A bicyclic peptide ligand specific for CD38

Abstract

The present invention relates to polypeptides covalently attached to a non-aromatic molecular scaffold such that two or more peptide loops are interposed between the attachment points to the scaffold. In particular, the present invention describes peptides that are high-affinity binders of CD38. The present invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and the use of the peptide ligands and drug conjugates in the prevention, suppression, or treatment of diseases or disorders mediated by CD38.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a polypeptide covalently attached to a non-aromatic molecular scaffold such that two or more peptide loops are interposed between the attachment points to the scaffold. In particular, the present invention describes peptides that are high affinity binders of CD38. The present invention also includes drug conjugates comprising the peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising the peptide ligands and drug conjugates, and the use of the peptide ligands and drug conjugates in the prevention, inhibition, or treatment of diseases or disorders mediated by CD38. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are able to bind protein targets with high affinity and target specificity and are therefore an attractive class of molecules for the development of therapeutic drugs. Indeed, several cyclic peptides have already been successfully used in the clinic, such as, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24). The excellent binding properties are due not only to the relatively large interaction surface formed between the peptide and the target, but also to the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are used with the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 (Wu et al., 2007), Science 330, 1066-71), a cyclic peptide (355 Å) with an Arg-Gly-Asp motif that binds to integrin αVb3 2 ) (Xiong et al., 2002, Science 296(5565), 151-5), or upain-1 (603 Å), a cyclic peptide inhibitor that binds to urokinase-type plasminogen activator. 2; Zhao et al. (2007), J Struct Biol 160(1), 1-10), which bind to surfaces measuring several hundred square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides and experience less entropy loss when binding to targets, resulting in higher binding affinity. The reduced flexibility also results in a target-specific conformational fixation, increasing binding specificity compared to linear peptides. This effect is exemplified by a 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 containing 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 the rapid and quantitative cyclization of multiple peptide loops onto synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al., 2005, ChemBioChem). Methods for producing candidate drug compounds, where the compounds are generated by linking cysteine-containing polypeptides to a molecular scaffold such as, for example, 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 random six amino acid regions (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] (Summary of the invention) According to a first aspect of the present invention there is provided a peptide ligand specific for CD38 comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a non-aromatic molecular scaffold which forms covalent bonds with the cysteine ​​residues 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 a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0008] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0009] According to a further aspect of the invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by CD38. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description of the Invention In one embodiment, the loop sequence comprises 2 or 7 amino acids.

[0011] In a further embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, one of two amino acids and the other of seven amino acids.

[0012] In one embodiment, the peptide ligand is [ka] (wherein X1 to X5 represent any amino acid residue, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof.

[0013] In one embodiment, the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of seven amino acids and the second of which consists of two amino acids, and the peptide ligand comprises [ka] (wherein X1 to X6 represent any amino acid residue, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof.

[0014] In a further embodiment, [ka] The peptide ligand is [ka] The peptide ligands include:

[0015] In a further embodiment, [ka] The peptide ligands are represented by SEQ ID NOs: 1 to 22: [ka] (Here, C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutical acceptable salt thereof.

[0016] In a further embodiment, [ka] The peptide ligand is A-(SEQ ID NO:1)-A (referred to herein as 66-50-01-N001); A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:3)-A (referred to herein as 66-50-03-N001); A-(SEQ ID NO:4)-A (referred to herein as 66-50-04-N001); A-(SEQ ID NO:5)-A (referred to herein as 66-50-05-N001); A-(SEQ ID NO:6)-A (referred to herein as 66-50-06-N001); A-(SEQ ID NO:7)-A (referred to herein as 66-50-07-N001); A-(SEQ ID NO:8)-A (referred to herein as 66-50-08-N001); A-(SEQ ID NO:9)-A (referred to herein as 66-50-09-N001); A-(SEQ ID NO:10)-A (referred to herein as 66-50-10-N001); A-(SEQ ID NO:11)-A (referred to herein as 66-50-11-N001); A-(SEQ ID NO:12)-A (referred to herein as 66-50-12-N001); A-(SEQ ID NO:13)-A (referred to herein as 66-50-13-N001); A-(SEQ ID NO:14)-A (referred to herein as 66-50-14-N001); A-(SEQ ID NO:15)-DST (referred to herein as 66-50-15-T01-N001); A-(SEQ ID NO:15)-EAD (referred to herein as 66-50-15-T02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:16)-DTS (referred to herein as 66-50-22-T01-N001) ASDN-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T01-N001); ARNE-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T02-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); A-(SEQ ID NO:19)-DVP (referred to herein as 66-50-19-T01-N001); A-(SEQ ID NO:20)-TEN (referred to herein as 66-50-19-T02-N001); A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001); A-(SEQ ID NO:22)-EEP (referred to herein as 66-50-21-T01-N001); and The amino acid sequence is selected from:

[0017] In one embodiment, the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and the peptide ligand is A-(SEQ ID NO:1)-A (referred to herein as 66-50-01-N001); A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:3)-A (referred to herein as 66-50-03-N001); A-(SEQ ID NO:4)-A (referred to herein as 66-50-04-N001); A-(SEQ ID NO:5)-A (referred to herein as 66-50-05-N001); A-(SEQ ID NO:6)-A (referred to herein as 66-50-06-N001); A-(SEQ ID NO:7)-A (referred to herein as 66-50-07-N001); A-(SEQ ID NO:8)-A (referred to herein as 66-50-08-N001); A-(SEQ ID NO:9)-A (referred to herein as 66-50-09-N001); A-(SEQ ID NO:10)-A (referred to herein as 66-50-10-N001); A-(SEQ ID NO:11)-A (referred to herein as 66-50-11-N001); A-(SEQ ID NO:12)-A (referred to herein as 66-50-12-N001); A-(SEQ ID NO:13)-A (referred to herein as 66-50-13-N001); A-(SEQ ID NO:14)-A (referred to herein as 66-50-14-N001); A-(SEQ ID NO:15)-DST (referred to herein as 66-50-15-T01-N001); A-(SEQ ID NO:15)-EAD (referred to herein as 66-50-15-T02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:16)-DTS (referred to herein as 66-50-22-T01-N001) ASDN-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T01-N001); ARNE-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T02-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); A-(SEQ ID NO:19)-DVP (referred to herein as 66-50-19-T01-N001); A-(SEQ ID NO:20)-TEN (referred to herein as 66-50-19-T02-N001); A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001); A-(SEQ ID NO:22)-EEP (referred to herein as 66-50-21-T01-N001); and The amino acid sequence is selected from:

[0018] In a further embodiment, the molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) and the peptide ligand is A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); and A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001) The scaffold / peptide ligand of this embodiment exhibited excellent CD38 competitive binding as shown in Table 1 herein.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, for example, peptide chemistry, cell culture, and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetics, and biochemistry methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc., incorporated herein by reference).

[0020] (Nomenclature) (Numbering) When referring to amino acid residue positions within the peptides of the invention, a cysteine ​​residue (C i , C ii , and C iii ) are invariant, they are omitted from the numbering and therefore the numbering of amino acid residues within the peptides of the invention is referred to as follows: -C i -Y1-W2-N3-P4-F5-M6-G7-C ii -Y8-T9-C iii - (SEQ ID NO: 1) .

[0021] For the purposes of this description, all bicyclic peptides are considered to be cyclized with 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA) to give trisubstituted structures.i , C ii , and C iii This occurs above.

[0022] (Molecular Format) N- or C-terminal extensions 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 βAla-Sar10-Ala tail is: βAla-Sar10-A-(SEQ ID NO:X) This is expressed as:

[0023] (Reverse peptide sequence) In view of the disclosure in Nair et al. (2003) J Immunol 170(3), 1362-1373, it is envisioned that the peptide sequences disclosed herein will also find utility in their retro-inverso forms, for example where the sequence is reversed (i.e., N-terminus becomes C-terminus and C-terminus becomes N-terminus) and the stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids and L-amino acids become D-amino acids).

[0024] (Peptide Ligand) A peptide ligand as referred to herein refers to a peptide covalently attached to a molecular scaffold. Typically, such a peptide contains 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, called a loop sequence, because the peptide forms a loop when it is attached to the scaffold. In this case, the peptide contains at least three cysteine ​​residues (herein referred to as C i , C ii , and C iii ), and forms at least two loops on the scaffold.

[0025] (Advantages of peptide ligands) Certain bicyclic peptides of the present invention have several advantageous properties that make them suitable drug-like molecules 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 ideally demonstrate stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastrointestinal proteases, pulmonary surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic lead candidates can not only be developed in animal models but also be administered with confidence to humans; - a desirable solubility profile, which is a function of charged and hydrophilic to hydrophobic residue ratios and intra / inter-molecular H-bonds, which are important for formulation and absorption purposes; - Optimal plasma half-life in the circulation. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides for short-term exposure in acute disease management settings or to develop bicyclic peptides with enhanced retention in the circulation, making them optimal for managing more chronic disease states. Other factors driving a desirable plasma half-life are the requirement for sustained exposure for maximum therapeutic efficacy and the associated toxicities of sustained exposure of the drug; and -Selectivity. Certain peptide ligands of the present invention exhibit better selectivity than other CDs.

[0026] (Pharmaceutical acceptable salts) It will be understood that the salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of that ligand.

[0027] The salts of the present invention can be synthesized from the parent compound which 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, page 388, August 2002. Typically, such salts can be prepared by reacting the free acid or base form of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0028] 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, Cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic 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, Examples of suitable salts include mono- or di-salts formed with acids selected from the group consisting of acetic 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, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0029] 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 (mesylic acid), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One particular salt is the hydrochloride salt. Another particular salt is the acetate salt.

[0030] The compound is anionic or has a functional group which may be anionic (e.g., -COOH can be -COO - In the case of cations such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 29 + , 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 those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0031] When the peptides of the invention contain amine functions, they may form quaternary ammonium salts, for example, by reaction with alkylating agents by methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the peptides of the invention.

[0032] (Modified derivative) It will be understood that modified derivatives of the peptide ligands defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; 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 spacer groups; 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 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; replacement of one or more peptide bonds with surrogate bonds; modification of the peptide backbone length; Modifications include one or more selected from: 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 acid / aspartic acid, and tyrosine with suitable amine-, thiol-, carboxylic acid-, and phenol-reactive reagents to functionalize the amino acids; and introduction or substitution of amino acids that introduce orthogonal reactivity suitable for functionalization, e.g., amino acids with azide or alkyne groups that allow functionalization with alkyne- or azide-bearing moieties, respectively.

[0033] In one embodiment, the modified derivative comprises an N-terminal and / or a C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using a suitable amino reaction chemistry and / or a C-terminal modification using a suitable carboxy reaction chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radioactive chelator, or a chromophore.

[0034] 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, an N-terminal cysteine ​​group (referred to herein as C iThe N-terminal acetyl group (referred to as "acetyl group") is capped with acetic anhydride or other suitable reagent during peptide synthesis to yield an N-terminally acetylated molecule. This embodiment offers the advantage of eliminating a potential recognition point for aminopeptidases, avoiding possible degradation of the bicyclic peptide.

[0035] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates the conjugation of an effector group and retention of potency of the bicyclic peptide against its target.

[0036] 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 cysteine ​​group (herein referred to as C iii The C-terminally amidated group, referred to as the C-terminus, is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment offers the advantage of eliminating a potential recognition point for carboxypeptidases, reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0037] In one embodiment, the modified derivatives include the substitution of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids may be selected that have isosteric / isoelectronic side chains that are not recognized by degradative proteases or have any adverse effect on targeting efficacy.

[0038] Alternatively, unnatural amino acids with constrained amino acid side chains may be used such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, these concern proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cycloamino acids that are simple derivatives of amino-cyclopropyl carboxylic acids.

[0039] 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

[0040] In one embodiment, the modified derivative comprises the substitution of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises the substitution of a tryptophan residue with a naphthylalanine or alanine residue. This embodiment provides the resulting bicyclic peptide ligand with the advantage of improved pharmaceutical stability profile.

[0041] 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 affect the degree of plasma protein binding and thus the concentration of the available free fraction in plasma, while charged amino acid residues (especially arginine) can affect the interaction of the peptide with the phospholipid membrane of the cell surface. The combination of the two can affect the half-life, volume of distribution, and exposure of the peptide drug, which can be adjusted depending on the clinical endpoint. Furthermore, the correct combination and number of charged and hydrophobic amino acid residues can reduce irritation at the injection site (if the peptide drug is administered subcutaneously).

[0042] In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to enhance proteolytic stability due to steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0043] In one embodiment, the modified derivative comprises the removal of any amino acid residue and replacement with alanine. This embodiment has the advantage of removing potential sites of proteolytic attack.

[0044] 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 sites resulting in lower dissociation rates so that higher affinities are achieved; - taking advantage of long-range ionic interactions, resulting in faster association rates and incorporating charged groups resulting in higher affinity (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - building further constraints into the peptide, for example by constraining the amino acid side chains correctly so that the entropy loss is minimal upon target binding, constraining the backbone torsion angles so that the entropy loss is minimal upon target binding, and introducing further cyclizations 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).

[0045] (Isotopic Variation) 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 to which a metal chelating group capable of bearing a relevant (radio)isotope has been attached (referred to as "effectors"), and peptide ligands of the invention in which a particular functional group has been covalently replaced with a relevant (radio)isotope or an isotopically labeled functional group.

[0046] Examples of isotopes suitable for inclusion in the peptide ligands of the present 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, isotopes of fluorine, e.g. 18 F, an isotope of iodine, e.g. 123 I, 125 I, and 131 I, isotopes 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, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, gallium isotopes, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y, as well as isotopes of lutetium, e.g. 177 Lu, as well as isotopes of bismuth, e.g. 213 Contains Bi.

[0047] Certain isotopically labeled peptide ligands of the invention, e.g., those incorporating a radioisotope, are useful in drug and / or substrate tissue distribution studies and for clinically assessing the presence and / or absence of CD38 targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may use compounds that are labeled with labeling agents, such as, for example, 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 given its ease of incorporation and the means of detection available.

[0048] 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 in vivo half-life or reduced dosage requirements, and therefore may be preferred in some circumstances.

[0049] 11 C. 18 F, 15 O, and 13 Substitution with positron emitting isotopes, such as N, can be useful in Positron Emission Topography (PET) studies for examining target occupancy.

[0050] Isotopically labeled compounds of the peptide ligands of the present invention can generally be prepared by conventional techniques known to those of skill in the art, or by processes similar to those described in the accompanying Examples, substituting appropriate isotopically labeled reagents for previously utilized non-labeled reagents.

[0051] (Non-aromatic molecular scaffolds) References herein to the term "non-aromatic molecular scaffold" refer to any molecular scaffold as defined herein that does not contain an aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring system.

[0052] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0053] As described in the aforementioned documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0054] 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.

[0055] 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.

[0056] The molecular scaffold can include 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.

[0057] 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).

[0058] (Effectors and Functional Groups) According to a further aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0059] The effectors and / or functional groups can be attached, for example, to the N- and / or C-terminus of the polypeptide, to amino acids within the polypeptide, or to a molecular scaffold.

[0060] Suitable effector groups include antibodies and portions or fragments thereof. For example, an effector group 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. An effector group can include an antibody hinge region (such a region typically found between the CH1 and CH2 domains of an IgG molecule).

[0061] 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, a peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion with 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, a peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion with a tβ half-life of 1 day or more.

[0062] Functional groups generally include linking groups, reactive groups for attachment of drugs or other entities, functional groups which aid in cellular uptake of the macrocyclic peptide, and the like.

[0063] The ability of peptides to penetrate into cells allows peptides to be effective against intracellular targets. Targets that can be accessed by peptides with the ability to penetrate into cells include intracellular signaling molecules such as transcription factors, tyrosine kinases, and molecules involved in apoptotic pathways. Functional groups that allow cell penetration include chemical groups added to either the peptide or the peptide or molecular scaffold. For example, peptides such as those derived from VP22, HIV-Tat, Drosophila homeobox protein (Antennapedia), etc., as described in Chen and Harrison, Biochemical Society Transactions (2007), Vol. 35, Part 4, p. 821; Gupta et al., Advanced Drug Discovery Reviews (2004), Vol. 57, p. 9637. Examples of short peptides that have been shown to be efficient at translocating through cell membranes include the 16 amino acid penetratin peptide from the Drosophila antennapedia protein (Derossi et al. (1994) J. Biol. Chem. 269:10444), the 18 amino acid "model amphipathic peptide" (Oehlke et al. (1998) Biochim Biophys Acts 1414:127), 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 attached to biomolecules (Okuyama et al. (2007) Nature Methods 4:153). Another chemical strategy, the addition of guanidinium groups to molecules, also enhances cell penetration (Elson-Scwab et al. (2007) J. Biol. Chem 282:13585). Small molecular weight molecules such as steroids can be added to the molecular scaffold to enhance cellular uptake.

[0064] One class of functional groups that can be attached to the peptide ligands includes antibodies and binding fragments thereof, such as Fab, Fv, or single domain fragments. In particular, antibodies that bind to proteins that can increase the half-life of the peptide ligand in vivo can be used.

[0065] In one embodiment, a 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, a peptide ligand-effector group or composition according to the invention has a tβ half-life in the range of 12 hours to 60 hours. In a further embodiment, it has a tβ half-life of 1 day or more. In a still further embodiment, it is in the range of 12 to 26 hours.

[0066] In one particular embodiment of the invention, the functional group is selected from metal chelators that are suitable for complexing medicamentously relevant metal radioisotopes.

[0067] Possible effector groups also include enzymes such as carboxypeptidase G2 for use in enzyme / prodrug therapy, where peptide ligands replace antibodies in ADEPT.

[0068] In one particular embodiment of the present invention, functional group is selected from drugs such as cytotoxic agents for cancer therapy.Suitable examples include: alkylating agents such as cisplatin and carboplatin, and oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids such as vincristine, vinblastine, vinorelbine, and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, originally known as taxol; topoisomerase inhibitors including camptothecin; irinotecan and topototecan, and type II inhibitors including amsacrine, etoposide, etoposide phosphate, and teniposide. Additional agents may include the immunosuppressant dactinomycin (used in kidney transplants), antitumor antibiotics including doxorubicin, epirubicin, bleomycin, calicheamicin, and the like.

[0069] In one further specific embodiment of the invention, the cytotoxic agent is selected from a maytansinoid (eg, DM1) or a monomethylauristatin (eg, MMAE).

[0070] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine and has the following structure: [ka] .

[0071] Monomethylauristatin E (MMAE) is a synthetic anti-neoplastic agent having the following structure: [ka] .

[0072] In one embodiment, the cytotoxic agent is linked to the bicyclic peptide by a cleavable bond, such as a disulfide bond or a protease-sensitive bond, hi a further embodiment, the groups adjacent to the disulfide bond are modified to control the rate of disruption of the disulfide bond and, thereby, cleavage and concomitant release of the cytotoxic agent.

[0073] Published studies have established the possibility of modifying the susceptibility of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance reduces the rate of reduction by intracellular glutathione and also by extracellular (systemic) reducing agents, and consequently reduces the ease with which the toxin is released both inside and outside the cell. Thus, the selection of optimal conditions for disulfide stability in circulation (which minimizes the unwanted side effects of the toxin) versus optimizing efficient release in the intracellular environment (which maximizes the therapeutic effect) can be achieved by careful selection of the degree of hindrance on either side of the disulfide bond.

[0074] The disorder on either side of the disulfide bond is adjusted by introducing one or more methyl groups into either the targeting entity (here, the bicyclic peptide) or the toxin side of the molecular construct.

[0075] In one embodiment, the cytotoxic agents and linkers are selected from any combination of those described in WO 2016 / 067035, which cytotoxic agents and linkers thereof are incorporated herein by reference.

[0076] (synthesis) The peptides of the present invention can be synthetically produced by standard techniques and then reacted with molecular scaffolds in vitro. Standard chemistry can be used to carry this out. This allows for rapid large-scale preparation of soluble material for further downstream experimentation or validation. Such methods can be achieved using conventional chemistry such as that disclosed in Timmerman et al. (supra).

[0077] Thus, the present invention also relates to the production of a polypeptide or conjugate selected as described herein, which production comprises any further steps as explained below, in one embodiment these steps are carried out on the final polypeptide / conjugate produced by chemical synthesis.

[0078] Optionally, amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

[0079] The peptide can also be extended, for example, to incorporate additional loops and thus introduce multiple specificities.

[0080] To extend a peptide, it may simply be chemically extended at its N- or C-terminus or within a loop using orthogonally protected lysines (and analogues) using standard solid- or solution-phase chemistries. Activated or activatable N- or C-termini may be introduced using standard (bio)conjugation techniques. Alternatively, the addition may be performed enzymatically by fragment condensation or native chemical ligation as described, for example, in (Dawson et al., 1994, Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779) or using subtiligase as described, for example, in (Chang et al., Proc Natl Acad Sci US A. 1994 Dec 20; 91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, Vol. 18, No. 22, 15 Nov. 2008, pp. 6000-6003).

[0081] 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 reducing environment of cells. In this case, a molecular scaffold can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; then, an additional cysteine ​​or thiol can be added to the N- or C-terminus of the first peptide, so that this cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0082] Similar techniques apply equally to the synthesis / coupling of two bicyclic bispecific macrocycles, potentially giving rise to a tetraspecific molecule.

[0083] Furthermore, the addition of other functional or effector groups may be achieved in the same manner, coupling at the N- or C-terminus or via a side chain using appropriate chemistry, hi one embodiment, the coupling is carried out in such a way as not to block the activity of either entity.

[0084] Pharmaceutical Composition According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharma- ceutically acceptable excipients.

[0085] Usually, the peptide ligands 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, and lactated Ringer's. Suitable physiologically acceptable adjuvants, if necessary to keep the polypeptide complex in suspension, may be selected from viscosity-increasing agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates.

[0086] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose, and preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents, and inert gases (Mack, 1982, Remington's Pharmaceutical Sciences, 16th ed.).

[0087] The peptide ligands of the invention may be used as a separately administered composition or in conjunction with other drugs. These may include antibodies, antibody fragments, and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin, or cisplatin, and immunotoxins. Pharmaceutical compositions may include "cocktails" of various cytotoxic or other drugs in conjunction with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities, such as polypeptides selected with different targeting ligands, whether pooled or not prior to administration.

[0088] The route of administration of the pharmaceutical composition according to the invention may be any 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 parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or also suitably by direct injection using a catheter. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dosage and frequency of administration will depend on the age, sex, and condition of the patient, the simultaneous administration of other drugs, contraindications, and other parameters taken into account by the clinician.

[0089] The peptide ligands of the present invention can be lyophilized before storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be utilized. It will be understood by those skilled in the art that lyophilization and reconstitution may result in varying degrees of activity loss, and that levels may need to be adjusted upwards to compensate.

[0090] Compositions containing the peptide ligands or cocktails thereof of the present invention can be administered for prophylactic and / or therapeutic treatments. In certain therapeutic applications, an amount sufficient to achieve at least partial inhibition, suppression, modulation, killing, or some other measurable parameter of a selected population of cells is defined as a "therapeutically effective dose". The amount required to achieve this dosage will depend on the severity of the disease and the general state of the patient's own immune system, but generally ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05 to 2.0 mg / kg being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof may also be administered in similar or slightly lower dosages.

[0091] Compositions containing peptide ligands according to the invention can be utilized in prophylactic and therapeutic settings to aid in the alteration, inactivation, killing, or elimination of selected target cell populations in a mammal. Additionally, the peptide ligands described herein can be selectively used ex vivo or in vitro to selectively kill, deplete, or otherwise effectively remove target cell populations from heterogeneous populations of cells. Blood from a mammal can be combined ex vivo with the selected peptide ligand, thereby killing or otherwise removing undesirable cells from the blood for return to the mammal according to standard techniques.

[0092] (therapeutic use) The bicyclic peptides of the invention have particular utility as CD38 binding agents.

[0093] CD38 is a 45 kD type II transmembrane glycoprotein with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 protein is a bifunctional ectoenzyme that can catalyze the conversion of NAD+ to cyclic ADP-ribose (cADPR) and also hydrolyze cADPR to ADP-ribose. During ontogeny, CD38 appears on CD34+ committed stem cells and lineage committed precursors of lymphoid, erythroid, and myeloid cells. CD38 expression persists in most cases in the lymphoid lineage, and expression levels vary at different stages of T and B cell development.

[0094] CD38 is upregulated in many hematopoietic malignancies and in cell lines derived from various hematopoietic malignancies, including non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myelogenous leukemia (CML). On the other hand, most primitive pluripotent stem cells of the hematopoietic system are CD38-. CD38 expression in hematopoietic malignancies and its correlation with disease progression make CD38 an attractive target for antibody therapy.

[0095] CD38 is Ca 2+mobilization (Morra et al. (1998) FASEB J. 12; 581-592; Zilber et al. (2000) Proc Natl Acad Sci USA 97, 2840-2845), and signaling through tyrosine phosphorylation of numerous signaling molecules, including phospholipase C-gamma, ZAP-70, syk, and c-cbl, in lymphoid and myeloid cells or cell lines (Funaro et al. (1993) Eur J Immunol 23, 2407-2411; Morra et al. (1998) supra; Funaro et al. (1990) J Immunol 145, 2390-2396; Zubiaur et al. (1997) J Immunol 159, 193-205; Deaglio et al. (2003) Blood 102, 2146-2155; Todisco et al. (2000) Blood 95, 535-542; Konopleva et al. (1998) J Immunol 161, 4702-4708; Zilber et al. (2000) Proc Natl Acad Sci USA 97, 2840-2845; Kitanaka et al. (1997) J Immunol 159, 184-192; Kitanaka et al. (1999) J Immunol 162, 1952-1958; Mallone et al. (2001) Int Immunol 13, 397-409). Based on these observations, CD38 has been proposed to be a key signaling molecule in the maturation and activation of lymphoid and myeloid cells during their normal development.

[0096] Notably, most of these signaling studies have used cell lines that ectopically overexpress CD38 and anti-CD38 monoclonal antibodies, which are non-physiological ligands, so the exact role of CD38 in signaling and hematopoiesis remains unclear. CD38 protein is involved in Ca 2+CD38 has the enzymatic activity to generate cADPR, a molecule that can induce Ca mobilization in lymphocytes (Lee et al. (1989) J Biol Chem 264, 1608-1615; Lee and Aarhus (1991) Cell Regul 2, 203-209). CD38 ligation with monoclonal antibodies increases the production of cADPR, thereby promoting Ca mobilization in lymphocytes. 2+ It has been proposed that CD38 induces recruitment and signaling (Lee et al. (1997) Adv Exp Med Biol 419, 411-419). In contrast to this hypothesis, truncation and point mutation analysis of the CD38 protein has shown that neither its cytoplasmic tail nor its enzymatic activity is required for signaling mediated by anti-CD38 antibodies (Kitanaka et al. (1999) J Immunol 162, 1952-1958; Lund et al. (1999) J Immunol 162, 2693-2702; Hoshino et al. (1997) J Immunol 158, 741-747).

[0097] The most compelling evidence for the function of CD38 comes from CD38- / - knockout mice, which have defective innate immunity and reduced T-cell dependent humoral responses due to defective dendritic cell migration (Partida-Sanchez et al. (2004) Immunity 20, 279-291; Partida-Sanchez et al. (2001) Nat Med 7, 1209-1216). However, it is unclear whether the function of CD38 in mice is identical to that of CD38 in humans, since the patterns of CD38 expression during hematopoiesis are significantly different between humans and mice: a) unlike immature progenitor stem cells in humans, similar progenitor stem cells in mice express high levels of CD38 (Randall et al. (1996) Blood 87, 4057-4067; Dagher et al. (1998) Biol Blood Marrow Transplant 4, 69-74); b) in human B cell development, high levels of CD38 expression are found on germinal center B cells and plasma cells (Uckun (1990) Blood 76, 1908-1923; Kumagai et al. (1995) J Exp Med 181, 1101-1110), whereas in mice the corresponding cells express lower levels of CD38 (Oliver et al. (1997) J Immunol 158, 1108-1115; Ridderstad and Tarlington (1998) J Immunol 160, 4688-4695).

[0098] Several anti-human CD38 antibodies with different growth characteristics against various tumor cells and cell lines have been described in the literature. For example, the chimeric OKT10 antibody with mouse Fab and human IgG1 Fc mediates highly efficient antibody-dependent cell-mediated cytotoxicity (ADCC) against lymphoma cells in the presence of peripheral blood mononuclear effector cells from either MM patients or normal individuals (Stevenson et al. (1991) Blood 77, 1071-1079). The CDR-grafted humanized version of the anti-CD38 antibody AT13 / 5 has been shown to have strong ADCC activity against CD38-positive cell lines (U.S. Patent Application Serial No. 09 / 797,941). Human monoclonal anti-CD38 antibodies have been shown to mediate in vitro killing of CD38-positive cell lines by ADCC and / or complement-dependent cytotoxicity (CDC) and to delay tumor growth in SCID mice bearing the MM cell line RPMI-8226 (WO 2005 / 103083). On the other hand, several anti-CD38 antibodies, IB4, SUN-4B7, and OKT10, but not IB6, AT1, or AT2, induced proliferation of peripheral blood mononuclear cells (PBMCs) from normal individuals (Ausiello et al. (2000) Tissue Antigens 56, 539-547).

[0099] Some of the prior art antibodies have been shown to be able to induce apoptosis in CD38+ B cells. However, they can only do so in the presence of stromal cells or stromal-derived cytokines. An agonistic anti-CD38 antibody (IB4) has been reported to prevent apoptosis in human germinal center (GC) B cells (Zupo et al. (1994) Eur J Immunol 24, 1218-1222) and to induce proliferation of KG-1 and HL-60 AML cells (Konopleva et al. (1998) J Immunol 161, 4702-4708), but not induce apoptosis in Jurkat T lymphoblastoid cells (Morra et al. (1998) FASEB J 12, 581-592). Another anti-CD38 antibody, T16, induced apoptosis in immature lymphoid cells and leukemic lymphoblast cells from ALL patients (Kumagai et al. (1995) J Exp Med 181, 1101-1110) and in leukemic myeloblast cells from AML patients (Todisco et al. (2000) Blood 95, 535-542), but only in the presence of stromal cells or stromal-derived cytokines (IL-7, IL-3, stem cell factor).

[0100] Polypeptide ligands selected by the methods of the invention can be used in in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assay and reagent applications, etc. Ligands with a selected level of specificity are useful in applications involving testing in non-human animals where cross-reactivity is desirable, or in diagnostic applications where cross-reactivity with homologs or paralogs must be carefully controlled. In some applications, such as vaccine applications, the ability to elicit an immune response against a range of antigens can be exploited to tailor vaccines to specific diseases and pathogens.

[0101] Substantially pure peptide ligands having at least 90-95% homogeneity are preferred for administration to mammals, with 98-99% or greater homogeneity being most preferred for pharmaceutical uses, particularly where the mammal is a human. Once purified, partially or to homogeneity as desired, the selected polypeptides can be used diagnostically or therapeutically (including in vitro), or in the development and implementation of assay procedures, immunofluorescence staining, and the like (Lefkovite and Pernis, 1979 and 1981, Immunological Methods, Vols. I and II, Academic Press, NY).

[0102] According to a further aspect of the invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or disorder mediated by CD38.

[0103] According to a further aspect of the present invention there is provided a method for the prevention, inhibition or treatment of a disease or disorder mediated by CD38 comprising administering to a patient in need thereof an effector group and drug conjugate of a peptide ligand defined herein.

[0104] In one embodiment, the CD38 is mammalian CD38. In a further embodiment, the mammalian CD38 is human CD38 (hCD38).

[0105] In one embodiment, the disease or disorder mediated by CD38 is selected from cancer.

[0106] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), such as tumors of the bladder and urinary tract, breast, gastrointestinal tract (including the esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, cancers of the nasal cavity, nasal cavity, and paranasal sinuses), ovary, fallopian tube, peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin, and adnexal cancers (melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and borderline malignancies including premalignant hematological disorders and hematological malignancies and related disorders of lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [D-cell lymphoma], LBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and related disorders of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], hypereosinophilic syndromes, myeloproliferative disorders, e.g., polycythemia vera, essential thrombocytopenia, myeloma, myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia; tumors of mesenchymal origin, e.g., sarcomas of the soft tissue, bone, or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma, meningioma, ependymoma, pineal tumor, and Schwannoma);These include, but are not limited to, endocrine tumors (e.g., pituitary tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid); ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas); and pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumors); or congenital or other syndromes that predispose a patient to malignancies (e.g., xeroderma pigmentosum);

[0107] In further embodiments, the cancer is selected from, e.g., non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin's lymphoma (HL), and chronic myelogenous leukemia (CML).

[0108] References herein to the term "prevention" include administration of the protective composition prior to the induction of disease. "Suppression" refers to administration of the composition after an inductive event but prior to clinical appearance of the disease. "Treatment" includes administration of the protective composition after disease symptoms are manifest.

[0109] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protecting against or treating disease, and 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.

[0110] The invention will now be further described with reference to the following examples. EXAMPLES

[0111] (Example) Materials and Methods (Peptide synthesis) Peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer manufactured by Peptide Instruments and a Syro II synthesizer from MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) were used with appropriate side chain protection groups: where applicable, standard coupling conditions were used in each case, followed by deprotection using standard methodologies. Peptides were purified using HPLC and after isolation they were modified with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma). For this, linear peptides were diluted to about 35 mL with 50:50 MeCN:H2O, about 500 μL of 100 mM TATA in acetonitrile was added and the reaction was started with 5 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed for about 30 to 60 min at RT and, once complete (as judged by MALDI), was lyophilized to dryness. Once complete, 1 ml of 1M L-cysteine ​​hydrochloride monohydrate (Sigma) in H2O was added to the reaction for approximately 60 min at RT to quench excess TATA.

[0112] After lyophilization, the modified peptide was purified as above, except that the Luna C8 was replaced with a Gemini C18 column (Phenomenex) and the acid was changed to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA-modified material were pooled, lyophilized, and kept at -20°C for storage.

[0113] Unless otherwise noted, all amino acids were used in the L-configuration.

[0114] Optionally, the peptide is converted to an activated disulfide and then coupled to a free thiol group of the toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in anhydrous DMSO (1.25 mol equiv.) was added to a solution of peptide (20 mM) in anhydrous DMSO (1 mol equiv.). The reaction was mixed well and DIPEA (20 mol equiv.) was added. The reaction was monitored by LC / MS until completion.

[0115] (Biological Data) (1. CD38 Competitive Binding Assay) The affinity (Ki) of the peptides of the invention for human CD38 was determined using the method reported by Lea et al. (Expert Opin Drug Discov. 2011 6(1): 17-3) and the following fluorescently labeled peptides for the TATA derivatives: [ka] (where Fl is a fluorescein molecule).

[0116] The peptide ligands of the invention were tested in the CD38 competitive binding assay described above, and the results are shown in Table 1: Table 1: Biological assay data for peptide ligands of the invention [Table 1] TIFF0007645801000012.tif154170 The present application provides the following aspects of the invention. (Aspect 1) A peptide ligand specific for CD38, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a non-aromatic molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold. (Aspect 2) 2. The peptide ligand of embodiment 1, wherein the loop sequence comprises 2 or 7 amino acids. (Aspect 3) (chemical 1) TIFF0007645801000013.tif9170 (where X 1 ~X 5 represents any amino acid residue, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. 3. The peptide ligand of embodiment 1 or embodiment 2, comprising an amino acid sequence selected from: (Aspect 4) the loop sequence comprises three cysteine ​​residues separated by two loop sequences, the first of which consists of seven amino acids and the second of which consists of two amino acids, and the peptide ligand (Case 2) TIFF0007645801000014.tif6170 (where X 1 ~X 5 represents any amino acid residue, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. 4. The peptide ligand according to any one of aspects 1 to 3, comprising an amino acid sequence selected from: (Aspect 5) The above (3) TIFF0007645801000015.tif7170 The peptide ligand is represented by SEQ ID NOs: 1 to 22: (C4) TIFF0007645801000016.tif150170 (Here, C i 、C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively. or a pharma- ceutical acceptable salt thereof; for example: A-(SEQ ID NO:1)-A (referred to herein as 66-50-01-N001); A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:3)-A (referred to herein as 66-50-03-N001); A-(SEQ ID NO:4)-A (referred to herein as 66-50-04-N001); A-(SEQ ID NO:5)-A (referred to herein as 66-50-05-N001); A-(SEQ ID NO:6)-A (referred to herein as 66-50-06-N001); A-(SEQ ID NO:7)-A (referred to herein as 66-50-07-N001); A-(SEQ ID NO:8)-A (referred to herein as 66-50-08-N001); A-(SEQ ID NO:9)-A (referred to herein as 66-50-09-N001); A-(SEQ ID NO:10)-A (referred to herein as 66-50-10-N001); A-(SEQ ID NO:11)-A (referred to herein as 66-50-11-N001); A-(SEQ ID NO:12)-A (referred to herein as 66-50-12-N001); A-(SEQ ID NO:13)-A (referred to herein as 66-50-13-N001); A-(SEQ ID NO:14)-A (referred to herein as 66-50-14-N001); A-(SEQ ID NO:15)-DST (referred to herein as 66-50-15-T01-N001); A-(SEQ ID NO:15)-EAD (referred to herein as 66-50-15-T02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:16)-DTS (referred to herein as 66-50-22-T01-N001) ASDN-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T01-N001); ARNE-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T02-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); A-(SEQ ID NO:19)-DVP (referred to herein as 66-50-19-T01-N001); A-(SEQ ID NO:20)-TEN (referred to herein as 66-50-19-T02-N001); A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001); A-(SEQ ID NO:22)-EEP (referred to herein as 66-50-21-T01-N001); and 5. The peptide ligand of embodiment 3 or embodiment 4, comprising an amino acid sequence selected from: (Aspect 6) The molecular scaffold is selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and the peptide ligand is A-(SEQ ID NO:1)-A (referred to herein as 66-50-01-N001); A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:3)-A (referred to herein as 66-50-03-N001); A-(SEQ ID NO:4)-A (referred to herein as 66-50-04-N001); A-(SEQ ID NO:5)-A (referred to herein as 66-50-05-N001); A-(SEQ ID NO:6)-A (referred to herein as 66-50-06-N001); A-(SEQ ID NO:7)-A (referred to herein as 66-50-07-N001); A-(SEQ ID NO:8)-A (referred to herein as 66-50-08-N001); A-(SEQ ID NO:9)-A (referred to herein as 66-50-09-N001); A-(SEQ ID NO:10)-A (referred to herein as 66-50-10-N001); A-(SEQ ID NO:11)-A (referred to herein as 66-50-11-N001); A-(SEQ ID NO:12)-A (referred to herein as 66-50-12-N001); A-(SEQ ID NO:13)-A (referred to herein as 66-50-13-N001); A-(SEQ ID NO:14)-A (referred to herein as 66-50-14-N001); A-(SEQ ID NO:15)-DST (referred to herein as 66-50-15-T01-N001); A-(SEQ ID NO:15)-EAD (referred to herein as 66-50-15-T02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:16)-DTS (referred to herein as 66-50-22-T01-N001) ASDN-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T01-N001); ARNE-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T02-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); A-(SEQ ID NO:19)-DVP (referred to herein as 66-50-19-T01-N001); A-(SEQ ID NO:20)-TEN (referred to herein as 66-50-19-T02-N001); A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001); A-(SEQ ID NO:22)-EEP (referred to herein as 66-50-21-T01-N001); and An amino acid sequence selected from: for example: A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); and A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001) 2. The peptide ligand of embodiment 1, comprising an amino acid sequence selected from: (Aspect 7) 7. The peptide ligand according to any one of aspects 1 to 6, wherein the pharma- ceutical acceptable salt is selected from the free acid or the sodium, potassium, calcium, ammonium salt. (Aspect 8) The peptide ligand according to any one of embodiments 1 to 7, wherein the CD38 is human CD38. (Aspect 9) A drug conjugate comprising the peptide ligand according to any one of aspects 1 to 8 conjugated to one or more effectors and / or functional groups. (Aspect 10) A drug conjugate comprising the peptide ligand according to any one of embodiments 1 to 8 conjugated to one or more cytotoxic agents. (Aspect 11) A pharmaceutical composition comprising the peptide ligand according to any one of aspects 1 to 8 or the drug conjugate according to aspect 9 or aspect 10 in combination with one or more pharma- ceutically acceptable excipients. (Aspect 12) A peptide ligand according to any one of aspects 1 to 8 or a drug conjugate according to aspect 9 or aspect 10 for use in the prevention, inhibition or treatment of a disease or disorder mediated by CD38.

Claims

1. 1. A peptide ligand specific for CD38, or a pharma- ceutically acceptable salt thereof, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences and a non-aromatic molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA); and The peptide ligand is selected from SEQ ID NOs: 1-22: 【Chemistry 1】 (wherein C i , C ii , and C iii represent the first, second, and third cysteine ​​residues, respectively). One of or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from the group consisting of:

2. The peptide ligand is A-(SEQ ID NO:1)-A (referred to herein as 66-50-01-N001); A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:3)-A (referred to herein as 66-50-03-N001); A-(SEQ ID NO:4)-A (referred to herein as 66-50-04-N001); A-(SEQ ID NO:5)-A (referred to herein as 66-50-05-N001); A-(SEQ ID NO:6)-A (referred to herein as 66-50-06-N001); A-(SEQ ID NO:7)-A (referred to herein as 66-50-07-N001); A-(SEQ ID NO:8)-A (referred to herein as 66-50-08-N001); A-(SEQ ID NO:9)-A (referred to herein as 66-50-09-N001); A-(SEQ ID NO:10)-A (referred to herein as 66-50-10-N001); A-(SEQ ID NO:11)-A (referred to herein as 66-50-11-N001); A-(SEQ ID NO:12)-A (referred to herein as 66-50-12-N001); A-(SEQ ID NO:13)-A (referred to herein as 66-50-13-N001); A-(SEQ ID NO:14)-A (referred to herein as 66-50-14-N001); A-(SEQ ID NO:15)-DST (referred to herein as 66-50-15-T01-N001); A-(SEQ ID NO:15)-EAD (referred to herein as 66-50-15-T02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:16)-DTS (referred to herein as 66-50-22-T01-N001) ASDN-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T01-N001); ARNE-(SEQ ID NO:17)-A (referred to herein as 66-50-17-T02-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); A-(SEQ ID NO:19)-DVP (referred to herein as 66-50-19-T01-N001); A-(SEQ ID NO:20)-TEN (referred to herein as 66-50-19-T02-N001); A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001); and A-(SEQ ID NO:22)-EEP (referred to herein as 66-50-21-T01-N001) 2. The peptide ligand of claim 1, or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from:

3. The peptide ligand is A-(SEQ ID NO:2)-A (referred to herein as 66-50-02-N001); A-(SEQ ID NO:16)-END (referred to herein as 66-50-16-T01-N001); A-(SEQ ID NO:18)-FSCDDD (referred to herein as 66-50-18-T01-N001); and A-(SEQ ID NO:21)-EPD (referred to herein as 66-50-20-T01-N001) 3. The peptide ligand of claim 2, or a pharma- ceutical acceptable salt thereof, comprising an amino acid sequence selected from:

4. 4. The peptide ligand or a pharma- ceutical acceptable salt thereof according to any one of claims 1 to 3, wherein the pharma- ceutical acceptable salt is selected from sodium, potassium, calcium, or ammonium salts.

5. The peptide ligand or a medicamentously acceptable salt thereof according to any one of claims 1 to 4, wherein the CD38 is human CD38.

6. A drug conjugate comprising the peptide ligand or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 5 conjugated to one or more effectors and / or functional groups.

7. A drug conjugate comprising the peptide ligand or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 5, conjugated to one or more cytotoxic agents.

8. A pharmaceutical composition comprising the peptide ligand according to any one of claims 1 to 5 or a pharma- ceutical acceptable salt thereof, or the drug conjugate according to claim 6 or claim 7.

9. 9. The pharmaceutical composition of claim 8 for preventing, suppressing, or treating a disease or disorder mediated by CD38.

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