阅读说明：本技术 特异于结合素-4的双环肽配体 (Bicyclic peptide ligands specific for bindin-4 ) 是由 P·比伊克 L·陈 G·E·马德 P·帕克 K·范·里茨霍滕 M·里格比 于 2019-06-21 设计创作，主要内容包括：本发明涉及与分子支架共价结合的多肽,其使得两个或更多个肽环在支架的连接点之间相对。特别地,本发明描述了作为结合素-4(Nectin-4)的高亲和力结合子的肽。本发明还包括药物缀合物,其包含缀合至一个或多个效应物和/或官能团的所述肽、包含所述肽配体和药物缀合物的药物组合物、以及所述肽配体和药物缀合物在预防、抑制或治疗结合素-4介导的疾病或病症中的用途。(The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide loops are opposed between attachment points of the scaffold. In particular, the present invention describes peptides that are high affinity binders of bindin-4 (Nectin-4). The 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 a bindin-4 mediated disease or disorder.)

1. A peptide ligand specific for bindin-4 comprising a polypeptide comprising at least three cysteine residues separated by at least two loop sequences and a molecular scaffold which forms a covalent bond with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises the amino acid sequence:

C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO：1)；

wherein 1Nal represents 1-naphthylalanine, HARg represents homoarginine, HyP represents hydroxyproline and C_i、C_iiAnd C_iiiRespectively, represents a first, second and third cysteine residue, or a pharmaceutically acceptable salt thereof.

2. The peptide ligand as defined in claim 1, comprising an amino acid sequence selected from the group consisting of:

[ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8234);

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122);

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126);

(SEQ ID NO: 1) (hereinafter referred to as BCY 8116);

fluorescein- (SEQ ID NO: 1) (hereinafter referred to as BCY 8205); and

[ PYA ] [ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8846).

3. The peptide ligand as defined in claim 1 or claim 2, which comprises an amino acid sequence selected from the group consisting of:

[ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8234);

Ac-[B-Ala][Sar_s]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122);

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126);

(SEQ ID NO: 1) (hereinafter referred to as BCY 8116); and

fluorescein- (SEQ ID NO: 1) (hereinafter referred to as BCY 8205).

4. The peptide ligand as defined in any one of claims 1 to 3, comprising an amino acid sequence selected from the group consisting of:

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122);

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126); and

(SEQ ID NO: 1) (hereinafter referred to as BCY 8116).

5. The peptide ligand as defined in any one of claims 1 to 4, comprising an amino acid sequence selected from the group consisting of:

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122); and

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126).

6. The peptide ligand as defined in any one of claims 1 to 3, comprising an amino acid sequence selected from the group consisting of:

[ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8234).

7. The peptide ligand as defined in any one of claims 1 to 6, wherein the molecular scaffold is selected from 1,1',1 "- (1,3, 5-triazinan-1, 3, 5-triyl) tripropyl-2-en-1-one (TATA).

8. The peptide ligand as defined in any one of claims 1 to 7, wherein the pharmaceutically acceptable salt is selected from the group consisting of the free acid or the sodium, potassium, calcium, ammonium salt.

9. The peptide ligand as defined in any one of claims 1 to 8, wherein bindin-4 is human bindin-4.

10. A drug conjugate comprising a peptide ligand as defined in any one of claims 1 to 9 conjugated to one or more effectors and/or functional groups.

11. A drug conjugate as defined in claim 10, conjugated to one or more cytotoxic agents.

12. A drug conjugate as defined in claim 11, wherein the cytotoxic agent is selected from MMAE or DM1, in particular MMAE.

13. A drug conjugate as defined in claim 12, 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- β Ala-, such as-PABC-Cit-Val-glutaryl-, wherein PABC stands for p-aminobenzyl carbamate.

14. A drug conjugate as defined in claim 13, wherein the cytotoxic agent is DM1, and the conjugate further comprises a linker which is-SPDB- (SO)₃H) -, wherein SPDB represents N-succinimidyl 3- (2-pyridyldithio) propionate.

15. A drug conjugate as defined in any one of claims 10 to 14, selected from: BCY8245 or BCY8549, in particular BCY 8245.

16. A pharmaceutical composition comprising a peptide ligand according to any one of claims 1 to 9 or a drug conjugate according to any one of claims 10 to 15, in combination with one or more pharmaceutically acceptable excipients.

17. The pharmaceutical composition as defined in claim 16, further comprising one or more therapeutic agents.

18. A drug conjugate as defined in any one of claims 10 to 15 for use in the prevention, inhibition or treatment of a disease or condition mediated by bindin-4.

19. A method of preventing, inhibiting or treating cancer comprising administering a drug conjugate as defined in any one of claims 10 to 15 to a patient in need thereof, wherein the patient is identified as having an increased Copy Number Variation (CNV) of bindin-4.

Technical Field

The present invention relates to polypeptides covalently bound to a molecular scaffold such that two or more peptide loops are opposed between attachment points of the scaffold (sutten). In particular, the present invention describes peptides that are high affinity binders of bindin-4. The 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 a bindin-4 mediated disease or disorder.

Background

Cyclic peptides are capable of binding 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 used successfully clinically, such as the antibacterial peptide vancomycin, the immunosuppressant Drug cyclosporin or the anticancer Drug octreotide (draggers et al (2008), Nat Rev Drug Discov 7(7), 608-24). Good binding properties result from the relatively large interaction surface formed between the peptide and the target and the reduced conformational flexibility of the cyclic structure. Typically, macrocycles are bound to surfaces of several hundred square angstroms, for example the cyclic peptide CXCR4 antagonist CVX15(Wu et al (2007), Science330,1066-71), Cyclic peptides with an Arg-Gly-Asp motif that bind to integrin α Vb3(Xiong et al (2002), Science 296(5565), 151-5), or the cyclic peptide inhibitor upain-1 (bound to urokinase-type plasminogen activator)Zhao et al (2007), J Structure Biol 160(1), 1-10).

Because of its cyclic configuration, the peptidic macrocycle is less flexible than a linear peptide, resulting in less loss of entropy upon binding to the target and higher binding affinity. The reduced flexibility also results in locking in the target-specific conformation, increasing the binding specificity compared to the linear peptide. This effect has been exemplified by a potent, selective matrix metalloproteinase 8(MMP-8) inhibitor which loses selectivity for other MMPs when its ring is opened (Cherney et al (1998), J Med Chem 41(11), 1749-51). The advantageous binding properties obtained by macrocyclization are more pronounced in polycyclic peptides having more than one peptide ring, for example in vancomycin, nisin and actinomycin.

Different research groups have previously attached polypeptides with cysteine residues to synthetic molecular structures (Kemp and McNamara (1985), J.Org.Chem; Timmerman et al (2005), ChemBioChem). Meloen and colleagues have used tris (bromomethyl) benzene and related molecules to rapidly and quantitatively cyclize multiple peptide loops onto synthetic scaffolds for structural simulation of protein surfaces (Timmerman et al (2005), ChemBiochem). Methods of producing a drug candidate compound by linking a cysteine-containing polypeptide to a molecular scaffold such as TATA (1, 1' - (1,3, 5-triazinan-1, 3, 5-triyl) tripropyl-2-en-1-one are disclosed, Heinis et al Angew Chem, Int Ed. 2014; 53: 1602-.

Combinatorial methods based on phage display have been developed to produceLarge libraries of bicyclic peptides directed against the target of interest were generated and screened (Heinis et al (2009), Nat Chem Biol 5(7), 502-7 and WO 2009/098450). Briefly, a linear peptide containing three cysteine residues and two regions of six random amino acids (Cys- (Xaa) was displayed on phage₆-Cys-(Xaa)₆-Cys) and circularization by covalent attachment of cysteine side chains to a small molecule scaffold.

Disclosure of Invention

According to a first aspect of the present invention there is provided a peptide ligand specific for bindin-4 (Nectin-4) comprising a polypeptide comprising at least three cysteine residues separated by at least two loop sequences and a molecular scaffold forming a covalent bond with the cysteine residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the peptide ligand comprises the amino acid sequence:

C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO：1)，

wherein 1Nal represents 1-naphthylalanine, HARg represents homoarginine, HyP represents hydroxyproline, and C_i、C_iiAnd C_iiiRespectively, represents a first, second and third cysteine residue, or a pharmaceutically acceptable salt thereof.

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.

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.

According to a further aspect of the present invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a bindin-4 mediated disease or condition.

Drawings

Error bars represent Standard Error (SEM) of mean values when present in the figures.

Fig. 1 and 2: tumor volume trajectory following administration of BCY8245 to female BALB/c nude mice bearing NCI-H292 xenografts.

Fig. 3 and 4: tumor volume trajectory following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts.

FIG. 5: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing Pan2.13 xenografts.

FIG. 6: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing MDA-MB-468 xenografts.

FIG. 7: tumor volume trajectory following administration of BCY8549 (with BCY8245 as control) to female BALB/c nude mice bearing NCI-H292 xenografts.

FIG. 8: gating strategy for bindin-4 in mammary gland (T-47D and MDA-MB-468).

FIG. 9: gating strategy for bindin-4 in NCI-H292 and NCI-H322.

Fig. 10 and 11: gating strategy for bindin-4 in NCI-H526 and HT1080, respectively.

FIGS. 12-16: bindin-4 gating strategies in bladder cancer (HT 1376; FIG. 12), breast cancer (MDA-MB-468; FIG. 13), colorectal cancer (HT-29; FIGS. 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 (Panc 02.13; FIG. 16), respectively.

FIG. 17: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing a549 xenograft.

FIG. 18: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing HCT116 xenografts.

FIG. 19: tumor volume trajectory following administration of BCY8245 to female CB17-SCID mice bearing HT-1376 xenografts.

FIG. 20: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing MDA-MB-468 xenografts.

FIG. 21: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing NCI-H292 xenografts.

FIG. 22: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing NCI-H526 xenografts.

FIG. 23: tumor volume trajectory following administration of BCY8245 to female Balb/c nude mice bearing Pan2.13 xenografts.

FIG. 24: tumor volume trajectories following administration of BCY8245 or a combination of BCY8245 and BCY8234 to female Balb/c nude mice bearing MDA-MB-468 xenografts.

FIG. 25: tumor volume trajectories following administration of BCY8245 alone or BCY8245 in combination with BCY8234 to female Balb/c nude mice bearing MDA-MB-468 xenografts.

Fig. 26 to 31: tumor volume trajectories in Lu-01-0412, LU-01-0007, CTG-1771, CTG-1171, CTG-1106, and CTG-0896PDX xenografts.

FIG. 32: BT8009 (i.e., BCY8245) therapeutic efficacy correlates with CDX/PDX xenograft expression. Xenografts with low/no expression of binding element-4 showed a decrease in tumor growth rate. Xenografts expressing binding element-4 showed tumor regression. This analysis included PDX and CDX models, with values sorted out from various reports.

FIG. 33: MDA-MB-468 cells expressed Binder-4 and showed prolonged retention of MMAE in tumors.

FIG. 34: HCS-data analysis on MDA-MB-468 cell line.

Detailed Description

In one embodiment, C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8234);

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122);

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126);

(SEQ ID NO: 1) (hereinafter referred to as BCY 8116);

fluorescein- (SEQ ID NO: 1) (hereinafter referred to as BCY 8205); and

[ PYA ] [ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8846).

In another embodiment, C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

[ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8234);

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122);

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126);

(SEQ ID NO: 1) (hereinafter referred to as BCY 8116); and

fluorescein- (SEQ ID NO: 1) (hereinafter referred to as BCY 8205).

In yet another embodiment, C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO: 1) the skin ligand comprises an amino acid sequence selected from the group consisting of:

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122);

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126); and

(SEQ ID NO: 1) (hereinafter referred to as BCY 8116).

The data are shown herein in table 2, which shows that the peptide ligands of this embodiment show excellent levels of binding to human bindin-4 as demonstrated by SPR binding data.

In yet another embodiment, C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO: 1) comprises an amino acid sequence selected from the group consisting of:

Ac-[B-Ala][Sar₅]- (SEQ ID NO: 1) (hereinafter referred to as BCY 8122); and

ac- (SEQ ID NO: 1) (hereinafter referred to as BCY 8126).

The data are shown herein in table 1, which shows that the peptide ligands of this embodiment show excellent levels of binding to human bindin-4 as demonstrated by the competitive binding data.

In yet another embodiment, C_iP[1Nal][dD]C_iiM[HArg]DWSTP[HyP]WC_iii(SEQ ID NO: 1) peptide ligands comprising an amino group selected fromThe sequence is as follows:

[ B-Ala ] [ Sar10] - (SEQ ID NO: 1) (hereinafter referred to as BCY 8234).

The data are shown herein in table 3, which demonstrates that the peptide ligands of this embodiment, when conjugated to cytotoxic agents, show excellent binding levels (< 10nM) to human bindin-4 as demonstrated by SPR binding data.

In one embodiment, the molecular scaffold is 1,1' - (1,3, 5-triazinan-1, 3, 5-triyl) tripropyl-2-en-1-one (TATA).

Unless defined otherwise, 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 biological, genetic and biochemical methods (see Sambrook et al, Molecular Cloning: A Laboratory Manual, third edition, 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al, Short Protocols in Molecular Biology (1999) fourth edition, John Wiley & Sons, Inc.), which are incorporated herein by reference.

Nomenclature

Numbering

When referring to amino acid residue positions within the peptides of the invention, cysteine residues are omitted from the numbering (C) since they are invariant_i、C_iiAnd C_iii) Thus, the numbering of amino acid residues within the peptides of the invention is referenced as follows:

C_i-P₁-[1Nal]₂-[dD]₃-C_ii-M₄-[HArg]₅-D₆-W₇-S₈-T₉-P₁₀-[HyP]₁₁-W₁₂-C_iii(SEQ ID NO：1)。

for the purposes of this description, it is assumed that all bicyclic peptides are cyclized with 1,1' - (1,3, 5-triazinan-1, 3, 5-triyl) tripropyl-2-en-1-one (TATA) to produce a trisubstituted structure. Cyclization with TATA takes place at C_i、C_iiAnd C_iiiThe above.

Molecular form

N-or C-terminal extensions of the bicyclic core sequence are added to the left or right side of the sequence and are separated by a hyphen. For example, the N-terminal (. beta. -Ala) -Sar₁₀the-Ala tail will be represented as:

βAla-Sar10-A-(SEQ ID NO：X)。

reverse peptide sequence

It is envisaged that the peptide sequences disclosed herein will also be used in their reverse form as disclosed in Nair et al (2003) J Immunol 170(3), 1362-1373. For example, the order is reversed (i.e., N-terminal to C-terminal, and vice versa), and their stereochemistry is reversed (i.e., D-amino acid to L-amino acid, and vice versa).

Peptide ligands

Peptide ligands as referred to herein refer to peptides covalently bound to a molecular scaffold. Typically, such peptides comprise two or more reactive groups (i.e. cysteine residues) capable of forming covalent bonds with the scaffold, and a sequence (referred to as a loop sequence) opposite between the reactive groups, which is referred to as a loop sequence, because when the peptide is bound to the scaffold it forms a loop. In the context of the present application, a peptide comprises at least three cysteine residues (referred to herein as C)_i、C_iiAnd C_iii) Which forms at least two rings on the stent.

Advantages of peptide ligands

Certain bicyclic peptides of the present invention have a number of advantageous properties that make them considered drug-like molecules suitable for injection, inhalation, nasal, ocular, oral or topical administration. These advantageous properties include:

species cross-reactivity. This is a typical requirement for preclinical efficacy and pharmacokinetic assessment;

-protease stability. Bicyclic peptide ligands should ideally exhibit stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, and the like. Protease stability should be maintained between different species so that bicyclic lead candidates can be developed in animal models and reliably administered to humans;

-ideal solubility curve. This is a function of the ratio of charged hydrophilic residues to hydrophobic residues and intramolecular/intermolecular H bonds, which is important for formulation and absorption purposes;

optimal plasma half-life in circulation. Depending on the clinical indication and treatment regimen, it may be desirable to develop bicyclic peptides with short exposures in acute disease management settings, or with enhanced retention in circulation, and thus be preferred for management of more chronic disease states. Other factors driving the desired plasma half-life are the continuous exposure required to achieve maximum therapeutic efficiency versus the toxicology attendant to continuous exposure to the agent; and

-selectivity. Certain peptide ligands of the invention exhibit good selectivity for other binders.

Pharmaceutically acceptable salts

It will be understood that salt forms are within the scope of the invention and reference to peptide ligands includes salt forms of the ligands.

The Salts of the invention may be prepared by conventional chemical methods, such as Pharmaceutical Salts: properties, Selection, and Use, p.heinrich Stahl (ed.), camile g.wermuth (ed.), ISBN: 3-90639-026-8, Hardcover, page 388, 8.2002, synthesized from the parent compound containing a base or acid moiety. In general, these salts can be prepared by reacting the free acid or base forms of these compounds with the appropriate base or acid in water or in an organic solvent or in a mixture of the two.

Acid addition salts (mono-or di-salts) can be formed with a variety of acids (inorganic and organic). Examples of acid addition salts include mono-or di-salts with acids selected from: acetic acid, 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, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), alpha-oxoglutaric acid, glycolic acid, hippuric acid, hydrohalic acid (e.g., hydrobromic acid, hydrochloric acid, citric acid, malic acid, Hydroiodic acid), isethionic acid, lactic acid (e.g., (+) -L-lactic acid, (+ -) -DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-) -L-malic acid, malonic acid, (+ -) -DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1, 5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, 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.

One particular group of 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 (methanesulfonate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propionic acid, butyric acid, malonic acid, glucuronic acid and lactobionic acid. One specific salt is the hydrochloride salt. Another specific salt is acetate.

If the compound is anionic or has a functional group which may be anionic (for example, -COOH may be-COO^-) Salts may be formed with organic or inorganic bases which generate the appropriate cations. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Li⁺、Na⁺And K⁺Alkaline earth metal cations such as Ca²⁺And Mg²⁺And other cations such as Al³⁺Or Zn⁺. Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e., NH)₄ ⁺) And substituted ammonium ions (e.g., NH)₃R⁺、NH₂R₂ ⁺、NHR₃ ⁺、NR₄ ⁺). 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, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N (CH)₃)₄ ⁺。

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

Modified derivatives

It is to be understood that modified derivatives of the peptide ligands as defined herein are within the scope of the invention. Examples of such suitable modified derivatives comprise one or more modifications selected from: n-terminal and/or C-terminal modifications; substitution of one or more amino acid residues with one or more non-natural amino acid residues (e.g., substitution of one or more polar amino acid residues with one or more isosteric or isoelectronic amino acids; substitution of one or more non-polar amino acid residues with other non-natural isosteric or isoelectronic amino acids); adding a spacer group; replacing one or more oxidation-sensitive amino acid residues with one or more oxidation-tolerant amino acid residues; (ii) one or more amino acid residues are replaced with alanine, one or more L-amino acid residues are replaced with one or more D-amino acid residues; n-alkylation of one or more amide bonds in a bicyclic peptide ligand; replacing one or more peptide bonds with a surrogate bond; peptide backbone length modification; substitution of one or more amino acid residues with another chemical group for a hydrogen on the alpha-carbon, modification of amino acids such as cysteine, lysine, glutamic/aspartic acid and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents to functionalize the amino acids, and introduction or substitution of amino acids to introduce orthogonal reactivity suitable for functionalization, e.g., amino acids bearing an azide or alkyne group allow functionalization with an alkyne or azide bearing moiety, respectively.

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

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 (this group is referred to herein as C)_i) Capping with acetic anhydride or other suitable reagent produces an N-terminally acetylated molecule. This embodiment provides the advantage of removing potential recognition sites for aminopeptidases and avoiding the possibility of degradation of bicyclic peptides.

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

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 (this 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 potential recognition sites for carboxypeptidases and reducing the potential for proteolytic degradation of bicyclic peptides.

In one embodiment, the modified derivative comprises the replacement of one or more amino acid residues with one or more non-natural amino acid residues. In this embodiment, unnatural amino acids can be selected that have isosteric/isoelectronic side chains that are not recognized by degradative proteases nor produce any side effects on target potency.

Alternatively, unnatural amino acids with constrained amino acid side chains can be used such that proteolysis of adjacent peptide bonds is conformationally and sterically hindered. In particular, these relate to proline analogs, bulky side chains, C α -disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and cyclic amino acids, simple derivatives that are amino-cyclopropyl carboxylic acids.

In one embodiment, the modified derivative comprises the addition of a spacer group. In a further embodiment, the modified derivative comprises a cysteine (C) towards the N-terminus_i) And/or a C-terminal cysteine (C)_iii) A spacer group is added.

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

In one embodiment, the modified derivative comprises the replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In alternative embodiments, the modified derivative comprises the replacement of 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 and thus the concentration of the available free fraction in plasma, whereas charged amino acid residues (in particular arginine) can affect the interaction of peptides with phospholipid membranes on cell surfaces. The combination of the two can affect the half-life, volume of distribution and exposure of the peptide drug and can be adjusted according to the clinical endpoint. In addition, the correct combination and number of charged amino acid residues and hydrophobic amino acid residues may reduce irritation at the injection site (if the peptide drug has been administered subcutaneously).

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

In one embodiment, the modified derivative comprises removing any amino acid residues and substituting with alanine. This embodiment provides the advantage of removing potential proteolytic attack sites.

It should be noted that each of the above mentioned modifications is used to intentionally improve the efficacy or stability of the peptide. Further efficacy improvement based on modification can be achieved by the following mechanisms:

incorporation of hydrophobic moieties that exhibit hydrophobic effects and lead to reduced dissociation rates, thereby achieving higher affinities;

incorporation of charged groups that exhibit a broad range of ionic interactions, leading to faster rates of binding and higher affinities (see, e.g., Schreiber et al, Rapid, electrophoretic associated association of proteins (1996), Nature Structure. biol.3, 427-31); and

incorporation of additional constraints in the peptide, for example by correctly constraining the side chains of the amino acids, so as to minimize the entropy loss after target binding; constraining the torsion angle of the scaffold, thereby minimizing entropy loss after target binding; and for the same reason introducing additional cyclization in the molecule.

(for review see Gentilucci et al, Current pharmaceutical Design, (2010), 16, 3185-.

Isotopic variation

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

Examples of isotopes suitable for inclusion in a peptide ligand of the invention include isotopes of hydrogen, such as²H, (D) and³h (T), isotopes of carbon, e.g.¹¹C、¹³C and¹⁴isotopes of C, chlorine, e.g.³⁶Cl, isotopes of fluorine, e.g.¹⁸F, isotopes of iodine, e.g.¹²³I、¹²⁵I and¹³¹i, isotopes of nitrogen, e.g.¹³N and¹⁵isotopes of N, oxygen, e.g.¹⁵O、¹⁷O and¹⁸o, isotopes of phosphorus, e.g.³²P, isotopes of sulfur, e.g.³⁵S, isotopes of copper, e.g.⁶⁴Isotopes of Cu and gallium, e.g.⁶⁷Ga or⁶⁸Isotopes of Ga, Y, e.g.⁹⁰Y, and isotopes of lutetium, e.g.¹⁷⁷Lu, and isotopes of bismuth, e.g.²¹³Bi。

Certain isotopically-labeled peptide ligands of the present invention, for example those incorporating a radioisotope, are useful in drug and/or substrate tissue distribution studies, and in clinical assessment of the presence and/or absence of a bindin-4 target in diseased tissue. The peptide ligands of the invention may further have valuable diagnostic properties as they may be used to detect or identify the formation of complexes between the marker compounds and other molecules, peptides, proteins, enzymes or receptors. The detection or identification method may use a compound labeled with a labeling agent such as a radioisotope, an enzyme, a fluorescent substance, a luminescent substance (e.g., luminol, a luminol derivative, luciferin, a luminescent protein, luciferase), or the like. Radioisotope tritium, i.e.³H (T) and carbon-14, i.e.¹⁴C, are particularly useful for this purpose in view of their ease of incorporation and convenient detection means.

With heavier isotopes such as deuterium (i.e.²Substitution of h (d)) may provide 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.

Using positron emitting isotopes (e.g. of the type¹¹C、¹⁸F、¹⁵O and¹³n) substitution can be used in Positron Emission Tomography (PET) studies to examine target occupancy.

Isotopically-labelled compounds of the peptide ligands of the present 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-labelled reagent in place of the non-labelled reagent previously used.

Molecular scaffold

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

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

As described in the previous document, the molecular scaffold may be a small molecule, such as an organic small molecule.

In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule consisting of amino acids, nucleotides, or carbohydrates.

In one embodiment, the molecular scaffold comprises a reactive group capable of reacting with a functional group of the polypeptide to form a covalent bond.

The molecular scaffold may include chemical groups that form links to peptides, such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

An example of a compound containing an α β unsaturated carbonyl group is 1,1',1 "- (1,3, 5-triazinan-1, 3, 5-triyl) tripropyl-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-.

Effectors and functional groups

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

The effector and/or functional group may be attached, for example, to the N and/or C terminus of the polypeptide, to an amino acid within the polypeptide, or to a molecular scaffold.

Suitable effector groups include antibodies and portions 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 comprise the hinge region of an antibody (such a region is typically found between the CH1 and CH2 domains of an IgG molecule).

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 t β half-life of one day or more, two days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. Most advantageously, the peptide ligand according to the invention comprises or consists of a peptide ligand Fc fusion having a t β half-life of one day or more.

Functional groups typically include binding groups, drugs, reactive groups for attachment of other entities, functional groups that facilitate uptake of the macrocyclic peptide into a cell, and the like.

The ability of the peptide to penetrate into the cell will allow the peptide to be effectively directed against the target within the cell. Targets that can be accessed by peptides with the ability to penetrate cells include transcription factors, intracellular signaling molecules such as tyrosine kinases, and molecules involved in apoptotic pathways. Functional groups capable of penetrating into cells include peptides or chemical groups that have been added to peptide or molecular scaffolds. Peptides, such as those derived from homeobox proteins such as VP22, HIV-Tat, Drosophila (Antennapedia), e.g., such as Chen and Harrison, Biochemical Society Transactions (2007) Vol.35, part 4, page 821; gupta et al, Advanced Drug Discovery Reviews (2004) 57, volume 9637. Examples of short peptides that have been shown to be effective in translocation across the plasma membrane include the 16 amino acid penetrating peptide from drosophila antennapedia protein (desossi et al (1994) J biol. chem. 269, p. 10444), the 18 amino acid "model amphipathic peptide" (Oehlke et al (1998) Biochim biophysis Acts, p. 1414, p. 127) and the arginine-rich region of the HIV TAT protein. Non-peptide Methods include the use of small molecule mimetics or SMOCs that can be readily attached to biomolecules (Okuyama et al (2007) Nature Methods, vol.4, p.153). Other chemical strategies that add guanidino groups to the molecule also enhance cell penetration (Elson-Scwab et al (2007) J Biol Chem, Vol.282, p.13585). Small molecular weight molecules (e.g., steroids) can be added to the molecular scaffold to enhance cellular uptake.

One class of functional groups that can be attached 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 increase the half-life of the peptide ligand in vivo may be used.

In one embodiment, a peptide ligand-effector group according to the present invention has a t β half-life selected from 12 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, or 20 days or more. Advantageously, a peptide ligand-effector group or composition according to the invention will have a t β half-life in the range of 12 to 60 hours. In a further embodiment, it will have a t β half-life of one day or more. In a still further embodiment, it will be in the range of 12 to 26 hours.

In a particular embodiment of the invention, the functional group is selected from metal chelators, which are suitable for complexing pharmaceutically relevant metal radioisotopes.

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

In a particular embodiment of the invention, the functional group is selected from drugs, e.g. cytotoxic agents for cancer therapy. Suitable examples include: alkylating agents such as cisplatin and carboplatin, and oxaliplatin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide; antimetabolites including the purine analogs azathioprine and mercaptopurine or pyrimidine analogs; plant alkaloids and terpenoids including vinca alkaloids, such as vincristine, vinblastine, vinorelbine, and vindesine; etoposide and teniposide, which are derivatives of podophyllotoxin; taxanes, including paclitaxel (paclitaxel), originally referred to as paclitaxel (Taxol); topoisomerase inhibitors include camptothecin: irinotecan and topotecan, and type II inhibitors, including ambridine, etoposide phosphate, and teniposide. Other agents may include antitumor antibiotics including the immunosuppressive agents actinomycin D (for kidney transplantation), doxorubicin, epirubicin, bleomycin, calicheamicin and the like.

In a further embodiment of the invention, the cytotoxic agent is selected from maytansinoids (e.g. DM1) or monomethyl auristatins (e.g. MMAE).

DM1 is a cytotoxic agent, which is a thiol-containing derivative of maytansinoids (maytansinoids), having the structure:

the data herein are shown in table 3, which demonstrates the effect of peptide ligands conjugated to DM 1-containing toxin.

Monomethyl auristatin e (mmae) is a synthetic antineoplastic agent having the following structure:

the data herein are shown in table 3, which demonstrates the effect of peptide ligands conjugated to MMAE-containing toxins.

In a still further specific embodiment of the invention, the cytotoxic agent is selected from monomethyl auristatin e (mmae).

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

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

The hindrance on 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.

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

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.

In one embodiment, the cytotoxic agent is selected from MMAE, and the drug conjugate further comprises a linker selected from the group consisting of: -PABC-Cit-Val-glutaryl-or-PABC-cyclobutyl-Ala-Cit- β Ala-, wherein PABC represents p-aminobenzyl carbamate. Details of the linker containing the cyclobutyl group can be found in Wei et al (2018) J.Med.chem.61,989-1000 in its entirety. In a further embodiment, the cytotoxic agent is selected from MMAE and the linker is-PABC-Cit-Val-glutaryl-.

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

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-.

This BDC is known herein as BCY8245, represented schematically as:

and also in a more detailed manner as follows:

as shown in table 3, the data provided herein demonstrate excellent binding of BCY8245 to human bindin-4 in SPR binding assays. The BDC also showed good antitumor activity in the non-small cell lung cancer model as shown in example 1, the bladder cancer model as shown in example 2, the pancreatic cancer model as shown in example 3, and the breast cancer model as shown in example 4.

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) -. This BDC is referred to herein as BCY 8549. As shown in table 3, the data provided herein demonstrate excellent binding of BCY8549 to human bindin-4 in the SPR binding assay.

In another embodiment, the bicyclic drug conjugate is selected from BCY8245 or BCY 8549. In another embodiment, the bicyclic drug conjugate is BCY 8245. As shown by the data described herein, the drug conjugate BCY8245 exhibited excellent dose-dependent antitumor activity.

Synthesis of

The peptides of the invention may be prepared synthetically by standard techniques and then reacted with the molecular scaffold in vitro. When doing so, standard chemical methods can be used. This enables rapid large-scale preparation of soluble materials for further downstream experiments or validation. Such a process can be accomplished using conventional chemistry such as that disclosed in Timmerman et al, supra.

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

When preparing the conjugate or complex, amino acid residues in the polypeptide of interest may optionally be substituted.

The peptide may also be extended to incorporate, for example, another loop, thus introducing multispecific properties.

For extension of the peptide, it can be chemically extended using standard solid or solution phase chemistry, using orthogonally protected lysines (and the like), simply at its N-or C-terminus or within a loop. The activated or activatable N-or C-terminus can be introduced using standard (bio) conjugation techniques. Alternatively, addition may be by fragment condensation or Native Chemical ligation (e.g., as described in (Dawson et al 1994.Synthesis of Proteins by Natural Chemical ligation. science 266: 776-.

Alternatively, the peptide may be extended or modified by further conjugation via a disulfide bond. This has the additional advantage of allowing the first and second peptides to be separated from one another once in the cell reducing environment. In this case, a molecular scaffold (e.g., TATA) may be added during the chemical synthesis of the first peptide to allow reaction with the three cysteine groups; an additional cysteine or thiol may then be added to the N-or C-terminus of the first peptide such 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.

Similar techniques are equally applicable to the synthesis/coupling of two bicyclic and bispecific macrocycles, potentially leading to tetraspecific molecules.

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

Pharmaceutical composition

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

Generally, the peptide ligands of the invention will be used in purified form together with a pharmacologically appropriate excipient or carrier. Typically, such excipients or carriers include aqueous or alcoholic/aqueous 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 agents. If it is desired to keep the polypeptide complex in suspension, suitable physiologically acceptable adjuvants may be selected from thickening agents such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates.

Intravenous carriers include liquid and nutritional supplements and electrolyte supplements such as those based on ringer's dextrose. Preservatives and other additives may also be present, such as antimicrobials, antioxidants, chelating agents and inert gases (Mack (1982) Remington's Pharmaceutical Sciences, 16 th edition).

The peptide ligands of the invention may be used as compositions administered alone or in combination with other agents. These may include antibodies, antibody fragments and various immunotherapeutic drugs, such as cyclosporine, methotrexate, doxorubicin or cisplatin and immunotoxins. Pharmaceutical compositions may include "cocktail mixtures" of various cytotoxins or other agents in combination with the protein ligands of the invention, or even combinations of polypeptides selected according to the invention with different specificities, e.g., polypeptides selected using different target ligands, whether or not combined prior to administration.

The route of administration of the pharmaceutical composition according to the present invention may be those generally known to those of ordinary skill in the art. For treatment, the peptide ligands of the invention may be administered to any patient according to standard techniques. Administration may be by any suitable mode, including parenteral, intravenous, intramuscular, intraperitoneal, transdermal, pulmonary routes, or also suitably by direct catheter infusion. Preferably, the pharmaceutical composition according to the invention is administered by inhalation. The dose and frequency of administration depends on the age, sex and condition of the patient, concurrent administration of other drugs, contraindications and other parameters to be considered by the clinician.

The peptide ligands of the invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has proven effective and lyophilization and reconstitution techniques known in the art can be employed. Those skilled in the art will appreciate that lyophilization and reconstitution can result in varying degrees of loss of activity, and that levels may have to be adjusted upward to compensate.

Compositions containing the peptide ligands of the invention or cocktail thereof can be administered for prophylactic and/or therapeutic treatment. In certain therapeutic applications, a sufficient amount to effect at least partial inhibition, suppression, modulation, 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 will generally range from 0.005 to 5.0mg of the selected peptide ligand per kilogram of body weight, with doses of from 0.05 to 2.0 mg/kg/dose being more common. For prophylactic use, compositions containing the peptide ligands of the invention or cocktail thereof can also be administered at similar or slightly lower doses.

Compositions comprising peptide ligands according to the invention may be used to help alter, inactivate, kill or eliminate selected target cell populations in mammals in both prophylactic and therapeutic settings. In addition, the peptide ligands described herein can be selectively used to kill, deplete, or otherwise effectively remove a target cell population from a heterogeneous collection of cells in vitro (extracorporeally) or in vitro (in vitro). Blood from the mammal may be combined in vitro with selected peptide ligands, whereby undesirable cells are killed or otherwise removed from the blood and returned to the mammal according to standard techniques.

Coadministration with one or more other therapeutic agents

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

In some embodiments, the invention provides a method of treating a disclosed disease or condition, comprising administering to a patient in need thereof an effective amount of a compound disclosed herein, or a pharmaceutically acceptable salt thereof, and concurrently 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 an additional therapeutic agent. In some embodiments, the method comprises co-administering two additional therapeutic agents. In some embodiments, the combination of the disclosed compounds and other therapeutic agent or agents exerts a synergistic effect.

The compounds of the invention may also be used in combination with known therapeutic methods (e.g., administration of hormones or radiation). In certain embodiments, the compounds provided are useful as radiosensitizers, particularly for treating tumors that are poorly sensitive to radiotherapy.

The compounds of the invention can be administered alone or in combination with one or more other therapeutic compounds, possible combination therapies taking the form of fixed combinations, or administration of the compounds of the invention and one or more other therapeutic compounds staggered or independently of one another, or administration of a fixed combination in combination with one or more other therapeutic compounds. The compounds of the present invention may also be administered additionally or alternatively in combination with chemotherapy, radiotherapy, immunotherapy, phototherapy, surgical intervention, or a combination thereof, in particular for the treatment of tumors. As mentioned above, long-term therapy has equal potential as adjuvant therapy in the context of other treatment strategies. Other possible treatments are therapy to maintain the patient's state after tumor regression, even chemopreventive therapy, for example in patients at risk.

One or more additional therapeutic agents may be administered separately from a compound or composition of the invention as part of a multiple dose regimen. Alternatively, one or more additional therapeutic agents may be part of a single dosage form, mixed together with the compounds of the present invention in a single composition. If administered in a multi-dose regimen, one or more additional therapeutic agents and a compound or composition of the invention may be administered simultaneously, sequentially, or within a time interval of one another, e.g., 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 of one another. In some embodiments, the one or more additional therapeutic agents and the compound or composition of the invention are administered in a multiple dose regimen over an interval of greater than 24 hours.

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

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

In those compositions that comprise one or more additional therapeutic agents, the one or more additional therapeutic agents and the compounds of the present invention may act synergistically. Thus, the amount of the one or more additional therapeutic agents in such a composition can be less than that required for a monotherapy employing only that therapeutic agent. In such compositions, one or more other therapeutic agents may be administered at a dose of 0.01 to 1,000g/kg body weight/day.

The amount of the one or more additional therapeutic agents present in the compositions of the present invention may not exceed the amount typically administered in compositions comprising the therapeutic agent as the only active agent. Preferably, the amount of the one or more additional therapeutic agents in the compositions of the present disclosure will generally range from about 50% to 100% of the amount typically present in a composition comprising the therapeutic agent as the sole therapeutically active agent. In some embodiments, one or more additional therapeutic agents are administered at a dose that is about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the amount of the agent that is typically administered. As used herein, the term "generally administered" refers to the amount of FDA-approved therapeutic agent provided according to the FDA label insert for administration.

The compounds of the present invention, or pharmaceutical compositions thereof, may also be incorporated into compositions for coating implantable medical devices, such as prostheses, prosthetic valves, vascular grafts, stents and catheters. For example, vascular stents have been used to overcome stenosis (restenosis of the vessel wall after injury). However, patients using stents or other implantable devices are at risk of developing blood clots or activated platelets. These adverse effects can be prevented or reduced by pre-coating the device with a pharmaceutically acceptable composition comprising a kinase inhibitor. Implantable devices coated with the compounds of the invention are another embodiment of the invention.

Exemplary other therapeutic Agents

In some embodiments, the one or more additional therapeutic agents is a Poly ADP Ribose Polymerase (PARP) inhibitor. In some embodiments, the PARP inhibitor is selected from olaparib (olaparib) (iii)AstraZeneca); ruocaparib (rucaparib) ((Rucaparib))Clovis Oncology); nilaparib (niraparib) ((niraparib))Tesaro); tazoparib (MDV3800/BMN 673/LT00673, Medivation/Pfizer/Biomarin); veliparib (ABT-888, AbbVie); and BGB-290(BeiGene, Inc.).

In some embodiments, the one or more additional therapeutic agents is a Histone Deacetylase (HDAC) inhibitor. In some embodiments, the HDAC inhibitor is selected from vorinostat (vorinostat) ((r))Merck); romidepsin (romidepsin) ((R))Celgene); panobinostat (panobinostat) (panobinostat)Novartis); belinostat (belinostat) (belinostat)Spectra Pharmaceuticals); entinostat (SNDX-275, Syndax Pharmaceuticals) (NCT 00866333); and formamide (chidamide) ((ii))HBI-8000, Chipscreen Biosciences, China).

In some embodiments, the one or more additional therapeutic agents is a CDK inhibitor, e.g., a CDK4/CDK6 inhibitor. In some embodiments, the CDK 4/6 inhibitor is selected from pabociclib (palbociclib) ((r))Pfizer); ribociclib (Ribociclib) ((R))Novartis); aberrali (abemaciciclib) (Ly2835219, Eli Lilly); and trilicib (G1T28, G1 Therapeutics).

In some embodiments, the one or more additional therapeutic agents is a phosphatidylinositol 3 kinase (PI3K) inhibitor. In some embodiments, the PI3K inhibitor is selected from idarasib (idelalisib) (ii) aGilead), apexib (antipelisib) (BYL719, Novartis), taselisib (taselisib) (GDC-0032, Genentech/Roche); (ii) Pertixib (pictilisb) (GDC-0941, Genentech/Roche); panisib (copenlisib) (BAY806946, Bayer); duveliib (original name IPI-145, Infinity Pharmaceuticals); PQR309(Piqur Therapeutics, Switzerland); and TGR1202 (original name RP5230, TG Therapeutics).

In some embodiments, the one or more other therapeutic agents are platinum-based therapeutic agents, also known as platinum (platins). Platinum causes DNA cross-linking primarily in rapidly proliferating cells (e.g., cancer cells), thereby inhibiting DNA repair and/or DNA synthesis. In some embodiments, the platinum-based therapy is selected from cisplatin (cissplatin) ((r))Bristol-Myers Squibb); carboplatin (carboplatin)Bristol-Myers Squibb; and Teva; pfizer); oxaliplatin (oxaliplatin) ((oxaliplatin))Sanofi-Aventis); nedaplatin (nedaplatin)Shionogi), picoplatin(picoplatin) (ponirad Pharmaceuticals); and satraplatin (JM-216, Agennix).

In some embodiments, the one or more additional therapeutic agents is a taxane compound that causes the destruction of microtubules, which are essential for cell division. In some embodiments, the taxane is selected from the group consisting of paclitaxel (paclitaxel) (paclitaxel)Bristol-Myers Squibb), docetaxel (docetaxel) ((docetaxel)Sanofi-Aventis；Sun Pharmaceutical); paclitaxel conjugated with albumin (Abraxis/Celgene); cabazitaxel (Cabazitaxel) ((R))Sanofi-Aventis) and SID530(SK Chemicals, Co.) (NCT 00931008).

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 cells that will inhibit rapid proliferation.

In some embodiments, the nucleoside inhibitor is selected from the group consisting of trabectedin (guanidine alkylating agent,janssen Oncology), methoxyethylamine (mechloroethylamine) (alkylating agent,aktelion Pharmaceuticals), vincristine (vincristine)Eli Lilly；Teva Pharmaceuticals；Talon Therapeutics); temozolomide (temozolomide) (prodrug of alkylating agent 5- (3-methyltriaza-1-yl) -imidazole-4-carboxamide (MTIC),merck); cytarabine (cytarabine) injection (ara-C, antimetabolite cytidine analog, Pfizer); lomustine (lomustine) (alkylating agent,Bristol-Myers Squibb；NextSource Biotechnology); azacitidine (a pyrimidine nucleoside analog of cytosine,celgene); omacitabine methylsuccinate (omacetaxine mepesuccinate) (ceftazidime octyl ester) (protein synthesis inhibitor,teva Pharmaceuticals); and asparaginase from Erwinia chrysanthemi (Erwinia chrysanthemi) (enzyme for removing asparagine,Lundbeck；EUSA Pharma); eribulin mesylate (microtubule inhibitor, tubulin-based antimitotic agent,eisai); cabazitaxel (cabazitaxel) (microtubule inhibitors, tubulin-based antimitotic agents,Sanofi-Aventis); capecitabine (capacetrine) (thymidylate synthase inhibitor,genentech); bendamustine (a bifunctional ethylamine derivative, believed to form interchain DNA crosslinks,Cephalon/Teva); ixabepilone (a semisynthetic analog of epothilone B, a microtubule inhibitor, a tubulin-based antimitotic agent,Bristol-Myers Squibb); nelarabine (nelarabine), a prodrug of a deoxyguanosine analog, an inhibitor of nucleoside metabolism,novartis); clofarabine (clorafabine), a prodrug of ribonucleotide reductase inhibitor, a competitive inhibitor of deoxycytidine,Sanofi-Aventis); and trifluoropyridine and tipiracil (tipiracil) (thymidine based nucleoside analogs and thymidine phosphorylase inhibitors,Taiho Oncology)。

in some embodiments, the one or more additional therapeutic agents is a kinase inhibitor or a VEGF-R antagonist. Approved VEGF and kinase inhibitors useful in the present invention include: bevacizumab (bevacizumab) (Bevacizumab)Genentech/Roche), an anti-VEGF monoclonal antibody; ramucirumab (ramucirumab) ((R))Eli Lilly), an anti-VEGFR-2 antibody and aflibercept (ziv-aflibercept), also known as VEGF Trap (r) ((r)Regeneron/Sanofi). VEGFR inhibitors, e.g. regorafenib (R.) (Bayer); vandetanib (vandetanib) ((vandetanib))AstraZeneca); axitinib (axitinib) ((a))Pfizer); levatinib (Levatinib) (Levatinib)Eisai); raf inhibitors, e.g. sorafenib (sorafenib) ((R))Bayer AG and Onyx); dabrafenib (dabrafenib) ((b))Novartis); and vemurafenib (vemurafenib) ((v))Genentech/Roche); MEK inhibitors, e.g. cobimetinib (cobimetinib) (II)Exelexis/Genentech/Roche); trametinib (trametinib) ((R))Novartis); Bcr-Abl tyrosine kinase inhibitors, e.g. imatinib (imatinib) (I) or (II) and (III)Novartis); nilotinib (nilotinib) ((NILOTIB))Novartis); dasatinib (dasatinib) (Dasatinib)Bristol myerssquibb); bosutinib (bosutinib) (B)Pfizer); and pinatinib (ponatinib) (ii)Ariad Pharmaceuticals); her2 and EGFR inhibitors, e.g. gefitinib (gefitinib) ((R))AstraZeneca); erlotinib (erlotinib) ((ii))Genentech/Roche/astella); lapatinib (lapatinib) (la)Novartis); afatinib (afatinib) (afatinib)Boehringer Ingelheim); ocitinib (osimertinib) (targeting activated EGFR,AstraZeneca); and brigatinib (brigatinib) (ii)Ariad Pharmaceuticals); c-Met and VEGFR2 inhibitors, e.g. cabozantinib (Cabozanitib) (C-Met) and VEGFR2Exellexis); and multi-kinase inhibitors, such as sunitinib (sunitinib) ((b))Pfizer); pazopanib (pazopanib) ((pazopanib))Novartis); ALK inhibitors, e.g. crizotinib (Pfizer); ceritinib (ceritinib) ((ii))Novartis); and Alletinib (alectinib) ((II))Genentech/Roche); bruton's tyrosine kinase inhibitors, e.g. ibrutinib (ibrutinib) (B)Pharmacyclics/Janssen); and Flt3 receptor inhibitors, such as midostaurin (midostaurin) ((Novartis)。

Other kinase inhibitors and VEGF-R antagonists that are being developed and may be used in the present invention include: tivozanib (tivozanib) (Aveo pharmaceuticals); vartanib (vatalanib) (Bayer/Novartis); lucitanib (Clovis Oncology); dovitinib (TKI258, Novartis); georgenic (chiaanib) (Chipscreen Biosciences); CEP-11981 (Cephalon); linifanib (linifanib) (Abbott Laboratories); naphthaleneLatinib (neratinib) (HKI-272, Puma Biotechnology); radotinib (raditinib) ((R))IY5511, Il-Yang Pharmaceuticals, s.korea); ruxolitinib (ruxolitinib) ((r))Incyte Corporation); PTC299(PTC Therapeutics); CP-547,632 (Pfizer); forrinib (foretinib) (exellesis, GlaxoSmithKline); quinazatinib (quinzartinib) (Daiichi Sankyo) and motesanib (Amgen/Takeda).

In some embodiments, the one or more additional therapeutic agents is an mTOR inhibitor that inhibits cell proliferation, angiogenesis, and glucose uptake. In some embodiments, the mTOR inhibitor is everolimus (everolimus), (la, lb), (lbNovartis); temsirolimus (temsirolimus) (ii)Pfizer); and sirolimus (sirolimus) ((ii))Pfizer)。

In some embodiments, the one or more additional therapeutic agents are proteasome inhibitors. Approved proteasome inhibitors useful in the present invention include bortezomib (bortezomib) ((b))Takeda) and carfilzomib (carfilzomib) ((R)Amgen); and ixazomi (ixazomib) (ii)Takeda)。

In some embodiments, the one or more additional therapeutic agents is a growth factor antagonist, such as an antagonist of Platelet Derived Growth Factor (PDGF), or an antagonist of Epidermal Growth Factor (EGF) or an antagonist of its receptor (EGFR). Approved PDGF antagonists useful in the present invention include olaratumab (olaratumab), (la) (e.g., a PDGF antagonist)Eli Lilly). Approved EGFR antagonists useful in the present invention include: cetuximab (Eli Lilly); nimotuzumab (necitumumab) ((b))Eli Lilly), panitumumab (panitumumab) ((ii)Amgen); and ositinib (osimertinib) (targeting activating EGFR,AstraZeneca)。

in some embodiments, the one or more additional therapeutic agents are aromatase inhibitors. In some embodiments, the aromatase inhibitor is selected from: exemestane (exemestane) ((II))Pfizer); anastazozole (anastazole) ((R))AstraZeneca) and letrozole (letrozole) ((R)Novartis)。

In some embodiments, the one or more additional therapeutic agents are antagonists of the hedgehog pathway. Approved hedgehog pathway inhibitors that may be used in the present invention include:sonedgi (sonidegib) ((iii))Sun Pharmaceuticals); and vismodegib (vismodegib) (vi)Genentech), all for the treatment of basal cell carcinoma.

In some embodiments, the one or more additional therapeutic agents is a folate inhibitor. Approved folate inhibitors for use in the present invention include pemetrexed (pemetrexed) ((R))Eli Lilly)。

In some embodiments, the one or more additional therapeutic agents is a CC chemokine receptor 4(CCR4) inhibitor. CCR4 inhibitors that are being investigated for use in the present invention include mogamuzumab (mogamulizumab) ((mogamulizumab))Kyowa Hakko Kirin, japan).

In some embodiments, the one or more additional therapeutic agents is an Isocitrate Dehydrogenase (IDH) inhibitor. IDH inhibitors that are under investigation for use in the present invention include: AG120 (Celgene; NCT 02677922); AG221(Celgene, NCT 02677922; NCT 02577406); BAY1436032(Bayer, NCT 02746081); IDH305(Novartis, NCT 02987010).

In some embodiments, the one or more additional therapeutic agents are arginase inhibitors. Arginase inhibitors under investigation that may be used in the present invention include: AEB1102 (pegylated recombinant arginase, Aeglea Biotherapeutics), is being studied in phase 1 clinical trials against acute myeloid leukemia and myelodysplastic syndrome (NCT02732184) and solid tumors (NCT 02561234); and CB-1158(Calithera Biosciences).

In some embodiments, the one or more additional therapeutic agents is a glutaminase inhibitor. Glutaminase inhibitors under investigation which may be used in the present invention include CB-839(Calithera Biosciences).

In some embodiments, the one or more additional therapeutic agents are antibodies that bind to a tumor antigen (i.e., a protein expressed on the cell surface of a tumor cell). Approved antibodies that bind to tumor antigens that can be used in the present invention include: rituximab (rituximab) ((R))Genentech/BiogenIdec); ofatumumab (anti-CD 20,GlaxoSmithKline); obinutuzumab (obinutuzumab) (anti-CD 20,genentech), ibritumomab (ibritumomab) (anti-CD 20 and Yttrium (Yttrium) -90,spectra Pharmaceuticals); daratumumab (daratumumab) (anti-CD 38,janssen Biotech), dintuximab (anti-glycolipid GD2,united Therapeutics); trastuzumab (trastuzumab) (anti-HER 2,genentech); trastuzumab metanoxin (ado-trastuzumab emtansine) (anti-HER 2, fused to metanoxin,genentech); and pertuzumab (pertuzumab) (anti-HER 2,Genentech); and brentuximab vedotin (anti-CD-30 drug conjugate,Seattle Genetics)。

in some embodiments, the one or more additional therapeutic agents are topoisomerase inhibitors. Approved topoisomerase inhibitors useful in the present invention include: irinotecan (irinotecan) ((R))Merrimack Pharmaceuticals); and topotecan (topotecan) ((C))GlaxoSmithKline). The topoisomerase inhibitors under investigation which may be used in the present invention include piroctone (pixantrone) ((R))CTI Biopharma)。

In some embodiments, the one or more additional therapeutic agents are inhibitors of anti-apoptotic proteins, such as BCL-2. Approved anti-apoptotic agents useful in the present invention include: venetocclax (Venetocclax)AbbVie/Genentech); and Bonatumumab (blinatumomab) (bleb)Amgen). Other therapeutic agents that have been clinically tested and that can be used in the present invention to target apoptotic proteins include neviratoxclax (ABT-263, Abbott), BCL-2 inhibitor (NCT 02079740).

In some embodiments, the one or more additional therapeutic agents is an androgen receptor inhibitor. Approved androgen receptor inhibitors useful in the present invention include enzalutamide (enzalutamide) ((r))Astellas/Medivation); approved androgen synthesis inhibitors include abiraterone (aberratone) ((R))Centocor/Ortho); approved gonadotropin releasing hormone (GnRH) receptor antagonists (degarelix,Ferring Pharmaceuticals)。

in some embodiments, the one or more additional therapeutic agents are Selective Estrogen Receptor Modulators (SERMs), which interfere with the synthesis or activity of estrogen. Approved SERMs useful in the present invention include raloxifene (raloxifene) ((R))Eli Lilly)。

In some embodiments, the one or more additional therapeutic agents are inhibitors of bone resorption. An approved therapeutic agent for inhibiting bone resorption is Denosumab (Denosumab) ((r))Amgen), an antibody that binds to RANKL, which prevents 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 for inhibiting bone resorption include bisphosphonates, such as zoledronic acid (zoledronic acid) ((R))Novartis)。

In some embodiments, the one or more additional therapeutic agents are two primary inhibitors of p53 that inhibit the interaction between the proteins MDMX and MDM 2. Inhibitors of the p53 inhibitor protein under investigation that may be used in the present invention include ALRN-6924(Aileron), a stapled peptide that equivalently binds to and disrupts the interaction of MDMX and MDM2 with p 53. ALRN-6924 is currently being evaluated in clinical trials for the treatment of AML, advanced myelodysplastic syndrome (MDS) and peripheral T-cell lymphoma (PTCL) (NCT 02909972; NCT 02264613).

In some embodiments, the one or more additional therapeutic agents are inhibitors of transforming growth factor-beta (TGF-beta or TGF beta). Inhibitors of TGF-beta protein under study that may be used in the present invention include NIS793(Novartis), an anti-TGF-beta antibody that is clinically tested for the treatment of various cancers including breast, lung, hepatocellular, colorectal, pancreatic, prostate, and renal cancers (NCT 02947165). In some embodiments, the inhibitor of TGF- β protein is fraserumumab (fresolimumab) (GC 1008; Sanofi-Genzyme) which is studied for melanoma (NCT 00923169); renal cell carcinoma ((NCT 00356460); and non-small cell lung cancer (NCT 02581787.) additionally, in some embodiments, an additional therapeutic agent is a TGF- β trap, e.g., described in Connolly et al (2012) Int' L J.biological Sciences 8: 964-978. one therapeutic compound currently used in clinical trials to treat solid tumors is M7824(Merck KgaA-original MSB0011459X), which is a bispecific anti-PD-L1/TGF- β trap compound (NCT02699515), and (NCT 025198). 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 TGF- β "trap").

In some embodiments, the one or more additional therapeutic agents are selected from the group consisting of glimba velutin-monomethyl auristatin E (MMAE) (Celldex), an anti-glycoprotein nmb (gpnmb) antibody linked to a cytotoxic MMAE (CR 011). gpNMB is a protein overexpressed by various tumors that is associated with the metastatic capacity of various cancer cells.

In some embodiments, the one or more additional therapeutic agents are antiproliferative compounds. Such antiproliferative compounds include, but are not limited to: an aromatase inhibitor; an antiestrogen; a topoisomerase I inhibitor; a topoisomerase II inhibitor; a microtubule active compound; an alkylating compound; (ii) a histone deacetylase inhibitor; compounds that induce a cellular differentiation process; a cyclooxygenase inhibitor; an MMP inhibitor; mTOR inhibitionAn agent; an antineoplastic antimetabolite; a platinum compound; compounds that target/reduce protein or lipid kinase activity and other anti-angiogenic compounds; a compound that targets, reduces or inhibits protein or lipid phosphatase activity; a gonadotropin releasing hormone agonist; an antiandrogen; methionine aminopeptidase inhibitors; a matrix metalloproteinase inhibitor; a bisphosphonate; a biological response modifier; an anti-proliferative antibody; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; a telomerase inhibitor; a proteasome inhibitor; compounds for use in the treatment of hematological malignancies; compounds that target, decrease or inhibit Flt-3 activity; hsp90 inhibitors, such as 17-AAG (17-allylaminogeldanamycin, NSC330507) from Conforma Therapeutics, 17-DMAG (17-dimethylaminoethylamino) -17-demethoxy-geldanamycin, NSC707545), IPI-504, CNF1010, CNF2024, CNF 1010; temozolomide (temozolomide)Kinesin spindle protein inhibitors, such as SB715992 or SB743921 by GlaxoSmithKline, or pentamidine (pentamidine)/chlorpromazine by CombinatoRx; MEK inhibitors, for example ARRY142886 from Array BioPharma, AZd from AstraZeneca₆244, PD181461 from Pfizer, and leucovorin (leucovorin).

As used herein, the term "aromatase inhibitor" relates to a compound that inhibits estrogen production, for example, converting the substrates androstenedione and testosterone to estrone and estradiol, respectively. The term includes, but is not limited to steroids, especially adamantane, exemestane and formalstein, especially non-steroids, especially aminoglutamine, rogelimide, pyridine-glutamine imide, trichlosartan, testosterone, ketoconazole, vorozole, fadorozole, anastrozole and letrozole. Exemestane (exemestane) is available under the trade name Aromasin^TM. Formalin (formastane) under the trade name Lentaron^TMAnd (5) selling. Fadarozole (Fadarzole) is available under the trade name Afema^TMAnd (5) selling. Anastrozole (Anastrozole) is available under the trade name Arimidex^TMAnd (5) selling. Letrozole (letrozole) is marketed under the name Femara^TMOr Femar^TMAnd (5) selling. Aminoglutamine (aminoglutethimide) under the trade name Orimeten^TMAnd (5) selling. The combinations of the invention comprising chemotherapeutic agents as aromatase inhibitors are particularly useful for the treatment of hormone receptor positive tumors, such as breast tumors.

The term "antiestrogen" as used herein relates to compounds that antagonize the effects of estrogen at the estrogen receptor level. The term includes, but is not limited to, tamoxifen (tamoxifen), fulvestrant (fulvestrant), raloxifene (raloxifene) and raloxifene hydrochloride (raloxifene hydrochloride). Tamoxifen is sold under the trade name Nolvadex^TMAnd (5) selling. Raloxifene hydrochloride under the trade name Evsta^TMAnd (5) selling. Fulvestrant is available under the trade name Faslodex^TMApplication is carried out. The combinations of the invention comprising a chemotherapeutic agent which is an antiestrogen are particularly useful for the treatment of estrogen receptor positive tumors, such as breast tumors.

The term "anti-androgen" as used herein relates to any substance 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, Aberelix (abarelix), goserelin (goserelin) and goserelin acetate (goserelin acetate). Goserelin may be sold under the tradename Zoladex^TMAnd (4) application.

The term "topoisomerase I inhibitor" as used herein includes, but is not limited to, topotecan (topotecan), gemitemcan (gimatecan), irinotecan (irinotecan), camptothecin and its analogs, 9-nitrocamptothecin, and the macromolecular camptothecin conjugate PNU-166148. Irinotecan can be administered, e.g., in its marketed form, e.g., at Camptosar^TMUnder the trademark. Topotecan is available under the trade name Hycamptin^TMAnd (5) selling.

The term "topoisomerase II inhibitor" as used herein includes, but is not limited to, anthracyclines, such as doxorubicin (including liposomal formulations, e.g., Caelyx)^TM) Daunorubicin, epirubicin, idarubicin, neorubicin, anthraquinone mitoxantroneQuinones and losoxanthraquinones, and podophyllotoxins etoposide and teniposide. Etoposide is sold under the trade name Etopophos^TMAnd (5) selling. Teniposide is sold under the trade name VM 26-Bristol. Adriamycin is sold under the trade name Acribilastin^TMOr Adriamycin^TMAnd (5) selling. Epirubicin is available under the trade name Farmorubicin^TMAnd (5) selling. Idarubicin is available under the trade name Zavedos^TMAnd (5) selling. Mitoxantrone is sold under the trade name Novantron.

The term "microtubule active agent" relates 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 derivatives thereof. Taxol is available under the trade name Taxol^TMAnd (5) selling. Docetaxel having the trade name Taxotere^TMAnd (5) selling. Vinblastine sulfate is available under the trade name Vinblastatin R.P^TMAnd (5) selling. Vincristine sulfate is sold under the trade name Farmistin^TMAnd (5) selling.

The term "alkylating agent" as used herein includes, but is not limited to, cyclophosphamide, ifosfamide, melphalan, or nitrosourea (BCNU or Gliadel). Cyclophosphamide under the trade name Cyclostin^TMAnd (5) selling. Ifosfamide is sold under the trade name of Holoxan^TMAnd (5) selling.

The term "histone deacetylase inhibitor" or "HDAC inhibitor" relates to a compound that inhibits histone deacetylase and has antiproliferative activity. This includes, but is not limited to suberoylanilide hydroxamic acid (SAHA).

The term "anti-tumor antimetabolites" 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 sold under the trade name Xeloda^TMAnd (5) selling. Gemcitabine is available under the trade name Gemzar^TMAnd (5) selling.

The term "platinum compound" as used herein includes, but is not limited toLimited to carboplatin, cisplatin and oxaliplatin. Carboplatin can be present, for example, as under the trademark Carboplat^TMThe following forms of administration. Oxaliplatin may be formulated, for example, as described under the trademark Eloxatin^TMThe following forms of administration.

The term "target/reduce protein or lipid kinase activity as used herein; or a protein or lipid phosphatase activity; or other anti-angiogenic compounds "including but not limited to protein tyrosine kinase and/or serine and/or threonine kinase inhibitors or lipid kinase inhibitors, such as: a) compounds that target, decrease or inhibit Platelet Derived Growth Factor Receptor (PDGFR) activity, such as compounds that target, decrease or inhibit PDGFR activity, particularly compounds that inhibit PDGF receptors, for example, N-phenyl-2-pyrimidine-amine derivatives, such as imatinib, SU101, SU6668 and GFB-111; b) compounds that target, decrease or inhibit Fibroblast Growth Factor Receptor (FGFR) activity; c) a compound that targets, decreases or inhibits the activity of insulin-like growth factor receptor I (IGF-IR), for example a compound that targets, decreases or inhibits the activity of IGF-IR, especially a compound that inhibits the kinase activity of IGF-I receptor, or an antibody that targets the extracellular domain of IGF-1 receptor or a growth factor thereof; d) a compound that targets, decreases or inhibits the activity of the Trk receptor tyrosine kinase family, or an ephrin B4 inhibitor; e) a compound that targets, reduces or inhibits the activity of the AxI receptor tyrosine kinase family; f) compounds that target, decrease or inhibit Ret receptor tyrosine kinase activity; g) compounds that target, decrease or inhibit the activity of Kit/SCFR receptor tyrosine kinases, such as imatinib; h) compounds that target, decrease or inhibit the activity of the C-Kit receptor tyrosine kinase that is part of the PDGFR family, e.g. compounds that target, decrease or inhibit the activity of the C-Kit receptor tyrosine kinase family, in particular compounds that inhibit the C-Kit receptor, e.g. imatinib; i) compounds that target, decrease or inhibit the activity of c-Abl family members, their gene fusion products (e.g., BCR-Abl kinase), and mutants, e.g., compounds that target, decrease or inhibit the activity of c-Abl family members and their gene fusion products, e.g., N-phenyl-2-pyrimidineA pyridine-amine derivative, such as imatinib or nilotinib (AMN 107); PD 180970; AG 957; NSC 680410; PD173955 from ParkeDavis; or dasatinib (BMS-354825); j) compounds that target, decrease or inhibit the activity of protein kinase c (pkc) and members of the Raf family of serine/threonine kinases, MEK, SRC, JAK/pan-JAK, FAK, PDK1, PKB/Akt, Ras/MAPK, PI3K, SYK, TYK2, BTK and TEC family members, and/or members of the cyclin-dependent kinase family (CDK) (including staurosporine derivatives such as midostaurin); examples of other compounds include UCN-01, safrog (safingol), BAY 43-9006, Bryostatin (Bryostatin)1, Perifosine (Perifosine); rifampicin (llmofosine); RO 318220 and RO 320432; GO 6976; lsis 3521; LY333531/LY 379196; an isoquinoline compound; FTI; PD184352 or QAN697(P13K inhibitor) or AT7519(CDK inhibitor); k) compounds that target, decrease or inhibit the activity of protein tyrosine kinase inhibitors, for example compounds that target, decrease or inhibit the activity of protein-tyrosine kinase inhibitors, including imatinib mesylate (Gleevec)^TM) Or Tyrphostin (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) epidermal growth factor family (EGFR which is homodimer or heterodimer) targeting, reducing or inhibiting receptor tyrosine kinases₁ErbB2, ErbB3, ErbB4), or mutants thereof, for example, a compound that targets, reduces or inhibits the activity of the epidermal growth factor receptor family, in particular a compound, protein or antibody that inhibits a member of the EGF receptor tyrosine kinase family, for example, EGF receptor, ErbB2, ErbB3 and ErbB4 or binds to EGF or to EGF-related ligands, CP 358774, ZD 1839, ZM 105180; trastuzumab (Herceptin)^TM) Cetuximab (Erbitux)^TM) Iressa, Tarceva(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]A pyrimidine derivative; m) compounds that target, decrease or inhibit the activity of the c-Met receptor, such as compounds that target, decrease or inhibit the activity of c-Met, in particular compounds that inhibit the kinase activity of the c-Met receptor, or antibodies that target the extracellular domain of c-Met or bind to HGF, n) compounds that target, decrease or inhibit the kinase activity of one or more JAK family members (JAK1/JAK2/JAK3/TYK2 and/or pan-JAK) including, but not limited to, PRT-062, SB-1578, barrecinib (baricitinib), palicinib (pacritib), morroniib (momelotinib), VX-509, AZD-1480, TG-101348, tofacitinib (tofacitinib), and ruxolitiib (ruxolitinib); o) compounds that target, decrease or inhibit the kinase activity of PI3 kinase (PI3K), including but not limited to ATU-027, SF-1126, DS-7423, PBI-05204, GSK-2126458, ZSTK-474, buparlisib (buparlisib), piperacillin (pictelisib), PF-4691502, BYL-719, Dasotinib (dacyliib), XL-147, XL-765, and idarasib (idelalisib); and p) compounds that target, reduce or inhibit the signaling effects of the hedgehog protein (Hh) or smooth receptor (SMO) pathway, including but not limited to cyclopamine, vismodegib (vismodegib), itraconazole (itraconazole), ibritumomab (eriodegib), and IPI-926 (saregib).

The term "PI 3K inhibitor" as used herein includes, but is not limited to, compounds having inhibitory activity against one or more enzymes of the phosphatidylinositol-3-kinase family, including, but not limited to, PI3K α, PI3K γ, PI3K δ, PI3K β, PI3K-C2 α, PI3K-C2 β, PI3K-C2 γ, Vps34, p110- α, p110- β, p110- γ, p110- δ, p85- α, p85- β, p55- γ, p150, p101, and p 87. 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 (buparlisib), piroxicam (pictelisib), PF-4691502, BYL-719, Dasotinib (dacyliib), XL-147, XL-765, and idaglissib.

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 (apocosypol), Ascenta pan-Bcl-2 inhibitor, curcumin (and its analogs), dual Bcl-2/Bcl-xL inhibitors (Infinity Pharmaceuticals/Novartis Pharmaceuticals), Genasense (G3139), HA14-1 (and its analogs; see WO 2008/118802), Nevetork (and its analogs, see US 7,390,799), NH-1(Shenayng Pharmaceutical), Oakba (obab) (and its analogs, see WO 2004/106328), S-001 (Pharmaceuticals), the TW series of compounds (Univitamin of Michio et al) and naphthalene. In some embodiments, the Bcl-2 inhibitor is a small molecule therapeutic. In some embodiments, the Bcl-2 inhibitor is a peptidomimetic.

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.

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

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

Further examples of SYK inhibiting compounds, as well as further examples of conditions that may be treated by such compounds in combination with the compounds of the present 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.

Further examples of PI3K inhibiting compounds, as well as further examples of conditions that may be treated by such compounds in combination with the compounds of the present invention, may 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.

Further examples of JAK inhibitory compounds, and of conditions that may be treated by such compounds in combination with the compounds of the present 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.

Other anti-angiogenic compounds include compounds with other mechanisms for their activity, e.g., not associated with protein or lipid kinase inhibition, such as thalidomide (Thalomid)^TM) And TNP-470.

Examples of proteasome inhibitors that may be used in combination with the compounds of the present invention include, but are not limited to, bortezomib, disulfiram (disulfiram), Epigallocatechin (EGCG) -3-gallate, salinospiramide a, carfilzomib, ONX-0912, CEP-18770, and MLN 9708.

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, e.g., okadaic acid or derivatives thereof.

Compounds that induce a cellular differentiation process include, but are not limited to, retinoic acid, alpha-gamma-or delta-tocopherol or alpha-gamma-or delta-tocotrienol.

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

As used herein, the term "bisphosphonate" includes, but is not limited to, etidronic acid (ethidonic), clodronic acid (clodronic), tiludronic acid (tildronic), pamidronic acid (pamidronic), alendronic acid (alendronic), ibandronic acid (ibandronic), risedronic acid (risedronic), and zoledronic acid (zoledronic acid). Etidronic acid is sold under the trade name Didronel^TMAnd (5) carrying out sale. Chlorophosphonic acids are sold under the trade name Bonefos^TMAnd (5) carrying out sale. Telophosphonic acid is available under the trade name Skelid^TMAnd (5) carrying out sale. Pamidronic acid is sold under the trade name Aredia^TMAnd (5) carrying out sale. Alendronic acid under the trade name Fosamax^TMAnd (5) carrying out sale. Ibandronic acid is sold under the trade name Bondranat^TMAnd (5) carrying out sale. Lituo phosphonic acid is sold under the trade name Actonel^TMAnd (5) carrying out sale. Zoledronic acid is sold under the tradename Zomet^TMAnd (5) carrying out sale. 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 ABT 578.

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

The term "inhibitor of Ras oncogenic isoform", such as H-Ras, K-Ras, or N-Ras, as used herein, refers to a compound that targets, decreases or inhibits Ras oncogenic activity. For example "farnesyl transferase inhibitors", for example L-744832, DK8G557 or R115777 (Zarnestra)^TM). The term "telomerase inhibitor" as used herein refers to compounds that target, decrease or inhibit telomerase activity. Compounds that target, decrease or inhibit telomerase activity are in particular compounds that inhibit the telomerase receptor, such as, for example, telomestatin.

The term "methionine aminopeptidase inhibitor" as used herein refers to a compound that targets, decreases or inhibits the activity of methionine aminopeptidase. A compound that targets, decreases or inhibits methionine aminopeptidase activity, including but not limited to bengamide or a derivative thereof.

The term "proteasome inhibitor" as used herein refers to a compound that targets, decreases or inhibits the activity of the proteasome. Targeting, reducing orCompounds that inhibit proteasome activity include, but are not limited to, bortezomib (Velcade)^TM) And MLN 341.

The term "matrix metalloproteinase inhibitor" or "MMP" inhibitor "as used herein includes, but is not limited to, collagen peptide mimetic and non-peptidomimetic inhibitors, tetracycline derivatives, e.g., the hydroxamate peptide mimetic inhibitor batimastat (batimastat), and its orally bioavailable analog marimastat (marimastat) (BB-2516), prinomastat (prinomastat) (AG3340), metamastat (metastat) (NSC 683551) BMS-279251, BAY 12-9566, TAA211, MMI270B, or AAJ 996.

The term "compound for use in the treatment of 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); interferon, 1-beta-D-arabinofuranosyl cytosine (ara-c) and busulfan (bisulfan); and ALK inhibitors, which are compounds that target, decrease or inhibit anaplastic lymphoma kinase.

Compounds which target, decrease or inhibit the activity of FMS-like tyrosine kinase receptors (Flt-3R) are in particular compounds, proteins or antibodies which inhibit members of the Flt-3R receptor kinase family, such as PKC412, midostaurin, staurosporine (staurosporine) derivatives, SU11248 and MLN 518.

The term "HSP 90 inhibitor" as used herein includes, but is not limited to, compounds that target, decrease or inhibit the intrinsic atpase activity of HSP 90; compounds that degrade, target, reduce or inhibit HSP90 client proteins through the ubiquitin proteosome pathway. Compounds that target, decrease or inhibit the intrinsic atpase activity of HSP90, in particular compounds, proteins or antibodies that inhibit the atpase activity of HSP90, e.g., 17-allylamine (allylamino), 17-demethoxygeldanamycin (17AAG), geldanamycin derivatives; other geldanamycin related compounds; radicicol (radicicol) and HDAC inhibitors.

The term "anti-proliferative antibody" as used herein includes, but is not limited to, trastuzumab (Herceptin)^TM) trastuzumab-DM 1,Cetuximab (erbitux), bevacizumab (Avastin)^TM) Rituximab (rituximab)PRO64553 (anti-CD 40) and 2C4 antibodies. An antibody refers to an intact monoclonal antibody, a polyclonal antibody, a multispecific antibody formed from at least two intact antibodies, and an antibody fragment, so long as they exhibit the desired biological activity.

For the treatment of Acute Myeloid Leukemia (AML), the compounds of the invention can be used in combination with standard leukemia therapies, especially in combination with therapies for the treatment of AML. Specifically, the compounds of the present invention may be administered in combination with, for example, farnesyl transferase inhibitors and/or other drugs useful for treating AML, such as daunorubicin, doxorubicin, Ara-C, VP-16, teniposide, Mitoxantrone (Mitoxantrone), Idarubicin (Idarubicin), carboplatin, and PKC 412.

Other anti-leukemic compounds include, for example, Ara-C, pyrimidine analogs, which are 2' - α -hydroxyribose (arabinoside) derivatives of deoxycytidine. Purine analogs of hypoxanthine, 6-mercaptopurine (6-MP) and fludarabine phosphate (fludarabine) are also included. Compounds that target, decrease or inhibit the activity of Histone Deacetylase (HDAC) inhibitors, such as sodium butyrate and suberoylanilide hydroxamic acid (SAHA), can inhibit the activity of enzymes known as histone deacetylases. Specific HDAC inhibitors include MS275, SAHA, FK228 (original name 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-propenamide, or a pharmaceutically acceptable salt thereof and N-hydroxy-3- [4- [ (2-hydroxyethyl) {2- (1H-indol-3-yl) ethyl ] -amino ] methyl ] phenyl ] -2E-2-propenamide, or a pharmaceutically acceptable salt thereof, particularly lactate. Somatostatin receptor antagonists, as used herein, refers to compounds that target, treat or inhibit somatostatin receptors, such as octreotide (octreotide) and SOM 230. Tumor cell destruction methods refer to methods such as ionizing radiation. The term "ionizing radiation" refers above and below 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, radiotherapy and is known in the art. See Hellman, Principles of Radiation Therapy, Cancer, in Principles and Practice of Oncology, Dexita et al, eds., fourth edition, volume 1, 248-.

Also included are EDG binding agents and ribonucleotide reductase inhibitors. As used herein, the term "EDG binding agent" refers to a class of immunosuppressive agents that modulate lymphocyte recirculation, such as FTY 720. The term "ribonucleotide reductase inhibitor" refers to pyrimidine or purine nucleoside analogs including, but not limited to, fludarabine and/or cytosine arabinoside (ara-C), 6-thioguanine, 5-fluorouracil, cladribine, 6-mercaptopurine (especially in combination with ara-C resistant to ALL) and/or pentostatin. Ribonucleotide reductase inhibitors are in particular hydroxyurea or 2-hydroxy-1H-isoindole-1, 3-dione derivatives.

Also included are compounds, proteins or monoclonal antibodies, particularly those of 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(ii) a Anthranilic acid amides; ZD 4190; zd₆474; SU 5416; SU 6668; bevacizumab; or anti-VEGF antibodies or anti-VEGF receptor antibodies, e.g., rhuMAb and RHUFab, or VEGF aptamers, e.g., Macugon; FLT-4 inhibitors, FLT-3 inhibitors, VEGFR-2IgGI antibodies, Angiozyme (RPI 4610) and bevacizumab (Avastin)^TM)。

As used herein, photodynamic therapy refers to therapy using certain chemical substances known as photosensitizing compounds to treat or prevent cancer. Examples of photodynamic therapy include treatment with compounds such as Visudyne^TMAnd sodium porphyrin.

Angiostatic steroids, as used herein, refers to compounds that block or inhibit angiogenesis, such as, for example, anecortave (anecortave), triamcinolone acetonide, hydrocortisone, 11-alpha-epihydrocortisone, 11-deoxycorticosterol, 17 alpha-hydroxyprogesterone, corticosterone, deoxycorticosterone, testosterone, estrone, and dexamethasone.

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

Other chemotherapeutic compounds include, but are not limited to: plant alkaloids, hormonal compounds and antagonists; biological response modifiers, preferably lymphokines or interferons; an antisense oligonucleotide or oligonucleotide derivative; shRNA or siRNA; or hybrid compounds, or compounds with other or unknown mechanisms of action.

The structure of The active compounds identified by number, generic or trade name can be taken from The actual version or database of The standard compilation "Merck Index", such as The Patents International (e.g., IMS World Publications).

Exemplary immunooncology agents

In some embodiments, the one or more additional therapeutic agents are immuno-oncology agents. As used herein, the term "immuno-neoplastic agent" refers to an agent effective to enhance, stimulate, and/or up-regulate an immune response in a subject. In some embodiments, administration of an immuno-neoplastic agent with a compound of the present invention has a synergistic effect in treating cancer.

The immuno-oncology agent may 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.

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

Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). An important family of membrane-bound ligands that bind to costimulatory 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 co-stimulatory or co-inhibitory receptors are the family of TNF molecules that bind to members of the homologous TNF receptor family, including CD40 and CD40L, OX-40L, CD70, CD27L, CD30, CD30L, 4-1BBL, CD137(4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAR 4, OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT β R, LIGHT, R3, VEGI/HVTL 1A, TRAMP/DR3, EDAR, EDA1, XEDA 2, TNFR1, LyFASR 1, TNFa β/TNFa β, TNFa 2, TNFa 599, TNFR β, TNFR 639, TNFa, TNFR β, TNFR 639, TNFR β, TNFR, TN.

In some embodiments, the immuno-tumor 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.

In some embodiments, the combination of a compound of the invention and an immuno-oncology agent may stimulate a T cell response. In some embodiments, the immuno-oncology agent is: (i) antagonists of proteins that inhibit T cell activation (e.g., immune checkpoint inhibitors), 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) agonists of proteins that stimulate T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS-L, OX40, OX40L, GITR, GITRL, CD70, CD27, CD40, DR3, and CD 28H.

In some embodiments, the immuno-neoplastic agent is an antagonist of an inhibitory receptor on NK cells, or an agonist of an activating receptor on NK cells. In some embodiments, the immuno-oncology agent is an antagonist of KIR, such as liriluzumab (lirilumab).

In some embodiments, the immuno-tumor agent is an agent that inhibits or depletes macrophages or monocytes, including but not limited to CSF-1R antagonists, such as CSF-1R antagonist antibodies, including RG7155(WO2011/70024, WO2011/107553, WO2011/131407, WO2013/87699, WO2013/119716, WO2013/132044) or FPA-008(WO 2011/140249; WO 2013/169264; WO 2014/036357).

In some embodiments, the immuno-oncology agent is selected from the following: agonists linked to positive co-stimulatory receptors, blockers, antagonists that attenuate signaling through inhibitory receptors, and one or more agents that systematically increase the frequency (frequency) of anti-tumor T cells, agents that overcome different immunosuppressive pathways in the tumor microenvironment (e.g., block inhibitory receptor involvement (e.g., PD-L1/PD-1 interaction)), deplete or inhibit tregs (e.g., using anti-CD 25 monoclonal antibodies (e.g., daclizumab, or by in vitro anti-CD 25 bead depletion), agents that inhibit metabolic enzymes (e.g., IDO), or reverse/prevent T cell energy or depletion), and agents that trigger innate immune activation and/or inflammation at the tumor site.

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

In some embodiments, the immuno-neoplastic agent is a PD-1 antagonist. In some embodiments, the PD-1 antagonist is administered by infusion. In some embodiments, the immuno-neoplastic 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; WO 2012/145493). In some embodiments, the immuno-oncology agent may be pidilizumab (pidilizumab) (CT-011). In some embodiments, the immune-neoplastic agent is a recombinant protein consisting of the extracellular domain of PD-L2(B7-DC) fused to the Fc portion of IgG1, referred to as AMP-224.

In some embodiments, the immuno-neoplastic agent is a PD-L1 antagonist. In some embodiments, the PD-L1 antagonist is an antagonistic PD-L1 antibody. In some embodiments, the PD-L1 antibody is MPDL3280A (RG 7446; WO2010/077634), de wagauumab (durvalumab) (MEDI4736), BMS-936559(WO2007/005874), and MSB0010718C (WO 2013/79174).

In some embodiments, the immuno-tumor agent is a LAG-3 antagonist. In some embodiments, the LAG-3 antagonist is an antagonistic LAG-3 antibody. In some embodiments, the LAG3 antibody is BMS-986016(WO2010/19570, WO2014/08218) or IMP-731 or IMP-321(WO2008/132601, WO 2009/44273).

In some embodiments, the immuno-neoplastic 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 urirumab (urelumab), or PF-05082566(WO 2012/32433).

In some embodiments, the immuno-tumor 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(WO 2011/028683).

In some embodiments, the immuno-oncology agent is an indoleamine (2, 3) -dioxygenase (IDO) antagonist. In some embodiments, the IDO antagonist is selected from: epacorstat (epacadostat) (INCB024360, Incyte); or indoximod (NLG-8189, New LinkGenetics corporation); carbopanitinib (capmanitib) (INC280, Novartis); GDC-0919 (Genentech/Roche); PF-06840003 (Pfizer); BMS: f001287(Bristol-Myers Squibb); phy906/KD108 (phytoeutica); kynurenine decomposing enzyme (Kynase, Kyn Therapeutics); and NLG-919(WO2009/73620, WO2009/1156652, WO2011/56652, WO 2012/142237).

In some embodiments, the immuno-neoplastic agent is an OX40 agonist. In some embodiments, the OX40 agonist is an agonist OX40 antibody. In some embodiments, the OX40 antibody is MEDI-6383 or MEDI-6469.

In some embodiments, the immuno-neoplastic 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(WO 2006/029879).

In some embodiments, the immuno-neoplastic agent is a CD40 agonist. In some embodiments, the CD40 agonist is an agonistic CD40 antibody. In some embodiments, the immuno-neoplastic 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 daclizumab (dacetuzumab).

In some embodiments, the immuno-neoplastic agent is a CD27 agonist. In some embodiments, the CD27 agonist is an agonistic CD27 antibody. In some embodiments, the CD27 antibody is galizumab (varluumab).

In some embodiments, the immuno-tumor agent is MGA271 (directed against B7H3) (WO 2011/109400).

In some embodiments, the immuno-tumor agent is abagomazumab (abagomomab), adakamab (adezeumab), afurtuzumab (afutuzumab), alemtuzumab (alemtuzumab), anatuzumab fentanil (anatuzumab mafentox), abebizumab (apilizumab), atizumab (atezolimab), aluzumab (avelumab), bonatumumab (blinatumumab), BMS-936559, rituximab (cataxomab), diluzumab (durvaluzumab), epradoptastapotatastat (epacostatstat), epratuzumab (epratuzumab), indole (indoximod), epratuzumab (inotuzumab) ozogamicin (zogagin), wumulumab (intetuzumab), epratuzumab (eputalizumab), epalizumab (epratuzumab), dolizumab (indomethacin), epuzumab (inotuzumab (indomituzumab), epuzumab), oxutazumab (inotuzumab (indomituzumab), oxutab (indomituzumab), omab (e), maculomab (e), maculob (e), maculob (e d-e), maculob (e), maculob), mac, Pidilizumab (pidilizumab), rituximab (rituximab), tikazumab (ticilimumab), samuzumab (samalizumab) or tremelimumab (tremelimumab).

In some embodiments, the immuno-neoplastic agent is an immunostimulatory agent. For example, blocking the inhibition of the axis by PD-1 and PD-L1Antibodies can release activated tumor-reactive T cells and have been shown in clinical trials to induce a durable anti-tumor response in an increasing number of tumor histologies, including some tumor types that are not generally considered sensitive to immunotherapy. See, e.g., Okazaki, T et al (2013) nat. immunol.14, 1212-1218; zuo et al, (2016) sci. trans. med.8. anti-PD-1 antibody nivolumab: (Bristol-Myers Squibb, also known as ONO-4538, MDX1106 and BMS-936558), shows potential in improving overall survival in patients with renal clear cell carcinoma (RCC) who have experienced disease progression during or after prior anti-angiogenic therapy.

In some embodiments, the immunomodulatory therapeutic specifically induces apoptosis of tumor cells. Approved immunomodulatory therapeutic agents that may be used in the present invention include: pomalidomide (Celgene); lenalidomide (lenalidomide) (II)Celgene); ingenol mebutrate (Ingenol Mebutate) ((R))LEO Pharma)。

In some embodiments, the immuno-oncology agent is a cancer vaccine. In some embodiments, the cancer vaccine is selected from the group consisting of: Simplex-T (sipuleucel-T) ((Dendreon/Valerant Pharmaceuticals) which have been approved for the treatment of asymptomatic, or minimally symptomatic, metastatic castration-resistant (hormone-refractory) prostate cancer; and talimogene laherparepvec (BioVex/Amgen, formerly 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 viral therapies, such as, for example, pexastimogen devicepvec (PexaVec/JX-594, SillaJen/original name Jennerex Biotherapeutics), thymidine kinase- (TK-) deficient vaccinia virus engineered to express GM-CSF for hepatocellular carcinoma (NCT02562755) and melanoma (NCT 00429312); perlaroprop (pelareorecept) (ii)Oncolytics Biotech), a variant of the respiratory tract enteroorphan virus (reovirus), is unable to replicate in cells that are not activated by the RAS in a variety of cancers, including colorectal cancer (NCT 01622543); prostate cancer (NCT 01619813); head and neck squamous cell carcinoma (NCT 01166542); pancreatic adenocarcinoma (NCT 00998322); 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 (NCT 02028117); metastatic or advanced epithelial tumors, e.g., colorectal cancer, bladder cancer, head and neck squamous cell carcinoma, and salivary gland carcinoma (NCT 02636036); ONCOS-102 (Targomax/original Oncos), an adenovirus engineered to express GM-CSF in melanoma (NCT 03003676); and peritoneal disease, colorectal cancer or ovarian cancer (NCT 02963831); 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 (NCT 02759588); or CG0070(Cold Genesys), an adenovirus engineered to express GM-CSF, in bladder cancer (NCT 02365818).

In some embodiments, the immuno-oncology agent is selected from: JX-929 (SillaJen/original name Jennerex Biotherapeutics), a TK-and vaccinia growth factor-deficient vaccinia virus engineered to express cytosine desaturationAn ammonia enzyme capable of converting the prodrug 5-fluorocytosine to the cytotoxic drug 5-fluorouracil; TG01 and TG02 (targomax/original name Oncos), peptide-based immunotherapeutics, targeting refractory RAS mutations; and an engineered adenovirus, TILT-123(TILT Biothereutetics), named: ad 5/3-E2F-delta 24-hTNF alpha-IRES-hIL 20; and VSV-GP (vira therapeutics), a Vesicular Stomatitis Virus (VSV) engineered to express Glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV), which may be further engineered to express CD8 with the aim of increasing antigen specificity⁺Antigens of T cell responses.

In some embodiments, the immuno-neoplastic agent is a T cell engineered to express a chimeric antigen receptor or CAR. T cells engineered to express such chimeric antigen receptors are referred to as CAR-T cells.

CARs have been constructed that consist of a binding domain that can be derived from: a natural ligand, a single chain variable fragment (scFv) of a monoclonal antibody specific for a cell surface antigen, an intracellular domain (endodomain) fused to the functional end of a T Cell Receptor (TCR), such as the CD 3-zeta signaling domain from the TCR, which is capable of generating an activation signal in T lymphocytes. Upon antigen binding, such CARs link to endogenous signaling pathways in effector cells and generate activation signals similar to those elicited by the TCR complex.

For example, in some embodiments, the CAR-T cell is one of those described in U.S. patent 8,906,682 (incorporated herein by reference in its entirety), which discloses that CAR-T cells are engineered to comprise an extracellular domain with an antigen binding domain (e.g., a domain that binds to CD 19), an intracellular signaling domain fused to the T cell antigen receptor complex zeta chain (e.g., CD3 zeta). When expressed in T cells, the CAR is capable of redirecting 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 underway in various indications [ https:// clinical trials. gov/ct2/resultster ═ clinical + anti + receptors & pg ═ 1 ].

In some embodiments, the immunostimulatory agent is an activator of retinoic acid receptor-associated orphan receptor gamma (ROR γ t). ROR γ T is a transcription factor that plays a key role in the differentiation and maintenance of the effector 17-type subgroup of CD4+ (Th17) and CD8+ (Tc17) T cells and the differentiation of a subset of IL-17-expressing innate immune cells such as NK cells. In some embodiments, the activator of ROR γ t is LYC-55716(Lycera), which is currently being evaluated in clinical trials for the treatment of solid tumors (NCT 02929862).

In some embodiments, the immunostimulatory agent is an agonist or activator of a toll-like receptor (TLR). Suitable activators of TLRs include agonists or activators of TLR9, such as SD-101 (Dynavax). SD-101 is an immunostimulatory CpG that is being investigated for use in B cells, follicular lymphoma and other lymphomas (NCT 02254772). Agonists or activators of TLR8 useful in the present invention include motomomod (VTX-2337, VentiRx Pharmaceuticals), which is being studied for head and neck squamous cell carcinoma (NCT02124850) and ovarian cancer (NCT 02431559).

Other immuno-oncology agents useful in the invention include: uruglizumab (BMS-663513, Bristol-Myers Squibb), an anti-CD 137 monoclonal antibody; (xxii) paliluzumab (varliumab) (CDX-1127, Celldex Therapeutics), an anti-CD 27 monoclonal antibody; BMS-986178(Bristol-Myers Squibb), an anti-OX 40 monoclonal antibody; rireluzumab (IPH2102/BMS-986015, Innate Pharma, Bristol-Myers Squibb), an anti-KIR monoclonal antibody; monatomicin (monatimab) (IPH2201, lnnate Pharma, AstraZeneca), an anti-NKG 2A monoclonal antibody; andecassimab (Andecaliximab) (GS-5745, Gilead Sciences), an anti-MMP 9 antibody; and MK-4166(Merck & Co.), an anti-GITR monoclonal antibody.

In some embodiments, the immunostimulatory agent is selected from the group consisting of elobizumab, mifamustine, an agonist or activator of toll-like receptors, and an activator of ROR γ t.

In some embodiments, the immunostimulatory therapeutic agent 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 (NCT 02689453). In some embodiments, the immunostimulant is recombinant human interleukin 12 (rhIL-12). In some embodiments, the IL-15-based immunotherapeutic is heterodimeric IL-15(hetIL-15, Novartis/admone), a fusion complex consisting of a synthetic form of endogenous IL-15 complexed with soluble IL-15 binding protein IL-15 receptor alpha chain (IL 15: 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 (NCT 02452268). In some embodiments, the recombinant human interleukin 12(rhIL-12) is NM-IL-12(Neumedicines, Inc.), NCT02544724, or NCT 02542124.

In some embodiments, the immuno-tumor agent is selected from those described in Jerry l.adams et al, "Big opportunities for small molecules in immuno-oncology," Cancer Therapy 2015, volume 14, 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 in Jerry l.adams et al in table 1. In some embodiments, the immuno-tumor agent is a small molecule that targets an immuno-tumor target selected from those listed in table 2 by Jerry l. In some embodiments, the immuno-oncology agent is a small molecule agent selected from those listed in table 2 by Jerry l.

In some embodiments, the immuno-tumoral agent is selected from the group consisting of Small molecule immuno-tumoral agents described in Peter L.Toogood, "Small molecule immuno-oncology therapeutic agents," Bioorganic & Medicinal Chemistry Letters 2018, Vol.28, p.319-329, the entire contents of which are incorporated herein by reference. In some embodiments, the immuno-tumor agent is an agent that targets a pathway as described by Peter l.

In some embodiments, the immuno-oncology agent is selected from Sandra L.Ross et al, "Bispecific T cell engageThe antisense constructs can be found in medium by cell kill ", PLoS ONE 12(8) e0183390, the entire contents of which are incorporated by referenceHerein. In some embodiments, the immune-tumor agent is a bispecific T cell engager (engage)An antibody construct. In some embodiments, bispecific T cell engagersThe antibody construct was a CD19/CD3 bispecific antibody construct. In some embodiments, bispecific T cell engagersThe antibody construct is an EGFR/CD3 bispecific antibody construct. In some embodiments, bispecific T cell engagersThe antibody construct activates T cells. In some embodiments, bispecific T cell engagersThe antibody construct activates T cells, which release cytokines that induce up-regulation of intercellular adhesion molecule 1(ICAM-1) and FAS on collateral cells (bystander cells). In some embodiments, bispecific T cell engagersThe antibody construct activates T cells, which results in induced paracellular lysis. In some embodiments, the paraneighbour cells are in a solid tumor. In some embodiments, the lysed paraneighboring cells are in close proximity to the quilt-activated T cells. In some embodiments, the paraneighboring cells comprise tumor-associated antigen (TAA) -negative cancer cells. In some embodiments, the paraneighbour cells comprise EGFR-negative cancer cells. In some embodiments, the immuno-neoplastic agent is an antibody that blocks the PD-L1/PD1 axis and/or CTLA 4. In thatIn some embodiments, the immuno-tumor agent is tumor-infiltrating T cells expanded ex vivo. In some embodiments, the immune-tumor agent is a bispecific antibody construct, or a Chimeric Antigen Receptor (CAR), that directly links T cells to a tumor-associated surface antigen (TAA).

Exemplary immune checkpoint inhibitors

In some embodiments, the immune-tumor agent is an immune checkpoint inhibitor described herein.

As used herein, the term "checkpoint inhibitor" relates to an agent that can be used to prevent cancer cells from escaping the immune system of a patient. One of the major mechanisms of anti-tumor immune subversion (subversion) is called "T-cell failure", which is due to up-regulation of inhibitory receptors as a result of chronic exposure to antigen. These inhibitory receptors serve as immune checkpoints to prevent uncontrolled immune responses.

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 (Mucin) domain-3 (Tim-3), lymphocyte activation gene-3 (Lag-3; CD223), others commonly referred to as checkpoint regulators.

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 this receptor from binding to the inhibitory ligand PDL-1, thereby overriding the ability of tumors to suppress host anti-tumor immune responses.

In one aspect, the checkpoint inhibitor is a biologic therapeutic 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 group consisting of: CTLA-4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof. In another aspect, the checkpoint inhibitor interacts with a ligand of a checkpoint protein selected from the group consisting of: CTLA-4, PDLl, PDL2, PDl, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligands, or combinations thereof. In one aspect, the checkpoint 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 particular aspect, the interleukin is glycosylated IL-7. In another aspect, the vaccine is a Dendritic Cell (DC) vaccine.

Checkpoint inhibitors include any agent that blocks or inhibits the inhibitory pathway of the immune system in a statistically significant manner. Such inhibitors may include small molecule inhibitors, or may include antibodies, or antigen-binding fragments thereof, that bind to and block or inhibit an immune checkpoint receptor, or antibodies that bind to and block or inhibit an immune checkpoint receptor ligand. 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 (belonging to the family of CD2 molecules and across all NK, γ δ and memory CD8⁺Expressed in (. alpha.beta.) T cells), CD160 (also known as BY55), CGEN-15049, CHK1 and CHK2 kinases, A2aR, and various B-7 family ligands. B7 family ligands include, but are not limited to, B7-1, B7-2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and B7-H7. Checkpoint inhibitors include antibodies, or antigen-binding fragments thereof, other binding proteins, biotherapeutics, or small molecules that bind to and block or inhibit the activity of one or more of 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-OX 40, PD-L1 monoclonal antibody (anti-B7-Hl; MEDI4736), MK-3475(PD-1 blocking agent), nivolumab (anti-PD 1 antibody), CT-011 (anti-PD 1 antibody), BY55 monoclonal antibody, AMP224 (anti-PDL 1 antibody), BMS-936559 (anti-PDL 1 antibody), MPLDL3280A (anti-PDL 1 antibody), MSB0010718C (anti-PDL 1 antibody), and ipilimumab (anti-CTLA-4 checkpoint inhibitor). Check point eggWhite ligands include, but are not limited to, PD-L1, PD-L2, B7-H3, B7-H4, CD28, CD86, and TIM-3.

In certain embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-1 antagonist, a PD-L1 antagonist, and a CTLA-4 antagonist. In some embodiments, the checkpoint inhibitor is selected from nivolumabIpilimumabAnd pembrolizumabIn 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); devolumab (durvalumab) (anti-PD-L1 antibody,AstraZeneca); and astuzumab (atezolizumab) (anti-PD-L1 antibody,Genentech)。

in some embodiments, the checkpoint inhibitor is selected from: lamborrelizumab (MK-3475), nivolumab (BMS-936558), piditumumab (CT-011), AMP-224, MDX-1105, MEDI4736, MPDL3280A, BMS-936559, ipilimumab, lilumab, IPH2101, pembrolizumabAnd tremelimumab.

In some embodiments, the immune checkpoint inhibitor is: REGN2810(Regeneron), an anti-PD-1 antibody, that has been found to have basal cell carcinoma (NCT 03132636); NSCLC (NCT 03088540); squamous cell carcinoma of skin (NCT 02760498); lymphoma (NCT 02651662); and melanoma (NCT 03002376); pidilizumab (CureTech), also known as CT-011, an antibody that binds to PD-1, for clinical trials in diffuse large B-cell lymphoma and multiple myeloma; afuzumab (A)Pfizer/Merck KGaA), also known as MSB0010718C), a fully human IgG1 anti-PD-L1 antibody, used in clinical trials: non-small cell lung cancer, merkel cell carcinoma, mesothelioma, solid tumors, kidney cancer, ovarian cancer, bladder cancer, head and neck cancer, and stomach 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. Tramezumab (CP-675,206; Astrazeneca) is a fully human monoclonal antibody against CTLA-4 and has been studied in clinical trials for a variety of indications, including: mesothelioma, colorectal, renal, breast, lung and non-small cell lung cancers, ductal pancreatic, germ cell, head and neck squamous cell, hepatocellular, prostate, endometrial, liver metastatic, liver, large B-cell lymphoma, ovarian, cervical, metastatic thyroid, urothelial, fallopian tube, multiple myeloma, bladder, soft tissue sarcoma, and melanoma. AGEN-1884(Agenus) is an anti-CTLA 4 antibody, and is undergoing phase 1 clinical trials against advanced solid tumors (NCT 02694822).

In some embodiments, the checkpoint inhibitor is an inhibitor of protein-3-containing T-cell immunoglobulin mucin (TIM-3). TIM-3 inhibitors useful in the present invention include TSR-022, LY3321367 and MBG 453. TSR-022(Tesaro) is an anti-TIM-3 antibody that is being studied in solid tumors (NCT 02817633). LY3321367(Eli Lilly) is an anti-TIM-3 antibody, which is being studied in solid tumors (NCT 03099109). MBG453(Novartis) is an anti-TIM-3 antibody that is being studied in advanced malignancies (NCT 02608268).

In some embodiments, the checkpoint inhibitor is an inhibitor of a T cell immune receptor with Ig and ITIM domains, or TIGIT (immune receptors on certain T cells and NK cells). TIGIT inhibitors useful in the present invention include BMS-986207(Bristol-Myers Squibb), anti-TIGIT monoclonal antibody (NCT 02913313); OMP-313M32 (Oncomed); and anti-TIGIT monoclonal antibody (NCT 03119428).

In some embodiments, the checkpoint inhibitor is an inhibitor of Lymphocyte Activation Gene-3 (LAG-3). LAG-3 inhibitors useful in the present invention include BMS-986016 and REGN3767 and IMP 321. BMS-986016(Bristol-Myers Squibb) is an anti-LAG-3 antibody, which is being studied in glioblastoma and glioma sarcoma (NCT 02658981). REGN3767(Regeneron) is also an anti-LAG-3 antibody, which is being studied in malignancies (NCT 03005782). IMP321(Immutep s.a.) is a LAG-3-Ig fusion protein, which is being studied: melanoma (NCT 02676869); adenocarcinoma (NCT 02614833); and metastatic breast cancer (NCT 00349934).

Checkpoint inhibitors useful in the present invention include OX40 agonists. OX40 agonists being studied in clinical trials include: PF-04518600/PF-8600(Pfizer), an agonistic anti-OX 40 antibody for metastatic renal cancer (NCT03092856) and advanced cancers and neoplasms (NCT 02554812; NCT 05082566); GSK3174998(Merck), an agonist anti-OX 40 antibody, in a stage 1 cancer test (NCT 02528357); MEDI0562 (Medmimmune/AstraZeneca), an agonist anti-OX 40 antibody for advanced solid tumors ((NCT02318394 and NCT 02705482); MEDI6469, an agonist anti-OX 40 antibody (Medmimmune/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 Squib), an agonist anti-OX 40 antibody for advanced cancer (NCT 02737475).

Checkpoint inhibitors useful in the present invention include CD137 (also referred to as 4-1BB) agonists. CD137 agonists being studied in clinical trials include: urotropin (utomicumab) (PF-05082566, Pfizer), an agonistic anti-CD 137 antibody, in diffuse large B-cell lymphoma (NCT02951156) and in advanced cancers and neoplasms (NCT02554812 and NCT 05082566); uruglizumab (BMS-663513, Bristol-Myers Squibb), an agonistic anti-CD 137 antibody, is used for melanoma and skin cancer (NCT02652455) and glioblastoma and gliosarcoma (NCT 02658981).

Checkpoint inhibitors useful in the present invention include CD27 agonists. CD27 agonists being studied in clinical trials include: malizumab (CDX-1127, Celldex Therapeutics), an agonistic anti-CD 27 antibody for head and neck squamous cell carcinoma, ovarian cancer, colorectal cancer, renal cell carcinoma, and glioblastoma (NCT 02335918); lymphoma (NCT 01460134); and glioma and astrocytoma (NCT 02924038).

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 agonistic anti-GITR antibody, for malignant melanoma and other malignant solid tumors (NCT01239134 and NCT 02628574); GWN323(Novartis), an agonistic anti-GITR antibody for solid tumors and lymphomas (NCT 02740270); INCAGN01876(Incyte/Agenus), an agonistic anti-GITR antibody, for advanced cancer (NCT02697591 and NCT 03126110); MK-4166(Merck), an agonistic anti-GITR antibody, for use in solid tumors (NCT02132754), and MEDI1873 (medimumne/AstraZeneca), an agonistic hexameric GITR-ligand molecule with the Fc domain of human IgG1, for use in advanced solid tumors (NCT 02583165).

Checkpoint inhibitors useful in the present invention include inducible T-cell costimulatory (ICOS, also known as CD278) agonists. ICOS agonists being studied in clinical trials include: MEDI-570(Medimmune), an agonistic anti-ICOS antibody, for use in lymphoma (NCT 02520791); GSK3359609(Merck), an agonistic anti-ICOS antibody, for phase 1 (NCT 02723955); and JTX-2011 (journal Therapeutics), an agonistic anti-ICOS antibody, for phase 1 (NCT 02904226).

Checkpoint inhibitors useful in the present invention include killer IgG-like receptor (KIR) inhibitors. KIR inhibitors being studied in clinical trials include: rireluzumab (IPH2102/BMS-986015, Innate Pharma/Bristol-Myers Squibb), an anti-KIR antibody, for use in leukemia (NCT01687387, NCT02399917, NCT02481297, NCT02599649), multiple myeloma (NCT02252263), and lymphoma (NCT 01592370); IPH2101(1-7F9, lnnate Pharma) for use in myeloma (NCT01222286 and NCT 01217203); and IPH4102 (lnnate Pharma), an anti-KIR antibody that binds to three domains of the long cytoplasmic tail (KIR3DL2), for use in lymphoma (NCT 02593045).

Checkpoint inhibitors useful in the present invention include CD47 inhibitors of the interaction between CD47 and signal-regulating protein alpha (SIRPa). CD47/SIRPa inhibitors being investigated in clinical trials include: ALX-148(Alexo Therapeutics), an antagonistic variant of (SIRPa) that binds to CD47 and prevents CD 47/SIRPa-mediated signaling, at stage 1 (NCT 03013218); TTI-621(SIRPa-Fc, Trillium Therapeutics), a soluble recombinant fusion protein produced by linking the N-terminal CD 47-binding domain of SIRPa to the Fc domain of human IgG1, which functions by binding to human CD47 and preventing it from delivering a "no-eat" signal to macrophages, in phase 1 clinical trials (NCT02890368 and NCT 02663518); CC-90002(Celgene), an anti-CD 47 antibody, in leukemia (NCT 02641002); and Hu5F9-G4(Forty Seven, Inc.), in colorectal and solid tumors (NCT02953782), acute myelogenous leukemia (NCT02678338), and lymphoma (NCT 02953509).

Checkpoint inhibitors useful in the present invention include CD73 inhibitors. CD73 inhibitors being studied in clinical trials include: MEDI9447(Medimmune), an anti-CD 73 antibody, in solid tumors (NCT 02503774); and BMS-986179(Bristol-Myers Squibb), an anti-CD 73 antibody, in solid tumors (NCT 02754141).

Checkpoint inhibitors useful in the present invention include agonists of stimulators of interferon gene proteins (STING, also known as transmembrane protein 173 or TMEM 173). STING agonists being studied in clinical trials include: MK-1454(Merck), an agonistic synthetic cyclic dinucleotide, in lymphoma (NCT 03010176); and ADU-S100(MIW815, Aduro Biotech/Novartis), an excitatory synthetic cyclic dinucleotide, in phase 1 (NCT02675439 and NCT 03172936).

Checkpoint inhibitors useful in the present invention include CSF1R inhibitors. CSF1R inhibitors being studied in clinical trials include: pexidartinib (pexidartinib) (PLX3397, Plexxikon), a CSF1R small molecule inhibitor for colorectal, pancreatic, metastatic and advanced cancer (NCT02777710) and melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, gastrointestinal stromal tumor (GIST), and ovarian cancer (NCT 02452424); and IMC-CS4(LY3022855, Lilly), an anti-CSF-1R antibody for pancreatic cancer (NCT03153410), melanoma (NCT03101254), and solid tumors (NCT 02718911); 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 (NCT 02829723).

Checkpoint inhibitors useful in the present invention include NKG2A receptor inhibitors. NKG2A receptor inhibitors being studied in clinical trials include monalizumab (IPH2201, lnnate Pharma), an anti-NKG 2A antibody for head and neck neoplasms (NCT02643550) and chronic lymphocytic leukemia (NCT 02557516).

In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, ipilimumab, afuzumab, de vacizumab, atuzumab, or pidlizumab.

Therapeutic uses

The bicyclic peptides of the invention have particular utility as a bindin-4 (Nectin-4) binding agent.

Binder-4 is a surface molecule belonging to the family of bindin proteins, comprising 4 members. Integrins are cell adhesion molecules that play key roles 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 either bindin-1 (PVRL1) or bindin-4 (PVRL4) result in ectodermal dysplasia syndromes associated with other abnormalities. Bindingtin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of the other members of the family. Bindin-4 is a tumor-associated antigen that accounts for 50%, 49% and 86% of breast, ovarian and lung cancers, respectively, and is predominantly on tumors with poor prognosis. Its expression was not detected in the corresponding normal tissues. In breast tumors, bindin-4 is expressed predominantly in triple negative and ERBB2+ cancers. In the sera of patients with these cancers, the detection of the soluble form of bindin-4 is associated with a poor prognosis. Serum bindin-4 levels were elevated during the transfer and decreased after treatment. These results indicate that bindin-4 may be a reliable target for the treatment of cancer. Thus, several anti-bindin-4 antibodies have been described in the prior art. In particular, Enfortumab Vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) targeted to bindin-4, and has been currently clinically studied to treat patients with solid tumors.

Polypeptide ligands selected according to the methods of the invention are useful for in vivo therapeutic and prophylactic applications, in vitro and in vivo diagnostic applications, in vitro assays and reagent applications, and the like. Ligands with selected levels of specificity may be used in assays involving non-human animals in which cross-reactivity is desired, or in diagnostic applications in which cross-reactivity with homologues or paralogues requires careful control. In some applications, such as vaccine applications, the ability to elicit an immune response to a predetermined range of antigens can be exploited to tailor vaccines against specific diseases and pathogens.

Substantially pure peptide ligands having at least 90 to 95% homogeneity are preferred for mammalian administration, and 98 to 99% or more homogeneity are most preferred for pharmaceutical administration (especially when the mammal is a human). After purification, partial purification, or to homogeneity as desired, the selected polypeptide may be used for diagnosis or therapy (including in vitro), or for development and execution of assay procedures, immunofluorescent staining, and the like (Lefkovite and Pernis, (1979 and 1981), Immunological Methods, volumes I and II, Academic Press, New York).

According to another aspect of the present invention there is provided a peptide ligand or drug conjugate as defined herein for use in the prevention, inhibition or treatment of a disease or condition mediated by bindin-4.

According to another aspect of the present invention there is provided a method of preventing, inhibiting or treating a disease or condition mediated by bindin-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.

In one embodiment, the bindin-4 is mammalian bindin-4. In another embodiment, the mammalian bindin-4 is human bindin-4.

In one embodiment, the disease or condition mediated by bindin-4 is selected from the group consisting of viral infections, ectodermal dysplastic syndrome and other abnormalities, breast, ovarian and lung cancer, metastatic progression and solid tumors.

In another embodiment, the disease or condition mediated by bindin-4 is selected from cancer.

Examples of cancers (and their benign counterparts) that can be treated (or inhibited) include, but are not limited to, tumors of epithelial origin (various types of adenomas and carcinomas, including adenocarcinomas, squamous carcinomas, transitional cell carcinomas, and other carcinomas), such as bladder and urinary tract cancers, breast cancers, gastrointestinal cancers (including esophagus, stomach, small intestine, colon, rectum, and anus), liver cancers (hepatocellular carcinoma), gallbladder and biliary tract system cancers, exocrine pancreatic cancers, kidney cancers, lung cancers (e.g., adenocarcinoma, small cell lung cancer, non-small cell lung cancer, bronchoalveolar carcinoma, and mesothelioma), head and neck cancers (e.g., tongue cancer, buccal cavity cancer, larynx cancer, pharynx cancer, nasopharynx cancer, tonsils cancer, salivary gland cancer, nasal cavity cancer, and paranasal sinus cancer), ovarian cancer, fallopian tube cancer, peritoneal cancer, vaginal cancer, vulval cancer, penile cancer, cervical cancer, myometrial cancer, endometrial cancer, and other cancers, Thyroid cancer (e.g., thyroid follicular cancer), adrenal cancer, prostate cancer, skin and adnexal cancers (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, keratoacanthoma, proliferative nevi); hematologic malignancies (i.e., leukemias, lymphomas) and precancerous hematologic diseases, as well as peripheral malignancies, including those associated with hematologic malignancies and lymphoid lineages (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 (Burkitt) lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [ NK ] cell lymphoma, Hodgkin's lymphoma, hairy cell leukemia, monoclonal agammaglobulinosis of unknown significance, plasmacytoma, multiple myeloma, and lymphoproliferative disease after transplantation), and those associated with hematologic malignancies and myeloid lineages (e.g., Acute Myelogenous Leukemia (AML), Chronic Myelogenous 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's sarcoma, ewing's sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytoma, and elevated skin fibrosarcoma; tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas, and glioblastoma, meningiomas, ependymomas, pinealomas, and schwannoma); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors, and medullary thyroid cancers); ocular and accessory tumors (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratoma, seminoma, dysgerminoma, hydatidiform mole, and choriocarcinoma); and pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, wilms' tumor, and primitive neuroectodermal tumors); or predispose a patient to a congenital or other form of syndrome of malignancy (e.g., xeroderma).

In another embodiment, the cancer is selected from hematopoietic malignancies, for example selected from: non-hodgkin's lymphoma (NHL), Burkitt's Lymphoma (BL), Multiple Myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T Acute Lymphocytic Leukemia (ALL), T Cell Lymphoma (TCL), Acute Myeloid Leukemia (AML), Hairy Cell Leukemia (HCL), Hodgkin's Lymphoma (HL), and Chronic Myeloid Leukemia (CML).

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

The term "prevention" as referred to herein relates to the administration of a protective composition prior to induction of disease. By "inhibit" is meant administration of the composition after an induction event, but prior to the clinical occurrence of the disease. "treatment" refers to the administration of a protective composition after symptoms of the disease become apparent.

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

In addition, the data provided herein demonstrate a correlation between Copy Number Variation (CNV) of bindin-4 from multiple tumor types and gene expression. Thus, according to another aspect of the present invention there is provided a method of preventing, inhibiting or treating cancer comprising administering to a patient in need thereof an effector group of a peptide ligand as defined herein and a drug conjugate, wherein the patient is identified as having an increased copy number change of bindin-4 (CNV).

In one embodiment, the cancer is selected from those identified herein as CNVs having increased bindin-4. In another embodiment, the cancer is selected from those cancers identified herein as having CNVs with increased bindin-4, i.e.: breast cancer, uterine cancer, bladder cancer, lung adenocarcinoma, lung squamous 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.

The invention is further described below with reference to the following examples.

Examples of the invention

Abbreviations

1,2,4-TriAz 3- (1,2, 4-triazol-1-yl) -alanine

1Nal 1-Naphthylalanine

2FuAla 2-Furalanine

2MePhe 2-methyl-phenylalanine

2Nal 2-Naphthylalanine

2Pal 2-pyridylalanine

3,3-DPA 3, 3-diphenylalanine

3MePhe 3-methyl-phenylalanine

3Pal 3-pyridylalanine

4,4-BPA 4, 4-biphenylalanine

4,4-DFP 4, 4-difluoroproline

4MePhe 4-methyl-phenylalanine

4Pal 4-pyridylalanine

4ThiAz beta- (4-thiazolyl) -alanine

5FTrp 5-fluoro-L-tryptophan

Agb 2-amino-4-guanidinobutyric acid

Aib Aminoisobutyric acid

Azatrp azatryptophan

Aze azetidines

C5A Cyclopentylglycine

Cha 3-cyclohexyl-alanine

Cpa Cyclopropylalanine

Cya cysteic acid

DOPA 3, 4-dihydroxyphenylalanine

HARG homoarginine

HGln homoglutamines

Hleu homoleucine

Hphe homophenylalanine

Hse (Me) homoserine (Me)

Hser homoserine

Hyp hydroxyproline

Lysine (Ac) lysine (acetyl)

Met (O2) methionine sulfone

Nle norleucine

Oic Octahydroindole Carboxylic acid

Oxa oxazolidine-4-carboxylic acid

pCoPhe p-carboxy-phenylalanine

PheOPhe 4-phenoxy-phenylalanine

Phg phenylglycine

Pip piperidine acid

Pro (4NH) 4-amino-proline

tBuAla tert-butyl-alanine

TetraZ Tetrazolylalanine

Thielanine by Thiediyl

THP (O) tetrahydropyran-4-propionic acid

THP (SO2) dioxo-4-tetrahydrothiopyranyl acetic acid

Trp (Me) methyltryptophan

Materials and methods

Peptide synthesis

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: in each case using the standard coupling conditions, and then using standard methods for deprotection.

Alternatively, the peptides were purified using HPLC and after isolation they were modified with 1,3, 5-triacryloylhexahydro-1, 3, 5-triazine (TATA, Sigma). For this, the linear peptide was purified using a 50:50 MeCN: h₂O diluted to-35 mL, added to-500. mu.L of 100mM TATA in acetonitrile and treated with 5mL of 1M NH₄HCO₃H of (A) to (B)₂The reaction is initiated by the O solution. The reaction was allowed to proceed at RT for about 30-60 minutes and lyophilized immediately upon completion of the reaction (judged by MALDI). After completion, 1ml of 1M L-cysteine hydrochloride monohydrate (Sigma) was taken in H₂The solution in O was added to the reaction at room temperature for-60 minutes to quench any excess TATA.

After lyophilization, the modified peptide was purified as above while replacing Luna C8 with a Gemini C18 column (Phenomenex) and changing the acid to 0.1% trifluoroacetic acid. Pure fractions containing the correct TATA-modifying substance were pooled, lyophilized and stored at-20 ℃.

Unless otherwise indicated, all amino acids are used in the L-configuration.

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

Preparation of bicyclic peptide drug conjugates

Preparation of BCY8549

Separation conditions are as follows: phase A: h₂0.075% TFA in O, phase B: MeCN

The separation method comprises the following steps: 18-48-55 min, RT 53.5 min

Separating the column: luna 200 x 25mm 10 μm, C18,110A and Gemin150 x 30mm, C18,5 μm,110A, ligation, 50 deg.C

The dissolving method comprises the following steps: DMF (dimethyl formamide)

Separation purity: 95 percent

BCY8234 was synthesized by solid phase synthesis.

Preparation of Compound 2

The peptide was synthesized using standard Fmoc chemistry.

1) DCM was added to a vessel containing CTC resin (5mmol, 4.3g,1.17mmol/g) and Fmoc-Cit-OH (2.0g,5mmol, 1.0eq) with N₂Bubbling.

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

3) MeOH (5mL) was added and mixed for 30 min.

4) The water was drained and washed 5 times with DMF.

5) 20% piperidine/DMF was added and reacted for 30 minutes.

6) The water was drained and washed 5 times with DMF.

7) Fmoc-amino acid solution was added and mixed for 30 seconds, then activation buffer, N, was added₂Bubbling was carried out for about 1 hour.

8) The above steps 4 to 7 were repeated to couple the following amino acids.

Note that:

#	material	Coupling agent
			1	Fmoc-Cit-OH(1.0eq)	DIEA(4.0eq)
2	1-ethoxycarbonylcyclobutanecarboxylic acid (3.0eq)	HATU (2.85eq) and DIEA (6.0eq)

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.

Peptide cleavage and purification:

1) lysis buffer (20% TFIP/80% DCM) was added to the flask containing the side chain protecting peptide at room temperature and stirred twice for 1 hour.

2) Filtered and the filtrate collected.

3) The solvent was removed by concentration.

4) The crude peptide was lyophilized to give the final product (1.4g, 85.0% yield).

Preparation of Compound 3

To a solution of compound 2(1.65g, 5.01mmol, 1.0eq) in DCM (30mL) and MeOH (15mL) was added EEDQ (2.48g, 10.02mmol, 2.0eq) and (4-aminophenyl) methanol (740.37mg,6.01mmol, 1.2 eq). The mixture was stirred at 15 ℃ for 16 hours. LC-MS showed complete consumption of Compound 2, with one major peak detected having the desired m/z. TLC indicated complete consumption of compound 2 and formation of many new spots. The reaction mixture was concentrated under reduced pressure to remove the solvent to give a residue. By flash chromatography on silica gel (80Silica Flash Column, gradient elution 0-15 DCM/MeOH @60 mL/min). Compound 3 was obtained as a yellow solid (1.3g,2.99mmol, 59.72% yield).

Preparation of Compound 4

To a solution of compound 3(1.3g,2.99mmol,1.0eq) in DMF (10mL) was added DIEA (2.32g,17.95mmol,3.13mL,6.0eq) and bis (4-nitrophenyl) carbonate (3.64g,11.97mmol,4.0 eq). The mixture was stirred at 15 ℃ for 1 hour. LC-MS showed complete consumption of compound 3 and detection of one major peak with the desired m/z. The residue was purified by preparative HPLC (neutral conditions). Compound 4(1.0g,1.67mmol, 55.74% yield) was obtained as a yellow solid.

Preparation of Compound 5

To a solution of compound 5(250.53mg,417.84 μmol,1.5eq) in DMF (5mL) was added HOBt (56.46mg,417.84 μmol,1.5eq) and DIEA (108.01mg,835.68 μmol,145.56 μ L,3.0eq), MMAE (0.200g,278.56 μmol,1.0 eq). The mixture was stirred at 35 ℃ for 12 hours. LC-MS showed complete consumption of MMAE and detection of one major peak with the desired m/z. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 5(0.180g,152.74 μmol, yield 54.83%) was obtained as a yellow solid.

Preparation of Compound 6

To compound 5(0.170g, 144.26. mu. mol,1.0eq) in THF (5mL) and H₂Adding LiOH.H into O (5mL) solution₂O (12.11mg, 288.51. mu. mol,2.0 eq). The mixture was stirred at 15 ℃ for 1 hour. LC-MS showed complete consumption of compound 5 and detection of one major peak with the desired m/z. PH 7 was adjusted by using AcOH, and THF was removed under reduced pressure to give a residue. The residue was purified by preparative HPLC (neutral conditions). Compound 6(0.185g, crude) was obtained as a yellow solid.

Preparation of BCY8549

To a solution of compound 6(0.100g,86.93 μmol,1.0eq) in DMA (4mL) was added HOSu (10.00mg,86.93 μmol,1.0eq) and EDCI (16.66mg,86.93 μmol,1.0 eq). After NHS ester formation, β -Ala-BCY8234(525.98mg, 173.85. mu. mol,2.0eq) and DIEA (33.70mg, 260.78. mu. mol, 45.42. mu.L, 3.0 eq). The mixture was stirred at 15 ℃ for 4 hours. LC-MS showed complete consumption of compound 6 and detection of one major peak with the desired m/z. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY8549 was obtained as a white solid (0.0528g,12.15 μmol, yield 13.98%, purity 95.70%). Retention time 11.48 minutes. Mass shown is 1386.4(M/3+ H).

Preparation of BCY8245

Separation conditions are as follows: phase A: h₂0.075% TFA in O, phase B: MeCN

The separation method comprises the following steps: 18-48-55 min, RT 53.5 min

Separating the column: luna 200 x 25mm 10um, C18,110A and Gemin150 x 30mm, C18,5um,110A, ligation, 50 deg.C

The dissolving method comprises the following steps: DMF (dimethyl formamide)

Separation purity: 95 percent

BCY8234 was synthesized by solid phase synthesis.

The reaction scheme of BCY8245 is shown below

Preparation of Compound 3

Compound 3 was synthesized by a solid phase method.

Preparation of Compound 4

To a solution of compound 3(1.3g,3.23mmol, 1.0eq) in DCM (10mL) and MeOH (5mL) was added EEDQ (1.60g,6.46mmol, 2.0eq) and (4-aminophenyl) methanol (517.16mg,4.20mmol, 1.3 eq). The mixture was stirred at 20 ℃ for 16 hours. LC-MS showed complete consumption of compound 3 and detection of one major peak with the desired m/z. Under reduced pressureThe solvent was removed. By flash chromatography on silica gel (40gSilica Flash Column, gradient elution 0-15% DCM/MeOH @40 mL/min) purification of the residue. Compound 4 was obtained as a yellow solid (0.950g,1.87mmol, 57.94% yield).

Preparation of Compound 5

To a solution of compound 4(0.950g,1.87mmol,1.0eq) in DMF (5mL) was added DIEA (1.21g,9.36mmol,1.63mL,5.0eq) and bis (4-nitrophenyl) carbonate (2.28g,7.49mmol,4.0 eq). The mixture was stirred at 20 ℃ for 1 hour. LC-MS showed complete consumption of compound 4 and detection of one major peak with the desired m/z. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 5 was obtained as a white solid (0.400g, 594.64 μmol, 31.77% yield).

Preparation of Compound 6

To a solution of compound 5(0.200g,297.32 μmol,1.0eq) in DMF (5mL) was added HOBt (52.23mg,386.51 μmol,1.3eq) and DIEA (115.28mg,891.95 μmol,155.36 μ L,3.0eq), MMAE (192.12mg,267.59 μmol,0.9 eq). The mixture was stirred at 20 ℃ for 16 hours. LC-MS showed complete consumption of compound 5 and detection of one major peak with the desired m/z. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 6(0.160g, 127.84. mu. mol, 43.00% yield) was obtained as a white solid.

Preparation of Compound 7

To compound 6(0.160g, 127.84. mu. mol,1.0eq) in THF (3mL) and H₂Adding LiOH.H into O (3mL) solution₂O (26.82mg, 639.21. mu. mol,5.0 eq). The mixture was stirred at 20 ℃ for 1 hour. LC-MS showed complete consumption of compound 6 and detection of one major peak with the desired m/z. THF was removed under reduced pressure and the pH was adjusted to 7 by AcOH and the mixture was lyophilized. Compound 7 was obtained as a white solid (0.130g,105.05 μmol, yield 82.17%).

Preparation of Compound 8

To a solution of compound 7(36.27mg,315.15 μmol,3.0eq) in DMA (6mL) and DCM (2mL) was added EDCI (60.41mg,315.15 μmol,3.0 eq). The mixture was stirred at 15 ℃ for 3 hours. LC-MS showed complete consumption of compound 7 and detection of one major peak with the desired m/z. DCM was removed under reduced pressure. The reaction was directly purified by preparative HPLC (neutral conditions). Compound 8(0.095g,71.18 μmol, 67.76% yield) was obtained as a white solid.

Preparation of BCY8245

To a solution of BCY8234(66.41mg, 22.48. mu. mol,1.0eq) in DMA (4mL) was added DIEA (8.72mg, 67.44. mu. mol, 11.75. mu.L, 3.0eq) and Compound 8(0.030g, 22.48. mu. mol,1.0 eq). The mixture was stirred at 20 ℃ for 16 hours. LC-MS showed complete consumption of BCY8234 and detected a major peak with the desired m/z or mass. The reaction was directly purified by preparative HPLC (TFA conditions). Compound BCY8245(0.0427g,10.16 μmol, 45.19% yield, 99.30% purity) was obtained as a white solid. Retention time 11.7 minutes. The display Mass (Mass found) is 1043.9(M/4+ H).

Biological data

Direct binding assay for bindin-4

The affinity (Ki) of the peptides of the invention for human bindin-4 was determined using fluorescence polarization assay according to the method disclosed in WO 2016/067035. The peptide of the present invention (fluorescein, SIGMA or Alexa Fluor 488) having a fluorescent tag is added^TMFisher Scientific) was diluted to 2.5nM in PBS (containing 0.01% tween 20), or 50mM HEPES (containing 100mM NaCl and 0.01% tween pH 7.4) (both referring to assay buffer). This was combined with titrated protein in the same assay buffer as the peptide to give a total volume of 25 μ L of 1nM peptide, typically 5 μ L assay buffer, 10 μ L protein then 10 μ L fluorescent peptide in a low binding capacity low capacity 384 well plate with black walls and bottom. Serial dilutions of 1 to 2 were used to give 12 different concentrations, with the highest concentrations ranging from 500nM for known high affinity binders to 10 μ M for low affinity binders and selectivity assays. Measurements were performed on a BMG PHERAStar FS equipped with an "FP 485520520" optics blockThe optical module was excited at 485nm and detected parallel and perpendicular emission at 520 nm. The PHERAstar FS was set at 25 ℃, flashed 200 times per well, with a positional delay of 0.1 seconds, measured at 5 to 10 minute intervals per well for 60 minutes. At the end of 60 minutes, when there was no protein in the wells, the gain for analysis was determined for each tracer. Data were analyzed using a Systat Sigmaplot version 12.0. The mP value is fitted to a user-defined quadratic equation to generate a Kd value: f ═ ymin + (ymax-ymin)/Lig [ ((x + Lig + Kd)/2-sqrt ((((x + Lig + Kd)/2) ^2) - (Lig ^ x)) ]). "Lig" is a defined value for the concentration of tracer used.

Binder-4 competitive binding assay

The competition sum of the non-fluorescently labeled peptide with ACPFGCHTDWSWPIWCA-Sar6-K (Fl) (SEQ ID NO:2) was tested (Kd 5 nM-determined using the protocol described above). Peptides were diluted to appropriate concentrations in assay buffer using a maximum of 5% DMSO, followed by serial dilutions at 1 to 2 as described for the direct binding assay. mu.L of the diluted peptide was added to the plate, followed by 10. mu.L of human bindin-4, and then 10. mu.L of the fluorescent peptide. The measurement was performed as a direct binding assay, although the gain was determined prior to the first measurement. Data were analyzed using a sysstat Sigmaplot version 12.0, where the mP values fit to a user-defined cubic equation to generate Ki values: f ═ ymin + (ymax-ymin)/Lig ((Lig ^2 ^3 ^ Kcomp + Lig + Prot ^ c) + (Kcomp ^0.5 ^ COS (ARCCO) (((2 ^ Klig + Kcomp + Lig + Comp Prot ^ c) + Klig ^ Kcomp ^3+ Kl-Proc) + (Kcomp + Kcomp ^0.5 ^ COS (ARCCO) ((Kcomp ^2 ^3+ Kcomp + Kl + Kpro ^ c) ((Klig + Kcomp + Kpro-c) & 3+ Klig + Kcomp ^3+ Kl-Prot ^ c) + Kcomp ^ 1 (Kcomp) ((Kcomp + Kl Kcomp ^ 2+ Kl Proc) ((Kcomp) + (Kpro-Prog) & Kl C) & Klig + (Kpro + Kcomp) & K2 +) ((Kpro + C) & K2K + C) & K + C + (Kpro ^ C) & K + C + (C) & K + C -Prot c ^2-3 (Kcomp ^ c) + Klig-Prot ^ c) + Klig-Kcomp ^0.5 ] COS (ARCCO ((-2 ^3+ Kcomp + Lig + Comp-Prot ^ c) + 3+9 ^ Klig + Kcomp + Lig + Comp-Prot ^ c) (-Kcomp + Prot ^ c) + Klig-Prot ^ c) + Kcomp ^ 27 ^ 1 [ Kcomp + (Kcomp) + Prot ^ c) (2 + ((Klig-Prot ^ c) + Klig + Kcomp) + Prot ^ c) + (Kcomp) + Kcomp) + Prot ^2 (Kcomp) + Kcomp) (Kcomp + Kcomp) + C) + Kcomp) (2K-Proc)) (Kcomp).

"Lig", "KLig" and "Prot" are defined values respectively associated with: fluorescent peptide concentration, Kd of fluorescent peptide, and bindin concentration.

BIACORE SPR BIOCORE BINDING ASSAY FOR BIOCORIN-4

Biacore experiments were performed to determine k for monomeric peptides that bind to human Necin-4 protein_a(M^-1s^-1)、k_d(s^-1)、K_D(nM) value (obtained from Charles River).

Standard Bac-to-Bac was used for human bindin-4 (residues Gly32-Ser 349; NCBI reference sequence: NP-112178.2) with gp67 signal sequence and C-terminal FLAG tag^TMThe protocol (Life Technologies) was cloned into pFastbac-1 and baculovirus. Infection of 1X10 in excel-420 medium (Sigma) with P1 Virus stock at MOI of 2 at 27 deg.C⁶ml^-1Sf21 cells, and the supernatant was harvested at 72 hours. The supernatant was combined in portions with anti-FLAG M2 affinity agarose resin (Sigma) washed in PBS for 1 hour at 4 ℃, after which the resin was transferred to a column and washed well with PBS. The protein was eluted with 100. mu.g/ml FLAG peptide. The eluted proteins were concentrated to 2ml and loaded onto an S-200Superdex column (GE Healthcare) in PBS at a rate of 1 ml/min. Fractions of 2ml were collected and fractions containing the bindin-4 protein were concentrated to 16 mg/ml.

According to the manufacturer's recommended operating scheme, EZ-Link is used^TMThe protein was randomly biotinylated in PBS by Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher). The protein was desalted sufficiently using a spin column to transfer unconjugated biotin into PBS.

For analysis of peptide binding, a Biacore 3000 instrument was used, which utilized a CM5 chip (GE Healthcare). Streptavidin was immobilized on the chip using standard amine coupling chemistry, HBS-N (10mM HEPES, 0.15M NaCl, pH 7.4) as running buffer at 25 ℃. Briefly, a mixture of 1:1 ratio of 0.4M 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)/0.1M N-hydroxysuccinimide (NHS) was injected at a flow rate of 10. mu.l/min for 7 minutes to activate the carboxymethyl dextran surface. To capture streptavidin, the protein was diluted to 0.2mg/ml in 10mM sodium acetate (pH 4.5) and captured by injecting 120. mu.l of streptavidin onto the activated chip surface. Residual activating groups were blocked by injection of 1M 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 diluted peptides were prepared in this buffer with a final DMSO concentration of 0.5%. The highest peptide concentration was 100nM and diluted 6 times in 2-fold dilutions. SPR analysis was run at 25 ℃ at a flow rate of 50 μ l/min with an association time of 60 seconds, depending on the dissociation time of each peptide of 400 to 1200 seconds. Data were corrected for DMSO exclusion volume effects. Both blank injections and double referencing of reference surfaces were performed on all data using standard processing procedures, and data processing and kinetic fitting were performed using scubber software version 2.0c (BioLogic software). Use of simple 1:1 combine the model fit data to achieve the mass transfer effect under appropriate circumstances.

Certain peptide ligands of the invention were tested in the above-described bindin-4 binding assay and the results are shown in table 1:

table 1: competitive binding data for selected peptide ligands of the invention

Double ring number	Ki(μM)	Experiment number
			BCY8122	0.003	2
BCY8126	0.0027	6

Certain bicyclic peptides of the invention were tested in the SPR assay described above and the results are shown in table 2:

table 2: SPR data for selected peptide ligands of the invention

n is the average number of experiments

Certain bicyclic peptides of the invention were conjugated to cytotoxic agents and tested in the SPR assay described above, with the results shown in table 3:

table 3: SPR data for BDC selected by the present invention

In vivo studies

In examples 1 to 5 and 9, the following methods were used for each study:

test and positive controls

Numbering	Physical description	Molecular weight	Purity of	Storage conditions
					BCY8245	Freeze-dried powder	4173.85	99.60％	Stored at-80 deg.C
BCY8549	Freeze-dried powder	4157.81	95.70％	Stored at-80 deg.C

Experimental methods and procedures

(i) Observation of

Procedures related to Animal handling, Care and treatment in all studies were performed according to guidelines approved by the Wuxi ApTec Institutional Animal Care and Use Committee (IACUC) and following the guidelines of the Association for the Assessment and characterization of Laboratory Animal Care (AAALAC). In routine monitoring, animals are examined daily for tumor growth and any effect of treatment on normal behavior, such as activity, food and water consumption (by observation only), weight gain/loss, eye/hair shine, and any abnormal effects as set forth in the protocol. Mortality and observed clinical signs were recorded based on the number of animals in each subset.

(ii) Tumor measurement and endpoint

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 using a caliper three times a week and in mm using the following formula³Representing volume：V＝0.5a x b²Wherein a and b are the major and minor diameters of the tumor, respectively. Tumor size was then used for T/C value calculation. The T/C value (percentage) indicates the antitumor effect; t and C are the average volumes of the treatment and control groups, respectively, on the indicated days.

TGI was calculated for each group using the following formula: TGI (%) - (1- (T)_i-T₀)/(V_i-V₀)]×100；T_iIs the mean tumor volume, T, of the treatment groups on the indicated day₀Is the mean tumor volume, V, of the treatment group on the day of treatment initiation_iIs a vehicle control group in combination with T_iMean tumor volume on the same day, V₀Is the mean tumor volume of the vehicle group on the day of treatment initiation.

(iii) Statistical analysis

A summary of statistical data including mean and Standard Error of Mean (SEM) for each group of tumor volumes at each time point is provided.

Statistical analysis of tumor volume differences between groups was performed based on data obtained at the optimal treatment time point after the final dose.

One-way ANOVA was performed to compare tumor volumes between groups, using the Games-Howell test when significant F-statistics (treatment variance versus error variance) were obtained. All data were analyzed using GraphPad Prism 5.0. P < 0.05 was considered statistically significant.

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

1. Purpose of study

The objective of this study was to evaluate the in vivo antitumor efficacy of BCY8245 in the NCI-H292 xenograft model in the treatment of BALB/c nude mice.

2. Design of experiments

Note that: n: numbering the animals; administration volume: the administration volume was adjusted to 10. mu.L/g based on body weight.

3. Material

3.1 animal and feeding conditions

3.1.1. Animal(s) production

Species: little mouse (Mus Musculus)

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: 18 mice for BCY8245 plus spares

Animal suppliers: shanghai LC Laboratory Animal Co., LTD.

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

3.2 test and Positive controls

4. Experimental methods and procedures

4.1 cell culture

NCI-H292 tumor cells were maintained in culture medium supplemented with 10% heat-inactivated fetal bovine serum, maintained at 37 ℃ in an atmosphere of 5% CO2 in air. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of NCI-H292 tumor cells (10X 10) in PBS⁶) To allow for tumor growth. When the average tumor volume reaches about 158-406 mm³At that time, animals were randomized and treatment was initiated. The experimental design table below shows the experimental administration and the number of animals per group.

4.3 preparation of test substance preparations

4.4 sample Collection

At the end of the study, plasma was collected 5 min, 15 min, 30 min, 60 min and 120 min after the last dose.

5. Results

5.1 tumor growth Curve

Tumor growth curves are shown in figures 1 and 2.

5.2 tumor volume trajectory

The average tumor volume over time for female Balb/c nude mice carrying NCI-H292 xenografts is shown in the table below:

table 4: trajectory of tumor volume over time

Table 5: trajectory of tumor volume over time

5.3 tumor growth inhibition assay

Based on tumor volume measurements, the tumor growth inhibition rate of BCY8245 in the NCI-H292 xenograft model at day 14 was calculated.

Table 6: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

Table 7: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

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

On day 14, the mean tumor size of vehicle-treated mice reached 879mm³。

BCY8245 at 1mg/kg did not produce significant antitumor activity, and after increasing the dose to 3mg/kg from day 7, the test article showed significant antitumor activity, but when the dose was increased to 5mg/kg at day 21, the therapeutic effect was not further improved. 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 substances.

BCY8245 and 3mg/kg,qw(TV＝149mm³,TGI＝101.4％,p<0.001),3mg/kg,biw(TV＝65mm³,TGI＝112.2％,p<0.001) and 5mg/kg, qw (TV 83 mm)³,TGI＝109.8％,p<0.001) produces significant antitumor activity.

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

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of the test substances in the treatment of HT-1376 xenografts in CB17-SCID mice.

2. Design of experiments

a 1mg/kg for the first week, 3mg/kg for the next 2 weeks

3. Material

3.1 animal and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: CB17-SCID

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: 21-41 mice were added for use

Animal suppliers: shanghai LC Laboratory Animal Co., LTD.

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

HT-1376 tumor cells were maintained as monolayer cultures in vitro in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37 ℃ with 5% CO in air₂Is maintained in the atmosphere of (2). Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of HT-1376 tumor cells (5X 10) in PBS with matrigel (1:1)⁶) To allow for tumor growth. When the average tumor volume reaches 153-³Animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3 preparation of test substance preparations

4.4 sample Collection

At the end of the study, group 2 plasma was collected at 5, 15, 30, 60 and 120 minutes after the last dose. Plasma was collected from group 6 at 5 min, 15 min, 30 min, 60 min and 120 min after the last dose. Group 6 tumors were collected 2 hours after the last dose. Tumors from groups 4 and 5 were collected 2 hours after the last dose.

5. Results

5.1 tumor growth Curve

Tumor growth curves are shown in fig. 3 and 4.

5.2 tumor volume trajectory

Table 8 and table 9 show the change in mean tumor volume over time in female CB17-SCID mice carrying HT-1376 xenografts.

Table 9: trajectory of tumor volume over time

5.3 tumor growth inhibition assay

Tumor growth inhibition rates of the test agents in the HT-1376 xenograft model were calculated based on tumor volume measurements at day 21 after treatment initiation.

Table 10: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

Table 11: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

Group 1 and group 2

In this study, the therapeutic efficacy of test substances in the HT-1376 xenograft model was evaluated. Fig. 3 and tables 8 and 10 show tumor volumes for all treatment groups measured at different time points.

On day 21, the mean tumor size of vehicle-treated mice reached 884mm³. BCY8245 at 1mg/kg produced slight antitumor activity, and better therapeutic effect was found after increasing the dose to 3mg/kg from day 7.

In this study, some mice treated with 3mg/kg of test substance lost more than 10% of their body weight.

Groups 3 to 6

In this study, the therapeutic efficacy of test substances in the HT-1376 xenograft model was evaluated. Fig. 4 and tables 9 and 11 show tumor volumes for all treatment groups measured at different time points.

BCY8245 at 3mg/kg, qw (TV 603 mm)³,TGI＝50.9％,p<0.01),3mg/kg,biw(TV＝407mm³,TGI＝72.3％,p<0.001) and 5mg/kg, qw (TV 465 mm)³,TGI＝66.0％,p<0.001) produces significant antitumor activity.

In this study, 5mg/kg qw of BCY8245 caused more than 10% weight loss in the animals over the treatment period.

Example 3: in vivo efficacy study of BCY8245 in treatment of Panc2.13 xenografts (pancreatic cancer model) in Balb/c nude mice

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of the test agents in panc2.13 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1 animal and feeding conditions

3.1.1 animals

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

Animal suppliers: shanghai LC Laboratory Animal Co., LTD.

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

Pan2.13 tumor cells were maintained in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units/ml human recombinant insulin at 37 ℃ in air5％CO₂Is maintained in the atmosphere of (2). Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml Pan2.13 tumor cells (5X 10) in PBS (1:1) containing matrigel⁶) To effect tumor formation. When the average tumor volume reaches 149mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3 preparation of test substance preparations

4.4 sample Collection

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

5. Results

5.1 tumor growth Curve

Tumor growth curves are shown in FIG. 5

5.2 tumor volume trajectory

The mean tumor volume of female Balb/c nude mice bearing Panc2.13 xenografts is shown in Table 12 below as a function of time.

Table 12: trajectory of tumor volume over time

5.3 tumor growth inhibition assay

The tumor growth inhibition rate of the test article in panc2.13 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 13: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of the test substances in the panc2.13 xenograft model was evaluated. Fig. 5 and tables 12 and 13 show tumor volumes for all treatment groups measured at different time points.

BCY8245 at 3mg/kg, qw (TV 271 mm)³,TGI＝69.2％,p<0.01)，3mg/kg,biw(TV＝231mm³,TGI＝79.1％,p<0.001) and 5mg/kg, qw (TV-238 mm)³,TGI＝77.5％,p<0.001) produces significant antitumor activity.

Example 4: in vivo efficacy study of BCY8245 in MDA-MB-468 xenograft (breast cancer model) in treatment of Balb/c nude mice

1. Purpose of study

The objective 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.

2. Design of experiments

3. Material

3.1 animal and feeding conditions

3.1.1 animals

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

Animal suppliers: shanghai LC Laboratory Animal Co., LTD.

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

Tumor cells were maintained in Leibovitz's L-15 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 ℃ with 5% CO in air₂Is maintained in the atmosphere of (2). Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right abdomen with MDA-MB-468 tumor cells (10X 10) in 0.2ml of PBS supplemented with 50% matrigel⁶) For use in tumor growth. When the average tumor volume reaches 196mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3 preparation of test substance preparations

4.4 sample Collection

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

5. Results

5.1 tumor growth Curve

Tumor growth curves are shown in FIG. 6

5.2 tumor volume trajectory

The change in mean tumor volume over time in female Balb/c nude mice carrying MDA-MB-468 xenografts is shown in tables 14 and 15 below.

5.3 tumor growth inhibition assay

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

Table 16: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of the test substances in the MDA-MB-468 xenograft model was evaluated. Fig. 6 and tables 14-16 show tumor volumes for all treatment groups measured at different time points.

BCY8245 at 3mg/kg, qw (TV 85 mm)³,TGI＝144.2％,p<0.001),3mg/kg,biw(TV＝22mm³,TGI＝169.8％,p<0.001) and 5mg/kg, qw (TV ═ 29 mm)³,TGI＝168.4％,p<0.001) in a dose-or dose-frequency dependent manner to produce significant antitumor activity.

The 5mg/kg group dosing was suspended from day 21 and the tumors did not show any recurrence during the additional three week monitoring period.

Example 5: BCY8549 in vivo efficacy test of NCI-H292 xenografts (non-small cell lung cancer (NSCLC) model) in the treatment of BALB/c nude mice.

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of BCY8549 in treating NCI-H292 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1 animal and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: 43 mice were added for use

Animal suppliers: shanghai Lingchang Biotechnology Experimental Animal Co.Ltd

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or4 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

NCI-H292 tumor cells were cultured in vitro as a monolayer in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum at 37 ℃ with 5% CO in air₂Is maintained in the atmosphere of (2). Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of NCI-H292 tumor cells in PBS (10X 10)⁶) To allow for tumor growth. When the average tumor volume reaches 168mm³At time, 43 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

Test article preparation

4.4 sample Collection

At the end of the study, group 2 plasma was collected 5 min, 15 min, 30 min, 1h and 2h after the last dose.

5. Results

5.1 tumor growth Curve

Tumor growth curves are shown in FIG. 7

5.2 tumor volume trajectory

The mean tumor volume of female Balb/c nude mice bearing NCI-H292 xenografts is shown in Table 17 below as a function of time.

Table 17: trajectory of tumor volume over time

5.3 tumor growth inhibition assay

The tumor growth inhibition rate of the test article in the NCI-H292 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 18: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of BCY and the like in the NCI-H292 xenograft model was evaluated. Fig. 7 and tables 17 and 18 show tumor volumes for all treatment groups measured at different time points.

On day 14, the mean tumor size of vehicle-treated mice reached 843mm³. BCY8549 at 3mg/kg showed significant antitumor activity. All mice maintained body weights well in this study.

Example 6: investigation of the correlation between Copy Number Variation (CNV) of Bindin-4 and Gene expression from multiple tumor types

Method

1. Selected in cBioPortal(http://www.cbioportal.org/)All studies in (a) and search for NECTIN 4.

(a) The provisional study was removed.

(b) Deselecting studies with overlapping samples to prevent sample bias (based on warnings in cbioport) — if selectable, the PanCancer study is always retained.

(c) The study was selected for analysis (table 19).

Table 19: analytical study from cBioPortal and research cell

2. CNV and RNA expression data were derived from cbioport.

3. It was tested whether CNV was statistically significantly associated with changes in bindin-4 mRNA expression (log 2 was not applied).

(a) A non-parametric Kruskal-Wallis test (significance threshold: p <0.01) was run in GraphPad Prism (7.04) and R/R bench (studio).

(i) GraphPad Prism: set up the list, run non-parametric tests that do not match or pair and do not employ a gaussian distribution.

(ii) Software package used in R:

1.XLConnect

2.dplyr

Kruskal-Wallis rank sum test: kruskal test.

4. Multiple comparisons (all possible comparisons included even if n in the group is 1) were adjusted in the R/R bench using the Dunn test (significance threshold: p < 0.025).

(a) Test is "bonferonni" using multiple comparison methods.

Results

The results are shown in table 20 below. In the 41 publicly available TCGA datasets reporting bindin-4 tumor CNV and mRNA gene expression data, there were many cases in which indications have been reported with increased (2-3 copies) or amplification (>3 copies) of the bindin-4 copy number. In addition, individual cases with shallow deletions (<2 copies) have been shown to rarely report tumors containing deep deletions (deep deletions), consistent with more than 1 copy loss or biallelic (biallelic) binder-4 loss. The most commonly detected indications for expansion 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 lung squamous carcinoma. These data indicate that there is a range of CNVs within and between the tumor indications, and that there is a diversity in copy number patterns between different indications.

The median bindin-4 mRNA expression levels for each indication, except CNV within the TCGA dataset, covered approximately 2¹⁰And (4) a range. Thus, given the range of bindin-4 mRNA expression levels and CNVs observed between and among tumor types, statistical tests were performed to identify potential correlations between bindin-4 mRNA levels and bindin-4 CNVs in the various TCGA datasets/indications. Tumors for each indication were classified into 1 of 5 classes:

a) deep deletion;

b) shallow deletion;

c) diploid;

d) increasing; or

e) And (5) amplification.

A Kruskall-Wallis test was then performed to determine whether the distribution of the expression values of each mRNA class was different between classes (P < 0.01). For those TCGA datasets with P <0.01, post-hoc tests were performed by calculating Z-statistics and calculating adjusted P-values (Bonferonni) to identify which classes differ from each other. For simplicity of explanation, pairwise comparisons against diploids were performed for each indication (although P values were calculated for all pairings). 18/41TCGA studies reached Kruskall-Wallis P <0.01 and Bonferonni P <0.025 in increasing relative diploid and/or amplifying relative diploid comparisons, indicating that increased bindin-4 mRNA expression correlates with increased bindin-4 copy number. These 18 studies represent 14 independent tumor histologies:

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

In addition, 6 studies had reduced mRNA expression associated with copy number loss. Four of these six studies showed not only a correlation between CNV loss and decreased expression, but also reported that increased CNV was associated with high expression:

gastric, squamous lung, colon and thyroid cancers.

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

These observed tumor CNV losses and increases and mRNA expression levels can represent the mechanism behind the expression of the bindin-4 tumor protein in those indications where this correlation is observed. Clearly, there are indications (e.g., hepatocellular carcinoma) where CNV does not appear to affect mRNA expression levels in a predictable pattern. The in vivo preclinical therapeutic efficacy of certain bindin-4 bicyclic drug conjugates of the present invention has been demonstrated to correlate with the expression of the bindin-4 protein as measured by IHC. Thus, tumor patients who are formally likely to have an increased copy number (gain or amplification) are more likely to respond to the bindin-4 bicyclic drug conjugates of the present invention if the tumor bindin-4 CNV correlates with mRNA levels and predicts protein expression levels. If a patient with increased CNV in bindin-4 can be identified, this information can be used to select patients for treatment with the bindin-4 bicyclic drug conjugates of the invention.

Example 7: analysis of the expression of Bindin-4 in 6 cell lines

1. Purpose of study

The aim of the study was to assess the expression of bindin-4 by flow cytometry in 6 cell lines, including 2 breast cancer (T-47D, MDA-MB-468), 3 lung cancer (NCI-H292, NCI-H322, NCI-H526) and one fibrosarcoma (HT-1080) cell line.

2. Group design

Subgroups for FCM in T-47D, MDA-MB-468, NCI-H292, NCI-H322 and HT-1080

Fluorescent dyes	Blank space	Isoforms	Group of
				PE	-	Isotype controls	Binding element-4

Group of NCI-H526

Fluorescent dyes	Blank space	Isoforms	Group of
				PE	-	Isotype controls	Binding element-4
BV421	Live/dead	Live/dead	Live/dead

3. Material

1.1. Sample(s)

List of cell lines

3.2. Reagent

Antibodies and kits for flow cytometry analysis

DPBS(Corning-21-031-CV)

Staining buffer (eBioscience-00-4222)

Fixing buffer (BD-554655)

3.3. Instrument for measuring the position of a moving object

Eppendorf Centrifuge 5810R

BD FACS Canto flow cytometer (BD)

4. Experimental methods and procedures

4.1 sample Collection

Cell lines in exponential growth phase were collected. Cells were counted by hemocytometer using trypan blue staining. Cells were centrifuged at 400 Xg for 5 min at 4 ℃, washed twice with staining buffer, and then resuspended to 1X10 in staining buffer⁷/mL。

4.2 antibody staining

1) 100 μ L of the cell suspension was aliquoted into each well of a 96-well V-plate.

2) Isotype control or antibody was added to the suspension cells and incubated for 30 minutes at 4 ℃ in the dark.

3) Cells were washed 2X by centrifugation at 400 Xg for 5 min at 4 ℃ and the supernatant discarded.

4) 100 μ L of fixation buffer was used to resuspend the cells and incubated for 30 min at 4 ℃ in the dark.

5) Cells were washed 2X by centrifugation at 300 Xg for 5 min at 4 ℃ and the supernatant was removed.

6) Resuspend cells in 400. mu.L staining buffer.

7) FACS data was analyzed using FlowJo V10 software.

4.3 data analysis

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

5. Results

5.1 subgroup gating strategies

The gating strategy for bindin-4 is shown in FIGS. 8-11

5.2 data analysis

5.2.1. Viability of cell lines

The viability of the cell lines was as follows.

5.2.2. Positive expression of Bindin-4 in cell lines

Positive expression of Bindin-4 and MFI in 6 cell lines are tabulated

6. Discussion of the related Art

Bindin-4 was highly expressed as follows: breast cancer T-47D (99.0%), MDA-MB-468 (99.0%), and lung cancer NCI-H292 (97.9%), NCI-H322 (99.1%). No expression of bindin-4 was found in NCI-H526 and HT-1080.

Example 8: analysis of Bindin-4 expression in 9 CDX cell lines by flow cytometry

1. Purpose of study

The objective of this project was to assess surface expression of Bindin-4 (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 cancers (HCT-116, HT-29) and 1 bladder cancer (HT1376) cell lines.

2. Group design

Panel of FCMs in 9 cell lines

Fluorescent dyes	Blank space	Isoforms	Group of
				PE	-	Isotype control IgG2b	Binding element-4
BV421	Live/dead	Live/dead	Live/dead

3. Material

3.1 samples

List of cell lines

3.2. Reagent

1)DPBS(Corning,21-031-CV)

2) Trypsin 0.25% (Invitrogen-25200072)

3) Staining buffer (eBioscience,00-4222)

4) Fixing buffer (BD,554655)

5) Antibodies

Fluorescence	Marker substance	Directory number	Suppliers of goods	Description of the invention
					PE	Binding element-4	FAB2659P	R&D	AAAO0217021
PE	Mouse IgG2b	IC0041P	R&D
					BV421	Live/dead	L34964	Invitrogen	-

3.3. Instrument for measuring the position of a moving object

Eppendorf Centrifuge 5810R

BD FACS Canto flow cytometer (BD)

4. Experimental methods and procedures

4.1 cell culture

Cell thawing

1) The frozen vials (final) were cleaned with 70% alcohol and then rapidly thawed in a 37 ℃ water bath.

2) The cell suspension was centrifuged at about 1000rpm for 5 minutes, the supernatant was removed, and the pre-warmed medium was added to the flask.

3) At 37 ℃ 5% CO₂The flasks were incubated in an incubator.

Cell passage

1) The medium and trypsin were incubated in a 37 ℃ water bath.

2) The culture medium was removed and the cell layer was washed with DPBS.

3) 5mL of 0.25% trypsin solution was added to the flask and the trypsin was diluted with 5mL of medium.

4) The cell suspension was centrifuged at 1000rpm for 5 minutes.

5) 15mL of fresh medium was added and the cells were resuspended by gentle pipetting.

6) The appropriate cell suspension was added to the fresh culture flask.

7) At 37 ℃ 5% CO₂The culture flasks were incubated in an incubator.

4.2. Sample collection

Harvesting at the fingerCell lines grown in several growth phases. Cells were counted using trypan blue staining. Cells were centrifuged at 400 Xg for 5 min at 4 ℃, washed twice with staining buffer, and then resuspended to 5X 10 in staining buffer⁶/mL。

4.3. Antibody staining

100 μ L of the cell suspension was aliquoted into each well of a 96-well V-plate. Isotype control or antibody was added to the suspension cells and incubated for 30 minutes at 4 ℃ in the dark. Cells were washed 2 times by centrifugation at 400 Xg for 5 minutes at 4 ℃ and the supernatant discarded. Resuspend cells in 300. mu.L staining buffer. FACS data was analyzed using Flow Jo V10 software.

4.4. Data analysis

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

5. Results

5.1. Group gating strategy

The gating strategy for bindin-4 is shown in FIGS. 12-16.

5.2. Data analysis

Positive expression of bindin-4 and MFI in 9 cell lines are tabulated.

6. Discussion of the related Art

Bindin-4 was highly expressed as follows: bladder cancer HT-1376 (92.4%), breast cancer MDA-MB-468 (97.1%), and lung cancer NCI-H358 (90.1%). Bindingtin-4 was found to be expressed in: HT-29 (40.0%), NCI-H292 (71.1%), and Panc02.13 (51.9%). No bindin-4 was found to be expressed in HCT-116, A549, and NCI-526. This data will be used to guide model selection for efficacy studies.

Example 9: study of in vivo efficacy

Example 9.1: in-vivo efficacy study of A549 xenograft of test object in treatment of Balb/c nude mice

1. Purpose of study

The objective of this study was to evaluate the in vivo anti-tumor efficacy of test agent treatment of a549 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

Animals and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1. Cell culture

5% CO in air at 37 ℃ in F-12K medium supplemented with 10% heat-inactivated fetal bovine serum₂As monolayer cultures, a549 tumor cells were maintained in vitro in the atmosphere of (a). Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of A549 tumor cells (5X 10) in PBS⁶) To effect tumor formation. When the average tumor volume reaches 158mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

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

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 17.

5.2. Tumor volume trajectory

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

Table 21: trajectory of tumor volume over time

5.3. Tumor growth inhibition assay

The tumor growth inhibition rate of the test article in the a549 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 22: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of test substances in an a549 xenograft model was evaluated. Fig. 17 and tables 21 and 22 show tumor volumes for all treatment groups measured at different time points.

On day 14, the mean tumor size of vehicle-treated mice reached 568mm³. BCY8245 at 3mg/kg, qw (TV 356 mm)³,TGI＝51.4％,p<0.05),3mg/kg,biw(TV＝194mm³,TGI＝90.8％,p<0.01) and 5mg/kg, qw (TV 228 mm)³,TGI＝82.6％,p<0.001) in a dose-or dose-frequency dependent manner to produce significant antitumor activity.

Animals in BCY8245 group maintained body weight well. In this cell line, minimal expression of bindin-4 was shown in FACS studies, tumor growth was limited by BCY8245, but tumors did not regress, emphasizing the targeted driving requirement for optimal therapeutic efficacy.

Example 9.2: in vivo efficacy study of test substance in treatment of HCT116 xenograft in Balb/c nude mice

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of test agents in treating HCT116 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1. Animals and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

5% CO in air at 37 ℃ in a medium supplemented with 10% heat-inactivated fetal bovine serum₂The atmosphere of (a) maintained HCT116 cells. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of HCT116 tumor cells (5.0X 10) in PBS⁶) To perform tumorigenesis. When the average tumor volume reachesTo 166mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

Tumors were collected for FFPE from group 1 and group 2 at the end of the study on day 14. For group 4, plasma was collected at 5, 15, 30, 60 and 120 minutes post-dose. Tumors were also collected and stored at-80 ℃.

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 18.

5.2. Tumor volume trajectory

The mean tumor volume of female Balb/c nude mice bearing HCT116 xenografts is shown in Table 23 below as a function of time.

Table 23: trajectory of tumor volume over time

5.3. Tumor growth inhibition assay

The tumor growth inhibition rate of the test substance in the HCT116 xenograft model was calculated based on the tumor volume measurement at day 14 after the start of the treatment.

Table 24: tumor growth inhibition assay

a. Mean ± SEM; 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).

6. Results summary and discussion

In this study, the therapeutic efficacy of test substances in the HCT116 xenograft model was evaluated. Fig. 18 and tables 23 and 24 show tumor volumes for all treatment groups measured at different time points.

On day 14 after the start of treatment, the mean tumor size of vehicle-treated mice reached 769mm³. BCY8245 at 3mg/kg, qw (TV 425 mm)³,TGI＝57.1％,p<0.001),3mg/kg,biw(TV＝197mm³,TGI＝94.9％,p<0.001) and 5mg/kg, qw (TV ═ 134 mm)³,TGI＝105.2％,p<0.001) in a dose-or dose-frequency dependent manner to produce significant antitumor activity.

In this study, the animals lost on average more than 10% of their body weight in all 5mg/kg qw groups.

In this cell line, bindin-4 was shown to be minimally expressed in FACS studies, tumor growth was limited by BCY8245, but tumors did not regress, emphasizing the targeted drive requirement for optimal therapeutic efficacy.

Example 9.3: study of the in vivo efficacy of test Agents in the treatment of HT-1376 xenografts in CB17-SCID mice

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of test agents in HT-1376 xenograft treatment in CB17-SCID mice.

2. Design of experiments

3. Material

3.1 animal and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: CB17-SCID

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

5% CO in air at 37 ℃ in EMEM medium supplemented with 10% heat-inactivated fetal bovine serum₂HT-1376 tumor cells were maintained in the atmosphere. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml HT-1376 tumor cells (5X 10) in PBS containing matrigel (1:1)⁶) To effect tumor formation. When the average tumor volume reaches 153mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

At the end of the study, group 4 plasma was collected at 5, 15, 30, 60 and 120 minutes after the last dose. Group 4 tumors were collected 2 hours after the last dose. Tumors from groups 1,2 and 3 were collected 2 hours after the last dose.

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 19.

5.2. Tumor volume trajectory

Table 25 shows the mean tumor volume as a function of time in female CB17-SCID mice carrying HT-1376 xenografts.

Table 25: trajectory of tumor volume over time

5.3. Tumor growth inhibition assay

Tumor growth inhibition rates of the test agents in the HT-1376 xenograft model were calculated based on tumor volume measurements at day 14 after treatment initiation.

Table 26: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of test substances in the HT-1376 xenograft model was evaluated. Fig. 19 and tables 25 and 26 show tumor volumes for all treatment groups measured at different time points.

On day 14, vehicle treatmentThe average tumor size of the mice reaches 1069mm³. BCY8245 at 3mg/kg, qw (TV 603 mm)³,TGI＝50.9％,p<0.01)，3mg/kg,biw(TV＝407mm³,TGI＝72.3％,p<0.001) and 5mg/kg, qw (TV 465 mm)³,TGI＝66.0％,p<0.001) produces significant antitumor activity. In this study, 5mg/kg qw of BCY8245 caused more than 10% weight loss in the animals over the treatment period.

Example 9.4: in-vivo efficacy study of test substance in treatment of MDA-MB-468 xenograft in Balb/c nude mice

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of test agents in treating MDA-MB-468 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1 animal and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4 Experimental methods and procedures

4.1 cell culture

5% CO in air at 37 ℃ in Lebovitz L-15 medium supplemented with 10% heat inactivated fetal bovine serum₂In an atmosphere of (4), tumor cells are maintained. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2 tumor inoculation

Each mouse was inoculated subcutaneously in the right abdomen with MDA-MB-468 tumor cells (10X 10) in 0.2ml of PBS supplemented with 50% matrigel⁶) For use in tumor formation. When the average tumor volume reaches 196mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3 preparation of test substance preparations

4.4 sample Collection

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

5 results

5.1 tumor growth Curve

The tumor growth curve is shown in fig. 20.

5.2 tumor volume trajectory

The change in mean tumor volume over time in female Balb/c nude mice carrying MDA-MB-468 xenografts is shown in tables 27 and 28 below.

5.3 tumor growth inhibition assay

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

Table 28: trajectory of tumor volume over time (day 23 to day 42)

Table 29: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of the test substances in the MDA-MB-468 xenograft model was evaluated. Fig. 20 and tables 27 to 29 show tumor volumes for all treatment groups measured at different time points.

On day 21, mean tumor size of vehicle-treated mice reached 447mm³. BCY8245 at 3mg/kg, qw (TV 85 mm)³,TGI＝144.2％,p<0.001),3mg/kg,biw(TV＝22mm³,TGI＝169.8％,p<0.001) and 5mg/kg, qw (TV ═ 29 mm)³,TGI＝168.4％,p<0.001) in a dose-or dose-frequency dependent manner to produce significant antitumor activity.

The 5mg/kg group dosing was suspended from day 21 and the tumors did not show any recurrence during the additional three week monitoring period. In this cell line, which showed high expression of bindin-4 in FACS studies, BCY8245 caused tumor regression, emphasizing the targeted driving property for optimal therapeutic efficacy.

Example 9.5: in vivo efficacy study of NCI-H292 xenograft in treatment of Balb/c nude mice by test substance

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of the test substances in treating NCI-H292 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1. Animals and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1. Cell culture

5% CO in air at 37 ℃ in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum₂In vitro as monolayer cultures, NCI-H292 tumor cells were maintained. Tumor cells were routinely passaged twice weekly by trypsin-EDTA treatment. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of NCI-H292 tumor cells (10X 10) in PBS⁶) To effect tumor formation. When the average tumor volume reaches 162mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

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

5. Results

5.1 tumor growth Curve

The tumor growth curve is shown in fig. 21.

5.2. Tumor volume trajectory

The mean tumor volume as a function of time in female Balb/c nude mice carrying NCI-H292 xenografts is shown in Table 30 below.

Table 30: trajectory of tumor volume over time

5.3. Tumor growth inhibition assay

The tumor growth inhibition rate of the test article in the NCI-H292 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 31: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the efficacy of treatment of the test substances in the NCI-H292 xenograft model was evaluated. Fig. 21 and tables 30 and 31 show tumor volumes for all treatment groups measured at different time points.

On day 14, the mean tumor size of vehicle-treated mice reached 948mm³. BCY8245 at 3mg/kg, qw (TV 149 mm)³,TGI＝101.4％,p<0.001),3mg/kg,biw(TV＝65mm³,TGI＝112.2％,p<0.001) and 5mg/kg, qw (TV 83 mm)³,TGI＝109.8％,p<0.001) produces significant antitumor activity.

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

The body weight of mice in all groups was well maintained in this study.

In this cell line, which showed high expression of bindin-4 in FACS studies, BCY8245 caused tumor regression, emphasizing the targeted driving property of optimal efficacy.

Example 9.6: study of the in vivo efficacy of the test Agents in treating the NCI-H526 xenograft in B alb/c nude mice

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of the test substances in treating NCI-H526 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1. Animals and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: 21 mice were added for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

At supplement with 10% heat-inactivated fetal bovine serum in air at 37 deg.C with 5% CO₂The atmosphere of (a) was maintained with NCI-H526 cells. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml of NCI-H526 tumor cells (5.0X 10) in PBS⁶) To effect tumor formation. When the average tumor volume reaches 181mm³At time, 21 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

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

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 22.

5.2. Tumor volume trajectory

The mean tumor volume as a function of time in female Balb/c nude mice carrying NCI-H526 xenografts is shown in Table 32 below.

Table 32: trajectory of tumor volume over time

5.3. Tumor growth inhibition assay

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

Table 33: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the efficacy of the test agents in the NCI-H526 xenograft model was evaluated. Fig. 22 and tables 32 and 33 show tumor volumes for all treatment groups measured at different time points.

On day 14 after initiation of treatment, the mean tumor size of vehicle-treated mice reached 1365³。BCY8245(3mg/kg,qw)(TV＝1205mm³TGI 13.4%, p > 0.05) and BCY8245(3mg/kg, biw) (TV 1109mm³,TGI＝21.6％,p>0.05) showed mild antitumor activity, BCY8245(5mg/kg, qw) (TV 476mm³,TGI＝75.0％,p<0.01) showed significant antitumor activity. In this study, BCY8245(5mg/kg biw) caused more than 10% of the animals' weight loss. In this cell line, which showed minimal expression of bindin-4 in FACS studies, tumor growth was restricted by BCY8245, but tumors did not regress, emphasizing the targeted driving requirement for optimal therapeutic efficacy.

Example 9.7: in vivo efficacy study of test substance on Panc2.13 xenograft in treatment of Balb/c nude mice

1. Purpose of study

The purpose of this study was to evaluate the in vivo anti-tumor efficacy of the test agents in treating panc2.13 xenografts in Balb/c nude mice.

2. Design of experiments

3. Material

3.1. Animals and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: add 41 mice for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 3 or 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1. Cell culture

5% CO in air at 37 ℃ in RMPI1640 medium supplemented with 15% heat-inactivated fetal bovine serum and 10 units/ml human recombinant insulin₂In an atmosphere of panc2.13 tumor cells. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right flank with 0.2ml Pan2.13 tumor cells (5X 10) in PBS (1:1) containing matrigel⁶) To effect tumor formation. When the average tumor volume reaches 149mm³At time, 41 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

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

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 23.

5.2. Tumor volume trajectory

The mean tumor volume as a function of time in female Balb/c nude mice carrying Panc2.13 xenografts is shown in Table 34 below.

Table 34: trajectory of tumor volume over time

5.3. Tumor growth inhibition assay

The tumor growth inhibition rate of the test article in panc2.13 xenograft model was calculated based on tumor volume measurements at day 14 after the start of treatment.

Table 35: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

6. Results summary and discussion

In this study, the therapeutic efficacy of the test substances in the panc2.13 xenograft model was evaluated. Fig. 23 and tables 34 and 35 show tumor volumes for all treatment groups measured at different time points.

On day 14, vehicle treatmentThe average tumor size of the mice reaches 545mm³。BCY8245，3mg/kg,qw，(TV＝271mm³,TGI＝69.2％,p<0.01),3mg/kg,biw(TV＝231mm³,TGI＝79.1％,p<0.001) and 5mg/kg, qw (TV-238 mm)³,TGI＝77.5％,p<0.001) produces significant antitumor activity. In this study, all animals in the 5mg/kg qw group lost weight on average more than 15%. In this cell line, which showed only moderate expression of bindin-4 in FACS studies, tumor growth was restricted by BCY8245, but tumors did not regress.

Example 9.8: in-vivo efficacy study of test substance in treatment of MDA-MB-468 xenograft in Balb/c nude mice

1. Purpose of study

The objective of this study was to evaluate the in vivo anti-tumor efficacy of BCY8245 and the combination of BCY8245 and BCY8234 in treating MDA-MB-468 xenografts in Balb/c nude mice to determine the role of targeted binding in the best efficacy.

2. Design of experiments

Note that: n, number of animals per group

3. Material

3.1. Animals and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: 36 mice were added for use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 4 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1. Cell culture

0% CO in air at 37 ℃ in Lebovitz L-15 medium supplemented with 10% heat inactivated fetal bovine serum₂In an atmosphere of (4), tumor cells are maintained. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right abdomen with MDA-MB-468 tumor cells (10X 10) in 0.2ml PBS supplemented with 50% matrigel⁶) For use in tumor formation. When the average tumor volume reaches 186mm³At time, 36 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

On study day 21, tumors from groups 5 and 6 were collected for FFPE. At the end of the study, group 3 tumors were collected for FFPE.

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 24.

5.2. Tumor volume trajectory

The mean tumor volume as a function of time in female Balb/c nude mice carrying MDA-MB-468 xenografts is shown in tables 36 to 38 below.

5.3. Tumor growth inhibition assay

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

6. Results summary and discussion

In this study, the therapeutic efficacy of the test substances in the MDA-MB-468 xenograft model was evaluated. Fig. 24 and tables 36-39 show tumor volumes for all treatment groups measured at different time points.

On day 21, the mean tumor size of vehicle-treated mice reached 420mm³. BCY8245 at 1mg/kg, qw (TV 204 mm)³,TGI＝92.1％,p<0.001),3mg/kg,qw(TV＝27mm³,TGI＝164.9％,p<0.001) to produce significant antitumor activity in a dose-dependent manner. BCY8245, 0.3mg/kg qw or biw did not produce any antitumor activity.

BCY8245,1mg/kg, qw and BCY8245,3mg/kg, qw in combination with BCY8234 (toxin-free homologous peptide), 300mg/kg, qw yielded significant antitumor activity (TV 242 mm)³,TGI＝75.4％,p<0.01), resulting in significant antitumor activity. The antitumor activity of 3mg/kg BCY8245 was antagonized by 300mg/kg BCY8234 when compared to BCY8245 alone (p)<0.001). The importance of targeted binding for optimal therapeutic efficacy is demonstrated by the decrease in therapeutic efficacy caused by competing non-toxin peptides. In the following monitoring schedule, mice treated with BCY8245,1mg/kg, qw showed significant tumor recurrence, whereas mice treated with BCY8245,3mg/kg, qw did not show any tumor recurrence.

Table 37: trajectory of tumor volume over time (day 23 to day 32)

Table 38: trajectory of tumor volume over time (day 35 to day 91)

Table 39: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

Example 9.9: in-vivo efficacy study of test substance in treatment of MDA-MB-468 xenograft in Balb/c nude mice

1. Purpose of study

The objective of this study was to evaluate the in vivo antitumor efficacy of BCY8245 alone or BCY8245 in combination with BCY8234 in treating MDA-MB-468 xenografts in Balb/c nude mice.

2. Design of experiments

Note that: n, number of animals per group.

3. Material

3.1 animal and feeding conditions

3.1.1. Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22 g

Animal number: adding 20 mice for later use

3.1.2. Feeding conditions

Mice were kept in separate ventilated cages at constant temperature and humidity, 5 animals per cage.

Temperature: 20-26 ℃.

Humidity 40-70%.

Cage: is made of polycarbonate. The dimensions are 300mm by 180mm by 150 mm. The bedding material was corncobs, changed twice a week.

Diet: throughout the study period, animals were free to eat radiation sterilized dry particulate foods.

Drinking water: animals can freely drink sterile drinking water.

Cage identification: the identification tag of each cage contains the following information: animal number, sex, species, date of receipt, treatment, study number, group number, and date of treatment initiation.

Animal identification: the animals were marked with ear codes.

4. Experimental methods and procedures

4.1 cell culture

0% CO in air at 37 ℃ in Lebovitz L-15 medium supplemented with 10% heat inactivated fetal bovine serum₂In an atmosphere of (4), tumor cells are maintained. Tumor cells were routinely subcultured twice a week. Cells grown in the exponential growth phase were collected and counted for tumor inoculation.

4.2. Tumor inoculation

Each mouse was inoculated subcutaneously in the right abdomen with MDA-MB-468 tumor cells (10X 10) in 0.2ml PBS supplemented with 50% matrigel⁶) For use in tumor formation. When the average tumor volume reaches 464mm³At time, 20 animals were randomized. Test article administration and animal numbers for each group are shown in the experimental design table.

4.3. Test article preparation

4.4. Sample collection

At the end of the study, group 3 tumors were collected for FFPE.

5. Results

5.1. Tumor growth curve

The tumor growth curve is shown in fig. 25.

5.2. Tumor volume trajectory

The mean tumor volume as a function of time in female Balb/c nude mice carrying MDA-MB-468 xenografts is shown in tables 40 to 42.

5.3. Tumor growth inhibition assay

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

6. Results summary and discussion

In this study, the therapeutic efficacy of the test substances in the MDA-MB-468 xenograft model was evaluated. Tumor volumes for all treatment groups measured at different time points are shown in figure 25 and tables 40-43.

The initial tumor starting size was intentionally made larger than previously used to determine if BCY8245 showed efficacy at this larger size. On day 28, the mean tumor size of vehicle-treated mice reached 773mm³。BCY8245(1mg/kg,qw)(TV＝384mm³,TGI＝126.6％,p<0.001) and BCY8245(3mg/kg, qw) (TV ═ 50mm³,TGI＝234.6％,p<0.001) to produce significant antitumor activity in a dose-dependent manner at day 28. Among these, mice treated with BCY8245(3mg/kg qw) showed some tumor recurrence after discontinuation of the treatment, and continued administration from day 76 did not serve to completely regress the tumor.

BCY8245(3mg/kg, qw) in combination with BCY8234(300mg/kg, qw) yielded significant antitumor activity on day 28 (TV 55 mm)³,TGI＝234.0％,p<0.001), and the tumor did not show any recurrence throughout the monitoring period.

Mice in the vehicle group treated with 10mg/kg of either the bindin-4 ADC or 5mg/kg of BCY8245, and in group 2 (BCY8245,1mpk, qw) treated with 5mg/kg of BCY8245 at PG-D28, showed effective tumor regression for the next 3 weeks, after which tumors showed regrowth for the next 4 weeks after drug withdrawal.

BCY8245 can be at about 450mm³But the initial tumor volume of the previously loaded vehicle was about 770mm³May also trigger tumor regression when administered in the group (2).

Table 41: trajectory of tumor volume over time (day 30 to day 75)

Table 42: trajectory of tumor volume over time (day 79 to day 103)

Table 43: tumor growth inhibition assay

a. Mean. + -. SEM.

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).

Example 10: in vivo PK Studies

MDA-MB-468 xenograft animals were injected with BCY8245(BT8009) at 3 mg/kg. At different time points, animals were euthanized, plasma and tumors were taken and snap frozen. Samples were analyzed for MMAE. Plasma levels of BT8009(BCY8245) were from historical PK studies. MMAE concentrations in plasma, MMAE concentrations in tumors, and BT8009 concentrations in plasma are shown in figure 33. MMAE has a longer retention time in tumors than in plasma, supporting the following hypothesis: systemic exposure was significantly less than tumor exposure.

Example 11: and (4) HCS determination.

The HCS assay was used in the bindin-4 BDC binding study. Cells are incubated with test reagents and then washed. Fluorescent antibody detection by MMAE. MDA-MB-468 cells showed moderate bindin-4 expression, with 20000 cells giving the best image. NCI-H292 cells showed low expression in this assay, even though MMAE detection was weak in 20000 cells. HCS data in the MDA-MB-468 cell line are shown in FIG. 34 and Table 44.

Watch 44

Test article	Maximum fluorescence intensity	Kd(nM)	History Kd (nM)
				Bindin-4 ADC	33.63	0.2	0.28±0.07
BCY8245	13.34	3.52	5.18
				MMAE	2.95	>10000	>10000

The bindin-4 ADC and BCY8245 were retained on the cells and co-localized with membrane staining. BCY8781 and MMAE showed minimal retention. Kd of all compounds for the MDA-MB-468 cell line was consistent with historical data. The bindin-4 ADC showed detectable binding affinity to the MDA-MB-468 cell line. BCY8425 showed a one-digit nanomolar affinity with Bmax lower than the bindin-4 ADC. This reduced maximum fluorescence intensity was due to the fact that the bindin-4 ADC had an MMAE to drug ratio of 4, whereas BCY8245 had an MMAE to drug ratio of 1. BCY8781 showed only very weak binding affinity for the MDA-MB-468 cell line, whereas MMAE showed little detectable binding affinity for the MDA-MB-468 cell line.

Example 12: in vivo efficacy of BCY8245 in two PDX models of lung cancer

Purpose(s) to

To evaluate the efficacy of BCY8245 in PDX models of squamous cell non-small cell carcinoma and adenocarcinoma, both non-small cell carcinomas.

Animal(s) production

Species: little mouse

And (2) breeding: balb/c nude mice

The week age is as follows: 6-8 weeks

Sex: female

Weight: 18-22

Test reagent

BCY8245 and Binder-4 ADC or BCY8781

Animal before study

Each mouse was subcutaneously inoculated with LU-01-000 in the right abdomen7 or LU-01-0412 tumor fragment (. about.30 mm)³) To effect tumor formation. When the average tumor volume reaches 161mm³(LU-01-0007) or 147mm³(LU-01-0412), animals were randomized.

Living measurement and endpoint

Animals were examined daily for any effect of tumor growth and treatment on normal behavior, such as motility, food and water consumption (by observation only), weight gain/loss, eye/hair shine, and any other abnormal effect stated in the protocol. Mortality 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 delayed or if the mice could be cured. Tumor volume was measured in two dimensions using a caliper three times a week and in mm using the following formula³Represents the volume: v ═ 0.5a x b²Wherein a and b are the major and minor diameters of the tumor, respectively. Tumor size was then used for the calculation of T/C values. The T/C value (percentage) indicates the antitumor effect; t and C are the average volume of the treatment and control groups, respectively, on a given day.

TGI was calculated for each group using the following formula: TGI (%) - (1- (T)_i-T₀)/(V_i-V₀)]×100；T_iIs the mean tumor volume, T, of the treatment groups on a given number of days₀Is the mean tumor volume of the treatment group on the day of treatment initiation, Vi is the ratio of T to T_iMean tumor volume of vehicle control group on same day, and V₀Is the mean tumor volume of the vehicle group on the day of treatment initiation.

The results of these studies are shown in fig. 26 and 27.

Lu-01-0412 (fig. 26) BCY8245 produced dose-related efficacy in this PDX model, with a decrease in tumor growth rate at 1mg/kg qw, but significant regression of tumors to baseline at 3mg/kg qw. After discontinuation of dosing (day 21), 5/6 animals showed no tumor regrowth until 105 days after study initiation. One animal that showed regrowth responded to 3mg/kg of BCY8245 and showed a return to baseline. BCY8781, unbound BDC, produced stable disease at 3mg/kg, and tumors grew rapidly at the same rate with vehicle treated groups after discontinuation of dosing, underscoring that the binding of avidin-4 provided enhanced therapeutic efficacy for these agents. Large tumors (vehicle treated group) regressed in response to a single dose of BCY8245 or BCY 8781.

LU-01-0007 (FIG. 27). BCY8245 produced dose-related efficacy, 1mg/kg qw produced stable disease and 3mg/kg produced complete regression. Dosing must be maintained until day 56 until complete regression (when dosing is stopped). There was no tumor regrowth in this group beyond day 56 (the latter was maintained until day 126 after study initiation). The bindin-4 ADC gave similar degree of efficacy. The 1mg/kg stable disease group responded to the increase in dosing (3 and 5mg/kg), indicating that the low dose of BCY8245 did not result in resistance development.

Example 13: BCY8245 in vivo evaluation in a low-generation PDX model of human breast, esophageal and bladder cancer in immunocompromised mice.

Purpose(s) to

To evaluate the antitumor activity of the bicyclic agents in a Low-generation champion tumor transplantation model (Low Passage times tumor lung graff Models) of human breast, esophageal, and bladder cancer in immunocompromised mice.

Test system

Species: mouse

And (2) breeding: athymic Nude-Foxn1nu (immune compromised)

The source is as follows: envigo: indianapolis, indiana

Sex: female

Target week age at start of dosing: at least 6-8 weeks old

Target body weight at the start of dosing: at least 18g

An adaptation period: 3 days

Design of experiments

Animals before study when enough stock animals (stock animals) reached 1.0-1.5 cm³At this time, tumors were harvested for reimplantation into pre-study animals. The animals before the study were implanted unilaterally on the left flank with tumor fragments harvested from stock animals. Each animal was implanted from a specific passage batch and recorded.

Study animals: the pre-study tumor volume for each experiment was recorded starting seven to ten days after implantation. When the tumor reaches 150-300mm³Will be divided into treatment groups or control groups for administration according to tumor volume, and administration will be started on day 0.

Test reagent

BCY8245 and the bindin-4 antibody drug conjugate were compared to the vehicle control. A standard-of-care agent docetaxel may be included. All agents were administered by intravenous route qw, the doses indicated in the figure.

Life measurement

Effective tumor volume was measured twice weekly. The day the study reached the endpoint final tumor volumes will be collected. If possible, final tumor volume will be collected if the animal is found to be moribund.

Effective animal weight animals were weighed twice a week. The final weight will be collected, if possible, on the day the study reached the endpoint or if the animal was found to be dying. Animals showing a weight loss of 10% or more will receive any food intake compared to day 0Net weight loss over a period of 7 days>Any animal that is 20%, or a mouse that shows a net weight loss of more than 30% compared to day 0, will be considered moribund and will be euthanized.

Data analysis

Toxicity of the reagent: starting on day 0, animals were observed daily and weighed twice weekly using a digital scale; data included individual and average grammage (average We ± SEM), with the average percent weight change (% vD0) from day 0 recorded for each group and the% vD0 plotted at study completion. Animal deaths were recorded daily and assigned as drug-related (D), technical-related (T), tumor-related (B), or unknown (U) based on weight loss and visual observation; the single agent or combination group reporting a mean% vD0> 20% and/or > 10% mortality will be considered higher than the Maximum Tolerated Dose (MTD) of the treatment in the evaluation protocol. The maximum mean% vD0 (weight nadir) was reported for each treatment group at the end of the study.

Therapeutic effect of reagent

Tumor growth inhibition tumor size was measured twice weekly by electronic calipers, starting on day 0, and the data included individual and mean estimated tumor volume (mean TV ± SEM) for each group record; tumor volume was calculated using equation (1), equation (1): TV is 2 width × length × 0.52. After completion of the study, initial (i) and final (f) tumor measurements will be taken, and the percent tumor growth inhibition (% TGI) values for each treatment group (T) versus control group (C) calculated and reported using equation (2), equation (2): % TGI ═ 1- (Tf-Ti)/(Cf-Ci). Individual mice with tumor volumes ≦ 30% of the measurements on day 0 measured in two consecutive measurements will be considered Partial Responders (PR). Lack of significant tumor (0.00 mm in two consecutive measurements)³) Will be classified as a Complete Responder (CR); CR, which persists until completion of the study, will be considered to be tumor-free survivors (TFS). Tumor Doubling Time (DT) was determined for vehicle treated groups using the formula DT ═ (Df-Di) × log 2/(logTVf-logTVi), where D ═ day, and TV ═ tumor volume. All data collected in this study is managed electronically and stored on redundant server systems.

The results of these studies are shown in fig. 28 to 31.

BCY8245 was tested in four low passage PDX models: the model represents bladder cancer (CTG-1771), estrogen-and progestin-negative Her 2-positive breast cancer (CTG-1171), triple-negative breast cancer (CTG-1106), and esophageal cancer (CTG-0896). In all these models, BCY8245 showed excellent therapeutic effects, causing tumor regression, and fully regressing tumors to baseline in three of the four models. In all cases, the efficacy was comparable to ADC and better than or equal to docetaxel SOC. BCY8245 tolerability was superior to docetaxel in all models.

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