve cellular uptake.

[0136] One class of functional groups that can be attached to the peptide ligands includes antibodies and their binding fragments, such as Fab, Fv or single domain fragments. In particular, antibodies that bind to proteins that can increase the in vivo half-life of the peptide ligand can be used.

[0137] In one embodiment, the peptide ligand-effector group according to the invention has a tβ half-life selected from the group consisting of: 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more or 20 days or more. Advantageously, the peptide ligand-effector group or composition according to the invention has a tβ half-life in the range of 12 to 60 hours. In a further embodiment, it has a tβ half-life of 1 day or more. In a further embodiment, it is in the range of 12 to 26 hours.

[0138] In one particular embodiment of the invention, the functional group is selected from metal chelators suitable for complexing medicamentously relevant metal radioisotopes.

[0139] Possible effector groups also include enzymes, for example carboxypeptidase G2, used in enzyme / pharmacotherapy, where a peptide ligand replaces an antibody in ADEPT.

[0140] In one embodiment, the multimeric binding complex of the present invention comprises a cleavable bond, such as a disulfide bond or a protease-sensitive bond. Without being bound by theory, it is believed that such a cleavable moiety inactivates the complex until it reaches the tumor microenvironment. The advantage of this embodiment is that the size of the complex is reduced after binding to the target. In a further embodiment, the groups adjacent to the disulfide bond are modified to control the inhibition of the disulfide bond, thereby controlling the rate of cleavage and the subsequent release of the binding agent.

[0141] Published studies have established that the susceptibility of disulfide bonds to reduction can be modified by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). Greater degrees of steric hindrance decrease the rate of reduction by extracellular (systemic) reducing agents as well as intracellular glutathione, thereby decreasing the ease with which toxins can be released both inside and outside the cell.

[0142] Thus, optimal selection of disulfide stability in circulation (minimizing unwanted side effects of the toxin) and efficient release in the intracellular environment (maximizing therapeutic efficacy) can be achieved by carefully selecting the degree of blockage on either side of the disulfide bond.

[0143] The barrier on both sides of the disulfide bond is modified by introducing one or more methyl groups into the targeting entity (here, the bicyclic peptide ligand).

[0144] Pharmaceutical Compositions According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand, multimeric binding complex or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0145] Generally, the peptide ligands of the present invention are utilized in purified form together with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and the like. Suitable physiologically acceptable adjuvants, if necessary to keep the polypeptide complex in suspension, may be selected from viscosity enhancing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0146] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0147] The peptide ligands of the invention may be used as a composition administered separately or in combination with other agents. These may include antibodies, antibody fragments, and various immunotherapeutic agents, such as cyclosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. In addition, examples of other agents that may be administered separately or in combination with the peptide ligands of the invention include cytokines, lymphokines, other hematopoietic factors, thrombolytic factors, antithrombotic factors, etc. Pharmaceutical compositions may include "cocktails" of various cytotoxic or other agents in combination with the protein ligands of the invention, or may also include combinations of selected polypeptides according to the invention with different specificities, such as polypeptides selected with different targeting ligands, whether or not pooled prior to administration.

[0148] The route of administration of the pharmaceutical composition according to the invention may be one generally known to those skilled in the art. For therapy, the peptide ligand of the invention can be administered to any patient according to standard techniques. Administration can be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, via the pulmonary route, and preferably by direct injection using a catheter. Preferably, the pharmaceutical composition according to the invention is administered intravenously. The dosage and frequency of administration depend on the age, sex and condition of the patient, the co-administration of other drugs, contraindications and other parameters that should be taken into account by the clinician.

[0149] The peptide ligand of the present invention can be freeze-dried for storage and reconstituted in suitable carrier before use.This technique has been shown to be effective, and can employ freeze-drying and reconstitution techniques known in the art.Those skilled in the art will understand that freeze-drying and reconstitution may cause varying degrees of activity loss, and may need to adjust levels upward to compensate.

[0150] Compositions containing the peptide ligands or cocktails thereof of the present invention can be administered for prophylactic and / or therapeutic treatments. In a particular therapeutic application, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a population of selected cells is defined as a "therapeutically effective dose". The amount required to achieve this dose depends on the severity of the disease, but generally 0.005-5.0 mg of the selected peptide ligand per kg of body weight is used, with doses of 0.05-2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof of the present invention may be administered at similar or slightly lower doses.

[0151] Compositions containing peptide ligands according to the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing or removal of selective target cell populations in mammals. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to kill, deplete or otherwise effectively remove target cell populations from heterogeneous cell populations. Blood from a mammal can be combined ex vivo with a selected peptide ligand, whereby undesirable cells are killed or removed from the blood and returned to the mammal according to standard techniques.

[0152] therapeutic use The bicyclic peptides of the invention have specific utility as P-selectin binding agents. According to a further aspect of the invention there is provided a peptide ligand, multimeric binding complex, drug conjugate or pharmaceutical composition as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by cell adhesion molecules such as P-selectin, E-selectin and L-selectin.

[0153] P-selectin is a cell adhesion molecule expressed on the surface of activated endothelial cells and platelets. In non-activated endothelial cells and platelets, P-selectin is stored in granules. P-selectin is involved in leukocyte recruitment during inflammation and platelet aggregation following vascular injury. Upon platelet activation by thrombin, α-granules and dense granules are released, exposing the inner wall of the cell to the outside, a process known as "membrane flipping." Platelet aggregation is promoted by P-selectin through platelet-fibrin and platelet-platelet binding.

[0154] P-selectin has also been shown to have a role in tumor metastasis, similar to E-selectin (Kohler et al., British Journal of Cancer 2010, 102:3). Expression of P-selectin on the surface of activated endothelial cells and platelets also aids in the infiltration of cancer cells into the bloodstream. The formation of complexes between activated platelets and tumor cells in the vasculature prevents tumor cell recognition by macrophages, shielding the tumor cells from recognition by the immune system. Reduction of circulating platelets has been shown to reduce cancer metastasis (Gasic GL, Cancer Metastasis Reviews 1984, 3:2). Furthermore, a related cell adhesion molecule, E-selectin, has been shown to be constitutively expressed on the surface of bone marrow endothelium, indicating a preferential route for tumor cells to metastasize to bone.

[0155] Roles of P-selectin in cardiovascular diseases such as cardiac inflammation and fibrosis, coronary heart disease, hypertension and atrial fibrillation have also been reported (Liu et al., Molecular Medicine Reports 2016, 13(6) and Blann et al., European Heart Journal 2003, 24(24)). For example, increased expression of P-selectin has been demonstrated in active atherosclerotic plaques, and animals lacking P-selectin have been found to have a reduced tendency to form atherosclerotic plaques. Furthermore, the P-selectin antagonist inclacumab reduced myocardial damage and inhibited platelet-leukocyte aggregation after percutaneous coronary intervention in patients with non-ST-segment elevation myocardial infarction (Stahli et al., Journal of the American Heart Association 2016, 16:5(11) and Schmitt et al., Jornal of Cardiovascular Pharmacology 2015, 65(6)). Thus, antagonism or blocking of P-selectin may be of therapeutic benefit.

[0156] In one embodiment, the P-selectin is a mammalian P-selectin. In a further embodiment, the mammalian P-selectin is a human P-selectin.

[0157] P-selectin peptides are primarily used (but not limited to) to agonistically block P-selectin or related cell adhesion molecules (e.g., E-selectin and / or L-selectin) and, as a result, activate endothelial cells and platelets. It is understood that such antagonism is particularly useful in preventing, inhibiting and / or treating vaso-occlusive crisis or other diseases / disorders associated with sickle cell disease or sickle cell anemia.

[0158] P-selectin peptides can also be used to agonistically block P-selectin or related cell adhesion molecules (e.g., E-selectin and / or L-selectin) binding to glycoprotein or glycolipid ligands expressed on cancer cells or cancers, and thus block metastasis or tumor invasion of said cancer cells or cancers. Such cancers include, but are not limited to, early or late stage human malignancies, including solid tumors such as non-small cell lung cancer (NSCLC), breast cancer, including triple negative breast cancer (TNBC), ovarian cancer, prostate cancer, bladder cancer, urothelial cancer, colorectal cancer, head and neck cancer, squamous cell carcinoma of the head and neck (SCCHN), melanoma, pancreatic cancer, and other advanced solid tumors. The cancer may also be selected from hematological cancers.

[0159] The term "solid tumor" as used herein is defined as an abnormal growth of tissue that does not contain significant liquid mass, whereas non-solid tumors are generally dispersed cancers that do not contain any solid mass or do not contain significant solid mass. Examples of solid tumors include carcinomas, sarcomas and lymphomas. Blood cancers (leukemias), such as acute myeloid leukemia (AML), acute lymphocytic (or lymphoblastic) leukemia (ALL), are non-solid tumors. The P-selectin peptides or complexes defined herein can be used as monotherapy in the aforementioned cancer indications to block, prevent or reduce metastasis of tumor cells. In addition to use as monotherapy in cancer, the P-selectin peptides defined herein, as well as complexes and conjugates thereof, can be used in combination with immunotherapeutic agents such as anti-PD-1 agents and anti-CTLA4 agents. Further therapeutic applications of the P-selectin peptides, complexes and conjugates thereof include, but are not limited to, monotherapy or combination therapy with radiation cancer treatment, and cancer vaccines. Non-cancer therapeutic applications of P-selectin peptides as monotherapy or combination therapy include, but are not limited to, diseases and disorders associated with sickle cell disease / anemia, such as vaso-occlusive crisis.

[0160] It will be appreciated that P-selectin peptides are useful in the treatment of conditions characterized by cell-cell adhesion mediated by P-selectin or related cell adhesion molecules (eg, E-selectin and / or L-selectin). Such conditions include, but are not limited to, post-ischemic leukocyte-mediated tissue damage (reperfusion injury), e.g., associated with myocardial infarction or transplantation; myocardial infarction; bacterial or viral infection; metastatic conditions; inflammatory disorders, e.g., arthritis; acute respiratory distress syndrome (ARDS); asthma; emphysema; delayed type hypersensitivity reactions; systemic lupus erythematosus (SLE); burns; autoimmune thyroiditis; experimental allergic encephalomyelitis (EAE); multiple sclerosis (MS); multiple organ injury syndrome secondary to trauma; diabetes; Raynaud's syndrome; neutrophilic dermatosis (Sweet's syndrome); inflammatory bowel disease (IBD); Graves' disease; glomerulonephritis; gingivitis; periodontitis; hemolytic uremic syndrome; ulcerative colitis; Crohn's disease; necrotizing enterocolitis; granulocyte transfusion associated syndrome; and cytokine-induced toxicity. Further examples of conditions or procedures in which P-selectin peptides may be useful include organ transplantation (including preparation of organs prior to transplantation and mitigation of transplant rejection); hemodialysis; and leukopheresis.

[0161] Thus, in one embodiment, the bicyclic peptides of the present invention find utility in and can be used to treat vaso-occlusive crisis. In a further embodiment, the bicyclic peptides can be used to treat or prevent metastasis. In a further embodiment, the bicyclic peptides can be used to treat or prevent cardiac inflammation, fibrosis and atherosclerotic plaque.

[0162] It is understood that the multimeric binding complexes defined herein, comprising at least one peptide ligand that binds to a cell adhesion molecule, such as P-selectin on endothelial cells, are also useful in the treatment of cancer and non-cancerous diseases and disorders, such as vascular occlusive crisis, cardiac inflammation, fibrosis and atherosclerotic plaque.

[0163] It is understood that the multimeric binding complexes defined herein, comprising at least one peptide ligand that binds to a cell adhesion molecule, such as P-selectin on endothelial cells, are also useful for the treatment of COVID-19.

[0164] COVID-19 is primarily a respiratory disease, but is also considered a multi-organ disease. Mounting evidence has revealed that the SARS-CoV-2 virus attacks organs and tissues expressing angiotensin-converting enzyme 2 (ACE2) receptors, including the heart, kidneys, liver, pancreas, gastrointestinal tract, brain, and endothelial cells, subsequently triggering an inflammatory cascade and blood clotting. COVID-19 can cause severe vascular complications, resulting in endothelial dysfunction, cytokine overproduction, and pathological cell-cell interactions. Red blood cells (RBCs) are the most abundant cellular component in blood and are responsible for transporting oxygen, removing waste products, and delivering nutrients throughout the human body. Previous studies have suggested that SARS-CoV-2 infection also damages RBC membranes, reduces hemoglobin (Hb) concentrations, and impedes red blood cell (RBC) formation, which leads to severe anemia and irreversible organ damage. Thus, one plausible pathogenic mechanism of the "post-COVID syndrome" is the combination of red blood cell abnormalities and accumulation of inflammatory factors, which may contribute, at least in part, to the prolonged symptoms observed in a significant portion of COVID patients (leading to insufficient oxygenation, hypercoagulability, and vascular complications, including chronic fatigue, excise intolerance, joint pain, chest pain, and brain fog). Since P-selectin dysregulation is a key factor in the pathology of COVID-19, inhibiting inflammation-driven P-selectin-mediated adhesion of blood cells to the endothelium may be a promising therapeutic strategy to alleviate adverse symptoms, promote functional recovery, and improve long-term health for millions of people affected by the disease.

[0165] As used herein, the term "prevention" includes administration of the protective composition prior to the induction of disease. "Suppression" refers to administration of the composition after an inductive event, prior to the clinical appearance of disease. "Treatment" includes administration of the protective composition after symptoms of disease are manifest.

[0166] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease. The use of animal model systems is facilitated by the present invention, which allows for the development of polypeptide ligands that can cross-react with human and animal targets, and allows for the use of animal models.

[0167] The invention will now be further described with reference to the following examples. EXAMPLES

[0168] material and method Preparation of monomeric bicyclic peptide ligands (general method) Bicyclic peptides were synthesized on Rink amide resin using standard Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis with manual coupling (for large scale) or on a Biotage SyroII automated peptide synthesizer (for small scale). After TFA-based cleavage from the resin, the peptides were precipitated with diethyl ether and dissolved in 50:50 acetonitrile / water. The crude peptides (~1 mM concentration) were then cyclized with 1.3 equivalents of the scaffold using ammonium bicarbonate (100 mM) as the base. Completion of the cyclization was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) or LC-MS. Once complete, the cyclization reaction was quenched with N-acetylcysteine ​​(10 equivalents relative to peptide) and the solution was lyophilized. The residue was dissolved in an appropriate solvent and purified by RP-HPLC. Peptide fractions of sufficient purity and correct molecular weight (verified by MALDI-TOF and HPLC or LC-MS) were pooled and lyophilized, and concentrations were determined by UV absorption at 280 nm with extinction coefficients based on Trp / Tyr content.

[0169] Unless otherwise noted, all amino acids were used in the L-configuration.

[0170] Preparation of dimeric / tandem binding complexes (exemplified with BCY5455) Synthesis of hinge GTA (PEG23-N3): [ka]

[0171] To a solution of compound 1 (10 mg, 59.17 μmol, 7.58 μL, 1 eq) in DCM (2 mL) was added triethylamine (17.96 mg, 177.51 μmol, 24.71 μL, 3 eq). Compound 2 (132.0 mg, 120.08 μmol, 2.03 eq.) was then added. The mixture was stirred at 30 °C for 2 hr. LC-MS showed that compound 1 was completely consumed and one main peak with the desired m / z was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent to obtain the residue. The residue was purified by preparative HPLC. GTA(PEG23-N3) (82 mg, 35.02 μmol, 59.19% yield, 98% purity) was obtained as a white solid. (MW: 2294.69. Observed m / z: 1156.31 [M+H3O+H] 2+ ).

[0172] Preparation of BCY5455: [ka]

[0173] To a solution of GTA(PEG23-N3) (10 mg, 4.36 μmol, 1 eq.), BCY10910 (20.00 mg, 9.72 μmol, 2.23 eq.) and THPTA (0.4 M, 10.89 μL, 1 eq.) in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged with N2 three times), CuSO4 (0.4 M, 10.89 μL, 1 eq.) and VcNa (0.4 M, 21.79 μL, 2 eq.) were added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (1:1 in t-BuOH / H2O), turning the solution pale yellow. The reaction mixture was stirred at 40 °C under N2 atmosphere for 12 h. LC-MS showed that compound 3 was completely consumed and one main peak of the desired m / z was detected. The residue was purified by preparative HPLC. BCY5455 (13.6 mg, 2.07 μmol, 47.56% yield, 97.70% purity) was obtained as a white solid (MW: 6411.26 observed m / z: 1603.1 [M+4H] 4+ , 1282.4 [M+5H]5+ , 1068.95 [M+6H] 6+ ).

[0174] Preparation of trimeric binding complexes (exemplified by BCY12259) Synthesis of Hinge TCA (PEG23-N3): [ka]

[0175] To a solution of compound 1 (32 mg, 79.63 μmol, 1.0 eq, HCl) and compound 2 (264.00 mg, 240.15 μmol, 3.02 eq.) in DMF (2 mL), HOBt (35.84 mg, 265.24 μmol, 3.33 eq.), EDCI (51.20 mg, 267.08 μmol, 3.35 eq.), DMAP (29.19 mg, 238.90 μmol, 3.0 eq.) and DIPEA (61.75 mg, 477.81 μmol, 83.23 μL, 6.0 eq.) were added. The mixture was stirred at 25-30 °C for 12 hr. LC-MS showed that compound 1 was completely consumed and one main peak of the desired m / z was detected. The reaction mixture was concentrated under reduced pressure and the residue was obtained by removing the solvent. The residue was purified by preparative HPLC. TCA(PEG23-N3) (214 mg, 53.36 μmol, 67.01% yield, 90% purity) was obtained as a pale yellow oil (MW: 3609.23 observed m / z: 1214 [M+2H3O+H] 3+ ).

[0176] Preparation of BCY12259: [ka]

[0177] To a solution of TCA(PEG23-N3) (10 mg, 2.77 μmol, 1 eq.), BCY12025 (17 mg, 8.41 μmol, 3.03 eq.) and THPTA (0.4 M, 6.93 μL, 1 eq.) in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged with N2 three times), CuSO4 (0.4 M, 6.93 μL, 1 eq.) and VcNa (0.4 M, 13.85 μL, 2 eq.) were added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), turning the solution pale yellow. The reaction mixture was stirred at 40 °C under N2 for 12 hr. LC-MS showed that compound 3 was completely consumed and one main peak of the desired m / z was detected. The residue was purified by preparative HPLC. BCY12259 (14 mg, 1.34 μmol, 48.28% yield, 92.46% purity) was obtained as a white solid (MW: 9676.10, observed m / z: 1382.2 [M+7H]). 7+ , 1209.3 [M+8H] 8+ , 1075.1 [M+9H] 9+ , 967.3 [M+10H] 10+ ).

[0178] Preparation of tetrameric binding complexes (exemplified by BCY5464) Synthesis of hinge-TET(PEG10-N3): [ka]

[0179] A mixture of compound 1 (52.0 mg, 122.53 μmol, 1 eq.), compound 2 (273.0 mg, 518.40 μmol, 4.23 eq.), EDCI (117.5 mg, 612.64 μmol, 5.0 eq.) and HOBT (72.8 mg, 539.12 μmol, 4.08 eq.) was dissolved in DMF (2 mL), then DIPEA (126.7 mg, 980.22 μmol, 170.74 μL, 8.0 eq.) was added and the solution was mixed well. The resulting mixture was stirred at 25 °C for 12 hr. LC-MS showed one main peak of the desired m / z. The reaction mixture was concentrated under reduced pressure to remove the solvent to give the residue. The residue was purified by preparative HPLC. TET(PEG10-N3) (147.8 mg, 55.79 μmol, 45.53% yield, 92.81% purity) was obtained as a white solid. (MW: 2458.81. Observed m / z: 810.7 [M-28+3H] 3+ ).

[0180] Preparation of BCY5464: [ka]

[0181] A solution of TET(PEG10-N3) (5 mg, 2.03 μmol, 1 eq.), BCY10911 (19 mg, 8.81 μmol, 4.33 eq.) and THPTA (0.4 M, 5.0 μL, 1 eq.) in t-BuOH / H2O (1:1, 2 mL, previously degassed and purged with N2) was prepared. CuSO4 (0.4 M, 5.0 μL, 1 eq.) and VcNa (0.4 M, 10.0 μL, 2 eq.) were added under N2. The pH of the solution was adjusted to 8 by dropwise addition of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), turning the solution pale yellow. The reaction mixture was stirred at 40 °C under N2 atmosphere for 16 hr. LC-MS showed that compound 3 was completely consumed and one main peak of the desired m / z was detected. The crude reactions were combined and purified by preparative HPLC. BCY5464 (12.6 mg, 92.6% purity) was obtained as a white solid (MW: 11088.49 observed m / z: 1233.6 [M+9H] 9+ , 1387.2 [M+8H] 8+ ).

[0182] In general, it will be understood that all multimeric binding complexes of the invention can be prepared according to these methods using the appropriate bicyclic peptide ligand.

[0183] Analysis data The following multimeric binding complexes of the invention were analyzed using mass spectrometry and HPLC, and the data are presented in Tables 4 and 5 below.

[0184] The HPLC settings were as follows: Methods A, B, and E: Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 1.0ml / min Column: Gemini-NX C18 5μm 110A 150*4.6mm Instrument: Agilent 1200 HPLC-BE(1-614) gradient: [Table 7]

[0185] Method C: Mobile phase: A: 0.1% TFA in H2O B: 0.1% TFA in ACN Flow: 0.4ml / min Column: Kintex 1.7μm C18 100A 2.1mm*150mm Equipment: Agilent UPLC 1290 Column temperature: 30℃ gradient: [Table 8]

[0186] Method D: Mobile phase: 0.1% TFA AcN / water Flow: 1.00ml / min Column:KINETEXPSC18 Equipment: Aglient LC2 Gradient: 5 to 95% B in 15.5 min [Table 9]

[0187] [Table 10]

[0188] Biological Data 1.SPR Binding Assay Biacore experiments were performed to measure the ka (M-1s-1), kd (s-1), and KD (nM) values ​​of monomeric peptides binding to human selectin protein. Recombinant biotinylated human P-selectin protein was used. Binding was analyzed using a Biacore 3000 or S200 instrument equipped with a CM5 sensor chip (GE Healthcare). For preparation of the sensor chip surface, streptavidin was immobilized on the chip using standard amine-coupling chemistry at 25 °C in HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. The carboxymethyldextran surface was activated by injecting 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a 1:1 ratio at a flow rate of 10 μl / min for 12 min. To capture streptavidin, proteins were diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 70 μl over the activated chip surface. Residual activated groups were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 min. Biotinylated protein stocks were diluted 1:100 in HBS-N and captured on one flow cell at 5 μl / min to a level of 1000-1500 RU. The buffer was changed to PBS / 0.05% Tween 20 and a dilution series of peptides was prepared in this buffer with a final DMSO concentration of 0.5%. The highest concentration of analyte varied from 200 nM to 50 μM depending on the predicted affinity interaction. In all cases, titrations were performed with seven 2-fold dilutions or five 3-fold dilutions. SPR analyses were performed at 25 °C, with a flow rate of 50–80 μl / min, 60 s association and appropriate dissociation periods (100–900 s). Data were corrected for volume effects excluding DMSO. All data were double-referenced against blank injections and a reference surface using standard processing procedures, and data processing and kinetic fitting were performed with Scrubber software, version 2.0c (BioLogic Software). Data were fitted using a mass transfer model that took into account mass transfer effects where necessary.

[0189] [Table 11]

[0190] 2. Receptor-ligand Inhibition Assay The bicyclic test sample, SelG1 (Crizanlizumab), was serially diluted in assay buffer (25 mM HEPES, 121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 6 mM NaHCO2, 5.5 mM glucose, 1% BSA, pH 7.4) while maintaining 0.5% DMSO and transferred to a 384-well clear bottom plate (Corning, 3712) in a final volume of 6 μL. Six microliters of P-selectin in imaging buffer (25 mM HEPES, 121 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 1.8 mM CaCl2, 6 mM NaHCO2, 5.5 mM glucose, pH 7.4) was added to a final concentration of 5 nM and incubated for 15 min at room temperature. HL-60 cells (ATCC, CCL-240) mixed with 1 μg / mL far-red labeled wheat germ agglutinin (WGA, ThermoFisher W21404) and 1 μg / mL AlexaFluor 488-streptavidin (Invitrogen S11223) were added to the plate and incubated for 2 h at room temperature without light. Staining and antibody binding were quantified by simultaneously scanning the microplate with the 488 nm and 640 nm lasers of a mirror-ball microplate cytometer, triggering AF488 in FL2 to AF647-WGA in FL4. Data were analyzed in Dotmatics using a four-parameter logistic fit to generate IC50 values ​​and percent response. Percent inhibition was calculated using a 100-% response at the highest concentration. Selected peptides were tested in this assay and the results (IC 50 and % inhibition data) are shown in Table 7, where data shown are geometric means of n=3.

[0191] [Table 12]

[0192] 3. Cell Adhesion Bioassay Characterization of BCY12262 background In the flow adhesion assay (Figure 1), a microfluidic channel is coated with an endothelium-mimicking protein that contains a specific binding receptor of interest (e.g., P-selectin). A sample of cells and test article is then flowed through the channel under physiologically relevant flow conditions, and the number of cells attached to the channel surface is quantified. P-selectin is a cell adhesion molecule found on the surface of activated endothelial cells. White blood cells (WBCs) contribute substantially to adhesion to P-selectin in whole blood. WBCs are isolated from blood samples and the concentration is normalized to 5 million / mL.

[0193] methodology We utilized a commercially available well-plate microfluidic flow adhesion system, BioFlux 1000Z (Fluxion Bio, San Francisco, CA, USA). Isolated WBC suspensions (5 million / ml in Hank's Balanced Salt Solution buffer) from one convalescent COVID-19 patient were passed through a microfluidic channel (width 350 μm × height 70 μm) coated with P-selectin (5 μg / ml) at pulsatile (1–67 Hz) shear stress (1 dyne / cm). 2 ) and non-adherent cells were removed by washing with buffer at the same flow rate. Images were acquired with a high-resolution CCD camera and analyzed with Montage imaging software (Molecular Devices, Downingtown, PA, USA). For each sample, an adhesion index (AI) was established by quantifying adherent cells within a standard observation area (cells / mm 2 ).

[0194] result In Figure 2, where the results of this analysis are shown, it can be seen that significant inhibition of leukocyte adhesion was observed with BCY12262 compared to the positive control antibody (crizanlizumab). BCY12262 showed a strong dose-response relationship, reaching approximately 70 percent inhibition at higher doses (100 nM to 10 μM). The negative control peptide BCY17800, which does not bind to P-selectin, showed no inhibition in the flow adhesion assay at 1 μM.

Claims

1. A peptide ligand specific for P-selectin, comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

2. The peptide ligand of claim 1 , wherein the reactive group comprises a cysteine ​​residue.

3. 2. The peptide ligand of claim 1, wherein the peptide ligand comprises the motif WCDV, or a modified derivative thereof.

4. The peptide ligand of claim 1 , wherein the loop sequence comprises four or six amino acids.

5. 2. The peptide ligand of claim 1, wherein the loop sequence comprises three cysteine ​​residues separated by two loop sequences, a first loop sequence of four amino acids and a second loop sequence of six amino acids.

6. The peptide ligand is C i -X 1 -X 2 -X 3 -X 4 -C ii -DVX 5 -X 6 -X 7 -X 8- C iii (SEQ ID NO:1) [In the sequence, X 1 indicates D or Y; X 2 indicates A or M; X 3 indicates D or E; X 4 indicates W, 1Nal or Trp(Me); X 5 indicates P or T; X 6 indicates S or D; X 7 indicates L or Y; X 8 indicates P or G; (where: 1Nal represents 1-naphthylalanine, Trp(Me) denotes methyl-tryptophan, and C i , C ii and C iii represent the first cysteine ​​residue, the second cysteine ​​residue, and the third cysteine ​​residue, respectively. or a modified derivative thereof, or a pharma- ceutically acceptable salt thereof.

7. X 4 The peptide ligand of claim 6, wherein: represents W.

8. C i -X 1 -X 2 -X 3 -X 4 -C ii -DVX 5 -X 6 -X 7 -X 8- C iii The peptide ligand of SEQ ID NO:1 C i DAD[1Nal]C ii DVPSLPC iii (SEQ ID NO:2); C i DADWC ii DVPSLPC iii (SEQ ID NO:3); C i YME[1Nal]C ii DVTDYGC iii (SEQ ID NO:4); C i YME[Trp(Me)]C ii DVTDYGC iii (SEQ ID NO:5); and C i YMEWC ii DVTDYGC iii (SEQ ID NO:6); [In the sequence, C i , C ii and C iii represent the first cysteine ​​residue, the second cysteine ​​residue, and the third cysteine ​​residue, respectively. or a modified derivative thereof, or a pharma- ceutically acceptable salt thereof, such as: A-(SEQ ID NO:2)-A (referred to herein as BCY12027); H 2 NA-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY12026); A-(SEQ ID NO:3)-A (referred to herein as BCY11648); H 2 NA-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY12025); Ac-A-(SEQ ID NO:4)-A (referred to herein as BCY11279); A-(SEQ ID NO:4)-A-[K(PYA)] (referred to herein as BCY11890); Ac-A-(SEQ ID NO:5)-A (referred to herein as BCY11281); Ac-(SEQ ID NO:6) (referred to herein as BCY9717); A-(SEQ ID NO:6)-A (referred to herein as BCY10194); A-(SEQ ID NO:6)-A-[K(PYA)] (referred to herein as BCY18041); [PYA]-A-(SEQ ID NO:6)-A-NH 2 (referred to herein as BCY10910); and Ac-A-(SEQ ID NO:6)-[K(PYA)]-NH 2 (referred to herein as BCY10911), [wherein PYA represents propargyl-acid], especially: A-(SEQ ID NO:2)-A (referred to herein as BCY12027); H 2 NA-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY12026); H 2 NA-(SEQ ID NO:3)-A-[K(PYA)] (referred to herein as BCY12025); and A-(SEQ ID NO:3)-A (referred to herein as BCY11648); more specifically: A-(SEQ ID NO:2)-A (referred to herein as BCY12027); and H 2 NA-(SEQ ID NO:2)-A-[K(PYA)] (referred to herein as BCY12026); most particularly; A-(SEQ ID NO:2)-A (referred to herein as BCY12027).

9. 2. The peptide ligand of claim 1, wherein the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) and 1,1',1''-(1,4,7-triazonane-1,4,7-triyl)tris(2-chloroethan-1-one) (TCAZ) or the bromo derivative 1,1',1''-(1,4,7-triazonane-1,4,7-triyl)tris(2-bromoethan-1-one) (TBAZ).

10. 2. The peptide ligand of claim 1, wherein the pharma- ceutically acceptable salt is selected from the free acid or a sodium, potassium, calcium or ammonium salt.

11. 1. A multimeric binding complex comprising at least two peptide ligands, wherein at least one peptide ligand is a peptide ligand specific for P-selectin as described in claim 1, said peptide ligands being the same or different, each comprising a polypeptide comprising at least three reactive groups separated by at least two loop sequences and a molecular scaffold that forms covalent bonds with the reactive groups of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold.

12. 12. The multimeric binding complex of claim 11, wherein each peptide ligand is attached to the central hinge portion by a spacer group.

13. Formula (I): 【Chemistry 1】 [In the formula, CHM indicates the central hinge part; S 1 represents a spacer group; The bicyclic ring represents the peptide ligand of claim 1; and m represents an integer selected from 2 to 10.

12. The multimeric binding complex of claim 11, comprising the compound:

14. The multimeric binding complex according to claim 11, wherein said peptide ligands are specific for the same target, in particular P-selectin.

15. The multimeric binding complex of claim 11, wherein the multimeric binding complex comprises at least two identical peptide ligands.

16. The multimeric binding complex of claim 15, wherein the multimeric binding complex comprises two identical peptide ligands and is selected from BCY5454, BCY5455, BCY5456, BCY5457, BCY12257, BCY12258, BCY19243 and BCY19240, such as BCY5454, BCY5455, BCY5456, BCY5457, BCY12257 and BCY12258.

17. The multimeric binding complex of claim 15, wherein the multimeric binding complex comprises three identical peptide ligands and is selected from BCY5458, BCY5459, BCY5460, BCY5461, BCY12259, BCY12260, BCY19242 and BCY19239, such as BCY5458, BCY5459, BCY5460, BCY5461, BCY12259 and BCY12260.

18. The multimeric binding complex of claim 15, wherein the multimeric binding complex comprises four identical peptide ligands and is selected from BCY5462, BCY5463, BCY5464, BCY5465, BCY12261, BCY12262, BCY11903 and BCY19238, such as BCY5462, BCY5463, BCY5464, BCY5465, BCY12261 and BCY12262.

19. The multimeric binding complex of claim 11, wherein the multimeric binding complex comprises at least two different peptide ligands.

20. A drug conjugate comprising the peptide ligand of claim 1 conjugated to one or more effector groups and / or functional groups.

21. A drug conjugate comprising the multimeric binding complex of claim 11 conjugated to one or more effector groups and / or functional groups.

22. A pharmaceutical composition comprising the peptide ligand of claim 1 in combination with one or more pharma- ceutically acceptable excipients.

23. A pharmaceutical composition comprising the multimeric binding complex of claim 11 in combination with one or more pharma- ceutically acceptable excipients.

24. A pharmaceutical composition comprising the drug conjugate of claim 20 in combination with one or more pharma- ceutically acceptable excipients.

25. A pharmaceutical composition comprising the drug conjugate of claim 21 in combination with one or more pharma- ceutically acceptable excipients.

26. The pharmaceutical composition according to any one of claims 22 to 25, for preventing, inhibiting or treating a disease or disorder mediated by a cell adhesion molecule.

27. 27. The pharmaceutical composition of claim 26, wherein the cell adhesion molecule is P-selectin.

28. 27. The pharmaceutical composition of claim 26, wherein the disease or disorder is a vaso-occlusive crisis.

29. 27. The pharmaceutical composition of claim 26, wherein the disease or disorder is related to sickle cell disease or sickle cell anemia.

30. The pharmaceutical composition according to any one of claims 22 to 25, for preventing, suppressing or treating cancer.

31. 31. The pharmaceutical composition of claim 30, wherein the prevention, suppression or treatment of cancer comprises blocking, preventing or suppressing cancer or cancer cell metastasis.

32. 27. The pharmaceutical composition of claim 26, wherein the disease or disorder is COVID-19.

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