Bicyclic peptide ligands specific for CD38

Abstract

To provide peptides which are high affinity binders of CD38.SOLUTION: The present invention provides 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.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention provides a method for preparing a peptide having two or more peptide loops interspersed between attachment points to a scaffold. In particular, the present invention relates to polypeptides covalently attached to non-aromatic molecular scaffolds. The invention describes peptides that are high affinity binders of CD38. A drug conjugate comprising the peptide conjugated to an effector and / or functional group. and a pharmaceutical composition comprising said peptide ligand and drug conjugate, and a method for detecting a CD38-dependent The peptide ligands and drugs in the prevention, inhibition, or treatment of diseases or disorders mediated by This also includes the use of bioconjugates. [Background technology]

[0002] BACKGROUND OF THEINVENTION Cyclic peptides are capable of binding to protein targets with high affinity and target specificity; It is therefore an attractive class of molecules for therapeutic development. Indeed, several cyclic peptides The tides include, for example, the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine, or the anticancer drug Like the cancer drug octreotide, it is already being used successfully in the clinic (Driggers et al. (2008), Nat Rev Drug Discov 7(7), 608-24. The excellent binding properties of the peptide and the target The conformational flexibility of the ring structure as well as the relatively large interaction surface formed between the ring and the target Generally, macrocycles are used as cyclic peptide CXCR4 antagonists, such as CVX15 (400 Å 2 (Wu et al., 2007), Science 330, 1066-71), Arg-Gly that binds to integrin αVb3 -Asp motif cyclic peptide (355 Å 2 )(Xiong et al. (2002), Science 296(5565), 151-5), or cyclic peptide inhibitors that bind to urokinase-type plasminogen activator Upain-1 (603Å 2 (Zhao et al., 2007), J Struct Biol 160(1), 1-10), It bonds to a surface of square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides. This results in a smaller entropy loss upon binding to the target, resulting in higher binding The reduced flexibility also leads to the fixation of the target-specific conformation, and the linear peptide This effect is due to the fact that when the ring is opened, it increases the binding specificity compared to other MM peptides. A potent and selective matrix metalloproteinase 8 (MMP-8) loses its selectivity for P This has been exemplified by specific inhibitors (Cherney et al., 1998, J Med Chem 41(11), 1749- 51) The advantageous binding properties achieved by macrocyclization have been demonstrated in, for example, vancomycin, nicotinamide, and nicotinamide-containing drugs. In polycyclic peptides, such as actinomycin and cyclopentadiene, Even more pronounced.

[0004] Various research teams have previously synthesized polypeptides containing cysteine ​​residues into synthetic molecular structures. (Kemp and McNamara, 1985, J. Org. Chem; Timmerman et al., 2005, Meloen and coworkers have reported that tris(bromomethyl)benzene and related molecules Synthesis of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces It was used for rapid and quantitative cyclization (Timmerman et al., 2005, ChemBioChem). A compound, wherein the compound is capable of converting a cysteine-containing polypeptide, e.g., 1,1',1''-(1,3,5- Molecular scaffolding such as triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA) The method for the preparation of the 1,2-diaminodiphenyl ether (1,2-diphenyl ether) is described in Heinis et al. (2014) Angewandt et al. e Chemie, International Edition 53(6) 1602-1606).

[0005] Creating and screening large libraries of bicyclic peptides against targets of interest A phage display-based combinatorial approach has been developed to (Heinis et al., 2009, Nat Chem Biol 5(7), 502-7 and WO 2009 / 098450). This results in a linear peptide containing three cysteine ​​residues and two random six-amino acid regions. A combinatorial library of peptides (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage. The cysteine ​​side chains were then covalently attached to a small molecule scaffold to effect cyclization. Summary of the Invention

[0006] (Summary of the invention) According to a first aspect of the present invention, at least two loop sequences separated by at least two loop sequences are provided. A polypeptide containing three cysteine ​​residues and a polypeptide having the cysteine ​​residues The compound comprises a non-aromatic molecular scaffold that forms a bond with at least two aromatic groups. A peptide loop is formed on the molecular scaffold, and the peptide is specific for CD38. Gund is provided.

[0007] According to a further aspect of the invention, the compound is conjugated to one or more effectors and / or functional groups. and a drug conjugate comprising the peptide ligand as defined herein. .

[0008] According to a further aspect of the invention, a peptide ligand or drug conjugate as defined herein is Pharmaceutical compositions are provided that include the adjugate in combination with one or more pharma- ceutical acceptable excipients. can be.

[0009] According to a further aspect of the present invention, a method for preventing, inhibiting, or preventing a disease or disorder mediated by CD38 is provided. or a peptide ligand or drug conjugate as defined herein for use in therapy. A gate is provided. 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 is one of two amino acids, The other has three cysteines separated by two loop sequences of seven amino acids. Contains amine residues.

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

[0013] In one embodiment, the loop sequences are, the first of which consists of 7 amino acids, The second one is a three-system protein separated by two loop sequences 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 are the first , the second, and the third cysteine ​​residues) or a pharma- ceutically acceptable salt thereof.

[0014] In a further embodiment, [ka] The peptide ligand of [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. vinegar) or a pharma- ceutical acceptable salt thereof.

[0016] In a further embodiment, [ka] The peptide ligand of 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 1,1',1''-(1,3,5-triazinane-1,3,5 -triyl)triprop-2-en-1-one (TATA), and the peptide ligand is selected from 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 further embodiments, the molecular scaffold is 1,1',1''-(1,3,5-triazinane -1,3,5-triyl)triplo-2-en-1-one (TATA), and the peptide ligand is selected from 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 comprises an amino acid sequence selected from: These antibodies exhibited excellent CD38 competitive binding as shown in Table 1 herein.

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

[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 are not included in the peptides of the present invention. The numbering of amino acid residues in 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 represented as 1,1',1''-(1,3,5-triazinane-1,3,5- It is believed that the cyclization with TATA (triyl) triprop-2-en-1-one results in a trisubstituted structure. Cyclization by TATA is i , C ii , and Ciii This occurs above.

[0022] (Molecular Format) N- or C-terminal extensions to the bicyclic core sequence may be placed to the left or right 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, The peptide sequences disclosed in also have utility in their retro-inverso forms. For example, it is envisaged that the sequence may be reversed (i.e., the N-terminus becomes the C-terminus, and so on). the terminus becomes N-terminus), and the stereochemistry is reversed as well (i.e., D-amino acids become L-amino acids). acids, and L-amino acids become D-amino acids).

[0024] (Peptide Ligand) The peptide ligands referred to herein are covalently attached to a molecular scaffold. Typically, such peptides are covalently bonded to a scaffold. The peptide has two or more reactive groups (i.e., cysteine ​​residues) that can form a sucrose residue. The sequence between the reactive groups is called a loop sequence because it forms a loop when it binds to the nucleic acid structure. In this case, the peptide contains at least three cysteine ​​residues (as in the present invention). In the fine print, C i , C ii , and C iii (called the "scaffold") and Both form two loops.

[0025] (Advantages of peptide ligands) Certain bicyclic peptides of the present invention can be administered by injection, inhalation, nasal, ocular, oral, or topical administration. These compounds have several advantageous properties that make them suitable drug-like molecules for administration. Such advantageous properties include: -species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation ; - Protease stability. The bicyclic peptide ligands are ideally suited to inhibit plasma proteases, epidermal ("membrane-anchored") proteases, gastrointestinal proteases, pulmonary surface proteases, intracellular proteases Protease stability should be demonstrated for bicyclic lead candidates. Not only can we develop them in animal models, but we can also confidently administer them to humans. should be maintained among different species; - Desirable solubility profile. This is important for formulation and absorption purposes, as it does not contain charged residues and and the ratio of hydrophilic to hydrophobic residues and intra- / inter-molecular H-bonds; - Optimal plasma half-life in the circulation. Depending on clinical indications and treatment regimens, acute disease management settings Develop bicyclic peptides for fixed, short-term exposure or to develop bicyclic peptides with enhanced retention in the circulation. It may be necessary to develop novel peptides that are best suited to managing more chronic disease states. Other factors driving new plasma half-lives are the need for sustained exposure for maximum therapeutic efficacy and the need for drug Concomitant toxicity with sustained exposure; and -Selectivity. Certain peptide ligands of the present invention exhibit better selectivity than other CDs.

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

[0027] The salts of the present invention can be prepared in a manner similar to that described in conventional chemical methods, e.g., Pharmaceutical Salts: Properties, Selection, and Uses. ical Salts: Properties, Selection, and Use), P. Heinrich Stahl (editor), Camille G. Wermuth (editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002 They can be synthesized from a parent compound that contains a basic or acidic moiety by the following methods: Typically, such salts are prepared by reacting the free acid or base form of these compounds with an appropriate base or acid and water. They can be prepared by reacting them in aqueous or organic solvents, or in a mixture of the two. This can be done.

[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, ascorbyl acetate, and the like. Ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid Acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphorsulfonic acid, (+) -(1S)-Camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, Enoic acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-Hydroxyethanesulfonic acid, formic acid, fumaric acid, mucic acid, gentisic acid, glucoheptan D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid, α-oxoglutaric acid, glycolic acid, hippuric acid, halogenated water Hydrochloric acid (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, malo (±)-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, oro Tonic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyrogenic acid glutamic 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, and acylated amino acids and cation exchange resins. Examples of the salt include mono-salts and di-salts formed with acids.

[0029] One particular group of salts is 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, naphtha Sulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactate. It comprises a salt formed from biotic acid. One particular salt is the hydrochloride salt. Another particular salt is , acetate.

[0030] The compound is anionic or has a functional group which may be anionic (e.g., -CO OH is -COO - In the case where the cation is a cation of the formula (I), the salt is formed with an organic or inorganic base to provide a suitable cation. Examples of suitable inorganic cations include Li + , Na +, and K + Alkali such as Metal ions, Ca 2+ and Mg 2+ Alkaline earth metal cations such as Al 3+ or Zn + And more Examples of suitable organic cations include, but are not limited to, the cations Ammonium ion (i.e., NH4 + ) and substituted ammonium ions (e.g., NHR + , NH2R 2 + , NHR3 + , NR4 + Some suitable substituted ammonium salts include, but are not limited to, Examples of nium ions are methylamine, ethylamine, diethylamine, propylamine, amine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, Ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzene amine, choline, meglumine, and tromethamine, as well as lysine and arginine. Examples of common quaternary ammonium ions include those derived from amino acids: N(C H3)4 + It is.

[0031] When the peptides of the invention contain amine functions, these can be, for example, substituted or unsubstituted amines, according to methods well known to those skilled in the art. By reaction with alkylating agents according to the method, quaternary ammonium salts can be formed. Such quaternary ammonium compounds are within the scope of the peptides of the present invention.

[0032] (Modified derivative) Modified derivatives of the peptide ligands defined herein are considered to be within the scope of the present invention. It will be appreciated that examples of suitable such modified derivatives include N-terminal and / or C-terminal End modifications; substitution of one or more amino acid residues with one or more non-naturally occurring amino acid residues (e.g., one or more Substitution of one or more polar amino acid residues with isosteric or isoelectronic amino acids; replacement of the amino acid residues with other non-natural isosteric or isoelectronic amino acids; addition of spacer groups; or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; substitution of one or more L-amino acid residues by one or more D-amino acid residues; substitution at one or more amide bonds in the bicyclic peptide ligand; N-alkylation of one or more peptide Replacement of doped bonds with surrogate bonds; modification of peptide backbone length; modification of the α-carbon of one or more amino acid residues replacement of hydrogen with another chemical group, cysteine, lysine, glutamic acid / aspartic acid, and the appropriate amine, thiol, etc. of the amino acid, such as tyrosine, to functionalize the amino acid. Direct modification and functionalization with phenol-, carboxylic acid-, and phenol-reactive reagents The amino acid that introduces the cross-linking activity, e.g., an alkyne or azide-containing moiety, respectively, may be used. Introduction or substitution of amino acids with azide or alkyne groups that allow functionalization of The modifications include one or more of the following modifications:

[0033] In one embodiment, the modified derivatives include N-terminal and / or C-terminal modifications. In embodiments, the modified derivatives include N-terminal modifications using suitable amino reaction chemistries, and and / or C-terminal modification using suitable carboxy reaction chemistry. The N- or C-terminal modifications may include, but are not limited to, cytotoxic agents, radioactive chelators, or It involves the addition of effector groups, including chromophores.

[0034] In a further embodiment, the modified derivative comprises an N-terminal modification. In this embodiment, the N-terminal modification comprises an N-terminal acetyl group. A cyclic group (referred to herein as C i The group called the aryl group is reacted with acetic anhydride or other suitable This embodiment results in a molecule that is capped with a reagent and acetylated at the N-terminus. This offers the advantage of eliminating potential recognition points for aminopeptidases, and is a promising method for the synthesis of bicyclic peptides. Avoid possible decomposition.

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

[0036] In a further embodiment, the modified derivative comprises a C-terminal modification. In this embodiment, the C-terminal modification comprises an amide group. In the specification, C iii The C-terminated group, called This embodiment results in a terminally amidated molecule. This offers the advantage of eliminating potential recognition points and reducing the proteolytic potential of bicyclic peptides. To decrease.

[0037] In one embodiment, the modified derivatives include one or more unnatural amino acid residues of one or more amino acid residues. In this embodiment, the substitution includes a group that is recognized by a degradative protease. have isosteric / isoelectronic side chains that are not toxic or have any adverse effect on targeting efficacy Unnatural amino acids may also be selected.

[0038] Alternatively, proteolytic hydrolysis of nearby peptide bonds may result in conformational and steric To be disruptive, unnatural amino acids with constrained amino acid side chains may be used. In particular, these include proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisopropyl ethers, butyric acid, Aib), and cycloamino, a simple derivative of amino-cyclopropyl carboxylic acid. Concerning acids.

[0039] In one embodiment, the modified derivative comprises the addition of a spacer group. In the present invention, the modified derivatives are i ) and / or a C-terminal cysteine ​​(C iii ) to This includes the addition of a spacer group.

[0040] In one embodiment, the modified derivatives include one or more oxidation-resistant modifications of one or more oxidation-sensitive amino acid residues. In a further embodiment, the modified derivative comprises a substitution with a tripeptide-binding amino acid residue. This embodiment includes the replacement of the tophan residue with a naphthylalanine or alanine residue. The resulting bicyclic peptide ligands provide the advantage of improved pharmaceutical stability profiles. do.

[0041] In one embodiment, the modified derivatives include one or more hydrophobic amino acids of one or more charged amino acid residues. In an alternative embodiment, the modified derivatives include one or more hydrophobic acid residues. The amino acid residues are replaced by one or more charged amino acid residues. The correct balance of 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 therefore the available free radicals in plasma. This affects the concentration of the isolated fraction, whereas charged amino acid residues (especially arginine) affect the concentration of the peptide. The combination of these two may affect the interaction of the phospholipid membranes on the cell surface. This may affect the half-life, volume of distribution, and exposure of peptide drugs, and may have clinical implications. In addition, the amount of charged and hydrophobic amino acids can be adjusted according to the endpoint. The correct combination and number of residues reduces irritation at the injection site (if the peptide drug is administered subcutaneously). It can be reduced.

[0042] In one embodiment, the modified derivative comprises one or more D-amino acid residues of one or more L-amino acid residues. This embodiment includes substitution by steric hindrance and stabilization of the β-turn conformation. The tendency of D-amino acids to form complexes is thought to enhance proteolytic stability (Tugyi et al. Reference (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 eliminating potential proteolytic attack sites. do.

[0044] Each of the above modifications serves to purposefully improve the potency or stability of the peptide. It should be noted that further improvement in potency based on the modification is achieved by the following mechanism: Can: -Utilizes the hydrophobic effect, resulting in a lower dissociation rate so that higher affinity is achieved Incorporating a hydrophobic moiety; -Long-range ionic interactions are utilized, resulting in faster association rates and higher affinity Incorporating charged groups into proteins (e.g., Schreiber et al., Rapid Electrostatic Assisted Synthesis of Proteins) Rapid, electrostatically assisted association of proteins (1996), Nature St. ruct. Biol. 3, 427-31); and - For example, amino acid side chains should be correctly oriented so that entropy loss is minimized upon target binding. The torsion angles of the backbone should be tightly constrained so that entropy loss is minimized upon target binding. By restricting the degree of cyclization and for the same reason introducing further cyclization into the molecule, Incorporating additional constraints into peptides (For a review, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 31 85-203, and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0045] (Isotopic Variation) The present invention relates to a compound in which one or more atoms have the same atomic number but different atomic masses or amounts commonly found in nature. is replaced by an atom having a different atomic mass or mass number from that of the present invention. All known pharma- ceutically acceptable (radio)isotope-labeled peptide ligands, as well as related The peptide of the present invention is provided with a metal chelating group capable of carrying a (radioactive) isotope. A peptide ligand (called an "effector"), as well as a specific functional group associated with it (radioactive Peptides of the invention covalently substituted with isotopes or isotopically labeled functional groups Contains a ligand.

[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. 3 6 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. Lin Isotopes of, e.g. 32 P, sulfur isotopes, e.g. 35 S, isotopes of copper, e.g. 64 Cu, Galli Isotopes of uranium, e.g. 67 Ga or 68 Ga, yttrium isotopes, e.g. 90 Y and Ru Isotopes of tethium, 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., incorporate a radioisotope. These include in drug and / or substrate tissue distribution studies and in the presence of CD38 targets on diseased tissues. The peptide ligands of the present invention are useful for clinically assessing the presence and / or absence of Formation of a complex between a labeled compound and another molecule, peptide, protein, enzyme, or receptor It also has valuable diagnostic properties in that it can be used to detect or identify Detection or identification methods may include, for example, radioisotopes, enzymes, fluorescent substances, luminescence Luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferin) Compounds that are labeled with a labeling agent such as a radioisotope may be used. Tritium in the body, i.e. 3 H(T) and carbon-14, i.e. 14 C is easy to implement It is particularly useful for this purpose given the means for detection and detection available.

[0048] Deuterium, i.e. 2 Substitution with heavier isotopes such as H(D) results in greater metabolic stability. , for example, as a result of increased in vivo half-life or reduced dosage requirements. may provide additional therapeutic benefits and therefore may be preferred in some circumstances. do.

[0049] 11 C. 18 F, 15 O, and 13 Substitution with positron emitting isotopes such as N is a promising method to investigate target occupancy. It may be useful in positron emission tomography (PET) studies for

[0050] Isotopically labeled compounds of the peptide ligands of the present invention are typically prepared using conventional techniques known to those of skill in the art. or by using appropriate isotopically labeled reagents instead of the previously utilized non-labeled reagents. These compounds can be prepared by processes similar to those described in the accompanying examples.

[0051] (Non-aromatic molecular scaffolds) Reference herein to the term "non-aromatic molecular scaffold" refers to an aromatic ( i.e., any of the ring systems defined herein that do not contain (unsaturated) carbocyclic or heterocyclic ring systems. This refers to the molecular scaffold of

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

[0053] As described in the aforementioned documents, molecular scaffolds are small molecules, such as small organic molecules. It may be a child.

[0054] In one embodiment, the molecular scaffold may be a polymer. In the present invention, the molecular scaffold is composed of amino acids, nucleotides, or carbohydrates. It is a polymer.

[0055] In one embodiment, the molecular scaffold reacts with a functional group of the polypeptide to form a covalent bond. It contains a reactive group capable of forming a bond.

[0056] The molecular scaffold contains chemical groups that form bonds with peptides, e.g., amines, thiol groups, etc. alcohols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, Alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and halides. It may contain acyl halogenides.

[0057] An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1''-(1,3,5-triazinane-1,3,5-triphenylphosphine) (yl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition, on(2014), 53(6), 1602-1606).

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

[0059] The effector and / or functional group can be, for example, a polypeptide at the N- and / or C-terminus of the polypeptide. It can be attached to an amino acid within a polypeptide or to a molecular scaffold.

[0060] Suitable effector groups include antibodies and portions or fragments thereof. For example, the effector group In addition to one or more constant region domains, the antibody light chain constant region (CL), antibody CH1 heavy chain domain, The antibody may comprise an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof. The effector group is attached to the hinge region of the antibody (between the CH1 and CH2 domains of the IgG molecule). Such regions may include those commonly found in

[0061] In a further embodiment of this aspect of the invention the effector group according to the invention is Advantageously, the peptide ligand-effector group according to the invention is tβ half-life of ≥ 2 days, ≥ 3 days, ≥ 4 days, ≥ 5 days, ≥ 6 days, or ≥ 7 days Most preferably, the peptide ligand Fc fusion comprises or consists of a peptide ligand Fc fusion having the The peptide ligand of the present invention is a peptide ligand Fc fusion protein having a tβ half-life of one day or more. It comprises or consists of a compound.

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

[0063] The ability of a peptide to penetrate into cells determines the effectiveness of the peptide against its intracellular target. This allows the target to be accessed by peptides that have the ability to penetrate into cells. In particular, transcription factors, tyrosine kinases, and other intracellular signaling molecules, as well as the apoptotic pathway, are involved in the pathogenesis of apoptosis. Functional groups that allow cell penetration include peptides or These include chemical groups attached to either the peptide or the molecular scaffold. See, for example, Chen and Harrison, Biochemical Society Transactions (2007), Vol. 35, no. 4, p. 821; Gupta et al., Advanced Drug Discovery Reviews (2004), Vol. 57, p. 9637 For example, VP22, HIV-Tat, Drosophila homeobox protein ( Peptides such as those derived from the ubiquitin complex (Antennapedia) that are efficient at moving through cell membranes Examples of short peptides that have been shown to The 16 amino acid penetratin peptide derived from protein (Derossi et al., 1994, J. Biol. Chem. 2003, 144:1311-1323) m. vol. 269, p. 10444), and the 18 amino acid "model amphipathic peptide" (Oehlke et al., 1998 ) Biochim Biophys Acts, Vol. 1414, p. 127) The arginine-rich region of the HIV TAT protein Non-peptide approaches include the use of peptides that can be easily attached to biomolecules. Examples include the use of small molecule mimetics, or SMOCs, that can s, vol. 4, p. 153). Another chemical strategy, adding guanidinium groups to molecules, also enhances cell penetration. (Elson-Scwab et al., 2007, J. Biol. Chem., vol. 282, p. 13585). Steroids, etc. It is possible to add low molecular weight molecules to the molecular scaffold to enhance their uptake into cells. can.

[0064] One class of functional groups that can be attached to peptide ligands is antibodies and Binding fragments thereof include, for example, Fab, Fv, or single domain fragments. In particular, peptides Using antibodies that bind to proteins that can increase the half-life of ligands in vivo It is possible.

[0065] In one embodiment, a peptide ligand effector group according to the invention is administered for: 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 Above, 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-enantiomer according to the invention has a tβ half-life selected from the group consisting of: The effector group or composition has a tβ half-life ranging from 12 hours to 60 hours. In an embodiment, it has a tβ half-life of 1 day or more. It ranges from 12 to 26 hours.

[0066] In one particular embodiment of the invention, the functional group complexes a medicamentously relevant metal radioisotope. The metal chelating agent is selected from those suitable for chelating the metal.

[0067] Possible effector groups include, for example, peptide ligands that act as substitutes for antibodies in ADEPT. Alternative Enzyme / Prodrug Therapy for Use with Carboxypeptidase G2 and Other Enzymes Any enzyme can be mentioned.

[0068] In one particular embodiment of the invention, the functional group is a cytotoxic agent, such as a cytotoxic agent for cancer therapy. Suitable examples include: cisplatin and carboplatin, and oxaliplatin. Saliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide alkylating agents such as the purine analogs azathioprine and mercaptopurine or pyrimidinyl Antimetabolites, including vinoxacin analogs; vincristine, vinblastine, vinorelbine, and vinoxacin analogs. Plant alkaloids and terpenoids, including vinca alkaloids such as desine; podophyllo Toxin and its derivatives etoposide and teniposide; originally known as taxol , taxanes, including paclitaxel; topoisomerase inhibitors, including camptothecin; Notecan and Topototecan, as well as Amsacrine, Etoposide, Etoposide Phosphate, and Further drugs include the immunosuppressant dactyly. cerebrospinal fluid (used in kidney transplants), doxorubicin, epirubicin, bleomycin, Examples of such antitumor antibiotics include cyclosporine, calicheamicin, and the like.

[0069] In one further particular embodiment of the invention, the cytotoxic agent is a maytansinoid (e.g. For example, DM1) or monomethyl auristatin (for example, MMAE).

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

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

[0072] In one embodiment, the cytotoxic agent is a cytotoxic agent that is a bond such as a disulfide bond or a protease sensitive bond. In a further embodiment, The groups adjacent to the disulfide bond are responsible for disruption of the disulfide bond and, therefore, cytotoxicity. The active agent may be modified to control the rate of cleavage and concomitant release of the active agent.

[0073] Published studies have demonstrated that by introducing steric hindrance on either side of the disulfide bond, has established the possibility of modifying the susceptibility of disulfide bonds to reduction (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). A greater degree of steric hindrance may lead to increased cell It slows down the rate of reduction by endogenous glutathione and also by extracellular (systemic) reducing agents, resulting in This reduces the ease with which toxins are released both inside and outside the cell. , versus optimizing efficient release in the intracellular environment, which maximizes therapeutic efficacy. Disulfide stability in circulation, which minimizes unwanted side effects of the toxin The selection of optimal conditions involves careful selection of the degree of disorder on either side of the disulfide bond. This can be achieved by:

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

[0075] In one embodiment, the cytotoxic agent and the linker are described in WO 2016 / 067035. (The cytotoxic agents and their linkers are incorporated herein by reference in their entirety.) (Incorporated herein).

[0076] (synthesis) The peptides of the invention can be synthetically produced by standard techniques and then subjected to in vitro molecular sequencing. This can be accomplished using standard chemistries. This allows for rapid release of soluble material for further downstream experimentation or validation. Such a method is disclosed in Timmerman et al. (supra). This can be accomplished using conventional chemistries such as those described in

[0077] Thus, the present invention also relates to a polypeptide selected as described herein. or the preparation of a conjugate, wherein the preparation is carried out as described below. In one embodiment, these steps include any further steps such as by chemical synthesis. The procedure is carried out on the final product polypeptide / conjugate produced by the method of the present invention.

[0078] Optionally, the amino acid residues in the polypeptide of interest are capable of forming a conjugate or complex. may be replaced when manufacturing.

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

[0080] To extend the peptide, it is simply performed using standard solid or solution phase chemistry: Orthogonally protected lysines (and analogues) are used at their N- or C-termini or within loops. Standard (bio)conjugation techniques can be used to chemically extend the active Alternatively, additions may be made, for example, by introducing a modified or activatable N- or C-terminus. awson et al., 1994, Synthesis of Proteins by Native Chemical Ligation (Synthesis of Proteins by Native Chemical Ligation). Hesis of Proteins by Native Chemical Ligation. Science 266:776-779) by fragment condensation or native chemical ligation, 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. Literature, Bioorganic & Medicinal Chemistry Letters, Volume 18, Issue 22, November 15, 2008, This may also be carried out enzymatically using subtiligase, as described in US Pat. No. 6,319,992 (pp. 6000-6003).

[0081] Alternatively, the peptides can be conjugated via further disulfide bonds. This is because the first and second peptides may be extended or modified by one another in the reducing environment of the cell. In this case, the molecular scaffold has the additional advantage of allowing the scaffold to dissociate into The cysteine ​​residues are added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups. then an additional cysteine ​​or thiol can be added to the N- or C-terminus of the first peptide; A cysteine ​​or thiol can be added to the second peptide so that the cysteine ​​or thiol can be released from the second peptide. It reacts only with cysteine ​​or thiol of the peptide to form a disulfide-bonded bicyclic peptide. A tide conjugate was formed.

[0082] A similar technique can be used to synthesize two bicyclic bispecific macrocycles, potentially giving rise to tetraspecific molecules. Applies equally to the synthesis / coupling of molecules.

[0083] Furthermore, the addition of other functional or effector groups can be accomplished using appropriate chemistry via N- or C- Coupling at the terminus or via a side chain may be accomplished in the same manner. In this manner, the coupling is performed in such a way as not to block the activity of either entity. do.

[0084] Pharmaceutical Composition According to a further aspect of the invention, a peptide ligand or drug conjugate as defined herein is Pharmaceutical compositions are provided that include the adjugate in combination with one or more pharma- ceutical acceptable excipients. can be.

[0085] Typically, the peptide ligand is administered in purified form together with a pharmacologically appropriate excipient or carrier. Typically, these excipients or carriers are saline and / or buffered The medium may include an aqueous or alcoholic / aqueous solution, an emulsion, or a suspension. Oral vehicles include sodium chloride solution, Ringer's dextrose, dextrose , and sodium chloride, and lactated Ringer's. Adjuvants, such as carboxymethylcellulose, may be used as needed to keep the polypeptide complex in suspension. From thickeners such as ethyl cellulose, polyvinylpyrrolidone, gelatin, and alginates may be selected.

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

[0087] The peptide ligands of the invention may be administered either as a separate composition or in combination with other agents. These may be used include antibodies, antibody fragments, as well as various immunotherapeutic agents, e.g. , cyclosporine, methotrexate, adriamycin, or cisplatin, and immunosuppressants. Pharmaceutical compositions include a variety of toxins in combination with the protein ligands of the present invention. "Cocktails" of cytotoxic or other drugs, or drugs that are pooled prior to administration or pooled polypeptides selected using different targeting ligands, whether or not they are It may even include a combination of selected polypeptides according to the invention having different specificities. Cut.

[0088] The route of administration of the pharmaceutical composition according to the present invention may be any of those commonly known to those skilled in the art. For therapy, the peptide ligands of the invention may be administered to any patient according to standard techniques. Administration can be parenteral, intravenous, intramuscular, intraperitoneal, transdermal, or pulmonary. or, equally appropriately, by direct injection using a catheter. Preferably, the pharmaceutical composition according to the present invention is The dosage and frequency of administration depend on the age, sex, and condition of the patient, as well as on other medications. The clinical outcome is determined by the concurrent administration of drugs, contraindications, and other parameters considered by the clinician. circle.

[0089] The peptide ligands of the present invention may be lyophilized prior to storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective and is compatible with freezing methods known in the art. Freeze-drying and reconstitution techniques can be used. Freeze-drying and reconstitution can result in varying degrees of activity. This may result in losses and the levels may need to be adjusted upwards to compensate. It will be understood by those skilled in the art.

[0090] Compositions containing the peptide ligands of the present invention or a cocktail thereof are useful for prophylactic and / or therapeutic purposes. In certain therapeutic applications, the concentration of selected cells can be increased. At least partial inhibition, suppression, regulation, killing, or some other measurable parameter of the group. An amount sufficient to achieve this goal is defined as a "therapeutically effective dose." The amount required to achieve this will depend on the severity of the disease and the general state of the patient's own immune system. The dose depends on the individual, but generally, 0.005-5.0 mg of the selected peptide ligase per kilogram of body weight is used. The range is from 0.05 to 2.0 mg / kg / day, with doses of 0.05 to 2.0 mg / kg / day being more commonly used. In addition, compositions containing the peptide ligands or cocktails thereof also have similar or slightly Small dosages may be administered.

[0091] Compositions containing peptide ligands according to the invention can be used in prophylactic and therapeutic settings. Assisting in the alteration, inactivation, killing, or elimination of selected target cell populations in mammals In addition, the peptide ligands described herein can be selected ex vivo or in vitro. selectively used to selectively kill or deplete a target cell population from a heterogeneous collection of cells. The blood from the mammal can be filtered or otherwise effectively removed. The peptide ligand can then be combined in vitro and then expressed in mammalian cells according to standard techniques. Killing or otherwise removing undesirable cells from the blood for return to the animal .

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

[0093] CD38 is a 45 kD type II molecule with a long C-terminal extracellular domain and a short N-terminal cytoplasmic domain. CD38 is a transmembrane glycoprotein that converts NAD+ to cyclic ADP-ribose (cADPR). It can catalyze the conversion and hydrolyze cADPR to ADP-ribose. During ontogeny, CD38 is expressed by CD34+ committed stem cells as well as lymphoid CD38 expression is expressed on most lineage-committed precursors of myeloid, erythroid, and myeloid cells. In either case, it persists in the lymphoid lineage and expression levels vary during different stages of T and B cell development. different.

[0094] CD38 is expressed in many hematopoietic malignancies, as well as in non-Hodgkin's lymphoma (NHL), Burkitt's lymphoma (BMLL), and leukemia (LE). B-chronic lymphocytic leukemia (B-CLL), B and T acute lymphoblastic leukemia (TCL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), pathocytic leukemia (ALL), T-cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL) ), Hodgkin's lymphoma (HL), and chronic myeloid leukemia (CML). On the other hand, most of the primitive pluripotent stem cells of the hematopoietic system are upregulated in the cell lines from which they are derived. The cells are CD38-. CD38 expression in hematopoietic malignancies and its correlation with disease progression , making 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 lymphoid and myeloid cells or cell lines. Numerous signaling molecules, including phospholipase C-γ, ZAP-70, syk, and c-cbl, in Signal transduction via tyrosine phosphorylation of F. cerevisiae (Funaro et al., 1993) Eur J Immunol 23, 24 07-2411; Morra et al. (1998), supra; Funaro et al. (1990) J Immunol 145, 2390-239 6; 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, 2840-284 5; Kitanaka et al. (1997) J Immunol 159, 184-192; Kitanaka et al. (1999) J Immun ol 162, 1952-1958; Mallone et al. (2001) Int Immunol 13, 397-409) Based on these observations, CD38 is essential for the normal development of lymphoid tissue. and has been proposed to be an important signaling molecule in the maturation and activation of myeloid cells.

[0096] Notably, the majority of these signaling studies were performed using cell lines ectopically overexpressing CD38. Since we use a non-physiological ligand, anti-CD38 monoclonal antibody, the signal transduction The exact role of CD38 in hematopoiesis and hematopoiesis remains unclear. 2+ Because it has the enzymatic activity to produce cADPR, a molecule that can induce mobilization (Lee et al. (1989) J Biol Chem 264, 1608-1615; Lee and Aarhus (1991) Cell Regul 2, 203-209), CD38 ligation by monoclonal antibodies increases cADPR production By this, Ca in lymphocytes 2+ It has been proposed that this mechanism 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 revealed that its cytoplasmic tail also modulates its enzymatic activity. was also shown to be dispensable for anti-CD38 antibody-mediated signal transduction (Kitanaka (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 is its role in T-cell proliferation through defects in innate immunity and defective dendritic cell migration. CD38 knockout mice have a reduced cytoplasmic humoral response (Pa Partida-Sanchez et al. (2004) Immunity 20, 279-291; Partida-Sanchez et al. (2001) Nat Med 7, 1209-1216). However, the expression pattern of CD38 during hematopoiesis differs significantly between humans and mice. Therefore, it is unclear whether the function of CD38 in mice is identical to that of CD38 in humans. a) Unlike immature progenitor stem cells in humans, similar progenitor stem cells in mice The cells express high levels of CD38 (Randall et al. (1996) Blood 87, 4057-4067; Dagh et al. (1998) Blood 87, 4057-4067). (1998) Biol Blood Marrow Transplant 4, 69-74), b) in human B cell development High levels of CD38 expression are seen on germinal center B cells and plasma cells (Uckun, 1990). d 76, 1908-1923; Kumagai et al. (1995) J Exp Med 181, 1101-1110), whereas in mice, The expression level of CD38 in the corresponding cells is low (Oliver et al. (1997) J Immunol 158, 1108-1119). 1115; Ridderstad and Tarlington (1998) J Immunol 160, 4688-4695).

[0098] Several anti-human CD38 antibodies have different proliferation characteristics against various tumor cells and cell lines. For example, the chimeric OKT10 antibody having a mouse Fab and a human IgG1 Fc has been described in the literature. The results showed that MM-related markers were expressed in the presence of peripheral blood mononuclear effector cells derived from either MM patients or normal individuals. Mediates antibody-dependent cell-mediated cytotoxicity (ADCC) highly efficiently against lymphoma cells (Stevenson (1991) Blood 77, 1071-1079). The CDR-grafted humanized version of the anti-CD38 antibody AT13 / 5 is It has been shown to have potent ADCC activity against CD38-positive cell lines (U.S. Pat. App. No. 6,313,363). Human monoclonal anti-CD38 antibodies have been shown to mediate ADCC and / or complement-dependent cytotoxicity (CD C) mediates in vitro killing of CD38 positive cell lines, as well as the MM cell line RPMI-8226 It has been shown to slow tumor growth in SCID mice bearing On the other hand, some anti-CD38 antibodies, IB4, SUN-4B7, and OKT10, inhibited peripheral blood from normal individuals. IB6, AT1, or AT2 did not induce proliferation of mononuclear cells (PBMCs) ( Ausiello et al. (2000) Tissue Antigens 56, 539-547).

[0099] Some of the prior art antibodies are capable of inducing apoptosis in CD38+ B cells. However, these have not been reported to be related to stromal cells or stromal-derived cytokines. The agonistic anti-CD38 antibody (IB4) inhibits human germinal center (GC) B cells. Preventing cell apoptosis (Zupo et al. (1994) Eur J Immunol 24, 1218-1222), as well as It also induces proliferation of KG-1 and HL-60 AML cells (Konopleva et al., 1998, J Immunol 16). 1, 4702-4708), and failure to induce apoptosis in Jurkat T lymphoblastic cells (Morr (1998) FASEB J 12, 581-592). Another anti-CD38 antibody, T16, has been reported to inhibit the inflammatory bowel disease of ALL patients. Apoptosis of immature lymphoid cells and leukemic lymphoblast cells from patients (Kumagai et al., 2013). (1995) J Exp Med 181, 1101-1110), and the allogeneic leukemic myeloblast cells derived from AML patients. T16 induced apoptosis in stromal cells (Todisco et al., 2000, Blood 95, 535-542). or induces apoptosis only in the presence of stromal-derived cytokines (IL-7, IL-3, stem cell factor) I didn't.

[0100] Polypeptide ligands selected by the methods of the invention can be used in in vivo therapeutic and prophylactic applications. It can be used for in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, etc. Ligands with a selected level of specificity can be used to target non-human immunocytes for which cross-reactivity is desired. For applications involving testing in human animals or where cross-reactivity with homologs or paralogs is of concern, It is useful in diagnostic applications where the concentration of the ion channel is carefully controlled. In this application, the ability to induce an immune response to a range of antigens is exploited to develop a vaccine. The treatment can be tailored to specific diseases and pathogens.

[0101] Substantially pure peptide ligands having at least 90-95% homogeneity are administered to mammals. A homogeneity of 98-99% or more is preferred for pharmaceutical use, particularly for administration to mammals. Once refined, either partially or until homogeneous, as desired, it is most preferred. Once produced, the selected polypeptides may be used diagnostically or therapeutically (including in vitro), or It can be used in the development and implementation of assay procedures, immunofluorescence staining, etc. (Lefkovi te and Pernis (1979 and 1981), Immunological Methods, Vols. I and II, Academic Press. ic Press, NY).

[0102] According to a further aspect of the present invention, a method for preventing, inhibiting or treating a disease or disorder mediated by CD38 is provided. The present invention relates to a peptide ligand or drug conjugate as defined herein for use in therapy. A gate is provided.

[0103] According to a further aspect of the present invention, a method for preventing, inhibiting or treating a disease or disorder mediated by CD38 is provided. The present invention relates to a method of treatment comprising administering to a patient in need thereof a peptide linker as defined herein. and administering to the subject a drug conjugate and an effector group thereof. do.

[0104] In one embodiment, the CD38 is mammalian CD38. The animal 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 can be treated (or inhibited) include tumors of epithelial origin (adenocarcinoma Various types of adenomas and carcinomas, including squamous cell carcinoma, transitional cell carcinoma, and other carcinomas, such as Examples include bladder and urinary tract, breast, gastrointestinal tract (esophagus, stomach (gastric), small intestine, colon, rectum). , and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidneys, lungs (e.g. , adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g. Cancer of the tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and paranasal sinuses), ovaries, fallopian tubes, Peritoneum, vagina, vulva, penis, cervix, myometrium, endometrium, thyroid (e.g., thyroid follicles) cancer), adrenal gland, prostate, skin, and adnexal cancer (melanoma, basal cell carcinoma, squamous cell carcinoma, keratinocyte carcinoma, acanthoma, dysplastic nevi); hematologic malignancies (i.e., leukemia, lymphoma) and premalignant hematologic disorders Borderline malignancies, including hematologic malignancies of the lymphoid lineage and related diseases (e.g., acute lymphoblastic leukemia, Myeloid leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large cell lymphoma DLBCL, follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma 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 disorders), and hematological malignancies and related disorders of the myeloid lineage (e.g. For example, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), Hypereosinophilic syndromes, myeloproliferative disorders such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndromes, myelodysplastic syndromes, and promyelocytic leukemia); mesenchymal Tumor of origin, e.g., soft tissue, bone, or cartilage sarcoma, 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 protruding Dermatofibrosarcoma; tumors of the central or peripheral nervous system (e.g., astrocytoma, glioma, and glioblastoma) endocrine tumors (e.g., pituitary tumors, meningiomas, ependymomas, pineal tumors, and schwannomas); tumors, adrenal tumors, pancreatic islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary carcinoma of the thyroid gland ocular and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, spermatozoa) epithelioma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and pediatric and embryonal tumors (e.g. , medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or Congenital or other syndromes that predispose to tumors (e.g., xeroderma pigmentosum) These include, but are not limited to:

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

[0108] References herein to the term "prophylaxis" refer to the administration of a protective composition prior to the induction of disease. "Suppression" includes administration of a composition after an inductive event but before clinical manifestation of the disease. "Treatment" includes administration of a protective composition after disease symptoms are manifest.

[0109] Screening for the efficacy of peptide ligands in protecting against or treating disease There are animal model systems available that can be used to study the effects of Allows for the development of polypeptide ligands that can cross-react with human and animal targets This is facilitated by the present invention.

[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 performed using a Symphony peptide synthesizer manufactured by Peptide Instruments. The synthesis was based on Fmoc chemistry using a MultiSynTech Syro II synthesizer. Acids (Sigma, Merck) with appropriate side-chain protecting groups were used: standard coupling Ring-protection conditions were used in each case, followed by deprotection using standard methodology. The peptide was purified and isolated using HPLC, and then was converted to 1,3,5-triacryloylhexahydro-1 To this end, the linear peptide was reacted with 50:50 MeCN: Dilute to approximately 35 mL with HO, add approximately 500 μL of 100 mM TATA in acetonitrile, and dilute to approximately 35 mL with HO. The reaction was initiated with L of 1M NH4HCO3. The reaction was allowed to proceed at RT for approximately 30 to 60 min and then diluted with (MALD When the reaction was complete (as judged by I), it was lyophilized. Cysteine ​​hydrochloride monohydrate (Sigma) was added to the reaction mixture for approximately 60 min at RT to quench excess TATA. I punched him.

[0112] After lyophilization, the modified peptides were purified as described above, while Luna C8 was purified using Gemini C18. The column was replaced with a Phenomenex column and the acid was changed to 0.1% trifluoroacetic acid. Pure fractions containing 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 purified using the following method to synthesize the toxin. 4-Methyl(succinimidyl) 4-(2-pyridylthio) (E) pentanoate) (100 mM) in anhydrous DMSO (1.25 mol equivalents) The reaction mixture was mixed thoroughly 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 peptide of the present invention for human CD38 was determined according to the method described by Lea et al. (Expert Opin Drug Discovery). iscov. 2011 6(1): 17-3) and for the following TATA derivatives: Fluorescently labeled peptides [ka] (where Fl is a fluorescein molecule) was used to determine the Ta.

[0116] The peptide ligands of the present invention were tested in the CD38 competitive binding assay described above. The results are shown in Table 1. As shown: Table 1: Biological assay data for peptide ligands of the invention [Table 1] TIFF2025023938000012.tif154170

Claims

[Claim 1] The novel products, methods and processes substantially as herein described.