Bicyclic peptide ligand specific for nectin-4

Abstract

The present invention relates to polypeptides covalently attached to a molecular scaffold such that two or more peptide loops are taut between attachment points to the scaffold. In particular, the present invention describes peptides that are high-affinity binders of Nectin-4. 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 preventing, suppressing, or treating diseases or disorders mediated by Nectin-4.

Description

[Technical field]

[0001] The present invention relates to a polypeptide covalently attached to a molecular scaffold such that two or more peptide loops are stretched between attachment points to the scaffold. In particular, the present invention describes peptides that are high affinity binders of Nectin-4. The present invention also includes drug conjugates comprising said peptides conjugated to one or more effectors and / or functional groups, pharmaceutical compositions comprising said peptide ligands and drug conjugates, and the use of said peptide ligands and drug conjugates in preventing, inhibiting or treating diseases or disorders mediated by Nectin-4. [Background technology]

[0002] Cyclic peptides are able to bind with high affinity and specifically target protein targets and are therefore an attractive class of molecules for the development of therapeutics. Indeed, some cyclic peptides have already been successfully used in the clinic, for example the antimicrobial peptide vancomycin, the immunosuppressant cyclosporine or the anticancer drug ocretide (Driggers et al. (2008), Nat Rev Drug Discov7(7), 608-24). The good binding properties are due to the relatively large interaction surface formed between the peptide and the target, as well as the reduced conformational flexibility of the cyclic structure. Typically, for example, the cyclic peptide CXCR4 antagonist CVX15 (400 Å 2 ;Wu et al.(2007), Science 330, 1066-71), integrin αVb3 (355Å 2 ) (Xiong et al. (2002), Science 296(5565), 151-5) or the cyclic peptide inhibitor upain-1 (603 Å) that binds to urokinase-type plasminogen activator. 2 Macrocycles such as those shown in Fig. 1; Zhao et al. (2007), J Struct Biol 160(1), 1-10) bind to surfaces measuring hundreds of square angstroms.

[0003] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropy loss when binding to targets and higher binding affinity. Reduced flexibility also leads to a more fixed target-specific conformation and increased binding specificity compared to linear peptides. This effect is exemplified by the potent and selective inhibitor of matrix metalloproteinase 8 (MMP-8), whose selectivity is lost over other MMPs when the ring is opened (Cherney et al. (1998), J Med Chem 41(11), 1749-51). The favorable binding properties achieved through macrocyclization are even more pronounced in polycyclic peptides with more than one peptide ring, such as vancomycin, nisin and actinomycin.

[0004] Different research teams have already conjugated polypeptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and coworkers have used tris(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds to structurally mimic protein surfaces (Timmerman et al. (2005), ChemBioChem). A method for generating candidate drug compounds, the compounds being generated by conjugating cysteine-containing polypeptides to molecular scaffolds, such as TATA (1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one Heinis et al. Angew Chem, Int Ed. 2014;53:1602-1606).

[0005] Phage display-based combinatorial approaches have 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 two regions of three cysteine ​​residues and six random amino acids (Cys-(Xaa)6-Cys-(Xaa)6-Cys) was displayed on phage and cyclized by covalently linking the cysteine ​​side chains to a small molecule scaffold. Summary of the Invention

[0006] According to a first aspect of the present invention, there is provided a peptide ligand specific for Nectin-4, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a 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 peptide ligand having the amino acid sequence: C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:1); (In the formula, 1NaI represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, and C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively), or a pharma- ceutically acceptable salt thereof.

[0007] According to a further aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as described 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 described herein in combination with one or more pharma- ceutically acceptable excipients.

[0009] According to a further aspect of the present invention, there is provided a peptide ligand or drug conjugate as described herein for use in preventing, inhibiting or treating a disease or disorder mediated by Nectin-4.

[0010] In the figures, error bars, when present, represent standard error of the mean (SEM). [Brief description of the drawings]

[0011] [Figure 1] Tumor volume tracing after administration of BCY8245 to female BALB / c nude mice bearing NCI-H292 xenografts. [Diagram 2] Tumor volume tracing after administration of BCY8245 to female BALB / c nude mice bearing NCI-H292 xenografts. [Diagram 3] Tumor volume tracing following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts. [Figure 4] Tumor volume tracing following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts. [Diagram 5] Tumor volume tracing after administration of BCY8245 to female Balb / c nude mice bearing Panc2.13 xenografts. [Figure 6] Tumor volume tracing following administration of BCY8245 to female Balb / c nude mice bearing MDA-MB-468 xenografts. [Figure 7] Tumor volume tracings following administration of BCY8549 (and BCY8245 as a control) to female BALB / c nude mice bearing NCI-H292 xenografts. [Figure 8]Gating strategy for Nectin-4 in breast (T-47D and MDA-MB-468). [Figure 9] Gating strategy for Nectin-4 in NCI-H292 and NCI-H322. [Figure 10] Gating strategies for nectin-4 in NCI-H526 and HT1080, respectively. [Figure 11] Gating strategies for nectin-4 in NCI-H526 and HT1080, respectively. [Figure 12] Gating strategy for Nectin-4 in bladder cancer (HT1376). [Figure 13] Gating strategy for Nectin-4 in breast cancer (MDA-MB-468). [Figure 14A] Gating strategy for Nectin-4 in colorectal cancer (HT-29). [Figure 14B] Gating strategy for Nectin-4 in colorectal cancer (HCT-116). [Figure 15A] Gating strategy for nectin-4 in lung cancer (A549). [Figure 15B] Gating strategy for Nectin-4 in lung cancer (NCI-H292). [Figure 15C] Gating strategy for Nectin-4 in lung cancer (NCI-H358). [Figure 15D] Gating strategy for Nectin-4 in lung cancer (NCI-526). [Figure 16] Gating strategy for Nectin-4 in pancreatic cancer (Panc02.13). [Figure 17] Tumor volume tracing after administration of BCY8245 to female Balb / c nude mice bearing A549 xenografts. [Figure 18]Tumor volume tracing following administration of BCY8245 to female Balb / c nude mice bearing HCT116 xenografts. [Figure 19] Tumor volume tracing following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts. [Figure 20] Tumor volume tracing following administration of BCY8245 to female Balb / c nude mice bearing MDA-MB-468 xenografts. [Figure 21] Tumor volume tracing after administration of BCY8245 to female BALB / c nude mice bearing NCI-H292 xenografts. [Figure 22] Tumor volume tracing after administration of BCY8245 to female Balb / c nude mice bearing NCI-H526 xenografts. [Diagram 23] Tumor volume tracing after administration of BCY8245 to female Balb / c nude mice bearing Panc2.13 xenografts. [Figure 24] Tumor volume tracings following administration of BCY8245 or a combination of BCY8245 and BCY8234 to female Balb / c nude mice bearing MDA-MB-468 xenografts. [Diagram 25] Tumor volume tracings following administration of BCY8245 alone or in combination with BCY8245 and BCY8234 to female Balb / c nude mice bearing MDA-MB-468 xenografts. [Figure 26] Tumor volume tracing in Lu-01-0412 PDX xenografts. [Figure 27] Tumor volume tracing in LU-01-0007 PDX xenografts. [Figure 28] Tumor volume tracing in CTG-1771 PDX xenografts. [Figure 29] Tumor volume tracing in CTG-1171 PDX xenografts. [Diagram 30] Tumor volume tracing in CTG-1106 PDX xenografts. [Diagram 31] Tumor volume tracing in CTG-0896 PDX xenografts. [Diagram 32] The efficacy of BT8009 (i.e. BCY8245) correlates with expression in CDX / PDX xenografts. Xenografts with little / no Nectin-4 expression show reduced tumor growth rate. Xenografts expressing Nectin-4 show tumor regression. Both PDX and CDX models are included in this analysis and values ​​are collated from various reports. [Diagram 33] MDA-MB-468 cells express Nectin-4 and demonstrate long-term retention of MMAE in tumors. [Diagram 34] HCS-data analysis for MDA-MB-468 cell line. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In one embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) is [B-Ala][Sar10]-(SEQ ID NO:1) (hereinafter referred to as BCY8234); Ac-[B-Ala][Sar5]-(SEQ ID NO:1) (hereinafter referred to as BCY8122); Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126); (SEQ ID NO:1) (hereinafter referred to as BCY8116); Fluorescein-(SEQ ID NO:1) (hereinafter referred to as BCY8205); and [PYA][B-Ala][Sar10]-(SEQ ID NO:1) (hereinafter referred to as BCY8846) The amino acid sequence is selected from the group consisting of:

[0013] In a further embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) is [B-Ala][Sar10]-(SEQ ID NO:1) (hereinafter referred to as BCY8234); Ac-[B-Ala][Sar5]-(SEQ ID NO:1) (hereinafter referred to as BCY8122); Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126); (SEQ ID NO:1) (hereinafter referred to as BCY8116); and Fluorescein-(SEQ ID NO:1) (hereinafter referred to as BCY8205) The amino acid sequence is selected from the group consisting of:

[0014] In a further embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) is Ac-[B-Ala][Sar5]-(SEQ ID NO:1) (hereinafter referred to as BCY8122); Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126); and (SEQ ID NO: 1) (hereinafter referred to as BCY8116) The amino acid sequence is selected from the group consisting of:

[0015] Data shown in Table 2 herein demonstrate that the peptide ligands of this embodiment exhibited excellent levels of binding to human Nectin-4, as evidenced by SPR binding data.

[0016] In a further embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) is Ac-[B-Ala][Sar5]-(SEQ ID NO:1) (hereinafter referred to as BCY8122); and Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126) The amino acid sequence is selected from the group consisting of:

[0017] Presented in Table 1 herein are data demonstrating that the peptide ligands of this embodiment exhibited superior levels of binding to human Nectin-4, as evidenced by competitive binding data.

[0018] In a further embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) is [B-Ala][Sar10]-(SEQ ID NO:1) (hereinafter referred to as BCY8234) The amino acid sequence is selected from the group consisting of:

[0019] Presented in Table 3 herein are data demonstrating that the peptide ligands of this embodiment, when conjugated to a cytotoxic agent, exhibited excellent levels of binding to human Nectin-4 (<10 nM), as evidenced by SPR binding data.

[0020] In one embodiment, the molecular scaffold is 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, e.g., in the art of peptide chemistry, cell culture and phage display, nucleic acid chemistry and biochemistry. Standard techniques are used for molecular biology, genetic and biochemical methods (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) 4). thed., John Wiley & Sons, Inc.), which is incorporated herein by reference.

[0022] <Nomenclature> Numbering When referring to the position of an amino acid residue in a peptide of the present invention, a cysteine ​​residue (C i , C ii and C iii ) are excluded from the numbering since they are invariant, so that the numbering of amino acid residues in the peptides of the present invention is C i -P1-[1Nal]2-[dD]3-C ii -M4-[HArg]5-D6-W7-S8-T9-P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO:1) It is regarded as follows.

[0023] For the purposes of this description, it is assumed that all bicyclic peptides are cyclized with 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplo-2-an-1-one (TATA) to give the trisubstituted structures. Cyclization with TATA results in the C i , C ii , and C iii This occurs in.

[0024] Molecular Format N- or C-terminal extensions to the bicyclic core sequence are added to the left or right of the sequence and separated by a hyphen. For example, an N-terminal βAla-Sar10-Ala tail is βAla-Sar10-A-(SEQ ID NO:X) It will be shown as:

[0025] Inverted 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 when a sequence is inverted (i.e., N-terminus becomes C-terminus, and vice versa), its stereochemistry is likewise inverted (i.e., D-amino acids become L-amino acids, and vice versa).

[0026] <Peptide Ligand> A peptide ligand as referred to herein refers to a peptide that is covalently attached to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) that can form a covalent bond to the scaffold, and a sequence defined between said reactive groups, referred to as a loop sequence, for forming a loop when the peptide is attached to the scaffold. In this case, the peptide comprises at least three cysteine ​​residues (referred to herein as C i , C ii and C iii ), which forms at least two loops on the scaffold.

[0027] <Advantages of peptide ligands> Certain bicyclic peptides of the invention have several advantageous properties that allow them to be considered suitable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include: - species cross-reactivity, which is a typical prerequisite for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability. Bicyclic peptide ligands should ideally demonstrate stability against plasma proteases, epithelial ("membrane anchored") proteases, gastrointestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicyclic lead candidates can be developed in animal models and administered to humans with confidence; - a desirable solubility profile, which is a function of charge and the ratio of hydrophilic to hydrophobic residues and intra / inter-molecular H-bonds, which is important for formulation and absorption purposes; - Optimal plasma half-life in the circulation. Depending on the clinical application and treatment regimen, it may be necessary to develop a bicyclic peptide for short-term exposure in the acute disease management setting, or to develop a bicyclic peptide with enhanced retention in the circulation, and therefore more optimal for the management of chronic disease conditions. Other factors driving the desired plasma half-life are the requirement of sustained exposure for maximum therapeutic efficacy versus the concomitant toxicology resulting from sustained exposure of the drug; and - Selectivity. Certain peptide ligands of the invention demonstrate better selectivity than other nectins.

[0028] <Pharmaceutically acceptable salts> It will be understood that salt forms are within the scope of the present invention and that a reference to a peptide ligand includes the salt form of said ligand.

[0029] The salts of the present invention may be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent, or in a mixture of the two.

[0030] Acid addition salts (mono or di-salts) may be formed with a 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, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, and acetic acid. Acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g. D-glucuronic acid), glutamic acid (e.g. L-glutamic acid), α-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acids (e.g. hydrobromic acid, salts acid, hydroiodic acid), isethionic acid, lactic acid (e.g. (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid The mono- or di-salts formed with acids selected from the group consisting of carboxylic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid, valeric acid, as well as with acylated amino acids and cation exchange resins.

[0031] One particular group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, 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.

[0032] The compound is or can be anionic (e.g., -COOH becomes -COO - In the case of compounds having functional groups, salts may be formed with organic or inorganic bases which generate suitable cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions, such as Li + , Na + and K. + , alkaline earth metal cations, e.g. Ca 2+ and Mg 2+ , and other cations, e.g. Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH + ) and substituted ammonium ions (e.g., NHR + , NH2R2 + , NHR3 + , NR4 + ) are some suitable substituted ammonium ions, such as methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as those derived from amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH3)4 + It is.

[0033] When the peptides of the invention contain amine functions, they may be formed into quaternary ammonium salts, for example by reaction with alkylating agents, according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the invention.

[0034] <Modified Derivatives> It will be understood that modified derivatives of the peptide ligands described herein are within the scope of the present invention. Examples of 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; replacement of one or more L-amino acid residues with one or more D-amino acid residues in a bicyclic peptide ligand. or N-alkylation of multiple amide bonds; replacement of one or more peptide bonds with a surrogate bond; modification of the peptide backbone length; replacement of the hydrogen on the alpha-carbon of one or more amino acid residues with another chemical group, modification of amino acids, e.g., cysteine, lysine, glutamic acid / aspartate and tyrosine, to functionalize said amino acids with suitable amine-, thiol-, carboxylic acid- and phenol-reactive reagents, introduction or replacement 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-containing moieties.

[0035] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, the modified derivative comprises an N-terminal modification using suitable amino-reactive chemistry and / or a C-terminal modification using suitable carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector group, including but not limited to a cytotoxic agent, a radiochelator, or a chromophore.

[0036] 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 (as defined herein as C i The 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 potential recognition points for aminopeptidases and avoids possible degradation of the bicyclic peptide.

[0037] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates retention of the potency of the bicyclic peptide for conjugating an effector group and its target.

[0038] 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 (referred to herein as C iii The C-terminal amidated group (referred to as "C-terminal amidated group") 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.

[0039] In one embodiment, the engineered derivative comprises 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 and do not have any adverse effect on targeting potential.

[0040] Alternatively, unnatural amino acids may be used that have constrained amino acid side chains such that proteolytic hydrolysis of nearby peptide bonds is conformationally and sterically hindered. In particular, these relate to proline analogues, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and simple derivatives that are cycloamino acids, aminocyclopropyl carboxylic acids.

[0041] In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises the addition of a spacer group to the N-terminal cysteine ​​(Ci) and / or the C-terminal cysteine ​​(Ciii).

[0042] 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.

[0043] 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 vs. hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues may 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) may affect the interaction of the peptide with phospholipid membranes on cell surfaces. The combination of the two may affect the half-life, volume of distribution and exposure of the peptide drug, which can be adjusted according to clinical endpoints. Furthermore, the correct combination and number of charged vs. hydrophobic amino acid residues may reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0044] In one embodiment, the modified derivative comprises the substitution of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability through steric hindrance and the tendency of D-amino acids to stabilize β-turn conformations (Tugyi et al (2005) PNAS, 102(2), 413-418).

[0045] In one embodiment, the modified derivative comprises the removal and replacement of any amino acid residue with alanine. This embodiment offers the advantage of removing potential proteolytic attack sites.

[0046] It should be noted that each of the above mentioned modifications serves to purposefully improve the potency or stability of the peptide. Further improvements in potency based on the modifications may be achieved by: - incorporating hydrophobic moieties that exploit the hydrophobic effect and result in low off rates so that high affinity is achieved; - incorporating charged groups that exploit long range ionic interactions, enhancing speed and providing high affinity (see, e.g., Schreiber et al, Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and - incorporating additional constraints in the peptide, for example by precisely constraining the side chains of the amino acids, constraining the torsion angles of the backbone so that the entropic loss is minimal upon target binding, and introducing additional cyclizations in 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); This may be achieved via the mechanism

[0047] <Isotope Variation> The present invention includes all pharma- ceutically acceptable (radioactive) isotope-labeled peptide ligands of the invention, in which one or more atoms are replaced by an atom having the same atomic number but an atomic mass or mass number different from that normally found in nature, and the peptide ligands of the invention (referred to as "effectors") to which metal chelating groups are attached may carry a suitable (radioactive) isotope, and the peptide ligands of the invention, in which certain functional groups are covalently replaced with a suitable (radioactive) isotope or isotopically labeled functional group.

[0048] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention are hydrogen, e.g. 2 H(D) and 3 H(T), carbon, e.g. 11 C. 13 C and 14 C, chlorine, e.g. 36 Cl, fluorine, e.g. 18 F, iodine, e.g. 123 I, 125 I and 131 I, nitrogen, e.g. 13 N and 15 N, oxygen, e.g. 15 O. 17 O and 18 O, phosphorus, e.g. 32 P, sulfur, e.g. 35 S, copper, e.g. 64 Cu, gallium, e.g. 67 Ga or 68 Ga, yttrium, e.g. 90 Y and lutetium, e.g. 177 Lu, and bismuth, e.g. 213 Contains isotopes of Bi.

[0049] 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 evaluating the presence and / or absence of Nectin-4 targets on diseased tissues. The peptide ligands of the invention may further have valuable diagnostic properties in that they may be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods may use compounds labeled with labeling agents, e.g., radioisotopes, enzymes, fluorescent substances, luminescent substances (e.g., luminol, luminol derivatives, luciferin, aequorin, and luciferase), and the like. 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 ready means of detection.

[0050] Heavier isotopes, such as deuterium, i.e. 2 Substitution with H(D) may confer certain therapeutic advantages due to increased metabolic stability, e.g., increased in vivo half-life or reduced dosage required, and therefore may be preferred in some circumstances.

[0051] Positron-emitting isotopes, e.g. 11 C. 18 F, 15 O and 13 Substitution with N may be useful in positron emission tomography (PET) studies to examine target occupancy.

[0052] Isotopically labeled compounds of the peptide ligands of the present invention may generally be prepared by conventional techniques known to those of skill in the art or by methods similar to those described in the accompanying Examples, substituting appropriate isotopically labeled reagents for previously used non-labeled reagents.

[0053] <Molecular scaffold> In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. Reference herein to a "non-aromatic molecular scaffold" refers to any molecular scaffold described herein that does not contain an aromatic (i.e., unsaturated) carbocyclic or heterocyclic ring system.

[0054] Suitable examples of non-aromatic molecular scaffolds are described in Heinis et al (2014) Angewandte Chemie, International Edition 53(6)1602-1606.

[0055] As described in the aforementioned documents, the molecular scaffold may be a small molecule, for example a small organic molecule.

[0056] 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.

[0057] In one embodiment, the molecular scaffold comprises reactive groups that can react with functional groups of a polypeptide to form a covalent bond.

[0058] The molecular scaffold may 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.

[0059] 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).

[0060] <Effectors and functional groups> According to a further aspect of the present invention there is provided a drug conjugate comprising a peptide ligand as described herein conjugated to one or more effectors and / or functional groups.

[0061] The effectors and / or functional groups may be attached, for example, to the N- and / or C-terminus of the polypeptide, to an amino acid within the polypeptide, or to a molecular scaffold.

[0062] Suitable effector groups include antibodies and portions or fragments thereof. For example, effector groups may include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination, in addition to one or more constant region domains. Effector groups may also include the hinge region of an antibody (such a region is usually found between the CH1 and CH2 domains of an IgG molecule).

[0063] In a further embodiment of this aspect of the invention, the effector group according to the invention is the Fc region of an IgG molecule. Advantageously, 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.

[0064] The functional groups typically include linking groups, drugs, reactive groups for attachment to other entities, and functional groups that aid in cellular uptake of the macrocyclic peptide.

[0065] The ability of peptides to enter cells will allow them to be effective against intracellular targets.Targets that can be accessed by peptides with the ability to enter cells include transcription factors, intracellular signaling molecules, such as tyrosine kinases and molecules involved in apoptosis pathways.Functional groups that allow cell entry include peptides, or chemical groups that are either peptides or molecular scaffolds attached to them, peptides, such as those described in Chen and Harrison, Biochemical Society Transactions (2007) Volume 35, part 4, p821; Gupta et al. in Advanced Drug Discovery Reviews (2004) Volume 57 9637, for example, VP22, HIV-Tat, and those derived from the homeobox protein of Drosophila (Antennapedia). Examples of short peptides that have been shown to be efficient for translocation through the plasma membrane include the 16 amino acid penetratin peptide from the Drosophila melanogaster antennapedia protein (Derossi et al (1994) J Biol. Chem. Volume 269 p10444), the 18 amino acid "model amphipathic peptide" (Oehlke et al (1998) Biochim Biophys Acts Volume 1414 p127) and the arginine-rich region of the HIV TAT protein. Non-peptide approaches include the use of small molecule mimetics or SMOCs that can be easily attached to biomolecules (Okuyama et al (2007) Nature Methods Volume 4 p153). Other chemical strategies for adding guanidinium groups to molecules also enhance cell entry (Elson-Scwab et al (2007) J Biol Chem Volume 282 p13585). Small molecular weight molecules, such as steroids, may be added to the molecular scaffold to enhance uptake into cells.

[0066] One class of functional groups that can be attached to the peptide ligands are antibodies and their binding fragments, 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 may be used.

[0067] 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 will have a tβ half-life in the range of 12 to 60 hours. In a further embodiment, it will have a tβ half-life of 1 day or more. In a further embodiment, it will be in the range of 12-26 hours.

[0068] In one particular embodiment of the invention, the functional group is selected from metal chelators suitable for complexing medicamentously relevant metal radioisotopes.

[0069] Possible effector groups also include enzymes, for example carboxypeptidase G2 for use in enzyme / prodrug therapy, where peptide ligands replace antibodies in ADEPT.

[0070] In one particular embodiment of the present invention, functional group is selected from drugs, for example cytotoxic agents for cancer therapy.Suitable examples include alkylating agents, such as cisplatin and carboplatin, as well as oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; metabolite antagonists, 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 camptothecins: irinotecan and topotecan, and type II inhibitors, including amsacrine, etoposide, etoposide phosphate, and teniposide. Additional drugs may include the immunosuppressants dactinomycin (which is used in kidney transplants), antitumor antibiotics including doxorubicin, epirubicin, bleomycin, calicheamicin, and the like.

[0071] In one further particular embodiment of the invention, the cytotoxic agent is selected from a maytansinoid (eg, DM1) or a monomethylauristatin (eg, MMAE).

[0072] DM1 is a cytotoxic agent and is a thiol-containing derivative of maytansine, having the following structure:

[0073] [ka] has.

[0074] Data demonstrating the efficacy of peptide ligands conjugated to toxins including DM1 are presented herein in Table 3.

[0075] Monomethylauristatin E (MMAE) is a synthetic anticancer drug having the following structure:

[0076] [ka] has.

[0077] Data demonstrating the efficacy of peptide ligands conjugated to toxins including MMAE are presented herein in Table 3.

[0078] In one further particular embodiment of the invention, the cytotoxic agent is selected from monomethylauristatin E (MMAE).

[0079] In one embodiment, the cytotoxic agent is attached to the bicyclic peptide by a cleavable bond, such as a disulfide bond or a protease-sensitive bond, hi a further embodiment, groups proximal to the disulfide bond are modified to control the rate of disruption of the disulfide bond and thus cleavage and concomitant release of the cytotoxic agent.

[0080] 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). As the degree of steric hindrance increases, the rate of reduction by intracellular glutathione, as well as extracellular (systemic) reducing agents, decreases, and the toxin is released both inside and outside the cell. Thus, the selection of the optimum conditions between disulfide stability in circulation (minimizing unwanted side effects of the toxin) and efficient release in the intracellular environment (maximizing therapeutic efficacy) may be achieved by carefully choosing the degree of hindrance on either side of the disulfide bond.

[0081] Disruption to either side of the disulfide bond is modulated via the introduction of one or more methyl groups on either the targeting entity (here, the bicyclic peptide) or the toxin side of the molecular construct.

[0082] In one embodiment, the cytotoxic agents and linkers are selected from any combination of those described in WO 2016 / 067035, which are incorporated herein by reference.

[0083] In one embodiment, the linker between the cytotoxic agent and the bicyclic peptide comprises one or more amino acid residues. Examples of amino acid residues suitable for suitable linkers include Ala, Cit, Lys, Trp and Val.

[0084] In one embodiment, the cytotoxic agent is selected from MMAE and the drug conjugate further comprises a linker selected from -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, where PABC represents p-aminobenzyl carbamate. Full details of the cyclobutyl containing linkers can be found in Wei et al (2018) J. Med. Chem. 61, 989-1000. In a further embodiment, the cytotoxic agent is selected from MMAE and the linker is -PABC-Cit-Val-glutaryl-.

[0085] In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate further comprises a linker that is -SPDB-(SO3H)-, where SPDB represents N-succinimidyl 3-(2-pyridyldithio)propionate.

[0086] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8234 described herein, and the linker is selected from -PABC-Cit-Val-glutaryl-. This BDC is known herein as BCY8245,

[0087] [ka] It is represented diagrammatically as

[0088] [ka] It may also be shown in a more detailed format as

[0089] Data are presented herein demonstrating superior binding to human Nectin-4 for BCY8245 in an SPR binding assay as shown in Table 3. This BDC has also demonstrated good anti-tumor activity in a non-small cell lung cancer model as shown in Example 1, a bladder cancer model as shown in Example 2, a pancreatic cancer model as shown in Example 3, and a breast cancer model as shown in Example 4.

[0090] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8234 described herein, and the linker is selected from -PABC-cyclobutyl-(B-Ala)-. This BDC is known herein as BCY8549. Presented herein are data demonstrating superior binding to human nectin-4 in an SPR binding assay for BCY8549, as shown in Table 3.

[0091] In a further embodiment, the bicyclic drug conjugate is selected from BCY8245 or BCY8549. In a further embodiment, the bicyclic drug conjugate is BCY8245. The drug conjugate BCY8245 has demonstrated superior dose-dependent antitumor activity as demonstrated by the data provided herein.

[0092] <Synthesis> The peptides of the present invention may be synthetically produced by standard techniques followed by reaction with molecular scaffolds in vitro. Standard chemistry may be used to do this. This allows for rapid, large-scale preparation of soluble material for further downstream realization or validation. Such methods may be accomplished using conventional chemistry, such as that disclosed in Timmerman et al. (see above).

[0093] The present invention therefore also relates to the production of a polypeptide or conjugate selected as indicated herein, comprising optional further steps as explained below, which in one embodiment are carried out on the final polypeptide / conjugate produced by chemical synthesis.

[0094] Optionally, amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

[0095] The peptide may be extended, for example to incorporate additional loops, thus introducing multiple specificities.

[0096] To extend a peptide, standard solid-phase or solution-phase chemistry may be used to simply chemically extend it at its N-terminus or C-terminus or within a loop using orthogonally protected lysines (and analogues). Standard (bio)conjugation techniques may be used to introduce activated or activatable N- or C-termini. Alternatively, the addition may be performed 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 enzymatically, using subtiligases, as described, for example, in (Chang et al. Proc Natl Acad Sci USA. 1994 Dec 20;91(26):12544-8 or Hikari et al Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003).

[0097] Alternatively, peptides can be extended or modified by further conjugation via disulfide bonds. This has the added advantage that the first and second peptides are dissociated from each other once in the cellular reducing environment. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide, so that it reacts with three cysteine ​​groups; then, a further 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, forming a disulfide-bonded bicyclic peptide-peptide conjugate.

[0098] Similar techniques could be equally applied to the synthesis / coupling of two bicyclic and bispecific macrocycles to potentially generate tetraspecific molecules.

[0099] Furthermore, the addition of other functional or effector groups may be accomplished 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 performed in such a manner as not to block the activity of either entity.

[0100] <Pharmaceutical Composition> According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide ligand or a drug conjugate as described herein, in combination with one or more pharma- ceutically acceptable excipients.

[0101] Generally, the peptide ligands of the present invention will be 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 may be selected from viscosity enhancing agents, such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginates, if necessary to maintain the polypeptide complex in suspension.

[0102] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. Preservatives and other additives may also be present, such as antibacterial agents, antioxidants, chelating agents, and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0103] The peptide ligands of the invention may be used as compositions administered separately or together with other agents. These may include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, adriamycin or cisplatinum, and immunotoxins. Pharmaceutical compositions include "cocktails" of various cytotoxic or other agents in combination with the protein ligands of the invention, or even combinations of selected polypeptides according to the invention with different specificities, such as polypeptides selected using different targeting ligands, whether or not these are pooled prior to administration.

[0104] The route of administration of the pharmaceutical composition according to the invention may be any of those generally known to those skilled in the art. For therapeutic purposes, the peptide ligand of the invention may be administered to any patient according to standard techniques. It may be administered by any suitable mode, including parenterally, intravenously, intramuscularly, intraperitoneally, transdermally, via the pulmonary route, or by direct injection using a catheter. Preferably, the pharmaceutical composition according to the invention will be administered by inhalation. The dosage and frequency of administration will depend on the age, sex and condition of the patient, the co-administration of other drugs, contraindications and other parameters that will be taken into account by the clinician.

[0105] The peptide ligands of the present invention may be lyophilized for storage and reconstitution in a suitable carrier prior to use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art may be used. Those skilled in the art will appreciate that lyophilization and reconstitution may result in varying degrees of activity loss, the levels of which may be adjusted upward to offset.

[0106] 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, the appropriate amount to achieve at least partial inhibition, suppression, modulation, lethality, or some other measurable parameter of a selected cell population 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 status of the patient's own immune system, but will generally range from 0.005-5.0 mg of the selected peptide ligand per kilogram of body weight, with doses of 0.05-2.0 mg / kg / dose being more commonly used. For prophylactic applications, compositions containing the peptide ligands or cocktails thereof of the present invention may also be administered at the same or slightly lower dosages.

[0107] Compositions containing peptide ligands according to the invention may 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 may be selectively used ex vivo or in vitro to kill, deplete or otherwise effectively remove target cell populations from a heterogeneous collection of cells. Blood from a mammal may be combined ex vivo with the selected peptide ligand, whereby undesired cells are killed or otherwise removed from the blood and returned to the mammal according to standard techniques.

[0108] Coadministration with one or more other therapeutic agents Depending on the particular condition or disease to be treated, additional therapeutic agents that are normally administered to treat that condition may also be present in the compositions of the present invention. Thus, in one embodiment, the pharmaceutical composition further comprises one or more therapeutic agents. As used herein, additional therapeutic agents that are normally administered to treat a particular disease or condition are known as "disease or condition, appropriate for treatment."

[0109] In some embodiments, the present invention provides a method for treating a disclosed disease or condition, comprising administering an effective amount of a compound disclosed herein or a pharma- ceutically acceptable salt thereof to a patient in need thereof, and simultaneously or sequentially co-administering an effective amount of one or more additional therapeutic agents, such as those described herein. In some embodiments, the method comprises co-administering one additional therapeutic agent. In some embodiments, the method comprises co-administering two additional therapeutic agents. In some embodiments, the combination of the disclosed compound and the additional therapeutic agent or agent acts synergistically.

[0110] The compounds of the present invention may also be used in combination with known therapeutic processes, such as the administration of hormones or radiation. In certain embodiments, provided compounds are used as radiosensitizers, particularly for treating tumors that exhibit poor sensitivity to radiation therapy.

[0111] The compound of the present invention may be administered alone or in combination with one or more other therapeutic compounds, or in combination therapy in the form of fixed combination, or in the form of staggered administration of the compound of the present invention and one or more other therapeutic compounds, or in combination administration of fixed combination and one or more other therapeutic compounds.The compound of the present invention may be administered additionally or further for tumor therapy, in particular in combination with chemotherapy, radiation therapy, immunotherapy, phototherapy, surgical intervention, or a combination thereof.Long-term therapy is possible as well as adjuvant therapy in the context of other treatment strategies as mentioned above.Other possible treatments are therapy to maintain the patient's condition after tumor reduction, or even chemopreventive therapy, for example in patients at risk.

[0112] One or more other therapeutic agents may be administered separately with the compound or composition of the present invention as part of a multiple dose regimen. Alternatively, one or more other therapeutic agents may be part of a single dosage form mixed with the compound of the present invention in a single composition. When administered as a multiple dose regimen, one or more other therapeutic agents and the compound or composition of the present invention may be administered simultaneously, sequentially, or alternating within a time range, for example, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours. In some embodiments, one or more other therapeutic agents and the compound or composition of the present invention are administered as a multiple dose regimen that is more than 24 hours apart.

[0113] The term "combination", "combined" and related terms used herein refer to simultaneous or sequential administration of therapeutic agents according to the present invention.For example, the compound of the present invention may be administered simultaneously or sequentially with one or more other therapeutic agents in separate unit dosage forms or in a single unit dosage form.Therefore, the present invention provides a single unit dosage form that includes the compound of the present invention, one or more other therapeutic agents, and pharmaceutically acceptable carriers, adjuvants, or vehicles.

[0114] The amounts of the compounds of the present invention and one or more other therapeutic agents (compositions containing additional therapeutic agents as described above) that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. Preferably, the compositions of the present invention should be formulated so that a dosage of between 0.01-100 mg / kg of body weight / day of the compounds of the present invention can be administered.

[0115] In these compositions containing one or more other therapeutic agents, the one or more other therapeutic agents and the compound of the present invention may act synergistically. As a result, the amount of one or more other therapeutic agents in such compositions may be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions, a dosage of one or more other therapeutic agents of 0.01 to 1,000 μg / kg body weight / day may be administered.

[0116] The amount of one or more other therapeutic agents present in the compositions of the invention may be equal to or less than the amount that would normally be administered in a composition containing that therapeutic agent as the sole active agent. Preferably, the amount of one or more other therapeutic agents in the compositions disclosed herein will be in the range of about 50% to 100% of the amount that would normally be present in a composition containing that agent as the only therapeutic active agent. In some embodiments, the one or more other therapeutic agents are administered in a dosage of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount that would normally be administered for that agent. As used herein, the phrase "normally administered" refers to the amount of FDA approved therapeutic agent provided for administration according to the FDA label insert.

[0117] The compound of the present invention or its pharmaceutical composition may also be incorporated into a composition to coat implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents and catheters. For example, vascular stents have been used to overcome restenosis (re-narrowing of the vessel wall after injury). However, patients who use stents or other implantable devices are at risk of clot formation or platelet activation. These undesirable effects may be prevented or reduced by pre-coating the device with a pharma- ceutically acceptable composition that includes a kinase inhibitor. An implantable device coated with the compound of the present invention is another embodiment of the present invention.

[0118] <Exemplary Other Therapeutic Agents> In some embodiments, the one or more other therapeutic agents is a poly ADP ribose polymerase (PARP) inhibitor. In some embodiments, the PARP inhibitor is selected from olaparib (Lynparza®, AstraZeneca); rucaparib (Rubraca®, Clovis Oncology); niraparib (Zejula®, Tesaro); lazoparib (MDV3800 / BMN 673 / LT00673, Medivation / Pfizer / Biomarin); veliparib (ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.).

[0119] In some embodiments, the one or more other therapeutic agents are histone deacetylase (HDAC) inhibitors. In some embodiments, the HDAC inhibitor is selected from vorinostat (Zolinza®, Merck); romidepsin (Istodax®, Celgene); panobinostat (Farydak®, Novartis); belinostat (Beleodaq®, Spectrum Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and chidamide (Epidaza®, HBI-8000, Chipscreen BioSciences, China).

[0120] In some embodiments, the one or more other therapeutic agents are CDK inhibitors, such as CDK4 / CDK6 inhibitors. In some embodiments, the CDK4 / 6 inhibitor is selected from palbociclib (Ibrance®, Pfizer); ribociclib (Kisqali®, Novartis); abemaciclib (Ly2835219, Eli Lilly); and trilaciclib (G1T28, G1 THERAPEUTICS).

[0121] In some embodiments, the one or more other therapeutic agents are phosphatidylinositol 3-kinase (PI3K) inhibitors. In some embodiments, the PI3K inhibitor is selected from idelalisib (Zydelig®, Gilead), alpelisib (BYL719, Novartis), taselisib (GDC-0032, Genentech / Roche); pictilisib (GDC-0941, Genentech / Roche); copanlisib (BAY806946, Bayer); duvelisib (formerly IPI-145, Infinity Pharmaceuticals); PQR309 (Piqur Therapeutics, Switzerland); and TGR1202 (formerly RP5230, TG THERAPEUTICS).

[0122] In some embodiments, the one or more other therapeutic agents are platinum-based therapeutic agents, also referred to as platinum. Platinums result in DNA cross-linking, which inhibits DNA repair and / or DNA synthesis, primarily in rapidly reproducing cells, such as cancer cells. In some embodiments, the platinum-based therapeutic agent is selected from cisplatin (Platinol®, Bristol-Myers Squibb); carboplatin (Paraplatin®, Bristol-Myers Squibb; also Teva; Pfizer); oxaliplatin (Eloxitin®, Sanofi-Aventis); nedaplatin (Aqupla®, Shionogi & Co., Ltd.), picoplatin (Poniard Pharmaceuticals); and satraplatin (JM-216, Agennix).

[0123] In some embodiments, the one or more other therapeutic agents are taxane compounds, which result in the disruption of microtubules necessary for cell division. In some embodiments, the taxane compound is selected from paclitaxel (Taxol®, Bristol-Myers Squibb), docetaxel (Taxotere®, Sanofi-Aventis; Docefrez®, Sun Pharmaceutical), albumin-bound paclitaxel (Abraxane®; Abraxis / Celgene), cabazitaxel (Jevtana®, Sanofi-Aventis), and SID530 (SK Chemicals, Co.) (NCT00931008).

[0124] In some embodiments, the one or more other therapeutic agents are nucleoside inhibitors or therapeutic agents that interfere with normal DNA synthesis, protein synthesis, cell replication, or would otherwise inhibit rapidly proliferating cells.

[0125] In some embodiments, the nucleoside inhibitor is trabectedin (a guanidine alkylator, Yondelis®, Janssen Oncology); mechlorethamine (an alkylator, Valchlor®, Aktelion Pharmaceuticals); vincristine (Oncovin®, Eli Lilly; Vincasar®, Teva Pharmaceuticals; Marqibo®, Talon Therapeutics); temozolomide (a prodrug for the alkylator 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC), Temodar®, Merck); cytarabine injection (ara-C, an antimetabolic cytidine analog, Pfizer); lomustine (an alkylator, CeeNU®, Bristol-Myers Squibb; Gleostine®, NextSource). Biotechnology); azacitidine (a pyrimidine nucleoside analogue of cytidine, Vidaza®, Celgene); omacetaxine mepesuccinate (a cephalotaxine ester) (a protein synthesis inhibitor, Synribo®; Teva Pharmaceuticals); Erwinia chrysanthemi asparaginase (an enzyme for depleting asparagine, Elspar®, Lundbeck; Erwinaze®, EUSA Pharma); eribulin mesylate (microtubule inhibitor, tubulin-based mitosis inhibitor, Halaven®, Eisai Co., Ltd.); cabazitaxel (microtubule inhibitor, tubulin-based mitosis inhibitor, Jevtana®, Sanofi-Aventis); capacetrin (thymidylate synthase inhibitor, Xeloda®, Genentech); bendamustine (bifunctional mechlorethamine derivative, believed to form interstrand DNA crosslinks, Treanda®, Cephalon / Teva); ixabepilone (semi-synthetic analogue of epothilone B, microtubule inhibitor, tubulin-based mitosis inhibitor, Ixempra®, Bristol-Myers Squibb);Selected from nelarabine (prodrug of a deoxyguanosine analog, a nucleoside metabolism inhibitor, Arranon®, Novartis); clofarabine (prodrug of a ribonucleotide reductase inhibitor, a competitive inhibitor of deoxycytidine, Clolar®, Sanofi-Aventis); and trifluridine and tipiracil (thymidine-based nucleoside analog and thymidine phosphorylase inhibitor, Lonsurf®, Taiho Oncology);

[0126] In some embodiments, the one or more other therapeutic agents are kinase inhibitors or VEGF-R antagonists. Approved VEGF inhibitors and kinase inhibitors useful in the present invention include bevacizumab (Avastin®, Genentech / Roche), an anti-VEGF monoclonal antibody; ramucirumab (Cyramza®, Eli Lilly), an anti-VEGFR-2 antibody; and ziv-aflibercept, a VEGF inhibitor. also known as Trap (Zaltrap®; Regeneron / Sanofi), VEGFR inhibitors such as regorafenib (Stivarga®, Bayer); vandetanib (Caprelsa®, AstraZeneca); axitinib (Inlyta®, Pfizer); and lenvatinib (Lenvima®, Eisai Co., Ltd.); Raf inhibitors such as sorafenib (Nexavar®, Bayer AG and Onyx); dabrafenib (Tafinlar®, Novartis); and vemurafenib (Zelboraf®). ), Genentech / Roche; MEK inhibitors such as cobimetanib (Cotellic®, Exelexis / Genentech / Roche); trametinib (Mekinist®, Novartis); Bcr-Abl tyrosine kinase inhibitors such as imatinib (Gleevec®, Novartis); nilotinib (Tasigna®, Novartis); dasatinib (Sprycel®, BristolMyersSquibb); bosutinib (Bosulif®, Pfizer); and ponatinib (Inclusig®, Ariad Pharmaceuticals; Her2 and EGFR inhibitors such as gefitinib (Iressa®, AstraZeneca); erlotinib (Tarceeva®, Genentech / Roche / Astellas); lapatinib (Tykerb®, Novartis); afatinib (Gilotrif®, Boehringer Ingelheim);and brigatinib (Alunbrig®, Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors such as cabozantinib (Cometriq®, Exelexis); and multikinase inhibitors such as sunitinib (Sutent®, Pfizer); pazopanib (Votrient®, Novartis); ALK inhibitors such as crizotinib (Xalkori®, Pfizer); ceritinib (Zykadia®, Novartis); and alectinib (Alecenza®, Genentech / Roche); Bruton's tyrosine kinase inhibitors such as ibrutinib (Imbruvica®, Pharmacyclics / Janssen); and Flt3 receptor inhibitors such as midostaurin (Rydapt®, Novartis). ;

[0127] Other kinase inhibitors and VEGF-R antagonists under development that may be used in the present invention include tivozanib (Aveo Pharmaecuticals); vatalanib (Bayer / Novartis); lucitanib (Clovis Oncology); dovitinib (TKI258, Novartis); chiauanib (Chipscreen Biosciences); CEP-11981 (Cephalon); linifanib (Abbott Laboratories); neratinib (HKI-272, Puma Biotechnology); ruxolitinib (Supect®, IY5511, Il-Yang Pharmaceuticals, S.Korea); ruxolitinib (Jakafi®, Incyte Corporation); PTC299 (PTC Therapy); CP-547,632 (Pfizer); foretinib (Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo Co., Ltd.) and motesanib (Amgen / Takeda Pharmaceutical Co., Ltd.).

[0128] In some embodiments, the one or more other therapeutic agents are mTOR inhibitors, which inhibit cell proliferation, angiogenesis, and glucose uptake. In some embodiments, the mTOR inhibitor is everolimus (Afinitor®, Novartis); temsirolimus (Torisel®, Pfizer); and sirolimus (Rapamune®, Pfizer).

[0129] In some embodiments, the one or more other therapeutic agents are proteasome inhibitors. Approved proteasome inhibitors useful in the present invention include bortezomib (Velcade®, Takeda Pharmaceutical Co., Ltd.); carfilzomib (Kyprolis®, Amgen); and ixazomib (Ninlaro®, Takeda Pharmaceutical Co., Ltd.).

[0130] In some embodiments, the one or more other therapeutic agents are growth factor antagonists, such as platelet-derived growth factor (PDGF), or epidermal growth factor (EGF) or its receptor (EGFR) antagonists. Approved PDGF antagonists that may be used in the present invention include olaratumab (Lartruvo®; Eli Lilly). Approved EGFR antagonists that may be used in the present invention include cetuximab (Erbitux®, Eli Lilly); necitumumab (Portrazza®, Eli Lilly), panitumumab (Vectibix®, Amgen); and osimertinib (targets activated EGFR, Tagrisso®, AstraZeneca).

[0131] In some embodiments, the one or more other therapeutic agents is an aromatase inhibitor. In some embodiments, the aromatase inhibitor is selected from exemestane (Aromasin®, Pfizer); anastrozole (Arimidex®, AstraZeneca) and letrozole (Femara®, Novartis).

[0132] In some embodiments, the one or more other therapeutic agents are antagonists of the hedgehog pathway.Approved hedgehog pathway inhibitors that may be used in the present invention include sonidegib (Odomzo®, Sun Pharmaceutical); and vismodegib (Erivedge®, Genentech), both of which are for treating basal cell carcinoma.

[0133] In some embodiments, the one or more other therapeutic agents are folate inhibitors. Approved folate inhibitors useful in the present invention include pemetrexed (Alimta®, Eli Lilly).

[0134] In some embodiments, the one or more other therapeutic agents are CC chemokine receptor 4 (CCR4) inhibitors. Investigational CCR4 inhibitors that may be useful in the present invention include mogamulizumab (Poteligeo®, Kyowa Kirin Co., Ltd., Japan).

[0135] In some embodiments, the one or more other therapeutic agents are isocitrate dehydrogenase (IDH) inhibitors. Investigational IDH inhibitors that may be used in the present invention include AG120 (Celgene; NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer, NCT02746081); IDH305 (Novartis, NCT02987010).

[0136] In some embodiments, one or more other therapeutic agents are arginase inhibitors.The arginase inhibitors under investigation that may be used in the present invention include AEB1102 (pegylated recombinant arginase, Aeglea Biotherapeutics), which is under investigation in Phase 1 clinical trials for acute myeloid leukemia and myelodysplastic syndrome (NCT02732184) and solid tumors (NCT02561234); and CB-1158 (Calithera Biosciences).

[0137] In some embodiments, the one or more other therapeutic agents are glutaminase inhibitors. Investigational glutaminase inhibitors that may be used in the present invention include CB-839 (Calithera Biosciences).

[0138] In some embodiments, the one or more other therapeutic agents are antibodies that bind to tumor antigens, i.e., proteins expressed on the cell surface of tumor cells. Approved antibodies that bind to tumor antigens that may be used in the present invention include rituximab (Rituxan®, Genentech / BiogenIdec); ofatumumab (anti-CD20, Arzerra®, GlaxoSmithKline); obinutuzumab (anti-CD20, Gazyva®, Genentech), ibritumomab (anti-CD20 and yttrium-90, Zevalin®, Spectrum Pharmaceuticals); daratumumab (anti-CD38, Darzalex®, Janssen Biotech), dinutuximab (anti-glycolipid GD2, Unituxin®, United Therapeutics); trastuzumab (anti-HER2, Herceptin®, Genentech); ado-trastuzumab emtansine (anti-HER2, fused to emtansine, Kadcyla®, Genentech); and pertuzumab (anti-HER2, Perjeta®, Genentech); and brentuximab vedotin (anti-CD30-drug conjugate, Adcetris®, Seattle Genetics).

[0139] In some embodiments, the one or more other therapeutic agents are topoisomerase inhibitors. Approved topoisomerase inhibitors useful in the present invention include irinotecan (Onivyde®, Merrimack Pharmaceuticals); topotecan (Hycamtin®, GlaxoSmithKline). Investigational topoisomerase inhibitors that may be used in the present invention include pixantrone (Pixuvri®, CTI Biopharma).

[0140] In some embodiments, the one or more other therapeutic agents are anti-apoptotic protein inhibitors, such as BCL-2. Approved anti-apoptotic drugs that may be used in the present invention include venetoclax (Venclexta®, AbbVie / Genentech); and blinatumomab (Blincyto®, Amgen). Other therapeutic agents targeting apoptotic proteins that are undergoing clinical trials and may be used in the present invention include navitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740).

[0141] In some embodiments, the one or more other therapeutic agents are androgen receptor inhibitors. Approved androgen receptor inhibitors useful in the present invention include enzalutamide (Xtandi®, Astellas / Medivation); approved androgen synthesis inhibitors include abiraterone (Zytiga®, Centocor / Ortho); approved gonadotropin releasing hormone (GnRH) receptor antagonists include degarelix (Firmagon®, Ferring Pharmaceuticals).

[0142] In some embodiments, the one or more other therapeutic agents are selective estrogen receptor modulators (SERMs), which interfere with the synthesis or activity of estrogen. Approved SERMs useful in the present invention include raloxifene (Evista®, Eli Lilly).

[0143] In some embodiments, one or more other therapeutic agents are bone resorption inhibitors.An approved therapeutic agent that inhibits bone resorption is denosumab (Xgeva®, Amgen), which is an antibody that binds to RANKL and blocks its binding to its receptor RANK, which is found on the surface of osteoclasts, their precursor cells, and osteoclast-like giant cells that mediate bone pathology in solid tumors with bone metastasis.Other approved therapeutic agents that inhibit bone resorption include bisphosphonates, such as zoledronic acid (Zometa®, Novartis).

[0144] In some embodiments, the one or more other therapeutic agents are inhibitors of the interaction between MDMX and MDM2, two major p53 suppressor proteins.An investigational p53 suppressor protein inhibitor that may be used in the present invention is ALRN-6924 (Aileron), which binds equally to staple peptides MDMX and MDM2 and disrupts their interaction with p53.ALRN-6924 is currently being evaluated in clinical trials (NCT02909972; NCT02264613) for the treatment of AML, progressive myelodysplastic syndrome (MDS) and peripheral T-cell lymphoma (PTCL).

[0145] In some embodiments, the one or more other therapeutic agents are transforming growth factor-beta (TGF-beta or TGFβ) inhibitors. Investigational TGF-beta protein inhibitors that may be used in the present invention include NIS793 (Novartis), an anti-TGF-beta antibody undergoing clinical trials (NCT02947165) for the treatment of various cancers, including breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer. In some embodiments, the TGF-beta protein inhibitor is fresolimumab (GC1008; Sanofi-Genzyme), which is under investigation for melanoma (NCT00923169); renal cell carcinoma (NCT00356460); and non-small cell lung cancer (NCT02581787). Additionally, in some embodiments, the additional therapeutic agent is a TGF-beta trap, e.g., as described in Connolly et al. (2012) Int'l J.Biological Sciences 8:964-978. One therapeutic compound currently in clinical trials for the treatment of solid tumors is M7824 (Merck KgaA - formerly MSB0011459X), which is a bispecific anti-PD-L1 / TGFβ trap compound (NCT02699515) and (NCT02517398). M7824 consists of a fully human IgG1 antibody against PD-L1 fused to the extracellular domain of the human TGF-beta receptor II, which functions as a TGFβ "trap".

[0146] In some embodiments, the one or more other therapeutic agents are selected from glembatumumab vedotin-monomethyl auristatin E (MMAE) (Celldex), an anti-glycoprotein NMB (gpNMB) antibody conjugated to a cytotoxic MMAE (CR011). gpNMB is a protein overexpressed by multiple tumor types that is associated with the ability of cancer cells to metastasize.

[0147] In some embodiments, the one or more other therapeutic agents are antiproliferative compounds, including, but not limited to, aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active compounds; alkylating compounds; histone deacetylase inhibitors; compounds that induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platinum compounds; compounds that target / reduce protein or lipid kinase activity and further antiangiogenic compounds; compounds that target, reduce or inhibit the activity of protein or lipid phosphatases; gonadorelin agonists; antiandrogens; methionine aminopeptides. idase inhibitors;matrix metalloproteinase inhibitors;bisphosphonates;biological response modifiers;antiproliferative antibodies;heparanase inhibitors;Ras oncogenic isoform inhibitors;telomerase inhibitors;proteasome inhibitors;compounds for use in the treatment of hematological malignancies;compounds which target, decrease or inhibit the activity of Flt-3;Hsp90 inhibitors, such as 17-AAG (17-allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010, Conforma Therapeutics; temozolomide (Temodal®); kinesin spindle protein inhibitors, such as SB715992 or SB743921, from GlaxoSmithKline, or pentamidine / chlorpromazine, from CombinatoRx; MEK inhibitors, such as ARRY142886, from Array BioPharma, AZd6244, from AstraZeneca, PD181461, from Pfizer, and leucovorin.

[0148] The term "aromatase inhibitor" as used herein refers to a compound that inhibits estrogen production, for example the conversion of the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to, steroids, particularly atamestane, exemestane and formestane, and nonsteroids, particularly aminoglutethimide, rogletimide, pyridoglutethimide, trilostane, testolactone, ketoconazole, vorozole, fadrozole, anastrozole and letrozole. Exemestane is commercially available under the trade name Aromasin™. Formestane is commercially available under the trade name Lentaron™. Fadrozole is commercially available under the trade name Afema™. Anastrozole is commercially available under the trade name Arimidex™. Letrozole is commercially available under the trade name Femara™ or Femar™. Aminoglutethimide is commercially available under the trade name Orimeten.TM.. The combinations of the present invention which include a chemotherapeutic agent that is an aromatase inhibitor are particularly useful in the treatment of hormone receptor positive tumors, such as breast tumors.

[0149] The term "anti-estrogen" as used herein refers to a compound that antagonizes the effect of estrogen at the estrogen receptor level. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride. Tamoxifen is marketed under the trade name Nolvadex™. Raloxifene hydrochloride is marketed under the trade name Evista™. Fulvestrant may be administered under the trade name Faslodex™. The combination of the present invention that includes a chemotherapeutic agent that is an anti-estrogen is particularly useful for treating estrogen receptor positive tumors, such as breast tumors.

[0150] The term "antiandrogen" as used herein refers to any substance capable of inhibiting the biological effects of androgenic hormones, including, but not limited to, bicalutamide (Casodex™). The term "gonadorelin agonist" as used herein includes, but is not limited to, abarelix, goserelin and goserelin acetate. Goserelin may be administered under the trade name Zoladex™.

[0151] The term "topoisomerase I inhibitors" as used herein includes, but is not limited to, topotecan, gimatecan, irinotecan, camptothecin and its analogs, 9-nitrocamptothecin and the polymeric camptothecin conjugate PNU-166148. Irinotecan may be administered, for example, in the form in which it is marketed, for example, under the trade name Camptosar™. Topotecan is marketed under the trade name Hycamptin™.

[0152] The term "topoisomerase II inhibitors" as used herein includes, but is not limited to, anthracyclines such as doxorubicin (including liposomal formulations, e.g., Caelyx™), daunorubicin, epirubicin, idarubicin and nemorubicin, the anthraquinones mitoxantrone and rosoxantrone, and the podophyllotoxins etoposide and teniposide. Etoposide is commercially available under the trade name Etopophos™. Teniposide is commercially available under the trade name VM26-Bristol. Doxorubicin is commercially available under the trade name Acriblastin™ or Adriamycin™. Epirubicin is commercially available under the trade name Farmorubicin™. Idarubicin is commercially available under the trade name Zavedos™. Mitoxantrone is commercially available under the trade name Novantron.

[0153] The term "microtubule active agent" refers to microtubule stabilizing compounds, microtubule destabilizing compounds and microtubule polymerization inhibitors, including, but not limited to, taxanes such as paclitaxel and docetaxel; vinca alkaloids such as vinblastine or vinblastine sulfate, vincristine or vincristine sulfate, and vinorelbine; discodermolides; colchicine and epothilones and their derivatives. Paclitaxel is commercially available under the trade name Taxol™. Docetaxel is commercially available under the trade name Taxotere™. Vinblastine sulfate is commercially available under the trade name Vinblastin R.P™. Vincristine sulfate is commercially available under the trade name Farmistin™.

[0154] The term "alkylating agent" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide is commercially available under the trade name Cyclostin™. Ifosfamide is commercially available under the trade name Holoxan™.

[0155] The term "histone deacetylase inhibitors" or "HDAC inhibitors" relates to compounds which inhibit histone deacetylase and which possess antiproliferative activity, including, but not limited to, suberoylanilide hydroxamic acid (SAHA).

[0156] The term "antineoplastic antimetabolite" includes, but is not limited to, 5-fluorouracil or 5-FU, capecitabine, gemcitabine, DNA demethylating compounds such as 5-azacytidine and decitabine, methotrexate and edatrexate, and folate antagonists such as pemetrexed. Capecitabine is commercially available under the trade name Xeloda™. Gemcitabine is commercially available under the trade name Gemzar™.

[0157] The term "platinum compounds" as used herein includes, but is not limited to, carboplatin, cisplatin, cisplatinum and oxaliplatin. Carboplatin may be administered, e.g., in the form as it is marketed, e.g., under the trademark Carboplat™. Oxaliplatin may be administered, e.g., in the form as it is marketed, e.g., under the trademark Eloxatin™.

[0158] The term "compounds targeting / reducing protein or lipid kinase activity; or protein or lipid phosphatase activity; or further anti-angiogenic compounds" as used herein includes, but is not limited to, protein tyrosine kinase and / or serine and / or threonine kinase inhibitors, or lipid kinase inhibitors, such as: a) compounds which target, reduce or inhibit the activity of the platelet derived growth factor receptor (PDGFR), e.g. compounds which target, reduce or inhibit the activity of PDGFR, in particular compounds which inhibit the PDGF receptor, e.g. N-phenyl-2-pyrimidine-amine derivatives, e.g. imatinib, SU101, SU6668 and GFB-111; b) compounds which target, reduce or inhibit the activity of the fibroblast growth factor receptor (FGFR); c) compounds which target, reduce or inhibit the activity of the insulin-like growth factor receptor I (IGF-IR), e.g. compounds which inhibit the activity of IGF-IR. d) compounds which target, reduce or inhibit the activity of the Trk receptor tyrosine kinase family, or ephrin B4 inhibitors; e) compounds which target, reduce or inhibit the activity of the AxI receptor tyrosine kinase family; f) compounds which target, reduce or inhibit the activity of the Ret receptor tyrosine kinase; g) compounds which target, reduce or inhibit the activity of the Kit / SCFR receptor tyrosine kinase, such as imatinib; h) compounds which target, reduce or inhibit the activity of the C-kit receptor tyrosine kinase, which is part of the PDGFR family, such as compounds which target, reduce or inhibit the activity of the c-kit receptor tyrosine kinase family, in particular compounds which inhibit the c-kit receptor, such as imatinib;i) Compounds that target, reduce or inhibit the activity of members of the c-Abl family, their gene fusion products (e.g. BCR-Abl kinase) and mutants, such as compounds that target reducing or inhibiting the activity of c-Abl family members and their gene fusion products, such as N-phenyl-2-pyrimidine-amine derivatives, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC680410; PD173955, from Parke Davis; or dasatinib ( BMS-354825);j) compounds which target, decrease or inhibit the activity of members of protein kinase C (PKC), including staurosporine derivatives, e.g. midostaurin, and members of the Raf family of serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and the TEC family, and / or members of the cyclin-dependent kinase family (CDK); further compounds Examples of are UCN-01, safingol, BAY43-9006, bryostatin 1, perifosine; irmofosine; RO318220 and RO320432; GO6976; lsis3521; LY333531 / LY379196; isoquinoline compounds; FTIs; PD184352 or QAN697 (P13K inhibitors) or AT7519 (CDK inhibitors); k) compounds which target, decrease or inhibit the activity of protein tyrosine kinase inhibitors, such as imatinib mesylate (Gle evec™) or tyrphostins, such as Tyrphostin A23 / RG-50810; AG99; Tyrphostin AG213; Tyrphostin AG1748; Tyrphostin AG490; Tyrphostin B44; Tyrphostin B44 (+) enantiomer; Tyrphostin AG555; AG494; Tyrphostin AG556, AG957 and adaphostin (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester;NSC680410, adahostin); l) compounds which target, reduce or inhibit the activity of the epidermal growth factor family of receptor tyrosine kinases (EGFR1, ErbB2, ErbB3, ErbB4 as homo- or heterodimers) and mutants thereof, e.g. compounds which target, reduce or inhibit the activity of the epidermal growth factor receptor family, e.g. compounds which target, reduce or inhibit the activity of members of the EGF receptor tyrosine kinase family, e.g. EGF receptor, ErbB2, ErbB3 and ErbB4, or specific compounds, proteins or antibodies which bind to EGF or EGF-related ligands, CP358774, ZD1839, ZM105180; trastuzumab (Herceptin™), cetuximab (Erbitux™), Iressa, Tarceva, OSI-774, Cl-1033, EKB-569, GW-2016, E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 or E7.6.3, and 7H-pyrrolo-[2,3-d]pyrimidine derivatives; m) compounds which target, reduce or inhibit the activity of the c-Met receptor, for example compounds which target, reduce or inhibit the activity of c-Met, in particular compounds which inhibit the kinase activity of the c-Met receptor, or compounds which target the extracellular domain of c-Met. n) antibodies that target or bind to HGF, n) compounds that target, decrease or inhibit the kinase activity of one or more JAK family members (JAK1 / JAK2 / JAK3 / TYK2 and / or pan-JAK), including but not limited to PRT-062070, SB-1578, baricitinib, pacritinib, momelotinib, VX-509, AZD-1480, TG-101348, tofacitinib, and ruxolitinib;o) compounds that target, decrease or inhibit the kinase activity of PI3 kinase (PI3K), including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictorelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib; and p) compounds that target, decrease or inhibit the signaling effects of the Hedgehog protein (Hh) or Smoothened receptor (SMO) pathways, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib, and IPI-926 (salidegib);

[0159] The term "PI3K inhibitor" as used herein includes compounds that have inhibitory activity against one or more enzymes in the phosphatidylinositol-3-kinase family, including, but not limited to, PI3K α, PI3K γ, PI3K δ, PI3K β, PI3K-C2 α, PI3K-C2 β, PI3K-C2 γ, Vps34, p110-α, p110-β, p110-γ, p110-δ, p85-α, p85-β, p55-γ, p150, p101, and p87. Examples of PI3K inhibitors useful in the present invention include, but are not limited to, ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictorelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idelalisib.

[0160] As used herein, the term "Bcl-2 inhibitors" includes, but is not limited to, ABT-199, ABT-731, ABT-737, apogossypol, Ascenta's pan-Bcl-2 inhibitors, curcumin (and analogs thereof), dual Bcl-2 / Bcl-xL inhibitors (Infinity Pharmaceuticals / Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and analogs thereof; see WO 2008 / 118802), navitoclax (and analogs thereof, U.S. Pat. No. 7,390,799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and analogs thereof, WO 2004 / 106328), S-001 (Gloria The compounds include compounds having inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including, but not limited to, Bcl-2 inhibitors (Bio-Pharmaceuticals), TW series compounds (University of Michigan), and venetoclax. In some embodiments, the Bcl-2 inhibitor is a small molecule therapeutic. In some embodiments, the Bcl-2 inhibitor is a peptidomimetic.

[0161] As used herein, the term "BTK inhibitor" includes compounds that have inhibitory activity against Bruton's tyrosine kinase (BTK), including, but not limited to, AVL-292 and ibrutinib.

[0162] As used herein, the term "SYK inhibitors" includes compounds that have inhibitory activity against spleen tyrosine kinase (SYK), including, but not limited to, PRT-062070, R-343, R-333, Excellair, PRT-062607, and fostamatinib.

[0163] Further examples of BTK inhibitor compounds, and conditions treatable by such compounds in combination with the compounds of the invention, can be found in WO 2008 / 039218 and WO 2011 / 090760, which are hereby incorporated by reference in their entireties.

[0164] Further examples of SYK inhibitor compounds, and conditions that can be treated by such compounds in combination with the compounds of the present invention, can be found in WO 2003 / 063794, WO 2005 / 007623, and WO 2006 / 078846, the entireties of which are incorporated herein by reference.

[0165] Further examples of PI3K inhibitor compounds, and conditions treatable by such compounds in combination with the compounds of the invention, can be found in WO 2004 / 019973, WO 2004 / 089925, WO 2007 / 016176, U.S. Pat. No. 8,138,347, WO 2002 / 088112, WO 2007 / 084786, WO 2007 / 129161, WO 2006 / 122806, WO 2005 / 113554, and WO 2007 / 044729, which are incorporated by reference in their entireties.

[0166] Further examples of JAK inhibitor compounds, and conditions treatable by such compounds in combination with the compounds of the invention, can be found in WO 2009 / 114512, WO 2008 / 109943, WO 2007 / 053452, WO 2000 / 142246, and WO 2007 / 070514, the entireties of which are incorporated herein by reference.

[0167] Further anti-angiogenic compounds include compounds that have alternative mechanisms for their activity, eg, unrelated to protein or lipid kinase inhibition, such as thalidomide (Thalomid™) and TNP-470.

[0168] Examples of proteasome inhibitors useful in combination with the compounds of the invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salinosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0169] Compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase are eg inhibitors of phosphatase 1, phosphatase 2A or CDC25, such as okadaic acid or a derivative thereof.

[0170] Compounds that induce cell differentiation processes include, but are not limited to, retinoic acid, α-γ- or δ-tocopherol or α-γ- or δ-tocotrienol.

[0171] The term cyclooxygenase inhibitors as used herein includes, but is not limited to, Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acids and derivatives such as celecoxib (Celebrex™), rofecoxib (Vioxx™), etoricoxib, valdecoxib or 5-alkyl-2-arylaminophenylacetic acids such as 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenylacetic acid and lumiracoxib.

[0172] The term "bisphosphonate" as used herein includes, but is not limited to, etridonic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid and zoledronic acid. Etridonic acid is commercially available under the trade name Didronel™. Clodronic acid is commercially available under the trade name Bonefos™. Tiludronic acid is commercially available under the trade name Skelid™. Pamidronic acid is commercially available under the trade name Aredia™. Alendronic acid is commercially available under the trade name Fosamax™. Ibandronic acid is commercially available under the trade name Bondranat™. Risedronic acid is commercially available under the trade name Actonel™. Zoledronic acid is commercially available under the trade name Zometa™. The term "mTOR inhibitors" relates to compounds which inhibit the mammalian target of rapamycin (mTOR) and which have antiproliferative activity, such as sirolimus (Rapamune®), everolimus (Certican™), CCI-779 and ABT578.

[0173] As used herein, the term "heparanase inhibitor" refers to a compound that targets, reduces or inhibits heparin sulfate degradation. The term includes, but is not limited to, PI-88. As used herein, the term "biological response modifier" refers to lymphokines or interferons.

[0174] The term "Ras oncogenic isoform inhibitor", such as H-Ras, K-Ras, or N-Ras, as used herein refers to compounds that target, reduce or inhibit the oncogenic activity of Ras; for example, "phenesyltransferase inhibitors", such as L-744832, DK8G557, or R115777 (Zarnestra™). The term "telomerase inhibitor", as used herein, refers to compounds that target, reduce or inhibit telomerase activity. Compounds that target, reduce or inhibit telomerase activity are particularly compounds that inhibit telomerase receptor, such as telomestatin.

[0175] The term "methionine aminopeptidase inhibitor" as used herein refers to a compound that targets, decreases or inhibits the activity of methionine aminopeptidase. Compounds that target, decrease or inhibit the activity of methionine aminopeptidase include, but are not limited to, bengamide or its derivatives.

[0176] The term "proteasome inhibitor" as used herein refers to a compound that targets, reduces or inhibits the activity of proteasome. Compounds that target, reduces or inhibit proteasome activity include, but are not limited to, bortezomib (Velcade™) and MLN341.

[0177] The term "matrix metalloproteinase inhibitors" or ("MMP" inhibitors) as used herein includes, but is not limited to, collagen peptidomimetic and non-peptidomimetic inhibitors, tetracycline derivatives, such as the hydroxamate peptidomimetic inhibitor batimastat and its orally bioavailable analogs marimastat (BB-2516), prinomastat (AG3340), metastat (NSC683551) BMS-279251, BAY12-9566, TAA211, MMI270B or AAJ996.

[0178] As used herein, the term "compounds for use in the treatment of hematological malignancies" includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, decrease or inhibit the activity of the FMS-like tyrosine kinase receptor (Flt-3R); interferon, 1-β-D-arabinofuranylcytosine (ara-c) and bisulfan; and ALK inhibitors, which are compounds that target, decrease or inhibit anaplastic lymphoma kinase.

[0179] Compounds that target, decrease or inhibit the activity of the FLT-3R-like tyrosine kinase receptor are specific compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, staurosporine derivatives, SU11248 and MLN518.

[0180] The term "HSP90 inhibitor" as used herein includes, but is not limited to, compounds that target, reduce or inhibit the intrinsic ATPase activity of HSP90; compounds that degrade, target, reduce or inhibit HSP90 client proteins via the ubiquitin proteosome pathway. Compounds that target, reduce or inhibit the intrinsic ATPase activity of HSP90 are specific compounds, proteins or antibodies that inhibit the ATPase activity of HSP90, such as 17-allylamino, 17-demethoxygeldanamycin (17AAG), geldanamycin derivatives; other geldanamycin-related compounds; radicicol and HDAC inhibitors.

[0181] The term "antiproliferative antibody" as used herein includes, but is not limited to, trastuzumab (Herceptin™), trastuzumab-DM1, erbitux, bevacizumab (Avastin™), rituximab (Rituxan®), PRO64553 (anti-CD40) and 2C4 antibodies. Antibody refers to intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity.

[0182] For the treatment of acute myeloid leukemia (AML), the compound of the present invention may be used in combination with standard leukemia therapy, in particular in combination with the therapy used for the treatment of AML.In particular, the compound of the present invention may be administered in combination with, for example, phenesyltransferase inhibitors and / or other drugs useful for the treatment of AML, such as daunorubicin, adriamycin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatin and PKC412.

[0183] Other anti-leukemia compounds include, for example, Ara-C, a 2'-alpha-hydroxyribose (arabinoside) derivative of deoxycytidine, a pyrimidine analogue. Also included are the purine analogue of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate. Compounds that target, reduce or inhibit the activity of histone deacetylase (HDAC) inhibitors, for example, sodium butyrate and suberoylanilide hydroxamic acid (SAHA), inhibit the activity of enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (previously FR901228), trichostatin A, and compounds disclosed in U.S. Pat. No. 6,552,065, including but not limited to N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or its pharmaceutically acceptable salt, and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or its pharmaceutically acceptable salt, especially lactate.Somatostatin receptor antagonists as used herein refer to compounds that target, treat or inhibit somatostatin receptors, such as ocretide and SOM230.Tumor cell damage approach refers to approaches such as ionizing radiation. The term "ionizing radiation" referred to above and below means ionizing radiation that occurs either as electromagnetic rays (e.g., X-rays and gamma rays) or particles (e.g., alpha and beta particles). Ionizing radiation is provided in, but not limited to, radiation therapy and is known in the art. Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., Eds., 4 th Edition, Vol. 1, pp. 248-275 (1993).

[0184] Also included are EDG binders and ribonucleotide reductase inhibitors.The term "EDG binder" used herein refers to certain immunosuppressants that modulate lymphocyte recirculation, such as FTY720.The term "ribonucleotide reductase inhibitor" refers to pyrimidine or purine nucleoside analogs, including but not limited to fludarabine and / or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C for ALL) and / or pentostatin.Ribonucleotide reductase inhibitors are certain hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0185] In particular, VEGF compounds, proteins or monoclonal antibodies, such as 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine or a pharma- ceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate; Angiostatin™; Endostatin™; anthranilamide; ZD4190; Zd6474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, such as rhuMAb and RHUFab, VEGF aptamers, such as Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibodies, Angiozyme (RPI4610) and bevacizumab (Avastin™).

[0186] As used herein, photodynamic therapy refers to the treatment that uses certain chemicals known as photosensitizing compounds to treat or prevent cancer.Examples of photodynamic therapy include treatment with compounds such as Visudyne™ and porfimer sodium.

[0187] As used herein, antiangiogenic steroids refer to compounds that block or inhibit angiogenesis, such as anecortave, triamcinolone, hydrocortisone, 11-α-epihydrocortisol, cortexolone, 17α-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone and dexamethasone.

[0188] Implants containing corticosteroids refer to compounds such as fluocinolone and dexamethasone.

[0189] Other chemotherapeutic compounds include, but are not limited to, plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; antisense oligonucleotides or oligonucleotide derivatives; shRNA or siRNA; or miscellaneous compounds or compounds with other or unknown mechanisms of action.

[0190] The structures of the active compounds identified by code number, generic name or trade name may be obtained from the current edition of the standard compendium "Merck Index" or from databases such as Patents International (eg IMS World Publications).

[0191] Exemplary immuno-oncology agents In some embodiments, one or more other therapeutic agents are immuno-oncology agents.As used herein, the term "immuno-oncology agent" refers to an agent that is effective for enhancing, stimulating and / or upregulating immune response in subject.In some embodiments, the administration of immuno-oncology agent and the compound of the present invention has synergistic effect in the treatment of cancer.

[0192] The immuno-oncology agent may be, for example, a small molecule drug, an antibody, or a biological molecule or small molecule. Examples of biological immuno-oncology agents include, but are not limited to, cancer vaccines, antibodies, and cytokines. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody is a humanized or human antibody.

[0193] In some embodiments, the immuno-oncology agent is (i) an agonist of a stimulatory (including costimulatory) receptor or (ii) an antagonist of an inhibitory (including co-inhibitory) signal to T cells, both of which result in the amplification of antigen-specific T cell responses.

[0194] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). One important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, which include CD40 and CD40L, OX-40, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137 (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, and R. ANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, Including EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin α / TNFβ, TNFR2, TNFα, LTβR, lymphotoxin α1β2, FAS, FASL, RELT, DR6, TROY and NGFR.

[0195] In some embodiments, the immuno-oncological agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-β, VEGF, and other immunosuppressive cytokines) or a cytokine that stimulates T cell activation to stimulate an immune response.

[0196] In some embodiments, the combination of a compound of the present invention and an immuno-oncology agent is capable of stimulating a T cell response. In some embodiments, the immuno-oncology agent is (i) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4; or (ii) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD28H.

[0197] In some embodiments, the immuno-oncology agent is an antagonist of an inhibitory receptor on NK cells or an agonist of an activating receptor on NK cells. In some embodiments, the immuno-oncology agent is an antagonist of KIR, such as lirilumab.

[0198] In some embodiments, the immuno-oncology agent is an agent that inhibits or depletes macrophages or monocytes, including, but not limited to, a CSF-1R antagonist, e.g., a CSF-1R antagonist antibody, including RG7155 (WO 2011 / 70024, WO 2011 / 107553, WO 2011 / 131407, WO 2013 / 87699, WO 2013 / 119716, WO 2013 / 132044) or FPA-008 (WO 2011 / 140249; WO 2013 / 169264; WO 2014 / 036357).

[0199] In some embodiments, the immuno-oncological agent is selected from agonistic agents that ligate positive co-stimulatory receptors, blocking agents that attenuate signaling through inhibitory receptors, antagonists and one or more agents that systemically increase the frequency of anti-tumor T cells, agents that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction), deplete or inhibit Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion), inhibit metabolic enzymes, e.g., IDO, or reverse / prevent T cell activation or exhaustion, and agents that induce innate immune activation and / or inflammation at the tumor site.

[0200] In some embodiments, the immuno-oncology agent is a CTLA-4 antagonist. In some embodiments, the CTLA-4 antagonist is an antagonist CTLA-4 antibody. In some embodiments, the antagonist CTLA-4 antibody is YERVOY (ipilimumab) or tremelimumab.

[0201] In some embodiments, the immuno-oncology agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the immuno-oncology agent is an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is an antagonist PD-1 antibody. In some embodiments, the antagonist PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO 2012 / 145493). In some embodiments, the immuno-oncology agent may be pidilizumab (CT-011). In some embodiments, the immuno-oncology agent is a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, referred to as AMP-224.

[0202] In some embodiments, the immuno-oncology agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonist PD-L1 antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG7446; WO 2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO 2007 / 005874), and MSB0010718C (WO 2013 / 79174).

[0203] In some embodiments, the immuno-oncological agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonist LAG-3 antibody. In some embodiments, the LAG3 antibody is BMS-986016 (WO 2010 / 19570, WO 2014 / 08218), or IMP-731 or IMP-321 (WO 2008 / 132601, WO 2009 / 44273).

[0204] In some embodiments, the immuno-oncology agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is an agonist CD137 antibody. In some embodiments, the CD137 antibody is urelumab or PF-05082566 (WO 2012 / 32433).

[0205] In some embodiments, the immuno-oncological agent is a GITR agonist.In some embodiments, the GITR agonist is an agonist GITR antibody.In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO2006 / 105021, WO2009 / 009116) or MK-4166 (WO2011 / 028683).

[0206] In some embodiments, the immuno-oncology agent is an indoleamine (2,3)-dioxygenase (IDO) antagonist. In some embodiments, the IDO antagonist is selected from epacadostat (INCB024360, Incyte); indoximod (NLG-8189, NewLink Genetics Corporation); capmatinib (INC280, Novartis); GDC-0919 (Genentech / Roche); PF-06840003 (Pfizer); BMS:F001287 (Bristol-Myers Squibb); Phy906 / KD108 (Phytoceutica); an enzyme that breaks down kynurenine (Kynase, Kyn Therapeutics); and NLG-919 (WO 2009 / 73620, WO 2009 / 1156652, WO 2011 / 56652, WO 2012 / 142237).

[0207] In some embodiments, the immuno-oncological agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonist OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0208] In some embodiments, the immuno-oncological agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonist OX40 antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO 2006 / 029879).

[0209] In some embodiments, the immuno-oncological agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonist CD40 antibody. In some embodiments, the immuno-oncological agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonist CD40 antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[0210] In some embodiments, the immuno-oncological agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonist CD27 antibody. In some embodiments, the CD27 antibody is valilumab.

[0211] In some embodiments, the immuno-oncological agent is MGA271 (against B7H3) (WO 2011 / 109400).

[0212] In some embodiments, the immuno-oncology agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumumab mafenatox, apolizumab, atezolimab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvalumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelmumab, ipilimumab, isatuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

[0213] In some embodiments, the immuno-oncological agent is an immunostimulatory agent. For example, antibodies that block the PD-1 and PD-L1 inhibitory axis may release activated tumor-reactive T cells, and have been shown in clinical trials to induce durable anti-tumor responses in an increasing number of tumor tissues, including some tumor types not traditionally considered to be immunotherapy-sensitive. See, for example, Okazaki, T. et al. (2013) Nat. Immunol. 14, 1212-1218; Zou et al. (2016) Sci. Transl. Med. 8. The anti-PD-1 antibody nivolumab (Opdivo®, Bristol-Myers Squibb, also known as ONO-4538, MDX1106, and BMS-936558) has shown potential to improve overall survival in patients presenting with renal clear cell carcinoma (RCC) who have experienced disease progression during or after previous antiangiogenic therapy.

[0214] In some embodiments, the immunomodulating therapeutic agent specifically induces apoptosis of tumor cells. Approved immunomodulating therapies that may be used in the present invention include pomalidomide (Pomalyst®, Celgene); lenalidomide (Revlimid®, Celgene); ingenol mebutate (Picato®, LEOPharma).

[0215] In some embodiments, the immuno-oncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from sipuleucel-T (Provenge®, Dendreon / Valeant Pharmaceuticals), approved for treating asymptomatic or minimally symptomatic metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (Imlygic®, BioVex / Amgen, formerly known as T-VEC), a recombinant oncolytic virus therapeutic approved for treating unresectable cutaneous, subcutaneous and nodal regions in melanoma. In some embodiments, the immuno-oncology agent is an oncolytic viral therapeutic, e.g., pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly Jennerex), a thymidine kinase-(TK-) deficient vaccinia virus engineered to express GM-CSF, for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312). pelareorep (Reolysin®, Oncolytics Biotech), a variant of respiratory enteric orphan virus (reovirus) that does not replicate in cells without RAS activation in many cancers, including colorectal cancer (NCT01622543); prostate cancer (NCT01619813); head and neck squamous cell carcinoma (NCT01166542); pancreatic adenocarcinoma (NCT00998322); and non-small cell lung cancer (NSCLC) (NCT00861627); ovarian cancer (NCT02028117);enadenotucirev (NG-348, PsiOxus, formerly known as ColoAd1), an adenovirus engineered to express full-length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in metastatic or advanced epithelial tumors, such as colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); melanoma (NCT03003676); and peritoneal disease, colorectal cancer, or ovarian cancer (NCT02963831). ONCOS-102 (Targovax / formerly Oncos), an adenovirus engineered to express GM-CSF in peritoneal carcinoma (NCT01443260); GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH), a vaccinia virus engineered to express beta-galactosidase (beta-gal) / beta-glucoronidase or beta-gal / human sodium iodide symporter (hNIS), studied in fallopian tube and ovarian cancers, respectively (NCT02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF in bladder cancer (NCT02365818).

[0216] In some embodiments, the immuno-oncology agent is JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK and vaccinia growth factor deficient vaccinia virus engineered to express cytosine deaminase capable of converting the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapy agents targeting difficult-to-treat RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus displayed with Ad5 / 3-E2F-delta24-hTNFα-IRES-hIL20; and antigen-specific CD8 + Selected from VSV-GP (Vira Therapeutics), a vesicular stomatitis virus (VSV) that may be further engineered to express antigens designed to enhance T cell responses.

[0217] In some embodiments, the immuno-oncology agent is a T cell engineered to express a chimeric antigen receptor or CAR. Such T cells engineered to express a chimeric antigen receptor are referred to as CAR-T cells.

[0218] CARs have been constructed that consist of a binding domain, which may be derived from a natural ligand, an endodomain that is the functional end of the T cell receptor (TCR) that can generate an activation signal in T lymphocytes, such as a single chain variable fragment (scFv) derived from a monoclonal antibody specific for a cell surface antigen fused to the CD3-zeta signaling domain from the TCR. Upon antigen binding, such CARs link to endogenous signaling pathways in the effector cell and generate an activation signal similar to that initiated by the TCR complex.

[0219] For example, in some embodiments, the CAR-T cell is one of those described in U.S. Pat. No. 8,906,682 (incorporated herein by reference in its entirety), which discloses CAR-T cells engineered to include an extracellular domain with an antigen-binding domain (e.g., a domain that binds to CD19) fused to an intracellular signaling domain of the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). When expressed in T cells, CARs can redirect antigen recognition based on antigen-binding specificity. In the case of CD19, the antigen is expressed on malignant B cells. Over 200 clinical trials using CAR-T in a wide range of indications are currently underway [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0220] In some embodiments, the immunostimulatory agent is an activator of retinoic acid receptor-related orphan receptor gamma (RORγt). RORγt is a transcription factor that plays an important role in the differentiation and maintenance of type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) T cells, as well as in the differentiation of IL-17-expressing innate immune cell subpopulations, such as NK cells. In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials (NCT02929862) for the treatment of solid tumors.

[0221] In some embodiments, the immunostimulatory agent is a toll-like receptor (TLR) agonist or activator. Suitable activators of TLR include TLR9 agonists or activators, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG under investigation for B-cell, follicular and other lymphomas (NCT02254772). Agonists or activators of TLR8 that may be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals), under investigation for head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT02431559).

[0222] Other immuno-oncological agents that may be used in the present invention include urelumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; valilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirilumab (IPH2102 / BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monalizumab (IPH2201, Innate Pharma, AstraZeneca), an anti-NKG2A monoclonal antibody; andecaliximab (GS-5745, Gilead Sciences), an anti-MMP9 antibody; and MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[0223] In some embodiments, the immunostimulatory agent is selected from elotuzumab, mifamurtide, agonists or activators of toll-like receptors, and activators of RORγt.

[0224] In some embodiments, the immunostimulatory therapeutic is recombinant human interleukin 15 (rhIL-15). rhIL-15 is being tested in clinical trials as a treatment for melanoma and renal cell carcinoma (NCT01021059 and NCT01369888) and leukemia (NCT02689453). In some embodiments, the immunostimulatory agent is recombinant human interleukin 12 (rhIL-12). In some embodiments, the IL-15-based immunotherapeutic is heterodimeric IL-15 (hetIL-15, Novartis / Admune), which is composed of a synthetic form of a fusion complex of endogenous IL-15 complexed with a soluble IL-15 binding protein IL-15 receptor alpha chain (IL15:sIL-15RA), which is being tested in Phase 1 clinical trials for melanoma, renal cell carcinoma, non-small cell lung cancer, and head and neck squamous cell carcinoma (NCT02452268). In some embodiments, the recombinant human interleukin-12 (rhIL-12) is NM-IL-12 (Neumedicines, Inc.), NCT02544724, or NCT02542124.

[0225] In some embodiments, the immuno-oncological agent is selected from those described in Jerry L. Adams et al., "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, Vol. 14, pages 603-622, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncological agent is selected from the examples described in Table 1 of Jerry L. Adams et al. In some embodiments, the immuno-oncological agent is a small molecule that targets an immuno-oncological target selected from those listed in Table 2 of Jerry L. Adams et al. In some embodiments, the immuno-oncological agent is a small molecule agent selected from those listed in Table 2 of Jerry L. Adams et al.

[0226] In some embodiments, the immuno-oncology agent is selected from the small molecule immuno-oncology therapeutic agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, Vol. 28, pages 319-329, the contents of which are incorporated by reference in their entirety. In some embodiments, the immuno-oncology agent is a pathway-targeting agent as described in Peter L. Toogood.

[0227] In some embodiments, the immuno-oncology agent is selected from those described in Sandra L. Ross et al., "Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing", PLoS ONE 12(8):e0183390, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the immuno-oncology agent is a bispecific T cell engager (BiTE®) antibody construct. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates T cells. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates T cells that release cytokines and induce upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engager (BiTE®) antibody construct activates T cells that induce bystander cell lysis. In some embodiments, the bystander cells are in a solid tumor. In some embodiments, the lysed bystander cells are in close proximity to the BiTE®-activated T cells. In some embodiments, the bystander cells comprise tumor-associated antigen (TAA)-negative cancer cells. In some embodiments, the bystander cells comprise EGFR-negative cancer cells. In some embodiments, the immuno-oncological agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the immuno-oncological agent is ex vivo expanded tumor-infiltrating T cells.In some embodiments, the immuno-oncological agent is a bispecific antibody construct or a chimeric antigen receptor (CAR) that directly binds T cells to a tumor-associated surface antigen (TAA).

[0228] <Exemplary immune checkpoint inhibitors> In some embodiments, the immuno-oncology agent is an immune checkpoint inhibitor described herein.

[0229] The term "checkpoint inhibitor" used herein refers to the agent that is useful for preventing cancer cells from evading the immune system of patients.One of the main mechanisms of antitumor immune destruction is known as "T cell exhaustion" due to chronic exposure to antigen, which leads to the upregulation of inhibitory receptors.These inhibitory receptors act as immune checkpoints to prevent uncontrolled immune response.

[0230] PD-1 and co-inhibitory receptors, such as cytotoxic T-lymphocyte antigen 4 (CTLA-4, B- and T-lymphocyte attenuator (BTLA; CD272), T-cell immunoglobulin and mucin domain-3 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD223), etc., are often referred to as checkpoint regulators. They act as molecular "gatekeepers" that allow extracellular information to dictate whether cell cycle progression and other intracellular signaling processes should proceed.

[0231] In some embodiments, the immune checkpoint inhibitor is an antibody against PD-1, which binds to the programmed cell death 1 receptor (PD-1) and blocks the receptor from binding to the inhibitory ligand PDL-1, thus abolishing the ability of the tumor to suppress the host's anti-tumor immune response.

[0232] In one embodiment, the checkpoint inhibitor is a biotherapeutic agent or a small molecule. In another embodiment, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. In a further embodiment, the checkpoint inhibitor inhibits a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In additional embodiments, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, or a combination thereof. In embodiments, the checkpoint inhibitor is an immunostimulatory agent, a T cell growth factor, an interleukin, an antibody, a vaccine, or a combination thereof. In further embodiments, the interleukin is IL-7 or IL-15. In certain embodiments, the interleukin is glycosylated IL-7. In additional embodiments, the vaccine is a dendritic cell (DC) vaccine.

[0233] A checkpoint inhibitor includes any agent that blocks or inhibits an inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors can be small molecule inhibitors, or can be antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that may be targeted for blocking or inhibition include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belonging to the CD2 family of molecules, which inhibits all NK, gamma delta, and memory CD8 +(expressed on αβ)T cells), CD160 (also called BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, and CGEN-15049. Exemplary immune checkpoint inhibitors include tremelimumab (CTLA-4 blocking antibody), anti-OX40, PD-Ll monoclonal antibody (anti-B7-Hl; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PDl antibody), CT-011 (anti-PDl antibody), BY55 monoclonal antibody, AMP224 (anti-PDLl antibody), BMS-936559 (anti-PDLl antibody), MPLDL3280A (anti-PDLl antibody), MSB0010718C (anti-PDLl antibody), and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to, PD-Ll, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

[0234] In certain embodiments, the immune checkpoint inhibitor is selected from a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from the group consisting of nivolumab (Opdivo®), ipilimumab (Yervoy®), and pembrolizumab (Keytruda®). In some embodiments, the checkpoint inhibitor is selected from nivolumab (anti-PD-1 antibody, Opdivo®, Bristol-Myers Squibb); pembrolizumab (anti-PD-1 antibody, Keytruda®, Merck); ipilimumab (anti-CTLA-4 antibody, Yervoy®, Bristol-Myers Squibb); durvalumab (anti-PD-L1 antibody, Imfinzi®, AstraZeneca); and atezolizumab (anti-PD-L1 antibody, Tecentriq®, Genentech).

[0235] In some embodiments, the checkpoint inhibitor is selected from the group consisting of lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirilumab, IPH2101, pembrolizumab (Keytruda®), and tremelimumab.

[0236] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of REGN2810 (Regeneron), an anti-PD-1 antibody that has been studied in patients with basal cell carcinoma (NCT03132636); NSCLC (NCT03088540); cutaneous squamous cell carcinoma (NCT02760498); lymphoma (NCT02651662); and melanoma (NCT03002376); pidilizumab (CureTech), also known as CT-011, an antibody that binds to PD-1 in clinical trials for diffuse large B-cell lymphoma and multiple myeloma; avelumab (Bavencio®, also known as MSB0010718C, Pfizer / Merck), a fully human IgG1 anti-PD-L1 antibody in clinical trials for non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, kidney cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer. KGaA); or PDR001 (Novartis), an inhibitory antibody that binds to PD-1 in clinical trials for non-small cell lung cancer, melanoma, triple-negative breast cancer, and advanced or metastatic solid tumors. Tremelimumab (CP-675,206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4 that is being studied in clinical trials for several indications, including mesothelioma, colorectal cancer, renal cancer, breast cancer, lung and non-small cell lung cancer, pancreatic ductal adenocarcinoma, pancreatic cancer, germ cell cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, prostate cancer, endometrial cancer, metastatic cancer in the liver, liver cancer, large B-cell lymphoma, ovarian cancer, cervical cancer, metastatic anaplastic thyroid cancer, urothelial carcinoma, fallopian tube cancer, multiple myeloma, bladder cancer, soft tissue sarcoma, and melanoma. AGEN-1884 (Agenus) is an anti-CTLA4 antibody being investigated in Phase 1 clinical trials for advanced solid tumors (NCT02694822).

[0237] In some embodiments, the checkpoint inhibitor is an inhibitor of T cell immunoglobulin mucin containing protein-3 (TIM-3). TIM-3 inhibitors that may be used in the present invention include TSR-022, LY3321367, and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody under investigation in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody under investigation in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody under investigation in advanced malignancies (NCT02608268).

[0238] In some embodiments, the checkpoint inhibitor is an inhibitor of TIGIT, which is a T cell immunoreceptor having Ig and ITIM domains, or an immunoreceptor on certain T cells and NK cells.The TIGIT inhibitors that may be used in the present invention include BMS-986207 (Bristol-Myers Squibb), anti-TIGIT monoclonal antibody (NCT02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT03119428).

[0239] In some embodiments, the checkpoint inhibitor is a lymphocyte activation gene-3 (LAG-3) inhibitor. LAG-3 inhibitors that may be used in the present invention include BMS-986016 and REGN3767 and IMP321. BMS-986016 (Bristol-Myers Squibb), an anti-LAG-3 antibody, is being studied in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody, and is being studied in malignant tumors (NCT03005782). IMP321 (Immutep SA) is a LAG-3-Ig fusion protein that is being studied in melanoma (NCT02676869); adenocarcinoma (NCT02614833); and metastatic breast cancer (NCT00349934).

[0240] Checkpoint inhibitors that may be used in the present invention include OX40 agonists. OX40 agonists under investigation in clinical trials include agonist anti-OX40 antibodies PF-04518600 / PF-8600 (Pfizer) in metastatic kidney cancer (NCT03092856) and advanced cancers and neoplasms (NCT02554812; NCT05082566); agonist anti-OX40 antibody GSK3174998 (Merck) in phase 1 cancer clinical trials (NCT02528357); agonist anti-OX40 antibody GSK3174998 (Merck) in advanced solid tumors (NCT02318394 and NCT02705482); OX40 antibody MEDI0562 (Medimmune / AstraZeneca); MEDI6469 (Medimmune / AstraZeneca), an agonistic anti-OX40 antibody in patients with colorectal cancer (NCT02559024), breast cancer (NCT01862900), head and neck cancer (NCT02274155), and metastatic prostate cancer (NCT01303705); and BMS-986178 (Bristol-Myers Squibb), an agonistic anti-OX40 antibody in advanced cancers (NCT02737475).

[0241] The checkpoint inhibitors that may be used in the present invention include CD137 (also called 4-1BB) agonists.CD137 agonists that are being investigated in clinical trials include utomirumab (PF-05082566, Pfizer), an agonist anti-CD137 antibody, in diffuse large B-cell lymphoma (NCT02951156) and advanced cancer and neoplasms (NCT02554812 and NCT05082566); urelumab (BMS-663513, Bristol-Myers Squibb), an agonist anti-CD137 antibody, in melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981).

[0242] The checkpoint inhibitor that may be used in the present invention includes CD27 agonist.The CD27 agonist that is being studied in clinical trials includes valilumab (CDX-1127, Celldex Therapeutics), an agonist anti-CD27 antibody, in squamous cell head and neck cancer, ovarian carcinoma, colorectal cancer, renal cell carcinoma, and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0243] Checkpoint inhibitors that may be used in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists under investigation in clinical trials include TRX518 (Leap), an agonistic anti-GITR antibody, in melanoma and other malignant solid tumors (NCT01239134 and NCT02628574). Therapeutics); GWN323 (Novartis), an agonistic anti-GITR antibody in solid tumors and lymphomas (NCT02740270); INCAGN01876 (Incyte / Agenus), an agonistic anti-GITR antibody in advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonistic anti-GITR antibody in solid tumors (NCT02132754), and MEDI1873 (Medimmune / AstraZeneca), an agonistic hexameric GITR-ligand molecule with a human IgG1 Fc domain in advanced solid tumors (NCT02583165).

[0244] The checkpoint inhibitors that may be used in the present invention include inducible T cell costimulator (ICOS, also known as CD278) agonists.ICOS agonists under investigation in clinical trials include MEDI-570 (Medimmune), an agonist anti-ICOS antibody in lymphoma (NCT02520791);GSK3359609 (Merck), an agonist anti-ICOS antibody in phase 1 (NCT02723955);and JTX-2011 (Jounce Therapeutics), an agonist anti-ICOS antibody in phase 1 (NCT02904226).

[0245] Checkpoint inhibitors that may be used in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors under investigation in clinical trials include the anti-KIR antibody lirilumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb) in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma) in myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an antibody that binds to the three domains of the long cytoplasmic tail (KIR3DL2), in lymphoma (NCT02593045).

[0246] Checkpoint inhibitors that may be used in the present invention include CD47, an inhibitor of the interaction between CD47 and signal regulatory protein alpha (SIRPa). CD47 / SIRPa inhibitors under investigation in clinical trials include ALX-148 (Alexo Therapeutics), an antagonist variant that binds to CD47 and blocks CD47 / SIRPa-mediated signaling (SIRPa), in Phase 1 (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein generated by binding the N-terminal CD47-binding domain of SIRPa to the Fc domain of human IgG1, which acts through bound human CD47 to block it from delivering its "don't eat" signal to macrophages, in Phase 1 (NCT02890368 and NCT02663518) clinical trials. Therapeutics; CC-90002 (Celgene), an anti-CD47 antibody, in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.) in colorectal neoplasia and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[0247] Checkpoint inhibitors that may be used in the present invention include CD73 inhibitors.CD73 inhibitors under investigation in clinical trials include MEDI9447 (Medimmune), an anti-CD73 antibody in solid tumors (NCT02503774); and BMS-986179 (Bristol-Myers Squibb), an anti-CD73 antibody in solid tumors (NCT02754141).

[0248] Checkpoint inhibitors that may be used in the present invention include agonists of the stimulator of interferon genes protein (STING, also known as transmembrane protein 173, or TMEM173). STING agonists under investigation in clinical trials include MK-1454 (Merck), an agonist synthetic cyclic dinucleotide in lymphoma (NCT03010176); and ADU-S100 (MIW815, Aduro Biotech / Novartis), an agonist synthetic cyclic dinucleotide in Phase 1 (NCT02675439 and NCT03172936).

[0249] Checkpoint inhibitors that may be used in the present invention include CSF1R inhibitors. CSF1R inhibitors under investigation in clinical trials include the CSF1R small molecule inhibitor pexidartinib (PLX3397, Plexxikon) in colorectal cancer, pancreatic cancer, metastatic and advanced cancers (NCT02777710) and melanoma, non-small cell lung cancer, squamous cell head and neck cancer, gastrointestinal stromal tumor (GIST) and ovarian cancer (NCT02452424); and pancreatic cancer (NCT03153410), melanoma (NCT031012 54), and the anti-CSF-1R antibody IMC-CS4 (LY3022855, Lilly) in solid tumors (NCT02718911); and the orally available CSF1R inhibitor BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxyl]-pyridine-2-carboxylic acid methylamide, Novartis) in advanced solid tumors (NCT02829723).

[0250] Checkpoint inhibitors that may be used in the present invention include NKG2A receptor inhibitors.NKG2A receptor inhibitors under investigation in clinical trials include the anti-NKG2A antibody monalizumab (IPH2201, Innate Pharma) in head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0251] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avelumab, durvalumab, atezolizumab, or pidilizumab.

[0252] <Therapeutic use> The bicyclic peptides of the invention have particular utility as Nectin-4 binding agents.

[0253] Nectin-4 is a surface molecule that belongs to the nectin family of proteins, which includes four members. Nectins are cell adhesion molecules that play important roles in various biological processes, e.g., polarity, proliferation, differentiation and migration for epithelial, endothelial, immune and neuronal cells during development and adulthood. They are involved in several pathological processes in humans. They are the main receptors for poliovirus, herpes simplex virus and measles virus. Mutations in the genes encoding nectin-1 (PVRL1) or nectin-4 (PVRL4) result in ectodermal dysplasia syndromes associated with other abnormalities. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of the family. Nectin-4 is a tumor-associated antigen in the majority of tumors with poor prognosis, in 50%, 49% and 86% of breast, ovarian and lung carcinomas, respectively. Its expression is not detectable in the corresponding normal tissues. In breast tumors, nectin-4 is expressed mainly in triple-negative and ERBB2+ carcinomas. In the serum of patients presenting with these cancers, the detection of soluble forms of Nectin-4 is associated with poor prognosis. The level of serum Nectin-4 increases during metastatic progression and decreases after treatment. These results suggest that Nectin-4 may be a promising target for the treatment of cancer. Accordingly, several anti-Nectin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody drug conjugate (ADC) that targets Nectin-4 and is currently being clinically investigated for the treatment of patients suffering from solid tumors.

[0254] Polypeptide ligands selected according to the methods of the invention may 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 needs to 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 against specific diseases and pathogens.

[0255] Substantially pure peptide ligands of at least 90 to 95% homogeneity are preferred for administration to mammals, with 98 to 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 may be used diagnostically or therapeutically (including in vitro) or in the development and performance of assay procedures, immunofluorescence staining, and the like (Lefkovite and Pernis, (1979 and 1981) Immunological Methods, Volumes I and II, Academic Press, NY).

[0256] According to a further aspect of the present invention, there is provided a peptide ligand or drug conjugate as described herein for use in preventing, inhibiting or treating a disease or disorder mediated by Nectin-4.

[0257] According to a further aspect of the present invention, there is provided a method for preventing, inhibiting or treating a disease or disorder mediated by Nectin-4, comprising administering to a patient in need thereof a drug conjugate of an effector group and a peptide ligand as described herein.

[0258] In one embodiment, the nectin-4 is mammalian nectin-4. In a further embodiment, the mammalian nectin-4 is human nectin-4.

[0259] In one embodiment, the Nectin-4 mediated disease or disorder is selected from viral infections, ectodermal dysplastic syndromes and other abnormalities, breast, ovarian and lung carcinoma, metastatic progression, and solid tumors.

[0260] In a further embodiment, the Nectin-4 mediated disease or disorder is selected from cancer.

[0261] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, 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 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 bronchioalveolar carcinoma), and tumors of the bladder and urinary tract ... breast, gastrointestinal tract (including 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 bronchioalveolar carcinoma), and tumors of the bladder and urinary tract, such as tumors of the bladder and urinary tract, carcinomas of the head and neck (e.g., cancers of the tongue, buccal 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 follicular carcinoma), adrenal gland, prostate, skin and adnexa (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, dysplastic nevi); hematological malignancies (i.e., leukemia, lymphoma) and premalignant hematological disorders, and hematological malignancies and associated conditions of the lymphoid lineage (e.g., acute lymphoblastic leukemia, Hematologic malignancies and associated conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic lymphocytic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), and hematological malignancies and associated conditions of the myeloid lineage (e.g., acute myeloid leukemia [AML], chronic osteomyelitis [CO], and leukemias ... borderline malignant disorders including myeloid 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; tumors of mesenchymal origin such as sarcomas of soft tissue, bone, or cartilage, such as 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); endocrine tumors (e.g., pituitary tumor, adrenal tumor, pancreatic islet cell tumor, parathyroid tumor, carcinoid tumor and medullary carcinoma of the thyroid); eye and adnexal tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles and choriocarcinomas); and childhood and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumor); or congenital syndromes that otherwise predispose a patient to malignant tumors (e.g., xeroderma pigmentosum).

[0262] In further embodiments, the cancer is selected from a hematopoietic malignancy, e.g., selected from 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).

[0263] In further embodiments, the cancer is selected from lung cancer (e.g., non-small cell lung cancer), bladder cancer, pancreatic cancer, and breast cancer. Data are presented herein in Examples 1 to 5 demonstrating that selected bicyclic drug conjugates of the invention exhibited anti-tumor activity in these cancer models.

[0264] Reference herein to the term "prevention" involves administration of the protective composition before the disease is induced. "Suppression" refers to administration of the composition after an inducing event but prior to the clinical appearance of the disease. "Treatment" involves administration of the protective composition after symptoms of the disease appear.

[0265] Animal model systems are available that may be used to screen the efficacy of peptide ligands in protecting against or treating disease. The use of animal model systems is facilitated by the present invention, whereby polypeptide ligands capable of cross-reacting with human and animal targets are developed to enable the use of animal models.

[0266] Further, data are presented herein demonstrating an association between copy number variation (CNV) and gene expression for Nectin-4 from multiple tumor types. Thus, according to a further aspect of the present invention, there is provided a method of preventing, inhibiting or treating cancer, comprising administering to a patient in need thereof a drug conjugate of an effector group and a peptide ligand as described herein, wherein the patient has been identified as having increased copy number variation (CNV) of Nectin-4.

[0267] In one embodiment, the cancer is selected from those identified herein as having increased CNVs in Nectin-4. In a further embodiment, the cancer is selected from those identified herein as having increased CNVs in Nectin-4, namely: breast, uterine, bladder, lung adenocarcinoma, lung squamous cell, cervical, head and neck, pancreatic, thyroid, colorectal, thymoma, sarcoma, renal clear cell carcinoma (RCC), prostate and gastric cancer.

[0268] The invention is further described below with reference to examples. EXAMPLES

[0269] <abbreviation> 1,2,4-TriAz 3-(1,2,4-triazol-1-yl)-alanine 1Nal 1-Naphthylalanine 2FuAla 2-Furylalanine 2MePhe 2-Methyl-phenylalanine 2Nal 2-Naphthylalanine 2Pal 2-pyridylalanine 3,3-DPA 3,3-Diphenylalanine 3MePhe 3-Methyl-phenylalanine 3Pal 3-Pyridylalanine 4,4-BPA 4,4-Biphenylalanine 4,4-DFP 4,4-Difluoroproline 4MePhe 4-Methyl-phenylalanine 4Pal 4-Pyridylalanine 4ThiAz beta-(4-thiazolyl)-alanine 5FTrp 5-Fluoro-L-tryptophan Agb 2-amino-4-guanidinobutyric acid Aib Aminoisobutyric Acid AzaTrp Azatryptophan Aze Azetidine C5A Cyclopentylglycine Cha 3-Cyclohexyl-alanine Cpa Cyclopropylalanine Cya Cysteic acid DOPA 3,4-dihydroxy-phenylalanine HArg Homoarginine HGln Homoglutamine Hleu homoleucine Hphe homophenylalanine Hse(me) Homoserine(Me) HSer homoserine HyP Hydroxyproline Lys(Ac) Lysine (acetyl) Met(O2) methionine sulfone Nle Norleucine Oic Octahydroindole carboxylic acid Oxa Oxazolidine-4-carboxylic acid pCoPhe para-carboxy-phenylalanine PheOPhe 4-phenoxy-phenylalanine Phg Phenylglycine Pip Pipecolic acid Pro(4NH) 4-amino-proline tBuAla t-Butyl-alanine TetraZ Tetrazole Alanine Thi Thienyl-alanine THP(O) Tetrahydropyran-4-propanoic acid THP(SO2) Dioxo-4-tetrahydrothiopyranylacetic acid Trp(Me) Methyltryptophan

[0270] [material and method] Peptide synthesis Peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer from Peptide Instruments and a Syro II synthesizer from MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) with appropriate side chain protecting groups were used, where applicable standard coupling conditions were used, followed by deprotection using standard methodologies.

[0271] Alternatively, peptides were purified using HPLC and after isolation, they were modified with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma). For this, the linear peptide was diluted up to about 35 mL with 50:50 MeCN:H2O, about 500 μL of 100 mM TATA in acetonitrile was added, and the reaction was initiated with 35 mL of 1 M NH4HCO3 in H2O. The reaction was allowed to proceed at room temperature for about 30-60 minutes and, once the reaction was complete (as determined by MALDI), lyophilized. Once complete, 1 mL of 1 mL cysteine ​​hydrochloride monohydrate (Sigma) in H2O was added to the reaction at room temperature for about 60 minutes to quench any excess TATA.

[0272] 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 stored at -20°C.

[0273] All amino acids were used in the L-configuration unless otherwise noted.

[0274] In some cases, the peptides are converted to activated disulfides and then coupled to free thiol groups on the toxins using the following method: A solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol equiv.) was added to a solution of peptide (20 mM) in dry DMSO (1 mol equiv.). The reaction was mixed thoroughly and DIPEA (20 mol equiv.) was added. The reaction was monitored by LC / MS until completion.

[0275] Preparation of Bicyclic Peptide Drug Conjugates Preparation of BCY8549 Separation conditions: Phase A: 0.075% TFA in HO, Phase B: MeCN Separation method: 18~48~55 minutes, RT=53.5 minutes Separation columns: Luna 200×25mm 10μm, C18, 110A and Gemin 150×30mm, C18, 5μm, 110A, joint, 50℃ Dissolution method:DMF Separation purity: 95% BCY8234 was synthesized by solid phase synthesis.

[0276] [ka] [ka]

[0277] Preparation of compound 2 Peptides were synthesized using standard Fmoc chemistry. 1) DCM is added to a vessel containing CTC resin (5 mmol, 4.3 g, 1.17 mmol / g) and Fmoc-Cit-OH (2.0 g, 5 mmol, 1.0 equiv.) while bubbling N. 2) Add DIEA (4.0 eq.) dropwise and mix for 2 hours. 3) Add MeOH (5 mL) and mix for 30 minutes. 4) Drain and wash five times with DMF. 5) Add 20% piperidine / DMF and react for 30 minutes. 6) Drain and wash five times with DMF. 7) Add Fmoc-amino acid solution and mix for 30 seconds, then add activation buffer and bubble with N2 for approximately 1 hour. 8) Repeat steps 4 to 7 above to couple the following amino acids:

[0278] Notes: [Table 1] Fmoc deprotection was carried out using 20% ​​piperidine in DMF for 30 min. The coupling reaction was monitored by the ninhydrin test and the resin was washed five times with DMF.

[0279] Peptide cleavage and purification: 1) Add cleavage buffer (20% TFIP / 80% DCM) to the flask containing the side-chain protected peptide at room temperature and stir for 1 h twice. 2) Filter and collect the filtrate. 3) Concentrate to remove the solvent. 4) The crude peptide was lyophilized to give the final product (1.4 g, 85.0% yield).

[0280] Preparation of compound 3 To a solution of compound 2 (1.65 g, 5.01 mmol, 1.0 equiv) in DCM (30 mL) and MeOH (15 mL) was added EEDQ (2.48 g, 10.02 mmol, 2.0 equiv) and (4-aminophenyl)methanol (740.37 mg, 6.01 mmol, 1.2 equiv). The mixture was stirred at 15° C. for 16 h. LC-MS showed that compound 2 was completely consumed and one major peak with the desired m / z was detected. TLC showed that compound 2 was completely consumed and many new spots were formed. The reaction mixture was concentrated under reduced pressure to remove the solvent and give a residue. The residue was purified by flash silica gel chromatography (ISCO®; 80 SepaFlash® silica flash column, eluent of 0-15 DCM / MeOH gradient at 60 mL / min). Compound 3 (1.3 g, 2.99 mmol, 59.72% yield) was obtained as a yellow solid.

[0281] Preparation of compound 4 To a solution of compound 3 (1.3 g, 2.99 mmol, 1.0 equiv) in DMF (10 mL) was added DIEA (2.32 g, 17.95 mmol, 3.13 mL, 6.0 equiv) and bis(4-nitrophenyl)carbonate (3.64 g, 11.97 mmol, 4.0 equiv). The mixture was stirred at 15° C. for 1 h. LC-MS showed that compound 3 was completely consumed and one major peak with the desired m / z was detected. The residue was purified by prep-HPLC (neutral condition). Compound 4 (1.0 g, 1.67 mmol, 55.74% yield) was obtained as a yellow solid.

[0282] Preparation of compound 5 To a solution of compound 5 (250.53 mg, 417.84 μmol, 1.5 equiv.) in DMF (5 mL), HOBt (56.46 mg, 417.84 μmol, 1.5 equiv.) and DIEA (108.01 mg, 835.68 μmol, 145.56 μL, 3.0 equiv.), MMAE (0.200 g, 278.56 μmol, 1.0 equiv.) were added. The mixture was stirred at 35° C. for 12 h. LC-MS showed that MMAE was completely consumed and one major peak with the desired m / z was detected. The reaction was directly purified by prep-HPLC (neutral conditions). Compound 5 (0.180 g, 152.74 μmol, 54.83% yield) was obtained as a yellow solid.

[0283] Preparation of compound 6 To a solution of compound 5 (0.170 g, 144.26 μmol, 1.0 equiv.) in THF (5 mL) and HO (5 mL) was added LiOH.HO (12.11 mg, 288.51 μmol, 2.0 equiv.). The mixture was stirred at 15° C. for 1 h. LC-MS showed that compound 5 was completely consumed and one major peak with the desired m / z was detected. AcOH was used to adjust PH=7 and THF was removed under reduced pressure to give a residue. The residue was purified by prep-HPLC (neutral condition). Compound 6 (0.185 g, crude) was obtained as a yellow solid.

[0284] Preparation of BCY8549 To a solution of compound 6 (0.100 g, 86.93 μmol, 1.0 equiv.) in DMA (4 mL) was added HOSu (10.00 mg, 86.93 μmol, 1.0 equiv.) and EDCI (16.66 mg, 86.93 μmol, 1.0 equiv.). After the NHS ester was formed, β-Ala-BCY8234 (525.98 mg, 173.85 μmol, 2.0 equiv.) and DIEA (33.70 mg, 260.78 μmol, 45.42 μL, 3.0 equiv.) were added. The mixture was stirred at 15° C. for 4 h. LC-MS showed that compound 6 was completely consumed and one major peak with the desired m / z was detected. The reaction was directly purified by prep-HPLC (TFA conditions). Compound BCY8549 (0.0528 g, 12.15 μmol, 13.98% yield, 95.70% purity) was obtained as a white solid. Retention time = 11.48 min. Found mass = 1386.4 (M / 3+H).

[0285] Preparation of BCY8245 Separation conditions: Phase A: 0.075% TFA in HO, Phase B: MeCN Separation method: 18~48~55 minutes, RT=53.5 minutes Separation columns: Luna 200×25mm 10um, C18, 110A and Gemin 150×30mm, C18, 5um, 110A, joint, 50℃ Dissolution method:DMF Separation purity: 95%

[0286] BCY8234 was synthesized by solid phase synthesis. The reaction scheme for BCY8245 is shown below:

[0287] [ka] [ka]

[0288] Preparation of compound 3 Compound 3 was synthesized by solid phase methodology.

[0289] Preparation of compound 4 To a solution of compound 3 (1.3 g, 3.23 mmol, 1.0 equiv.) in DCM (10 mL) and MeOH (5 mL), EEDQ (1.60 g, 6.46 mmol, 2.0 equiv.) and (4-aminophenyl)methanol (517.16 mg, 4.20 mmol, 1.3 equiv.) were added. The mixture was stirred at 20° C. for 16 h. LC-MS showed that compound 3 was completely consumed and one major peak with the desired m / z was detected. The solvent was removed under reduced pressure. The residue was purified by flash silica gel chromatography (ISCO®; 40 g SepaFlash® silica flash column, eluent of 0-15% DCM / MeOH gradient at 40 mL / min). Compound 4 (0.950 g, 1.87 mmol, 57.94% yield) was obtained as a yellow solid.

[0290] Preparation of compound 5 To a solution of compound 4 (0.950 g, 1.87 mmol, 1.0 equiv) in DMF (5 mL) was added DIEA (1.21 g, 9.36 mmol, 1.63 mL, 5.0 equiv) and bis(4-nitrophenyl)carbonate (2.28 g, 7.49 mmol, 4.0 equiv). The mixture was stirred at 20° C. for 1 h. LC-MS showed that compound 4 was completely consumed and one major peak with the desired m / z was detected. The reaction was directly purified by prep-HPLC (neutral conditions). Compound 5 (0.400 g, 594.64 μmol, 31.77% yield) was obtained as a white solid.

[0291] Preparation of compound 6 To a solution of compound 5 (0.200 g, 297.32 μmol, 1.0 equiv.) in DMF (5 mL), HOBt (52.23 mg, 386.51 μmol, 1.3 equiv.) and DIEA (115.28 mg, 891.95 μmol, 155.36 μL, 3.0 equiv.), MMAE (192.12 mg, 267.59 μmol, 0.9 equiv.) were added. The mixture was stirred at 20° C. for 16 h. LC-MS showed that compound 5 was completely consumed and one major peak with the desired m / z was detected. The reaction was directly purified by prep-HPLC (neutral conditions). Compound 6 (0.160 g, 127.84 μmol, 43.00% yield) was obtained as a white solid.

[0292] Preparation of compound 7 To a solution of compound 6 (0.160 g, 127.84 μmol, 1.0 equiv.) in THF (3 mL) and HO (3 mL) was added LiOH.HO (26.82 mg, 639.21 μmol, 5.0 equiv.). The mixture was stirred at 20° C. for 1 h. LC-MS showed that compound 6 was completely consumed and one major peak with the desired m / z was detected. THF was removed under reduced pressure, pH=7 was adjusted with AcOH, and the mixture was lyophilized. Compound 7 (0.130 g, 105.05 μmol, 82.17% yield) was obtained as a white solid.

[0293] Preparation of Compound 8 To a solution of compound 7 (36.27 mg, 315.15 μmol, 3.0 equiv.) in DMA (6 mL) and DCM (2 mL) was added EDCI (60.41 mg, 315.15 μmol, 3.0 equiv.). The mixture was stirred at 15° C. for 3 h. LC-MS showed that compound 7 was completely consumed and one major peak with the desired m / z was detected. DCM was removed under reduced pressure. The reaction was directly purified by prep-HPLC (neutral conditions). Compound 8 (0.095 g, 71.18 μmol, 67.76% yield) was obtained as a white solid.

[0294] Preparation of BCY8245 To a solution of BCY8234 (66.41 mg, 22.48 μmol, 1.0 equiv) in DMA (4 mL) was added DIEA (8.72 mg, 67.44 μmol, 11.75 μL, 3.0 equiv) and compound 8 (0.030 g, 22.48 μmol, 1.0 equiv). The mixture was stirred at 20° C. for 16 h. LC-MS showed that BCY8234 was completely consumed and one major peak with the desired m / z or mass was detected. The reaction was directly purified by prep-HPLC (TFA conditions). Compound BCY8245 (0.0427 g, 10.16 μmol, 45.19% yield, 99.30% purity) was obtained as a white solid. Retention time = 11.7 min. Found mass = 1043.9 (M / 4+H)

[0295] <Biological data> Nectin-4 direct binding assay The affinity (Ki) of the peptides of the invention for human nectin-4 was determined using a fluorescence polarization assay according to the method disclosed in WO 2016 / 067035. The peptides of the invention with a fluorescent tag (either fluorescein, SIGMA or Alexa Fluor488™, Fisher Scientific) were diluted to 2.5 nM in PBS with 0.01% tween 20 or in 50 mM HEPES with 100 mM NaCl and 0.01% tween pH 7.4 (both referred to as assay buffer). This was combined with a protein titration in the same assay buffer as the peptide to give 1 nM peptide in a total volume of 25 μL, typically 5 μL assay buffer, 10 μL protein, and 10 μL fluorescent peptide in a black wall and bottom low binding low volume 384 well plate. Selection assays were performed using one in two serial dilutions to obtain 12 different concentrations, including the highest concentration, ranging from 500 nM for known high affinity binders to 10 μM for low affinity binders. Measurements were performed on a BMG PHERAstar FS equipped with an "FP 485 520520" optical module, exciting at 485 nm and detecting parallel and perpendicular emission at 520 nm. The PHERAstar FS was set at 25°C, 200 flashes per well, 0.1 second positioning delay, and each well was measured at 5 to 10 minute intervals for 60 minutes. In the absence of protein in the wells, the increase used for analysis was determined for each tracer at the end of 60 minutes. Data were analyzed using Systat Sigmaplot version 12.0. The mP values ​​were fitted to a user-defined quadratic equation to generate the Kd value: f=ymin+(ymax-ymin) / Lig×((x+Lig+Kd) / 2-sqrt((((x+Lig+Kd) / 2)^2)-(Lig×x))). "Lig" was the defined value of the concentration of the tracer used.

[0296] Nectin-4 competitive binding assay Peptides without fluorescent tags were tested in competition with ACPFGCHTDWSWPIWCA-Sar6-K(Fl) (SEQ ID NO:2) and (Kd=5nM-determined using the protocol above). Peptides were diluted to the appropriate concentration in assay buffer with up to 5% DMSO as described in the direct binding assay, then serially diluted 2-fold. 5 μL of diluted peptide was added to the plate, followed by 10 μL human nectin-4, then 10 μL fluorescent peptide. Measurements were performed as in the direct binding assay, but the increase was determined before the first measurement. Data analysis was performed with Systat Sigmaplot version 12.0 where mP values ​​were fitted to a user-defined cubic equation, and Ki values ​​were calculated as follows: f=ymin+(ymax-ymin) / Lig*((Lig*((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Pro t*c)+Klig*Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(K comp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*(((Klig+Kcomp+Lig+Com p-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp- Prot*c))) / ((3*Klig)+((2*((Klig+Kcomp+Lig+Comp-Prot*c)^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig *Kcomp))^0.5*COS(ARCCOS((-2*(Klig+Kcomp+Lig+Comp-Prot*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)*(Kcomp*(Lig -Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp)-27*(-1*Klig*Kcomp*Prot*c)) / (2*(((Klig+Kcomp+Lig+Comp-Prot*c) ^2-3*(Kcomp*(Lig-Prot*c)+Klig*(Comp-Prot*c)+Klig*Kcomp))^3)^0.5))) / 3))-(Klig+Kcomp+Lig+Comp-Prot*c)))) "Lig", "KLig" and "Prot" were all defined values ​​related to the fluorescent peptide concentration, the Kd of the fluorescent peptide and the nectin concentration, respectively.

[0297] Biacore SPR binding assay of Nectin-4 Biacore experiments were performed to determine the k of monomeric peptides that bind to human Nectin-4 protein (obtained from Charles River). a (M -1 s -1 ), k d (s -1 ), K D Values ​​(nM) were determined.

[0298] Human nectin-4 (residues Gly32-Ser349; NCBI Reference Sequence: NP_112178.2) with a gp67 signal sequence and a C-terminal FLAG tag was cloned into pFastbac-1 and baculovirus was generated using the standard Bac-to-Bac™ protocol (Life Technologies). 6 ml -1 Sf21 cells were infected with P1 virus stock at 2 MOI and supernatants were harvested at 72 hours. Supernatants were pooled and bound to anti-FLAGM2 affinity agarose resin (Sigma) washed in PBS for 1 hour at 4°C, after which the resin was transferred to a column and washed extensively with PBS. Proteins were eluted with 100 μg / ml FLAG peptide. Eluted protein was concentrated to 2 ml and loaded onto an S-200 Superdex column (GE Healthcare) in PBS at 1 ml / min. Fractions of 2 ml were collected and the fraction containing nectin-4 protein was concentrated to 16 mg / ml.

[0299] Proteins were randomly biotinylated in PBS using EZ-Link™ Sulfo-NHS-LC-LC-Biotin Reagent (Thermo Fisher) according to the manufacturer's suggested protocol. Proteins were extensively desalted and unbound biotin was removed in PBS using spin columns.

[0300] For peptide binding analysis, a Biacore 3000 instrument utilizing a CM5 chip (GE Healthcare) was used. Streptavidin was immobilized on the chip using standard amine coupling chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as running buffer. Briefly, the carboxymethyldextran surface was activated with a 7 min injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min. For streptavidin capture, proteins were diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 μl of streptavidin over the activated chip surface. The remaining activated groups were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5) to capture biotinylated nectin-4 to a level of 1,200–1,800 RU. The buffer was exchanged with PBS / 0.05% Tween 20 and a dilution series of the peptide was prepared in this buffer, resulting in a final DMSO concentration of 0.5%. The maximum peptide concentration was 100 nM, with six further 2-fold dilutions. SPR analysis was performed at a flow rate of 50 μl / min, 25 °C, and 60 s of binding and dissociation of peptides between 400 and 1,200 s depending on the individual peptide. Data were corrected for DMSO excluded volume effects. All data were double-referenced with respect to blank injections and reference surfaces using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Where necessary, data were fitted using a simple 1:1 binding model allowing for mass transfer effects.

[0301] Certain peptide ligands of the present invention were tested in the Nectin-4 binding assay described above and the results are shown in Table 1.

[0302] [Table 2]

[0303] Certain bicyclic peptides of the invention were tested in the SPR assay described above and the results are shown in Table 2.

[0304] [Table 3]

[0305] Certain bicyclic peptides of the invention were conjugated to cytotoxic agents and tested in the SPR assay described above, and the results are shown in Table 3.

[0306] [Table 4]

[0307] In vivo studies In Examples 1 to 5 and 9, the following methodology was used for each study: Test and positive control articles

[0308] [Table 5]

[0309] Experimental Methods and Procedures (i) Observation All procedures related to the handling, care and treatment of animals in the study were performed in accordance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) and approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec. At the designated monitoring time points, animals were checked daily for normal behavior, such as movement, food and water consumption (by appearance only), weight gain / loss, tumor growth and any effects of treatment on eye / hair mattness, and other abnormal effects as specified in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0310] (ii) Tumor Measurements and Endpoints The primary endpoint was to see whether tumor growth could be slowed or whether mice could be cured. Tumor volumes were measured three times a week in two dimensions using calipers and the volume was calculated according to the formula: V = 0.5a × b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 The tumor size was then used to calculate the T / C value. The T / C value (percent) is an index of antitumor efficacy; T and C are the mean volumes of the treatment and control groups, respectively, on a given day. TGI was calculated using the formula: TGI(%)=[1-(T i -T0) / (V i -V0)] × 100 for each group; T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the day treatment began, and V i is T i V0 was the mean tumor volume of the vehicle control group on the same day as treatment initiation, and V1 was the mean tumor volume of the vehicle group on the day treatment initiation was initiated.

[0311] (iii) Statistical analysis Summary statistics, including the mean and standard error of the mean (SEM), are provided for the tumor volume of each group at each time point.

[0312] Statistical analysis of the differences in tumor volume between groups was performed on the data obtained at the best treatment time point after the final dosing.

[0313] One-way ANOVA was performed to compare the tumor volumes between groups, and a significant F statistic (the ratio of treatment variance to error variance) was obtained. Comparisons between groups were made using the Games-Howell test. All data were analyzed using GraphPad Prism 5.0. P < 0.05 was considered statistically significant.

[0314] [Example 1] <In Vivo Efficacy Study of BCY8245 in the Treatment of NCI-H292 Xenografts (Non-Small Cell Lung Cancer (NSCLC) Model) in Balb / c Nude Mice> 1. Research Objective The purpose of the study was to evaluate the in vivo antitumor efficacy of BCY8245 in the treatment of a NCI-H292 xenograft model in Balb / c nude mice.

[0315] 2. Experimental Design

[0316]

Table 6

[0317] 3.Material 3.1 Animals and laboratory conditions 3.1.1.Animals Species: Mouse (Mus Musculus) Strain: Balb / c Nude Age: 6 - 8 weeks Sex: Female Weight: 18 - 22 g Number of animals: 18 mice + spares for BCY8245 Animal supplier:Shanghai LC Laboratory Animal Co.,LTD

[0318] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding. 3.2 Test and positive control articles

[0319] 4. Experimental methods and procedures 4.1 Cell culture NCI-H292 tumor cells will be maintained in medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells will be routinely passaged twice weekly. Exponentially growing cells will be harvested, counted, and tumors inoculated.

[0320] 4.2 Tumor inoculation Each mouse was inoculated with NCI-H292 tumor cells (10 × 10 6 ) was subcutaneously inoculated to induce tumors. The average tumor volume was approximately 158-406 mm. 3 Animals will be randomized and treatment will commence when the dose of test article is reached. The experimental design table below shows the number of animals in each group.

[0321] 4.3 Preparation of Test Article Formulations

[0322] [Table 7]

[0323] 4.4 Sample Collection At the end of the study, plasma was collected 5, 15, 30, 60 and 120 minutes after the last dose.

[0324] 5.Results 5.1 Tumor growth curves Tumor growth curves are shown in Figures 1 and 2.

[0325] 5.2 Tumor volume tracing The mean tumor volumes over time in female Balb / c nude mice bearing NCI-H292 xenografts are shown in the table below.

[0326] [Table 8]

[0327] [Table 9]

[0328] 5.3 Tumor growth inhibition assay Tumor growth inhibition rates for BCY8245 in the NCI-H292 xenograft model at day 14 were calculated based on tumor volume measurements.

[0329] [Table 10]

[0330] [Table 11]

[0331] 6. Summary and discussion of results In this study, the therapeutic efficacy of BCY8245 in the NCI-H292 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figures 1 and 2 and Tables 4 to 7.

[0332] The average tumor size of vehicle-treated mice reached 879 mm 3 on day 14.

[0333] BCY8245 at 1 mg / kg did not produce significant antitumor activity. The test article showed clear antitumor activity after increasing the dosage from 7 days to 3 mg / kg, but the efficacy was not further improved after increasing the dosage to 5 mg / kg on day 21. In this study, all treated animals showed continuous weight loss during the dosing schedule, which may be due to the systemic tumor tissue mass and the toxicity of the test article.

[0334] BCY8245 at 3 mg / kg once a week (TV = 149 mm 3 , TGI = 101.4%, p < 0.001), 3 mg / kg twice a week (TV = 65 mm 3 , TGI = 112.2%, p < 0.001) and 5 mg / kg once a week (TV = 83 mm 3 , TGI = 109.8%, p < 0.001) showed significant antitumor activity.

[0335] [Example 2] <In Vivo Efficacy Study of BCY7825, BCY8245, BCY8253, BCY8254 and BCY8255 in HT-1376 Xenograft (Bladder Cancer Model) of CB17-SCID Mice> 1. Research Objective The purpose of the study is to evaluate the in vivo antitumor efficacy of the test article in the treatment of HT-1376 xenograft in CB17-SCID mice.

[0336] 2. Experimental Design

[0337] [Table 12]

[0338] 3.Material 3.1 Animals and laboratory conditions 3.1.1.Animals Species: Mouse System: CB17-SCID Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 21-41 mice + spare Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[0339] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0340] 4. Experimental methods and procedures 4.1 Cell culture HT-1376 tumor cells were maintained in vitro as monolayer cultures in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice weekly by trypsin-EDTA treatment. Exponentially growing cells were harvested, counted, and tumors were inoculated.

[0341] 4.2 Tumor inoculation Each mouse was inoculated with HT-1376 tumor cells (5 × 10 6 ) was subcutaneously inoculated with Matrigel (1:1) to allow tumors to develop. The mean tumor volume was 153–164 mm 3 Animals were randomized when the dose of test article was reached. The administration of test article and the number of animals in each group are shown in the experimental design table.

[0342] 4.3 Preparation of Test Article Formulations

[0343] [Table 13]

[0344] 4.4 Sample Collection At the end of the study, plasma from group 2 was collected 5, 15, 30, 60, and 120 minutes after the last dose. Plasma from group 6 was collected 5, 15, 30, 60, and 120 minutes after the last dose. Tumors from group 6 were collected 2 hours after the last dose. Tumors from groups 4 and 5 were collected 2 hours after the last dose.

[0345] 5.Results 5.1 Tumor growth curves Tumor growth curves are shown in Figures 3 and 4.

[0346] 5.2 Tumor volume tracing The mean tumor volumes over time in female CB17-SCID mice bearing HT-1376 xenografts are shown in Tables 8 and 9.

[0347] [Table 14]

[0348] [Table 15]

[0349] 5.3 Tumor growth inhibition assay Tumor growth inhibition rates for the test articles in the HT-1376 xenograft model were calculated based on tumor volume measurements 21 days after the start of treatment.

[0350] [Table 16]

[0351] [Table 17]

[0352] 6. Summary and discussion of results Groups 1 and 2 In this study, the therapeutic efficacy of the test article was evaluated in the HT-1376 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 3 and Tables 8 and 10.

[0353] The average tumor size in vehicle-treated mice was 884 mm on day 21. 3 BCY8245 at 1 mg / kg showed modest antitumor activity, with better efficacy observed after increasing the dosage to 3 mg / kg from day 7 onwards.

[0354] In this study, some mice treated with 3 mg / kg of the test article exhibited weight loss of more than 10%.

[0355] Groups 3-6 In this study, the therapeutic efficacy of the test article in the HT-1376 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figure 4 and Tables 9 and 11.

[0356] BCY8245 at 3 mg / kg once a week (TV = 603 mm 3 , TGI = 50.9%, p < 0.01), 3 mg / kg twice a week (TV = 407 mm 3 , TGI = 72.3%, p < 0.001) and 5 mg / kg once a week (TV = 465 mm 3 , TGI = 66.0%, p < 0.001) showed significant antitumor activity.

[0357] In this study, BCY8245 at 5 mg / kg once a week resulted in weight loss of more than 10% of the animals during the treatment schedule.

[0358] [Example 3] <In Vivo Efficacy Study of BCY8245 in the Treatment of Panc2.13 Xenograft (Pancreatic Cancer Model) in Balb / c Nude Mice> 1. Research Objective The purpose of the study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of Panc2.13 xenograft in Balb / c nude mice.

[0359] 2. Experimental Design

[0360]

Table 18

[0361] 3.Material 3.1 Animals and laboratory conditions 3.1.1 Animals Species: Mouse Strain: Balb / c Nude Age: 6 - 8 weeks Sex: Female Body weight: 18 - 22 g Number of animals: 41 mice + spare Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[0362] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0363] 4. Experimental methods and procedures 4.1 Cell culture Panc2.13 tumor cells will be maintained in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units / ml human recombinant insulin in a 5% CO2 atmosphere at 37°C. Tumor cells will be routinely passaged twice weekly. Exponentially growing cells will be harvested, counted, and tumors inoculated.

[0364] 4.2 Tumor inoculation Each mouse was inoculated with Panc2.13 tumor cells (5 × 10 6 ) were inoculated subcutaneously with Matrigel (1:1) in 0.2 ml of PBS to allow tumors to develop. The mean tumor volume was 149 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0365] 4.3 Preparation of Test Article Formulations

[0366] [Table 19]

[0367] 4.4 Sample Collection At the end of the study, tumors in all groups were harvested 2 hours after the last dose.

[0368] 5.Results 5.1 Tumor growth curves The tumor growth curves are shown in FIG.

[0369] 5.2 Tumor volume tracing The mean tumor volumes over time in female Balb / c nude mice bearing Panc2.13 xenografts are shown in Table 12.

[0370] [Table 20]

[0371] 5.3 Tumor growth inhibition assay Tumor growth inhibition rates for the test articles in the Panc2.13 xenograft model were calculated based on tumor volume measurements 14 days after treatment initiation.

[0372] [Table 21]

[0373] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article was evaluated in the Panc2.13 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 5 and Tables 12 and 13.

[0374] 3 mg / kg, once a week (TV=271 mm3 , TGI = 69.2%, p < 0.01), 3 mg / kg, twice a week (TV = 231 mm 3 , TGI = 79.1%, p < 0.001) and 5 mg / kg, once a week (TV = 238 mm 3 , TGI = 77.5%, p < 0.001) of BCY8245 showed significant antitumor activity.

[0375] [Example 4] [In Vivo Efficacy Study of BCY8245 in the Treatment of MDA-MB-468 Xenografts (Breast Cancer Model) in Balb / c Nude Mice] 1. Research Objective The purpose of the study was to evaluate the in vivo antitumor efficacy of BCY8245 in the treatment of MDA-MB-468 xenografts in Balb / c nude mice.

[0376] 2. Experimental Design

[0377]

Table 22

[0378] 3.Material 3.1 Animals and laboratory conditions 3.1.1 Animals Species: Mouse Strain: Balb / c nude Age: 6 - 8 weeks Gender: Female Weight: 18 - 22 g Number of animals: 41 mice + spares Animal supplier: Shanghai LC Laboratory Animal Co., LTD.

[0379] 3.1.2.Indoor conditions The mice were housed three or five animals per cage and maintained in individual ventilated cages at a constant temperature and humidity. · Temperature: 20 - 26 °C · Humidity: 40 - 70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0380] 4. Experimental methods and procedures 4.1 Cell culture Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week. Exponentially growing cells were harvested, counted, and inoculated with tumors.

[0381] 4.2 Tumor inoculation Each mouse was inoculated with MDA-MB-468 tumor cells (10 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 196 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0382] 4.3 Preparation of Test Article Formulations

[0383] [Table 23]

[0384] 4.4 Sample Collection On day 21 of the study, plasma from group 2 was harvested 5, 15, 30, 60 and 120 minutes after the last dose. Tumors from groups 1 and 3 were harvested 2 hours after the last dose. Animals from group 4 were kept running for an additional 21 days without any doses, and tumors from these groups were harvested on day 42.

[0385] 5.Results 5.1 Tumor growth curves The tumor growth curves are shown in FIG.

[0386] 5.2 Tumor volume tracing The mean tumor volumes over time in female Balb / c nude mice bearing MDA-MB-468 xenografts are shown in Tables 14 and 15.

[0387] [Table 24]

[0388] [Table 25]

[0389] 5.3 Tumor growth inhibition assay Tumor growth inhibition rates for the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements 21 days after treatment initiation.

[0390] [Table 26]

[0391] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 6 and Tables 14 to 16.

[0392] 3mg / kg, once a week (TV=85mm 3, TGI = 144.2%, p < 0.001), 3 mg / kg, twice a week (TV = 22 mm 3 , TGI = 169.8%, p < 0.001) and 5 mg / kg, once a week (TV = 29 mm 3 , TGI = 168.4%, p < 0.001) of BCY8245 showed significant antitumor activity in a dose or dose - frequency dependent manner.

[0393] The 5 mg / kg dosing group was discontinued on day 21, and the tumors showed no recurrence during the additional 3 - week monitoring schedule.

[0394] [Example 5] <In - vivo Efficacy Study of BCY8549 in the Treatment of NCI - H292 Xenografts (Non - small Cell Lung Cancer (NSCLC) Model) in Balb / c Nude Mice> 1. Research Objective The purpose of the study was to evaluate the in - vivo antitumor efficacy of BCY8549 in the treatment of NCI - H292 xenografts in Balb / c nude mice.

[0395] 2. Experimental Design

[0396]

Table 27

[0397] 3.Material 3.1 Animals and laboratory conditions 3.1.1.Animals Species: Japanese white mouse Strain: Balb / c nude Age: 6 - 8 weeks Gender: Female Body weight: 18 - 22 g Number of animals: 43 mice + spares Animal supplier: Shanghai Lingchang Biotechnology Experimental Animal Co., Ltd 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 4 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0398] 4. Experimental methods and procedures 4.1 Cell culture NCI-H292 tumor cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells were subcultured twice a week by trypsin-EDTA treatment. Exponentially growing cells were harvested, counted, and tumors were inoculated.

[0399] 4.2 Tumor inoculation Each mouse was inoculated with NCI-H292 tumor cells (10 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 168 mm 3 43 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0400] Preparation of test article formulations

[0401] [Table 28]

[0402] 4.4 Sample Collection At the end of the study, plasma from the two groups of mice was collected 5 min, 15 min, 30 min, 1 h, and 2 h after the last dose.

[0403] 5.Results 5.1 Tumor growth curves The tumor growth curves are shown in FIG.

[0404] 5.2 Tumor volume tracing The mean tumor volumes over time in female Balb / c nude mice bearing NCI-H292 xenografts are shown in Table 17.

[0405] [Table 29]

[0406] 5.3 Tumor growth inhibition assay Tumor growth inhibition rates for the test articles in the NCI-H292 xenograft model were calculated based on tumor volume measurements 14 days after treatment initiation.

[0407] [Table 30]

[0408] 6. Summary and discussion of results In this study, the therapeutic efficacy of BCY was evaluated in the NCI-H292 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 7 and Tables 17 and 18.

[0409] The average tumor size in vehicle-treated mice was 843 mm on day 14. 3 BCY8549 at 3 mg / kg showed significant antitumor activity. All mice in this study maintained their body weight well.

[0410] [Example 6] <Investigation of the association between copy number variation (CNV) and gene expression of Nectin-4 from multiple tumor types> <Method> 1. Select all studies in cBioPortal (http: / / www.cbioportal.org / ) and search for Nectin-4. (a) Eliminate interim studies. (b) deselect studies with duplicate samples to prevent sample bias (based on cBioPortal warnings) – always keeping the PanCancer study where this is optional; (c) Select studies and conduct the analysis (Table 19).

[0411] [Table 31-1] [Table 31-2] [Table 31-3] [Table 31-4] 2. Export CNV and RNA expression data from cBioPortal. 3. Test whether the CNVs are statistically significantly associated with changes in mRNA expression for Nectin-4 (log2 not applied). (a) Non-parametric Kruskal-Wallis tests were performed in GraphPad Prism (7.04) and R / Rstudio (significance threshold: p<0.01). (i) GraphPad Prism: Set up column tables and perform non-parametric tests without matching or pairing, and do not assume Gaussian distribution. (ii) Packages used in R: 1.XLConnect 2. dplyr 3.Kruskal-Wallis Rank Sum Test:Kruskal.test 4. Adjust for multiple comparisons (including all possible comparisons even if n=1 within groups) in R / Rstudio using Dunn's test (significance threshold: p<0.025). (a) Dunn's test with multiple comparison method = "Bonferroni"

[0412] <Result> The results are shown in Table 20 below. Across 41 publicly available TCGA datasets reporting both tumor CNV and mRNA gene expression data for Nectin-4, there are many indications where cases have been reported with either copy number gain (2-3 copies) or amplification (>3 copies) of Nectin-4. Additionally, other cases have been documented to have shallow deletions (<2 copies), with rare reports of tumors containing greater than 1 copy loss or deeper deletions consistent with biallelic Nectin-4 loss. The indications where amplification was most frequently detected were breast cancer (10-22%), bladder cancer (20%), lung cancer (5-7%) and hepatocellular carcinoma (8%). The indications with the most frequent copy number loss were renal chromophobe (77%), renal clear cell carcinoma (RCC) (6.5%), sarcoma (10%), colon cancer (10%), head and neck cancer (7%) and lung squamous cell carcinoma. These data indicate that a variety of CNVs exist within and across tumor indications, with diversity in copy number patterns across different indications.

[0413] In addition to CNVs within the TCGA dataset, the median Nectin-4 mRNA expression level per indicator was approximately 2 10 As a result, given the range of Nectin-4 mRNA expression levels as well as the CNVs observed across and within tumor types, statistical tests were performed to identify potential associations between Nectin-4 mRNA levels and Nectin-4 CNVs within individual TCGA datasets / indications. Tumors per indication were assigned to one of five classes. a) deep deletion; b) shallow deletion; c) diploid; d) Increase; or e) Amplification A Kruskal-Wallis test was then performed to detect whether the distribution of mRNA expression values ​​per class differed between classes (P<0.01). For those TCGA datasets with P<0.01, to identify which classes were different from each other, a post-hoc test was performed by calculating Z-statistics with the adjusted P-values ​​calculated (Bonferroni). For ease of illustration, pairwise comparisons vs. diploid were reviewed per indicator (but all pairwise P-values ​​were calculated). 18 / 41 TCGA studies met Kruskal-Wallis P<0.01 and Bonferroni P<0.025 for the comparisons of gain vs. diploid and / or gain vs. diploid, indicating an association between increased nectin-4 mRNA expression and increased nectin-4 copy number. These 18 studies represent 14 independent tumor histologies: breast, uterine, bladder, lung adenocarcinoma, lung squamous, cervical, head and neck, pancreatic, thyroid, colorectal, thymoma, sarcoma, renal clear cell carcinoma (RCC), and stomach.

[0414] Furthermore, six studies found decreased mRNA expression associated with copy number loss. Four of these six studies: stomach, lung squamous epithelium, colon, and thyroid, not only showed an association between CNV loss and low expression, but also reported CNV gain associated with high expression.

[0415] However, only two indices, renal chromophobes and prostate cancer, reported an association between CNV loss and low transcript abundance, and there was another prostate cancer study (metastatic prostate cancer, SU2C / PCF Dream Team (Robinson et al., Cell 2015)) showing that copy number gain was associated with high expression (compared to diploidy).

[0416] These observations of tumor CNV loss and gain and mRNA expression levels may represent the mechanism behind Nectin-4 tumor protein expression in these indications where such associations were observed. There are indications where apparently CNV does not seem to affect mRNA expression levels in a predictable pattern, e.g., hepatocellular carcinoma. In vivo preclinical efficacy with certain Nectin-4 bicyclic drug conjugates of the present invention has been demonstrated to correlate with Nectin-4 protein expression as measured by IHC. Thus, if tumor Nectin-4 CNV is associated with mRNA levels and predicts protein expression levels, it may be formally possible that patients presenting with tumors containing increases (gains or amplifications) in copy number may be more likely to respond to the Nectin-4 bicyclic drug conjugates of the present invention. If patients can be identified with increased CNV in Nectin-4, then this information can be used to select patients and treat them with the Nectin-4 bicyclic drug conjugates of the present invention.

[0417] [Table 32-1] [Table 32-2] [Table 32-3] [Table 32-4] [Table 32-5] [Table 32-6] [Table 32-7] [Table 32-8] [Table 32-9] [Table 32-10] [Table 32-11] [Table 32-12] [Table 32-13]

[0418] [Example 7] <Expression analysis of Nectin-4 in six cell lines> 1. Research Objective The aim of the study was to evaluate the expression of Nectin-4 in six cell lines, including two breast cancer (T-47D, MDA-MB-468), three lung cancer (NCI-H292, NCI-H322, NCI-H526) and one fibrosarcoma (HT-1080) cell line, by flow cytometry.

[0419] 2. Panel design

[0420] Panels for FCM in T-47D, MDA-MB-468, NCI-H292, NCI-H322 and HT-1080 [Table 33]

[0421] Panel for NCI-H526 [Table 34]

[0422] 3.Material 3.1. Sample Cell Line List

[0423] [Table 35]

[0424] 3.2. Reagents Antibodies and kits for flow cytometry analysis

[0425] [Table 36] DPBS (Corning-21-031-CV) Staining buffer (eBioscience-00-4222) Fixation buffer (BD-554655)

[0426] 3.3.Equipment Eppendorf Centrifuge 5810R BD FACS Canto flow cytometer (BD)

[0427] 4. Experimental methods and procedures 4.1.Sample Collection Harvest cell lines growing in exponential growth phase. Count cells by hemocytometer using trypan blue staining. Centrifuge cells at 400×g for 5 min at 4° C., wash cells twice with staining buffer, suspend cells in staining buffer and count 1×10 7 / mL.

[0428] 4.2. Antibody staining 1) 100 μL of the cell suspension was dispensed into each well of a 96-well V-plate. 2) Isotype control or antibody was added to the suspension cells and incubated for 30 minutes at 4°C in the dark. 3) The cells were washed twice by centrifugation at 400 x g for 5 minutes at 4°C and the supernatant was discarded. 4) The cells were resuspended with 100 μL of fixation buffer and incubated in the dark at 4° C. for 30 minutes. 5) The cells were washed twice by centrifugation at 300 xg for 5 minutes at 4°C and the supernatant was removed. 6) The cells were resuspended in 400 μL of cell staining buffer. 7) FACS data was analyzed using FlowJo V10 software.

[0429] Data Analysis All FACS data was analyzed by Flowjo V10 software and GraphPad Prism or Excel software.

[0430] 5.Results 5.1. Panel Gating Strategy The gating strategy for nectin-4 is shown in Figures 8-11.

[0431] Data Analysis 5.2.1. Cell line viability The viability of the cell lines was as follows:

[0432] [Table 37]

[0433] 5.2.2. Positive expression of Nectin-4 in cell lines The positive expression and MFI of Nectin-4 in the six cell lines were as listed.

[0434] [Table 38]

[0435] 6. Discussion High expression of nectin-4 was observed in breast cancer T-47D (99.0%), MDA-MB-468 (99.0%), and lung cancer NCI-H292 (97.9%), NCI-H322 (99.1%). No expression of nectin-4 was observed in NCI-H526 and HT-1080.

[0436] [Example 8] <Flow cytometric analysis of Nectin-4 expression in nine CDX cell lines> 1. Research Objective The aim of the project was to evaluate the surface expression of nectin-4 (PVRL-4) in nine cell lines, including one breast cancer (MDA-MB-468), four lung cancer (NCI-H292, NCI-H358, NCI-H526, A549), one pancreatic cancer (Panc02.13), two colorectal cancer (HCT-116, HT-29) and one bladder cancer (HT1376) cell lines.

[0437] 2. Panel design Panel for FCM in 9 cell lines

[0438] [Table 39]

[0439] 3.Material 3.1 Sample Cell Line List

[0440] [Table 40]

[0441] 3.2. Reagents 1) DPBS (Corning, 21-031-CV) 2) 0.25% trypsin (Invitrogen-25200072) 3) Staining buffer (eBioscience, 00-4222) 4) Fixation buffer (BD, 554655) 5) Antibodies

[0442] [Table 41]

[0443] 3.3.Equipment Eppendorf Centrifuge 5810R BD FACS Canto flow cytometer (BD)

[0444] 4. Experimental methods and procedures 4.1 Cell culture Thawing cells 1) Wash the frozen vials with 70% alcohol and quickly thaw the vials in a 37°C water bath. 2) The cell suspension was centrifuged at approximately 1000 rpm for 5 minutes, the supernatant was removed, and pre-warmed medium was added to the flask. 3) The culture flask was incubated in a 5% CO2 incubator at 37°C.

[0445] Cell passaging 1) The medium and trypsin were warmed in a 37°C water bath. 2) The culture medium was removed and the cell layer was rinsed with DPBS. 3) 5 mL of 0.25% trypsin solution was added to the flask and the trypsin was diluted with 5 mL of medium. 4) The cell suspension was centrifuged at 1000 rpm for 5 minutes. 5) 15 mL of fresh medium was added and the cells were resuspended by gently pipetting. 6) The appropriate cell suspension was added to a new culture flask. 7) The culture flask was incubated in a 5% CO2 incubator at 37°C.

[0446] 4.2.Sample Collection Cell lines growing in exponential phase were harvested. Cells were counted using trypan blue staining. Cells were centrifuged at 400×g for 5 min at 4° C., cells were washed twice with staining buffer, and cells were diluted to 5×10 in staining buffer. 6 / mL.

[0447] 4.3. Antibody staining 100 μL of cell suspension was dispensed into each well of a 96-well V-plate. Isotype control or antibodies were added to the suspended cells and incubated for 30 min at 4°C in the dark. Cells were washed twice by centrifugation at 400×g for 5 min at 4°C and the supernatant was discarded. Cells were resuspended in 300 μL of staining buffer. FACS data was analyzed using Flow Jo V10 software.

[0448] Data Analysis All FACS data was analyzed by Flow Jo V10 software and GraphPad Prism or Excel software.

[0449] 5.Results 5.1. Panel Gating Strategy The gating strategy for nectin-4 is shown in Figures 12-16.

[0450] Data Analysis The positive expression and MFI of Nectin-4 in the nine cell lines were as listed.

[0451] [Table 42]

[0452] 6. Discussion High expression of Nectin-4 was observed in bladder cancer HT-1376 (92.4%), breast cancer MDA-MB-468 (97.1%) and lung cancer NCI-H358 (90.1%). Moderate expression of Nectin-4 was observed in HT-29 (40.0%), NCI-H292 (71.1%) and Panc02.13 (51.9%). No expression of Nectin-4 was observed in HCT-116, A549 and NCI-526. This data will be used to guide model selection for efficacy studies.

[0453] [Example 9] In Vivo Efficacy Study [Example 9.1] <In Vivo Efficacy Study of Test Article in the Treatment of A549 Xenograft in Balb / c Nude Mice> 1. Purpose of the Study The purpose of the study is to evaluate the in vivo antitumor efficacy of the test article in the treatment of A549 xenograft in Balb / c nude mice.

[0454] 2. Experimental Design

[0455] [Table 43]

[0456] 3.Material Animals and room conditions 3.1.1.Animals Species: Japanese white mouse Strain: Balb / c nude Age: 6 - 8 weeks Sex: Female Body weight: 18 - 22 g Number of animals: 41 mice + spares

[0457] 3.1.2.Indoor conditions The mice were housed in groups of 3 or 5 animals per cage and maintained in individual ventilated cages at a constant temperature and humidity. · Temperature: 20 - 26 °C · Humidity: 40 - 70% Cage: Made of polycarbonate. The size is 300 mm × 180 mm × 150 mm. The bedding material is corn cob, which is changed twice a week. Diet: The animals had free access to irradiated and sterilized dry pellet diet throughout the study period. Water: The animals had free access to sterilized drinking water. Cage identification: The identification labels for each cage contained information on the number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0458] 4. Experimental methods and procedures 4.1. Cell culture A549 tumor cells were maintained in vitro as monolayer cultures in F-12K medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Exponentially growing cells were harvested, counted, and inoculated into tumors.

[0459] Tumor inoculation Each mouse was inoculated with A549 tumor cells (5 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 158 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table. 4.3. Preparation of Test Article Formulations

[0460] [Table 44]

[0461] 4.4. Sample Collection At the end of the study, tumors in all groups were harvested 2 hours after the last dose.

[0462] 5.Results 5.1.Tumor growth curve Tumor growth curves are shown in FIG. 5.2.Tumor Volume Tracing The mean tumor volumes over time in female Balb / c nude mice bearing A549 xenografts are shown in Table 21.

[0463] [Table 45]

[0464] 5.3. Tumor Growth Inhibition Assay The tumor growth inhibition rate of the test article in the A549 xenograft model was calculated based on tumor volume measurement on the 14th day after the start of treatment.

[0465]

Table 46

[0466] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article in the A549 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figure 17 and Tables 21 and 22.

[0467] The average tumor size of vehicle-treated mice reached 568 mm 3 on day 14. BCY8245 at 3 mg / kg, once a week (TV = 356 mm 3 , TGI = 51.4%, p < 0.05), 3 mg / kg, twice a week (TV = 194 mm 3 , TGI = 90.8%, p < 0.01) and 5 mg / kg, once a week (TV = 228 mm 3 , TGI = 82.6%, p < 0.001) showed significant antitumor activity in a dose or dose-frequency dependent manner.

[0468] Animals in the BCY8245 group maintained their body weight well. In this cell line that shows minimal expression of nectin-4 in FACS studies, tumor growth is suppressed by BCY8245, but the tumors do not regress, highlighting the need for target-directedness for optimal efficacy.

[0469] [Example 9.2] [In Vivo Efficacy Study of the Test Article in the Treatment of HCT116 Xenograft in Balb / c Nude Mice] 1. Research Objective The purpose of the study is to evaluate the in vivo antitumor efficacy of the test article in the treatment of HCT116 xenograft in Balb / c nude mice.

[0470] 2. Experimental Design

[0471] [Table 47]

[0472] 3.Material 3.1. Animals and room conditions 3.1.1.Animals Species: Mus musculus Type: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 41 mice + spare

[0473] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0474] 4. Experimental methods and procedures 4.1. Cell culture HCT116 cells were maintained in 10% heat-inactivated medium supplemented with fetal bovine serum at 37°C in a 5% CO2 atmosphere. Tumor cells were routinely passaged twice weekly. Exponentially growing cells were harvested, counted, and inoculated with tumors.

[0475] Tumor inoculation Each mouse was inoculated with HCT116 tumor cells (5.0 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 166 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0476] 4.3. Preparation of Test Article Formulations

[0477] [Table 48]

[0478] 4.4. Sample Collection At the end of the study on day 14, tumors from groups 1 and 2 were harvested for FFPE. For group 4, plasma was harvested 5, 15, 30, 60 and 120 minutes after dosing. Tumors were also harvested and stored at -80°C.

[0479] 5.Results 5.1.Tumor growth curve Tumor growth curves are shown in FIG.

[0480] 5.2.Tumor Volume Tracing The mean tumor volumes over time in female Balb / c nude mice bearing HCT116 xenografts are shown in Table 23.

[0481] [Table 49]

[0482] 5.3. Tumor Growth Inhibition Assay The tumor growth inhibition rate of the test article in the HCT116 xenograft model was calculated based on tumor volume measurement on the 14th day after the start of treatment.

[0483]

Table 50

[0484] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article in the HCT116 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figure 18 and Tables 23 and 24.

[0485] The average tumor size of vehicle-treated mice reached 769 mm 3 on the 14th day after the start of treatment. BCY8245 at 3 mg / kg, once a week (TV = 425 mm 3 , TGI = 57.1%, p < 0.001), 3 mg / kg, twice a week (TV = 197 mm 3 , TGI = 94.9%, p < 0.001) and 5 mg / kg, once a week (TV = 134 mm 3 , TGI = 105.2%, p < 0.001) showed significant antitumor activity in a dose or dose-frequency dependent manner.

[0486] In this study, the body weights of all animals in the group receiving 5 mg / kg once a week decreased by more than 10% on average.

[0487] In this cell line that shows minimal expression of nectin-4 in FACS studies, tumor growth is inhibited by BCY8245, but the tumors do not regress, highlighting the need for target-directedness for optimal efficacy.

[0488] [Example 9.3] <In Vivo Efficacy Study of the Test Article in the Treatment of HT-1376 Xenograft in CB17-SCID Mice> 1. Research Objective The objective of the study is to evaluate the in vivo antitumor efficacy of the test article in treating HT-1376 xenografts in CB17-SCID mice.

[0489] 2. Experimental Design

[0490] [Table 51]

[0491] 3.Material 3.1 Animals and laboratory conditions 3.1.1.Animals Species: Mouse System: CB17-SCID Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 41 mice + spare

[0492] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: animal number, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0493] 4. Experimental methods and procedures 4.1 Cell culture HT-1376 tumor cells will be maintained in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells will be routinely passaged twice weekly. Exponentially growing cells will be harvested, counted, and tumors inoculated.

[0494] 4.2 Tumor inoculation Each mouse was inoculated with HT-1376 tumor cells (5 × 10 6 ) was inoculated subcutaneously with Matrigel (1:1) to allow tumors to develop. The mean tumor volume was 153 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0495] 4.3. Preparation of Test Article Formulations

[0496] [Table 52]

[0497] 4.4. Sample Collection At the end of the study, plasma from group 4 was collected 5, 15, 30, 60 and 120 minutes after the last dose. Tumors from group 4 were collected 2 hours after the last dose. Tumors from groups 1, 2 and 3 were collected 2 hours after the last dose.

[0498] 5.Results 5.1.Tumor growth curve Tumor growth curves are shown in FIG.

[0499] 5.2.Tumor Volume Tracing The mean tumor volume over time in female CB17-SCID mice bearing HT-1376 xenografts is shown in Table 25.

[0500] [Table 53]

[0501] 5.3. Tumor Growth Inhibition Assay The tumor growth inhibition rate of the test article in the HT-1376 xenograft model was calculated based on tumor volume measurement on the 14th day after the start of treatment.

[0502]

Table 54

[0503] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article in the HT-1376 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figure 19 and Tables 25 and 26.

[0504] The average tumor size of vehicle-treated mice reached 1069 mm 3 on the 14th day. BCY8245 at 3 mg / kg, once a week (TV = 603 mm 3 , TGI = 50.9%, p < 0.01), 3 mg / kg, twice a week (TV = 407 mm 3 , TGI = 72.3%, p < 0.001) and 5 mg / kg, once a week (TV = 465 mm 3 , TGI = 66.0%, p < 0.001) showed significant antitumor activity. In this study, BCY8245 at 5 mg / kg, once a week resulted in more than 10% reduction in body weight of the animals during the treatment schedule.

[0505] [Example 9.4] [In Vivo Efficacy Study of the Test Article in the Treatment of MDA-MB-468 Xenograft in Balb / c Nude Mice] 1. Research Objective The purpose of the study is to evaluate the in vivo antitumor efficacy of the test article in the treatment of MDA-MB-468 xenograft in Balb / c nude mice.

[0506] 2. Experimental Design

[0507] [Table 55]

[0508] 3.Material 3.1. Animals and room conditions 3.1.1.Animals Species: Mouse Type: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 41 mice + spare

[0509] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: animal number, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0510] 4. Experimental methods and procedures 4.1. Cell culture Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week. Exponentially growing cells were harvested, counted, and inoculated with tumors.

[0511] Tumor inoculation Each mouse was inoculated with MDA-MB-468 tumor cells (10 × 10 6 ) was inoculated subcutaneously to induce tumors. The average tumor volume was 196 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0512] 4.3. Preparation of Test Article Formulations

[0513] [Table 56]

[0514] 4.4. Sample Collection On day 21 of the study, plasma from group 2 was harvested 5, 15, 30, 60 and 120 minutes after the last dose. Tumors from groups 1 and 3 were harvested 2 hours after the last dose. Animals from group 4 were kept running for an additional 21 days without any medication, and tumors from these groups were harvested on day 42.

[0515] 5.Results 5.1.Tumor growth curve The tumor growth curves are shown in FIG.

[0516] 5.2.Tumor Volume Tracing The mean tumor volumes over time in female Balb / c nude mice bearing MDA-MB-468 xenografts are shown in Tables 27 and 28.

[0517] 5.3. Tumor Growth Inhibition Assay Tumor growth inhibition rates for the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements 21 days after treatment initiation.

[0518] [Table 57]

[0519]

Table 58

[0520]

Table 59

[0521] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article in the MDA-MB-468 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figure 20 and Tables 27 to 29.

[0522] The average tumor size of vehicle-treated mice reached 447 mm 3 on day 21. BCY8245 at 3 mg / kg, once a week (TV = 85 mm 3 , TGI = 144.2%, p < 0.001), 3 mg / kg, twice a week (TV = 22 mm 3 , TGI = 169.8%, p < 0.001) and 5 mg / kg, once a week (TV = 29 mm 3 , TGI = 168.4%, p < 0.001) showed significant antitumor activity in a dose or dose-frequency dependent manner.

[0523] The 5 mg / kg dosing group was discontinued on day 21 and the tumors showed no recurrence during the additional 3-week monitoring schedule. In this cell line showing high expression of nectin-4 in FACS studies, BCY8245 brought about tumor regression, highlighting the optimal efficacy and target-directed characteristics.

[0524] [Example 9.5] <In Vivo Efficacy Study of Test Article in the Treatment of NCI-H292 Xenograft in Balb / c Nude Mice> 1. Research Objective The purpose of the study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of NCI-H292 xenograft in Balb / c nude mice.

[0525] 2. Experimental Design

[0526] [Table 60]

[0527] 3.Material 3.1. Animals and room conditions 3.1.1.Animals Species: Mus musculus Type: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 41 mice + spare

[0528] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0529] 4. Experimental methods and procedures 4.1. Cell culture NCI-H292 tumor cells were maintained in vitro as monolayer cultures in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells were routinely subcultured twice a week by trypsin-EDTA treatment. Exponentially growing cells were harvested, counted, and tumors were inoculated.

[0530] Tumor inoculation Each mouse was inoculated with NCI-H292 tumor cells (10 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 162 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0531] 4.3. Preparation of Test Article Formulations

[0532] [Table 61]

[0533] 4.4. Sample Collection At the end of the study, tumors in all groups were harvested 2 hours after the last dose.

[0534] 5.Results 5.1.Tumor growth curve The tumor growth curves are shown in FIG.

[0535] 5.2.Tumor Volume Tracing The mean tumor volume over time in female Balb / c nude mice bearing NCI-H292 xenografts is shown in Table 30.

[0536] [Table 62]

[0537] 5.3. Tumor Growth Inhibition Assay Tumor growth inhibition rates for the test articles in the NCI-H292 xenograft model were calculated based on tumor volume measurements 14 days after treatment initiation.

[0538] [Table 63]

[0539] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article was evaluated in the NCI-H292 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 21 and Tables 30 and 31.

[0540] The average tumor size in vehicle-treated mice was 948 mm on day 14. 3 3 mg / kg, once a week (TV=149 mm 3 , TGI=101.4%, p<0.001), 3mg / kg, twice weekly (TV=65mm 3 , TGI=112.2%, p<0.001) and 5 mg / kg, once a week (TV=83 mm 3 , TGI=109.8%, p<0.001) BCY8245 showed significant antitumor activity.

[0541] Test articles at 3 mg / kg once weekly, 3 mg / kg twice weekly and 5 mg / kg once weekly all demonstrated comparable antitumor activity, and efficacy did not improve further with increasing dosage or dosing frequency.

[0542] In this study, mice in all groups maintained their body weight well.

[0543] In this cell line, which shows high expression of Nectin-4 in FACS studies, BCY8245 caused tumor regression, highlighting its targeting properties for optimal efficacy.

[0544] [Example 9.6] <In Vivo Efficacy Study of Test Article in the Treatment of NCI-H526 Xenografts in Balb / c Nude Mice> 1. Research Objective The purpose of the study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of NCI-H526 xenografts in Balb / c nude mice.

[0545] 2. Experimental Design

[0546]

Table 64

[0547] 3.Material 3.1. Animals and room conditions 3.1.1.Animals Species: Japanese white mouse Strain: Balb / c nude Age: 6 - 8 weeks Sex: Female Body weight: 18 - 22 g Number of animals: 21 mice + spares

[0548] 3.1.2.Indoor conditions The mice were maintained in individual ventilated cages at a constant temperature and humidity, with 3 animals per cage. · Temperature: 20 - 26 °C · Humidity: 40 - 70% Cage: Made of polycarbonate. Size: 300 mm × 180 mm × 150 mm. Bedding material was corn cob, which was changed twice a week. Diet: The animals had free access to irradiated and sterilized dry pellet diet throughout the study period. Water: The animals had free access to sterilized drinking water. Cage identification: The identification label for each cage contained information on the number of animals, sex, strain, date of receipt, treatment, study number, group number, and start date of treatment. Animal identification: The animals were marked by attaching a code to the ear.

[0549] 4. Experimental methods and procedures 4.1. Cell culture NCI-H526 cells were maintained in medium supplemented with 10% heat-inactivated fetal bovine serum at 37° C. in a 5% CO2 atmosphere. Tumor cells were routinely passaged twice a week. Exponentially growing cells were harvested, counted, and inoculated with tumors.

[0550] Tumor inoculation Each mouse was inoculated with NCI-H526 tumor cells (5.0 × 10 6 ) was inoculated subcutaneously to induce tumors. The average tumor volume was 181 mm 3 21 animals were randomized when the number of animals reached 1. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0551] 4.3. Preparation of Test Article Formulations

[0552] [Table 65]

[0553] 4.4. Sample Collection At the end of the study on day 14, all tumors were harvested for FFPE.

[0554] 5.Results 5.1.Tumor growth curve The tumor growth curves are shown in FIG.

[0555] 5.2.Tumor Volume Tracing The mean tumor volume over time in female Balb / c nude mice bearing NCI-H526 xenografts is shown in Table 32.

[0556] [Table 66]

[0557] 5.3. Tumor Growth Inhibition Assay Tumor growth inhibition rates for the test articles in the NCI-H526 xenograft model were calculated based on tumor volume measurements 14 days after treatment initiation.

[0558] [Table 67]

[0559] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article was evaluated in the NCI-H526 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 22 and Tables 32 and 33.

[0560] The average tumor size in vehicle-treated mice was 1365 mm on day 14 after treatment initiation. 3 3 mg / kg, once a week (TV=1205 mm 3 , TGI=13.4%, p>0.05) and 3 mg / kg, twice weekly (TV=1109 mm 3 , TGI=21.6%, p>0.05) BCY8245 showed slight antitumor activity, at 5 mg / kg once a week (TV=476 mm 3 BCY8245 demonstrated significant antitumor activity (TGI=75.0%, p<0.01). In this study, BCY8245 at 5 mg / kg twice weekly resulted in greater than 10% animal weight loss. In this cell line, which shows minimal expression of Nectin-4 in FACS studies, tumor growth was inhibited by BCY8245 but tumors did not regress, highlighting the need for targeting for optimal efficacy.

[0561] [Example 9.7] <In vivo efficacy study of the test article in the treatment of Panc2.13 xenografted Balb / c in nude mice> 1. Research Objective The objective of the study is to evaluate the in vivo antitumor efficacy of the test article in treating Panc2.13 xenografts in Balb / c nude mice.

[0562] 2. Experimental Design

[0563] [Table 68]

[0564] 3.Material 3.1. Animals and room conditions 3.1.1.Animals Species: Mus musculus Type: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 41 mice + spare

[0565] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 3 or 5 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0566] 4. Experimental methods and procedures 4.1. Cell culture Panc2.13 tumor cells will be maintained in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units / ml human recombinant insulin in a 5% CO2 atmosphere at 37°C. Tumor cells will be routinely passaged twice weekly. Exponentially growing cells will be harvested, counted, and tumors inoculated.

[0567] Tumor inoculation Each mouse was inoculated with Panc2.13 tumor cells (5 × 10 6 ) was inoculated subcutaneously with Matrigel (1:1) to allow tumors to develop. The mean tumor volume was 149 mm 3 41 animals were randomized when the number of animals in each group was reached. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0568] 4.3. Preparation of Test Article Formulations

[0569] [Table 69]

[0570] 4.4. Sample Collection At the end of the study, tumors in all groups were harvested 2 hours after the last dose.

[0571] 5.Results 5.1.Tumor growth curve Tumor growth curves are shown in FIG.

[0572] 5.2.Tumor Volume Tracing The mean tumor volumes over time in female Balb / c nude mice bearing Panc2.13 xenografts are shown in Table 34.

[0573] [Table 70]

[0574] 5.3. Tumor Growth Inhibition Assay The tumor growth inhibition rate of the test article in the Panc2.13 xenograft model was calculated based on tumor volume measurement on the 14th day after the start of treatment.

[0575]

Table 71

[0576] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article in the Panc2.13 xenograft model was evaluated. The measured tumor volumes of all treatment groups at various time points are shown in Figure 23 and Tables 34 and 35.

[0577] The average tumor size of vehicle-treated mice reached 545 mm 3 on the 14th day. BCY8245 at 3 mg / kg, once a week (TV = 271 mm 3 , TGI = 69.2%, p < 0.01), 3 mg / kg, twice a week (TV = 231 mm 3 , TGI = 79.1%, p < 0.001) and 5 mg / kg, once a week (TV = 238 mm 3 , TGI = 77.5%, p < 0.001) showed significant antitumor activity. In this study, the body weights of all animals in the 5 mg / kg, once-a-week group decreased by more than 15% on average. In this cell line that shows only moderate expression of nectin-4 in FACS studies, tumor growth is inhibited by BCY8245, but the tumors did not regress.

[0578] [Example 9.8] [In Vivo Efficacy Study of the Test Article in the Treatment of MDA-MB-468 Xenografts in Balb / c Nude Mice] 1. Research Objective The purpose of the study was to evaluate the in vivo antitumor efficacy of BCY8245 and the combination of BCY8245 and BCY8234 in the treatment of MDA-MB-468 xenografts in Balb / c nude mice and to determine the role that target binding plays in achieving optimal efficacy.

[0579] 2. Experimental Design

[0580] [Table 72]

[0581] 3.Material 3.1. Animals and room conditions 3.1.1.Animals Species: Mus musculus Type: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18~22g Number of animals: 36 mice + spare

[0582] 3.1.2.Indoor conditions Mice were maintained in individual ventilated cages at constant temperature and humidity with 4 animals in each cage. ·Temperature: 20~26℃ ·Humidity 40~70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0583] 4. Experimental methods and procedures 4.1. Cell culture Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum in an atmosphere of 0% CO2 in air at 37°C. Tumor cells were routinely passaged twice weekly. Exponentially growing cells were harvested, counted, and inoculated with tumors.

[0584] Tumor inoculation Each mouse was inoculated with MDA-MB-468 tumor cells (10 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 186 mm 3 36 animals were randomized when the number of animals reached 1. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0585] 4.3. Preparation of Test Article Formulations

[0586] [Table 73]

[0587] 4.4. Sample Collection Tumors in groups 5 and 6 were harvested for FFPE on day 21 of the study. Tumors in group 3 were harvested for FFPE at the end of the study.

[0588] 5.Results 5.1.Tumor growth curve Tumor growth curves are shown in FIG.

[0589] 5.2.Tumor Volume Tracing The mean tumor volumes over time in female Balb / c nude mice bearing MDA-MB-468 xenografts are shown in Tables 36 to 38.

[0590] 5.3. Tumor Growth Inhibition Assay Tumor growth inhibition rates for the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements 21 days after treatment initiation.

[0591] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 24 and Tables 36 to 39.

[0592] The average tumor size in vehicle-treated mice was 420 mm on day 21. 3 1 mg / kg, once a week (TV=204 mm 3 , TGI=92.1%, p<0.001), 3mg / kg, once a week (TV=27mm 3 BCY8245 at 0.3 mg / kg once or twice weekly (TGI=164.9%, p<0.001) showed significant antitumor activity in a dose-dependent manner. BCY8245 at 0.3 mg / kg once or twice weekly did not show any antitumor activity.

[0593] BCY8245 at 1 mg / kg, once weekly and at 3 mg / kg, once weekly in combination with BCY8234 (the toxin-free cognate peptide) at 300 mg / kg, once weekly showed significant antitumor activity (TV=242 mm 3 , TGI=75.4%, p<0.01). When compared to BCY8245 alone, the antitumor activity of 3 mg / kg BCY8245 was antagonized by 300 mg / kg BCY8234 (p<0.001). This reduction in efficacy by the competing toxin-free peptide demonstrates the importance of target binding for optimal efficacy. During the following monitoring schedule, mice treated with 1 mg / kg BCY8245 once a week showed obvious tumor recurrence, whereas mice treated with 3 mg / kg BCY8245 once a week at the same time did not show any tumor recurrence.

[0594] [Table 74]

[0595] [Table 75]

[0596]

Table 76

[0597]

Table 77

[0598] [Example 9.9] <In Vivo Efficacy Study of Test Article in the Treatment of MDA-MB-468 Xenograft in Balb / c Nude Mice> 1. Research Objective The purpose of the study is to evaluate the in vivo antitumor efficacy of BCY8245 alone or in combination with BCY8234 in the treatment of MDA-MB-468 xenograft in Balb / c nude mice.

[0599] 2. Experimental Design

[0600]

Table 78

[0601] 3.Material 3.1 Animals and laboratory conditions 3.1.1.Animals Species: Mouse Strain: Balb / c Nude Age: 6 - 8 weeks Sex: Female Weight: 18 - 22 g Number of animals: 20 mice + spares

[0602] 3.1.2.Indoor conditions The mice were housed in groups of 5 animals per cage and maintained in individual ventilated cages at a constant temperature and humidity. · Temperature: 20 - 26°C · Humidity: 40 - 70% Cage: Made of polycarbonate, size 300 mm x 180 mm x 150 mm, bedding material is corn cob, which is changed twice a week. Diet: Animals had free access to radiation-sterilized dry granular diet throughout the study period. Water: Animals had free access to sterile drinking water. Cage Identification: An identification label for each cage contained the following information: number of animals, sex, strain, date of receipt, treatment, study number, group number, and treatment start date. Animal Identification: Animals were marked by ear coding.

[0603] 4. Experimental methods and procedures 4.1 Cell culture Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum in an atmosphere of 0% CO2 in air at 37°C. Tumor cells were routinely passaged twice weekly. Exponentially growing cells were harvested, counted, and inoculated with tumors.

[0604] Tumor inoculation Each mouse was inoculated with MDA-MB-468 tumor cells (10 × 10 6 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 464 mm 3 Twenty animals were randomized when the number of animals reached 10. The administration of test articles and the number of animals in each group are shown in the experimental design table.

[0605] 4.3. Preparation of Test Article Formulations

[0606] [Table 79]

[0607] 4.4. Sample Collection At the end of the study, tumors from the three groups were harvested for FFPE.

[0608] 5.Results 5.1.Tumor growth curve Tumor growth curves are shown in FIG.

[0609] 5.2.Tumor Volume Tracing The mean tumor volumes over time in female Balb / c nude mice bearing MDA-MB-468 xenografts are shown in Tables 40 to 42.

[0610] 5.3. Tumor Growth Inhibition Assay Tumor growth inhibition rates for the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements 28 days after treatment initiation.

[0611] 6. Summary and discussion of results In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. The measured tumor volumes of all treatment groups at various time points are shown in Figure 25 and Tables 40 to 43.

[0612] The initial tumor starting size was intentionally larger than previously used to determine whether BCY8245 demonstrated efficacy at this larger size. The mean tumor size in vehicle-treated mice was 773 mm at day 28. 3 1 mg / kg, once a week (TV=384 mm 3 , TGI=126.6%, p<0.001) and 3 mg / kg, once a week (TV=50 mm 3 BCY8245 (TGI=234.6%, p<0.001) showed significant antitumor activity in a dose-dependent manner on day 28. Among these, mice treated with BCY8245 at 3 mg / kg once a week showed some tumor recurrence after treatment was discontinued, and further dosing from day 76 onwards was nearly ineffective in tumor regression.

[0613] BCY8245 at 3 mg / kg, once weekly in combination with BCY8234 at 300 mg / kg, once weekly showed significant antitumor activity at day 28 (TV=55 mm 3, TGI=234.0%, p<0.001), and the tumor did not show any recurrence throughout the entire monitoring schedule.

[0614] Against PG-D28, mice in the vehicle group treated with 10 mg / kg Nectin-4ADC or 5 mg / kg BCY8245 and two groups treated with 5 mg / kg BCY8245 (BCY8245, 1 mpk, once a week) showed effective tumor regression after 3 weeks, and tumors showed regrowth in the following 4 weeks when the drug was subsequently discontinued.

[0615] BCY8245 is approximately 450mm 3 When administered to a group previously administered vehicle, tumors of approximately 770 mm 3 It was also possible to induce tumor regression in tumors with starting volumes of

[0616] [Table 80]

[0617] [Table 81]

[0618] [Table 82]

[0619] [Table 83]

[0620] Example 10: In vivo PK studies MDA-MB-468 xenograft animals were injected with 3 mg / kg BCY8245 (BT8009). At various time points, animals were euthanized and plasma and tumors were collected and flash frozen. Samples were analyzed for MMAE. Plasma levels of BT8009 (BCY8245) were from a previous PK study. MMAE concentrations in plasma, MMAE in tumors, and BT8009 in plasma are shown in Figure 33. MMAE is retained longer in tumors than in plasma, supporting the hypothesis that systemic exposure is significantly lower than tumor exposure.

[0621] [Example 11: HCS Assay] HCS assay was used in Nectin-4 BDC binding study. Cells were incubated with test drugs and then washed. Detection was by fluorescent antibody against MMAE. MDA-MB-468 cells showed moderate Nectin-4 expression, with good images of 20000 cells. NCI-H292 cells showed low expression in this assay, with poor detection of MMAE even at 20000 cells. HCS data for MDA-MB-468 cell line is shown in Figure 34 and Table 44.

[0622] [Table 84]

[0623] Nectin-4ADC and BCY8245 were retained on cells and colocalized with membrane staining. BCY8781 and MMAE showed minimal retention. The Kd of all compounds for MDA-MB-468 cell line was consistent with previous data. Nectin-4ADC showed detectable binding affinity for MDA-MB-468 cell line. BCY8425 showed an order of magnitude lower nanomolar affinity at Bmax than that for Nectin-4ADC. This reduced fluorescence intensity is attributed to the MMAE to drug ratio of 4 for Nectin-4ADC, whereas the MMAE to drug ratio of 1 for BCY8245. BCY8781 showed only very weak affinity for MDA-MB-468 cell line, whereas MMAE showed almost no detectable binding affinity for MDA-MB-468 cell line.

[0624] [Example 12] In vivo efficacy of BCY8245 in two PDX models of lung cancer the purpose The objective of this study was to evaluate the efficacy of BCY8245 in PDX models of squamous non-small cell lung cancer and adenocarcinoma (both non-small cell carcinoma types).

[0625] animal Species: Mouse Type: Balb / c nude Age: 6-8 weeks Gender: Female Weight: 18-22

[0626] Drugs in the study BCY8245 and Nectin-4ADC or BCY8781

[0627] Pre-study animals Each mouse was implanted with LU-01-0007 or LU-01-0412 tumor fragments (approximately 30 mm) in the right flank. 3 ) was inoculated subcutaneously to allow tumors to develop. The average tumor volume was 161 mm 3 (LU-01-0007) or 147mm 3Animals were randomized when all 14 patients (LU-01-0412) were reached.

[0628] In-life measurements and endpoints Animals were checked daily for normal behavior, including movement, food and water consumption (by appearance only), weight gain / loss, tumor growth and any effects of treatment on eye / hair mattness, and other abnormal effects as defined in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

[0629] The primary endpoint was to see whether tumor growth could be slowed or whether mice could be cured. Tumor volumes were measured three times a week in two dimensions using calipers and the volume was calculated according to the formula: V = 0.5a × b 2 where a and b are the long and short diameters of the tumor, respectively, in mm 3 The tumor size was then used to calculate the T / C value. The T / C value (percentage) is an index of antitumor efficacy; T and C are the mean volumes of the treatment and control groups, respectively, on a given day.

[0630] TGI is calculated using the formula: TGI(%)=[1-(T i -T0) / (V i -V0)] × 100 for each group; T i is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the day treatment began, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 was the mean tumor volume of the vehicle group on the day treatment began.

[0631] The results of these studies are shown in Figures 26 and 27.

[0632] Lu-01-0412 (Figure 26): BCY8245 showed dose-related efficacy in this PDX model, with tumor growth rate slowed at 1 mg / kg once a week, but tumors significantly regressed to baseline at 3 mg / kg once a week. After dosing was discontinued (day 21), 5 / 6 animals showed no tumor regrowth until day 105 after the start of the study. The one animal that showed regrowth responded to 3 mg / kg BCY8245 with a return of regression to baseline. Disease was unchanged for BCY8781 non-binding BDC at 3 mg / kg, and tumors grew rapidly at the same rate as the vehicle-treated group when dosing was discontinued, highlighting the increased efficacy that Nectin-4 binding confers on these agents. Large tumors (vehicle-treated group) regressed in response to a single dose of BCY8245 or BCY8781.

[0633] LU-01-0007 (Figure 27): BCY8245 produced dose-related efficacy, with stable disease at 1 mg / kg once weekly and complete regression at 3 mg / kg. Dosing had to be maintained until day 56 to achieve complete regression (when dosing was discontinued). Tumors did not re-grow beyond that in this group (the latter was maintained until day 126 after study initiation). Nectin-4 ADC produced comparable efficacy. The stable disease group at 1 mg / kg responded to increasing dosing (3 and 5 mg / kg), suggesting that low doses of BCY8245 do not result in the development of resistance.

[0634] [Example 13] In vivo evaluation of BCY8245 in low-passage PDX models of human breast, esophageal and bladder cancer in immunocompromised mice the purpose To evaluate the antitumor activity of bicyclic agents in low-passage Champions Tumor Graft models of human breast, esophageal, and bladder cancer in immunocompromised mice.

[0635] Test system Species:Mouse Lineage: Athymic nude-Foxn1nu (immunocompromised) Ingredients: Envigo: Indianapolis, Indiana Gender: Female Target age for initiation of medication: at least 6-8 weeks of age Target weight at start of dosing: at least 18 grams Acclimatization period: 3 days

[0636] Experimental design Pre-study animals: Livestock animals with tumors between 1.0 and 1.5 cm 3 When tumors reached a sufficient level of 0.01 mg / kg, they were harvested and re-implanted into pre-study animals. Pre-study animals will be unilaterally implanted in the left flank with tumor fragments harvested from livestock animals. Each animal will be implanted from a specific passage lot and recorded.

[0637] Study Animals: Pre-study tumor volumes are recorded for each experiment beginning 7 to 10 days after implantation. Tumors should reach a mean tumor volume of 150-300 mm. 3 If tumor volume reaches 100%, animals will be matched by tumor volume into treatment or control groups and used for dosing, starting dosing on day 0.

[0638] Drugs in the study BCY8245 and Nectin-4 antibody drug conjugates are compared with vehicle control. Standard treatment drug Docetaxel may be included. All drugs will be administered once a week by intravenous route, doses are shown on the graph.

[0639] In-life measurements Effective tumor volumes: Tumor volumes will be obtained twice weekly. Final tumor volumes will be obtained on the day the study reaches its endpoint. If possible, final tumor volumes will be obtained when animals are moribund.

[0640] Effective animal weight: Animals will be weighed twice weekly. Final weights will be taken on the day the study reaches its endpoint or, if possible, when animals are found to be moribund. Animals showing 10% or more weight loss compared to day 0 will be given DietGel® ad libitum. Any animal showing more than 20% net weight loss for a sustained 7-day period or mice showing more than 30% net weight loss compared to day 0 will be considered moribund and euthanized.

[0641] Data analysis Drug Toxicity: Beginning on day 0, animals will be observed daily and weighed twice weekly using a digital scale; data including individual and mean gram body weights (mean body weight ± SEM), mean percent body weight change (%vD0) relative to day 0 will be recorded for each group, and %vD0 will be plotted at the completion of the study. Animal mortality will be recorded daily and designated as drug-related (D), technical (T), tumor-related (B), or unknown (U) based on weight loss and gross observation; single agent or combination groups reporting a mean %vD0 of greater than 20% and / or a mortality rate of greater than 10% will be considered to have exceeded the maximum tolerated dose (MTD) of that treatment in the regimen evaluated. The maximum mean %vD0 (lowest body weight) for each treatment group will be reported at the completion of the study.

[0642] Drug efficacy Tumor growth inhibition - Beginning on day 0, tumor dimensions will be measured twice weekly by digital calipers and data including individual and mean estimated tumor volumes (mean TV±SEM) will be recorded for each group; tumor volumes will be calculated using formula (1): TV=Width2×Length×0.52. Upon completion of the study, percent tumor growth inhibition (%TGI) values ​​will be calculated and reported for each treatment group (T) versus control (C) using the initial (i) and final (f) tumor measurements by formula (2): %TGI=1-(Tf-Ti) / (Cf-Ci). Individual mice reporting tumor volumes less than or equal to 30% of the day 0 measurements for two consecutive measurements will be considered partial responders (PR). No palpable tumors (0.00 mm for two consecutive measurements) were observed. 3 ) Individual mice will be classified as complete responders (CR); CRs that persist until completion of the study will be considered tumor free survivors (TFS). Tumor doubling time (DT) will be determined for the vehicle treated group using the formula DT=(Df-Di)×log2 / (logTVf-logTVi), where D=days and TV=tumor volume. All data collected in this study will be electronically managed and stored in a redundant server system.

[0643] The results of these studies are shown in Figures 28 to 31.

[0644] BCY8245 was tested in four low passage PDX models representing bladder cancer (CTG-1771), estrogen and progesterone negative Her2 positive breast cancer (CTG-1171), triple negative breast cancer (CTG-1106) and esophageal cancer (CTG-0896). In all of these models, BCY8245 demonstrated excellent efficacy in causing tumor regression, with three of four showing complete regression to baseline. Efficacy was comparable to ADC in all cases and was superior or equivalent to docetaxel SOC. In all models, BCY8245 was better tolerated than docetaxel.

Claims

1. A peptide ligand specific for Nectin-4, comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a 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 being selected from 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA), and having the amino acid sequence: C ｉ P[1Nal][dD]C ｉｉ M[HArg]DWSTP[HyP]WC ｉｉｉ (SEQ ID NO: 1) (wherein 1NaI represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, and C ｉ , C ｉｉ and C ｉｉｉ represent the first, second and third cysteine ​​residues, respectively. or a pharma- ceutically acceptable salt thereof.

2. [B-Ala][Sar10]-(SEQ ID NO:1) (hereinafter referred to as BCY8234); Ac-[B-Ala][Sar ５ ] - (SEQ ID NO: 1) (hereinafter referred to as BCY8122); Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126); (SEQ ID NO:1) (hereinafter referred to as BCY8116); and Fluorescein-(SEQ ID NO:1) (hereinafter referred to as BCY8205) The peptide ligand of claim 1 , comprising an amino acid sequence selected from:

3. Ac-[B-Ala][Sar ５ ] - (SEQ ID NO: 1) (hereinafter referred to as BCY8122); Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126); and (SEQ ID NO: 1) (hereinafter referred to as BCY8116) 3. The peptide ligand of claim 1 or 2, comprising an amino acid sequence selected from:

4. Ac-[B-Ala][Sar ５ ] - (SEQ ID NO: 1) (hereinafter referred to as BCY8122); and Ac-(SEQ ID NO:1) (hereinafter referred to as BCY8126) The peptide ligand of any one of claims 1 to 3, comprising an amino acid sequence selected from:

5. [B-Ala][Sar10]-(SEQ ID NO:1) (hereinafter referred to as BCY8234) 3. The peptide ligand of claim 1 or 2, comprising an amino acid sequence selected from:

6. The peptide ligand of any one of claims 1 to 5, wherein the pharma- ceutically acceptable salt is selected from sodium, potassium, calcium and ammonium salts.

7. The peptide ligand according to any one of claims 1 to 6, wherein the nectin-4 is human nectin-4.

8. A drug conjugate comprising a peptide ligand according to any one of claims 1 to 7 conjugated to one or more effectors and / or functional groups, wherein the one or more effectors and / or functional groups are selected from the group consisting of radioactive chelators, chromophores, antibodies or antigen-binding fragments thereof and / or metal chelators.

9. The drug conjugate of claim 8 conjugated to one or more cytotoxic agents.

10. 10. The drug conjugate of claim 9, wherein the cytotoxic agent is selected from MMAE or DM1.

11. 11. The drug conjugate of claim 10, wherein the cytotoxic agent is MMAE and the conjugate further comprises a linker selected from -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, where PABC represents p-aminobenzylcarbamate.

12. the cytotoxic agent is DM1 and the conjugate is -SPDB-(SO ３ 11. The drug conjugate of claim 10, further comprising a linker which is N-succinimidyl 3-(2-pyridyldithio)propionate.

13. BCY8245: 【Chemistry 1】 or a pharma- ceutically acceptable salt thereof, or BCY8549: 【Chemistry 2】 or a pharma- ceutically acceptable salt thereof The drug conjugate according to any one of claims 8 to 12, selected from the group consisting of 14. The drug conjugate of claim 8, which is BCY8245 (BCY00008245) or a pharma- ceutically acceptable salt thereof, wherein BCY8245 (BCY00008245) is: 【Chemistry 3】 where BCY00008234 has the sequence: [B-Ala][Sar10]- (SEQ ID NO: 1) is a polypeptide of the formula SEQ ID NO:1 is: C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1) wherein C i , C ii and C iii represent the first, second and third cysteine ​​residues, respectively, and the polypeptide of SEQ ID NO: 1 is cyclized at C i , C ii and C iii with 1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triplop-2-en-1-one (TATA).

15. 【Chemical 4】 or a pharma- ceutically acceptable salt thereof.

16. A pharmaceutical composition comprising a peptide ligand according to any one of claims 1 to 7 or a drug conjugate according to any one of claims 8 to 15 in combination with one or more pharma- ceutically acceptable excipients.

17. 17. The pharmaceutical composition of claim 16, further comprising one or more therapeutic agents.

18. The drug conjugate according to any one of claims 8 to 15 for use in preventing, suppressing or treating a disease or disorder mediated by Nectin-4.

19. The drug conjugate of any one of claims 8 to 15 for use in preventing, suppressing or treating cancer in a patient identified as having increased copy number alterations (CNV) of Nectin-4.

20. 20. The drug conjugate of claim 19, wherein the cancer is selected from breast, uterine, bladder, lung adenocarcinoma, lung squamous cell, cervical, head and neck, pancreatic, thyroid, colorectal, thymoma, sarcoma, renal clear cell carcinoma, prostate and gastric cancer.

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