Cancer treatment using identified adenosine fingerprints

Abstract

The present disclosure provides a method for treating cancer in a subject with an established adenosine fingerprint. The established adenosine fingerprint includes assessing the concentration of one or more adenosine machinery proteins in the blood, the enzymatic activity of one or more adenosine machinery proteins, and / or assessing the expression level of adenosine machinery proteins in a tumor. The method disclosed herein includes administering to the subject a therapeutic agent selected from the group consisting of an agent that targets the extracellular production of adenosine and an agent that antagonizes the activation of one of adenosine's receptors.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 826,728, filed March 29, 2019, the contents of which are incorporated herein by reference for all purposes.

[0002] STATEMENT REGARDING RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT Not applicable

[0003] Reference to a "Sequence Listing," table, or computer program listing attachment submitted on a compact disc Not applicable Summary of the Invention

[0004] In some embodiments, there is provided a method of treating cancer in a subject with an established adenosine fingerprint, comprising administering to the subject a therapeutic agent that targets the extracellular production of adenosine and / or antagonizes adenosine's activation of one of its receptors, The cancer in the subject is (i) an increase in the concentration of one or more adenosine machinery proteins in blood obtained from the subject compared to a typical concentration of one or more adenosine machinery proteins in blood obtained from a subject having the same type of cancer; (ii) an increase in the activity of CD73 or TNAP in blood obtained from the subject compared to the typical AMP hydrolysis activity of CD73 and / or TNAP in blood obtained from subjects with the same type of cancer, as determined by an AMP hydrolysis assay; (iii) a biopsy of the subject's cancer showing an increased amount of one or more adenosine machinery proteins compared to the typical amount of the one or more adenosine machinery proteins in a biopsy obtained from a subject with the same type of cancer, as determined by immunostaining for the one or more adenosine machinery proteins; and (iv) a biopsy of the subject's cancer showing upregulation of one or more adenosine machinery proteins compared to the typical amount of the one or more adenosine machinery proteins in a biopsy obtained from a subject with the same type of cancer, as determined by mRNA levels; A method is provided having at least one of the features selected from the group consisting of:

[0005] Some embodiments provided herein are kits and methods for detecting the concentration of soluble CD73 in blood and for determining CD73-mediated and / or TNAP-mediated adenosine monophosphate (AMP) hydrolysis activity in a sample.

[0006] Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from the following detailed description and drawings. [Brief explanation of the drawings]

[0007] [Figure 1] 1 is a schematic diagram outlining the principle of the CD73 ELISA assay. [Figure 2A] Figure 1 shows a robust assay for quantifying soluble CD73 in peripheral blood. (A) The concentrations of soluble CD73 in plasma and serum from healthy donors are plotted, showing a strong correlation. [Figure 2B] (B) A robust assay for quantifying soluble CD73 in peripheral blood. (C) Parallelism assessment plots identify the quantitative range of the assay. [Figure 2C] Figure 1 shows a robust assay for quantifying soluble CD73 in peripheral blood. (C) Plots soluble CD73 levels in healthy and cancer-bearing subjects, demonstrating that soluble CD73 levels are generally elevated in cancer patients. [Figure 3A] Figure 1 shows the determination of AMP hydrolysis in serum using AMP-Glo™ (A) shows a schematic diagram outlining the principle of the assay. [Figure 3B]Figure 1 shows the determination of AMP hydrolysis in serum using AMP-Glo™. (B) AMP hydrolysis in serum of healthy volunteers under different conditions (+ / - CD73 inhibitor and / or TNAP inhibitor) is plotted. [Figure 3C] Figure 1 shows the determination of AMP hydrolysis in serum using AMP-Glo™ (C) plots the correlation between CD73 protein concentration and AMP hydrolysis activity in serum of healthy volunteers and cancer patients. [Figure 4A] Figure 1 shows TCGA analysis of human tumors for CD73 and TNAP. Expression of CD73 (Panel A) and TNAP (Panel B) from RNA-seq is plotted in The Cancer Genome Atlas samples. Values ​​represent the ratio of log2 counts per million samples. Samples are ordered according to their CD73 / TNAP ratio, with tumors with higher CD73 expression on the left and tumors with higher TNAP expression on the right. [Figure 4B] Figure 1 shows TCGA analysis of human tumors for CD73 and TNAP. Expression of CD73 (Panel A) and TNAP (Panel B) from RNA-seq is plotted in The Cancer Genome Atlas samples. Values ​​represent the ratio of log2 counts per million samples. Samples are ordered according to their CD73 / TNAP ratio, with tumors with higher CD73 expression on the left and tumors with higher TNAP expression on the right. [Figure 5A-B] Figure 1 shows the detection and quantification of CD73 in human tumors using immunostaining. (A-D) Representative images of CD73 immunostaining (brown) in human FFPE tumor samples. The tumors shown are non-small cell lung cancer (NSCLC) (A, B), triple-negative breast cancer (TNBC) (C), and colorectal cancer (CRC) (D). [Figure 5C-D]Figure 1 shows the detection and quantification of CD73 in human tumors using immunostaining. (A-D) Representative images of CD73 immunostaining (brown) in human FFPE tumor samples. The tumors shown are non-small cell lung cancer (NSCLC) (A, B), triple-negative breast cancer (TNBC) (C), and colorectal cancer (CRC) (D). [Figure 5E]

[0023] Figure 1 shows detection and quantification of CD73 in human tumors using immunostaining. Panel E shows quantification of CD73 stained area as a percentage of total tumor area in the listed cancers. [Figure 5F] FIG. 1 shows the detection and quantification of CD73 in human tumors using immunostaining, and panel F plots the correlation between H-score and percent stained area. [Figure 6A-B] Figure 1 shows the detection and quantification of TNAP in human tumors using immunostaining. (A-D) Shown are representative images of TNAP immunostaining (brown) in human FFPE tumor samples. The tumors shown are ovarian cancer (A), non-small cell lung cancer (NSCLC) (B), breast cancer (C), and colorectal cancer (CRC) (D). [Figure 6C-D] Figure 1 shows the detection and quantification of TNAP in human tumors using immunostaining. (A-D) Shown are representative images of TNAP immunostaining (brown) in human FFPE tumor samples. The tumors shown are ovarian cancer (A), non-small cell lung cancer (NSCLC) (B), breast cancer (C), and colorectal cancer (CRC) (D). [Figure 6E]

[0023] Figure 1 shows the detection and quantification of TNAP in human tumors using immunostaining. Panel E shows quantification of TNAP-stained area as a percentage of total tumor area in the listed cancers. [Figure 7A-B]Figure 1 shows inhibition of CD73-mediated dephosphorylation of 13C5-AMP to 13C5-adenosine in human plasma. (A-C) show representative plots of percent remaining activity at certain test concentrations of Compound A. The data points and plots shown were used to calculate IC50s for Volunteer 1 (Panel A), Volunteer 2 (Panel B), and Volunteer 3 (Panel C). [Figure 7C] Figure 1 shows inhibition of CD73-mediated dephosphorylation of 13C5-AMP to 13C5-adenosine in human plasma. (A-C) show representative plots of percent remaining activity at certain test concentrations of Compound A. The data points and plots shown were used to calculate IC50s for Volunteer 1 (Panel A), Volunteer 2 (Panel B), and Volunteer 3 (Panel C). DETAILED DESCRIPTION OF THE INVENTION

[0008] I. Overview The present disclosure focuses on the discovery that assessing and determining a cancer adenosine fingerprint in a subject provides a method for more effectively treating cancer. In particular, determining a cancer adenosine fingerprint provides a means for identifying subjects that will respond more favorably to a particular treatment regimen.

[0009] II. Definition Unless otherwise indicated, the following terms have the meanings indicated below: Other terms are defined elsewhere throughout the specification.

[0010] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical having the specified number of carbon atoms (i.e., C 1~8 means 1 to 8 carbons). Alkyl can be any number of carbons, e.g., C 1~2 , C 1~3 , C 1~4 , C 1~5 , C 1~6 , C1~7 , C 1~8 , C 1~9 , C 1~10 , C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 3~4 , C 3~5 , C 3~6 , C 4~5 , C 4~6 and C 5~6 Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

[0011] The term "alkylene" refers to a straight-chain or branched-chain saturated aliphatic radical, i.e., a divalent hydrocarbon radical, having the indicated number of carbon atoms and linking at least two other groups. The two moieties linked to the alkylene can be linked to the same atom or different atoms of the alkylene group. For example, a straight-chain alkylene is -(CH2) n -, where n is 1, 2, 3, 4, 5, or 6. Representative alkylene groups include, but are not limited to, methylene, ethylene, propylene, isopropylene, butylene, isobutylene, secbutylene, pentylene, hexylene, and the like. Often referred to herein as X 1 group or X 2 The alkylene groups referred to as groups can be substituted or unsubstituted. X 1 or X 2 When a group comprising is optionally substituted, it is understood that the optional substitution may be on the alkylene portion of the moiety.

[0012] The term "cycloalkyl" refers to a group having the indicated number of ring atoms (e.g., C 3~6"Cycloalkyl" refers to a hydrocarbon ring that is fully saturated or has no more than one double bond between the ring vertices. "Cycloalkyl" is also intended to refer to bicyclic and polycyclic hydrocarbon rings, such as, for example, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, and the like. In some embodiments, the cycloalkyl compositions of the present disclosure include monocyclic C 3~6 It is a cycloalkyl moiety.

[0013] The term "heterocycloalkyl" refers to a cycloalkyl ring having the indicated number of ring vertices (or members) and having 1 to 5 heteroatoms replacing 1 to 5 carbon vertices selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. Cycloheteroalkyls may be monocyclic, bicyclic, or polycyclic ring systems. Non-limiting examples of cycloheteroalkyl groups include pyrrolidine, imidazolidine, pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin, dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine, thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone, tetrahydrofuran, tetrahydrothiophene, quinuclidine, and the like. A cycloheteroalkyl group can be attached to the remainder of the molecule through a ring carbon or heteroatom.

[0014] As used herein, a wavy line "" across a single bond, double bond, or triple bond in any chemical structure described herein is TIFF0007743309000001.tif9160" represents the point of attachment of a single, double, or triple bond to the rest of the molecule. Additionally, a bond extending into the center of a ring (e.g., a phenyl ring) is meant to indicate attachment at any available ring vertex. It will be apparent to one of skill in the art that multiple substituents shown attached to a ring will occupy ring vertices that result in a stable compound and are otherwise sterically compatible. Expressions in the case of divalent moieties are meant to include either orientation (forward or reverse). For example, the group "-C(O)NH-" is meant to include either the -C(O)NH- or -NHC(O)- attachment orientation; similarly, "-O-CHCH-" is meant to include both -O-CHCH- and -CHCH-O-.

[0015] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "C 1~4 "Haloalkyl" is meant to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0016] The term "aryl," unless otherwise specified, means a polyunsaturated, typically aromatic hydrocarbon group which may be a single ring or multiple rings (up to three rings) fused or covalently linked together. Non-limiting examples of aryl groups include phenyl, naphthyl, and biphenyl.

[0017] The term "heteroaryl" refers to an aryl group (or ring) containing 1 to 5 heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include pyridyl, pyridazinyl, pyrazinyl, pyrimidinyl, triazinyl, quinolinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzotriazinyl, purinyl, benzimidazolyl, benzopyrazolyl, benzotriazolyl, benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl, thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, imidazopyridine, benzothiazolyl, benzofuranyl, benzothienyl, indolyl, quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl, imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl, pyrrolyl, thiazolyl, furyl, thienyl, and the like. Substituents for each of the heteroaryl rings may be selected from the group of acceptable substituents described below.

[0018] The above terms (e.g., "alkyl," "aryl," and "heteroaryl") are optionally substituted in some embodiments. Selected substituents for each type of radical are provided below.

[0019] Optionally, substituents on an alkyl radical (including groups often referred to as alkylene, alkenyl, and alkynyl) are selected from the group consisting of -halogen, -OR', -NR'R'', -SR', -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R''', -NR' R', R'', and R''' may each independently represent a variety of groups selected from hydrogen; unsubstituted C(O)R; -NH-C(NH)=NH; -NR'C(NH)=NH; -NH-C(NH)=NR'; -S(O)R'; -S(O)R'; -S(O)NR'R'', -NR'S(O)R'', -CN(cyano), -NO, aryl, aryloxy, oxo, cycloalkyl, and heterocycloalkyl, where m' is the total number of carbon atoms in such radical. R', R'', and R''' each independently represent hydrogen; unsubstituted C 1~8 Alkyl; Unsubstituted Aryl; Aryl substituted with 1-3 halogens, C 1~8 Alkoxy or C 1~8 thioalkoxy group; or unsubstituted aryl-C 1~4 represents an alkyl group. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 3-, 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl.

[0020] Optionally, substituents on the cycloalkyl and heterocycloalkyl radicals can be alkyl optionally substituted with various groups selected from C(O)OR', -halogen, -OR', -NR'R'', -SR', -SiR'R''R''', -OC(O)R', -C(O)R', -COR', -CONR'R'', -OC(O)NR'R'', -NR''C(O)R', -NR'-C(O)NR''R'''', -NR''C(O)R', -NH-C(NH)=NH, -NR'C(NH)=NH, -NH-C(NH)=NR', -S(O)R', -S(O)R', -S(O)NR'R'', -NR'S(O)R'', -CN(cyano), -NO, aryl, aryloxy, and oxo. R', R'', and R''' are each independently hydrogen; unsubstituted C 1~8 Alkyl; Unsubstituted Aryl; Aryl substituted with 1-3 halogens, C 1~8 Alkoxy or C 1~8 thioalkoxy group; or unsubstituted aryl-C 1~4 represents an alkyl group.

[0021] Similarly, optional substituents on the aryl and heteroaryl groups are varied and generally selected from -halogen, -OR', -OC(O)R', -NR'R'', -SR', -R', -CN, -NO2, -C02R', -CONR'R'', -C(O)R', -OC(O)NR'R'', -NR''C(O)R', -NR''C(O)2R', -NR'-C(O)NR''R''', -NH-C(NH2)=NH, -NR'C(NH2)=NH, -NH-C(NH2)=NR', -S(O)R', -S(O)2R', -S(O)2NR'R'', -NR'S(O)2R'', -N3, perfluoro(C1-C4)alkoxy, and perfluoro(C1-C4)alkyl in a number ranging from zero to the total number of open valences on the aromatic ring system; R', R'', and R''' are independently hydrogen, C 1~8 Alkyl, C 1~8 Haloalkyl, C 3~6 Cycloalkyl, C 2~8 Alkenyl, and C 2~8alkynyl. Other suitable substituents include each of the above aryl substituents attached to a ring atom by an alkylene tether of 1 to 6 carbon atoms.

[0022] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be of the formula -TC(O)-(CH) q Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced by a substituent of the formula -A-(CR f R g ) r A and B are each independently -CH2-, -O-, -NH-, -S-, -S(O)-, -S(O)2-, -S(O)2NR'-, or a single bond; r is an integer of 1 to 3; and R f and R g are each independently H or a halogen. One of the single bonds in the new ring thus formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be a group of the formula -(CH2) s -X-(CH2) t In the formula, s and t are independently an integer of 0 to 3, and X is -O-, -NR'-, -S-, -S(O)-, -S(O)2-, or -S(O)2NR'-. The substituent R' in -NR'- and -S(O)2NR'- is hydrogen or an unsubstituted C 1~6 alkyl.

[0023] As used herein, the term "heteroatom" is intended to include oxygen (O), nitrogen (N), sulfur (S), and silicon (Si).

[0024] The term "pharmaceutically acceptable salts" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of the present invention possess relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either pure or in a suitable inert solvent. Examples of salts derived from pharmaceutically acceptable inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium, zinc, and the like. Salts derived from pharmaceutically acceptable organic bases include salts of primary, secondary, and tertiary amines, including substituted amines, cyclic amines, naturally occurring amines, etc., such as arginine, betaine, caffeine, choline, N,N'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purine, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, etc. When a compound of the present invention possesses a relatively basic functionality, an acid addition salt can be obtained by contacting the neutral form of such a compound, either neat or in a suitable inert solvent, with a sufficient amount of the desired acid.Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from relatively non-toxic organic acids such as acetic acid, propionic acid, isobutyric acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginate, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge, SM, et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19). Certain compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts.

[0025] The neutral forms of the compounds can be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for purposes of this invention. In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the invention. Furthermore, prodrugs can be converted to the compounds of the invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. Prodrugs are described in detail elsewhere herein.

[0026] In addition to salt forms, the present invention provides compounds in prodrug form. Prodrugs of the compounds described herein are compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Furthermore, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0027] Certain compounds of the present invention can exist in unsolvated and solvated forms, such as hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention can exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0028] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; racemates, diastereomers, geometric isomers, positional isomers, and individual isomers (e.g., separated enantiomers) are all intended to be encompassed within the scope of the present invention. When a stereochemical depiction is shown, it is intended to refer to a compound in which one of the isomers is present and the other isomer is substantially absent. "Substantially absent" of another isomer refers to a ratio of at least 80 / 20, more preferably 90 / 10, or 95 / 5 or greater, of the two isomers. In some embodiments, one of the isomers is present in an amount of at least 99%.

[0029] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. An unnatural proportion of an isotope can be defined as a range from the amount found in nature to the amount at which the atom in question constitutes 100%. For example, the compounds may contain radioactive isotopes, such as tritium ( 3 H), iodine-125( 125 I), or carbon-14 (14 C), or non-radioactive isotopes such as deuterium ( 2 H) or carbon-13 ( 13 C). Such isotopic variations may provide additional utilities to those described elsewhere in this application. For example, isotopic variants of the compounds of the present invention may find additional utilities, including, but not limited to, as diagnostic and / or imaging reagents or as cytotoxic / radiotoxic therapeutic agents. Furthermore, isotopic variants of the compounds of the present invention may have altered pharmacokinetic and pharmacodynamic properties that may contribute to enhanced safety, tolerability, or efficacy during treatment. All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention.

[0030] The terms "patient" and "subject" are used interchangeably to refer to a human or non-human animal (e.g., a mammal).

[0031] The terms "administration," "administering," and the like, when applied to, e.g., a subject, cell, tissue, organ, or bodily fluid, refer to the contact of, e.g., an inhibitor of A2aR / A2bR (or another inhibitor or antagonist described herein) or a pharmaceutical composition comprising same, with the subject, cell, tissue, organ, or bodily fluid. In the context of cells, administration includes contact of a reagent with the cell (e.g., in vitro or ex vivo), as well as contact of a reagent with a fluid, where the fluid is in contact with the cell.

[0032] The terms "treat," "treating," "treatment," and the like refer to a course of action (e.g., administering an inhibitor of A2aR / A2bR or another inhibitor or antagonist described herein) initiated, for example, after a disease, disorder, or condition or its symptoms has been diagnosed or observed, to temporarily or permanently eliminate, reduce, inhibit, alleviate, or ameliorate at least one of the underlying causes of the disease, disorder, or condition affecting a subject, or at least one of the symptoms associated with the disease, disorder, or condition affecting a subject. Thus, treatment includes preventing active disease (e.g., preventing the onset or further development of the disease, disorder, or condition or clinical symptoms associated therewith).

[0033] As used herein, the term "in need of treatment" refers to the judgment by a physician or other caregiver that a subject needs or would benefit from treatment. This judgment is made based on a variety of factors that are within the physician's or caregiver's area of ​​expertise.

[0034] The terms "prevent," "preventing," "prevention," and the like, generally in the context of a subject predisposed to a particular disease, disorder, or condition, refer to a course of action (e.g., administering an A2aR / A2bR inhibitor described herein or another inhibitor or antagonist) initiated in a manner (e.g., prior to the onset of the disease, disorder, condition, or its symptoms) that temporarily or permanently prevents, suppresses, arrests, or reduces the subject's risk of developing the disease, disorder, condition, etc. (e.g., as determined by the absence of clinical symptoms) or delays its onset. In certain instances, these terms also refer to slowing the progression of the disease, disorder, or condition, or to arresting its progression to a harmful or otherwise undesirable state.

[0035] As used herein, the term "in need of prevention" refers to the judgment by a physician or other caregiver that a subject needs or would benefit from preventative medical care. This judgment is made based on a variety of factors within the physician's or caregiver's area of ​​expertise.

[0036] The phrase "therapeutically effective amount" refers to the administration of an agent to a subject, alone or as part of a pharmaceutical composition, in an amount capable of having any detectable positive effect on any symptom, aspect, or characteristic of a disease, disorder, or condition when administered to a subject, either in a single dose or as part of a series of doses. A therapeutically effective amount can be ascertained by measuring the relevant physiological effect, which can be adjusted in conjunction with diagnostic analyses, such as dosing regimens and the subject's condition. For example, measuring serum levels of an A2aR / A2bR inhibitor (or another inhibitor or antagonist, e.g., as described herein) at a particular time after administration can indicate whether a therapeutically effective amount has been used.

[0037] The phrase "in an amount sufficient to effect a change" means that there is a detectable difference between the level of an indicator measured before (e.g., baseline level) and after administration of a particular therapy. Indicators include any objective parameter (e.g., serum concentration) or subjective parameter (e.g., a subject's sense of well-being).

[0038] The term "small molecule" refers to a compound having a molecular weight of less than about 10 kDa, less than about 2 kDa, or less than about 1 kDa. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, and synthetic molecules. Therapeutically, small molecules are more cell permeable, less susceptible to degradation, and less likely to elicit an immune response than large molecules.

[0039] The term "ligand" refers to, for example, a peptide, polypeptide, membrane-associated or membrane-bound molecule, or complex thereof, that can act as an agonist or antagonist of a receptor. Ligands include natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compositions derived from antibodies, as well as small molecules. The term also includes agents that are neither agonists nor antagonists but can bind to a receptor without significantly affecting its biological properties, such as signal transduction or adhesion. Furthermore, the term includes membrane-bound ligands that have been modified, for example, by chemical or recombinant methods, into soluble versions of the membrane-bound ligand. The ligand or receptor can be entirely intracellular, i.e., it can reside in the cytosol, nucleus, or some other intracellular compartment. The complex of a ligand and receptor is called a "ligand-receptor complex."

[0040] The terms "inhibitor" and "antagonist" or "activator" and "agonist" refer to molecules that have an inhibitory or activating effect, respectively, such as activation of a ligand, receptor, cofactor, gene, cell, tissue, or organ. An inhibitor is a molecule that, for example, decreases, blocks, prevents, delays activation, inactivates, desensitizes, or downregulates a gene, protein, ligand, receptor, or cell. An activator is a molecule that, for example, increases, activates, promotes, enhances activation, sensitizes, or upregulates a gene, protein, ligand, receptor, or cell. An inhibitor can also be defined as a molecule that reduces, blocks, or inactivates constitutive activity. An "agonist" is a molecule that interacts with a target to cause or promote increased activation of the target. An "antagonist" is a molecule that opposes the action of an agonist. An antagonist blocks, reduces, inhibits or neutralizes the activity of an agonist; an antagonist can also block, inhibit or reduce the constitutive activity of a target, e.g., a target receptor, even in the absence of a specified agonist.

[0041] The terms "modulate," "modulation," and the like refer to the ability of a molecule (e.g., an activator or inhibitor) to directly or indirectly increase or decrease the function or activity of an adenosine-related protein described herein. A modulator can act alone, or it can use a cofactor, such as a protein, metal ion, or small molecule. Examples of modulators include small molecule compounds and other bioorganic molecules. Numerous libraries of small molecule compounds (e.g., combinatorial libraries) are commercially available and can serve as a starting point for identifying modulators. One skilled in the art can develop one or more assays (e.g., biochemical or cell-based assays) that can screen such compound libraries to identify one or more compounds with desired properties. A skilled pharmaceutical scientist can then optimize such one or more compounds, for example, by synthesizing and evaluating analogs and derivatives thereof. Synthesis and / or molecular modeling studies can also be utilized in identifying activators.

[0042] The "activity" of a molecule can describe or refer to the molecule's binding to a ligand or receptor, catalytic activity, ability to stimulate gene expression or cell signaling, differentiation or maturation, antigenic activity, modulation of the activity of other molecules, etc. The term "proliferative activity" includes, for example, activity that promotes, is required for, or is specifically associated with normal cell division as well as cancer, tumors, dysplasia, cell transformation, metastasis, and angiogenesis.

[0043] As used herein, "equivalent," "equivalent activity," "equivalent activity to," "equivalent effect," "equivalent effect to," and the like are relative terms that can be viewed quantitatively and / or qualitatively. The meaning of these terms frequently depends on the context in which they are used. As an example, two agents that both activate a single receptor may be viewed as having equivalent effects from a qualitative perspective, but if one agent can only achieve 20% of the activity of the other agent as determined in an art-recognized assay (e.g., a dose-response assay) or art-recognized animal model, the two agents may be viewed as lacking equivalent effect from a quantitative perspective. When comparing one result to another (e.g., comparing one result to a reference standard), "equivalent" frequently (but not always) means that the result deviates from the reference standard by less than 35%, 30%, 25%, 20%, 15%, 10%, 7%, 5%, 4%, 3%, 2%, or 1%. In certain embodiments, a result is equivalent to a reference standard if it deviates from the reference standard by less than 15%, less than 10%, or less than 5%. By way of example and not limitation, activity or effect can refer to efficacy, stability, solubility, or immunogenicity.

[0044] "Substantially pure" indicates that the component comprises more than about 50% of the total content of the composition, and typically more than about 60% of the total polypeptide content. More typically, "substantially pure" refers to a composition in which the component of interest comprises at least 75%, at least 85%, at least 90%, or more of the total composition. In some cases, the polypeptide comprises more than about 90%, or more than about 95%, of the total content of the composition.

[0045] The terms "specifically bind" or "selectively bind," when referring to a ligand / receptor, antibody / antigen, or other binding pair, refer to a binding reaction that is determinative of the presence of a protein in a heterogeneous population of proteins and other biological materials. Thus, under specified conditions, a particular ligand will bind to a specific receptor and will not bind in significant amounts to other proteins present in a sample. The antibody or binding composition derived from the antigen-binding portion of an antibody of the contemplated method will bind to its antigen, or a variant or mutein thereof, with an affinity that is at least 2-fold greater, at least 10-fold greater, at least 20-fold greater, or at least 100-fold greater than the affinity by any other antibody or binding composition derived therefrom. In certain embodiments, the antibody binds with an affinity of about 10 times greater, as determined, for example, by Scatchard analysis. 9 It has an affinity of greater than liter / mol (Munsen, et al. 1980 Analyt. Biochem. 107:220-239).

[0046] The term "response," for example, of a cell, tissue, organ, or organism, encompasses a change in biochemical or physiological behavior, e.g., concentration, density, adhesion or migration, gene expression rate, or differentiation state within a biological compartment, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming. In certain contexts, the terms "activation," "stimulation," and the like refer to cellular activation regulated by internal mechanisms as well as external or environmental factors, while the terms "inhibition," "downregulation," and the like refer to the opposite effect.

[0047] The terms "adenosine machinery protein" or "adenosine machinery mRNA" or "adenosine machinery gene" refer to proteins, mRNAs, or encoding DNAs, respectively, involved in the extracellular production of adenosine and / or adenosine-mediated signaling pathways. Examples of these proteins and corresponding mRNAs include, but are not limited to, adenosine A2a receptor (A2aR), adenosine A2b receptor (A2bR), adenosine A1 receptor (A1R), CD26, adenosine deaminase (ADA), tissue-nonspecific alkaline phosphatase (TNAP), CD73, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), CD38, and / or CD39.

[0048] The term "agents that target the extracellular production of adenosine" refers to modulators of one or more proteins involved in the extracellular production of adenosine. Examples of modulators include small molecule compounds, antibodies, and interfering RNA. Proteins involved in the extracellular production of adenosine include, but are not limited to, tissue-nonspecific alkaline phosphatase (TNAP), CD73, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), CD38, and / or CD39. Therefore, modulators known to target these proteins are relevant to the present disclosure.

[0049] The term "agents that antagonize the activation of one of its receptors by adenosine" refers to antagonists that reduce or completely prevent adenosine from binding to adenosine receptor proteins, often integral membrane proteins. Protein receptors activated by adenosine include, but are not limited to, adenosine A1 receptor (A1R), adenosine A2a receptor (A2aR), and / or adenosine A2b receptor (A2bR). Thus, antagonists known to target these receptors are relevant to the present disclosure.

[0050] III. Detailed Description of the Embodiments Disclosed herein are methods for establishing an adenosine fingerprint of a subject's cancer as a means of identifying subjects who will respond more favorably to a particular treatment regimen. Establishing an adenosine fingerprint in a subject is further described herein and typically involves determining one or more of the following: the expression level of one or more adenosine machinery proteins in the subject's blood, the expression level (or mRNA level) of one or more adenosine machinery proteins from a biopsy of the subject's tumor, or the activity of specific adenosine machinery proteins in the subject's blood or tumor.

[0051] Accordingly, provided herein is a method of treating cancer in a subject with an established adenosine fingerprint, comprising administering to the subject a therapeutic agent that targets the extracellular production of adenosine and / or antagonizes adenosine's activation of one of its receptors, The cancer in the subject is (i) an increase in the concentration of one or more adenosine machinery proteins in blood obtained from the subject compared to a typical concentration of one or more adenosine machinery proteins in blood obtained from a subject having the same type of cancer; (ii) an increase in the activity of CD73 or TNAP in blood obtained from the subject compared to the typical AMP hydrolysis activity of CD73 and / or TNAP in blood obtained from subjects with the same type of cancer, as determined by an AMP hydrolysis assay; (iii) a biopsy of the subject's cancer showing an increased amount of one or more adenosine machinery proteins compared to the typical amount of the one or more adenosine machinery proteins in a biopsy obtained from a subject with the same type of cancer, as determined by immunostaining for the one or more adenosine machinery proteins; and (iv) a biopsy of the subject's cancer showing upregulation of one or more adenosine machinery proteins compared to the typical amount of the one or more adenosine machinery proteins in a biopsy obtained from a subject with the same type of cancer, as determined by mRNA levels; having at least one of the characteristics selected from the group consisting of: A method is also provided.

[0052] Adenosine machinery proteins Extracellular adenosine in the tumor microenvironment has been shown to have immunosuppressive effects in various tumor models. Therefore, proteins involved in the production of extracellular adenosine and / or adenosine signaling (adenosine machinery proteins) are potential candidates for blocking, reducing, or inhibiting the immunosuppressive effects of adenosine. Adenosine machinery proteins include, but are not limited to, adenosine A2a receptor (A2aR), adenosine A2b receptor (A2bR), adenosine A1 receptor (A1R), CD26, adenosine deaminase (ADA), tissue-nonspecific alkaline phosphatase (TNAP), CD73, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), CD38, and / or CD39.

[0053] Diagnostic tests that provide medical professionals with information about the expression and / or activity levels of adenosine machinery proteins provide a means of identifying subjects who will respond more favorably to particular treatment regimens. As described below, assessing the adenosine fingerprint of cancer can provide valuable information about adenosine machinery proteins.

[0054] Adenosine fingerprinting of cancer Adenosine fingerprints of cancer can be used to influence decisions for identifying and selecting appropriate treatments for subjects with cancer. Adenosine fingerprints can include evaluation of the expression level and / or activity of one or more adenosine machinery proteins. The expression level can be the amount of mRNA in a cancer sample, i.e., the amount of expressed protein in a cancer sample, the amount of expressed protein in the blood of a subject with cancer, or a combination of these evaluations. Activity evaluations generally utilize enzyme assays using blood samples or samples obtained from cancer to evaluate the catalytic activity of adenosine machinery proteins.

[0055] Assessing the blood concentration of adenosine machinery proteins. Determining the blood concentration of one or more adenosine machinery proteins provides information about the amount of related proteins expressed in a subject with cancer. Many methods are known for determining the concentration of an analyte in a blood sample. One such example is a sandwich ELISA assay. Example 1 provides a description of determining the amount of soluble CD73 in a subject's blood sample. Many other similar or related methods can be used to determine the concentration of one or more additional adenosine machinery proteins.

[0056] In some embodiments, when evaluating a subject's adenosine fingerprint, an increase in the concentration of one or more adenosine machinery proteins is considered relevant. The increase is determined relative to the typical concentration of one or more adenosine machinery proteins in blood obtained from subjects with the same type of cancer. In some embodiments, the typical concentration of a given adenosine machinery protein is a threshold value in blood obtained from subjects with the same type of cancer. In some embodiments, the typical concentration of a given adenosine machinery protein is the average concentration of one or more adenosine machinery proteins in blood obtained from subjects with the same type of cancer. In some embodiments, the increase is compared to the concentration of one or more adenosine machinery proteins in the subject before being diagnosed with cancer.

[0057] In some embodiments, when evaluating adenosine fingerprint, the concentration of soluble CD73 is considered to be a relevant threshold.For example, in some embodiments, the concentration of soluble CD73 in the blood of a subject is considered to be about 1 ng / mL as a relevant threshold.In some embodiments, the threshold is 3 ng / mL.In some embodiments, the threshold is 8 ng / mL.

[0058] In some embodiments, in the context of determining an adenosine fingerprint, elevated levels of adenosine machinery proteins are determined by measuring the relative increase compared to a reference level. The reference level can be a threshold or average level of a given protein in subjects with the same type of cancer. For example, in some embodiments, the concentration of soluble CD73 in blood from a subject with cancer is considered relevant if there is an increase of at least 1% compared to the reference level. In some embodiments, an increase of about 2, 3, 4, 5, 10, 15, 20, 25, or more percent compared to the reference level is considered relevant.

[0059] Assessing the enzymatic activity of adenosine machinery proteins using an AMP hydrolysis assay. Determining the enzymatic activity of one or more adenosine machinery proteins provides information about the activity of related proteins expressed in a subject with cancer. These determinations can be made from a blood sample taken from a subject with cancer. Many methods are known for determining the activity of proteins in a blood sample. One particularly relevant assay for measuring the activity of CD73 or TNAP in this disclosure is the AMP-Glo ​​hydrolysis assay, described in Example 2 of the present application.

[0060] The amount of AMP hydrolysis mediated by CD73, TNAP, or another protein can be reported in many different ways. In some embodiments, CD73- and / or TNAP-mediated hydrolysis in a sample is reported as a percentage of the total AMP hydrolysis activity in the sample. In the context of determining an adenosine fingerprint, the results of an AMP hydrolysis assay can indicate that treatment with one or more agents that target the extracellular production of adenosine or antagonize adenosine's activation of one of its receptors is appropriate.

[0061] In some embodiments, when assessing a subject's adenosine fingerprint via an AMP hydrolysis assay, an increase in CD73 and / or TNAP activity is considered relevant. The increase is determined relative to the typical AMP hydrolysis activity of CD73 and / or TNAP in blood from subjects with the same type of cancer. In some embodiments, the typical AMP hydrolysis activity of CD73 and / or TNAP is a threshold value in subjects with the same type of cancer. In some embodiments, the typical AMP hydrolysis activity of CD73 and / or TNAP is the average AMP hydrolysis activity of one or more adenosine machinery proteins in blood from subjects with the same type of cancer. In some embodiments, the increase is compared to the AMP hydrolysis activity of CD73 and / or TNAP in the subject's blood before they were diagnosed with cancer.

[0062] In some embodiments, when assessing a subject's adenosine fingerprint via the AMP-Glo ​​hydrolysis assay, a threshold level of AMP-mediated hydrolysis by adenosine machinery proteins is considered relevant. In some embodiments, such results include a value in which at least 10% of the total AMP hydrolysis activity in the subject's blood is mediated by CD73 or TNAP, a value in which at least 20% of the total AMP hydrolysis activity in the subject's blood is mediated by CD73 or TNAP, or a value in which at least 50% of the total AMP hydrolysis activity in the subject's blood is mediated by CD73 or TNAP.

[0063] In some embodiments, assessment of the enzymatic activity of the adenosine machinery protein is performed using the isotopic AMP hydrolysis assay described in Example 7 of the present application.

[0064] In some embodiments, when assessing a subject's adenosine fingerprint via an isotopic AMP hydrolysis assay, an increase in isotopic AMP hydrolysis activity of CD73 and / or TNAP is considered relevant. The increase is determined relative to the typical isotopic AMP hydrolysis activity of CD73 and / or TNAP in blood obtained from subjects with the same type of cancer. In some embodiments, the typical isotopic AMP hydrolysis activity of CD73 and / or TNAP is increased above a threshold value in subjects with the same type of cancer. In some embodiments, the typical isotopic AMP hydrolysis activity of CD73 and / or TNAP is the average isotopic AMP hydrolysis activity of one or more adenosine machinery proteins in blood obtained from subjects with the same type of cancer. In some embodiments, the increase is compared to the isotopic AMP hydrolysis activity of CD73 and / or TNAP in the subject's blood before they were diagnosed with cancer.

[0065] In some embodiments, when assessing a subject's adenosine fingerprint via an isotopic AMP hydrolysis assay, a threshold level of AMP-mediated hydrolysis by adenosine machinery proteins is considered relevant. In some embodiments, such results include a value in which at least 10% of the total AMP hydrolysis activity in the subject's blood is mediated by CD73 or TNAP, a value in which at least 20% of the total AMP hydrolysis activity in the subject's blood is mediated by CD73 or TNAP, or a value in which at least 50% of the total AMP hydrolysis activity in the subject's blood is mediated by CD73 or TNAP.

[0066] Assessing the expression levels of adenosine machinery proteins in tumors using immunostaining. Immunostaining is a well-established technique for identifying the presence of specific proteins and quantifying their relative abundance. Many methods are available for labeling the proteins for visualization and quantification. Typically, immunostaining involves obtaining a tumor biopsy from a subject with cancer and applying a labeled antibody that binds to the target of interest. Exemplary methods for determining the amount of CD73 and TNAP are described in Example 3. Those skilled in the art will recognize that additional adenosine machinery proteins can be assessed using techniques similar to those described in Example 3 or based on methods known in the art.

[0067] In some embodiments, when assessing a subject's adenosine fingerprint via immunostaining, an increase in an adenosine machinery protein is considered relevant. The increase is determined relative to the typical amount of such adenosine machinery protein in biopsies obtained from subjects with the same type of cancer. In some embodiments, the typical amount of a given adenosine machinery protein is a threshold amount in biopsies obtained from subjects with the same type of cancer. In some embodiments, the typical amount of a given adenosine machinery protein is the average amount of such adenosine machinery protein in biopsies obtained from subjects with the same type of cancer. In some embodiments, the increase is compared to the amount of the adenosine machinery protein in the same tissue of the subject before the cancer was diagnosed.

[0068] In some embodiments, when assessing a subject's adenosine fingerprint via immunostaining, the percentage of stained area for the analyte of interest is considered a relevant threshold. For example, in some embodiments, 1% stained area is considered a relevant threshold. In some embodiments, 7, 10, 20% or more stained area is considered a relevant threshold.

[0069] Assessment of adenosine machinery proteins by measuring mRNA levels. Many methods for identifying and quantifying relative mRNA levels in biological samples are known in the art, and each of these is suitable for assessing adenosine machinery mRNA levels. As intended herein, in some embodiments, the method for measuring mRNA levels is performed from tumor biopsies obtained from subjects. A typical method for determining the mRNA levels of adenosine machinery proteins is described in Example 4.

[0070] In some embodiments, when evaluating a subject's adenosine fingerprint through measuring mRNA level, upregulation of adenosine mechanism mRNA is considered relevant.Upregulation is determined by comparing with the typical amount of adenosine mechanism mRNA in biopsies obtained from subjects with the same type of cancer.In some embodiments, the typical amount of a given adenosine mechanism mRNA is a threshold value in biopsies obtained from subjects with the same type of cancer.In some embodiments, the typical amount of a given adenosine mechanism mRNA is the average amount of such adenosine mechanism mRNA in biopsies obtained from subjects with the same type of cancer.In some embodiments, the increase is compared with the amount of adenosine mechanism mRNA in the same tissue of the subject before being diagnosed with cancer.

[0071] Accordingly, in some embodiments, the present disclosure provides a method of treating cancer in a subject with an established adenosine fingerprint, comprising administering to the subject a therapeutic agent selected from the group consisting of an adenosine A2a receptor (A2aR) and / or an adenosine A2b receptor (A2bR) antagonist and a CD73 inhibitor, The cancer in the subject is (i) an increase in the concentration of soluble CD73 in blood obtained from the subject compared to the typical concentration of CD73 in blood obtained from a subject with the same type of cancer; (ii) an increase in the activity of CD73 in blood obtained from the subject compared to the typical AMP hydrolysis activity of CD73 in blood obtained from subjects with the same type of cancer, as determined by an AMP hydrolysis assay; (iii) a biopsy of the subject's cancer that shows an increased amount of CD73 compared to the typical amount of CD73 in a biopsy obtained from a subject with the same type of cancer, as determined by CD73 immunostaining; and (iv) a biopsy of the subject's cancer showing upregulation of CD73 as determined by mRNA levels, compared to the typical amount of CD73 in a biopsy obtained from a subject with the same type of cancer; and wherein the subject is administered a CD73 inhibitor if the subject has at least one of the characteristics selected from the group consisting of: The cancer in the subject is (a) an increase in the concentration of TNAP in blood obtained from the subject compared to the typical concentration of TNAP in blood obtained from a subject with the same type of cancer; (b) an increase in the activity of TNAP in blood obtained from the subject compared to the typical AMP hydrolysis activity of TNAP in blood obtained from subjects with the same type of cancer, as determined by an AMP hydrolysis assay; (c) a biopsy of the subject's cancer showing an increased amount of TNAP compared to the typical amount of TNAP in a biopsy obtained from a subject with the same type of cancer, as determined by TNAP immunostaining; and (d) A biopsy of a subject's cancer showing upregulation of TNAP compared to the typical amount of TNAP in a biopsy obtained from a subject with the same type of cancer, as determined by mRNA levels. adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist is administered to a subject when the subject has at least one of the characteristics selected from the group consisting of: A method is provided.

[0072] In some embodiments, the subject is administered a CD73 inhibitor if the cancer in the subject has at least two, three, or four of the characteristics selected from the group consisting of (i) through (iv); or The subject is administered an adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist if the cancer in the subject has at least two, three, or four of the characteristics selected from the group consisting of (a) through (d).

[0073] In some embodiments, a subject is administered only an adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist if the cancer in the subject exhibits at least one, two, three, or four of the characteristics selected from each of (i) through (iv) and (a) through (d).

[0074] In some embodiments, both an adenosine A2a receptor (A2aR) and / or adenosine A2b receptor (A2bR) antagonist and a CD73 inhibitor are administered when the cancer in the subject exhibits at least one, two, three, or four of the characteristics selected from each of (i) through (iv) and (a) through (d).

[0075] Assessment of one or more of the characteristics described herein can help identify subjects who will respond favorably to selected therapeutic agents, including agents that target the extracellular production of adenosine and agents that antagonize adenosine's activation of one of its receptors.

[0076] Drugs that target extracellular production of adenosine Many proteins are known to be involved in the extracellular production of adenosine in the body. For example, the primary pathway leading to the generation of extracellular adenosine is the sequential dephosphorylation of ATP by CD39, which hydrolyzes ATP to ADP and then to AMP, and CD73, which hydrolyzes AMP to adenosine. TNAP also contributes to the production of adenosine from AMP. An alternative mechanism leading to the generation of extracellular adenosine is the hydrolysis of NAD+ to ADPR by CD38 and the hydrolysis of ADPR to AMP by ENPP1. ENPP1 can also hydrolyze NAD+ to produce AMP. Therefore, proteins involved in the extracellular production of adenosine include, but are not limited to, tissue-nonspecific alkaline phosphatase (TNAP), CD73, ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1), CD38, and / or CD39.

[0077] As contemplated herein, the present disclosure provides methods of treating cancer in subjects with an established adenosine fingerprint using one or more agents that target the extracellular production of adenosine.

[0078] Tissue non-specific alkaline phosphatase (TNAP) inhibitor. Some TNAP inhibitors are known in the art. In some embodiments, the TNAP inhibitor useful in the described method is an agent disclosed in WO / 2013 / 126608, WO / 2006 / 039480 or WO / 2002 / 092020, the contents of each of which are incorporated herein by reference for all purposes. In some embodiments, the TNAP inhibitor is a compound of the formula: [ka] It has.

[0079] CD73 Inhibitors. In some embodiments, the CD73 inhibitors useful in the described methods are compounds of formula (i):

[0080] [ka] or a pharmaceutically acceptable salt, hydrate or solvate thereof; During the ceremony, Each R 1 is selected from hydrogen, optionally substituted C1-C6 alkyl, optionally substituted aryl, and —C(R 2 R 2 )-OC(O)-OR 3 or two R 1 groups are optionally combined to form a 5- to 7-membered ring; Each R 2 are independently selected from the group consisting of H and optionally substituted C1-C6 alkyl; Each R 3 are independently selected from the group consisting of H, C1-C6 alkyl, and optionally substituted aryl; R 5is selected from the group consisting of H and optionally substituted C1-C6 alkyl; X is selected from the group consisting of O, CH2 and S; A is selected from the group consisting of:

[0081] [ka] Each of them has 1 to 5 R 6 optionally substituted with a substituent; where the subscript n is an integer from 0 to 3; Z is CH2, CHR 6 , N.R. 6 and O, Each R 6 are independently selected from the group consisting of H, CH3, OH, CN, F, optionally substituted C1-C6 alkyl and OC(O)-C1-C6 alkyl, and optionally two R on adjacent ring vertices are 6 the groups are linked to form a 5- to 6-membered ring having at least one heteroatom as a ring vertex; Het is selected from the group consisting of:

[0082] [ka] where the wavy line indicates the point of attachment to the rest of the compound: R a are H, NH2, and NHR 7 , NHC(O)R 7 , N.R. 7 R 7 , R 7 , O.H., S.R. 7 and OR 7 is selected from the group consisting of R b is H, halogen, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH and OR 7 is selected from the group consisting of R c and R dis H, halogen, haloalkyl, NH2, NHR 7 , N.R. 7 R 7 , R 7 , OH, OR 7 , S.R. 7 , SO2R 7 , -X 1 -NH2, -X 1 -NHR 7 , -X 1 -NR 7 R 7 , -X 1 -OH, -X 1 -OR 7 , -X 1 -SR 7 and -X 1 -SO2R 7 are independently selected from the group consisting of R e and R f are independently selected from the group consisting of H, halogen, and optionally substituted C1-C6 alkyl; each X 1 is a C1-C4 alkylene, Each R 7 is C1 to C, substituted as necessary 10 Alkyl, optionally substituted C2-C 10 Alkenyl, optionally substituted C2-C 10 two R independently selected from the group consisting of alkynyl, optionally substituted C3-C7 cycloalkyl, optionally substituted C3-C7 cycloalkylC1-C4 alkyl, optionally substituted 4-7 membered cycloheteroalkyl, optionally substituted 4-7 membered cycloheteroalkylC1-C4 alkyl, optionally substituted aryl, optionally substituted arylC1-C4 alkyl, optionally substituted arylC2-C4 alkenyl, optionally substituted arylC2-C4 alkynyl, optionally substituted heteroaryl, optionally substituted heteroarylC1-C4 alkyl, optionally substituted heteroarylC1-C4 alkenyl, and optionally substituted heteroarylC2-C4 alkynyl, optionally bonded to the nitrogen atom; 7the groups are linked to form a 4- to 7-membered heterocyclic ring, which is optionally fused to an aryl ring; However, these compounds have the following combinations of X, A and Het:

[0083] [ka] other than a compound that results in In the formula, R g is H or two R g the groups combine to form an acetonide, and (1) R c and R e is hydrogen and R a is -OEt, -OCH2Ph, -SCH2Ph, -NH2, methylamino, ethylamino, dimethylamino, diethylamino, N-methyl-N-ethylamino, phenylamino, benzylamino, 2-phenylethylamino, N-benzyl-N-ethylamino, dibenzylamino, 4-aminobenzylamino, 4-chlorobenzylamino, 4-nitrobenzylamino or 4-sulfamoylbenzylamino, or (2) R c is hydrogen and R a is -NH2 and R e is bromo, chloro, aminomethyl or thioethyl, or (3) R c is hydrogen and R a is benzylamino and R e is bromo.

[0084] In some embodiments, the CD73 inhibitor is Compound A:

[0085] [ka] or a pharmaceutically acceptable salt thereof.

[0086] In some embodiments, the CD73 inhibitor is compound B:

[0087] [ka] or a pharmaceutically acceptable salt thereof.

[0088] In some embodiments, the CD73 inhibitor is compound C:

[0089] [ka] or a pharmaceutically acceptable salt thereof.

[0090] In some embodiments, the CD73 inhibitor is a molecule described in U.S. Patent Application Publication No. 2017 / 0267710 (see U.S. Patent Application No. 15 / 400,748, filed June 6, 2017), the contents of which are incorporated herein by reference for all purposes.

[0091] In some embodiments, the CD73 inhibitor is an agent disclosed in WO2015 / 164573, WO2017 / 120508, WO2018 / 183635, WO2018 / 094148, WO2018 / 119284, WO2018 / 183635, WO2018 / 208727, WO2018 / 208980, WO2017 / 098421, WO2017 / 153952, the contents of each of which are incorporated herein by reference for all purposes.

[0092] In some embodiments, the CD73 inhibitor is oleclumab (MEDI-9447), CPI-006, NZV930 / SRF373, BMS-986179, or TJ4309.

[0093] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitors. In some embodiments, an ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) inhibitor useful in the described methods is MV-626.

[0094] In some embodiments, ENPP1 inhibitors useful in the described methods are agents disclosed in WO2019 / 023635, the contents of which are incorporated herein by reference for all purposes.

[0095] CD38 Inhibitors. In some embodiments, the CD38 inhibitor useful in the described methods is daratumumab or isatuximab.

[0096] In some embodiments, the CD38 inhibitor is an agent disclosed in WO / 2019 / 034753, US2018 / 0298106, WO2019 / 034752, the contents of each of which are incorporated herein by reference for all purposes.

[0097] CD39 inhibitors. CD39 is also known as ectonucleoside triphosphate diphosphohydrolase-1. In some embodiments, the CD39 inhibitor useful in the described methods is IPH5201, SRF617, or TTX-030.

[0098] In some embodiments, the CD39 inhibitor is an agent disclosed in WO2012 / 085132, WO2017 / 089334, WO2009 / 095478, WO2011 / 154453, and WO2018 / 224685, the contents of each of which are incorporated herein by reference for all purposes.

[0099] The present disclosure includes pharmaceutically acceptable salts or derivatives of any of the above.

[0100] Drugs that antagonize adenosine activation of one of its receptors There are many receptors in the body that are activated by extracellular adenosine. That is, adenosine binding initiates enzymatic activity and / or propagates cellular signals. Activation by adenosine occurs through four G-coupled adenosine receptors: A1, A2a, A2b, and A3. Adenosine primarily signals through the A2a receptor (predominantly expressed on T cells) and the A2b receptor (expressed on myeloid cells), which, when stimulated by adenosine, leads to exacerbated T cell activation. Although less understood, the A1 receptor has been reported to be involved in the pathogenesis of cancers such as breast, colon, and gastric cancer, and the A3 receptor has been reported to be involved in colorectal and breast cancer. Overactivation of one or more of these receptors by adenosine in the tumor microenvironment can lead to immunosuppressive effects. Therefore, antagonists that can block or otherwise inhibit adenosine binding to these receptors are useful in cancer treatment. Relevant receptors include, but are not limited to, the adenosine A1 receptor (A1R), the adenosine A2a receptor (A2aR) and / or the adenosine A2b receptor, and the adenosine A3 receptor (A3R).

[0101] As contemplated herein, the present disclosure provides methods of treating cancer in a subject with an established adenosine fingerprint using one or more agents that antagonize adenosine's activation of one of its receptors.

[0102] Adenosine A1 receptor (A1R) antagonists. In some embodiments, the A1R antagonist useful in the described methods is FK352, KW-3902 (Rolofylline), SLV320, BG9719 (CVT-124), or BG9928 (Adentri).

[0103] Adenosine A2a Receptor (A2aR) and / or Adenosine A2b Receptor Antagonists. In some embodiments, the adenosine A2a receptor (A2aR) and / or adenosine A2b receptor (A2bR) antagonists that may be used in the described methods are compounds of formula (I):

[0104] [ka] or a pharmaceutically acceptable salt, hydrate or solvate thereof; During the ceremony, G 1 is N or CR 3a and G 2 is N or CR 3b and G 3 is N or CR 3c and R 3a , R 3b and R 3c each independently represents H or C 1~3 is alkyl, R 1a and R 1b teeth, i) H ii) 1 to 3 Rs 5 C optionally substituted with substituents 1~8 Alkyl, iii) 1 to 3 Rs 5 -X optionally substituted with a substituent 1 -OC 1~8 Alkyl, iv) -C-(O)R 6 , v) 1 to 3 R 7 Y optionally substituted with a substituent, and vi) 1 to 3 R 7 -X optionally substituted with a substituent 1 -Y, or vii) R 1a and R 1b are 1 to 3 R's together with the nitrogen to which they are attached. 8 forming a 5-6 membered heterocycloalkyl ring optionally substituted with substituents, wherein the heterocycloalkyl has 0-2 additional heteroatom ring vertices selected from the group consisting of O, N and S; Each Y is C 3~8cycloalkyl or a 4- to 6-membered heterocycloalkyl having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S; R 2 and R 4 are each independently H or C 1~3 is alkyl, Ar 1 is phenyl or a 5- to 6-membered heteroaryl, each of which is 1 to 3 R 9 , where necessary, Ar 2 is phenyl or a 5- to 6-membered heteroaryl, each of which is 1 to 3 R 10 , where necessary, Ar 1 and Ar 2 The 5- or 6-membered heteroaryl has 1 to 3 heteroatom ring vertices independently selected from the group consisting of O, N, and S; each X 1 is C 1~6 is alkylene, Each R 5 is hydroxyl, C 3~8 Cycloalkyl, phenyl, -O-phenyl, -C(O)OR a and oxo; Each R 6 is C 1~8 alkyl or Y, which are hydroxyl, -O-phenyl, phenyl and -OC 1~8 and optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl, Each R 7 is C 1~8 Alkyl, hydroxyl, -OC 1~8 Alkyl, oxo and C(O)OR a are independently selected from the group consisting of Each R 8 is C 1~8 independently selected from the group consisting of alkyl, hydroxyl, and oxo; Each R 9 is C 1~8 Alkyl, -OC 1~8 Alkyl, -X 1-OC 1~8 Alkyl, -OX 1 -OC 1~8 Alkyl, -X 1 -OX 1 -OC 1~8 Alkyl, -C(O)OR a , halogen, cyano, -NR b R c , Y, -X 1 -C 3~8 Cycloalkyl and -X 2 -Z; 2 is C 1~6 Alkylene, -C 1~6 alkylene-O-, -C(O)-, and -S(O)-; Z is a 4- to 6-membered heterocycloalkyl having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S; 9 Each of the substituents is 1 to 3 R 11 , where necessary, Each R 10 is C 1~8 Alkyl, halo, cyano, -OC 1~8 Alkyl, -X 1 -OC 1~8 Alkyl, -OX 1 -OC 1~8 Alkyl, -S(O)2-C 1~6 Alkyl, -C(O)NR d R e and 4- to 6-membered heteroaryl rings having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S; 10 Each of the substituents is 1 to 3 R 12 or Ar 2 Two R on adjacent ring vertices of 10 are optionally combined to form a 5-membered heterocycle optionally substituted with 1 to 2 halogens; Each R 11 -hydroxyl, halo, cyano, -NR d R e , -C(O)OR a , phenyl, C 3~8Cycloalkyl and C(O)OR a C substituted as needed 1~4 independently selected from the group consisting of alkyl, Each R 12 is halo, cyano, hydroxy, -C(O)OR a are independently selected from the group consisting of Each R a is H or C 1~6 is alkyl, Each R b and R c is H, C 1~8 Alkyl, -S(O)2-C 1~6 Alkyl, -C(O)OR a and -X 1 -C(O)OR a are independently selected from the group consisting of Each R d and R e is H, C 1~8 Alkyl, -S(O)2-C 1~6 independently selected from the group consisting of alkyl, However, G 1 and G 2 are N and G 3 is CH and R 2 is CH3 and R 1a and R 1b are each H, then Ar 2 is other than 2-thienyl, phenyl, 2-, 3- or 4-methoxyphenyl, 3- or 4-halophenyl, 2,4-dimethoxyphenyl, 2,4-dichlorophenyl or 2- or 4-methylphenyl.

[0105] In some embodiments, the adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist is Compound 1.

[0106] [ka] or a pharmaceutically acceptable salt thereof.

[0107] In some embodiments, the adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist is Compound 2.

[0108] [ka] or a pharmaceutically acceptable salt thereof.

[0109] In some embodiments, the adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist is Compound 3.

[0110] [ka] or a pharmaceutically acceptable salt thereof.

[0111] In some embodiments, the adenosine A2a receptor (A2aR) and / or adenosine A2b receptor (A2bR) antagonist is a molecule described in U.S. Patent Application Publication No. 2018 / 0215730 (see also U.S. Patent Application No. 15 / 875,106, filed June 19, 2018, the contents of which are incorporated herein by reference for all purposes).

[0112] In some embodiments, the A2a receptor (A2aR) and / or adenosine A2b receptor (A2bR) antagonist is AZD4635, ciforadenant (CPI-444), NIR178, or PBF-1129.

[0113] Adenosine A3 receptor (A3R) antagonists. In some embodiments, the A3R antagonists useful in the described methods are molecules described in WO2007 / 063539A1, US2003 / 0078232, the contents of each of which are incorporated herein by reference for all purposes.

[0114] Cancer type Those skilled in the art will recognize that the treatment methods described herein are independent of the origin of the tumor and rely on the evaluation of the tumor's adenosine fingerprint. Thus, the present disclosure provides methods that are not limited to a particular type of cancer. Accordingly, the present disclosure is useful in treating many different cancer types, including, but not limited to, cancers of the prostate, colorectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin (including melanoma and basal carcinoma), mesothelial lining, white blood cells (including lymphoma and leukemia), esophagus, breast (including triple-negative breast cancer), muscle, connective tissue, lung (including small cell lung cancer and non-small cell lung cancer), adrenal gland, thyroid, kidney, or bone, glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma (including Kaposi's sarcoma), choriocarcinoma, basal cell carcinoma of the skin, and testicular seminoma.

[0115] In some embodiments of the present disclosure, the cancer is melanoma, colon cancer, pancreatic cancer, breast cancer, prostate cancer, lung cancer, leukemia, brain cancer, lymphoma, sarcoma, ovarian cancer, head and neck cancer, cervical cancer, or Kaposi's sarcoma.

[0116] In some embodiments of the present disclosure, the cancer is cancer of the thyroid, adrenal gland, mesothelial lining, bile duct, pancreas, brain, kidney, esophagus, rectum, colon, stomach, head, neck, skin, testicle, ovary, lung, endometrium, eye, prostate, breast, or liver, or a glioblastoma, mesothelioma, or sarcoma.

[0117] In some embodiments of the present disclosure, the cancer is testicular, ovarian, lung, endometrial, or adrenal cancer.

[0118] In some embodiments of the present disclosure, the cancer is cancer of the eye, prostate, breast, kidney, liver, or lung.

[0119] In some embodiments, the present disclosure provides methods of treating a subject identified as having a particular type of cancer with an agent that targets the extracellular production of adenosine and / or an agent that antagonizes adenosine's activation of one of its receptors, and at least one additional therapeutic agent, examples of which are provided elsewhere herein.

[0120] Combination therapy The present disclosure contemplates the use of the therapeutic agents described herein alone or in combination with one or more other active therapeutic agents. The additional active therapeutic agent may be a small chemical molecule, a macromolecule, e.g., a protein, antibody, peptibody, peptide, DNA, RNA, or fragments of such macromolecules, or a cell or gene therapy. In such combination therapy, the various active agents often have different, complementary mechanisms of action. Such combination therapy may be particularly advantageous by allowing for a reduction in the dosage of one or more of the agents, thereby reducing or eliminating adverse effects associated with one or more of the agents. Furthermore, such combination therapy may exert a synergistic therapeutic or preventative effect against the underlying disease, disorder, or condition.

[0121] As used herein, "combination" is meant to include therapies that can be administered separately, e.g., formulated separately for separate administration (e.g., provided in a kit), as well as therapies that can be administered together in a single formulation (i.e., "co-formulation").

[0122] In certain embodiments, the therapeutic agents described herein are administered or applied sequentially, e.g., one agent is administered before one or more other agents. In other embodiments, the therapeutic agents described herein are administered simultaneously, e.g., two or more agents are administered simultaneously or near simultaneously, and the two or more agents can be present in two or more separate formulations or can be combined into a single formulation (i.e., co-formulated). Whether two or more agents are administered sequentially or simultaneously, they are considered to be administered in combination for purposes of the present invention.

[0123] The disclosed agents targeting the extracellular production of adenosine and / or agents antagonizing the activation of one of its receptors by adenosine can be used in combination with at least one other (active) agent in any manner appropriate under the circumstances. In one embodiment, treatment with at least one active agent and at least one additional therapeutic agent described herein is maintained for a period of time. In another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable) and treatment with a therapeutic agent described herein is maintained at a constant dosing regimen. In a further embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable) and treatment with a therapeutic agent described herein is reduced (e.g., a lower dose, less frequent dosing, or a shorter treatment regimen). In yet another embodiment, treatment with at least one active agent is reduced or discontinued (e.g., if the subject is stable) and treatment with a therapeutic agent described herein is increased (e.g., a higher dose, more frequent dosing, or a longer treatment regimen). In yet another embodiment, treatment with at least one active agent is maintained and treatment with a therapeutic agent described herein is reduced or discontinued (e.g., a lower dose, less frequent dosing, or a shorter treatment regimen). In yet another embodiment, treatment with at least one active agent and treatment with a therapeutic agent described herein are reduced or discontinued (e.g., a lower dose, less frequent dosing, or a shorter treatment regimen).

[0124] The present disclosure provides methods of treating and / or preventing cancer in a subject with an established adenosine fingerprint with an agent that targets the extracellular production of adenosine and / or an agent that antagonizes adenosine's activation of one of its receptors and at least one additional therapeutic or diagnostic agent. In some embodiments, the additional therapeutic agent is radiation, an immunomodulatory agent, or a chemotherapeutic agent. Suitable immunomodulatory agents that can be used in the present invention include CD4OL, B7, and B7RP1; activating monoclonal antibodies (mAbs) against stimulatory receptors, such as anti-CD40, anti-CD38, anti-ICOS, and 4-IBB ligand; dendritic cell antigen loading (in vitro or in vivo); anti-cancer vaccines such as dendritic cell cancer vaccines; cytokines / chemokines, such as ILL IL2, IL12, IL18, ELC / CCL19, SLC / CCL21, MCP-1, IL-4, IL-18, TNF, IL-15, MDC, IFNa / b, M-CSF, IL-3, GM-CSF, IL-13, and anti-IL-10; bacterial lipopolysaccharide (LPS); indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors, and immunostimulatory oligonucleotides.

[0125] In certain embodiments, the present disclosure includes the administration of a therapeutic agent described herein in combination with a signal transduction inhibitor (STI). As used herein, the term "signal transduction inhibitor" refers to an agent that selectively inhibits one or more steps in a signal transduction pathway. Signal transduction inhibitors (STIs) of the present invention include: (i) bcr / abl kinase inhibitors (e.g., Gleevec), (ii) epidermal growth factor (EGF) receptor inhibitors, including kinase inhibitors and antibodies, (iii) her-2 / neu receptor inhibitors (e.g., Herceptin), (iv) inhibitors of Akt family kinases or the Akt pathway (e.g., rapamycin), (v) cell cycle kinase inhibitors (e.g., flavopiridol), and (vi) phosphatidylinositol kinase inhibitors. Agents involved in immune regulation can also be used in combination with the therapeutic agents described herein to suppress tumor growth in cancer patients.

[0126] TMB is an effective tool for determining the total number of somatic mutations present in a subject's genome. This information can be used to identify and select viable treatment options. For example, in some embodiments, the tumor mutation burden (TMB) in a subject is used to identify patients who should receive additional chemotherapy. For example, in some embodiments, the methods provided herein include further administering a chemotherapy agent to the subject if the TMB is less than 2.0 as determined by whole exome sequencing (WES). In some embodiments, the subject is further administered a chemotherapy agent if the TMB is less than 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, or 10.

[0127] Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamime; nitrogen methylamines, amides, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine hydrochloride oxide, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, e.g., aclacinomycin, actinomycin, ausramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, kalambucil, Minomycin, Carzinophilin, Chromomycin, Dactinomycin, Daunorubicin, Detorubicin, 6-Diazo-5-oxo-L-norleucine, Doxorubicin, Epirubicin, Esorubicin, Idarubicin, Marcellomycin, Mitomycin, Mycophenolic Acid, Nogalamycin, Olivomycin, Peplomycin, Potfilomycin, Puromycin, Keramycin, Rhodorubicin, Streptonigrin, Streptozocin, Tubercidin, Ubenimex, Zinostatin, Zorubicin; Antimetabolites such as Methotrexate and 5- Fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone;Antiadrenal agents, such as aminoglutethimide, mitotane, trilostane; folic acid replacement, such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; fenamet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziconazole; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitolactol; Pipobroman; Gacytosine; Arabinoside (Ara-C); Cyclophosphamide; Thiotepa; Taxoids, such as paclitaxel and doxetaxel; Chlorambucil; Gemcitabine; 6-Thioguanine; Mercaptopurine; Methotrexate; Platinum and platinum coordination complexes, such as cisplatin, carboplatin, and oxaliplatin; Vinblastine; Etoposide (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Xeloda; Ibandronate; CPT11; topoisomerase inhibitors; difluoromethylornithine (DMFO); retinoic acid; esperamycin; capecitabine; anthracyclines; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0128] Chemotherapeutic agents also include antihormonal agents that regulate or inhibit hormone action on tumors, such as antiestrogens including tamoxifen, raloxifene, aromatase inhibitor 4(5)-imidazole, 4-hydroxytamoxifen, trioxifene, ketoxifene, onapristone and toremifene; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.In certain embodiments, combination therapy includes a chemotherapy regimen that includes one or more chemotherapy agents.In certain embodiments, combination therapy includes the administration of hormones or related hormonal agents.

[0129] Additional treatment modalities that can be used in combination with the therapeutic agents described herein include radiation therapy, monoclonal antibodies against tumor antigens, monoclonal antibody and toxin conjugates, T cell adjuvants, bone marrow transplantation, or antigen-presenting cells (e.g., dendritic cell therapy) containing TLR agonists used to stimulate such antigen-presenting cells.

[0130] In certain embodiments, the present disclosure contemplates the use of the therapeutic agents described herein in combination with adoptive cell therapy, a novel and promising form of personalized immunotherapy in which immune cells with anti-tumor activity are administered to cancer patients. Adoptive cell therapy has been explored, for example, using tumor-infiltrating lymphocytes (TILs) and T cells engineered to express chimeric antigen receptors (CARs) or T cell receptors (TCRs). Adoptive cell therapy generally involves collecting T cells from an individual, genetically modifying them to target specific antigens or enhance their anti-tumor effects, expanding them to sufficient numbers, and infusing the genetically modified T cells into the cancer patient. T cells can be collected from the patient (e.g., autologous) to which the expanded cells are later re-infused, or they can be collected from a donor patient (e.g., allogeneic).

[0131] In certain embodiments, the present disclosure contemplates the use of the compounds described herein in combination with the therapy based on RNA interference to suppress gene expression.RNAi begins with the cleavage of longer double-stranded RNA into small interfering RNA (siRNA).One strand of siRNA is incorporated into a ribonucleoprotein complex known as RNA-induced silencing complex (RISC), which is then used to identify the mRNA molecule that is at least partially complementary to the siRNA strand that is incorporated.RISC can bind to or cleave mRNA, both of which inhibit translation.

[0132] The present disclosure contemplates the use of inhibitors of the therapeutic agents described herein in combination with immune checkpoint inhibitors.

[0133] The vast array of genetic and epigenetic alterations characteristic of all cancers provides a diverse set of antigens that the immune system can use to distinguish tumor cells from their normal counterparts. In the case of T cells, the ultimate amplitude (e.g., level of cytokine production or proliferation) and quality (e.g., type of immune response generated, e.g., pattern of cytokine production) of the response initiated through antigen recognition by the T cell receptor (TCR) are regulated by the balance between costimulatory and inhibitory signals (immune checkpoints). Under normal physiological conditions, immune checkpoints are important for preventing autoimmunity (i.e., maintaining self-tolerance) and for protecting tissues from injury when the immune system is responding to pathogen infection. The expression of immune checkpoint proteins can be dysregulated by tumors as an important immune resistance mechanism.

[0134] T cells have been a major focus of efforts to therapeutically manipulate endogenous antitumor immunity due to i) their ability to selectively recognize peptides derived from proteins in all cellular compartments, ii) their ability to directly recognize and kill antigen-expressing cells (by CD8+ effector T cells, also known as cytotoxic T lymphocytes (CTLs)), and iii) their ability to orchestrate diverse immune responses by CD4+ helper T cells integrating adaptive and innate effector mechanisms.

[0135] In clinical settings, blockade of immune checkpoints leading to the amplification of antigen-specific T cell responses has shown to be a promising approach in human cancer therapy.

[0136] T cell-mediated immunity involves multiple sequential steps, each regulated by countervailing stimulatory and inhibitory signals to optimize the response. Almost all inhibitory signals in the immune response ultimately modulate intracellular signaling pathways, many of which are initiated through membrane receptors, whose ligands are membrane-bound or soluble (cytokines). While costimulatory and inhibitory receptors and ligands that regulate T cell activation are often not overexpressed in cancer compared with normal tissue, inhibitory ligands and receptors that regulate T cell effector function in tissues are commonly overexpressed on tumor cells or on non-transformed cells associated with the tumor microenvironment. The function of soluble and membrane-bound receptor-ligand immune checkpoints can be modulated using agonist antibodies (for costimulatory pathways) or antagonist antibodies (for inhibitory pathways). Thus, in contrast to most antibodies currently approved for cancer therapy, immune checkpoint-blocking antibodies do not directly target tumor cells but rather target lymphocyte receptors or their ligands to enhance intrinsic antitumor activity. (See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64).

[0137] Examples of immune checkpoints (ligands and receptors) that are candidates for blockade, some of which are selectively upregulated on various tumor cells, include PD1 (programmed cell death protein 1); PD-L1 (PD1 ligand); BTLA (B and T lymphocyte attenuator); CTLA4 (cytotoxic T-lymphocyte-associated antigen 4); TIM3 (T cell membrane protein 3); LAG3 (lymphocyte activation gene 3); TIGIT (T cell immunoreceptor with Ig and ITIM domains); and killer inhibitory receptors, which can be divided into two classes based on their structural features: i) killer cell immunoglobulin-like receptors (KIRs) and ii) C-type lectin receptors (members of the type II transmembrane receptor family). Other less well-defined immune checkpoints have been described in the literature, including both receptors (e.g., the 2B4 (also known as CD244) receptor) and ligands (e.g., certain B7 family inhibitory ligands, such as B7-H3 (also known as CD276) and B7-H4 (also known as B7-S1, B7x, and VCTN1)). (See Pardoll, (April 2012) Nature Rev. Cancer 12:252-64).

[0138] The present disclosure contemplates the use of the therapeutic agents described herein in combination with inhibitors of the aforementioned immune checkpoint receptors and ligands, as well as undescribed immune checkpoint receptors and ligands. Certain modulators of immune checkpoints are available today, and others are in development, including the PD1 and PD-L1 antibodies nivolumab (Bristol-Myers Squibb), pembrolizumab (Merck), cemiplimab (Sanofi and Regeneron), atezolizumab (Roche), durvalumab (AstraZeneca), and avelumab (Merck).

[0139] In some embodiments, the treatment methods described herein include administering a PD1 and PD-L1 inhibitor if a biopsy from a subject's cancer indicates that the cancer is PD-L1 positive. Methods for determining PD-L1 status are known in the art, and Example 6 of the present application references FDA-approved products for determining PD-L1 status. In some embodiments, a cancer can be considered PD-L1 positive if at least 1% of cells from the subject's cancer express PD-L1. In some embodiments, a cancer can be considered PD-L1 positive if at least 10% of cells from the subject's cancer express PD-L1. In some embodiments, a cancer can be considered PD-L1 positive if at least 50% of cells from the subject's cancer express PD-L1. Similar assays can be performed to determine the status of other immune checkpoints in a subject's tumor.

[0140] In one aspect of the present invention, the therapeutic agents described herein are combined with immuno-oncology agents that are (i) agonists of stimulatory (including costimulatory) receptors or (ii) antagonists of inhibitory (including co-inhibitory) signals on T cells, both of which result in the amplification of antigen-specific T cell responses. Certain stimulatory and inhibitory molecules are members of the immunoglobulin superfamily (IgSF). One important family of membrane-bound ligands that bind to costimulatory or co-inhibitory receptors is the B7 family, which includes B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3, B7-H4, B7-H5 (VISTA), and B7-H6. Another family of membrane-bound ligands that bind to costimulatory or coinhibitory receptors is the TNF family of molecules that bind to cognate TNF receptor family members, namely CD40 and CD4OL, OX-40, OX-40L, CD70, CD27L, CD30, CD3OL, 4-1BBL, and CD137. (4-1BB), TRAIL / Apo2-L, TRAILR1 / DR4, TRAILR2 / DR5, TRAILR3, TRAILR4, OPG, RANK, RANKL, TWEAKR / Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI, APRIL, BCMA, LT13R, LIGH Contains T, DcR3, HVEM, VEGI / TL1A, TRAMP / DR3, EDAR, EDA1, XEDAR, EDA2, TNFR1, lymphotoxin a / TNF13, TNFR2, TNFa, LT13R, lymphotoxin a1132, FAS, FASL, RELT, DR6, TROY, NGFR.

[0141] In another aspect, the immuno-oncology agent is a cytokine that inhibits T cell activation (e.g., IL-6, IL-10, TGF-B, VEGF and other immunosuppressive cytokines) or a cytokine that stimulates T cell activation to stimulate an immune response.

[0142] In one aspect, T cell responses can be stimulated by a combination of a Therapeutic Agent described herein and one or more of (i) an antagonist of a protein that inhibits T cell activation (e.g., an immune checkpoint inhibitor), such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, TIM-3, Galectin 9, CEACAM-1, BTLA, CD69, Galectin-1, TIGIT, CD113, GPR56, VISTA, 2B4, CD48, GARP, PD1H, LAIR1, TIM-1, and TIM-4, and / or (ii) an agonist of a protein that stimulates T cell activation, such as B7-1, B7-2, CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX4OL, GITR, GITRL, CD70, CD27, CD40, DR3, and CD2. Other agents that can be combined with the therapeutic agents described herein for the treatment of cancer include antagonists of inhibitory receptors on NK cells or agonists of activating receptors on NK cells. For example, the compounds herein can be combined with antagonists of KIR, such as lirilumab.

[0143] Still other agents for combination therapy include CSF-1R antagonists, for example, agents that inhibit or deplete macrophages or monocytes, including, but not limited to, CSF-1R antagonist antibodies, including RG7155 (WO11 / 70024, WO11 / 107553, WO11 / 131407, WO13 / 87699, WO13 / 119716, WO13 / 132044) or FPA-008 (WO11 / 140249, WO13169264, WO14 / 036357).

[0144] In another aspect, the disclosed agents that target the proteins / receptors described herein can be used with one or more of: agonist agents that ligate positive costimulatory receptors; blocking agents that attenuate signaling through inhibitory receptors; antagonists; and one or more agents that systemically increase the frequency of anti-tumor T cells; agents that overcome different immunosuppressive pathways within the tumor microenvironment (e.g., blocking inhibitory receptor engagement (e.g., PD-L1 / PD-1 interaction); depleting or inhibiting Tregs (e.g., using anti-CD25 monoclonal antibodies (e.g., daclizumab) or by ex vivo anti-CD25 bead depletion); or reversing / preventing T cell anergy or exhaustion); and agents that induce innate immune activation and / or inflammation at the tumor site.

[0145] In one embodiment, the immuno-oncology agent is a CTLA-4 antagonist, e.g., an antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include, for example, YERVOY (ipilimumab) or tremelimumab.

[0146] In another embodiment, the immuno-oncology agent is a PD-1 antagonist, e.g., an antagonistic PD-1 antibody. Suitable PD-1 antibodies include, for example, OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), MEDI-0680 (AMP-514, WO2012 / 145493), BGB-108, GB-226, PDR-001, mDX-400, SHR-1210, IBI-308, and PF-06801591. The immuno-oncology agent can also include pidilizumab (CT-011), although its specificity for PD-1 binding has been questioned. Another approach to targeting the PD-1 receptor is a recombinant protein called AMP-224, which consists of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1.

[0147] In another embodiment, the immuno-oncology agent is a PD-L1 antagonist, e.g., an antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for example, MPDL3280A (RG7446, WO2010 / 077634), durvalumab (MEDI4736), atezolizumab, avelumab, BMS-936559 (WO2007 / 005874), MSB0010718C (WO2013 / 79174), KD-033, CA-327, CA-170, ALN-PDL, TSR-042, and STI-1014.

[0148] In another embodiment, the immuno-oncology agent is a LAG-3 antagonist, e.g., an antagonistic LAG-3 antibody. Suitable LAG-3 antibodies include, for example, BMS-986016 (WO10 / 19570, WO14 / 08218) or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273).

[0149] In another embodiment, the immuno-oncology agent is a CD137 (4-1BB) agonist, e.g., an agonist CD137 antibody. Suitable CD137 antibodies include, for example, urelumab and PF-05082566 (WO12 / 32433).

[0150] In another embodiment, the immuno-oncology agent is a GITR agonist, e.g., an agonistic GITR antibody. Suitable GITR antibodies include, for example, BMS-986153, BMS-986156, TRX-518 (WO06 / 105021, WO09 / 009116), and MK-4166 (WO11 / 028683).

[0151] In another embodiment, the immuno-oncology agent is an OX40 agonist, e.g., an agonist OX40 antibody. Suitable OX40 antibodies include, for example, MEDI-6383 or MEDI-6469.

[0152] In another embodiment, the immuno-oncology agent is an OX4OL antagonist, e.g., an antagonistic OX40 antibody. Suitable OX4OL antagonists include, for example, RG-7888 (WO06 / 029879).

[0153] In another aspect, the immuno-oncology agent is a CD40 agonist, e.g., an agonist CD40 antibody. In yet another embodiment, the immuno-oncology agent is a CD40 antagonist, e.g., an antagonistic CD40 antibody. Suitable CD40 antibodies include, for example, lucatumumab or dacetuzumab.

[0154] In another embodiment, the immuno-oncology agent is a CD27 agonist, e.g., an agonist CD27 antibody. Suitable CD27 antibodies include, for example, varlilumab.

[0155] In another embodiment, the immuno-oncology agent is MGA271 (to B7H3) (WO11 / 109400).

[0156] dosage The disclosed agents that target the extracellular production of adenosine and / or antagonize the activation of one of its receptors by adenosine can be administered to a subject in an amount that depends, for example, on the goal of administration (e.g., the desired degree of resolution); the age, weight, sex, and health and physical condition of the subject to whom the formulation is administered; the route of administration; and the nature of the disease, disorder, condition, or symptoms thereof. The dosing regimen can also take into account the existence, nature, and extent of any adverse effects associated with the administered agent. Effective amounts and dosing regimens can be readily determined, for example, from safety and dose escalation studies, in vivo studies (e.g., animal models), and other methods known to those skilled in the art.

[0157] In general, dosing parameters should dictate that the dosage is below an amount that may be irreversibly toxic to the subject (maximum tolerated dose (MTD)) and at least the amount required to produce a measurable effect in the subject, such amounts being determined, for example, by ADME-relevant pharmacokinetic and pharmacodynamic parameters, taking into account the route of administration and other factors.

[0158] An effective dose (ED) is a dose or amount of a drug that produces a therapeutic response or desired effect in a proportion of subjects who receive it. The "median effective dose" or ED50 of a drug is a dose or amount of a drug that produces a therapeutic response or desired effect in 50% of the population to which it is administered. Although ED50 is commonly used as a measure of the reasonable expectation of a drug's effect, it may not necessarily be the dose that a clinician considers appropriate, taking all relevant factors into account. Thus, in some situations, the effective amount is higher than the calculated ED50, in other situations, the effective amount is less than the calculated ED50, and in still other situations, the effective amount is the same as the calculated ED50.

[0159] Furthermore, an effective dose of an agent targeting a therapeutic agent described herein can be an amount that, when administered in one or more doses to a subject, produces a desired result compared to a healthy subject. For example, for a subject experiencing a particular disorder, an effective dose can be one that improves a diagnostic parameter, measure, marker, etc. of that disorder by at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or more than 90%, where 100% is defined as the diagnostic parameter, measure, marker, etc. exhibited by a normal subject.

[0160] In certain embodiments, the therapeutic agents described herein can be administered (e.g., orally) at dosage levels of about 0.01 mg to about 50 mg per kg of subject body weight per day, or about 1 mg to about 25 mg per kg of subject body weight per day, one or more times per day to achieve the desired therapeutic effect.

[0161] For oral administration, the compositions can be provided in the form of tablets, capsules, and the like containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of active ingredient.

[0162] In addition to oral dosing, suitable routes of administration for certain agents described herein include parenteral (e.g., intramuscular, intravenous, subcutaneous (e.g., injection or implant), intraperitoneal, intracisternal, intraarticular, intraperitoneal, intracerebral (intraparenchymal), and intraventricular) and intraocular. Depot injections, typically administered subcutaneously or intramuscularly, can also be utilized to release the agents described herein for a defined period of time.

[0163] In certain embodiments, a dosage of a desired agent, a therapeutic agent described herein, is contained in a "unit dosage form." The phrase "unit dosage form" refers to physically discrete units, each unit containing a predetermined amount of a therapeutic agent described herein, alone or in combination with one or more additional agents, sufficient to produce a desired effect. It is understood that the parameters of a unit dosage form depend on the particular agent and the effect to be achieved.

[0164] Kits and detection methods The present disclosure also contemplates kits comprising the therapeutic agents and pharmaceutical compositions thereof described herein, generally in the form of a physical structure housing various components, as described below, that can be utilized, for example, in practicing the methods described above.

[0165] The kit can include one or more compounds disclosed herein, which may be in the form of a pharmaceutical composition suitable for administration to a subject (e.g., provided in a sterile container). The compounds described herein can be provided in a ready-to-use form (e.g., a tablet or capsule) or in a form that requires reconstitution or dilution, e.g., before administration (e.g., a powder). If the compounds described herein are in a form that requires reconstitution or dilution by the user, the kit can also include a diluent (e.g., sterile water), a buffer, a pharmaceutically acceptable excipient, etc., packaged together with or separately from the compounds described herein. If combination therapy is intended, the kit can contain several agents separately, or they may already be combined in the kit. Each component of the kit can be enclosed in an individual container, or all of the various containers can be in a single package. The kits of the present invention can be designed for the conditions (e.g., refrigeration or freezing) necessary to properly maintain the components contained therein.

[0166] The kit can contain a label or package insert containing identifying information and instructions for use for the components therein (e.g., clinical pharmacology of the active ingredients, including dosing parameters, mechanism of action, pharmacokinetics and pharmacodynamics, adverse effects, contraindications, etc.). The label or package insert can include manufacturer information, e.g., lot number and expiration date. The label or package insert can, for example, be incorporated into the physical structure that houses the component, be included separately in the physical structure, or be affixed to a component of the kit (e.g., an ampoule, tube, or vial).

[0167] The label or package insert may further comprise or be incorporated into a computer readable medium, such as a disk (e.g., hard disk, card, memory disk), optical disk, such as CD- or DVD-ROM / RAM, DVD, MP3, magnetic tape, or electronic storage medium, such as RAM and ROM, or a hybrid thereof, such as magnetic / optical storage medium, FLASH® media, or memory card. In some embodiments, the actual instructions are not present in the kit, but means are provided for obtaining the instructions from a remote source, for example, via the internet.

[0168] Kits and methods for detecting soluble CD73. In certain aspects, kits for determining soluble CD73 (sCD73) in a sample are also provided herein. The kits include an sCD73 capture antibody and a labeled sCD73 detection antibody. In some embodiments, the capture antibody is 7G2, Thermo Scientific #41-0200. In some embodiments, the labeled sCD73 detection antibody is AD2 clone, BioLegend #344017.

[0169] In some embodiments, the labeled sCD73 detection antibody is a biotinylated antibody, and the kit further comprises streptavidin-horseradish peroxidase and a horseradish peroxidase substrate detectable after enzymatic conversion with horseradish peroxidase. In some embodiments, the horseradish peroxidase substrate is 3,3',5,5'-tetramethylbenzidine (TMB) or 2,2'-azino-di-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS).

[0170] In some embodiments, the kit further comprises one or more of a coating buffer, a wash buffer, and a blocking buffer. In some embodiments, the kit further comprises a blocking buffer. In some embodiments, the kit further comprises a coating buffer. In some embodiments, the kit further comprises a wash buffer. In some embodiments, the coating buffer comprises about 0.2 M NaHCO3 buffer, pH 9.6. In some embodiments, the wash buffer comprises about 0.1% Tween 20 in PBS. In some embodiments, the blocking buffer comprises about 0.02% Tween 20 + 1% bovine serum albumin + 10 μg / ml bovine IgG + 10 μg / ml mouse IgG in PBS.

[0171] In a further aspect, also provided herein is a method for measuring the amount of soluble CD73 (sCD73) in a sample. The method comprises: a) contacting the sample with an immobilized anti-CD73 antibody to form captured sCD73; b) contacting the captured sCD73 with a labeled anti-CD73 antibody to form a sandwich of sCD73, wherein the labeled anti-CD73 antibody binds to a portion of sCD73 that is distinct from the portion bound by the immobilized anti-CD73 antibody; c) contacting the sandwiched sCD73 with an imaging solution to produce a detectable signal; d) measuring the detectable signal Includes:

[0172] In some embodiments, the capture antibody is 7G2, Thermo Scientific #41-0200. In some embodiments, the labeled sCD73 detection antibody is AD2 clone, BioLegend #344017.

[0173] Step a) involves an incubation period for the formation of a capture sCD73 complex between the immobilized anti-CD73 antibody and sCH73 in the sample. The incubation period can range from a few minutes to about 1, 2, 3, 4, 5, 6, 7, or 8 hours or more, or overnight. Longer periods are also permissible. Like step a), step b) also involves an incubation period that can range from a few minutes to about 1, 2, 3, 4, 5, 6, 7, or 8 hours or more, or overnight. In some embodiments, the incubation period for step b) is about 1 hour. Like steps a) and b), step c) also involves an incubation period that can range from a few minutes to about 1, 2, 3, 4, 5, 6, 7, or 8 hours or more, or overnight. In some embodiments, the incubation period for step c) is about 1 hour.

[0174] In some embodiments, the method further comprises one or more washing steps with a washing buffer after steps a), b), and c). The washing typically occurs after a desired incubation time. In some embodiments, the washing buffer comprises about 0.1% Tween 20 in PBS.

[0175] In some embodiments, the method further comprises a blocking buffer. In some embodiments, the blocking buffer is included in the sample prior to step a). In some embodiments, the blocking buffer is also included with the labeled anti-CD73 antibody in step b). In some embodiments, the blocking buffer is also included with the imaging solution in step c). In some embodiments, the blocking buffer comprises about 0.02% Tween 20 + 1% bovine serum albumin + 10 μg / ml bovine IgG + 10 μg / ml mouse IgG in PBS.

[0176] Many imaging solutions are useful in ELISA assays. The identity of the label on the labeled anti-CD73 antibody determines the appropriate imaging solution. Typically, the imaging solution contains a fluorescent or chemiluminescent reagent or substrate. For example, if the label on the labeled anti-CD73 antibody is biotin, the imaging solution generally contains avidin or streptavidin conjugated to a fluorophore or to an additional agent capable of binding to form a detectable signal or enzymatically converting a substrate into a detectable signal. In some embodiments, the avidin or biotin is conjugated to horseradish peroxidase. Suitable substrates for horseradish peroxidase (HRP), which form a signal upon conversion by HRP, include 3,3',5,5'-tetramethylbenzidine (TMB) or 2,2'-azino-di-[3-ethylbenzothiazoline-6-sulfonic acid] (ABTS).

[0177] In some embodiments, the sample is blood. In some embodiments, the sample is serum isolated from a blood sample. In some embodiments, the sample is plasma isolated from a blood sample.

[0178] Those skilled in the art will recognize that levels of different adenosine machinery proteins can be measured using similar sandwich ELISA methods.

[0179] Kits and methods for detecting soluble adenosine monophosphate (AMP) hydrolysis activity. In a further aspect, kits for determining CD73-mediated and / or TNAP-mediated adenosine monophosphate (AMP) hydrolysis activity in a sample are also provided herein. In some embodiments, the kit for determining CD73-mediated adenosine monophosphate (AMP) hydrolysis activity in a sample comprises a CD73 inhibitor and AMP-Glo™. In some embodiments, the kit for determining TNAP-mediated adenosine monophosphate (AMP) hydrolysis activity in a sample comprises a TNAP inhibitor and AMP-Glo™.

[0180] In some embodiments, the kits described herein further comprise an adenosine deaminase inhibitor, an SAH dehydrolase inhibitor, and an ADK inhibitor. These inhibitors can help reduce background adenosine degradation in the sample. In some embodiments, the adenosine deaminase inhibitor is EHNA. In some embodiments, the SAH dehydrolase inhibitor is aristromycin. In some embodiments, the ADK inhibitor is iodotubercidin.

[0181] In some embodiments, the kit further comprises adenosine monophosphate (AMP).

[0182] In a further aspect, also provided herein is a method for determining CD73-mediated adenosine monophosphate (AMP) hydrolysis activity in a sample, the method comprising: a) contacting the sample with a CD73 inhibitor, adenosine monophosphate (AMP), and AMP-Glo™ to form a CD73i sample; b) contacting a separate sample aliquot with adenosine monophosphate (AMP) and AMP-Glo™ to form a baseline sample; c) measuring the final RLU signal after a specific time for the CD73i sample and the baseline sample; and d) assessing the difference between the final RLU of the CD73i sample and the final RLU of the baseline sample to determine the CD73-mediated AMP hydrolysis activity in the sample; Includes:

[0183] In a further aspect, also provided herein is a method for determining TNAP-mediated adenosine monophosphate (AMP) hydrolysis activity in a sample, the method comprising: a) contacting a sample with a TNAP inhibitor, adenosine monophosphate (AMP), and AMP-Glo™ to form a TNAPi sample; b) contacting a separate sample aliquot with adenosine monophosphate (AMP) and AMP-Glo™ to form a baseline sample; c) measuring the final RLU signal after a specific time for the TNAPi sample and the baseline sample; and d) assessing the difference between the final RLU of the TNAPi sample and the final RLU of the baseline sample to determine the TNAP-mediated AMP hydrolysis activity in the sample; Includes:

[0184] In some embodiments, the CD73i sample, the TNAPi sample, and the baseline sample further comprise one or more of an adenosine deaminase inhibitor, an aristromycin dehydrolase inhibitor, and an ADK inhibitor. In some embodiments, the CD73i sample and / or the TNAPi sample further comprise an adenosine deaminase inhibitor, an SAH dehydrolase inhibitor, and an ADK inhibitor. In some embodiments, the adenosine deaminase inhibitor is EHNA. In some embodiments, the SAH dehydrolase inhibitor is aristromycin. In some embodiments, the ADK inhibitor is iodotubercidin.

[0185] The specific time for measuring the final RLU signal depends on several factors. Chief among these is the amount of AMP included with the sample (steps a and b), since RLU is a measure of the AMP remaining after a specific time has passed. When using 25 μM AMP, a reaction time in minutes is usually sufficient to observe a measurable change in signal. In some embodiments, the specific time is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more minutes. In some embodiments, the specific time is 8 minutes.

[0186] In some embodiments, the sample is blood, in some embodiments, the sample is serum, in some embodiments, the sample is plasma.

[0187] IV. Working Examples The following examples are presented so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention; they are not intended to represent that the experiments described below have been performed or are all that may be performed. It should be understood that exemplary descriptions written in the present tense have not necessarily been performed; rather, the descriptions may be performed to produce data of the nature described therein, etc. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for.

[0188] [Example 1] Determination of the amount of soluble CD73 in human blood via sandwich ELISA The method for determining the amount of soluble CD73 in human blood via sandwich ELISA involves coating the wells of a microtiter plate with a capture antibody, a sample containing the analyte of interest, a wash buffer, and a secondary antibody conjugated to biotin, which is used to detect the bound target. Secondary detection of the biotinylated antibody is performed using HRP-streptavidin followed by a chromogenic or chemiluminescent substrate. The principle of the CD73 ELISA assay is outlined in Figure 1.

[0189] Materials and Methods Reagents and equipment - sCD73 capture antibody (7G2, Thermo Scientific #41-0200) - Coated buffer: - 0.2M NaHCO3 buffer, pH 9.6 (Thermo Scientific #28382) - Washing buffer: - 0.1% Tween 20 in PBS - Block buffer: - 0.02% Tween 20 + 1% bovine serum albumin + 10 μg / ml bovine IgG (Jackson ImmunoResearch #001-000-003) + 10 μg / ml mouse IgG (Jackson ImmunoResearch #015-000-003) in PBS - CD73 protein standard - Biotinylated sCD73 detection antibody (AD2 clone, BioLegend #344017) - Streptavidin-horseradish peroxidase (Thermo Scientific #21130) - Chemiluminescent ELISA Reagent (Thermo Scientific #37069) - 96-well microtiter plate, flat bottom, white, high-binding polystyrene (Costar #3922) - Self-adhesive foil seals for plates - EnVision plate reader

[0190] protocol Plate coating: 1. Coat the wells of a microtiter plate with anti-sCD73 mAb (2 μg / ml, 100 μl) overnight at 4° C. Seal the plate for incubation. 2. Wash the wells four times with 0.1% Tween 20 in PBS (Tween / PBS, 350 μl each wash). 3. Block with blocking buffer (200 μl) for at least 4 hours at room temperature. 4. Wash the wells four times. - Use the plate immediately or store at 4°C with wash buffer in the wells and tightly sealed.

[0191] ELISA assay: 1. Dilute each sample in blocking solution (or make a dilution series for samples and standards), then add each sample (100 μl) to a well. 2. Seal the plate and incubate at 4°C overnight. 3. Wash the wells four times. 4. Add biotinylated anti-CD73 mAb (0.5 μg / ml, 100 μl in blocking solution). 5. Seal the plate and incubate at room temperature for 1 hour. 6. Wash the wells four times. 7. Add streptavidin-horseradish peroxidase (100 μl, diluted 1:10000 in blocking solution) to the wells. 8. Incubate again at room temperature for 1 hour. 9. Wash the wells four times. 10. Develop chemiluminescent ELISA reagent according to manufacturer's instructions. a. Mix equal parts luminol / enhancer and stable peroxide solution (solution is stable for 24 hours at room temperature - store in the dark). b. Add 100 μl of working solution to each well. c. Spin the plate at 1000 xg for 2 minutes to remove all foam. d. Measure the intensity of the chemiluminescent reaction in the wells with a luminometer (optimal range is 1-5 minutes).

[0192] Data analysis Raw luminescence data from the EnVision plate reader are interpolated against a standard curve to determine absolute sCD73 levels. The concentration of each undiluted sample is calculated from all wells in the dilution series, and these values ​​are compared to each other to assess the linearity of the dilutions.

[0193] Using the assay described above, healthy donor serum and sodium heparin plasma were evaluated to measure soluble CD73. As shown in Figure 2A, measurements were highly correlated. Parallel evaluation of samples was performed to determine the assay's quantitative range. Figure 2B demonstrates that the assay exhibits acceptable assay parallelism. Figure 2C shows a comparison of serum sCD73 between healthy subjects and subjects with cancer. CD73 levels were generally higher in cancer patients than in healthy donors.

[0194] [Example 2] Determination of the amount of soluble CD73 and / or TNAP in human blood via the AMP-Glo ​​hydrolysis assay The assay described herein allows for the determination of the amount of AMP hydrolysis activity by CD73 and / or TNAP in a blood sample. This determination is performed using the AMP-Glo™ assay in the presence of 25 μM AMP, a CD73 inhibitor, and / or a TNAP inhibitor. AMP-Glo™ is a homogeneous biochemical assay that generates a luminescent signal resulting from a biochemical reaction that produces AMP. The principle of the AMP Glo assay is outlined in Figure 3A.

[0195] Materials and Methods reagent - CD73 inhibitor (Compound A) - AMP-Glo™ (Catalog No. V5011, Promega Corporation) - PBS - Phosphate Buffered Saline (Cat. No. 10010023, Gibco) - TNAP inhibitor cocktail: - 625 μM TNAP inhibitor (CAS 496014-13-2, Calbiochem) 10 μM EHNA, an adenosine deaminase inhibitor (Cat. No. E114-25 mg, Sigma) - 4 μM aristromycin, a SAH dehydrolase inhibitor (Cat. No. SC-233890, Chem Cruz) 10 μM 5-iodotubercidin, an ADK inhibitor (Cat. No. I100-5MG, Sigma)

[0196] Materials and Equipment - 96-well assay plate (Corning Catalog No. 3992) - EnVision Leader

[0197] Determination of AMP hydrolytic activity in human serum Serum isolated from whole blood of healthy volunteers and cancer patients was stored at -80°C. 50 μl of serum from each donor was transferred into 4 × 500 μl aliquots. Samples were then labeled a–d for the following conditions: a) no Compound A, no TNAP inhibitor cocktail; b) 10 μM Compound A, no TNAP inhibitor cocktail; c) no Compound A, with TNAP inhibitor cocktail; and d) with 10 μM Compound A, with TNAP inhibitor cocktail. Conditions c and d received 1:200 TNAP inhibitor cocktail (0.25 μL), while conditions b and d (with 10 μM Compound A) received 1:100 Compound A (1 mM solution, final concentration 10 μM). The samples are then mixed and incubated at 37°C for 1 hour, after which 18 μL of sample per well is transferred to a low-volume 96-well AMP Glo™ assay plate containing 2 μL of AMP at a final concentration of 25 μM. Using a multichannel pipette, the samples are mixed 6–10 times and incubated at room temperature for 8 minutes, after which the reaction is stopped with 20 μL of the R1 solution provided by the kit. The 8-minute time point was chosen after a series of experiments to determine the time point after AMP spike-in that provided the best window for assays without adding CD73 inhibitors to human serum. Once the samples are mixed with R1, 40 μL of pre-prepared R2 solution + AMP Glo™ reagent is added to the wells. The final reaction mixture is incubated for 30 minutes and then read on a Wallac EnVision reader to measure luminescence.

[0198] Analysis of AMP Glo assay data Data from the AMP Glo assay can be analyzed in several ways. Raw data is in the form of Raw Luminescence Units (RLU) obtained from the Envision Reader. Data can be expressed as RLU values, percent AMP remaining (AMP remaining at the end of the 8 minute reaction time compared to 0 minutes), and hydrolytic activity (comparison of RLU in test samples with wells containing the highest amount of CD73 inhibitor in the presence and absence of TNAP inhibitor).

[0199] Using the above assay, serum from healthy volunteers was tested in the presence of a CD73 inhibitor and / or a TNAP inhibitor. The measured luminescence (RLU) values ​​are shown in Figure 3B. The correlation between the percent CD73 hydrolysis activity and CD73 protein concentration in healthy and cancer-bearing subjects is shown in Figure 3C.

[0200] [Example 3] Determination of the amount of CD73 and / or TNAP using immunostaining Ecto-5'-nucleotidase (CD73) and tissue-nonspecific alkaline phosphatase (TNAP) proteins were detected in formalin-fixed, paraffin-embedded (FFPE) tissues after antigen retrieval using Cell Signaling Technology's anti-NT5E / CD73 D7F9A antibody clone and Sino Biological's anti-alkaline phosphatase / ALPL R034 antibody clone. Detection of single-antibody staining was performed using anti-rabbit IgG conjugated to horseradish peroxidase (HRP) and 3,3'-diaminobenzidine (DAB) chromogen precipitation. Simultaneous detection of both CD73 and TNAP was performed in a multiplex fluorescent assay using the above antibody clones, and HRP was used to precipitate the fluorescent chromogen. The staining-positive area, percentage of staining-positive cells, H score, combined positive score (CPS), and tumor proportion score (TPS) were calculated using an image analysis program. For singleplex chromogen staining, the QuPath Quantitative Pathology & Bioimage Analysis program was used. For multiplex fluorescence, Indica Labs' HALO software was used. Figures 5A-D and 6A-D show representative images of CD73 and TNAP immunostaining, respectively. Figures 5E and 6E plot the quantification of CD73 and TNAP stained areas as a percentage of the total tumor area. Figure 5F plots the correlation between the percent stained area and H-score.

[0201] [Example 4] Determination of the amount of CD73, TNAP, or other adenosine machinery mRNA RNA extraction from formalin-fixed, paraffin-embedded (FFPE) tissue is performed using the Qiagen RNeasy FFPE kit (#73504). Tissue sections are scraped from microscope slides using a scalpel, or sections are placed directly into microcentrifuge tubes. NanoString analysis is performed on the extracted RNA using the nCounter Oncology or Immunology panel on the nCounter SPRINT profiler, followed by analysis using the nSolver software package. Real-time PCR is performed on cDNA generated from the extracted FFPE RNA using Taqman probes on an Applied Biosystems QuantStudio 6 Flex Real-time PCR system.

[0202] [Example 5] Determining the tumor mutation burden (TMB) in subjects The method for determining tumor mutational burden (TMB) in a subject described herein is published in Goodman et al. Tumor Mutational Burden as an Independent Predictor of Response to Immunotherapy in Diverse Cancers. Mol Cancer Ther. (2017). 16(11):2598-2608.

[0203] [Example 6] Determination of subject's PD-L1 status Many methods are known for determining a subject's PD-L1 status. One useful method is through use of the FDA-approved PD-L1 IHC 22C3 pharmDx instrument and method developed by Dako North America, Inc.

[0204] [Example 7] Determination of CD73 and / or TNAP levels using an isotopic AMP hydrolysis assay Assay design The activity of CD73 in human plasma was 13 From C5-AMP 13 The in vitro assay was performed using an LC-MS / MS method that monitored dephosphorylation to C5-adenosine. TNAP was blocked using an inhibitor cocktail consisting of 2,5-dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (TNAP inhibitor), erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, adenosine deaminase inhibitor), 5-iodotubercidin (adenosine kinase inhibitor), and aristeromycin (S-adenosyl-L-homocysteine ​​hydrolase inhibitor). 13 C5-adenosine was stabilized.

[0205] IC by nonlinear regression analysis 50 The value was estimated.

[0206] Assay conditions Human plasma (50 μL) at pH 7.4 was preincubated for 1 hour with the CD73 inhibitor (Compound A) (0 to 10 μM), as well as EHNA (10 μM), dimethoxy-N-(quinolin-3-yl)benzenesulfonamide (625 μM), 5-iodotubercidin (10 μM), and aristeromycin (4 μM). 13 The reaction was initiated with C5-AMP and allowed to proceed for 1 minute at 37°C in a shaking water bath. The reaction was stopped by adding 4 volumes of 0.4 M perchloric acid containing an internal standard (cIMP, 5 ng / mL). The sample was vortexed for 15 minutes and then centrifuged at 4200 rpm for 20 minutes at 10°C. The supernatant was analyzed by LC-MS / MS as described in the Analytical Methods section.

[0207] Analytical methods and data analysis The mass spectrometer was acquired and integrated with Applied Biosystems-Sciex Analyst software (version 1.6.3).

[0208] device: - API 4000 mass spectrometer (Applied Biosystems, Foster City, CA) - API 6500 mass spectrometer (Applied Biosystems, Foster City, CA) - Shimadzu Nexera X2 UHPLC System (Shimadzu Scientific Instruments, Canby, OR) Column: Atlantis dC18, 100 Å, 3.0 × 100 mm, 3 μm (Waters, Milford, MA) Injection volume: 0.5μL Flow rate: 0.80mL / min HPLC gradient:

[0209] TIFF0007743309000014.tif56162Ionization mode: Electrospray (ESI) Detection mode: Positive MRM (Q1 / Q3 transition: 13 m / z 273.14 / 136.10 for C5-adenosine and m / z 330.98 / 137.10 for cIMP)

[0210] Analysis of samples without a CD73 inhibitor provides information about CD73 activity in the sample. Performing the same assay with a CD73 inhibitor and / or a TNAP inhibitor allows assessment of the relative contributions of CD73- and TNAP-mediated dephosphorylation of AMPs.

[0211] Using titrations of CD73 inhibitors, IC was calculated using the algorithm included in GraphPad Prism Version 5.0 (GraphPad Software Inc., San Diego, CA) by fitting the data to the following four-parameter equation (with variable slope):50 The value was estimated.

[0212]

number

[0213] Representative ICs from two volunteers 50 The values ​​are shown in Table 1 below and in Figures 7A-C.

[0214] [Table 1]

[0215] Certain embodiments of the invention are described herein, including the best mode for carrying out the invention known to the inventors. It is expected that variations on the disclosed embodiments may become apparent to those skilled in the art upon reading the foregoing description, and that such variations may be employed by those skilled in the art as appropriate. Accordingly, it is intended that the invention be practiced otherwise than as specifically described herein, and that the invention include all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0216] All publications, patent applications, accession numbers, and other references cited herein are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In certain embodiments, for example, the following items are provided: (Item 1) 1. A method of treating cancer in a subject with an established adenosine fingerprint, comprising administering to the subject a therapeutic agent selected from the group consisting of an adenosine A2a receptor (A2aR) and / or an adenosine A2b receptor (A2bR) antagonist and a CD73 inhibitor; The cancer in the subject is (i) an increase in the concentration of soluble CD73 in blood obtained from the subject compared to the typical concentration of CD73 in blood obtained from a subject with the same type of cancer; (ii) an increase in the activity of CD73 in blood obtained from the subject compared to the typical AMP hydrolysis activity of CD73 in blood obtained from subjects with the same type of cancer, as determined by an AMP hydrolysis assay; (iii) a biopsy of the subject's cancer that shows an increased amount of CD73 compared to the typical amount of CD73 in a biopsy obtained from a subject with the same type of cancer, as determined by CD73 immunostaining; and (iv) a biopsy of the subject's cancer showing upregulation of CD73 as determined by mRNA levels, compared to the typical amount of CD73 in a biopsy obtained from a subject with the same type of cancer; and wherein the subject is administered a CD73 inhibitor if the subject has at least one of the characteristics selected from the group consisting of: The cancer in the subject is (a) an increase in the concentration of TNAP in blood obtained from the subject compared to the typical concentration of TNAP in blood obtained from a subject with the same type of cancer; (b) an increase in the activity of TNAP in blood obtained from the subject compared to the typical AMP hydrolysis activity of TNAP in blood obtained from subjects with the same type of cancer, as determined by an AMP hydrolysis assay; (c) a biopsy of the subject's cancer showing an increased amount of TNAP compared to the typical amount of TNAP in a biopsy obtained from a subject with the same type of cancer, as determined by TNAP immunostaining; and (d) a biopsy of a subject's cancer showing upregulation of TNAP as determined by mRNA levels, compared to the typical amount of TNAP in a biopsy obtained from a subject with the same type of cancer; adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist is administered to a subject when the subject has at least one of the characteristics selected from the group consisting of: method. (Item 2) the subject is administered a CD73 inhibitor when the cancer in the subject has at least two of the characteristics selected from the group consisting of (i) to (iv); If the cancer in the subject has at least two characteristics selected from the group consisting of (a) to (d), the subject is administered an adenosine A2a receptor (A2aR) or an adenosine A2b receptor (A2bR) antagonist; The method according to item 1. (Item 3) the subject is administered a CD73 inhibitor when the cancer in the subject has at least three characteristics selected from the group consisting of (i) to (iv); If the cancer in the subject has at least three characteristics selected from the group consisting of (a) to (d), the subject is administered an adenosine A2a receptor (A2aR) or an adenosine A2b receptor (A2bR) antagonist; The method according to item 1. (Item 4) and wherein the subject is administered a CD73 inhibitor when the cancer in the subject has four of the characteristics selected from the group consisting of (i) to (iv); the subject is administered an adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist if the cancer in the subject has four characteristics selected from the group consisting of (a) to (d); The method according to item 1. (Item 5) 5. The method of any one of items 1 to 4, wherein only an adenosine A2a receptor (A2aR) or adenosine A2b receptor (A2bR) antagonist is administered if the cancer in the subject exhibits at least one, two, three, or four of the characteristics selected from each of (i) to (iv) and (a) to (d). (Item 6) 5. The method of any one of items 1 to 4, wherein both an adenosine A2a receptor (A2aR) and / or adenosine A2b receptor (A2bR) antagonist and a CD73 inhibitor are administered when the cancer in the subject exhibits at least one, two, three, or four of the characteristics selected from each of (i) to (iv) and (a) to (d). (Item 7) 7. The method of any one of items 1 to 6, wherein the increase in the concentration of soluble CD73 in blood obtained from the subject exceeds a threshold of 1 ng / mL. (Item 8) 8. The method of item 7, wherein the concentration of soluble CD73 in blood obtained from the subject exceeds a threshold of 3 ng / mL. (Item 9) 9. The method of any one of items 1 to 8, wherein the increase in CD73 and / or TNAP activity in blood obtained from the subject is determined by an AMP-Glo ​​hydrolysis assay and exceeds a threshold value, wherein the threshold value for CD73 and / or TNAP-mediated AMP hydrolysis is at least 10% of the total AMP hydrolysis activity in the subject's blood. (Item 10) 10. The method of claim 9, wherein the threshold for CD73- and / or TNAP-mediated AMP hydrolysis is at least 20% of the total AMP hydrolysis activity in the subject's blood. (Item 11) 10. The method of any one of items 1 to 9, wherein the increased amount of CD73 and / or TNAP as determined by immunostaining is a measurement that exceeds the average amount of CD73 and / or TNAP in biopsies obtained from subjects with the same type of cancer. (Item 12) 10. The method of any one of items 1 to 9, wherein the increase in the amount of CD73 and / or TNAP determined by immunostaining exceeds a threshold value, said threshold value being at least 7% of the CD73 and / or TNAP stained area. (Item 13) 13. The method of any one of items 1 to 12, wherein the upregulation of CD73 and / or TNAP in a biopsy obtained from the subject, as determined by mRNA levels, measures greater than the average amount of CD73 and / or TNAP mRNA in biopsies obtained from subjects with the same type of cancer. (Item 14) 14. The method of any one of items 1 to 13, wherein the increase in CD73 and / or TNAP activity in blood obtained from the subject is determined by an isotopic AMP hydrolysis assay and exceeds a threshold value, wherein the threshold value for CD73 and / or TNAP-mediated AMP hydrolysis is at least 10% of the total AMP hydrolysis activity in the subject's blood. (Item 15) 15. The method of claim 14, wherein the threshold for CD73- and / or TNAP-mediated AMP hydrolysis is at least 20% of the total AMP hydrolysis activity in the subject's blood. (Item 16) 16. The method of any one of items 1 to 15, wherein the concentration of soluble CD73 or TNAP in blood obtained from the subject is determined by sandwich enzyme-linked immunosorbent assay (ELISA). (Item 17) 17. The method of any one of items 1 to 16, wherein the blood used to determine the soluble CD73 concentration in blood, the TNAP concentration in blood, the CD73-mediated adenosine monophosphate (AMP) hydrolysis activity in blood, and / or the TNAP-mediated AMP hydrolysis activity in blood is plasma or serum. (Item 18) 5. The adenosine A2a receptor (A2aR) and / or adenosine A2b receptor (A2bR) antagonist of formula (I):

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Claims

1. 1. A composition comprising a CD73 inhibitor for use in treating cancer in a human subject, wherein the subject is (i) an increase in the concentration of soluble CD73 in blood obtained from the subject, wherein the concentration of soluble CD73 in blood obtained from the subject exceeds a threshold of 3 ng / mL; and (ii) a biopsy of the subject's cancer that shows an increased amount of CD73 compared to the typical amount of CD73 in a biopsy obtained from a subject with the same cancer, as determined by CD73 immunostaining; and having at least one of the characteristics selected from the group consisting of: the CD73 inhibitor 【Chemistry 101】 or a pharmaceutically acceptable salt thereof, wherein the cancer is cancer of the prostate, colon, rectum, pancreas, cervix, stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck, skin, mesothelial lining, esophagus, breast, muscle, connective tissue, lung, adrenal gland, thyroid, kidney, or bone, or is glioblastoma, mesothelioma, renal cell carcinoma, gastric cancer, sarcoma, choriocarcinoma, basal cell carcinoma of the skin, or testicular seminoma.

2. 2. The composition of claim 1, further comprising an adenosine A2a receptor (A2aR) antagonist, an adenosine A2b receptor (A2bR) antagonist, or an A2aR and A2bR antagonist.

3. 3. The composition of claim 1, wherein the concentration of soluble CD73 in the blood obtained from the subject exceeds a threshold value of 8 ng / mL.

4. 4. The composition of claim 1, wherein the amount of CD73 determined by immunostaining is a measurement that exceeds the average amount of CD73 in biopsies obtained from subjects with the same cancer, or wherein the amount of CD73 determined by immunostaining exceeds a threshold value, the threshold being at least 7% of the CD73-stained area.

5. The A2aR antagonist, the A2bR antagonist, or the A2aR and A2bR antagonist has formula (I): 【Chemical 1】 or a pharmaceutically acceptable salt, hydrate or solvate thereof, During the ceremony, G 1 is N or CR 3a and G 2 is N or CR 3b and G 3 is N or CR 3c and R 3a , R 3b and R 3c each independently represents H or C 1～3 is alkyl, R 1a and R 1b teeth, i) H ii) 1 to 3 R 5 C optionally substituted with substituents 1～8 Alkyl, iii) 1 to 3 R 5 -X optionally substituted with a substituent 1 -OC 1～8 Alkyl, iv)-C(O)-R 6 、 v) 1 to 3 R's 7 Y optionally substituted with a substituent, and vi) 1 to 3 R 7 -X optionally substituted with a substituent 1 -Y, or vii)R 1a and R 1b are one to three R together with the nitrogen to which they are attached. 8 forming a 5-6 membered heterocycloalkyl ring optionally substituted with substituents, wherein the heterocycloalkyl has 0-2 additional heteroatom ring vertices selected from the group consisting of O, N and S; Each Y is C 3～8 cycloalkyl or a 4- to 6-membered heterocycloalkyl having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S; R 2 and R 4 are each independently H or C 1～3 is alkyl, Ar 1 are phenyl or 5- to 6-membered heteroaryl, each of which is 1 to 3 R 9 , where necessary, Ar 2 are phenyl or 5- to 6-membered heteroaryl, each of which is 1 to 3 R 10 , where necessary, Ar 1 and Ar 2 wherein the 5- to 6-membered heteroaryl has 1 to 3 heteroatom ring vertices each independently selected from the group consisting of O, N, and S; each X 1 is C 1～6 is alkylene, Each R 5 is hydroxyl, C 3～8 Cycloalkyl, phenyl, -O-phenyl, -C(O)OR a and oxo; Each R 6 is C 1～8 alkyl or Y, which are hydroxyl, -O-phenyl, phenyl and -OC 1～8 optionally substituted with 1 to 3 substituents selected from the group consisting of alkyl; Each R 7 is C 1～8 Alkyl, hydroxyl, -OC 1～8 Alkyl, oxo and C(O)OR a are independently selected from the group consisting of Each R 8 is C 1～8 independently selected from the group consisting of alkyl, hydroxyl, and oxo; Each R 9 is C 1～8 Alkyl, -OC 1～8 Alkyl, -X 1 -OC 1～8 Alkyl, -OX 1 -OC 1～8 Alkyl, -X 1 -OX 1 -OC 1～8 Alkyl, -C(O)OR a , halogen, cyano, -NR b R c , Y, -X 1 -C 3～8 Cycloalkyl and -X 2 -Z; 2 is C 1～6 Alkylene, -C 1～6 Alkylene -O-, -C(O)- and -S(O) 2 -, Z is a 4- to 6-membered heterocycloalkyl ring having 1 to 3 heteroatom ring vertices selected from the group consisting of O, N, and S, and said R 9 Each of the substituents is one to three R 11 , where necessary, Each R 10 is C 1～8 Alkyl, halo, cyano, -OC 1～8 Alkyl, -X 1 -OC 1～8 Alkyl, -OX 1 -OC 1～8 Alkyl, -S(O) 2 -C 1～6 Alkyl, -C(O)NR d R e and 4-6 membered heteroaryl having 1-3 heteroatom ring vertices selected from the group consisting of O, N, and S; 10 Each of the substituents is one to three R 12 or Ar 2 Two R on adjacent ring vertices of 10 are optionally combined to form a 5-membered heterocycle optionally substituted with 1 to 2 halogens; Each R 11 -hydroxyl, halo, cyano, -NR d R e , -C(O)OR a , phenyl, C 3～8 Cycloalkyl and C(O)OR a C substituted as needed 1～4 independently selected from the group consisting of alkyl, Each R 12 is halo, cyano, hydroxy, and -C(O)OR a are independently selected from the group consisting of Each R a is H or C 1～6 is alkyl, Each R b and R c is H, C 1～8 Alkyl, -S(O) 2 -C 1～6 Alkyl, -C(O)OR a and -X 1 -C(O)OR a are independently selected from the group consisting of Each R d and R e is H, C 1～8 Alkyl, and -S(O) 2 -C 1～6 independently selected from the group consisting of alkyl, However, G 1 and G 2 are N and G 3 is CH and R 2 is CH 3 and R 1a and R 1b are each H, then Ar 2 is other than 2-thienyl, phenyl, 2-, 3- or 4-methoxyphenyl, 3- or 4-halophenyl, 2,4-dimethoxyphenyl, 2,4-dichlorophenyl or 2- or 4-methylphenyl; 3. The composition of claim 2.

6. The A2aR antagonist, the A2bR antagonist, or the A2aR and A2bR antagonist is Compound 1. 【Chemistry 2】 or a pharmaceutically acceptable salt thereof; Compound 2 【Chemistry 3】 or a pharmaceutically acceptable salt thereof; or Compound 3 【Chemistry 4】 or a pharmaceutically acceptable salt thereof.

7. 3. The composition of claim 2, wherein the A2aR antagonist, A2bR antagonist, or A2aR and A2bR antagonist is selected from the group consisting of AZD4635, ciforadenant (CPI-444), NIR178, and PBF-1129.

8. 8. The composition of claim 1, further comprising a PD1 inhibitor and / or a PD-L1 inhibitor.

9. 9. The composition of claim 8, wherein the PD1 inhibitor and / or PD-L1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, MEDI-0680, BGB-108, GB-226, PDR-001, mDX-400, SHR-1210, IBI-308, PF-06801591, atezolizumab, durvalumab, avelumab, BMS-936559, KD-033, CA-327, CA-170, ALN-PDL, TSR-042, and STI-1014.

10. 10. The composition of any one of claims 1 to 9, further comprising a chemotherapeutic agent, optionally a platinum-based or anthracycline-based chemotherapeutic agent.

11. 11. The composition of any one of claims 1 to 10, wherein the cancer is pancreatic cancer.

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