Bicyclic peptide ligands specific for binding tin-4

Abstract

The present invention relates to bicyclic peptide ligands specific for binding tin-4. In particular, the present invention relates to polypeptides covalently bound to molecular scaffolds, such that two or more peptide rings oppose each other between the attachment points of the scaffold. In particular, the present invention describes peptides that are high affinity binders of Bintin-4 (Nectin-4). The invention also encompasses 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 the prevention, inhibition or treatment of binding group-4 mediated diseases or conditions.

Description

[0001] This application is a divisional application of Chinese patent application 2019800539643 "Bicyclic peptide ligands specific for bindin-4" filed on June 21, 2019. Technical Field

[0002] The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide loops subtend between the attachment points of the scaffold. In particular, the present invention describes peptides that are high affinity binders of integrin-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 uses of the peptide ligands and drug conjugates in preventing, inhibiting or treating integrin-4 mediated diseases or conditions. Background Art

[0003] Cyclic peptides can bind to protein targets with high affinity and target specificity, and are therefore an attractive class of molecules for the development of therapeutics. In fact, several cyclic peptides have been successfully used clinically, such as the antimicrobial peptide vancomycin, the immunosuppressant drug cyclosporin, or the anticancer drug octreotide (Driggers et al. (2008), Nat Rev Drug Discov 7 (7), 608-24). The good binding properties come from the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles bind to surfaces of hundreds of square angstroms, such as the cyclic peptide CXCR4 antagonist CVX15 ( Wu et al. (2007), Science 330, 1066-71), Cyclic peptide with Arg-Gly-Asp motif that binds to integrin αVb3 (Xiong et al. (2002), Science 296(5565), 151-5), or the cyclic peptide inhibitor upain-1 ( Zhao et al. (2007), J Struct Biol 160(1), 1-10).

[0004] Due to their cyclic configuration, peptide macrocycles are less flexible than linear peptides, resulting in less entropic loss when binding to a target and resulting in higher binding affinity. Reduced flexibility also results in locking of a target-specific conformation, increasing binding specificity compared to linear peptides. This effect has been exemplified by a potent, selective inhibitor of matrix metalloproteinase 8 (MMP-8) that loses selectivity for other MMPs when its ring is opened (Cherney et al. (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties obtained by macrocyclization are more pronounced in polycyclic peptides having more than one peptide ring, such as in vancomycin, nisin, and actinomycin.

[0005] Different research groups have previously attached peptides with cysteine ​​residues to synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al. (2005), ChemBioChem). Meloen and colleagues have used tri(bromomethyl)benzene and related molecules to rapidly and quantitatively cyclize multiple peptide rings to synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al. (2005), ChemBioChem). Methods for producing candidate drug compounds are disclosed, wherein the compounds are produced by attaching cysteine-containing peptides 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).

[0006] Combinatorial approaches based on phage display have been developed to generate and screen large libraries of bicyclic peptides against targets of interest (Heini et al. s et al. (2009), Nat Chem Biol 5(7), 502-7 and WO2009 / 098450). Briefly, a linear peptide containing three cysteine ​​residues and two regions of six random amino acids (Cys-(Xaa) 6 -Cys-(Xaa) 6 -Cys) and cyclized by covalently linking the cysteine ​​side chain to a small molecule scaffold. Summary of the invention

[0007] According to a first aspect of the present invention, a peptide ligand specific for nectin-4 is provided, comprising a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two loop sequences, and the molecular scaffold forms a covalent bond with the cysteine ​​residues of the polypeptide, so that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises the amino acid sequence:

[0008] C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO: 1),

[0009] Among them, 1Nal 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 pharmaceutically acceptable salt thereof.

[0010] According to another aspect of the present invention, there is provided a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0011] According to another aspect of the present invention, there is provided a pharmaceutical composition comprising a peptide ligand or drug conjugate as defined herein in combination with one or more pharmaceutically acceptable excipients.

[0012] According to another aspect of the present invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, inhibiting or treating a disease or condition mediated by Integrin-4. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Where present in the figures, error bars represent the standard error of the mean (SEM).

[0014] Figure 1 and Figure 2 : Tumor volume trajectory after administration of BCY8245 to female BALB / c nude mice bearing NCI-H292 xenografts.

[0015] Figure 3 and Figure 4 : Tumor volume trajectory following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts.

[0016] Figure 5 : Tumor volume trajectory after administration of BCY8245 to female Balb / c nude mice bearing Panc2.13 xenografts.

[0017] Figure 6 : Tumor volume trajectory following administration of BCY8245 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0018] Figure 7 : Tumor volume trajectory after administration of BCY8549 (with BCY8245 as control) to female BALB / c nude mice bearing NCI-H292 xenografts.

[0019] Figure 8 : Gating strategy for integrin-4 in mammary gland (T-47D and MDA-MB-468).

[0020] Fig. 9 : Gating strategy for henectin-4 in NCI-H292 and NCI-H322.

[0021] Fig.10 and Fig.11 : Gating strategies for integrin-4 in NCI-H526 and HT1080, respectively.

[0022] Figures 12 to 16 : respectively in bladder cancer (HT1376; Fig.12 ), breast cancer (MDA-MB-468; Fig.13 ), colorectal cancer (HT-29; Fig.14A and HCT-116; Fig. 14B ), lung cancer (A549; Fig.15A , NCI-H292; Fig. 15B , NCI-H358; Fig. 15C and NCI-526; Fig.15D ), and pancreatic cancer (Panc02.13; Fig.16 ) in the gating strategy of nexin-4.

[0023] Fig.17 : Tumor volume trajectory after administration of BCY8245 to female Balb / c nude mice bearing A549 xenografts.

[0024] Fig.18 : Tumor volume trajectory after administration of BCY8245 to female Balb / c nude mice bearing HCT116 xenografts.

[0025] Fig.19 : Tumor volume trajectory following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts.

[0026] Fig. 20: Tumor volume trajectory following administration of BCY8245 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0027] Fig.21 : Tumor volume trajectory following administration of BCY8245 to female Balb / c nude mice bearing NCI-H292 xenografts.

[0028] Fig. 22 : Tumor volume trajectory following administration of BCY8245 to female Balb / c nude mice bearing NCI-H526 xenografts.

[0029] Fig.23 : Tumor volume trajectory after administration of BCY8245 to female Balb / c nude mice bearing Panc2.13 xenografts.

[0030] Fig.24 : Tumor volume trajectory following administration of BCY8245 or the combination of BCY8245 and BCY8234 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0031] Fig.25 : Tumor volume trajectory following administration of BCY8245 alone or in combination with BCY8234 to female Balb / c nude mice bearing MDA-MB-468 xenografts.

[0032] Figure 26 to Figure 31 : Tumor volume trajectories in Lu-01-0412, LU-01-0007, CTG-1771, CTG-1171, CTG-1106, and CTG-0896 PDX xenografts.

[0033] Fig.32 : BT8009 (i.e. BCY8245) efficacy correlates with expression in CDX / PDX xenografts. Xenografts with little / no expression of integrin-4 showed reduced tumor growth rate. Xenografts expressing integrin-4 showed tumor regression. This analysis included both PDX and CDX models, collating values ​​from various reports.

[0034] Fig.33 : MDA-MB-468 cells express integrin-4 and show prolonged retention of MMAE in tumors.

[0035] Fig.34 : HCS-data analysis on MDA-MB-468 cell line. DETAILED DESCRIPTION

[0036] In one embodiment, C iP[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[0037] [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234);

[0038] Ac-[B-Ala][Sar 5 ]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);

[0039] Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126);

[0040] (SEQ ID NO: 1) (hereinafter referred to as BCY8116);

[0041] Fluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205); and

[0042] [PYA][B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8846).

[0043] In another embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[0044] [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234);

[0045] Ac-[B-Ala][Sar 5 ]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);

[0046] Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126);

[0047] (SEQ ID NO: 1) (hereinafter referred to as BCY8116); and

[0048] Fluorescein-(SEQ ID NO: 1) (hereinafter referred to as BCY8205).

[0049] In yet another embodiment, C i P[1Nal][dD]Cii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[0050] Ac-[B-Ala][Sar 5 ]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122);

[0051] Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126); and

[0052] (SEQ ID NO: 1) (hereinafter referred to as BCY8116).

[0053] The data are presented herein in Table 2, which demonstrates that the peptide ligands of this embodiment exhibit excellent levels of binding to human Bindex-4 as demonstrated by SPR binding data.

[0054] In yet another embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[0055] Ac-[B-Ala][Sar 5 ]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122); and

[0056] Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126).

[0057] The data are shown herein in Table 1, which demonstrates that the peptide ligands of this embodiment exhibit excellent levels of binding to human Bindex-4 as demonstrated by competition binding data.

[0058] In yet another embodiment, C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii The peptide ligand of (SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[0059] [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234).

[0060] The data are presented herein in Table 3, which demonstrates that the peptide ligands of this embodiment, when conjugated to a cytotoxic agent, exhibit excellent levels of binding to human Bindex-4 (<10 nM), as demonstrated by SPR binding data.

[0061] In one embodiment, the molecular scaffold is 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, e.g., in the fields of peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used for molecular biology, genetic, and biochemical methods (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., Short Protocols in Molecular Biology (1999) 4th ed., John Wiley & Sons, Inc.), which are incorporated herein by reference.

[0063] Nomenclature

[0064] serial number

[0065] When referring to amino acid residue positions within the peptides of the invention, cysteine ​​residues are omitted from the numbering since they are invariant (C i , C ii and C iii ), therefore, the numbering of the amino acid residues in the peptides of the present invention is as follows:

[0066] C i -P 1 -[1Nal] 2 -[dD] 3 -C ii -M 4 -[HArg] 5 -D 6 -W 7 -S 8 -T 9 -P 10 -[HyP] 11 -W 12 -C iii (SEQ ID NO: 1).

[0067] 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)triprop-2-en-1-one (TATA) to produce a trisubstituted structure. Cyclization with TATA occurs at C i , C ii and C iii superior.

[0068] Molecular form

[0069] The N- or C-terminal extension of the bicyclic core sequence is added to the left or right side of the sequence and separated by a hyphen. For example, N-terminal (β-Ala)-Sar 10 The -Ala tail will be represented as:

[0070] βAla-Sar10-A-(SEQ ID NO:X).

[0071] Reverse peptide sequence

[0072] According to Nair et al. (2003) J Immunol 170 (3), 1362-1373, it is envisaged that the peptide sequences disclosed herein will also be used in their inverse form. For example, the order is reversed (i.e., the N-terminus becomes the C-terminus and vice versa) and their stereochemistry is also inverted (i.e., D-amino acids become L-amino acids and vice versa).

[0073] Peptide ligand

[0074] The peptide ligand referred to herein refers to a peptide covalently bound to a molecular scaffold. Typically, such peptides contain two or more reactive groups (i.e., cysteine ​​residues) capable of forming a covalent bond with the scaffold, and a sequence (referred to as a loop sequence) relative to the reactive groups, which is referred to as a loop sequence because when the peptide is bound to the scaffold, it forms a ring. In the case of the present application, the peptide contains at least three cysteine ​​residues (referred to herein as C i , C ii and C iii ), which form at least two rings on the support.

[0075] Advantages of peptide ligands

[0076] Certain bicyclic peptides of the present invention have many favorable properties that make them considered as drug-like molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. These favorable properties include:

[0077] - Species cross-reactivity. This is a typical requirement for preclinical efficacy and pharmacokinetic evaluations;

[0078] - Protease stability. Bicyclic peptide ligands should ideally exhibit stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across species so that bicyclic lead candidates can be developed in animal models and reliably administered to humans;

[0079] - Ideal solubility profile. This is a function of the ratio of charged hydrophilic residues to hydrophobic residues and intra / inter-molecular H-bonds, which is important for formulation and absorption purposes;

[0080] - Optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides with short exposure in acute disease management settings, or to develop bicyclic peptides with enhanced retention in the circulation and therefore preferred for management of more chronic disease states. Other factors driving the desired plasma half-life are the sustained exposure required to achieve maximum therapeutic efficacy versus the toxicology associated with sustained exposure to the agent; and

[0081] - Selectivity. Certain peptide ligands of the present invention show good selectivity over other binding proteins.

[0082] Pharmaceutically acceptable salts

[0083] It will be understood that salt forms are within the scope of the present invention and reference to a peptide ligand includes salt forms of the ligand.

[0084] The salts of the present invention can be synthesized from parent compounds containing a base or acid moiety by conventional chemical methods, such as those described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (ed.), Camille G. Wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002. Typically, these salts can be prepared by reacting the free acid or base forms of these compounds with an appropriate base or acid in water or in an organic solvent or in a mixture of the two.

[0085] Acid addition salts (mono or di-salts) can be formed with various acids (inorganic and organic). Examples of acid addition salts include mono or di-salts formed with acids selected from the group consisting of 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, butyric acid, (+) camphoric acid, camphorsulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactonic 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 acid (such as hydrobromic acid, hydrochloric acid, hydroiodic acid), isethionic acid, lactic acid (such as (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyruvic acid, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, thiocyanic acid, p-toluenesulfonic acid, undecylenic acid and valeric acid, as well as acylated amino acids and cation exchange resins.

[0086] One group of specific salts includes salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One specific salt is the hydrochloride. Another specific salt is the acetate.

[0087] If the compound is anionic or has a functional group that can be anionic (e.g., -COOH can be -COO - ), then a salt may be formed with an organic or inorganic base that generates an appropriate cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li + 、Na + and K + , alkaline earth metal cations such as Ca 2+ and Mg 2+ , and other cations such as Al 3+ or Zn + Examples of suitable organic cations include, but are not limited to, ammonium ions (i.e., NH 4 + ) and substituted ammonium ions (e.g., NH 3 R+ NH 2 R 2 + 、NHR 3 + NR 4 + ). Some examples of suitable substituted ammonium ions are those derived from methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids such as lysine and arginine. Examples of common quaternary ammonium ions are N(CH 3 ) 4 + .

[0088] When the peptides of the invention contain amine functional groups, they can form quaternary ammonium salts, for example by reaction with an alkylating agent according to methods well known to those skilled in the art. Such quaternary ammonium compounds are within the scope of the present invention.

[0089] Modified derivatives

[0090] It will be appreciated that modified derivatives of the peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include one or more modifications selected from the following: N-terminal and / or C-terminal modification; 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, and replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; modification of the peptide backbone length; replacement of hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of amino acids such as cysteine, lysine, glutamate / aspartate and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introduction or replacement of amino acids to introduce orthogonal reactivity suitable for functionalization, such as amino acids carrying azide or alkyne groups allow functionalization with alkyne or azide carrying moieties, respectively.

[0091] In one embodiment, the modified derivative comprises an N-terminal and / or C-terminal modification. In a further embodiment, wherein the modified derivative comprises an N-terminal modification using a suitable amino-reactive chemistry, and / or a C-terminal modification using a suitable carboxyl-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.

[0092] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In this embodiment, during peptide synthesis, the N-terminal cysteine ​​group (which group is referred to herein as C i ) is capped with acetic anhydride or other suitable reagent to produce an N-terminally acetylated molecule. This embodiment provides the advantage of removing a potential recognition site for aminopeptidases and avoiding the possibility of degradation of the bicyclic peptide.

[0093] In an alternative embodiment, the N-terminal modification comprises the addition of a molecular spacer group that facilitates the conjugation of the effector group and maintains the potency of the bicyclic peptide to its target.

[0094] 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 (which group is referred to herein as C iii ) is synthesized as an amide during peptide synthesis, resulting in a C-terminally amidated molecule. This embodiment provides the advantage of removing a potential recognition site for carboxypeptidases and reducing the likelihood of proteolytic degradation of the bicyclic peptide.

[0095] In one embodiment, the modified derivative comprises replacing one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, non-natural amino acids can be selected that have isosteric / isoelectronic side chains that are not recognized by degradative proteases and do not produce any side effects on target efficacy.

[0096] Alternatively, unnatural amino acids with constrained amino acid side chains can be used so that proteolysis of adjacent peptide bonds is conformationally and sterically hindered. Specifically, these relate to proline analogs, bulky side chains, Cα-disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cyclic amino acids, simple derivatives of amino-cyclopropylcarboxylic acids.

[0097] 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 ​​(C i) and / or C-terminal cysteine ​​(C iii )Add a spacer group.

[0098] In one embodiment, the modified derivative comprises the replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues.

[0099] In one embodiment, the derivative of modification comprises replacing one or more charged amino acid residues with one or more hydrophobic amino acid residues. In an alternative embodiment, the derivative of modification comprises replacing one or more hydrophobic amino acid residues with one or more charged amino acid residues. The correct balance of charged amino acid residues and hydrophobic amino acid residues is an important feature of bicyclic peptide ligands. For example, hydrophobic amino acid residues affect the degree of plasma protein binding, therefore affecting the concentration of available free fractions in blood plasma, and charged amino acid residues (specifically arginine) can affect the interaction of peptide with the phospholipid membrane on the cell surface. Both combinations can affect the half-life, distribution volume and exposure of peptide drugs, and can be adjusted according to clinical endpoints. In addition, the correct combination and quantity of charged amino acid residues and hydrophobic amino acid residues can reduce the stimulation of injection site (if peptide drugs have been subcutaneously administered).

[0100] In one embodiment, the modified derivative comprises replacing one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and by the tendency of D-amino acids to stabilize the β-turn conformation (Tugyi et al. (2005) PNAS, 102(2), 413-418).

[0101] In one embodiment, the modified derivatives include removal of any amino acid residue and substitution with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

[0102] It should be noted that each of the modifications mentioned above is used to intentionally improve the potency or stability of the peptide. Further potency improvements based on modifications can be achieved through the following mechanisms:

[0103] - Incorporation of hydrophobic moieties that exhibit a hydrophobic effect and lead to a reduced off-rate, thereby achieving higher affinity;

[0104] - incorporation of charged groups that exhibit long-range ionic interactions, resulting in faster on-rates and higher affinities (see, e.g., Schreiber et al., Rapid, electrostatically assisted association of proteins (1996), Nature Struct. Biol. 3, 427-31); and

[0105] - Incorporating additional constraints into the peptide, for example by properly constraining the side chains of the amino acids to minimize entropy loss upon target binding; constraining the torsion angles of the backbone to minimize entropy loss upon target binding; and introducing additional cyclizations into the molecule for the same reason.

[0106] (For review, see Gentilucci et al., Curr. Pharmaceutical Design, (2010), 16, 3185-203 and Nestor et al., Curr. Medicinal Chem (2009), 16, 4399-418).

[0107] Isotope variation

[0108] The present invention includes all pharmaceutically acceptable (radio)isotope-labeled peptide ligands of the present invention, in which one or more atoms are replaced by atoms with the same atomic number, but the atomic mass or mass number is different from the atomic mass or mass number usually found in nature, and peptide ligands of the present invention, in which a metal chelating group (referred to as "effector") capable of accommodating the relevant (radio)isotope is attached, and certain functional groups in the peptide ligands of the present invention are covalently replaced by relevant (radio)isotopes or isotope-labeled functional groups.

[0109] Examples of isotopes suitable for inclusion in the peptide ligands of the present invention include isotopes of hydrogen, such as 2 H(D) and 3 H(T), isotopes of carbon, such as 11 C. 13 C and 14 C, isotopes of chlorine, such as 36 Cl, isotopes of fluorine, such as 18 F, isotopes of iodine, such as 123 I. 125 I and 131 I, isotopes of nitrogen, such as 13 N and 15 N, isotopes of oxygen, such as 15 O. 17 O and 18 O, isotopes of phosphorus, such as 32 P, isotopes of sulfur, such as 35 S, isotopes of copper, such as 64 Isotopes of Cu, gallium, such as 67 Ga or 68 Ga, an isotope of yttrium, such as 90 Y, and isotopes of lutetium, such as 177 Lu, and isotopes of bismuth, such as 213 Bi.

[0110] Certain isotopically labeled peptide ligands of the invention, such as those incorporating radioactive isotopes, can be used in drug and / or substrate tissue distribution studies and clinically assess the presence and / or absence of the integrin-4 target in diseased tissues. The peptide ligands of the invention can further have valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between labeled compounds and other molecules, peptides, proteins, enzymes, or receptors. Detection or identification methods can use compounds labeled with labeling agents such as radioactive isotopes, enzymes, fluorescent materials, luminescent materials (e.g., luminol, luminol derivatives, fluorescein, luminescent proteins, and luciferases), and the like. The radioactive isotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14 C is particularly useful for this purpose due to its ease of incorporation and convenient means of detection.

[0111] Using heavier isotopes such as deuterium (i.e. 2 Substitution of H(D)) may offer certain therapeutic advantages resulting from greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some circumstances.

[0112] Using positron emitting isotopes (such as 11 C. 18 F. 15 O and 13 N) substitutions can be used in positron emission tomography (PET) studies to examine target occupancy.

[0113] Isotopically-labeled compounds of the peptide ligands of the invention can generally be prepared by conventional techniques known to those skilled in the art or by processes analogous to those described in the accompanying examples, using an appropriate isotopically-labeled reagent in place of the non-labeled reagent previously employed.

[0114] Molecular scaffold

[0115] In one embodiment, the molecular scaffold comprises a non-aromatic molecular scaffold. "Non-aromatic molecular scaffold" referred to herein refers to any molecular scaffold as defined herein that does not contain an aromatic (ie, unsaturated) carbocyclic or heterocyclic ring system.

[0116] Examples of suitable non-aromatic molecular scaffolds are described in Heinis et al. (2014) Angewandte Chemie, International Edition 53(6) 1602-1606.

[0117] As described in previous documents, the molecular scaffold may be a small molecule, such as an organic small molecule.

[0118] In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0119] In one embodiment, the molecular scaffold comprises reactive groups capable of reacting with functional groups of the polypeptide to form covalent bonds.

[0120] The molecular scaffold may include chemical groups that form linkages with the peptide, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0121] An example of a compound containing an αβ unsaturated carbonyl group 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).

[0122] Effectors and functional groups

[0123] According to a further aspect of the invention, the invention provides a drug conjugate comprising a peptide ligand as defined herein conjugated to one or more effectors and / or functional groups.

[0124] Effectors and / or functional groups can be attached, for example, to the N- and / or C-terminus of a polypeptide, to amino acids within a polypeptide, or to a molecular scaffold.

[0125] Suitable effector groups include antibodies and parts or fragments thereof. For example, the effector group may include an antibody light chain constant region (CL), an antibody CH1 heavy chain domain, an antibody CH2 heavy chain domain, an antibody CH3 heavy chain domain, or any combination thereof, and one or more constant region domains. The effector group may also include the hinge region of an antibody (usually found between the CH1 and CH2 domains of an IgG molecule).

[0126] In a further embodiment of this aspect of the invention, the effector group according to the invention is the Fc region of an IgG molecule. Advantageously, the peptide ligand-effector group according to the invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of one day or more, two days or more, three days or more, four days or more, five days or more, six days or more, or seven days or more. Most advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion having a tβ half-life of one day or more.

[0127] Functional groups typically include binding groups, drugs, reactive groups for linking other entities, functional groups that facilitate uptake of the macrocyclic peptide into cells, and the like.

[0128] The ability of peptides to penetrate cells will allow peptides to effectively target intracellular targets. Targets that can be approached by peptides with the ability to penetrate cells include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in apoptosis pathways. Functional groups that can penetrate cells include peptides or chemical groups that have been added to peptides or molecular scaffolds. Peptides, such as those derived from homeobox proteins such as VP22, HIV-Tat, Drosophila (Antennapedia), for example, as Chen and Harrison, Biochemical Society Transactions (2007) Vol. 35, Part 4, p. 821; Gupta et al., Advanced Drug Discovery Reviews (2004) Vol. 57, 9637. The example of short peptides that has been shown to be effective in the translocation of plasma membrane includes 16 amino acid penetrating peptides (Derossi et al. (1994) J Biol.Chem. Vol. 269, p. 10444), 18 amino acid " model amphipathic peptides " (Oehlke et al. (1998) Biochim Biophys Acts Vol. 1414, p. 127) and HIV TAT protein arginine-rich regions. Non-peptide methods include using small molecule mimics or SMOC (Okuyama et al. (2007) Nature Methods Vol. 4, p. 153) that can be easily connected to biomolecules. Other chemical strategies by which guanidine groups are added to molecules also enhance cell penetration (Elson-Scwab et al. (2007) J Biol Chem Vol. 282, p. 13585). Small molecular weight molecules (such as steroids) can be added to molecular scaffolds to enhance cellular uptake.

[0129] One type of functional group that can be linked to a peptide ligand includes antibodies and binding fragments thereof, such as Fab, Fv or single domain fragments. In particular, antibodies that bind to proteins that can increase the half-life of the peptide ligand in vivo can be used.

[0130] In one embodiment, the peptide ligand-effector group according to the invention has a tβ half-life selected from 12 hours or longer, 24 hours or longer, 2 days or longer, 3 days or longer, 4 days or longer, 5 days or longer, 6 days or longer, 7 days or longer, 8 days or longer, 9 days or longer, 10 days or longer, 11 days or longer, 12 days or longer, 13 days or longer, 14 days or longer, 15 days or longer, or 20 days or longer. Advantageously, the 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 one day or longer. In a further embodiment, it will be in the range of 12 to 26 hours.

[0131] In a particular embodiment of the invention, the functional group is selected from metal chelators, which are suitable for complexing metal radioisotopes of pharmaceutical relevance.

[0132] Possible effector groups also include enzymes such as carboxypeptidase G2 for enzyme / prodrug therapy, where the peptide ligand replaces the antibody in ADEPT.

[0133] In a specific embodiment of the invention, the functional group is selected from drugs, such as cytotoxic agents for cancer treatment. Suitable examples include: alkylating agents such as cisplatin and carboplatin, as well as oxaliplatin, nitrogen mustard, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites, including purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids, including vinca alkaloids, such as vincristine, vinblastine, vinorelbine and vindesine; podophyllotoxin and its derivatives etoposide and teniposide; taxanes, including paclitaxel, originally known as Taxol; topoisomerase inhibitors including camptothecins: irinotecan and topotecan, and type II inhibitors, including amridine, etoposide, etoposide phosphate and teniposide. Other agents may include antitumor antibiotics, which include immunosuppressants actinomycin D (used in kidney transplantation), doxorubicin, epirubicin, bleomycin, calicheamicin, etc.

[0134] In a further embodiment of the invention, the cytotoxic agent is selected from maytansinoids (eg DM1) or monomethyl auristatins (eg MMAE).

[0135] DM1 is a cytotoxic agent that is a thiol-containing derivative of maytansine with the following structure:

[0136]

[0137] Data are shown herein in Table 3, which demonstrates the effect of peptide ligands conjugated to DM1 containing toxins.

[0138] Monomethyl auristatin E (MMAE) is a synthetic antineoplastic agent with the following structure:

[0139]

[0140] Data are shown herein in Table 3, which demonstrates the effect of peptide ligands conjugated to MMAE-containing toxins.

[0141] In a further specific embodiment of the present invention, said cytotoxic agent is selected from monomethyl auristatin E (MMAE).

[0142] In one embodiment, the cytotoxic agent is linked to the bicyclic peptide via a cleavable bond (e.g., a disulfide bond or a protease-sensitive bond). In a further embodiment, the groups adjacent to the disulfide bond are modified to control the hindrance of the disulfide bond and thereby control the rate of cleavage and the accompanying release of the cytotoxic agent.

[0143] Published work has identified the potential to alter the sensitivity of disulfide bonds to reduction by introducing steric hindrance on either side of the disulfide bond (Kellogg et al. (2011) Bioconjugate Chemistry, 22, 717). Greater degrees of steric hindrance reduce the rate of reduction by intracellular glutathione and extracellular (systemic) reducing agents, thereby reducing the ease of toxin release inside and outside the cell. Thus, an optimal choice of disulfide stability in circulation (which minimizes adverse side effects of the toxin) versus efficient release in the intracellular environment (which maximizes therapeutic effect) can be achieved by carefully selecting the degree of hindrance on either side of the disulfide bond.

[0144] The obstruction of either side of the disulfide bond is modulated by the introduction of one or more methyl groups on the targeting entity (here the bicyclic peptide) or toxin side of the molecular construct.

[0145] In one embodiment, the cytotoxic agent and linker are selected from any combination of those cytotoxic agents and linkers described in WO 2016 / 067035 (which cytotoxic agents and linkers are incorporated herein by reference).

[0146] 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 as suitable linkers include Ala, Cit, Lys, Trp and Val.

[0147] 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-, wherein PABC represents p-aminobenzylcarbamate. Full details of linkers containing cyclobutyl 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-.

[0148] In an alternative embodiment, the cytotoxic agent is DM1 and the drug conjugate further comprises a linker which is -SPDB-(SO 3 H)-, wherein SPDB represents N-succinimidyl 3-(2-pyridyldithio)propionate.

[0149] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8234 as defined herein, and the linker is selected from -PABC-Cit-Val-glutaryl-.

[0150] This BDC is known herein as BCY8245 and is schematically represented as:

[0151]

[0152] And a more detailed way is as follows:

[0153]

[0154] As shown in Table 3, the data provided herein demonstrate that BCY8245 binds very well to human bindin-4 in an SPR binding assay. The BDC also exhibited 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.

[0155] In an alternative embodiment, the cytotoxic agent is MMAE, the bicyclic peptide is selected from BCY8234 as defined herein, and the linker is selected from -PABC-cyclobutyl-(B-Ala)-. The BDC is referred to herein as BCY8549. As shown in Table 3, the data provided herein demonstrate that BCY8549 binds very well to human ligase-4 in an SPR binding assay.

[0156] In another embodiment, the bicyclic drug conjugate is selected from BCY8245 or BCY8549. In another embodiment, the bicyclic drug conjugate is BCY8245. As shown by the data described herein, the drug conjugate BCY8245 exhibits excellent dose-dependent anti-tumor activity.

[0157] synthesis

[0158] The peptides of the present invention can be prepared by standard technology synthesis, and then reacted with molecular scaffolds in vitro. When this operation is performed, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or verification. Such methods can be realized using conventional chemical methods disclosed in, for example, Timmerman et al. (supra).

[0159] Therefore, the present invention also relates to the preparation of a selected polypeptide or conjugate as described herein, wherein the preparation comprises the optional further steps as described below. In one embodiment, these steps are performed on a final product polypeptide / conjugate prepared by chemical synthesis.

[0160] When preparing conjugates or complexes, amino acid residues in the target polypeptide may optionally be substituted.

[0161] The peptides may also be extended to incorporate, for example, another loop, thereby introducing multispecificity.

[0162] To extend the peptide, standard solid phase or solution phase chemistry can be used, using orthogonally protected lysine (and analogs), simply chemically extended at its N-terminus or C-terminus or within a ring. Standard (bio) conjugation techniques can be used to introduce activated or activatable N- or C-termini. Alternatively, addition can be carried out by fragment condensation or native chemical ligation (for example, as described in (Dawson et al. 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266: 776-779)), or by enzymes, for example using subtiligase as described in (Chang et al. Proc Natl Acad Sci US A. 1994 Dec 20; 91 (26): 12544-8 or Hikari et al. Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, pages 6000-6003).

[0163] Alternatively, the peptides can be extended or modified by further conjugation via disulfide bonds. This has the additional advantage of allowing the first and second peptides to be separated from each other once in a cellular reducing environment. In this case, a molecular scaffold (e.g., TATA) can be added during the chemical synthesis of the first peptide to react with three cysteine ​​groups; additional cysteine ​​or thiol can then be attached to the N or C terminus of the first peptide so that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked bicyclic peptide-peptide conjugate.

[0164] Similar techniques are also applicable to the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially generating tetraspecific molecules.

[0165] Furthermore, the addition of other functional groups or effector groups can be accomplished in the same manner, using appropriate chemistry, at the N- or C-terminus or through side chain coupling. In one embodiment, coupling is performed in a manner that does not block the activity of either entity.

[0166] Pharmaceutical composition

[0167] According to a further aspect of the present invention, there is provided a pharmaceutical composition comprising the peptide ligand or drug conjugate as defined herein and one or more pharmaceutically acceptable excipients.

[0168] Generally, the peptide ligands of the present invention will be used in purified form together with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers include aqueous or alcohol / water solutions, emulsions or suspensions, including physiological saline and / or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's reagent. If it is necessary to maintain the polypeptide complex in a suspension, a suitable physiologically acceptable adjuvant can be selected from thickening agents such as carboxymethyl cellulose, polyvinyl pyrrolidone, gelatin and alginate.

[0169] Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers such as those based on Ringer's dextrose. Preservatives and other additives such as antimicrobials, antioxidants, chelating agents and inert gases may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th ed.).

[0170] The peptide ligands of the present invention can be used as compositions for single administration, or can be administered in combination with other agents. These can include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Pharmaceutical compositions can include a "cocktail mixture" of various cytotoxins or other agents combined with the protein ligands of the present invention, or even a combination of polypeptides selected according to the present invention with different specificities, such as polypeptides selected using different target ligands, whether or not combined before administration.

[0171] The route of administration of the pharmaceutical composition according to the present invention can be those generally known to those of ordinary skill in the art. For treatment, the peptide ligands of the present invention can be administered to any patient according to standard techniques. Administration can be by any appropriate mode, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary route, or also appropriately by direct catheter infusion. Preferably, the pharmaceutical composition according to the present invention is administered by inhalation. The dosage and frequency of administration depend on the age, sex and condition of the patient, the simultaneous administration of other drugs, contraindications and other parameters that the clinician will consider.

[0172] The peptide ligands of the present invention can be lyophilized for storage and reconstituted in a suitable carrier before use. This technique has been shown to be effective, and lyophilization and reconstitution techniques known in the art can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution may result in varying degrees of loss of activity, and levels may have to be adjusted upwards to compensate.

[0173] Compositions containing peptide ligands of the present invention or cocktails thereof can be administered for preventive and / or therapeutic treatments. In certain therapeutic applications, a sufficient amount to achieve at least partial inhibition, suppression, regulation, killing or some other measurable parameter of a selected cell population is defined as a "therapeutically effective dose". The amount required to achieve this dose will depend on the severity of the disease and the general state of the patient's own immune system, but typically ranges from 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, more commonly 0.05 to 2.0 mg / kg / dose. For preventive applications, compositions containing peptide ligands of the present invention or cocktails thereof can also be administered at similar or slightly lower doses.

[0174] Compositions comprising peptide ligands according to the present invention can be used to help change, inactivate, kill or remove target cell populations selected in mammals in preventive and therapeutic settings. In addition, peptide ligands as described herein can be selectively used to kill, consume or otherwise effectively remove target cell populations from heterogeneous collections of cells in vitro (extracorporeally) or in vitro (in vitro). Blood from mammals can be combined with selected peptide ligands in vitro, thereby killing or otherwise removing undesirable cells from the blood and returning to mammals according to standard techniques.

[0175] Co-administration with one or more other therapeutic agents

[0176] Depending on the specific condition, or disease to be treated, other therapeutic agents that are typically administered to treat that condition may also be present in the compositions of the invention. Thus, in one embodiment, the pharmaceutical composition additionally comprises one or more therapeutic agents. As used herein, other therapeutic agents that are typically administered to treat a specific disease, or condition are known as "suitable for the disease or condition being treated."

[0177] 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 pharmaceutically acceptable salt thereof, to a patient in need thereof, and simultaneously or sequentially administering an effective amount of one or more other therapeutic agents, such as those described herein. In some embodiments, the method comprises co-administering one other therapeutic agent. In some embodiments, the method comprises co-administering two other therapeutic agents. In some embodiments, the combination of the disclosed compound and one or more other therapeutic agents exerts a synergistic effect.

[0178] The compounds of the invention may also be used in combination with known treatment methods (eg, administration of hormones or radiation). In certain embodiments, provided compounds are useful as radiosensitizers, particularly for treating tumors that are poorly sensitive to radiation therapy.

[0179] The compounds of the present invention can be administered alone or in combination with one or more other therapeutic compounds, and possible combination therapies take the form of fixed combinations, or staggered administration or independent administration of the compounds of the present invention and one or more other therapeutic compounds, or combined administration of fixed combinations and one or more other therapeutic compounds. The compounds of the present invention may also be administered in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination thereof, in addition or in addition, particularly for tumor treatment. As described above, in the context of other treatment strategies, long-term therapy and adjuvant therapy have equal possibilities. Other possible treatments are therapies that maintain the patient's state after tumor regression, even chemoprevention, for example, in patients at risk.

[0180] As part of a multiple dose regimen, one or more other therapeutic agents can be used separately from the compounds or compositions of the present invention. Alternatively, one or more other therapeutic agents can be part of a single dosage form, mixed in a single composition with the compounds of this invention. If used in a multiple dose regimen, one or more other therapeutic agents and the compounds or compositions of the present invention can be used simultaneously, sequentially or within a period of time apart from each other, 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 apart from each other. In some embodiments, one or more other therapeutic agents and the compounds or compositions of the present invention are used in a multiple dose regimen greater than 24 hours apart.

[0181] As used herein, the terms "combination", "combined" and related terms refer to the simultaneous or sequential administration of therapeutic agents according to the present invention. For example, the compounds of the present invention can be administered with one or more other therapeutic agents simultaneously or sequentially in separate unit dosage forms or administered together in a single unit dosage form. Therefore, the present invention provides a single unit dosage form comprising a compound of the present invention, one or more other therapeutic agents, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.

[0182] The amount of the compound of the invention and one or more other therapeutic agents (in those compositions comprising other therapeutic agents as described above) that can be combined with carrier materials to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. Preferably, the compositions of the invention should be formulated so that a dosage of 0.01-100 mg / kg body weight / day of the compound of the invention can be administered.

[0183] In those compositions comprising one or more other therapeutic agents, the one or more other therapeutic agents and the compounds of the invention may act synergistically. Thus, the amount of one or more other therapeutic agents in such a composition may be less than the amount required for a monotherapy using only the therapeutic agent. In such a composition, one or more other therapeutic agents may be administered at a dose of 0.01–1,000 g / kg body weight / day.

[0184] The amount of one or more other therapeutic agents present in the composition of the present invention may not exceed the amount normally administered in a composition comprising the therapeutic agent as the sole active agent. Preferably, the amount of one or more other therapeutic agents in the composition of the present disclosure will be in the range of about 50% to 100% of the amount normally present in a composition comprising the therapeutic agent as the sole therapeutic active agent. In some embodiments, one or more other therapeutic agents are administered at 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 normally administered amount of the agent. As used herein, the term "normally administered" refers to the amount of an FDA-approved therapeutic agent for administration provided according to the FDA label insert.

[0185] The compounds of the present invention, or their pharmaceutical compositions, may also be incorporated into compositions for coating implantable medical devices, such as prostheses, artificial valves, vascular grafts, stents, and catheters. For example, vascular stents have been used to overcome stenosis (re-narrowing of the vessel wall after injury). However, patients using stents or other implantable devices have the risk of forming blood clots or activated platelets. These adverse effects may be prevented or mitigated by pre-coating the device with a pharmaceutically acceptable composition comprising a kinase inhibitor. An implantable device coated with a compound of the present invention is another embodiment of the present invention.

[0186] Exemplary Additional Therapeutic Agents

[0187] In some embodiments, the one or more additional therapeutic agents are poly ADP ribose polymerase (PARP) inhibitors. In some embodiments, the PARP inhibitor is selected from olaparib ( AstraZeneca; rucaparib ( Clovis Oncology; niraparib ( Tesaro); talazoparib (MDV3800 / BMN 673 / LT00673, Medivation / Pfizer / Biomarin); veliparib (ABT-888, AbbVie); and BGB-290 (BeiGene, Inc.).

[0188] 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 ( Merck; romidepsin ( Celgene; panobinostat ( Novartis; belinostat ( Spectrum Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals) (NCT00866333); and chidamide ( HBI-8000, Chipscreen Biosciences, China).

[0189] In some embodiments, the one or more additional therapeutic agents are CDK inhibitors, e.g., CDK4 / CDK6 inhibitors. In some embodiments, the CDK 4 / 6 inhibitor is selected from palbociclib ( Pfizer; ribociclib ( Novartis); abemaciclib (Ly2835219, Eli Lilly); and trilacicib (G1T28, G1 Therapeutics).

[0190] In some embodiments, the one or more additional therapeutic agents are phosphatidylinositol 3-kinase (PI3K) inhibitors. In some embodiments, the PI3K inhibitor is selected from idelalisib ( 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).

[0191] In some embodiments, one or more additional therapeutic agents are platinum-based therapeutic agents, also known as platins. Platinum primarily causes DNA crosslinking in rapidly multiplying cells (e.g., cancer cells), thereby inhibiting DNA repair and / or DNA synthesis. In some embodiments, the platinum-based therapy is selected from cisplatin ( Bristol-Myers Squibb; carboplatin ( Bristol-Myers Squibb; and Teva; Pfizer); oxaliplatin ( Sanofi-Aventis; nedaplatin ( Shionogi), picoplatin (Poniard Pharmaceuticals); and satraplatin (JM-216, Agennix).

[0192] In some embodiments, the one or more additional therapeutic agents is a taxane compound, which causes disruption of microtubules, which are essential for cell division. In some embodiments, the taxane compound is selected from paclitaxel ( Bristol-Myers Squibb, docetaxel ( Sanofi-Aventis; Sun Pharmaceutical); albumin-bound paclitaxel ( Abraxis / Celgene; Cabazitaxel( Sanofi-Aventis) and SID530 (SK Chemicals, Co.) (NCT00931008).

[0193] In some embodiments, the one or more additional therapeutic agents are nucleoside inhibitors, or therapeutic agents that interfere with normal DNA synthesis, protein synthesis, cell replication, or will inhibit rapidly proliferating cells.

[0194] In some embodiments, the nucleoside inhibitor is selected from trabectedin (a guanidinating agent, Janssen Oncology), mechlorethamine (alkylating agent, Aktelion Pharmaceuticals), vincristine ( Eli Lilly; Teva Pharmaceuticals; Talon Therapeutics; temozolomide (a prodrug of the alkylating agent 5-(3-methyltriaza-1-yl)-imidazole-4-carboxamide (MTIC), Merck); cytarabine injection (ara-C, an antimetabolite cytidine analog, Pfizer); lomustine (alkylating agent, Bristol-Myers Squibb; NextSource Biotechnology); azacitidine (a pyrimidine nucleoside analog of cytosine, Celgene; omacetaxine mepesuccinate (ceftazidime axetil) (protein synthesis inhibitor, Teva Pharmaceuticals); and Erwinia chrysanthemi asparaginase (an enzyme used to remove asparagine, Lundbeck; EUSA Pharma); eribulin mesylate (microtubule inhibitor, tubulin-based antimitotic agent, Eisai); cabazitaxel (microtubule inhibitor, tubulin-based antimitotic agent, Sanofi-Aventis; capecitabine (thymidylate synthase inhibitor, Genentech; bendamustine (a bifunctional methylethylamine derivative believed to form interstrand DNA crosslinks, Cephalon / Teva); ixabepilone (a semisynthetic analog of epothilone B, a microtubule inhibitor, a tubulin-based antimitotic agent, Bristol-Myers Squibb; nelarabine (prodrug of deoxyguanosine analog, nucleoside metabolism inhibitor, Novartis); clorafabine (ribonucleotide reductase inhibitor prodrug, competitive inhibitor of deoxycytidine, Sanofi-Aventis); and trifluridine and tipiracil (thymidine-based nucleoside analogs and thymidine phosphorylase inhibitors, Taiho Oncology).

[0195] In some embodiments, one or more additional therapeutic agents are kinase inhibitors or VEGF-R antagonists. Approved VEGF inhibitors and kinase inhibitors that can be used in the present invention include: bevacizumab ( Genentech / Roche), an anti-VEGF monoclonal antibody; ramucirumab ( Eli Lilly, an anti-VEGFR-2 antibody and ziv-aflibercept, also known as VEGF Trap ( Regeneron / Sanofi). VEGFR inhibitors, such as regorafenib ( Bayer; vandetanib ( AstraZeneca; axitinib ( Pfizer; lenvatinib ( Eisai; Raf inhibitors, such as sorafenib ( Bayer AG and Onyx); dabrafenib ( Novartis; and vemurafenib ( Genentech / Roche; MEK inhibitors, such as cobimetanib ( Exelexis / Genentech / Roche; trametinib ( Novartis); Bcr-Abl tyrosine kinase inhibitors, such as imatinib ( Novartis; nilotinib ( Novartis; dasatinib ( BristolMyersSquibb; bosutinib ( Pfizer; and ponatinib ( Ariad Pharmaceuticals); Her2 and EGFR inhibitors, such as gefitinib ( AstraZeneca; erlotinib ( Genentech / Roche / Astellas; Lapatinib ( Novartis; afatinib ( Boehringer Ingelheim; osimertinib (targeting activated EGFR, AstraZeneca; and brigatinib ( Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, such as Cabozantinib ( Exelexis; and multikinase inhibitors, such as sunitinib ( Pfizer; pazopanib ( Novartis; ALK inhibitors, such as crizotinib ( Pfizer; ceritinib ( Novartis; and alectinib ( Genentech / Roche; Bruton's tyrosine kinase inhibitors, such as ibrutinib ( Pharmacyclics / Janssen); and Flt3 receptor inhibitors, such as midostaurin ( Novartis).

[0196] Other kinase inhibitors and VEGF-R antagonists that are in development and 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); radotinib ( IY5511, Il-Yang Pharmaceuticals, S.Korea); ruxolitinib ( Incyte Corporation); PTC299 (PTC Therapeutics); CP-547,632 (Pfizer); foretinib (Exelexis, GlaxoSmithKline); quizartinib (Daiichi Sankyo) and motesanib (Amgen / Takeda).

[0197] In some embodiments, the one or more additional therapeutic agents are mTOR inhibitors, which inhibit cell proliferation, angiogenesis, and glucose uptake. In some embodiments, the mTOR inhibitor is everolimus ( Novartis; temsirolimus ( Pfizer; and sirolimus ( Pfizer).

[0198] In some embodiments, one or more additional therapeutic agents are proteasome inhibitors. Approved proteasome inhibitors useful in the present invention include bortezomib ( Takeda and carfilzomib ( Amgen; and ixazomib ( Takeda).

[0199] In some embodiments, the one or more additional therapeutic agents are growth factor antagonists, such as antagonists of platelet-derived growth factor (PDGF), or antagonists of epidermal growth factor (EGF) or its receptor (EGFR). Approved PDGF antagonists that can be used in the present invention include olaratumab ( Eli Lilly). Approved EGFR antagonists that can be used in the present invention include: Cetuximab ( Eli Lilly; necitumumab ( Eli Lilly, panitumumab ( Amgen); and osimertinib (targeting activated EGFR, AstraZeneca).

[0200] In some embodiments, the one or more additional therapeutic agents are aromatase inhibitors. In some embodiments, the aromatase inhibitor is selected from: exemestane ( Pfizer; anastazole ( AstraZeneca and letrozole ( Novartis).

[0201] In some embodiments, one or more of the other therapeutic agents is an antagonist of the hedgehog pathway. Approved hedgehog pathway inhibitors that can be used in the present invention include: sonidegib ( Sun Pharmaceuticals; and vismodegib ( Genentech), both used to treat basal cell carcinoma.

[0202] In some embodiments, the one or more additional therapeutic agents are folate inhibitors. Approved folate inhibitors for use in the present invention include pemetrexed ( Eli Lilly).

[0203] In some embodiments, the one or more additional therapeutic agents is a CC chemokine receptor 4 (CCR4) inhibitor. CCR4 inhibitors under investigation for use in the present invention include mogamulizumab ( Kyowa Hakko Kirin, Japan).

[0204] In some embodiments, one or more other therapeutic agents are isocitrate dehydrogenase (IDH) inhibitors. IDH inhibitors that are being studied and can be used in the present invention include: AG120 (Celgene; NCT02677922); AG221 (Celgene, NCT02677922; NCT02577406); BAY1436032 (Bayer, NCT02746081); IDH305 (Novartis, NCT02987010).

[0205] In some embodiments, one or more other therapeutic agents are arginase inhibitors. Arginase inhibitors under investigation that can be used in the present invention include: AEB1102 (PEGylated recombinant arginase, Aeglea Biotherapeutics), which is being studied in Phase 1 clinical trials for acute myeloid leukemia and myelodysplastic syndrome (NCT02732184) and solid tumors (NCT02561234); and CB-1158 (Calithera Biosciences).

[0206] In some embodiments, the one or more additional therapeutic agents is a glutaminase inhibitor. Investigational glutaminase inhibitors that may be used in the present invention include CB-839 (Calithera Biosciences).

[0207] 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 can be used in the present invention include: rituximab ( Genentech / BiogenIdec; ofatumumab (anti-CD20, GlaxoSmithKline; obinutuzumab (anti-CD20, Genentech), ibritumomab (anti-CD20 and Yttrium-90, Spectrum Pharmaceuticals; daratumumab (anti-CD38, Janssen Biotech), dinutuximab (anti-glycolipid GD2, United Therapeutics; trastuzumab (anti-HER2, Genentech; ado-trastuzumab emtansine (anti-HER2, fused to emtansine, Genentech; and pertuzumab (anti-HER2, Genentech); and brentuximab vedotin (anti-CD-30 drug conjugate, Seattle Genetics).

[0208] In some embodiments, one or more additional therapeutic agents are topoisomerase inhibitors. Approved topoisomerase inhibitors useful in the present invention include: irinotecan ( Merrimack Pharmaceuticals; and topotecan ( GlaxoSmithKline). Topoisomerase inhibitors under investigation that may be used in the present invention include pixantrone ( CTI Biopharma).

[0209] In some embodiments, one or more additional therapeutic agents are inhibitors of anti-apoptotic proteins, such as BCL-2. Approved anti-apoptotic agents that can be used in the present invention include: venetoclax ( AbbVie / Genentech; and blinatumomab ( Other therapeutic agents targeting apoptotic proteins that have been clinically tested and can be used in the present invention include navitoclax (ABT-263, Abbott), a BCL-2 inhibitor (NCT02079740).

[0210] In some embodiments, one or more additional therapeutic agents are androgen receptor inhibitors. Approved androgen receptor inhibitors that can be used in the present invention include enzalutamide ( Astellas / Medivation; approved androgen synthesis inhibitors include abiraterone ( Centocor / Ortho; approved gonadotropin-releasing hormone (GnRH) receptor antagonists (degaralix, Ferring Pharmaceuticals).

[0211] In some embodiments, one or more other therapeutic agents are selective estrogen receptor modulators (SERMs), which interfere with the synthesis or activity of estrogen. Approved SERMs that can be used in the present invention include raloxifene ( Eli Lilly).

[0212] In some embodiments, the one or more additional therapeutic agents are inhibitors of bone resorption. Approved therapeutic agents that inhibit bone resorption are Denosumab ( Amgen), an antibody that binds to RANKL, which blocks binding to its receptor RANK present on the surface of osteoclasts, their precursors, and osteoclast bone-like giant cells, which mediates bone pathology in solid tumors with bone metastases. Other approved therapeutic agents that inhibit bone resorption include bisphosphonates, such as zoledronic acid ( Novartis).

[0213] In some embodiments, one or more other therapeutic agents are inhibitors of the interaction between the two main p53 inhibitory proteins MDMX and MDM2. Inhibitors of p53 inhibitory proteins that can be used in the present invention include ALRN-6924 (Aileron), a stapled peptide that binds and disrupts the interaction of MDMX and MDM2 with p53. ALRN-6924 is currently being evaluated in clinical trials to treat AML, advanced myelodysplastic syndrome (MDS), and peripheral T-cell lymphoma (PTCL) (NCT02909972; NCT02264613).

[0214] In some embodiments, the one or more additional therapeutic agents are inhibitors of transforming growth factor-β (TGF-β or TGFβ). Inhibitors of TGF-β proteins under investigation that can be used in the present invention include NIS793 (Novartis), an anti-TGF-β antibody being clinically tested for the treatment of various cancers, including breast cancer, lung cancer, hepatocellular carcinoma, colorectal cancer, pancreatic cancer, prostate cancer, and renal cancer (NCT 02947165). In some embodiments, the inhibitor of TGF-β protein is fresolimumab (GC1008; Sanofi-Genzyme), which is being studied for use in melanoma (NCT00923169); renal cell carcinoma ((NCT00356460); and non-small cell lung cancer (NCT02581787). Additionally, in some embodiments, the additional therapeutic agent is a TGF-β 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 known as 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 human TGF-β receptor II (functioning as a TGFβ "trap").

[0215] In some embodiments, the one or more additional therapeutic agents are selected from glembatumumab vedotin-monomethyl auristatin E (MMAE) (Celldex), an anti-glycoprotein NMB (gpNMB) antibody linked to the cytotoxic MMAE (CR011). gpNMB is a protein overexpressed by a variety of tumors that is associated with the metastatic ability of a variety of cancer cells.

[0216] In some embodiments, one or more additional therapeutic agents are antiproliferative compounds. Such antiproliferative compounds include, but are 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; anti-tumor antimetabolites; platinum compounds; compounds that target / reduce protein or lipid kinase activity and other anti-angiogenic compounds; compounds that target, reduce or inhibit protein or lipid phosphatase activity; gonadotropin-releasing hormone agonists; anti-androgens; methionine aminopeptidase inhibitors; matrix metalloproteinase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; compounds for the treatment of hematological malignancies; compounds that target, reduce or inhibit Flt-3 activity; Hsp90 inhibitors, such as those from Conforma Therapeutics' 17-AAG (allylaminogeldanamycin, NSC330507), 17-DMAG (17-dimethylaminoethylamino-17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF1010; temozolomide Kinesin spindle protein inhibitors, such as GlaxoSmithKline's SB715992 or SB743921, or CombinatoRx's pentamidine / chlorpromazine; MEK inhibitors, such as Array BioPharma's ARRY142886, AstraZeneca's AZd 6 244, PD181461 from Pfizer and leucovorin.

[0217] As used herein, the term "aromatase inhibitor" relates to compounds that inhibit the production of estrogens, for example, by converting the substrates androstenedione and testosterone into estrone and estradiol, respectively. The term includes, but is not limited to, steroids, especially adamantane, exemestane and formamestane, and especially non-steroids, especially aminoglutamine, rosiglitazone, pyridine glutamate, triclosan, testolactone, ketoconazole, vorozole, faldrozole, anastrozole and letrozole. The trade name of exemestane is Aromasin TMFormestane is sold under the trade name Lentaron TM Fadrozole is sold under the trade name Afema TM Anastrozole is sold under the trade name Arimidex TM Letrozole is sold under the trade name Femara TM or Femar TM Aminoglutethimide is sold under the trade name Orimeten TM The combination of the invention comprising a chemotherapeutic agent which is an aromatase inhibitor is particularly useful in the treatment of hormone receptor positive tumors, such as breast tumors.

[0218] As used herein, the term "anti-estrogen" refers to compounds that antagonize the effects of estrogen at the level of the estrogen receptor. The term includes, but is not limited to, tamoxifen, fulvestrant, raloxifene, and raloxifene hydrochloride. Tamoxifen is sold under the trade name Nolvadex TM Raloxifene hydrochloride is sold under the trade name Evista TM Fulvestrant is sold under the trade name Faslodex TM The combination of the invention comprising a chemotherapeutic agent that is an anti-estrogen is particularly useful for treating estrogen receptor positive tumors, such as breast tumors.

[0219] As used herein, the term "anti-androgen" refers to any substance that is capable of inhibiting the biological effects of androgens, including but not limited to bicalutamide (Casodex TM ). The term "gonadotropin-releasing agonist" as used herein includes, but is not limited to, abarelix, goserelin, and goserelin acetate. Goserelin is available under the trade name Zoladex TM Application.

[0220] 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 macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, for example, in the form in which it is marketed, for example at Camptosar. TMTopotecan is sold under the trade name Hycamptin TM Sale.

[0221] As used herein, the term "topoisomerase II inhibitors" includes, but is not limited to, anthracyclines such as doxorubicin (including liposomal formulations such as Caelyx TM ), daunorubicin, epirubicin, idarubicin and neorubicin, the anthraquinones mitoxantrone and losoxantrone, and the podophillotoxines etoposide and teniposide. Etoposide is sold under the trade name Etopophos TM Teniposide is sold under the trade name VM 26-Bristol. Doxorubicin is sold under the trade name Acriblastin TM or Adriamycin TM Epirubicin is sold under the trade name Farmorubicin TM Idarubicin is sold under the trade name Zavedos TM Mitoxantrone is sold under the trade name Novantron.

[0222] The term "microtubule active agent" refers to microtubule stabilizing, microtubule destabilizing compounds and tubulin 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 marketed under the trade name Taxol TM Docetaxel is sold under the trade name Taxotere TM Vinblastine sulfate is sold under the trade name Vinblastin RP TM Vincristine sulfate is sold under the trade name Farmistin TM Sale.

[0223] The term "alkylating agent" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide is marketed under the trade name Cyclostin TM Ifosfamide is sold under the trade name Holoxan TM Sale.

[0224] The term "histone deacetylase inhibitor" or "HDAC inhibitor" relates to compounds that inhibit histone deacetylase and have antiproliferative activity. This includes, but is not limited to, suberoylanilide hydroxamic acid (SAHA).

[0225] 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 folic acid antagonists such as pemetrexed. Capecitabine is marketed under the trade name Xeloda TM Gemcitabine is sold under the trade name Gemzar TM Sale.

[0226] The term "platinum compound" as used herein includes, but is not limited to, carboplatin, cisplatin, cisplatin and oxaliplatin. Carboplatin can be marketed, for example, as in the trademark Carboplat TM Oxaliplatin can be administered in the form marketed under the trademark Eloxatin TM The following sales forms are applied.

[0227] The term "compounds that target / reduce protein or lipid kinase activity; or protein or lipid phosphatase activity; or other 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 that target, reduce or inhibit platelet-derived growth factor receptor (PDGFR) activity, such as compounds that target, reduce or inhibit PDGFR activity, in particular compounds that inhibit PDGF receptors, such as N-phenyl-2-pyrimidine-amine derivatives, such as imatinib, SU101, SU6668 and GFB-111; b) compounds that target, reduce or inhibit fibroblast growth factor receptor (FGFR) activity; c) compounds that target, reduce or inhibit insulin-like growth factor receptor I (IGF-IR) activity, such as compounds that target, reduce or inhibit IGF-IR activity, in particular compounds that inhibit IGF-I receptor kinase activity, or antibodies that target the extracellular domain of the IGF-1 receptor or its growth factor; d) compounds that target, reduce or inhibit the activity of the Trk receptor tyrosine kinase family , or ephrin B4 inhibitors; e) compounds that target, reduce or inhibit the activity of the AxI receptor tyrosine kinase family; f) compounds that target, reduce or inhibit the activity of the Ret receptor tyrosine kinase; g) compounds that target, reduce or inhibit the activity of the Kit / SCFR receptor tyrosine kinase, such as imatinib; h) compounds that target, reduce or inhibit the activity of the C-kit receptor tyrosine kinase, which is part of the PDGFR family, such as compounds that target, reduce or inhibit the c-Kit receptor tyrosine kinase. kinase family activity, in particular compounds that inhibit c-Kit receptors, such as imatinib; i) compounds that target, reduce or inhibit the activity of c-Abl family members, their gene fusion products (e.g., BCR-Abl kinase), and mutants, for example, compounds that target, reduce or inhibit the activity of c-Abl family members and their gene fusion products, for example, N-phenyl-2-pyrimidine-amine derivatives, such as imatinib or nilotinib (AMN107); PD180970; AG957; NSC 680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825); j) compounds that target, decrease or inhibit the activity of members of the Raf family of protein kinase C (PKC) and serine / threonine kinases, MEK, SRC, JAK / pan-JAK, FAK, PDK1, PKB / Akt, Ras / MAPK, PI3K, SYK, TYK2, BTK and TEC families, and / or members of the cyclin-dependent kinase family (CDK), including staurosporine derivatives such as midostaurin;Examples of other compounds include UCN-01, safingol, BAY 43-9006, Bryostatin 1, Perifosine; llmofosine; RO 318220 and RO 320432; GO 6976; lsis 3521; LY333531 / LY379196; isoquinoline compounds; FTIs; PD184352 or QAN697 (P13K inhibitors) or AT7519 (CDK inhibitors); k) compounds that target, decrease or inhibit the activity of protein tyrosine kinase inhibitors, such as compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, including imatinib mesylate (Gleevec; TM ), or tyrosine phosphorylation inhibitors (tyrphostin), such as Tyrphostin A23 / RG-50810; AG 99; Tyrphostin AG 213; Tyrphostin AG 1748; Tyrphostin AG 490; Tyrphostin B44; Tyrphostin B44(+) enantiomer; Tyrphostin AG 555; AG 494; Tyrphostin AG 556, AG957 and adenosine deaminase phosphorylation inhibitor (adaphostin) (4-{[(2,5-dihydroxyphenyl)methyl]amino}-benzoic acid adamantyl ester; NSC 680410, adenosine deaminase phosphorylation inhibitor); l) targeting, decreasing or inhibiting the epidermal growth factor family of receptor tyrosine kinases (EGFR homodimers or heterodimers) 1 Compounds that inhibit the activity of ErbB2, ErbB3, ErbB4, or a mutant thereof, such as compounds that target, reduce or inhibit the activity of the epidermal growth factor receptor family, specifically compounds, proteins or antibodies that inhibit members of the EGF receptor tyrosine kinase family, such as EGF receptor, ErbB2, ErbB3 and ErbB4 or bind to EGF or bind to an EGF-related ligand, CP358774, ZD 1839, ZM 105180; trastuzumab (Herceptin TM ), Erbitux TM), 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-pyrrole-[2,3-d]pyrimidine derivatives; m) compounds that target, decrease or inhibit the activity of c-Met receptors, such as compounds that target, decrease or inhibit the activity of c-Met, especially compounds that inhibit the activity of c-Met receptor kinase, or antibodies targeting the extracellular domain of c-Met or antibodies that bind to HGF, n) compounds that target, reduce 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-1 o) compounds that target, reduce or inhibit the activity of PI3 kinase (PI3K) kinase, including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib, pictrelisib, PF-4691502, BYL-719, dabrafenib, dapoxetine, tadalafil, styrene, styrene-binding protein ... p) compounds that target, reduce or inhibit the signaling effects of the hedgehog protein (Hh) or smoothened receptor (SMO) pathway, including but not limited to cyclopamine, vismodegib, itraconazole, erismodegib and IPI-926 (saridegib).

[0228] The term "PI3K inhibitor" as used herein includes, but is not limited to, compounds that have inhibitory activity on one or more enzymes in the phosphatidylinositol-3-kinase family, including, but not limited to, PI3Kα, PI3Kγ, PI3Kδ, PI3Kβ, PI3K-C2α, PI3K-C2β, PI3K-C2γ, 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, pictrelisib, PF-4691502, BYL-719, dactolisib, XL-147, XL-765, and idarisib.

[0229] The term "Bcl-2 inhibitor" as used herein includes, but is not limited to, compounds having inhibitory activity against B-cell lymphoma 2 protein (Bcl-2), including, but not limited to, ABT-199, ABT-731, ABT-737, apogossypol, Ascenta's pan-Bcl-2 inhibitor, curcumin (and its analogs), dual Bcl-2 / Bcl-xL inhibitor (Infinity Pharmaceuticals / Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and its analogs; see WO 2008 / 118802), nevitoclax (and its analogs, see US 7,390,799), NH-1 (Shenayng Pharmaceutical University), obatoclax (and its analogs, see WO 2004 / 106328), S-001 (Gloria Pharmaceuticals), TW series compounds (Univ. 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.

[0230] As used herein, the term "BTK inhibitor" includes, but is not limited to, compounds having inhibitory activity on Bruton's tyrosine kinase (BTK), including, but not limited to, AVL-292 and ibrutinib.

[0231] As used herein, the term "SYK inhibitor" includes, but is not limited to, compounds having inhibitory activity on spleen tyrosine kinase (SYK), including, but not limited to, PRT-062070, R-343, R-333, Excellair, PRT-062607, and fostamatinib.

[0232] Further examples of BTK inhibiting compounds, and further examples of conditions that may be treated by such compounds in combination with the compounds of the invention, can be found in WO 2008 / 039218 and WO 2011 / 090760, the entire contents of which are incorporated herein by reference.

[0233] Further examples of SYK inhibiting compounds, and further examples of conditions that may be treated by such compounds in combination with the compounds of the invention, may be found in WO 2003 / 063794, WO 2005 / 007623 and WO 2006 / 078846, the entire contents of which are incorporated herein by reference.

[0234] Further examples of PI3K inhibitory compounds, and further examples of conditions that can be treated by such compounds in combination with the compounds of the invention, can be found in WO 2004 / 019973, WO 2004 / 089925, WO 2007 / 016176, US 8,138,347, WO 2002 / 088112, WO 2007 / 084786, WO 2007 / 129161, WO 2006 / 122806, WO 2005 / 113554, and WO 2007 / 044729, the entire contents of which are incorporated herein by reference.

[0235] Further examples of JAK inhibitory compounds, and further examples of disorders that can be treated 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 entire contents of which are incorporated herein by reference.

[0236] Other anti-angiogenic compounds include compounds that have other mechanisms for their activity, for example, not related to protein or lipid kinase inhibition, such as thalidomide. TM ) and TNP-470.

[0237] Examples of proteasome inhibitors that can be used in combination with the compounds of the invention include, but are not limited to, bortezomib, disulfiram, epigallocatechin-3-gallate (EGCG), salisosporamide A, carfilzomib, ONX-0912, CEP-18770, and MLN9708.

[0238] Compounds that target, decrease or inhibit the activity of a protein or lipid phosphatase are, for example, inhibitors of phosphatase 1, phosphatase 2A, or CDC25, for example, okadaic acid or a derivative thereof.

[0239] 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.

[0240] The term cyclooxygenase inhibitors as used herein include, but are not limited to, Cox-2 inhibitors, 5-alkyl substituted 2-arylaminophenylacetic acid and its derivatives, such as celecoxib (Celebrex TM ), rofecoxib (Vioxx TM ), etoricoxib, valdecoxib or 5-alkyl-2-arylaminophenylacetic acid, for example, 5-methyl-2-(2'-chloro-6'-fluoroanilino)phenylacetic acid and lumiracoxib.

[0241] As used herein, the term "bisphosphonate" includes, but is not limited to, etridonic, clodronic, tiludronic, pamidronic, alendronic, ibandronic, risedronic, and zoledronic acid. TM Clodronic acid is sold under the trade name Bonefos TM Tiludronic acid is sold under the trade name Skelid TM Pamidronate is sold under the trade name Aredia TM Alendronate is sold under the trade name Fosamax TM Ibandronic acid is sold under the trade name Bondranat TM Actonel TM Zoledronic acid is sold under the trade name ZometaTM The term "mTOR inhibitor" relates to compounds that inhibit the mammalian target of rapamycin (mTOR) and have antiproliferative activity, such as sirolimus ), everolimus (Certican TM ), CCI-779 and ABT578.

[0242] The term "heparanase inhibitor" as used herein refers to a compound that targets, reduces or inhibits the degradation of heparan sulfate. The term includes, but is not limited to, PI-88. The term "biological response modifier" as used herein refers to a lymphokine or an interferon.

[0243] As used herein, the term "inhibitor of an oncogenic isoform of Ras", such as H-Ras, K-Ras, or N-Ras, refers to a compound that targets, decreases, or inhibits the oncogenic activity of Ras. For example, a "farnesyl transferase inhibitor", such as L-744832, DK8G557, or R115777 (Zarnestra TM ). The term "telomerase inhibitor" as used herein refers to a compound that targets, reduces or inhibits telomerase activity. Compounds that target, reduce or inhibit telomerase activity are specifically compounds that inhibit telomerase receptors, such as telomestatins.

[0244] The term "methionine aminopeptidase inhibitor" as used herein refers to a compound that targets, reduces or inhibits the activity of methionine aminopeptidase. Compounds that target, reduce or inhibit the activity of methionine aminopeptidase include, but are not limited to, bengamide or its derivatives.

[0245] The term "proteasome inhibitor" as used herein refers to a compound that targets, decreases or inhibits proteasome activity. Compounds that target, decrease or inhibit proteasome activity include, but are not limited to, bortezomib (Velcade TM ) and MLN341.

[0246] As used herein, the term "matrix metalloproteinase inhibitors" or ("MMP" inhibitors) includes, but is not limited to, collagen peptide mimetic and non-peptide mimetic inhibitors, tetracycline derivatives, for example, the hydroxamate peptide mimetic inhibitor batimastat, and its orally bioavailable analogs marimastat (BB-2516), prinomastat (AG3340), metastat (NSC 683551) BMS-279251, BAY 12-9566, TAA211, MMI270B or AAJ996.

[0247] The term "compounds for treating hematological malignancies" as used herein includes, but is not limited to, FMS-like tyrosine kinase inhibitors, which are compounds that target, decrease or inhibit the activity of FMS-like tyrosine kinase receptor (Flt-3R); interferons, 1-β-D-arabinofuranosylcytosine (ara-c) and bisulfan; and ALK inhibitors, which are compounds that target, decrease or inhibit anaplastic lymphoma kinase.

[0248] Compounds that target, decrease or inhibit the activity of FMS-like tyrosine kinase receptor (Flt-3R) are specifically compounds, proteins or antibodies that inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, staurosporine derivatives, SU11248 and MLN518.

[0249] 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 through the ubiquitin proteosome pathway. Compounds that target, reduce or inhibit the intrinsic ATPase activity of HSP90, in particular compounds, proteins or antibodies that inhibit the ATPase activity of HSP90, for example, 17-allylamino, 17-demethoxygeldanamycin (17AAG), geldanamycin derivatives; other compounds related to geldanamycin; radicicol and HDAC inhibitors.

[0250] As used herein, the term "anti-proliferative antibodies" includes, but is not limited to, trastuzumab (Herceptin TM ), trastuzumab-DM1, cetuximab (erbitux), bevacizumab (Avastin TM ), rituximab PRO64553 (anti-CD40) and 2C4 antibodies. Antibodies refer to intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies, and antibody fragments, as long as they exhibit the desired biological activity.

[0251] In order to treat acute myeloid leukemia (AML), the compounds of the present invention can be used in combination with standard leukemia therapy, especially in combination with a therapy for the treatment of AML. Specifically, the compounds of the present invention can be co-administered with, for example, a farnesyl transferase inhibitor and / or other drugs that can be used to treat AML, such as daunorubicin, doxorubicin, Ara-C, VP-16, teniposide, mitoxantrone, idarubicin, carboplatin, and PKC412.

[0252] Other anti-leukemic compounds include, for example, Ara-C, a pyrimidine analog, which is a 2'-α-hydroxyribose (arabinoside) derivative of deoxycytidine. Also included are purine analogs of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate. Compounds that target, reduce or inhibit the activity of histone deacetylase (HDAC) inhibitors, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA), can inhibit the activity of an enzyme called histone deacetylase. Specific HDAC inhibitors include MS275, SAHA, FK228 (formerly known as FR901228), trichostatin A and compounds disclosed in US6,552,065, including but not limited to, N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-acrylamide, or a pharmaceutically acceptable salt thereof and N-hydroxy-3-[4-[(2-hydroxyethyl){2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-acrylamide, or a pharmaceutically acceptable salt thereof, particularly lactate. Somatostatin receptor antagonists used herein refer to compounds that target, treat or inhibit somatostatin receptors, such as octreotide and SOM230. Tumor cell destruction methods refer to methods such as ionizing radiation. The term "ionizing radiation" above and below refers to ionizing radiation that occurs 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. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Devita et al., eds., 4th edition, volume 1, pages 248-275 (1993).

[0253] Also included are EDG binders and ribonucleotide reductase inhibitors. As used herein, the term "EDG binder" refers to a class of immunosuppressants that regulate 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 anti-ALL ara-C) and / or pentostatin. Ribonucleotide reductase inhibitors are especially hydroxyurea or 2-hydroxy-1H-isoindole-1,3-dione derivatives.

[0254] Also included are compounds, proteins or monoclonal antibodies, particularly those to VEGF, such as: 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine, or a pharmaceutically acceptable salt thereof, 1-(4-chloroanilino)-4-(4-pyridylmethyl)phthalazine succinate; Angiostatin TM ;Endostatin TM ; anthranilamide; ZD4190; Zd 6 474; SU5416; SU6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, such as rhuMAb and RHUFab, or VEGF aptamers, such as Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2 IgGI antibodies, Angiozyme (RPI 4610) and bevacizumab (Avastin TM ).

[0255] As used herein, photodynamic therapy refers to therapy that uses certain chemicals known as photosensitive compounds to treat or prevent cancer. Examples of photodynamic therapy include treatment with compounds such as Visudyne TM and sodium porphyrin.

[0256] As used herein, angiostatic steroids refer to compounds that block or inhibit angiogenesis, such as, for example, anecortave, triamcinolone acetonide, hydrocortisone, 11-α-epihydrocortisol, 11-deoxycortisol, 17α-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone, and dexamethasone.

[0257] Implants containing corticosteroids refer to compounds such as fluocinolone and dexamethasone.

[0258] Other chemotherapeutic compounds include, but are not limited to: plant alkaloids, hormone 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.

[0259] The structures of active compounds identified by number, generic or trade name may be taken from actual editions of the standard compilation "The Merck Index" or from databases such as Patents International (eg IMS World Publications).

[0260] Example Immuno-Oncology Drugs

[0261] 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 effectively enhances, stimulates, and / or upregulates an immune response in a subject. In some embodiments, the immuno-oncology agent is administered together with the compounds of the invention to have a synergistic effect in treating cancer.

[0262] The immuno-oncology agent can be, for example, a small molecule drug, an antibody, or a biological 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 humanized or human.

[0263] In some embodiments, the immuno-oncology agent is (i) an agonist of a stimulatory (including co-stimulatory) receptor, or (ii) an antagonist of an inhibitory (including co-inhibitory) signal on T cells, both of which result in amplification of antigen-specific T cell responses.

[0264] Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to co-stimulatory or co-inhibitory receptors is the B7 family, including 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 co-stimulatory or co-inhibitory receptors is the family of TNF molecules that bind to members of the homologous TNF receptor family, including 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, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LTβR, LIGHT, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1 , XEDAR, EDA2, TNFR1, Lymphotoxinα / TNFβ, TNFR2, TNFα, LTβR, Lymphotoxinα1β2, FAS, FASL, RELT, DR6, TROY and NGFR.

[0265] In some embodiments, the immuno-oncology 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 for stimulating an immune response.

[0266] In some embodiments, the combination of the compound of the present invention and immune-tumor agent can stimulate T cell response. In some embodiments, immune-tumor agent is: (i) antagonist (for example, immune checkpoint inhibitor) of protein that suppresses T cell activation, 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) agonist of 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.

[0267] In some embodiments, the immuno-oncology agent is an antagonist of an inhibitory receptor on a NK cell, or an agonist of an activating receptor on a NK cell. In some embodiments, the immuno-oncology agent is an antagonist of a KIR, such as lirilumab.

[0268] 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, such as a CSF-1R antagonist antibody, including RG7155 (WO2011 / 70024, WO2011 / 107553, WO2011 / 131407, WO2013 / 87699, WO2013 / 119716, WO2013 / 132044) or FPA-008 (WO2011 / 140249; WO2013 / 169264; WO2014 / 036357).

[0269] In some embodiments, the immuno-oncology agent is selected from the following: agonists that bind positive co-stimulatory receptors, blockers 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 in the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interactions)), agents that deplete or inhibit Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab, or depletion by anti-CD25 beads in vitro), agents that inhibit metabolic enzymes (e.g., IDO), or reverse / prevent T cell energy or exhaustion), and agents that trigger innate immune activation and / or inflammation at the tumor site.

[0270] 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.

[0271] 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 a programmed death-1 (PD-1) receptor and inhibits PD-1 activity. In some embodiments, the PD-1 antagonist is an antagonistic PD-1 antibody. In some embodiments, the antagonistic PD-1 antibody is OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680 (AMP-514; WO2012 / 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.

[0272] 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; WO2010 / 077634), durvalumab (MEDI4736), BMS-936559 (WO2007 / 005874), and MSB0010718C (WO2013 / 79174).

[0273] In some embodiments, the immuno-oncology 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 (WO2010 / 19570, WO2014 / 08218) or IMP-731 or IMP-321 (WO2008 / 132601, WO2009 / 44273).

[0274] In some embodiments, the immuno-oncology agent is a CD137 (4-1BB) agonist. In some embodiments, the CD137 (4-1BB) agonist is an agonistic CD137 antibody. In some embodiments, the CD137 antibody is urelumab, or PF-05082566 (WO2012 / 32433).

[0275] In some embodiments, the immuno-oncology agent is a GITR agonist. In some embodiments, the GITR agonist is an agonistic GITR antibody. In some embodiments, the GITR antibody is BMS-986153, BMS-986156, TRX-518 (WO2006 / 105021, WO2009 / 009116), or MK-4166 (WO2011 / 028683).

[0276] 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); or indoximod (NLG-8189, New Link Genetics Corporation); capmanitib (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 (WO2009 / 73620, WO2009 / 1156652, WO2011 / 56652, WO2012 / 142237).

[0277] In some embodiments, the immuno-oncology agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonistic OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

[0278] In some embodiments, the immuno-oncology agent is an OX40L antagonist. In some embodiments, the OX40L antagonist is an antagonistic OX40 antibody. In some embodiments, the OX40L antagonist is RG-7888 (WO2006 / 029879).

[0279] In some embodiments, the immuno-oncology agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonistic CD40 antibody. In some embodiments, the immuno-oncology agent is a CD40 antagonist. In some embodiments, the CD40 antagonist is an antagonistic CD40 antibody. In some embodiments, the CD40 antibody is lucatumumab or dacetuzumab.

[0280] In some embodiments, the immuno-oncology agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonistic CD27 antibody. In some embodiments, the CD27 antibody is varlilumab.

[0281] In some embodiments, the immuno-oncology agent is MGA271 (directed against B7H3) (WO2011 / 109400).

[0282] In some embodiments, the immuno-oncology agent is abagovomab, adecatumumab, afutuzumab, alemtuzumab, anatumomab mafenatox, apolizumab, atezolimab, avelumab, blinatumomab, BMS-936559, catumaxomab, durvaluumab, epacadostat, epratuzumab, indoximod, inotuzumab ozogamicin, intelumumab, ipilimumab, isaximab tuximab, lambrolizumab, MED14736, MPDL3280A, nivolumab, obinutuzumab, ocaratuzumab, ofatumumab, olatatumab, pembrolizumab, pidilizumab, rituximab, ticilimumab, samalizumab, or tremelimumab.

[0283] In some embodiments, the immuno-oncology agent is an immunostimulatory agent. For example, antibodies that block the PD-1 and PD-L1 inhibitory axis can release activated tumor-reactive T cells and have been shown in clinical trials to induce durable anti-tumor responses in a growing number of tumor histologies, including some tumor types that are not generally considered to be sensitive to immunotherapy. 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 ( Bristol-Myers Squibb, also known as ONO-4538, MDX1106, and BMS-936558), showed potential in improving overall survival in patients with renal clear cell carcinoma (RCC) who had experienced disease progression during or after prior anti-angiogenic therapy.

[0284] In some embodiments, the immunomodulatory therapeutic agent specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutic agents that can be used in the present invention include: pomalidomide ( Celgene; lenalidomide ( Celgene; Ingenol Mebutate ( LEO Pharma).

[0285] In some embodiments, the immuno-oncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from: sipuleucel-T ( Dendreon / Valeant Pharmaceuticals), which has been approved for the treatment of asymptomatic, or minimally symptomatic, metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec ( BioVex / Amgen, formerly known as T-VEC), a genetically modified oncolytic virus therapy approved for the treatment of unresectable skin, subcutaneous and nodular lesions in melanoma. In some embodiments, the immuno-oncology agent is selected from oncolytic virus therapies, such as pexastimogene devacirepvec (PexaVec / JX-594, SillaJen / formerly known as Jennerex Biotherapeutics), a thymidine kinase-(TK-) deficient vaccinia virus engineered to express GM-CSF for hepatocellular carcinoma (NCT02562755) and melanoma (NCT00429312); pelareorep ( Oncolytics Biotech, a variant of a respiratory enteric orphan virus (reovirus), is unable to replicate in cells that are not activated by RAS in a variety of 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) (NCT 00861627); enadenotucirev (NG-348, PsiOxus, formerly ColoAd1), an adenovirus engineered to express full-length CD80 and an antibody fragment specific for the T-cell receptor CD3 protein, in ovarian cancer (NCT02028117); metastatic or advanced epithelial tumors, such as colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland cancer (NCT02636036); ONCOS-102 (Targovax / formerly Oncos), an engineered Adenoviruses engineered to express GM-CSF were studied in melanoma (NCT03003676); and peritoneal disease, colorectal cancer, or ovarian cancer (NCT02963831); GL-ONC1 (GLV-1h68 / GLV-1h153, Genelux GmbH), vaccinia virus engineered to express β-galactosidase (β-gal) / β-glucuronidase or β-gal / human sodium iodide cotransporter (hNIS), respectively, were studied in peritoneal cancer (NCT01443260), fallopian tube cancer, ovarian cancer (NCT02759588); or CG0070 (Cold Genesys), an adenovirus engineered to express GM-CSF, was studied in bladder cancer (NCT02365818).

[0286] In some embodiments, the immuno-oncology agent is selected from: JX-929 (SillaJen / formerly Jennerex Biotherapeutics), a TK- and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine deaminase, which is capable of converting the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TG01 and TG02 (Targovax / formerly Oncos), peptide-based immunotherapeutics that target difficult-to-treat RAS mutations; and TILT-123 (TILT Biotherapeutics), an engineered adenovirus named: Ad5 / 3-E2F-δ24-hTNFα-IRES-hIL20; and VSV-GP (Vira Therapeutics), a vesicular stomatitis virus (VSV) engineered to express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which can be further engineered to express a protein designed to increase antigen-specific CD8 + Antigens to which T cells respond.

[0287] In some embodiments, the immuno-oncology agent is a T cell engineered to express a chimeric antigen receptor or CAR. T cells engineered to express such a chimeric antigen receptor are called CAR-T cells.

[0288] A CAR consisting of a binding domain has been constructed, which can be derived from the following: a natural ligand, a single-chain variable fragment (scFv) of a monoclonal antibody specific for a cell surface antigen, fused to an intracellular domain (endodomain) as the functional end of a T cell receptor (TCR), such as a CD3-ζ signaling domain from a TCR, which can generate activation signals in T lymphocytes. After antigen binding, such CARs are connected to endogenous signaling pathways in effector cells and generate activation signals similar to those triggered by the TCR complex.

[0289] 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 that the CAR-T cell is engineered to include an extracellular domain having an antigen binding domain (e.g., a domain that binds to CD19), fused to an intracellular signaling domain of a T cell antigen receptor complex ζ chain (e.g., CD3ζ). When expressed in T cells, the CAR is able to redirect antigen recognition based on antigen binding specificity. For CD19, the antigen is expressed on malignant B cells. Currently, more than 200 clinical trials using CAR-T are ongoing in various indications [https: / / clinicaltrials.gov / ct2 / results?term=chimeric+antigen+receptors&pg=1].

[0290] 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 a key role in the differentiation and maintenance of type 17 effector subsets of CD4+ (Th17) and CD8+ (Tc17) T cells, as well as the differentiation of IL-17-expressing innate immune cell subsets such as NK cells. In some embodiments, the activator of RORγt is LYC-55716 (Lycera), which is currently being evaluated in clinical trials for the treatment of solid tumors (NCT02929862).

[0291] In some embodiments, the immunostimulant is an agonist or activator of a toll-like receptor (TLR). Suitable activators of TLR include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG that is being studied for use in B cells, follicular lymphomas, and other lymphomas (NCT02254772). Agonists or activators of TLR8 that can be used in the present invention include motolimod (VTX-2337, VentiRx Pharmaceuticals), which is being studied for use in head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT02431559).

[0292] Other immuno-oncology agents that can be used in the present invention include: urerulumab (BMS-663513, Bristol-Myers Squibb), an anti-CD137 monoclonal antibody; varlilumab (CDX-1127, Celldex Therapeutics), an anti-CD27 monoclonal antibody; BMS-986178 (Bristol-Myers Squibb), an anti-OX40 monoclonal antibody; lirelumab (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) Sciences), an anti-MMP9 antibody; and MK-4166 (Merck & Co.), an anti-GITR monoclonal antibody.

[0293] In some embodiments, the immunostimulatory agent is selected from evolozumab, mifamortide, an agonist or activator of a toll-like receptor, and an activator of RORγt.

[0294] In some embodiments, the immunostimulatory therapeutic is recombinant human interleukin 15 (rhIL-15). rhIL-15 has been clinically tested as a therapy 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), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed with a soluble IL-15 binding protein IL-15 receptor alpha chain (IL15:sIL-15RA), which has been 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.

[0295] In some embodiments, the immuno-oncology 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, pp. 603-622, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-oncology agent is selected from the examples described by Jerry L. Adams et al. in Table 1. In some embodiments, the immuno-oncology agent is a small molecule targeting those immuno-oncology targets listed in Table 2 by Jerry L. Adams et al. In some embodiments, the immuno-oncology agent is a small molecule selected from those listed in Table 2 by Jerry L. Adams et al.

[0296] In some embodiments, the immuno-oncology agent is selected from the small molecule immuno-oncology agents described in Peter L. Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, volume 28, pages 319-329, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-oncology agent is an agent targeting a pathway as described by Peter L. Toogood.

[0297] In some embodiments, the immuno-oncology agent is selected from Sandra L. Ross et al., "Bispecific T cell engager Antibody constructs can mediate bystander tumor cell killing”, PLoS ONE 12(8):e0183390, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-oncology agent is a bispecific T cell engager Antibody Constructs. In some embodiments, the bispecific T cell engager The antibody construct is a CD19 / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager The antibody construct is an EGFR / CD3 bispecific antibody construct. In some embodiments, the bispecific T cell engager Antibody constructs activate T cells. In some embodiments, the bispecific T cell engager The antibody construct activates T cells, which release cytokines that induce upregulation of intercellular adhesion molecule 1 (ICAM-1) and FAS on bystander cells. In some embodiments, the bispecific T cell engager The antibody construct activates T cells, which results in induced bystander cell lysis. In some embodiments, the bystander cell is in a solid tumor. In some embodiments, the lysed bystander cell is adjacent to the bystander cell. -activated T cells. In some embodiments, the adjacent cells include tumor-associated antigen (TAA) negative cancer cells. In some embodiments, the adjacent cells include EGFR-negative cancer cells. In some embodiments, the immune-tumor agent is an antibody that blocks the PD-L1 / PD1 axis and / or CTLA4. In some embodiments, the immune-tumor agent is a tumor-infiltrating T cell amplified ex vivo. In some embodiments, the immune-tumor agent is a bispecific antibody construct or a chimeric antigen receptor (CAR) that directly connects T cells to tumor-associated surface antigens (TAA).

[0298] Exemplary Immune Checkpoint Inhibitors

[0299] In some embodiments, the immuno-oncology agent is an immune checkpoint inhibitor described herein.

[0300] As used herein, the term "checkpoint inhibitor" refers to agents that can be used to prevent cancer cells from escaping the patient's immune system. One of the main mechanisms of anti-tumor immune subversion is called "T-cell exhaustion", which is caused by upregulation of inhibitory receptors due to chronic exposure to antigens. These inhibitory receptors serve as immune checkpoints to prevent uncontrolled immune responses.

[0301] 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-containing-3 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD223), and others are commonly referred to as checkpoint regulators. They act as molecular “gatekeepers” to allow extracellular information to determine whether cell cycle progression and other intracellular signaling processes should proceed.

[0302] In some embodiments, the immune checkpoint inhibitor is an antibody directed against PD-1. PD-1 binds to the programmed cell death 1 receptor (PD-1) to prevent the receptor from binding to the inhibitory ligand PDL-1, thereby overriding the tumor's ability to suppress the host's anti-tumor immune response.

[0303] On the one hand, the checkpoint inhibitor is a biotherapeutic agent or a small molecule. In another aspect, the checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof. In another aspect, the checkpoint inhibitor inhibits a checkpoint protein selected from the following: 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 another aspect, the check point inhibitor interacts with a ligand selected from the following check point proteins: 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 ligands or a combination thereof. On the one hand, the check point inhibitor is an immunostimulant, a T cell growth factor, an interleukin, an antibody, a vaccine or a combination thereof. In another aspect, the interleukin is IL-7 or IL-15. In a specific aspect, the interleukin is a glycosylated IL-7. In another aspect, the vaccine is a dendritic cell (DC) vaccine.

[0304] Checkpoint inhibitors include any agent that blocks or inhibits immune system inhibitory pathways in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies that bind to and block or inhibit immune checkpoint receptors, or antigen binding fragments thereof, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands. Exemplary checkpoint molecules that may be targeted to block or inhibit include, but are not limited to, CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, GAL9, LAG3, TIM3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and in all NK, γδ and memory CD8 +(αβ) T cells), CD160 (also known as BY55), CGEN-15049, CHK 1 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 the following: 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-L1 monoclonal antibody (anti-B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD1 antibody), CT-011 (anti-PD1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL1 antibody), BMS-936559 (anti-PDL1 antibody), MPLDL3280A (anti-PDL1 antibody), MSB0010718C (anti-PDL1 antibody) and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Checkpoint protein ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86 and TIM-3.

[0305] 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 nivolumab Ipilimumab and pembrolizumab In some embodiments, the checkpoint inhibitor is selected from: nivolumab (anti-PD-1 antibody, Bristol-Myers Squibb; Pembrolizumab (anti-PD-1 antibody, Merck); Ipilimumab (anti-CTLA-4 antibody, Bristol-Myers Squibb; durvalumab (anti-PD-L1 antibody, AstraZeneca; and atezolizumab (anti-PD-L1 antibody, Genentech).

[0306] In some embodiments, the checkpoint inhibitor is selected from: lambrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lirlumab, IPH2101, pembrolizumab and tremelimumab.

[0307] In some embodiments, the immune checkpoint inhibitor is: REGN2810 (Regeneron), an anti-PD-1 antibody that has been tested 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 ( Pfizer / Merck KGaA), also known as MSB0010718C), a fully human IgG1 anti-PD-L1 antibody in clinical trials for: non-small cell lung cancer, Merkel cell carcinoma, mesothelioma, solid tumors, renal cancer, ovarian cancer, bladder cancer, head and neck cancer, and gastric cancer; 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 has been studied in clinical trials for multiple indications, including: mesothelioma, colorectal cancer, renal cancer, breast cancer, lung cancer 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, liver metastases, hepatocellular carcinoma, 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 that is being studied in a Phase 1 clinical trial for advanced solid tumors (NCT02694822).

[0308] In some embodiments, the checkpoint inhibitor is an inhibitor of T-cell immunoglobulin mucin (TIM-3) containing protein-3. TIM-3 inhibitors that can be used in the present invention include TSR-022, LY3321367 and MBG453. TSR-022 (Tesaro) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT02817633). LY3321367 (Eli Lilly) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT03099109). MBG453 (Novartis) is an anti-TIM-3 antibody that is being studied in advanced malignancies (NCT02608268).

[0309] In some embodiments, the checkpoint inhibitor is an inhibitor of a T cell immunoreceptor with Ig and ITIM domains, or TIGIT (an immunoreceptor on certain T cells and NK cells). TIGIT inhibitors that can 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).

[0310] In some embodiments, the checkpoint inhibitor is an inhibitor of Lymphocyte Activation Gene-3 (LAG-3). LAG-3 inhibitors that can be used in the present invention include BMS-986016, REGN3767, and IMP321. BMS-986016 (Bristol-Myers Squibb) is an anti-LAG-3 antibody that is being studied in glioblastoma and gliosarcoma (NCT02658981). REGN3767 (Regeneron) is also an anti-LAG-3 antibody that 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).

[0311] Checkpoint inhibitors that can be used in the present invention include OX40 agonists. OX40 agonists being studied in clinical trials include: PF-04518600 / PF-8600 (Pfizer), an agonistic anti-OX40 antibody for metastatic renal cancer (NCT03092856) and advanced cancers and neoplasms (NCT02554812; NCT05082566); GSK3174998 (Merck), an agonistic anti-OX40 antibody in a Phase 1 cancer trial (NCT02528357); MEDI0562 (Medimmune / AstraZenec a), an agonist anti-OX40 antibody for advanced solid tumors ((NCT02318394 and NCT02705482); MEDI6469, an agonist anti-OX40 antibody (Medimmune / AstraZeneca) for 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 agonist anti-OX40 antibody for advanced cancers (NCT02737475).

[0312] Checkpoint inhibitors that can be used in the present invention include CD137 (also known as 4-1BB) agonists. CD137 agonists being studied in clinical trials include: utomilumab (PF-05082566, Pfizer), an agonistic anti-CD137 antibody, in diffuse large B-cell lymphoma (NCT02951156) and advanced cancers and neoplasms (NCT02554812 and NCT05082566); Urecitumab (BMS-663513, Bristol-Myers Squibb), an agonistic anti-CD137 antibody, for melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT02658981).

[0313] Checkpoint inhibitors that can be used in the present invention include CD27 agonists. CD27 agonists being studied in clinical trials include: Valirumab (CDX-1127, Celldex Therapeutics), an agonistic anti-CD27 antibody for head and neck squamous cell carcinoma, ovarian cancer, colorectal cancer, renal cell carcinoma, and glioblastoma (NCT02335918); lymphoma (NCT01460134); and glioma and astrocytoma (NCT02924038).

[0314] Checkpoint inhibitors useful in the present invention include glucocorticoid-induced tumor necrosis factor receptor (GITR) agonists. GITR agonists being studied in clinical trials include: TRX518 (Leap Therapeutics), an agonist anti-GITR antibody for malignant melanoma and other solid tumors (NCT01239134 and NCT02628574); GWN323 (Novartis), an agonist anti-GITR antibody for solid tumors and lymphomas (NCT02740270); INCAGN01876 (Incyte / Agenus), an agonist anti-GITR antibody for advanced cancers (NCT02697591 and NCT03126110); MK-4166 (Merck), an agonist anti-GITR antibody for solid tumors (NCT02132754), and MEDI1873 (Medimmune / AstraZeneca), an agonist anti-GITR antibody with human IgG1 An agonistic hexameric GITR-ligand molecule with an Fc domain for use in advanced solid tumors (NCT02583165).

[0315] Checkpoint inhibitors that can be used in the present invention include inducible T-cell co-stimulator (ICOS, also known as CD278) agonists. ICOS agonists being studied in clinical trials include: MEDI-570 (Medimmune), an agonistic anti-ICOS antibody, used in lymphoma (NCT02520791); GSK3359609 (Merck), an agonistic anti-ICOS antibody, used in Phase 1 (NCT02723955); and JTX-2011 (Jounce Therapeutics), an agonistic anti-ICOS antibody, used in Phase 1 (NCT02904226).

[0316] Check point inhibitors useful in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors being studied in clinical trials include: lirelumab (IPH2102 / BMS-986015, Innate Pharma / Bristol-Myers Squibb), an anti-KIR antibody, for leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT01592370); IPH2101 (1-7F9, Innate Pharma), for myeloma (NCT01222286 and NCT01217203); and IPH4102 (Innate Pharma), an anti-KIR antibody that binds to three domains of the long cytoplasmic tail (KIR3DL2), for lymphoma (NCT02593045).

[0317] Checkpoint inhibitors useful in the present invention include CD47 inhibitors of the interaction between CD47 and signal regulatory protein alpha (SIRPα). CD47 / SIRPa inhibitors being studied in clinical trials include: ALX-148 (Alexo Therapeutics), an antagonist variant of (SIRPa) that binds to CD47 and blocks CD47 / SIRPa-mediated signaling, in Phase 1 (NCT03013218); TTI-621 (SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein generated by linking the N-terminal CD47-binding domain of SIRPa to the Fc domain of human IgG1, which works by binding to human CD47 and blocking it from delivering a “don’t eat” signal to macrophages, in Phase 1 (NCT02890368 and NCT02663518) clinical trials; CC-90002 (Celgene), an anti-CD47 antibody, in leukemia (NCT02641002); and Hu5F9-G4 (Forty Seven, Inc.), in colorectal tumors and solid tumors (NCT02953782), acute myeloid leukemia (NCT02678338), and lymphoma (NCT02953509).

[0318] Checkpoint inhibitors that can be used in the present invention include CD73 inhibitors. CD73 inhibitors being studied 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).

[0319] Checkpoint inhibitors useful in the present invention include agonists of stimulators of interferon gene proteins (STING, also known as transmembrane protein 173 or TMEM173). STING agonists being studied in clinical trials include: MK-1454 (Merck), an agonist synthetic cyclic dinucleotide, in lymphoma (NCT03010176); and ADU-S100 (MIW815, AduroBiotech / Novartis), an agonist synthetic cyclic dinucleotide, in Phase 1 (NCT02675439 and NCT03172936).

[0320] Checkpoint inhibitors that can be used in the present invention include CSF1R inhibitors. CSF1R inhibitors being studied in clinical trials include: pexidartinib (PLX3397, Plexxikon), a CSF1R small molecule inhibitor for colorectal cancer, pancreatic cancer, metastatic and advanced cancer (NCT02777710) and melanoma, non-small cell lung cancer, head and neck squamous cell carcinoma, gastrointestinal stromal tumor (GIST) and ovarian cancer (NCT02452424); and IMC-CS4 (LY3022855 , Lilly), an anti-CSF-1R antibody for pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT02718911); and BLZ945 (4-[2((1R,2R)-2-hydroxycyclohexylamino)-benzothiazol-6-yloxy]-pyridine-2-carboxylic acid carboxamide, Novartis), an orally available CSF1R inhibitor for advanced solid tumors (NCT02829723).

[0321] Checkpoint inhibitors useful in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors being studied in clinical trials include monazumab (IPH2201, Innate Pharma), an anti-NKG2A antibody for head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT02557516).

[0322] In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, avastin, durvalumab, atuzumab, or pidilizumab.

[0323] Therapeutic Uses

[0324] The bicyclic peptide of the present invention has a specific use as a nectin-4 binding agent.

[0325] Integrin-4 is a surface molecule that belongs to the integrin protein family, which contains 4 members. Integrins are cell adhesion molecules that play a key role in various biological processes (e.g. polarity, proliferation, differentiation and migration) of 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 integrin-1 (PVRL1) or integrin-4 (PVRL4) lead to ectodermal dysplasia syndrome associated with other abnormalities. Integrin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of the family. Integrin-4 is a tumor-associated antigen that accounts for 50%, 49% and 86% of breast, ovarian and lung cancers, respectively, mainly on tumors with poor prognosis. Its expression is not detected in the corresponding normal tissues. In breast tumors, integrin-4 is mainly expressed in triple-negative and ERBB2+ carcinomas. In the serum of patients with these cancers, soluble forms of integrins-4 were detected and were associated with poor prognosis. Serum integrins-4 levels increased during metastasis and decreased after treatment. These results suggest that integrins-4 may be a reliable target for the treatment of cancer. Therefore, several anti-integrins-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeting integrins-4, which is currently being studied clinically for the treatment of patients with solid tumors.

[0326] Polypeptide ligands selected according to the methods of the present invention can be used for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, etc. Ligands with selected specificity levels can be used in tests involving non-human animals where cross-reactivity is desired, 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 to a predetermined range of antigens can be used to tailor vaccines against specific diseases and pathogens.

[0327] For mammalian administration, substantially pure peptide ligands having at least 90 to 95% homogeneity are preferred, and for pharmaceutical administration (especially when the mammal is a human), 98 to 99% or higher homogeneity is most preferred. After purification, partial purification, or to the desired homogeneity, the selected polypeptide can be used for diagnosis or treatment (including in vitro), or for the development and implementation of assay procedures, immunofluorescence staining, etc. (Lefkovite and Pernis, (1979 and 1981), Immunological Methods, Volumes I and II, Academic Press, New York).

[0328] According to another aspect of the present invention, there is provided a peptide ligand or drug conjugate as defined herein for use in preventing, inhibiting or treating a disease or condition mediated by Integrin-4.

[0329] According to another aspect of the present invention, there is provided a method of preventing, inhibiting or treating a disease or condition mediated by Integrin-4, the method comprising administering to a patient in need thereof an effector group of a peptide ligand as defined herein and a drug conjugate.

[0330] In one embodiment, the binding protein-4 is mammalian binding protein-4. In another embodiment, the binding protein-4 is human binding protein-4.

[0331] In one embodiment, the disease or condition mediated by Integrin-4 is selected from viral infection, ectodermal dysplasia syndrome and other abnormalities, breast, ovarian and lung cancer, metastatic progression and solid tumors.

[0332] In another embodiment, the disease or condition mediated by Integrin-4 is selected from cancer.

[0333] Examples of cancers (and their benign counterparts) that may be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (adenomas and carcinomas of various types, including adenocarcinomas, squamous carcinomas, transitional cell carcinomas, and others), such as bladder and urinary tract cancer, breast cancer, gastrointestinal cancer (including esophagus, stomach (gastric), small intestine, colon, rectum, and anus), liver cancer (hepatocellular carcinoma), gallbladder and biliary system cancer, exocrine pancreatic cancer, kidney cancer, lung cancer (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancer (e.g., tongue cancer, buccal cancer, laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, tonsil cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulvar cancer, penis cancer, cervical cancer, cancers of the uterus, myometrium, endometrium, thyroid (e.g., follicular thyroid carcinoma), adrenal gland, prostate, skin and appendages (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, hyperplastic nevus); hematologic malignancies (i.e., leukemias, lymphomas) and precancerous hematologic disorders, as well as borderline malignancies, including hematologic malignancies and related disorders of the lymphoid lineage (e.g., acute lymphocytic leukemia [ALL], chronic lymphocytic leukemia [CLL], B-cell lymphomas such as diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt's lymphoma, mantle cell lymphoma, T-cell lymphomas and leukemias, natural killer (NK) NK-cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of undetermined significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorder), as well as hematologic malignancies and related disorders of the myeloid lineage (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML), hypereosinophilic syndrome, myelodysplastic disorders, such as polycythemia vera, essential thrombocythemia, and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome, 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 sarcoma, Ewing sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and cutaneous fibrosarcoma protuberans; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas, and glioblastomas, meningiomas, ependymomas, pinealomas, and schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid carcinoma); tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma);and pediatric and embryonal tumors (e.g., medulloblastoma, neuroblastoma, Wilms' tumor, and primitive neuroectodermal tumor); or congenital or other syndromes that predispose patients to malignancy (e.g., xeroderma).

[0334] In another embodiment, the cancer is selected from hematopoietic malignancies, for example selected from: non-Hodgkin lymphoma (NHL), Burkitt 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 lymphoma (HL), and chronic myeloid leukemia (CML).

[0335] In yet another embodiment, the cancer is selected from lung cancer (eg, non-small cell lung cancer), bladder cancer, pancreatic cancer, and breast cancer. The data herein are presented in Examples 1 to 5, which demonstrate that selected bicyclic drug conjugates of the present invention exhibit anti-tumor activity in these cancer models.

[0336] The term "prevention" as referred to herein involves administration of a protective composition prior to induction of a disease. "Suppression" refers to administration of a composition after an induction event but prior to clinical appearance of the disease. "Treatment" involves administration of a protective composition after symptoms of the disease become apparent.

[0337] Animal model systems are available for screening peptide ligands for effectiveness in preventing or treating disease. The present invention facilitates the use of animal model systems, which allow the development of polypeptide ligands that can cross-react with both human and animal targets, thereby allowing the use of animal models.

[0338] In addition, the data provided herein demonstrate the association between copy number variation (CNV) and gene expression of binding factor-4 from multiple tumor types. Therefore, according to another aspect of the present invention, a method for preventing, inhibiting or treating cancer is provided, which comprises administering to a patient in need thereof an effector group and a drug conjugate of a peptide ligand defined herein, wherein the patient is identified as having an increased copy number variation (CNV) of binding factor-4.

[0339] In one embodiment, the cancer is selected from those cancers identified herein as having an increased CNV of integrin-4. In another embodiment, the cancer is selected from those cancers identified herein as having an increased CNV of integrin-4, namely: breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, head and neck cancer, pancreatic cancer, thyroid cancer, colorectal cancer, thymoma, sarcoma, renal clear cell carcinoma (RCC), prostate cancer, and gastric cancer.

[0340] The present invention is further described below with reference to the following examples.

[0341] Example

[0342] Abbreviations

[0343] 1,2,4-TriAz 3-(1,2,4-Triazol-1-yl)-alanine

[0344] 1Nal 1-naphthylalanine

[0345] 2FuAla 2-furanolalanine

[0346] 2MePhe 2-methyl-phenylalanine

[0347] 2Nal 2-naphthylalanine

[0348] 2Pal 2-pyridylalanine

[0349] 3,3-DPA 3,3-diphenylalanine

[0350] 3MePhe 3-Methyl-phenylalanine

[0351] 3Pal 3-pyridylalanine

[0352] 4,4-BPA 4,4-Biphenylalanine

[0353] 4,4-DFP 4,4-difluoroproline

[0354] 4MePhe 4-Methyl-phenylalanine

[0355] 4Pal 4-pyridylalanine

[0356] 4ThiAz β-(4-thiazolyl)-alanine

[0357] 5FTrp 5-Fluoro-L-tryptophan

[0358] Agb 2-amino-4-guanidinobutyric acid

[0359] Aib aminoisobutyric acid

[0360] AzaTrp Azatryptophan

[0361] Aze Azetidine

[0362] C5A Cyclopentylglycine

[0363] Cha 3-cyclohexyl-alanine

[0364] Cpa Cyclopropyl Alanine

[0365] Cya Cysteic Acid

[0366] DOPA 3,4-dihydroxyphenylalanine

[0367] HArg Homoarginine

[0368] HGln High Glutamine

[0369] Hleu Homoleucine

[0370] Hphe Homophenylalanine

[0371] Hse(me) Homoserine(Me)

[0372] Hser Homoserine

[0373] HyP Hydroxyproline

[0374] Lysine(Ac) Lysine(Acetyl)

[0375] Met(O2) Methionine sulfone

[0376] Nle Norleucine

[0377] Oic Octahydroindole carboxylic acid

[0378] Oxa Oxazolidine-4-carboxylic acid

[0379] pCoPhe p-Carboxy-phenylalanine

[0380] PheOPhe 4-phenoxy-phenylalanine

[0381] Phg Phenylglycine

[0382] Pip pipecolic acid

[0383] Pro(4NH) 4-amino-proline

[0384] tBuAla tert-Butyl-alanine

[0385] TetraZ Tetrazolyl Alanine

[0386] Thi Thiamine

[0387] THP(O) Tetrahydropyran-4-propionic acid

[0388] THP(SO2) 4-tetrahydrothiopyranyl acetic acid

[0389] Trp(Me) Methyltryptophan

[0390] Materials and methods

[0391] Peptide synthesis

[0392] Peptide synthesis was based on Fmoc chemistry using a Symphony peptide synthesizer produced by Peptide Instruments and a Syro II synthesizer produced by MultiSynTech. Standard Fmoc-amino acids (Sigma, Merck) were used with appropriate side chain protecting groups: standard coupling conditions were used in each case as appropriate, followed by deprotection using standard methods.

[0393] Alternatively, peptides were purified using HPLC and modified after separation with 1,3,5-triacryloylhexahydro-1,3,5-triazine (TATA, Sigma). To this end, the linear peptides were treated with 50:50 MeCN:H 2 O was diluted to 35 mL, 500 μL of 100 mM TATA in acetonitrile was added, and 5 mL of 1 M NH 4 HCO 3 H 2 The reaction was initiated with 1 mL of 1 M L-cysteine ​​hydrochloride monohydrate (Sigma) in H 2 O was added to the reaction for ~60 min at room temperature to quench any excess TATA.

[0394] After lyophilization, the modified peptide was purified as above, while using a Gemini C18 column (Phenomenex) instead of Luna C8 and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA-modified material were pooled, lyophilized and stored at -20°C.

[0395] Unless otherwise stated, all amino acids were used in the L-configuration.

[0396] In some cases, the peptide was converted to an activated disulfide prior to coupling to a free thiol group of the toxin using the following method; a solution of 4-methyl(succinimidyl 4-(2-pyridylthio)pentanoate) (100 mM) in dry DMSO (1.25 mol eq) was added to a solution of the peptide (20 mM) in dry DMSO (1 mol eq). The reaction was mixed well and DIPEA (20 mol eq) was added. The reaction was monitored by LC / MS until completion.

[0397] Preparation of bicyclic peptide drug conjugates

[0398] Preparation of BCY8549

[0399] Separation conditions: Phase A: H 2 0.075% TFA in O, Phase B: MeCN

[0400] Separation method: 18-48-55 minutes, RT = 53.5 minutes

[0401] Separation columns: Luna 200*25mm 10μm, C18, 110A and Gemin150*30mm, C18, 5μm, 110A, connected, 50℃

[0402] Dissolution method: DMF

[0403] Separation purity: 95%

[0404] BCY8234 was synthesized by solid phase synthesis.

[0405]

[0406]

[0407] Preparation of compound 2

[0408] The peptide was synthesized using standard Fmoc chemistry.

[0409] 1) DCM was added to a container containing CTC resin (5 mmol, 4.3 g, 1.17 mmol / g) and Fmoc-Cit-OH (2.0 g, 5 mmol, 1.0 eq) and heated with N 2 Bubbling.

[0410] 2) DIEA (4.0 eq) was added dropwise and mixed for 2 hours.

[0411] 3) Add MeOH (5 mL) and mix for 30 minutes.

[0412] 4) Drain and wash with DMF 5 times.

[0413] 5) Add 20% piperidine / DMF and react for 30 minutes.

[0414] 6) Drain and wash 5 times with DMF.

[0415] 7) Add Fmoc-amino acid solution and mix for 30 seconds, then add activation buffer, N 2 Bubble for about 1 hour.

[0416] 8) Repeat steps 4 to 7 above to couple the following amino acids.

[0417] Notice:

[0418]

[0419] 20% piperidine in DMF was used for Fmoc deprotection for 30 min. The coupling reaction was monitored by ninhydrin test and the resin was washed 5 times with DMF.

[0420] Peptide cleavage and purification:

[0421] 1) Add lysis buffer (20% TFIP / 80% DCM) to the flask containing the side-chain protected peptide at room temperature and stir twice for 1 hour.

[0422] 2) Filter and collect the filtrate.

[0423] 3) Concentrate to remove the solvent.

[0424] 4) The crude peptide was lyophilized to obtain the final product (1.4 g, 85.0% yield).

[0425] Preparation of compound 3

[0426] EEDQ (2.48 g, 10.02 mmol, 2.0 eq) and (4-aminophenyl)methanol (740.37 mg, 6.01 mmol, 1.2 eq) were added to a solution of compound 2 (1.65 g, 5.01 mmol, 1.0 eq) in DCM (30 mL) and MeOH (15 mL). The mixture was stirred at 15 ° C for 16 hours. LC-MS showed that compound 2 was completely consumed and a main 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 to obtain a residue. The product was purified by flash silica gel chromatography ( 80 The residue was purified by silica flash column, gradient elution (0-15 DCM / MeOH@60 mL / min) to obtain compound 3 (1.3 g, 2.99 mmol, yield 59.72%) as a yellow solid.

[0427] Preparation of compound 4

[0428] DIEA (2.32 g, 17.95 mmol, 3.13 mL, 6.0 eq) and bis(4-nitrophenyl) carbonate (3.64 g, 11.97 mmol, 4.0 eq) were added to a DMF (10 ml) solution of compound 3 (1.3 g, 2.99 mmol, 1.0 eq). The mixture was stirred at 15 ° C for 1 hour. LC-MS showed that compound 3 was completely consumed, and a main peak with the desired m / z was detected. The residue was purified by preparative HPLC (neutral conditions). Compound 4 (1.0 g, 1.67 mmol, yield 55.74%) was obtained as a yellow solid.

[0429] Preparation of compound 5

[0430] HOBt (56.46 mg, 417.84 μmol, 1.5 eq) and DIEA (108.01 mg, 835.68 μmol, 145.56 μL, 3.0 eq) and MMAE (0.200 g, 278.56 μmol, 1.0 eq) were added to a DMF (5 mL) solution of compound 5 (250.53 mg, 417.84 μmol, 1.5 eq). The mixture was stirred at 35 ° C for 12 hours. LC-MS showed that MMAE was completely consumed, and a main peak was detected with the desired m / z. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 5 (0.180 g, 152.74 μmol, yield 54.83%) was obtained as a yellow solid.

[0431] Preparation of compound 6

[0432] Compound 5 (0.170 g, 144.26 μmol, 1.0 eq) in THF (5 mL) and H 2 Add LiOH.H O (5 mL) solution 2 O (12.11 mg, 288.51 μmol, 2.0 eq). The mixture was stirred at 15 ° C for 1 hour. LC-MS showed that compound 5 was completely consumed, and a main peak was detected with the desired m / z. The pH was adjusted to 7 using AcOH, and THF was removed under reduced pressure to obtain a residue. The residue was purified by preparative HPLC (neutral conditions). Compound 6 (0.185 g, crude product) was obtained as a yellow solid.

[0433] Preparation of BCY8549

[0434] To a solution of compound 6 (0.100 g, 86.93 μmol, 1.0 eq) in DMA (4 mL) was added HOSu (10.00 mg, 86.93 μmol, 1.0 eq) and EDCI (16.66 mg, 86.93 μmol, 1.0 eq). After the NHS ester formation, β-Ala-BCY8234 (525.98 mg, 173.85 μmol, 2.0 eq) and DIEA (33.70 mg, 260.78 μmol, 45.42 μL, 3.0 eq). The mixture was stirred at 15 °C for 4 hours. LC-MS showed that compound 6 was completely consumed, and a major peak with the desired m / z was detected. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY8549 (0.0528 g, 12.15 μmol, yield 13.98%, purity 95.70%) was obtained as a white solid. Retention time = 11.48 minutes. Displayed mass = 1386.4 (M / 3+H).

[0435] Preparation of BCY8245

[0436] Separation conditions: Phase A: H 2 0.075% TFA in O, Phase B: MeCN

[0437] Separation method: 18-48-55 minutes, RT = 53.5 minutes

[0438] Separation column: Luna 200*25mm 10um, C18, 110A and Gemin150*30mm, C18, 5um, 110A, connected, 50℃

[0439] Dissolution method: DMF

[0440] Separation purity: 95%

[0441] BCY8234 was synthesized by solid phase synthesis.

[0442] The reaction diagram of BCY8245 is shown below

[0443]

[0444]

[0445] Preparation of compound 3

[0446] Compound 3 was synthesized by a solid phase method.

[0447] Preparation of compound 4

[0448] EEDQ (1.60 g, 6.46 mmol, 2.0 eq) and (4-aminophenyl)methanol (517.16 mg, 4.20 mmol, 1.3 eq) were added to a solution of compound 3 (1.3 g, 3.23 mmol, 1.0 eq) in DCM (10 mL) and MeOH (5 mL). The mixture was stirred at 20 ° C for 16 hours. LC-MS showed that compound 3 was completely consumed, and a major peak with the desired m / z was detected. The solvent was removed under reduced pressure. The product was purified by flash silica gel chromatography ( 40g The residue was purified by silica flash column, gradient elution 0-15% DCM / MeOH @ 40 mL / min) to obtain compound 4 (0.950 g, 1.87 mmol, yield 57.94%) as a yellow solid.

[0449] Preparation of compound 5

[0450] DIEA (1.21 g, 9.36 mmol, 1.63 mL, 5.0 eq) and bis(4-nitrophenyl) carbonate (2.28 g, 7.49 mmol, 4.0 eq) were added to a DMF (5 ml) solution of compound 4 (0.950 g, 1.87 mmol, 1.0 eq). The mixture was stirred at 20 ° C for 1 hour. LC-MS showed that compound 4 was completely consumed, and a main peak with the desired m / z was detected. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 5 (0.400 g, 594.64 μmol, yield 31.77%) was obtained as a white solid.

[0451] Preparation of compound 6

[0452] To a DMF (5 mL) solution of compound 5 (0.200 g, 297.32 μmol, 1.0 eq) was added HOBt (52.23 mg, 386.51 μmol, 1.3 eq) and DIEA (115.28 mg, 891.95 μmol, 155.36 μL, 3.0 eq), MMAE (192.12 mg, 267.59 μmol, 0.9 eq). The mixture was stirred at 20 ° C for 16 hours. LC-MS showed that compound 5 was completely consumed, and a main peak was detected with the desired m / z. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 6 (0.160 g, 127.84 μmol, yield 43.00%) was obtained as a white solid.

[0453] Preparation of compound 7

[0454] Compound 6 (0.160 g, 127.84 μmol, 1.0 eq) in THF (3 mL) and H 2 Add LiOH.H O (3 mL) solution 2 O (26.82 mg, 639.21 μmol, 5.0 eq). The mixture was stirred at 20 ° C for 1 hour. LC-MS showed that compound 6 was completely consumed, and a main peak with the desired m / z was detected. THF was removed under reduced pressure, and pH=7 was adjusted by AcOH, and the mixture was lyophilized. Compound 7 (0.130 g, 105.05 μmol, yield 82.17%) was obtained as a white solid.

[0455] Preparation of compound 8

[0456] EDCI (60.41 mg, 315.15 μmol, 3.0 eq) was added to a solution of compound 7 (36.27 mg, 315.15 μmol, 3.0 eq) in DMA (6 mL) and DCM (2 mL). The mixture was stirred at 15 ° C for 3 hours. LC-MS showed that compound 7 was completely consumed, and a main peak with the desired m / z was detected. DCM was removed under reduced pressure. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 8 (0.095 g, 71.18 μmol, yield 67.76%) was obtained as a white solid.

[0457] Preparation of BCY8245

[0458] DIEA (8.72 mg, 67.44 μmol, 11.75 μL, 3.0 eq) and compound 8 (0.030 g, 22.48 μmol, 1.0 eq) were added to a solution of BCY8234 (66.41 mg, 22.48 μmol, 1.0 eq) in DMA (4 mL). The mixture was stirred at 20 ° C for 16 hours. LC-MS showed that BCY8234 was completely consumed, and a main peak was detected with the desired m / z or desired mass. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY8245 (0.0427 g, 10.16 μmol, yield 45.19%, purity 99.30%) was obtained as a white solid. Retention time = 11.7 minutes. Display mass (Mass found) = 1043.9 (M / 4+H).

[0459] Biological data

[0460] Integrin-4 direct binding assay

[0461] According to the method disclosed in WO 2016 / 067035, the affinity (Ki) of the peptides of the present invention for human nectin-4 was determined using fluorescence polarization assay. The peptides of the present invention with fluorescent tags (fluorescein, SIGMA or Alexa Fluor 488 TM, Fisher Scientific) in PBS (containing 0.01% Tween 20), or in 50mM HEPES (containing 100mM NaCl and 0.01% Tween pH 7.4) (both referred to as assay buffer) to 2.5nM. This was combined with the titrated protein in the same assay buffer as the peptide to give 1 nM peptide in a total volume of 25μL, typically 5μL assay buffer, 10μL protein and then 10μL fluorescent peptide in a black walled and bottomed low binding low volume 384-well plate. Serial dilutions of 1 to 2 were used to give 12 different concentrations, with the highest concentration ranging from 500nM for known high affinity binders to 10μM for low affinity binders and selectivity assays. Measurements were performed on a BMG PHERAstar FS equipped with an "FP 485 520 520" optical module that excites at 485nm and detects parallel and perpendicular emission at 520nm. The PHERAstar FS was set at 25°C, 200 flashes per well, a localization delay of 0.1 seconds, and each well was measured for 60 minutes at intervals of 5 to 10 minutes. At the end of 60 minutes, the gain for the analysis was determined for each tracer in the absence of protein in the well. The data were analyzed using Systat Sigmaplot version 12.0. The mP values ​​were fitted to a user-defined quadratic equation to generate Kd values: f = ymin + (ymax - ymin) / Lig * ((x + Lig + Kd) / 2 - sqrt ((((x + Lig + Kd) / 2) ^ 2) - (Lig * x))). "Lig" is the defined value for the tracer concentration used.

[0462] Bindase-4 competition binding assay

[0463] Peptides without fluorescent tags were tested for competition with ACPFGCHTDWSWPIWCA-Sar6-K(Fl) (SEQ ID NO:2) (Kd = 5 nM - determined using the protocol described above). Peptides were diluted to the appropriate concentration in assay buffer using a maximum of 5% DMSO followed by a 1 in 2 serial dilution as described for the direct binding assay. 5 μL of diluted peptide was added to the plate followed by 10 μL of human ligase-4 and then 10 μL of fluorescent peptide. Measurements were performed as in the direct binding assay, however the gain was determined prior to the first measurement. Data were analyzed using Systat Sigmaplot version 12.0 where mP values ​​were fit to a user defined cubic equation to generate Ki values:

[0464] 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+CompProt*c)^3+9*(Klig+Kcomp+Lig+Comp-Prot*c)'(Kc omp*(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-P rot*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)))).

[0465] "Lig", "KLig" and "Prot" are defined values ​​related to: fluorescent peptide concentration, Kd of fluorescent peptide and binding protein concentration, respectively.

[0466] Integrin-4 Biacore SPR binding assay

[0467] Biacore experiments were performed to determine the k of monomeric peptides binding to human Necin-4 protein. a (M-i s -1 ), k d (s -1 ), K D (nM) values ​​(obtained from Charles River).

[0468] Human integrin-4 (residues Gly32-Ser349; NCBI reference sequence: NP_112178.2) with a gp67 signal sequence and a C-terminal FLAG tag was transfected into the 5′-cell suspension using standard Bac-to-Bac TM Protocol (Life Technologies) cloned into pFastbac-1 and baculovirus. P1 virus stock was used to infect 1x10 cells in Excell-420 medium (Sigma) at 27°C with an M01 of 2. 6 m1 -1 Sf21 cells were added and the supernatant was harvested at 72 hours. The supernatant was combined with anti-FLAG M2 affinity agarose resin (Sigma) washed in PBS for 1 hour in batches at 4 ° C, and then the resin was transferred to the column and fully washed with PBS. The protein was eluted with 100pg / ml of FLAG peptide. The eluted protein was concentrated to 2ml and loaded onto a S-200 Superdex column (GE Healthcare) in PBS at a speed of 1ml / min. 2ml fractions were collected and the fractions containing the binding factor-4 protein were concentrated to 16mg / ml.

[0469] Follow the manufacturer's recommended procedures to use EZ-Link TM Proteins were randomly biotinylated using Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) in PBS. Proteins were extensively desalted using spin columns to remove uncoupled biotin into PBS.

[0470] For analysis of peptide binding, a Biacore 3000 instrument was used, which utilizes a CM5 chip (GE Healthcare). 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, a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) was injected at a flow rate of 10 gl / min for 7 minutes to activate the carboxymethyl dextran surface. For capture of streptavidin, the protein was diluted to 0.2 mg / ml in 10 mM sodium acetate (pH 4.5) and captured by injecting 120 gl of streptavidin onto the activated chip surface. Residual activated groups were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 minutes, and biotinylated bindin-4 was captured to a level of 1,200-1,800 RU. The buffer was changed to PBS / 0.05% Tween 20, and a series of peptide dilutions were prepared in this buffer with a final DMSO concentration of 0.5%. The highest peptide concentration was 100 nM, and it was diluted 6 times in 2-fold dilutions. SPR analysis was run at 25°C at a flow rate of 50 μl / min, with an association time of 60 seconds and dissociation times of 400 to 1200 seconds depending on the individual peptides. Data were corrected for DMSO exclusion volume effects. All data were double-referenced 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). Data were fit using a simple 1:1 binding model to account for mass transfer effects where appropriate.

[0471] Certain peptide ligands of the invention were tested in the above described binding protein-4 binding assay and the results are shown in Table 1:

[0472] Table 1: Competition binding data of selected peptide ligands of the present invention

[0473] Double Ring <![CDATA[K i (μM)]]> Experiment No. BCY8122 0.003 2 BCY8126 0.0027 6

[0474] Certain bicyclic peptides of the invention were tested in the above SPR assay and the results are shown in Table 2:

[0475] Table 2: SPR data of selected peptide ligands of the present invention

[0476]

[0477] n = average number of experiments

[0478] Certain bicyclic peptides of the invention were conjugated to cytotoxic agents and tested in the above SPR assay, the results are shown in Table 3:

[0479] Table 3: SPR data of BDC selected by the present invention

[0480]

[0481] In vivo studies

[0482] In Examples 1 to 5 and 9, each study employed the following methods:

[0483] Test and Positive Controls

[0484] Table 4-1

[0485]

[0486]

[0487] Experimental methods and procedures

[0488] (i) Observation

[0489] All procedures related to animal handling, care, and treatment in the studies were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec and followed the guidance of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). During routine monitoring, animals were checked daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain / loss, eye / hair gloss, and any other abnormal effects stated in the operating procedures. Deaths and observed clinical signs were recorded based on the number of animals in each subset.

[0490] (ii) Tumor measurements and endpoints

[0491] The primary endpoint was to see if tumor growth could be delayed or if the mice could be cured. Tumor volume was measured in two dimensions three times a week using calipers and expressed in mm using the following formula: 3 Indicates volume: V = 0.5axb 2 , where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used to calculate the T / C value. The T / C value (percentage) represents the antitumor effect; T and C are the average volumes of the treatment group and the control group, respectively, on the specified day.

[0492] TGI was calculated for each group using the following formula: TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )]×100;T i is the mean tumor volume of the treatment group on a given day, T 0 is the mean tumor volume of the treatment group on the day of treatment initiation, V i The vehicle control group was compared with T i The average tumor volume on the same day, V 0 is the mean tumor volume of the vehicle group on the day of treatment initiation.

[0493] (iii) Statistical analysis

[0494] A statistical summary of tumor volume for each group at each time point is provided, including the mean and standard error of the mean (SEM).

[0495] The differences in tumor volume between the groups were statistically analyzed based on the data obtained at the optimal treatment time point after the final dose.

[0496] One-way ANOVA was performed to compare tumor volumes between groups, and when a significant F-statistic (ratio of treatment difference to error difference) was obtained, the Games-Howell test was used for comparisons between groups. All data were analyzed using GraphPadPrism 5.0. P < 0.05 was considered statistically significant.

[0497] Example 1: In vivo efficacy testing of BCY8245 treatment of NCI-H292 xenografts (non-small cell lung cancer (NSCLC) model) in BALB / c nude mice.

[0498] 1. Research objectives

[0499] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY8245 in treating the NCI-H292 xenograft model in BALB / c nude mice.

[0500] 2. Experimental Design

[0501] Table 4-2

[0502]

[0503] Note: n: animal number; Dosing volume: 10 μL / g based on body weight adjustment.

[0504] 3. Materials

[0505] 3.1 Animals and housing conditions

[0506] 3.1.1. Animals

[0507] Species: House mouse (Mus Musculus)

[0508] Strain: Balb / c nude mice

[0509] Age: 6-8 weeks

[0510] Gender: Female

[0511] Weight: 18-22 g

[0512] Number of animals: 18 mice for BCY8245 plus spare

[0513] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[0514] 3.1.2. Rearing conditions

[0515] Mice were kept in individually ventilated cages at constant temperature and humidity, with 3 animals per cage.

[0516] Temperature: 20-26℃.

[0517] Humidity 40-70%.

[0518] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[0519] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[0520] Drinking water: Animals had free access to sterile drinking water.

[0521] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[0522] Animal identification: Tagging animals with ear codes.

[0523] 3.2 Test and Positive Control

[0524] 4. Experimental methods and procedures

[0525] 4.1 Cell culture

[0526] NCI-H292 tumor cells were maintained in culture medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO2 in air. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0527] 4.2 Tumor inoculation

[0528] Each mouse was inoculated subcutaneously on the right abdomen with NCI-H292 tumor cells (10 × 10 6 ) for tumor growth. When the average tumor volume reached approximately 158–406 mm 3 At 14:00, animals were randomized and treatments were started. The experimental design table below shows the test article administration and the number of animals in each group.

[0529] 4.3 Preparation of test article

[0530] Table 4-

[0531]

[0532] Table 4-4

[0533]

[0534] 4.4 Sample collection

[0535] At the end of the study, plasma was collected 5, 15, 30, 60 and 120 minutes after the last dose.

[0536] 5. Results

[0537] 5.1 Tumor growth curve

[0538] Tumor growth curve Figure 1 and Figure 2 shown.

[0539] 5.2 Tumor volume trajectory

[0540] The following table shows the mean tumor volume over time in female Balb / c nude mice bearing NCI-H292 xenografts:

[0541] Table 4-5: Trajectory of tumor volume changes over time

[0542]

[0543]

[0544] Table 5: Trajectory of tumor volume changes over time

[0545]

[0546] 5.3 Tumor Growth Inhibition Analysis

[0547] Tumor growth inhibition rate of BCY8245 in the NCI-H292 xenograft model was calculated on day 14 based on tumor volume measurements.

[0548] Table 6: Tumor growth inhibition analysis

[0549]

[0550] a. Mean ± SEM.

[0551] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0552] Table 7-1: Tumor growth inhibition analysis

[0553]

[0554] a. Mean ± SEM.

[0555] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0556] 6. Summary and discussion of results

[0557] In this study, the therapeutic efficacy of BCY8245 was evaluated in the NCI-H292 xenograft model. Figure 1 and Figure 2 And Tables 4-5 to 7-1 show the tumor volumes measured for all treatment groups at various time points.

[0558] On day 14, the mean tumor size in vehicle-treated mice reached 879 mm 3 .

[0559] BCY8245 at 1 mg / kg did not produce significant antitumor activity, and the test substance showed significant antitumor activity after increasing the dose to 3 mg / kg starting from day 7, but the efficacy was not further improved when the dose was increased to 5 mg / kg on day 21. In this study, all treated animals showed sustained weight loss during the dosing regimen, which may be due to tumor burden and toxicity of the test substance.

[0560] BCY8245 was 3mg / kg, qw (TV = 149mm 3 ,TGI=101.4%,p<0.001),3mg / kg,biw

[0561] (TV=65mm 3 , TGI=112.2%, p<0.001) and 5mg / kg, qw (TV=83mm 3 , TGI=109.8%, p<0.001) produced significant anti-tumor activity.

[0562] Example 2: In vivo efficacy testing of BCY7825, BCY8245, BCY8253, BCY8254 and BCY8255 in the treatment of HT-1376 xenografts (bladder cancer model) in CB17-SCID mice

[0563] 1. Research objectives

[0564] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of HT-1376 xenografts in CB17-SCID mice.

[0565] 2. Experimental Design

[0566] Table 7-2

[0567]

[0568]

[0569] a 1 mg / kg in the first week, 3 mg / kg in the next 2 weeks

[0570] 3. Materials

[0571] 3.1 Animals and housing conditions

[0572] 3.1.1. Animals

[0573] Species: House mouse

[0574] Germline: CB17-SCID

[0575] Age: 6-8 weeks

[0576] Gender: Female

[0577] Weight: 18-22 g

[0578] Number of animals: 21-41 mice plus spare

[0579] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[0580] 3.1.2. Rearing conditions

[0581] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[0582] Temperature: 20-26℃.

[0583] Humidity 40-70%.

[0584] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[0585] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[0586] Drinking water: Animals had free access to sterile drinking water.

[0587] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[0588] Animal identification: Tagging animals with ear codes.

[0589] 4. Experimental methods and procedures

[0590] 4.1 Cell culture

[0591] HT-1376 tumor cells were maintained in vitro as monolayer cultures in EMEM supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0592] 4.2 Tumor inoculation

[0593] Each mouse was inoculated subcutaneously on the right flank with 0.2 ml of HT-1376 tumor cells (5 × 10 6 ) for tumor growth. When the average tumor volume reached 153-164mm 3 The animals were randomized at 1:10. The experimental design table shows the test article administration and the number of animals in each group.

[0594] 4.3 Preparation of test article

[0595] Table 7-3

[0596]

[0597] Table 7-4

[0598]

[0599] 4.4 Sample collection

[0600] At the end of the study, plasma was collected from Group 2 at 5, 15, 30, 60 and 120 minutes after the last dose. Plasma was collected from Group 6 at 5, 15, 30, 60 and 120 minutes after the last dose. Tumors were collected from Group 6 at 2 hours after the last dose. Tumors were collected from Groups 4 and 5 at 2 hours after the last dose.

[0601] 5. Results

[0602] 5.1 Tumor growth curve

[0603] Tumor growth curve Figure 3 and Figure 4 shown.

[0604] 5.2 Tumor volume trajectory

[0605] Tables 8 and 9 show the change in mean tumor volume over time in female CB17-SCID mice bearing HT-1376 xenografts.

[0606]

[0607] Table 9: Trajectory of tumor volume changes over time

[0608]

[0609] 5.3 Tumor Growth Inhibition Analysis

[0610] The tumor growth inhibition rate of the test articles in the HT-1376 xenograft model was calculated based on tumor volume measurements on day 21 after the start of treatment.

[0611] Table 10: Tumor Growth Inhibition Analysis

[0612]

[0613] a. Mean ± SEM.

[0614] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0615] Table 11-1: Tumor growth inhibition analysis

[0616]

[0617] a. Mean ± SEM.

[0618] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0619] 6. Summary and discussion of results

[0620] Group 1 and Group 2

[0621] In this study, the therapeutic efficacy of the test article was evaluated in the HT-1376 xenograft model. Figure 3 and Tables 8 and 10 show the tumor volumes of all treatment groups measured at different time points.

[0622] On day 21, the mean tumor size in vehicle-treated mice reached 884 mm 3 BCY8245 at 1 mg / kg produced mild antitumor activity, and better efficacy was found after increasing the dose to 3 mg / kg from day 7 onwards.

[0623] In this study, some mice treated with 3 mg / kg of the test article lost more than 10% of their body weight.

[0624] Groups 3-6

[0625] In this study, the therapeutic efficacy of the test article was evaluated in the HT-1376 xenograft model. Figure 4 And Table 9 and Table 11-1 show the tumor volumes of all treatment groups measured at different time points.

[0626] BCY8245 was 3mg / kg, qw (TV = 603mm 3 ,TGI=50.9%,p<0.01),3mg / kg,biw

[0627] (TV=407mm 3 , TGI=72.3%, p<0.001) and 5mg / kg, qw (TV=465mm 3 , TGI=66.0%, p<0.001) produced significant anti-tumor activity.

[0628] In this study, BCY8245 at 5 mg / kg qw caused greater than 10% body weight loss in animals over the treatment period.

[0629] Example 3: In vivo efficacy study of BCY8245 in treating Panc2.13 xenografts (pancreatic cancer model) in Balb / c nude mice

[0630] 1. Research objectives

[0631] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of Panc2.13 xenografts in Balb / c nude mice.

[0632] 2. Experimental Design

[0633] Table 11-2

[0634]

[0635] 3. Materials

[0636] 3.1 Animals and housing conditions

[0637] 3.1.1 Animals

[0638] Species: House mouse

[0639] Strain: Balb / c nude mice

[0640] Age: 6-8 weeks

[0641] Gender: Female

[0642] Weight: 18-22 g

[0643] Number of animals: 41 mice plus spare

[0644] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[0645] 3.1.2. Rearing conditions

[0646] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[0647] Temperature: 20-26℃.

[0648] Humidity 40-70%.

[0649] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[0650] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[0651] Drinking water: Animals had free access to sterile drinking water.

[0652] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[0653] Animal identification: Tagging animals with ear codes.

[0654] 4. Experimental methods and procedures

[0655] 4.1 Cell culture

[0656] Panc2.13 tumor cells were maintained in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units / ml human recombinant insulin at 37°C in an atmosphere of 5% CO 2 The cells were maintained in an atmosphere of 40 °C. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0657] 4.2 Tumor inoculation

[0658] Each mouse was inoculated subcutaneously in the right abdomen with 0.2 ml of PBS (1:1) containing Matrigel and Panc2.13 tumor cells (5 × 10 6 ) for tumor formation. When the average tumor volume reached 149mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[0659] 4.3 Preparation of test article

[0660] Table 11-3

[0661]

[0662] 4.4 Sample collection

[0663] At the end of the study, tumors from all groups were collected 2 h after the last dose.

[0664] 5. Results

[0665] 5.1 Tumor growth curve

[0666] exist Figure 5 Tumor growth curves are shown in

[0667] 5.2 Tumor volume trajectory

[0668] The mean tumor volume over time in female Balb / c nude mice bearing Panc2.13 xenografts is shown in Table 12 below.

[0669] Table 12: Trajectory of tumor volume changes over time

[0670]

[0671] 5.3 Tumor Growth Inhibition Analysis

[0672] The tumor growth inhibition rate of the test articles in the Panc2.13 xenograft model was calculated based on tumor volume measurements on day 14 after the start of treatment.

[0673] Table 13-1: Tumor growth inhibition analysis

[0674]

[0675] a. Mean ± SEM.

[0676] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0677] 6. Summary and discussion of results

[0678] In this study, the therapeutic efficacy of the test article was evaluated in the Panc2.13 xenograft model. Figure 5 And Table 12 and Table 13-1 show the tumor volumes of all treatment groups measured at different time points.

[0679] BCY8245 was 3mg / kg, qw (TV = 271mm 3 ,TGI=69.2%, p<0.01), 3mg / kg,biw

[0680] (TV=231mm 3 , TGI=79.1%, p<0.001) and 5mg / kg, qw (TV=238mm 3 , TGI=77.5%, p<0.001) produced significant anti-tumor activity.

[0681] Example 4: In vivo efficacy study of BCY8245 in treating MDA-MB-468 xenografts (breast cancer model) in Balb / c nude mice

[0682] 1. Research objectives

[0683] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY8245 in the treatment of MDA-MB-468 xenografts in Balb / c nude mice.

[0684] 2. Experimental Design

[0685] Table 13-2

[0686]

[0687] 3. Materials

[0688] 3.1 Animals and housing conditions

[0689] 3.1.1 Animals

[0690] Species: House mouse

[0691] Strain: Balb / c nude mice

[0692] Age: 6-8 weeks

[0693] Gender: Female

[0694] Weight: 18-22 g

[0695] Number of animals: 41 mice plus spare

[0696] Animal supplier: Shanghai LC Laboratory Animal Co.,LTD.

[0697] 3.1.2. Rearing conditions

[0698] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[0699] Temperature: 20-26℃.

[0700] Humidity 40-70%.

[0701] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[0702] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[0703] Drinking water: Animals had free access to sterile drinking water.

[0704] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[0705] Animal identification: Tagging animals with ear codes.

[0706] 4. Experimental methods and procedures

[0707] 4.1 Cell culture

[0708] Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 The cells were maintained in an atmosphere of 40 °C. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0709] 4.2 Tumor inoculation

[0710] Each mouse was inoculated subcutaneously in the right flank with MDA-MB-468 tumor cells (10 × 10 6 ) for tumor growth. When the average tumor volume reached 196mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[0711] 4.3 Preparation of test article

[0712] Table 13-3

[0713]

[0714] 4.4 Sample collection

[0715] On day 21 of the study, plasma was collected from group 2 at 5, 15, 30, 60 and 120 minutes after the last dose. Tumors were collected 2 h after the last dose from groups 1 and 3. Animals in group 4 were kept for another 21 days without any dosing, and tumors from these groups were collected on day 42.

[0716] 5. Results

[0717] 5.1 Tumor growth curve

[0718] exist Figure 6 Tumor growth curves are shown in

[0719] 5.2 Tumor volume trajectory

[0720] The mean tumor volume over time in female Balb / c nude mice bearing MDA-MB-468 xenografts is shown below in Tables 14 and 15.

[0721]

[0722] 5.3 Tumor Growth Inhibition Analysis

[0723] The tumor growth inhibition rate of the test articles in the MDA-MB-468 xenograft model was calculated based on tumor volume measurements on day 21 after the start of treatment.

[0724] Table 16-1: Tumor growth inhibition analysis

[0725]

[0726] a. Mean ± SEM.

[0727] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0728] 6. Summary and discussion of results

[0729] In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. Figure 6 and Tables 14 to 16-1 show the tumor volumes of all treatment groups measured at different time points.

[0730] BCY8245 was 3mg / kg, qw (TV = 85mm 3 ,TGI=144.2%,p<0.001),3mg / kg,biw

[0731] (TV=22mm 3 , TGI=169.8%, p<0.001) and 5mg / kg, qw (TV=29mm 3 , TGI=168.4%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[0732] Dosing for the 5 mg / kg group was suspended from day 21, and the tumor did not show any recurrence during an additional three-week monitoring period.

[0733] Example 5: In vivo efficacy testing of BCY8549 in treating NCI-H292 xenografts (non-small cell lung cancer (NSCLC) model) in BALB / c nude mice.

[0734] 1. Research objectives

[0735] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY8549 in the treatment of NCI-H292 xenografts in Balb / c nude mice.

[0736] 2. Experimental Design

[0737] Table 16-2

[0738]

[0739] 3. Materials

[0740] 3.1 Animals and housing conditions

[0741] 3.1.1. Animals

[0742] Species: House mouse

[0743] Strain: Balb / c nude mice

[0744] Age: 6-8 weeks

[0745] Gender: Female

[0746] Weight: 18-22 g

[0747] Number of animals: 43 mice plus spare

[0748] Animal supplier: Shanghai Lingchang Biotechnology Experimental Animal Co. Ltd

[0749] 3.1.2. Rearing conditions

[0750] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 4 animals per cage.

[0751] Temperature: 20-26℃.

[0752] Humidity 40-70%.

[0753] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[0754] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[0755] Drinking water: Animals had free access to sterile drinking water.

[0756] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[0757] Animal identification: Tagging animals with ear codes.

[0758] 4. Experimental methods and procedures

[0759] 4.1 Cell culture

[0760] NCI-H292 tumor cells were cultured as monolayers in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0761] 4.2 Tumor inoculation

[0762] Each mouse was inoculated subcutaneously in the right abdomen with NCI-H292 tumor cells (10×10 6 ) for tumor growth. When the average tumor volume reached 168mm 3 43 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[0763] Test article preparation

[0764] Table 16-3

[0765]

[0766] 4.4 Sample collection

[0767] At the end of the study, plasma from Group 2 was collected at 5 min, 15 min, 30 min, 1 h and 2 h after the last dose.

[0768] 5. Results

[0769] 5.1 Tumor growth curve

[0770] exist Figure 7 Tumor growth curves are shown in

[0771] 5.2 Tumor volume trajectory

[0772] The mean tumor volume over time in female Balb / c nude mice bearing NCI-H292 xenografts is shown below in Table 17.

[0773] Table 17: Trajectory of tumor volume changes over time

[0774]

[0775] 5.3 Tumor Growth Inhibition Analysis

[0776] Tumor growth inhibition rates of the test articles in the NCI-H292 xenograft model were calculated based on tumor volume measurements on day 14 after the start of treatment.

[0777] Table 18: Tumor Growth Inhibition Analysis

[0778]

[0779] a. Mean ± SEM.

[0780] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0781] 6. Summary and discussion of results

[0782] In this study, the therapeutic efficacy of BCY et al. was evaluated in the NCI-H292 xenograft model. Figure 7 and Tables 17 and 18 show the tumor volumes of all treatment groups measured at different time points.

[0783] On day 14, the mean tumor size in vehicle-treated mice reached 843 mm 3 BCY8549 at 3 mg / kg showed significant antitumor activity. In this study, the body weight of all mice was well maintained.

[0784] Example 6: Investigating the correlation between copy number variation (CNV) and gene expression of integrin-4 from multiple tumor types

[0785] method

[0786] 1. Select cBioPortal (http: / / www.cbioportal.org / ) All studies in and search NECTIN4.

[0787] (a) Removal of temporary studies.

[0788] (b) Deselect studies with overlapping samples to prevent sample bias (based on warnings in cBioPortal) - always keep the PanCancer study if you can choose.

[0789] (c) Studies were selected for analysis (Table 19).

[0790] Table 19: Analyzed studies from cBioPortal and research units

[0791]

[0792]

[0793]

[0794] 2. Export CNV and RNA expression data from cBioPortal.

[0795] 3. Test whether CNV is statistically significantly associated with changes in integrin-4 mRNA expression (log2 not applied).

[0796] (a) Nonparametric Kruskal-Wallis test (significance threshold: p<0.01) was run in GraphPad Prism (7.04) and R / R workbench (studio).

[0797] (i) GraphPad Prism: Set up the list and run nonparametric tests that are unmatched or unpaired and do not use a Gaussian distribution.

[0798] (ii) Software packages used in R:

[0799] 1.XLConnect

[0800] 2. dplyr

[0801] 3.Kruskal-Wallis rank-sum test: Kruskal test.

[0802] 4. Dunn's test was used in R / R Workbench to adjust for multiple comparisons (including all possible comparisons even if n=1 in a group) (significance threshold: p<0.025).

[0803] (a) dunn.test = "bonferonni" using the multiple comparison method.

[0804] result

[0805] The results are shown in Table 20-1 below. Among the 41 publicly available TCGA datasets reporting tumor CNV and mRNA gene expression data for bindin-4, many indications have reported cases with increased (2-3 copies) or amplified (>3 copies) copies of bindin-4. In addition, individual cases with shallow deletions (<2 copies) have been shown to rarely report tumors containing deep deletions, which is consistent with more than 1 copy loss or biallelic bindin-4 loss. The most commonly detected indications for amplification are breast cancer (10-22%), bladder cancer (20%), lung cancer (5-7%), and hepatocellular carcinoma (8%). The most frequent signs of copy number loss are renal chromophobe carcinoma (77%), renal clear cell carcinoma (RCC) (6.5%), sarcoma (10%), colon cancer (10%), head and neck cancer (7%), and squamous cell carcinoma of the lung. These data suggest a range of CNVs within and between tumor indications, and diversity in copy number patterns between different indications.

[0806] In addition to the CNVs within the TCGA dataset, the median integrin-4 mRNA expression level for each indication covers approximately 2 10 Therefore, given the range of integrin-4 mRNA expression levels and the CNVs observed between and among tumor types, statistical tests were performed to identify potential correlations between integrin-4 mRNA levels and integrin-4 CNVs in each TCGA dataset / indication. Tumors for each indication were classified into 1 of 5 categories:

[0807] a) Deep deletion;

[0808] b) Shallow deletion;

[0809] c) diploid;

[0810] d) increase; or

[0811] e) Amplification.

[0812] The Kruskall-Wallis test was then performed to test whether the distribution of mRNA expression values ​​for each category differed between the categories (P < 0.01). For those TCGA datasets with P < 0.01, post hoc tests were performed to identify which categories differed from each other by calculating the Z-statistic and calculating the adjusted P value (Bonferonni). To simplify interpretation, pairwise comparisons relative to diploid were performed for each indication (although P values ​​were calculated for all pairs). 18 / 41 TCGA studies achieved Kruskall-Wallis P < 0.01 and Bonferonni P < 0.025 in the increase relative diploid and / or expansion relative diploid comparisons, indicating that increased integrin-4 mRNA expression was associated with increased integrin-4 copy number. These 18 studies represented 14 independent tumor histologies:

[0813] Breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous cell carcinoma, cervical cancer, head and neck cancer, pancreatic cancer, thyroid cancer, colorectal cancer, thymoma, sarcoma, renal clear cell carcinoma (RCC) and gastric cancer.

[0814] In addition, six studies had decreased mRNA expression associated with copy number loss. Four of these six studies not only showed an association between CNV loss and decreased expression, but also reported that CNV gain was associated with high expression:

[0815] Gastric cancer, squamous cell lung cancer, colon cancer and thyroid cancer.

[0816] The only two indications reported to be associated with CNV loss and low transcript abundance are renal chromophobe carcinoma and prostate cancer. In addition, there is a separate prostate cancer study (metastatic prostate cancer, SU2C / PCF Dream Team (Robinson et al., Cell 2015)) showing that copy number gain is associated with high expression (relative to diploid).

[0817] These observed tumor CNV losses and gains with mRNA expression levels may represent the mechanisms behind the expression of the integrin-4 tumor protein in those indications where such correlations are observed. Clearly, there are indications (e.g., hepatocellular carcinoma) where CNVs do not appear to affect mRNA expression levels in a predictable pattern. The in vivo preclinical efficacy of certain integrin-4 bicyclic drug conjugates of the present invention has been shown to correlate with integrin-4 protein expression as measured by IHC. Therefore, if tumor integrin-4 CNVs correlate with mRNA levels and predict protein expression levels, then patients with tumors that may formally have an increase in copy number (gain or amplification) are more likely to respond to the integrin-4 bicyclic drug conjugates of the present invention. If patients with increased CNVs in integrin-4 can be identified, then this information can be used to select patients for treatment with the integrin-4 bicyclic drug conjugates of the present invention.

[0818]

[0819]

[0820]

[0821]

[0822]

[0823]

[0824]

[0825]

[0826]

[0827] Example 7: Expression analysis of integrin-4 in 6 cell lines

[0828] 1. Research objectives

[0829] The aim of the study was to evaluate the expression of integrin-4 by flow cytometry 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 lines.

[0830] 2. Group Design

[0831] Panels for FCM in T-47D, MDA-MB-468, NCI-H292, NCI-H322, and HT-1080:

[0832]

[0833] NCI-H526 Group:

[0834]

[0835] 3. Materials

[0836] 1.1. Sample

[0837] Cell Series List

[0838] Table 20-2

[0839]

[0840] Reagents

[0841] Antibodies and kits for flow cytometry analysis

[0842] Table 20-3

[0843]

[0844] DPBS (Corning-21-031-CV)

[0845] Staining buffer (eBioscience-00-4222)

[0846] Fixation Buffer (BD-554655)

[0847] 3.3. Instruments

[0848] Eppendorf Centrifuge 5810R

[0849] BD FACS Canto flow cytometer (BD)

[0850] 4. Experimental methods and procedures

[0851] 4.1 Sample collection

[0852] Collect cell lines in the exponential growth phase. Count cells using trypan blue staining on a hemocytometer. Centrifuge cells at 400 × g for 5 min at 4 °C, wash cells twice with staining buffer, and then resuspend cells to 1 × 10 7 / mL.

[0853] 4.2 Antibody staining

[0854] 1) Aliquot 100 μL of cell suspension into each well of a 96-well V plate.

[0855] 2) Add isotype control or antibody to the suspended cells and incubate at 4°C in the dark for 30 minutes.

[0856] 3) Wash cells 2× by centrifugation at 400×g for 5 minutes at 4°C and discard the supernatant.

[0857] 4) Resuspend the cells with 100 μL of fixation buffer and incubate at 4°C in the dark for 30 minutes.

[0858] 5) Wash cells 2× by centrifugation at 300×g for 5 minutes at 4°C and remove the supernatant.

[0859] 6) Resuspend cells in 400 μL staining buffer.

[0860] 7) FACS data were analyzed using FlowJo V10 software.

[0861] 4.3 Data Analysis

[0862] All FACS data were analyzed using FlowJo V10 software and Graphpad Prism or Excel software.

[0863] 5. Results

[0864] 5.1 Gating strategy for the group

[0865] exist Figure 8-Figure 11 Gating strategy for integrin-4 is shown in

[0866] 5.2 Data Analysis

[0867] 5.2.1. Viability of cell lines

[0868] The viability of the cell lines is as follows.

[0869] Table 20-4

[0870]

[0871]

[0872] 5.2.2. Positive expression of integrin-4 in cell lines

[0873] The positive expression and MFI of integrin-4 in the six cell lines are listed as follows.

[0874] Table 20-5

[0875] serial number Cell lines Integrin-4 MFI-isotype MFI-Group 1 T-47D 99.0％ 132 1808 2 MDA-MB-468 99.0％ 184 2324 3 NCI-H292 97.9％ 180 729 4 NCI-H322 99.1％ 145 1655 5 NCI-H526 0.21％ 104 91.3 6 HT1080 1.53％ 134 134

[0876] 6. Discussion

[0877] Integrin-4 was highly expressed in breast cancer T-47D (99.0%), MDA-MB-468 (99.0%) and lung cancer NCI-H292 (97.9%), NCI-H322 (99.1%). Integrin-4 was not found to be expressed in NCI-H526 and HT-1080.

[0878] Example 8: Analysis of Integrin-4 Expression in 9 CDX Cell Lines by Flow Cytometry

[0879] 1. Research objectives

[0880] The aim of this project was to evaluate the surface expression of PVRL-4 in 9 cell lines, including 1 breast cancer (MDA-MB-468), 4 lung cancer (NCI-H292, NCI-H358, NCI-H526, A549), 1 pancreatic cancer (Panc02.13), 2 colorectal cancer (HCT-116, HT-29) and 1 bladder cancer (HT1376) cell lines.

[0881] 2. Group Design

[0882] Panel of FCM in nine cell lines.

[0883] Table 20-6

[0884] Fluorescent dyes blank Isotype group PE - Isotype Control IgG2b Integrin-4 BV421 Live / Dead Live / Dead Live / Dead

[0885] 3. Materials

[0886] 3.1 Sample

[0887] Cell series list:

[0888] Table 20-7

[0889]

[0890] Reagents

[0891] 1) DPBS (Corning, 21-031-CV)

[0892] 2) Trypsin 0.25% (Invitrogen-25200072)

[0893] 3) Staining buffer (eBioscience, 00-4222)

[0894] 4) Fixation buffer (BD, 554655)

[0895] 5) Antibodies

[0896] Table 20-8

[0897] Fluorescence Markers Catalog Number supplier illustrate PE Integrin-4 FAB2659P R&D AAAO0217021 PE Mouse IgG2b IC0041P R&D BV421 Live / Dead L34964 Invitrogen -

[0898] 3.3. Instruments

[0899] Eppendorf Centrifuge 5810R

[0900] BD FACS Canto flow cytometer (BD)

[0901] 4. Experimental methods and procedures

[0902] 4.1 Cell culture

[0903] Cell Thawing

[0904] 1) Clean the frozen vials with 70% alcohol and then quickly thaw the vials in a 37°C water bath.

[0905] 2) Centrifuge the cell suspension at about 1000 rpm for 5 minutes, remove the supernatant, and add pre-warmed culture medium to the flask.

[0906] 3) At 37°C, 5% CO 2 Incubate the flask in an incubator.

[0907] Cell passaging

[0908] 1) Incubate the medium and trypsin in a 37°C water bath.

[0909] 2) Remove the culture medium and rinse the cell layer with DPBS.

[0910] 3) Add 5 mL of 0.25% trypsin solution to the flask and dilute the trypsin with 5 mL of medium.

[0911] 4) Centrifuge the cell suspension at 1000 rpm for 5 minutes.

[0912] 5) Add 15 mL of fresh medium and resuspend the cells by gently pipetting.

[0913] 6) Add appropriate cell suspension to the new culture flask.

[0914] 7) At 37°C, 5% CO 2 Incubate the culture flask in an incubator.

[0915] 4.2. Sample collection

[0916] Harvest cell lines growing in the exponential growth phase. Count cells using trypan blue staining. Centrifuge cells at 400 × g for 5 min at 4 °C, wash cells twice with staining buffer, and then resuspend cells to 5 × 10 6 / mL.

[0917] Antibody staining

[0918] Aliquot 100 μL of cell suspension into each well of a 96-well V-plate. Add isotype control or antibody to the suspended cells and incubate at 4°C for 30 minutes in the dark. Wash cells twice by centrifugation at 400 × g for 5 minutes at 4°C and discard the supernatant. Resuspend cells in 300 μL of staining buffer. Analyze FACS data using Flow Jo V10 software.

[0919] Data analysis

[0920] All FACS data were analyzed using FlowJo V10 software and Graphpad Prism or Excel software.

[0921] 5. Results

[0922] 5.1. Gating strategy for the group

[0923] exist Figure 12-16 The gating strategy for nectin-4 is shown in FIG.

[0924] Data analysis

[0925] The positive expression and MFI of integrin-4 in the nine cell lines are tabulated.

[0926] Table 20-9

[0927]

[0928]

[0929] 6. Discussion

[0930] Bindex-4 was highly expressed in bladder cancer HT-1376 (92.4%), breast cancer MDA-MB-468 (97.1%), and lung cancer NCI-H358 (90.1%). Bindex-4 was found to be expressed in HT-29 (40.0%), NCI-H292 (71.1%), and Panc02.13 (51.9%). Bindex-4 was not found to be expressed in HCT-116, A549, and NCI-526. This data will be used to guide model selection for efficacy studies.

[0931] Example 9: In vivo efficacy study

[0932] Example 9.1: In vivo efficacy study of test substances in treating A549 xenografts in Balb / c nude mice

[0933] 1. Research objectives

[0934] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in treating A549 xenografts in Balb / c nude mice.

[0935] 2. Experimental Design

[0936]

[0937] 3. Materials

[0938] Animals and housing conditions

[0939] 3.1.1. Animals

[0940] Species: House mouse

[0941] Strain: Balb / c nude mice

[0942] Age: 6-8 weeks

[0943] Gender: Female

[0944] Weight: 18-22 g

[0945] Number of animals: 41 mice plus spare

[0946] 3.1.2. Rearing conditions

[0947] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[0948] Temperature: 20-26℃.

[0949] Humidity 40-70%.

[0950] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[0951] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[0952] Drinking water: Animals had free access to sterile drinking water.

[0953] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[0954] Animal identification: Tagging animals with ear codes.

[0955] 4. Experimental methods and procedures

[0956] Cell culture

[0957] The cells were cultured in F-12K medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 A549 tumor cells were maintained in vitro as monolayer cultures in an atmosphere of . Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[0958] Tumor inoculation

[0959] Each mouse was inoculated subcutaneously on the right abdomen with A549 tumor cells (5 × 10 6 ) for tumor formation. When the average tumor volume reached 158mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[0960] 4.3. Preparation of test article preparation

[0961] Table 20-10

[0962]

[0963] 4.4. Sample collection

[0964] At the end of the study, tumors from all groups were collected 2 h after the last dose.

[0965] 5. Results

[0966] Tumor growth curve

[0967] exist Fig.17 Tumor growth curves are shown in .

[0968] Tumor volume trajectory

[0969] The mean tumor volume over time in female Balb / c nude mice bearing A549 xenografts is shown below in Table 21.

[0970] Table 21: Trajectory of tumor volume changes over time

[0971]

[0972]

[0973] Tumor growth inhibition analysis

[0974] The tumor growth inhibition rate of the test articles in the A549 xenograft model was calculated based on tumor volume measurements on day 14 after the start of treatment.

[0975] Table 22-1: Tumor growth inhibition analysis

[0976]

[0977] a. Mean ± SEM.

[0978] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[0979] 6. Summary and discussion of results

[0980] In this study, the therapeutic efficacy of the test articles was evaluated in the A549 xenograft model. Fig.17 And Table 21 and Table 22-1 show the tumor volumes of all treatment groups measured at different time points.

[0981] On day 14, the mean tumor size in vehicle-treated mice reached 568 mm 3 BCY8245 at 3mg / kg, qw (TV = 356mm 3 ,TGI=51.4%,p<0.05),3mg / kg,biw(TV=194mm 3 , TGI=90.8%, p<0.01) and 5mg / kg, qw (TV=228mm 3 , TGI=82.6%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[0982] Animals in the BCY8245 group maintained body weight well. In this cell line, which showed minimal expression of integrin-4 in FACS studies, tumor growth was limited by BCY8245, but tumors did not regress, emphasizing the need for target-driven treatment for optimal efficacy.

[0983] Example 9.2: In vivo efficacy study of test substances in treating HCT116 xenografts in Balb / c nude mice

[0984] 1. Research objectives

[0985] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of HCT116 xenografts in Balb / c nude mice.

[0986] 2. Experimental Design

[0987] Table 22-2

[0988]

[0989] 3. Materials

[0990] 3.1. Animals and housing conditions

[0991] 3.1.1. Animals

[0992] Species: House mouse

[0993] Strain: Balb / c nude mice

[0994] Age: 6-8 weeks

[0995] Gender: Female

[0996] Weight: 18-22 g

[0997] Number of animals: 41 mice plus spare

[0998] 3.1.2. Rearing conditions

[0999] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1000] Temperature: 20-26℃.

[1001] Humidity 40-70%.

[1002] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1003] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1004] Drinking water: Animals had free access to sterile drinking water.

[1005] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1006] Animal identification: Tagging animals with ear codes.

[1007] 4. Experimental methods and procedures

[1008] 4.1 Cell culture

[1009] The cells were cultured in medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 HCT116 cells were maintained in an atmosphere of . Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1010] Tumor inoculation

[1011] Each mouse was inoculated subcutaneously on the right abdomen with 0.2 ml of HCT116 tumor cells (5.0 × 10 6) for tumorigenesis. When the average tumor volume reached 166 mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[1012] 4.3. Preparation of test article preparation

[1013] Table 22-3

[1014]

[1015] 4.4. Sample collection

[1016] Tumors were collected for FFPE from Groups 1 and 2 at the end of the study on Day 14. Plasma was collected at 5, 15, 30, 60, and 120 minutes post-dose for Group 4. Tumors were also collected and stored at -80°C.

[1017] 5. Results

[1018] Tumor growth curve

[1019] exist Fig.18 Tumor growth curves are shown in .

[1020] Tumor volume trajectory

[1021] The mean tumor volume over time of female Balb / c nude mice bearing HCT116 xenografts is shown in Table 23 below.

[1022] Table 23: Trajectory of tumor volume changes over time

[1023]

[1024] Tumor growth inhibition analysis

[1025] The tumor growth inhibition rate of the test articles in the HCT116 xenograft model was calculated based on tumor volume measurements on day 14 after the start of treatment.

[1026] Table 24-1: Tumor growth inhibition analysis

[1027]

[1028] a. Mean ± SEM; b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treatment group by the group mean tumor volume of the control group (T / C).

[1029] 6. Summary and discussion of results

[1030] In this study, the therapeutic efficacy of the test article was evaluated in the HCT116 xenograft model. Fig.18 And Table 23 and Table 24-1 show the tumor volumes of all treatment groups measured at different time points.

[1031] On day 14 after the start of treatment, the mean tumor size in vehicle-treated mice reached 769 mm 3 BCY8245 was 3mg / kg, qw (TV = 425mm 3 ,TGI=57.1%,p<0.001),3mg / kg,biw(TV=197mm 3 , TGI=94.9%, p<0.001) and 5mg / kg, qw (TV=134mm 3 , TGI=105.2%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[1032] In this study, animals in all 5 mg / kg qw groups lost an average of more than 10% body weight.

[1033] In this cell line, in which integrin-4 was minimally expressed in FACS studies, tumor growth was restricted by BCY8245, but tumors did not regress, emphasizing the need for targeted drive for optimal efficacy.

[1034] Example 9.3: In vivo efficacy study of test substances in treating HT-1376 xenografts in CB17-SCID mice

[1035] 1. Research objectives

[1036] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of HT-1376 xenografts in CB17-SCID mice.

[1037] 2. Experimental Design

[1038] Table 24-2

[1039]

[1040] 3. Materials

[1041] 3.1 Animals and housing conditions

[1042] 3.1.1. Animals

[1043] Species: House mouse

[1044] Germline: CB17-SCID

[1045] Age: 6-8 weeks

[1046] Gender: Female

[1047] Weight: 18-22 g

[1048] Number of animals: 41 mice plus spare

[1049] 3.1.2. Rearing conditions

[1050] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1051] Temperature: 20-26℃.

[1052] Humidity 40-70%.

[1053] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1054] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1055] Drinking water: Animals had free access to sterile drinking water.

[1056] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1057] Animal identification: Tagging animals with ear codes.

[1058] 4. Experimental methods and procedures

[1059] 4.1 Cell culture

[1060] The cells were cultured in EMEM supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 HT-1376 tumor cells were maintained in an atmosphere of . Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1061] 4.2 Tumor inoculation

[1062] Each mouse was inoculated subcutaneously on the right flank with 0.2 ml of PBS containing Matrigel (1:1) and HT-1376 tumor cells (5 × 10 6 ) for tumor formation. When the average tumor volume reached 153mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[1063] 4.3. Preparation of test article preparation

[1064] Table 24-3

[1065]

[1066] 4.4. Sample collection

[1067] At the end of the study, plasma was collected at 5, 15, 30, 60 and 120 minutes after the last dose for Group 4. Tumors were collected 2 hours after the last dose for Group 4. Tumors were collected 2 hours after the last dose for Groups 1, 2 and 3.

[1068] 5. Results

[1069] Tumor growth curve

[1070] exist Fig.19 Tumor growth curves are shown in .

[1071] Tumor volume trajectory

[1072] Table 25 shows the change in mean tumor volume over time in female CB17-SCID mice bearing HT-1376 xenografts.

[1073] Table 25: Trajectory of tumor volume changes over time

[1074]

[1075] Tumor growth inhibition analysis

[1076] Tumor growth inhibition rates of the test articles in the HT-1376 xenograft model were calculated based on tumor volume measurements on day 14 after the start of treatment.

[1077] Table 26-1: Tumor growth inhibition analysis

[1078]

[1079] a. Mean ± SEM.

[1080] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1081] 6. Summary and discussion of results

[1082] In this study, the therapeutic efficacy of the test articles was evaluated in the HT-1376 xenograft model. Fig.19 And Table 25 and Table 26-1 show the tumor volumes of all treatment groups measured at different time points.

[1083] On day 14, the mean tumor size in vehicle-treated mice reached 1069 mm 3 BCY8245 was 3mg / kg, qw (TV = 603mm3 ,TGI=50.9%, p<0.01), 3mg / kg, biw (TV=407mm 3 , TGI=72.3%, p<0.001) and 5mg / kg, qw (TV=465mm 3 , TGI = 66.0%, p < 0.001) produced significant antitumor activity. In this study, 5 mg / kg qw of BCY8245 caused more than 10% weight loss in animals during the treatment period.

[1084] Example 9.4: In vivo efficacy study of test substances in treating MDA-MB-468 xenografts in Balb / c nude mice

[1085] 1. Research objectives

[1086] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of MDA-MB-468 xenografts in Balb / c nude mice.

[1087] 2. Experimental Design

[1088] Table 26-2

[1089]

[1090] 3. Materials

[1091] 3.1 Animals and housing conditions

[1092] 3.1.1. Animals

[1093] Species: House mouse

[1094] Strain: Balb / c nude mice

[1095] Age: 6-8 weeks

[1096] Gender: Female

[1097] Weight: 18-22 g

[1098] Number of animals: 41 mice plus spare

[1099] 3.1.2. Rearing conditions

[1100] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1101] Temperature: 20-26℃.

[1102] Humidity 40-70%.

[1103] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1104] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1105] Drinking water: Animals had free access to sterile drinking water.

[1106] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1107] Animal identification: Tagging animals with ear codes.

[1108] 4 Experimental methods and procedures

[1109] 4.1 Cell culture

[1110] The cells were cultured in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 Tumor cells were maintained in an atmosphere of 5% CO. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1111] 4.2 Tumor inoculation

[1112] Each mouse was inoculated subcutaneously in the right flank with MDA-MB-468 tumor cells (10 × 10 6 ) for tumor formation. When the average tumor volume reached 196 mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[1113] 4.3 Preparation of test article

[1114] Table 26-3

[1115]

[1116] 4.4 Sample collection

[1117] On day 21 of the study, plasma was collected from Group 2 at 5, 15, 30, 60 and 120 minutes after the last dose. Tumors were collected 2 h after the last dose from Groups 1 and 3. Animals from Group 4 were kept for another 21 days without any dosing, and tumors from these groups were collected on day 42.

[1118] 5 Results

[1119] 5.1 Tumor growth curve

[1120] exist Fig. 20 Tumor growth curves are shown in .

[1121] 5.2 Tumor volume trajectory

[1122] The mean tumor volume over time in female Balb / c nude mice bearing MDA-MB-468 xenografts is shown below in Tables 27 and 28.

[1123] 5.3 Tumor Growth Inhibition Analysis

[1124] Tumor growth inhibition rates of the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements on day 21 after the start of treatment.

[1125]

[1126] Table 28: Trajectory of tumor volume changes over time (Day 23 to Day 42)

[1127]

[1128] Table 29-1: Tumor growth inhibition analysis

[1129]

[1130] a. Mean ± SEM.

[1131] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1132] 6. Summary and discussion of results

[1133] In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. Fig. 20 And Table 27 to Table 29-1 show the tumor volumes of all treatment groups measured at different time points.

[1134] On day 21, the mean tumor size in vehicle-treated mice reached 447 mm 3 BCY8245 was 3mg / kg, qw (TV = 85mm 3 ,TGI=144.2%,p<0.001),3mg / kg,biw(TV=22mm 3 , TGI=169.8%, p<0.001) and 5mg / kg, qw (TV=29mm 3, TGI=168.4%, p<0.001) produced significant antitumor activity in a dose- or dose-frequency-dependent manner.

[1135] Dosing was suspended in the 5 mg / kg group from day 21, and the tumors did not show any recurrence during an additional three-week monitoring period. In this cell line, which showed high expression of integrin-4 in FACS studies, BCY8245 resulted in tumor regression, emphasizing the target-driven nature of optimal efficacy.

[1136] Example 9.5: In vivo efficacy study of test substances in treating NCI-H292 xenografts in Balb / c nude mice

[1137] 1. Research objectives

[1138] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of NCI-H292 xenografts in Balb / c nude mice.

[1139] 2. Experimental Design

[1140] Table 29-2

[1141]

[1142] 3. Materials

[1143] 3.1. Animals and housing conditions

[1144] 3.1.1. Animals

[1145] Species: House mouse

[1146] Strain: Balb / c nude mice

[1147] Age: 6-8 weeks

[1148] Gender: Female

[1149] Weight: 18-22 g

[1150] Number of animals: 41 mice plus spare

[1151] 3.1.2. Rearing conditions

[1152] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1153] Temperature: 20-26℃.

[1154] Humidity 40-70%.

[1155] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1156] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1157] Drinking water: Animals had free access to sterile drinking water.

[1158] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1159] Animal identification: Tagging animals with ear codes.

[1160] 4. Experimental methods and procedures

[1161] Cell culture

[1162] The cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 NCI-H292 tumor cells were maintained in vitro as monolayer cultures in an atmosphere of . Tumor cells were routinely passaged twice a week by trypsin-EDTA treatment. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1163] Tumor inoculation

[1164] Each mouse was inoculated subcutaneously on the right abdomen with NCI-H292 tumor cells (10 × 10 6 ) for tumor formation. When the average tumor volume reached 162mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[1165] 4.3. Preparation of test article preparation

[1166] Table 29-3

[1167]

[1168] 4.4. Sample collection

[1169] At the end of the study, tumors from all groups were collected 2 h after the last dose.

[1170] 5. Results

[1171] 5.1 Tumor growth curve

[1172] exist Fig.21 Tumor growth curves are shown in .

[1173] Tumor volume trajectory

[1174] The mean tumor volume over time in female Balb / c nude mice bearing NCI-H292 xenografts is shown in Table 30 below.

[1175] Table 30: Trajectory of tumor volume changes over time

[1176]

[1177] Tumor growth inhibition analysis

[1178] Tumor growth inhibition rates of the test articles in the NCI-H292 xenograft model were calculated based on tumor volume measurements on day 14 after the start of treatment.

[1179] Table 31-1: Tumor growth inhibition analysis

[1180]

[1181]

[1182] a. Mean ± SEM.

[1183] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1184] 6. Summary and discussion of results

[1185] In this study, the therapeutic efficacy of the test articles was evaluated in the NCI-H292 xenograft model. Fig.21 And Table 30 and Table 31-1 show the tumor volumes of all treatment groups measured at different time points.

[1186] On day 14, the mean tumor size in vehicle-treated mice reached 948 mm 3 BCY8245 was 3mg / kg, qw (TV = 149mm 3 ,TGI=101.4%,p<0.001),3mg / kg,biw(TV=65mm 3 , TGI=112.2%, p<0.001) and 5mg / kg, qw (TV=83mm 3 , TGI=109.8%, p<0.001) produced significant anti-tumor activity.

[1187] All of these test substances showed comparable antitumor activity at 3 mg / kg, qw, 3 mg / kg, biw, and 5 mg / kg, qw, with no further improvement in efficacy when increasing the dose or dose-frequency.

[1188] In this study, mice in all groups maintained their body weight well.

[1189] In this cell line, which showed high expression of integrin-4 in FACS studies, BCY8245 resulted in tumor regression, emphasizing the target-driven nature of optimal efficacy.

[1190] Example 9.6: In vivo efficacy study of test substances in treating NCI-H526 xenografts in Balb / c nude mice

[1191] 1. Research objectives

[1192] The aim of this 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.

[1193] 2. Experimental Design

[1194] Table 31-2

[1195]

[1196] 3. Materials

[1197] 3.1. Animals and housing conditions

[1198] 3.1.1. Animals

[1199] Species: House mouse

[1200] Strain: Balb / c nude mice

[1201] Age: 6-8 weeks

[1202] Gender: Female

[1203] Weight: 18-22 g

[1204] Number of animals: 21 mice plus spare

[1205] 3.1.2. Rearing conditions

[1206] Mice were kept in individually ventilated cages at constant temperature and humidity, with 3 animals per cage.

[1207] Temperature: 20-26℃.

[1208] Humidity 40-70%.

[1209] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1210] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1211] Drinking water: Animals had free access to sterile drinking water.

[1212] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1213] Animal identification: Tagging animals with ear codes.

[1214] 4. Experimental methods and procedures

[1215] 4.1 Cell culture

[1216] The cells were cultured in medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 5% CO 2 NCI-H526 cells were maintained in an atmosphere of 4% CO. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1217] Tumor inoculation

[1218] Each mouse was inoculated subcutaneously on the right flank with 0.2 ml of NCI-H526 tumor cells (5.0 × 10 6 ) for tumor formation. When the average tumor volume reached 181mm 3 At 14:00, 21 animals were randomized. The experimental design table shows the test article administration and the number of animals in each group.

[1219] 4.3. Preparation of test article preparation

[1220] Table 31-2

[1221]

[1222] 4.4. Sample collection

[1223] At the end of the study on day 14, all tumors were collected for FFPE.

[1224] 5. Results

[1225] Tumor growth curve

[1226] exist Fig. 22 Tumor growth curves are shown in .

[1227] Tumor volume trajectory

[1228] The mean tumor volume over time in female Balb / c nude mice bearing NCI-H526 xenografts is shown below in Table 32.

[1229] Table 32: Trajectory of tumor volume changes over time

[1230]

[1231] Tumor growth inhibition analysis

[1232] The tumor growth inhibition rate of the test articles in the NCI-H526 xenograft model was calculated based on tumor volume measurements on day 14 after the start of treatment.

[1233] Table 33-1: Tumor growth inhibition analysis

[1234]

[1235] a. Mean ± SEM.

[1236] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1237] 6. Summary and discussion of results

[1238] In this study, the therapeutic efficacy of the test articles was evaluated in the NCI-H526 xenograft model. Fig. 22 And Table 32 and Table 33-1 show the tumor volumes of all treatment groups measured at different time points.

[1239] On day 14 after the start of treatment, the average tumor size in vehicle-treated mice reached 1365 3 . BCY8245(3mg / kg,qw)(TV=1205mm 3 , TGI=13.4%, p>0.05) and BCY8245 (3mg / kg, biw) (TV=1109mm 3 , TGI=21.6%, p>0.05) showed mild antitumor activity, BCY8245 (5mg / kg, qw) (TV=476mm 3 , TGI = 75.0%, p < 0.01) showed significant antitumor activity. In this study, BCY8245 (5 mg / kg biw) caused more than 10% weight loss in animals. In this cell line, which showed minimal expression of integrin-4 in FACS studies, tumor growth was limited by BCY8245, but tumors did not regress, emphasizing the need for target-driven treatment for optimal efficacy.

[1240] Example 9.7: In vivo efficacy study of test substances in treating Panc2.13 xenografts in Balb / c nude mice

[1241] 1. Research objectives

[1242] The aim of this study was to evaluate the in vivo antitumor efficacy of the test article in the treatment of Panc2.13 xenografts in Balb / c nude mice.

[1243] 2. Experimental Design

[1244] Table 33-2

[1245]

[1246] 3. Materials

[1247] 3.1. Animals and housing conditions

[1248] 3.1.1. Animals

[1249] Species: House mouse

[1250] Strain: Balb / c nude mice

[1251] Age: 6-8 weeks

[1252] Gender: Female

[1253] Weight: 18-22 g

[1254] Number of animals: 41 mice plus spare

[1255] 3.1.2. Rearing conditions

[1256] Mice were kept in individual ventilated cages at constant temperature and humidity, with 3 or 5 animals per cage.

[1257] Temperature: 20-26℃.

[1258] Humidity 40-70%.

[1259] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1260] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1261] Drinking water: Animals had free access to sterile drinking water.

[1262] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1263] Animal identification: Tagging animals with ear codes.

[1264] 4. Experimental methods and procedures

[1265] Cell culture

[1266] The cells were cultured in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units / ml human recombinant insulin at 37°C in an atmosphere of 5% CO 2 Panc2.13 tumor cells were maintained in an atmosphere of 4% CO. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1267] Tumor inoculation

[1268] Each mouse was inoculated subcutaneously on the right flank with 0.2 ml of PBS containing Matrigel (1:1) containing Panc2.13 tumor cells (5 × 10 6 ) for tumor formation. When the average tumor volume reached 149mm 3 41 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[1269] 4.3. Preparation of test article preparation

[1270] Table 33-3

[1271]

[1272] 4.4. Sample collection

[1273] At the end of the study, tumors from all groups were collected 2 h after the last dose.

[1274] 5. Results

[1275] Tumor growth curve

[1276] exist Fig.23 Tumor growth curves are shown in .

[1277] Tumor volume trajectory

[1278] The mean tumor volume changes over time in female Balb / c nude mice bearing Panc2.13 xenografts are shown in Table 34 below.

[1279] Table 34: Trajectory of tumor volume changes over time

[1280]

[1281]

[1282] Tumor growth inhibition analysis

[1283] Tumor growth inhibition rates of the test articles in the Panc2.13 xenograft model were calculated based on tumor volume measurements on day 14 after the start of treatment.

[1284] Table 35-1: Tumor Growth Inhibition Analysis

[1285]

[1286] a. Mean ± SEM.

[1287] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1288] 6. Summary and discussion of results

[1289] In this study, the therapeutic efficacy of the test article was evaluated in the Panc2.13 xenograft model. Fig.23 And Table 34 and Table 35-1 show the tumor volumes of all treatment groups measured at different time points.

[1290] On day 14, the mean tumor size in vehicle-treated mice reached 545 mm 3 . BCY8245, 3mg / kg, qw, (TV=271mm 3 ,TGI=69.2%,p<0.01),3mg / kg,biw(TV=231mm 3 , TGI=79.1%, p<0.001) and 5mg / kg, qw (TV=238mm 3 , TGI = 77.5%, p < 0.001) produced significant antitumor activity. In this study, all animals in the 5 mg / kg qw group lost an average of more than 15% body weight. In this cell line, which showed only moderate expression of integrin-4 in FACS studies, tumor growth was restricted by BCY8245, but the tumors did not regress.

[1291] Example 9.8: In vivo efficacy study of test substances in treating MDA-MB-468 xenografts in Balb / c nude mice

[1292] 1. Research objectives

[1293] The purpose of this 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 to determine the role of targeted binding in optimal efficacy.

[1294] 2. Experimental Design

[1295] Table 35-2

[1296]

[1297] Note: N, number of animals per group

[1298] 3. Materials

[1299] 3.1. Animals and housing conditions

[1300] 3.1.1. Animals

[1301] Species: House mouse

[1302] Strain: Balb / c nude mice

[1303] Age: 6-8 weeks

[1304] Gender: Female

[1305] Weight: 18-22 g

[1306] Number of animals: 36 mice plus spare

[1307] 3.1.2. Rearing conditions

[1308] Mice were kept in individually ventilated cages at constant temperature and humidity, with 4 animals per cage.

[1309] Temperature: 20-26℃.

[1310] Humidity 40-70%.

[1311] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1312] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1313] Drinking water: Animals had free access to sterile drinking water.

[1314] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1315] Animal identification: Tagging animals with ear codes.

[1316] 4. Experimental methods and procedures

[1317] Cell culture

[1318] The cells were cultured in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 0% CO 2 Tumor cells were maintained in an atmosphere of 5% CO. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1319] Tumor inoculation

[1320] Each mouse was inoculated subcutaneously in the right flank with MDA-MB-468 tumor cells (10 × 10 6 ) for tumor formation. When the average tumor volume reached 186mm 3 36 animals were randomized at 1:1. The experimental design table shows the test article administration and the number of animals in each group.

[1321] 4.3. Preparation of test article preparation

[1322] Table 35-3

[1323]

[1324] 4.4. Sample collection

[1325] Tumors from Groups 5 and 6 were harvested for FFPE on study day 21. Tumors from Group 3 were harvested for FFPE at the end of the study.

[1326] 5. Results

[1327] Tumor growth curve

[1328] exist Fig.24 Tumor growth curves are shown in .

[1329] Tumor volume trajectory

[1330] The mean tumor volume over time in female Balb / c nude mice bearing MDA-MB-468 xenografts is shown below in Tables 36 to 38.

[1331] Tumor growth inhibition analysis

[1332] Tumor growth inhibition rates of the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements on day 21 after the start of treatment.

[1333] 6. Summary and discussion of results

[1334] In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. Fig.24 And Table 36 to Table 39-1 show the tumor volumes of all treatment groups measured at different time points.

[1335] On day 21, the mean tumor size in vehicle-treated mice reached 420 mm 3 BCY8245 was 1mg / kg, qw (TV = 204mm 3,TGI=92.1%, p<0.001),3mg / kg,qw(TV=27mm 3 , TGI=164.9%, p<0.001), produced significant antitumor activity in a dose-dependent manner. BCY8245, 0.3mg / kg qw or biw, did not produce any antitumor activity.

[1336] BCY8245, 1 mg / kg, qw and BCY8245, 3 mg / kg, qw combined with BCY8234 (homologous peptide without toxin), 300 mg / kg, qw produced significant antitumor activity (TV = 242 mm 3 , TGI=75.4%, p<0.01), resulting in significant antitumor activity. When compared to BCY8245 alone, the antitumor activity of 3 mg / kg BCY8245 was antagonized by 300 mg / kg BCY8234 (p<0.001). The importance of targeted binding for optimal efficacy was demonstrated by the reduced efficacy caused by competing non-toxic peptides. In the following monitoring schedule, mice treated with BCY8245, 1 mg / kg, qw showed obvious tumor recurrence, while mice treated with BCY8245, 3 mg / kg, qw did not show any tumor recurrence.

[1337]

[1338] Table 37: Trajectory of tumor volume changes over time (Day 23 to Day 32)

[1339]

[1340] Table 38: Trajectory of tumor volume changes over time (Day 35 to Day 91)

[1341]

[1342] Table 39-1: Tumor Growth Inhibition Analysis

[1343]

[1344] a. Mean ± SEM.

[1345] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1346] Example 9.9: In vivo efficacy study of test substances in treating MDA-MB-468 xenografts in Balb / c nude mice

[1347] 1. Research objectives

[1348] The aim of this study was to evaluate the in vivo antitumor efficacy of BCY8245 alone or in combination with BCY8245 and BCY8234 in the treatment of MDA-MB-468 xenografts in Balb / c nude mice.

[1349] 2. Experimental Design

[1350] Table 39-2

[1351]

[1352] Note: N, number of animals per group.

[1353] 3. Materials

[1354] 3.1 Animals and housing conditions

[1355] 3.1.1. Animals

[1356] Species: House mouse

[1357] Strain: Balb / c nude mice

[1358] Age: 6-8 weeks

[1359] Gender: Female

[1360] Weight: 18-22 g

[1361] Number of animals: 20 mice plus spare

[1362] 3.1.2. Rearing conditions

[1363] Mice were kept in individually ventilated cages at constant temperature and humidity, with 5 animals per cage.

[1364] Temperature: 20-26℃.

[1365] Humidity 40-70%.

[1366] Cage: Made of polycarbonate. Dimensions are 300mm×180mm×150mm. Bedding material is corn cob, which is changed twice a week.

[1367] Diet: Animals had free access to irradiated sterilized dry pelleted food throughout the study.

[1368] Drinking water: Animals had free access to sterile drinking water.

[1369] Cage Identification: Each cage had an identification tag containing the following information: number of animals, sex, strain, date received, treatment, study number, group number, and treatment start date.

[1370] Animal identification: Tagging animals with ear codes.

[1371] 4. Experimental methods and procedures

[1372] 4.1 Cell culture

[1373] The cells were cultured in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in an atmosphere containing 0% CO 2 Tumor cells were maintained in an atmosphere of 5% CO. Tumor cells were routinely subcultured twice a week. Cells growing in the exponential growth phase were collected and counted for tumor inoculation.

[1374] Tumor inoculation

[1375] Each mouse was inoculated subcutaneously in the right flank with MDA-MB-468 tumor cells (10 × 10 6 ) for tumor formation. When the average tumor volume reached 464mm 3 At 1 hr, 20 animals were randomized. The experimental design table shows the test article administration and the number of animals in each group.

[1376] 4.3. Preparation of test article preparation

[1377] Table 39-3

[1378]

[1379]

[1380] 4.4. Sample collection

[1381] At the end of the study, tumors from Group 3 were collected for FFPE.

[1382] 5. Results

[1383] Tumor growth curve

[1384] exist Fig.25 Tumor growth curves are shown in .

[1385] Tumor volume trajectory

[1386] The mean tumor volume changes over time in female Balb / c nude mice bearing MDA-MB-468 xenografts are shown in Tables 40 to 42.

[1387] Tumor growth inhibition analysis

[1388] Tumor growth inhibition rates of the test articles in the MDA-MB-468 xenograft model were calculated based on tumor volume measurements on day 28 after the start of treatment.

[1389] 6. Summary and discussion of results

[1390] In this study, the therapeutic efficacy of the test article was evaluated in the MDA-MB-468 xenograft model. Fig.25 And Tables 40 to 43 show the tumor volumes of all treatment groups measured at different time points.

[1391] The initial tumor starting size was intentionally larger than previously used to determine whether BCY8245 showed efficacy at this larger size. On day 28, the mean tumor size in vehicle-treated mice reached 773 mm 3 . BCY8245(1mg / kg,qw)(TV=384mm 3 , TGI=126.6%, p<0.001) and BCY8245(3mg / kg,qw)(TV=50mm 3 , TGI = 234.6%, p < 0.001), and produced significant antitumor activity in a dose-dependent manner on day 28. Among them, mice treated with BCY8245 (3 mg / kg qw) showed some tumor recurrence after stopping the treatment, and continued administration from day 76 did not play a role in complete tumor regression.

[1392] BCY8245 (3 mg / kg, qw) combined with BCY8234 (300 mg / kg, qw) produced significant antitumor activity on day 28 (TV = 55 mm 3 , TGI=234.0%, p<0.001), and the tumor did not show any recurrence during the entire monitoring period.

[1393] Mice in the vehicle group treated with 10 mg / kg of Bindex-4 ADC or 5 mg / kg of BCY8245, as well as mice in group 2 (BCY8245, 1 mpk, qw) treated with 5 mg / kg of BCY8245 at PG-D28, showed effective tumor regression over the next 3 weeks, after which the tumors showed regrowth over the next 4 weeks when the drug was stopped.

[1394] BCY8245 can be used at about 450mm 3 induced tumor regression in tumors with a starting volume of approximately 770 mm in tumors that had previously received vehicle. 3 It can also induce tumor regression when administered in the group.

[1395]

[1396] Table 41: Trajectory of tumor volume changes over time (Day 30 to Day 75)

[1397]

[1398] Table 42: Trajectory of tumor volume changes over time (Day 79 to Day 103)

[1399]

[1400] Table 43: Tumor Growth Inhibition Analysis

[1401]

[1402]

[1403] a. Mean ± SEM.

[1404] b. Tumor growth inhibition was calculated by dividing the group mean tumor volume of the treated group by the group mean tumor volume of the control group (T / C).

[1405] Example 10: In vivo PK study

[1406] MDA-MB-468 xenograft animals were injected with BCY8245 (BT8009) at 3 mg / kg. At various time points, animals were euthanized, plasma and tumors were collected and snap frozen. Samples were analyzed for MMAE. Plasma levels of BT8009 (BCY8245) were from historical PK studies. Fig.33 MMAE concentration in plasma, MMAE concentration in tumor, and BT8009 concentration in plasma are shown in Figure 2. MMAE is retained longer in tumors than in plasma, which supports the hypothesis that systemic exposure is significantly less than tumor exposure.

[1407] Example 11: HCS assay.

[1408] The HCS assay was used in the integrin-4 BDC binding study. Cells were incubated with the test reagents and then washed. Detection was by fluorescent antibody with MMAE. MDA-MB-468 cells showed moderate integrin-4 expression, with 20,000 cells giving the best images. NCI-H292 cells showed low expression in this assay, and even at 20,000 cells MMAE detection was weak. Fig.34 HCS data in the MDA-MB-468 cell line are shown in Table 44.

[1409] Table 44

[1410] Test object Maximum fluorescence intensity Kd(nM) Historical Kd(nM) Bindex-4ADC 33.63 0.2 0.28±0.07 BCY8245 13.34 3.52 5.18 MMAE 2.95 >10000 >10000

[1411] Binding element-4ADC and BCY8245 are retained on cells and co-localized with membrane staining. BCY8781 and MMAE show minimum retention. The Kd of all compounds to MDA-MB-468 cell lines is consistent with historical data. Binding element-4ADC shows detectable binding affinity to MDA-MB-468 cell lines. BCY8425 shows a single-digit nanomolar affinity, and its Bmax is lower than binding element-4ADC. The maximum fluorescence intensity of this reduction is because binding element-4ADC has MMAE to drug ratio of 4, but BCY8245 has MMAE to drug ratio of 1. BCY8781 shows only very weak binding affinity to MDA-MB-468 cell lines, while MMAE shows almost no detectable binding affinity to MDA-MB-468 cell lines.

[1412] Example 12: In vivo efficacy of BCY8245 in two PDX models of lung cancer

[1413] Purpose

[1414] To evaluate the efficacy of BCY8245 in PDX models of squamous cell non-small cell carcinoma and adenocarcinoma (both non-small cell carcinoma).

[1415] animal

[1416] Species: House mouse

[1417] Strain: Balb / c nude mice

[1418] Age: 6-8 weeks

[1419] Gender: Female

[1420] Weight: 18-22

[1421] Test reagents

[1422] BCY8245 and integrin-4 ADC or BCY8781

[1423] Animals before the study

[1424] Each mouse was inoculated subcutaneously in the right flank with LU-01-0007 or LU-01-0412 tumor fragments (~30 mm 3 ) for tumor formation. When the average tumor volume reached 161mm 3 (LU-01-0007) or 147mm 3 Animals were randomized at the time of the study (LU-01-0412).

[1425] Life Measures and Endpoints

[1426] Animals were examined daily for tumor growth and any effects of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain / loss, eye / hair gloss, and any other abnormal effects stated in the protocol. Deaths and observed clinical signs were recorded based on the number of animals in each subset. The primary endpoint was to see if tumor growth could be slowed or if mice could be cured. Tumor volume was measured in two dimensions three times a week using calipers and expressed in mm using the following formula: 3 Indicates volume: V = 0.5axb 2 , where a and b are the long and short diameters of the tumor, respectively. The tumor size is then used to calculate the T / C value. The T / C value (percentage) indicates the anti-tumor effect; T and C are the average volumes of the treatment group and the control group, respectively, on a given day.

[1427] TGI was calculated for each group using the following formula: TGI (%) = [1-(T i -T 0 ) / (V i -V 0 )]×100;T i is the mean tumor volume of the treatment group on a given day, T 0 is the average tumor volume of the treatment group on the day of treatment initiation, Vi is the i The mean tumor volume of the vehicle control group on the same day, and V 0 is the mean tumor volume of the vehicle group on the day of treatment initiation.

[1428] The results of these studies are Fig.26 and Fig. 27 shown.

[1429] Lu-01-0412( Fig.26 ): BCY8245 produced dose-related efficacy in this PDX model, with a reduction in tumor growth rate at 1 mg / kg qw, but at 3 mg / kg qw, tumors significantly regressed to baseline. After cessation of dosing (day 21), 5 / 6 animals showed no tumor regrowth until 105 days after the start of the study. The one animal showing regrowth responded to 3 mg / kg of BCY8245 and showed recovery and regression to baseline. BCY8781, a non-binding BDC, produced stable disease at 3 mg / kg, and after cessation of dosing, tumors grew rapidly at the same rate as the vehicle-treated group, emphasizing that binding to bindin-4 may provide enhanced efficacy for these agents. Large tumors (vehicle-treated group) regressed in response to a single dose of BCY8245 or BCY8781.

[1430] LU-01-0007( Fig. 27): BCY8245 produces a dose-related effect, with 1 mg / kg qw producing stable disease and 3 mg / kg producing complete regression. Dosing must be maintained until day 56 until complete regression (when dosing is stopped). There was no tumor regrowth after more than 56 days in this group (the latter was maintained until 126 days after the start of the study). Binding element-4 ADC gave a similar degree of efficacy. The 1 mg / kg stable disease group responded to an increase in dosing (3 and 5 mg / kg), indicating that low doses of BCY8245 do not lead to the formation of resistance.

[1431] Example 13: In vivo evaluation of BCY8245 in low-passage PDX models of human breast, esophageal, and bladder cancers in immunocompromised mice.

[1432] Purpose

[1433] To evaluate the antitumor activity of bicyclic agents in low passage champion tumor graft models of human breast cancer, esophageal cancer, and bladder cancer in immunocompromised mice.

[1434] Test system

[1435] Species: Mouse

[1436] Germline: Athymic Nude-Foxn1nu (immune compromised)

[1437] Source: Envigo: Indianapolis, Indiana

[1438] Gender: Female

[1439] Target age at start of dosing: At least 6-8 weeks

[1440] Target weight at start of dosing: at least 18 g

[1441] Adaptation period: 3 days

[1442] Experimental design

[1443] Animals before study: When sufficient stock animals reach 1.0–1.5 cm 3 Tumors were harvested for reimplantation into pre-study animals at 1:10 pm. Tumor fragments harvested from reserve animals were implanted unilaterally in the left flank of pre-study animals. Each animal was implanted from a specific passage batch and recorded.

[1444] Study Animals: Pre-study tumor volume for each experiment was recorded starting seven to ten days after implantation. When tumors reached 150-300 mm 3When the mean tumor volume is , the animals will be divided into treatment or control groups according to the tumor volume for dosing, and dosing will begin on day 0.

[1445] Test Reagents

[1446] BCY8245 and Bindex-4 antibody drug conjugates were compared to vehicle control. Standard of care agent docetaxel may be included. All agents were administered qw via the intravenous route at doses indicated in the figures.

[1447] Life Measurement

[1448] Effective tumor volume: Tumor volume will be measured twice a week. The final tumor volume will be collected on the day the study reaches the end point. If possible, the final tumor volume will be collected if the animal is found to be moribund.

[1449] Effective Animal Weights: Animals will be weighed twice weekly. If possible, final weights will be collected on the day the study reaches endpoint or if an animal is found moribund. Animals that show a ≥10% weight loss compared to Day 0 will receive ad libitum food. Any animal with a net body weight loss of >20% over a 7-day period, or mice showing a net body weight loss of more than 30% compared to day 0, will be considered moribund and will be euthanized.

[1450] Data analysis

[1451] Agent toxicity: Starting from day 0, animals were observed daily and weights were measured twice a week using a digital scale; data included individual and average gram weights (mean We ± SEM), and the average percentage weight change (% vD0) relative to day 0 was recorded for each group, and % vD0 was plotted at the completion of the study. Animal deaths were recorded daily and designated as drug-related (D), technology-related (T), tumor-related (B), or unknown (U) based on weight loss and macroscopic observation; single agent or combination groups reporting average % vD0> 20% and / or> 10% mortality will be considered to be above the maximum tolerated dose (MTD) of the treatment in the evaluation scheme. The maximum average % vD0 (nadir of body weight) for each treatment group was reported at the end of the study.

[1452] Reagent efficacy

[1453] Tumor growth inhibition: Starting on day 0, tumor size was measured twice a week by electronic calipers, and the data included individual and average estimated tumor volumes (mean TV ± SEM) recorded for each group; tumor volume was calculated using formula (1), formula (1): TV = width 2 × length × 0.52. After the completion of the study, initial (i) and final (f) tumor measurements will be performed, and the percentage of tumor growth inhibition (%TGI) values ​​for each treatment group (T) and control group (C) will be calculated and reported using formula (2), formula (2): %TGI = 1-(Tf-Ti) / (Cf-Ci). Individual mice with tumor volumes measured twice in a row ≤ 30% of the measurement result on day 0 will be considered partial responders (PR). Lack of obvious tumors (0.00mm for two consecutive measurements) 3 ) will be classified as complete responders (CR); CRs that persist until study completion will be considered tumor-free survivors (TFS). Tumor doubling time (DT) for vehicle-treated groups was determined using the formula DT = (Df–Di)*log2 / (logTVf–logTVi), where D = days and TV = tumor volume. All data collected in this study were managed electronically and stored on redundant server systems.

[1454] The results of these studies are Figure 28 to Figure 31 shown.

[1455] BCY8245 was tested in four low-passage PDX models: the models represent 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 showed excellent efficacy, causing tumor regression, and in three of the four models, complete tumor regression to baseline. In all cases, the efficacy was comparable to the ADC and was superior to or equal to docetaxel SOC. In all models, BCY8245 was better tolerated than docetaxel.

Claims

1. A peptide ligand specific for binding protein-4, which is: (a) A peptide ligand comprising a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two loop sequences, and the molecular scaffold forms covalent bonds with the cysteine ​​residues of the polypeptide, so that at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide comprises the amino acid sequence: C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (SEQ ID NO:1); in, 1Nal stands for 1-naphthylalanine, HArg stands for homoarginine, HyP stands for hydroxyproline, and C i 、Ci i and Cii i represents the first, second and third cysteine ​​residues, respectively, or a pharmaceutically acceptable salt thereof; or (b) A modified derivative of the peptide ligand described in (a) or a pharmaceutically acceptable salt thereof.

2. A peptide ligand according to claim 1, wherein the modified derivative described in (b) comprises one or more modifications selected from the following: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues; replacement of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids; addition of a spacer group; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with alanine, and replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds in the bicyclic peptide ligand; replacement of one or more peptide bonds with alternative bonds; modification of the peptide backbone length; replacement of hydrogen on the α-carbon of one or more amino acid residues with another chemical group, modification of cysteine ​​with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acid, introduction or replacement of amino acids to introduce orthogonal reactivity suitable for functionalization; replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues; and, replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues.

3. The peptide ligand according to claim 1, wherein the polypeptide in (a) comprises an amino acid sequence selected from the group consisting of: [B-Ala][Sar10]-(SEQ IS 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).

4. The peptide ligand according to claim 1, wherein the polypeptide in (a) comprises an amino acid sequence selected from the group consisting of: [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).

5. The peptide ligand according to claim 1, wherein the polypeptide in (a) comprises an amino acid sequence selected from the group consisting of: 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).

6. The peptide ligand according to claim 1, wherein the polypeptide in (a) comprises an amino acid sequence selected from the group consisting of: Ac-[B-Ala][Sar5]-(SEQ ID NO: 1) (hereinafter referred to as BCY8122); and Ac-(SEQ ID NO: 1) (hereinafter referred to as BCY8126).

7. The peptide ligand according to claim 1, wherein the polypeptide in (a) comprises an amino acid sequence selected from the group consisting of: [B-Ala][Sar10]-(SEQ ID NO: 1) (hereinafter referred to as BCY8234).

8. The peptide ligand according to claim 1, wherein the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

9. The peptide ligand according to claim 1, wherein the pharmaceutically acceptable salt is selected from the group consisting of free acid, sodium, potassium, calcium and ammonium salts.

10. The peptide ligand according to claim 1, wherein the binding protein-4 is human binding protein-4.

11. A drug conjugate comprising a peptide ligand as defined in any one of claims 1 to 10 conjugated to one or more effectors and / or functional groups.

12. A drug conjugate comprising the peptide ligand as defined in any one of claims 1 to 10 or a modified derivative thereof, which is conjugated to a radiochelator, a chromophore or a metal chelator.

13. A drug conjugate comprising a peptide ligand as defined in any one of claims 1 to 10 or a modified derivative thereof, conjugated to one or more cytotoxic agents.

14. The drug conjugate as defined in claim 13, wherein the cytotoxic agent is selected from MMAE or DM1.

15. The drug conjugate as defined in claim 14, wherein the cytotoxic agent is MMAE, and the conjugate further comprises a linker selected from the group consisting of: -PABC-Cit-Val-glutaryl- or -PABC-cyclobutyl-Ala-Cit-βAla-, wherein PABC represents p-aminobenzylcarbamate.

16. The drug conjugate as defined in claim 14, wherein the cytotoxic agent is DM1, and the conjugate further comprises a linker, which is -SPDB-(SO3H)-, wherein SPDB represents N-succinimidyl 3-(2-pyridyldithio)propionate.

17. A pharmaceutical composition comprising the drug conjugate according to any one of claims 11 to 16, and one or more pharmaceutically acceptable excipients.

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

19. Use of the drug conjugate as defined in any one of claims 11 to 16 in the preparation of a medicament for preventing, inhibiting or treating a disease or condition mediated by integrin-4.

20. Use of a drug conjugate as defined in any one of claims 11 to 16 for the preparation of a medicament for preventing, inhibiting or treating cancer in a patient, wherein the patient is identified as having an increased copy number variation (CNV) of Bindex-4.

21. A high-affinity binder of binding factor-4, comprising a peptide ligand, wherein the peptide ligand comprises a peptide covalently bound to a molecular scaffold, the peptide comprising two or more reactive groups capable of forming covalent bonds with the scaffold, and a sequence between the reactive groups that forms a ring relative to the scaffold.

22. The compound represented by formula 8:

23. A method for synthesizing the compound of formula 8, The method comprises: - Add EDCI to a solution of the compound of formula 7 in DMA and DCM; - stirring the mixture at 15°C for 3 hours; and - DCM was removed under reduced pressure and the reaction mixture was purified by preparative HPLC to give the compound of formula 8.

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