Biomarkers for methods of treating cancer

Abstract

Methods of using biomarker levels to improve treatment outcome by A2R antagonists in oncology are provided. Embodiments of the present disclosure also provide methods of treating a subject with a cancer having high expression of the disclosed biomarkers with an A2R antagonist as well as methods of improving standard treatment outcomes in patients having high expression levels of the biomarkers.

Description

BIOMARKERS FOR METHODS OF TREATING CANCER CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 693,600, filed September 11, 2024, and titled “BIOMARKERS FOR METHODS OF TREATING CANCER,” U.S. Provisional Patent Application No.63 / 716,049, filed November 4, 2024, and titled “BIOMARKERS FOR METHODS OF TREATING CANCER,” and U.S. Provisional Patent Application No. 63 / 862,534, filed August 12, 2025, and titled “BIOMARKERS FOR METHODS OF TREATING CANCER.” Each application is incorporated herein by reference in its entirety. FIELD

[0002] The present disclosure relates to improved methods of treating cancer based on biomarker expression in a subject. BACKGROUND

[0003] The following discussion is provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

[0004] The tumor microenvironment produces high levels of adenosine triphosphate (ATP) due in part to cell death caused by therapeutic agents (e.g., chemotherapy). ATP is subsequently converted to adenosine monophosphate (AMP) and then adenosine, in part through the enzymatic activity of CD39 and CD73, respectively. Once formed, adenosine can bind to adenosine receptors (A2AR and A2BR) on different cell types, including T cells, dendritic cells, tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), and fibroblasts, causing downregulation of, e.g., effector function, cytokine production, and T-cell stimulation, thereby resulting in adenosine-mediated immune suppression. This immune suppression can negatively impact the efficacy of cancer therapies. Accordingly, there is a need to develop effective treatment regimens that deliver meaningful clinical efficacy and means for identifying patients and predicting which patients will respond. SUMMARY

[0005] The instant disclosure provides methods of treating a human subject with a cancer having a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, 1 299641857and TOX1, said methods comprising administering to the subject a therapeutically effective amount of an A2R antagonist, wherein the high expression of the one or more biomarkers is characterized by a baseline tumor sample obtained from the subject having an increased expression level of the one or more biomarkers, compared to a reference level.

[0006] Also provided are methods of identifying a human subject with a cancer that is responsive to treatment with an A2R antagonist, the methods comprising measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 from a baseline tumor sample obtained from the subject, wherein responsiveness is characterized by an increased expression of the one or more biomarkers compared to a reference level.

[0007] Also provided are methods of improving treatment response to a therapy in a human subject having a cancer with a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1, said methods comprising administering an A2R antagonist to the subject.

[0008] Also provided are methods of quantifying an A2R antagonist responsive biomarker in a tumor sample, the methods comprising measuring expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX in a tumor sample.

[0009] Also provided are methods of treating cancer in a human subject, the methods comprising (a) comparing the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3 and TOX1 in a tumor sample from the subject to a reference level of the one or more biomarkers; and (b) administering an A2R antagonist to the subject if the tumor sample from the subject has an increased expression of the one or more biomarkers compared to the reference level.

[0010] Still also provided are methods of increasing efficacy of a therapy in a human subject having cancer, the methods comprising the steps of (a) measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX in a tumor sample from the subject; (b) comparing the expression of the one or more biomarkers with the expression of the one or more biomarkers in a reference sample; and (c) for a subject with increased expression of the one or more biomarkers in said tumor sample as compared to said 2 299641857reference sample, treating the subject with an A2R antagonist in combination with the therapy, thereby increasing the efficacy of the therapy.

[0011] Also provided are methods of treating cancer in a human subject, the method comprising a step for measuring one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 from a tumor sample from the subject, and administering an A2R antagonist to the subject if the expression of the one or more biomarkers is higher than a reference level.

[0012] Also provided are methods for indirectly measuring adenosine levels in a tumor sample, the method comprising measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in the tumor sample.

[0013] Also provided are methods of measuring the response of a cancer in a human subject to treatment with an A2R antagonist said method comprising: (a) measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 from a first tumor sample obtained from the subject prior to administration of the A2R antagonist, (b) administering the A2R antagonist to the subject; and (c) measuring the expression of the one or more biomarkers from a second tumor sample obtained from the subject after administration of the A2R antagonist; wherein a response is characterized by a decrease in the expression of the one or more biomarkers in the second tumor sample compared to the first tumor sample.

[0014] Also provided are methods of treating cancer in a human subject said method comprising: (a) measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 from a first tumor sample obtained from the subject prior to administration of the A2R antagonist, (b) administering a A2R antagonist to the subject for one or more than one dosing cycles (e.g., 1, 2, 3, 4 or more); (c) measuring the expression of the one or more biomarkers from a second tumor sample obtained from the subject after administration of the A2R antagonist, and (d) administering the A2R antagonist to the subject for additional dosing cycles (e.g., 1, 2, 3, 4 or more) when the measurement in (c) is characterized by a decrease in the expression of the one or more biomarkers in the second tumor sample compared to the first tumor sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIG. 1A and FIG. 1B depict the relationship between the negative log10(p-value) and the log10(hazard ratio) for overall survival (OS) (FIG. 1A) and progression free survival 3 299641857(PFS) (FIG. 1B) for a set of 88 gene signatures. Of the 88 gene signatures in patients in the BEP subset of the pooled Q100 Q(±Z)+G / nP cohort of the ARC-8 trial evaluated, a gene signature consisting of NR4A1, NR4A2, NR4A3, and TOX (the “NR4A / TOX signature”) was most predictive of treatment benefit. Each point represents a gene set that is tested. The further away a gene set is from zero on the x-axis, the larger the hazard ratio (HR) magnitude and the higher a gene set sits on the y-axis, the more significant that relationship. The dotted line represents a p-value of 0.05, and gene sets above that line show a significant relationship.

[0016] FIG.2A and FIG.2B depict Kaplan-Meier curves comparing patients stratified by high vs low baseline NR4A / TOX scores against OS (FIG.2A) or PFS (FIG.2B) in patients in the BEP subset of the pooled Q100 Q(±Z)+G / nP cohort of the ARC-8 trial. Patients were stratified using a “best-cut” approach, which identifies the optimal cutpoint by maximizing the logrank statistic (Kassambara A., rdrr.io / cran / survminer / man / surv_cutpoint.html). To prevent outliers driving the statistic, at least 25% of the patients needed to be in either the high or low group. This resulted in a threshold of -0.19 (high ≥ threshold, low < threshold). Hazard ratios (HR) were calculated using Cox regression and p-values were calculated using the log-rank statistic. Pr(OS) = probability of overall survival; Pr(PFS) = probability of progression free survival.

[0017] FIG. 2C and FIG. 2D depict hazard ratios of Cox regression models that compare the rate of OS (FIG. 2C) or PFS (FIG. 2D) against continuous values of select biomarkers in patients in the BEP subset of the pooled Q100 Q(±Z)+G / nP cohort of the ARC-8 trial. The biomarkers are NR4A1, NR4A2, and NR4A3 (NR4A Family), Sidders_ADO (see Table 1) and Willingham_ADO (see Table 1). A hazard ratio < 1 indicates a more favorable outcome in patients with higher biomarker expression. A hazard ratio >1 indicates a less favorable outcome in patients with higher biomarker expression.

[0018] FIG. 3A and FIG. 3B depict hazard ratios of Cox regression models that compare the rate of OS (FIG. 3A) or PFS (FIG. 3B) against continuous values of select biomarkers in patients in the BEP subset of the pooled Q100 Q(±Z)+G / nP cohort of the ARC-8 trial (left panel) or the G / nP + nivolumab (nivo) cohort of the PRINCE trial (right panel). The biomarkers are NR4A1, NR4A2, NR4A3, and TOX (NR4A / TOX signature); NR4A1, NR4A2, and NR4A3 (NR4A Family), and NR4A1, NR4A2, NR4A3, and TOX each individually. A hazard 4 299641857ratio < 1 indicates a more favorable outcome in patients with higher biomarker expression. A hazard ratio >1 indicates a less favorable outcome in patients with higher biomarker expression.

[0019] FIG. 4A, FIG. 4B, and FIG. 4C depict NR4A1, NR4A2, and NR4A3 (“NR4A family”) expression data (ssGSEA scores) in baseline samples versus on-treatment samples obtained from patients in the ARC-8 clinical trial BEP subset of the pooled Q100 Q(±Z)+G / nP cohort. FIG. 4A depicts data from all available samples. Rectangles in FIG. 4A pre-treatment samples identify patients with high baseline NR4A family expression (upper rectangle in black) and low baseline NR4A expression (lower rectangle in grey), where “high” is defined as levels equal to or above the median and “low” is less than the median. These groups are then separately shown in Figs. 4b and 4c, which depict the changes in NR4A family expression scores for subjects identified as having high baseline NR4A family expression (FIG. 4B) and for patients identified as having low baseline NR4A family expression (FIG.4C). Patients with the highest baseline levels of NR4A family expression exhibit the greatest decrease in NR4A family expression in their corresponding on-treatment samples.

[0020] FIG. 5A, FIG. 5B, and FIG. 5C depict NR4A1, NR4A2, and NR4A3 (“NR4A family”) expression (ssGSEA scores) in baseline samples versus on-treatment samples obtained from patients in the ARC-9 clinical trial EZFB cohort. FIG. 5A depicts data from all available samples. Rectangles in FIG. 5A pre-treatment samples identify patients with high baseline NR4A family expression (upper rectangle in black) and low baseline NR4A expression (lower rectangle in grey), where “high” is defined as levels equal to or above the median and “low” is less than the median. These groups are then separately shown in Figs. 5b and 5c, which depict the changes in NR4A family expression scores for patients identified as having high baseline NR4A family expression (FIG. 5B) and for patients identified as having low baseline NR4A family expression (FIG. 5C). Patients with the highest baseline levels of NR4A family expression exhibit the greatest decrease in NR4A family expression in their corresponding on-treatment samples.

[0021] FIG. 6A depicts the baseline and on-treatment T cell-inflamed gene expression profile (GEP) ssGSEA scores for patients in the ARC-8 BEP subset of the pooled Q100 Q(±Z)+G / nP cohort (left panel) and ARC-9 EZFB cohort (right panel). The data demonstrate significant increase in immune cell infiltration for on-treatment samples compared to baseline samples in both trials. 5 299641857

[0022] FIG. 6B depicts the median gene expression change (y-axis) of T-cell lineage / activation and effector genes (x-axis) upon treatment with a quemliclustat-containing regimen. The bars denote median log2 change of on-treatment minus pre-treatment biopsies of either all patients (grey), patients with maximal NR4A decrease (black), or patients with minimal NR4A decrease (white). An asterisk denotes statistically significant on-treatment minus pre-treatment changes, which are observed for six genes only for the patients with max NR4A decrease.

[0023] FIG. 7A depicts a Kaplan-Meier curve comparing the overall survival (OS) of biomarker evaluable patients from the ARC-8 clinical trial pooled Q100 Q(±Z)+G / nP cohort stratified by median change in NR4A family expression after treatment. Data for patients with the highest decrease of NR4A family expression are in light grey. Data for patients with the lowest decrease are in dark grey. Subjects with the highest decrease in NR4A family expression post-treatment were associated with longer OS benefit. Pr(OS) = probability of overall survival.

[0024] FIG. 7B depicts a Kaplan-Meier curve comparing the progression free survival (PFS) of biomarker evaluable patients from the ARC-8 clinical trial pooled Q100 Q(±Z)+G / nP cohort stratified by median change in NR4A family expression after treatment. High and low are defined as in FIG.7A. Patients with the highest decrease in NR4A family expression post- treatment were associated with longer progression free survival benefit. Pr(PFS) = probability of progression free survival.

[0025] FIG.8 depicts a waterfall plot analysis showing the relationship between tumor size changes (y-axis) and NR4A family expression changes (gray scale) after treatment in samples obtained from the biomarker evaluable patients of the EZFB cohort of the ARC-9 clinical trial. Patients exhibiting the highest decrease in NR4A family expression experienced significant tumor size reductions.

[0026] FIG. 9A depicts a dot plot showing the expression of NR4A family gene signature and its individual family members (NR4A1, NR4A2, and NR4A3) in each columnar box for specific cell types (y-axis) from surgical resections from PDAC patients with or without receiving a neoadjuvant chemoradiotherapy regimen (x-axis). The size of the dot shows the percent of cells expressing that gene or signature and the color, from light to dark depicts the degree of expression. 6 299641857

[0027] FIG. 9B depicts graphs showing that forskolin induced NR4A1 (top left), NR4A2 (top middle), and NR4A3 (top right) expression through PKA in a dose-dependent manner, and AMP induced NR4A1 (bottom left), NR4A2 (bottom middle), and NR4A3 (bottom right) expression is prevented in the presence of a CREB inhibitor (CREBi). For each graph, the y- axis depicts relative mRNA levels for the NR4A family member (e.g., NR4A1, NR4A2 or NR4A3). Different experimental conditions are across the x-axis. Two cell lines were tested: PANC-1 (left) and HCT-116 (right).

[0028] FIG. 9C depicts a model for adenosine regulation of NR4A family expression. AMP, adenosine monophosphate; CAF, cancer-associated fibroblasts; CREB, cyclic AMP response element binding protein; PKA, protein kinase A.

[0029] FIG. 10A depicts the correlation between RNA and protein levels for NR4A1 (left panel) and NR4A2 (right panel).

[0030] FIG. 10B depicts the correlation between RNA and protein levels for NR4A3 (left panel) and TOX (right panel). DETAILED DESCRIPTION

[0031] The present disclosure is drawn to biomarkers useful in numerous contexts, particularly regarding identifying a patient for treatment of a disease, particularly cancer, with a therapy comprising an A2R antagonist. Also provided are methods for determining a prognosis for a patient who has or who is yet to receive a therapy comprising an A2R antagonist. The present disclosure is also directed to technologies for the treatment of a patient who has been identified as having cancer, comprising measuring the expression level of one or more biomarkers in a sample obtained from the patient, and administering to the patient a therapy comprising an A2R antagonist when the sample obtained from the patient comprises a particular biomarker expression profile. The present disclosure is directed to the discovery that the expression profile of certain biomarkers, including NR4A1, NR4A2, NR4A3 (i.e., a NR4A family member) and / or TOX1, may be used to determine the likelihood that a patient will be responsive or non-responsive to a therapy comprising an A2R antagonist. In the Examples described herein, it was surprisingly observed that certain biomarker expression profiles can be used to predict treatment efficacy for a therapy comprising an A2R antagonist. It is also shown herein that the biomarker expression profiles of the present disclosure may be used as a surrogate measure for adenosine levels, and their down regulation following treatment with a 7 299641857CD73 inhibitor (e.g., quemliclustat) and an A2R antagonist (e.g., etrumadenant) is associated with treatment benefit. This suggests that adenosine modulation by either mechanism of action plays a role in the treatment’s efficacy, and illustrates the utility of the biomarker expression profiles of the present disclosure for other therapeutic agents with a mechanism of action similar to etrumadenant (e.g., other A2R antagonist). Exemplary A2R antagonists include small molecule antagonists of A2R. Definitions

[0032] Unless otherwise defined, all terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this disclosure pertains.

[0033] The term “about” as used herein has its original meaning of approximately and is to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In general, the term “about” refers to the usual error range for the respective value readily known to the skilled person in this technical field. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. Where ranges are provided, they are inclusive of the boundary values.

[0034] The “amount,” “level,” or “expression level,” used herein interchangeably, of a biomarker (e.g., NR4A1, NR4A2, NR4A3, or TOX1) is a detectable level in a biological sample. The term “amount” as used herein encompasses an absolute amount of a biomarker and a relative amount of a biomarker. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the said biomarker by direct measurements. Moreover, encompassed are all values or parameters which are obtained by indirect measurements specified elsewhere in this description. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained by all standard mathematical operations. “Expression” generally refers to the process by which information (e.g., gene-encoded) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide or translation into a polypeptide. Fragments of the transcribed polynucleotide, the translated polypeptide, shall also be regarded as expressed whether they originate from a transcript 8 299641857generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs). Expression levels may be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of a biomarker (e.g., NR4A1, NR4A2, NR4A3, and / or TOX1) may be used to identify / characterize a cancer that may be likely to respond to, or benefit from, a particular therapy.

[0035] The term “biomarker” as used herein refers to an indicator, e.g., predictive, diagnostic, and / or prognostic, which can be detected in a sample. The biomarker may serve as an indicator of a particular subtype of a disease or disorder characterized by certain, molecular, pathological, histological, and / or clinical features. Biomarkers include, but are not limited to, polypeptides, and polynucleotides (e.g., DNA, and / or RNA).

[0036] The terms “detecting” and “detection” are used herein in the broadest sense to include both qualitative and quantitative measurements of a target molecule. Detecting includes identifying the mere presence of the target molecule in a sample as well as determining whether the target molecule is present in the sample at detectable levels. Detecting may be direct or indirect.

[0037] The term “sample,” as used herein, refers to a composition that is obtained or derived from a subject that contains a biomarker that is to be identified and / or characterized, for example based on physical, biochemical, chemical and / or physiological characteristics. For example, the phrase “tumor sample,” “disease sample,” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the biomarker that is to be characterized. Other samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, stool, whole lymph nodes or lymph node biopsies, tissue from biopsies or resections, tumor biopsies, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, cellular extracts, and combinations thereof. 9 299641857

[0038] A “reference sample,” “reference cell,” “reference tissue,” “control sample,”“control cell,” or “control tissue,” as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes. In one embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissue or cells) of the same subject. For example, healthy and / or non-diseased cells or tissue that is / are adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor). In another embodiment, a reference sample is obtained from an untreated tissue and / or cell of the body of the same subject. In yet another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and / or non-diseased part of the body (e.g., tissues or cells) of a subject who is not the subject. In even another embodiment, a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and / or cell of the body of an individual who is not the subject.

[0039] A “tumor cell,” as used herein, refers to a cancerous cell.

[0040] A “tumor sample” refers to a sample comprising tumor cells. Typically, a tumorsample obtained from a solid tumor (e.g., tissue from biopsies or resections) comprises tumor cells and other cells of the tumor microenvironment (e.g., immune cells, stromal cells, fibroblasts, etc.).

[0041] The term “positive cell fraction” is the percentage of viable cells showing positivestaining for a protein of interest in one or more cellular location (e.g., membrane, cytoplasm) at any intensity in a sample, following staining of the sample, e.g., in an immunohistochemical (IHC) assay. A positive cell fraction may be reported for all cell types or a certain subtype or subtypes (e.g., tumor cells, immune cells, other cells of the tumor microenvironment, etc.). The term “positive tumor cell fraction,” which may be abbreviated “% TC,” is the percentage of viable tumor cells showing positive staining for a protein of interest in one or more cellular location. A “tumor cell,” as used herein, refers to a cancerous cell. Accordingly, a positive tumor cell fraction may be calculated using the formula , wherein all positive-staining non- tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) are excluded from evaluation and scoring. The term “positive immune cell fraction,” which may 10 299641857be abbreviated “% IC” is the percentage of viable immune cells in a sample or specified area of a sample showing positive staining for a protein of interest in one or more cellular location.

[0042] An “H-Score” is another means for characterizing expression level of a protein andcaptures both the intensity and the proportion of the biomarker of interest from an image (e.g., IHC). Individual viable cells (e.g., tumor cells, immune cells, other cells of the tumor microenvironment, and combinations thereof), and in some embodiments their sub-cellular compartments (e.g., nucleus, cytoplasm, cell membrane, etc.), are first detected and based on the relative expression of the biomarker of interest in the cell, or one or more sub-cellular compartments, the cells are classified as either positive or negative. The positive cells are further classified into high (3+), medium (2+), or low (1+) based on the biomarker signal intensity. The H-score (range of 0 to 300) is calculated using the following equation: (3 x % cells staining at 3+) + (2 x % cells staining at 2+) + (1 x % cells staining at 1+). A tumor H- score refers to an H-score calculated based on the relative expression of a biomarker of interest in viable tumor cells.

[0043] Another means for characterizing expression level of a protein that captures boththe intensity and proportion of the biomarker of interest from an image (e.g., IHC) is “2+ or 3+ % TC,” which is the percentage of viable tumor cells showing positive staining for a protein of interest in one or more cellular location (e.g., cell membrane, cytoplasm) with a signal intensity of either medium (2+) or high (3+). Accordingly, 2+ or 3+ % TC may be calculated using the formula , wherein all positive-staining non-tumor cells (e.g., tumor-infiltrating immune cells, normal cells, necrotic cells, and debris) are excluded from evaluation and scoring.

[0044] The term “protein,” as used herein, encompasses “full-length,” unprocessed proteinas well as any form of the protein that results from processing in the subject (e.g., processing that may occur intracellularly or extracellularly within the body). The term also encompasses naturally occurring variants of the protein, e.g., splice variants or allelic variants.

[0045] “Polynucleotide” or “nucleic acid,” as used interchangeably herein, refers topolymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or 11 299641857any substrate that can be incorporated into a polymer by DNA or RNA polymerase, or by a synthetic reaction. Thus, for instance, polynucleotides as defined herein include, without limitation, single- and double-stranded DNA, DNA including single- and double-stranded regions, single- and double-stranded RNA, and RNA including single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or include single- and double-stranded regions. In addition, the term “polynucleotide” as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple- helical region often is an oligonucleotide. The terms “polynucleotide” and “nucleic acid” specifically includes mRNA and cDNAs.

[0046] A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after synthesis, such as by conjugation with a label. Other types of modifications include, for example, “caps,” substitution of one or more of the naturally-occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, and the like) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, and the like), those containing pendant moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, and the like), those with intercalators (e.g., acridine, psoralen, and the like), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, and the like), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids), as well as unmodified forms of the polynucleotide(s). Further, any of the hydroxyl groups ordinarily present in the sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5′ and 3′ terminal OH can be phosphorylated or substituted with amines or organic capping group moieties of from 1 to 20 carbon atoms. Other hydroxyls may also be derivatized to standard protecting groups. Polynucleotides can also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2′-O- 12 299641857methyl-, 2′-O-allyl-, 2′-fluoro-, or 2′-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs, and abasic nucleoside analogs such as methyl riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, embodiments wherein phosphate is replaced by P(O)S (“thioate”), P(S)S (“dithioate”), “(O)NR2 (“amidate”), P(O)R, P(O)OR′, CO or CH2 (“formacetal”), in which each R or R′ is independently H or substituted or unsubstituted alkyl (1-20 C) optionally containing an ether (—O—) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a polynucleotide need be identical. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0047] The terms “patient” or “subject” are used interchangeably to refer to a human.

[0048] The terms “treat,” “treating,” “treatment” and the like refer to a course of action that eliminates, reduces, suppresses, mitigates, ameliorates, or prevents the worsening of, either temporarily or permanently, a disease, disorder or condition to which the term applies, or at least one of the symptoms associated therewith. Treatment includes alleviation of symptoms, diminishment of extent of disease, inhibiting (e.g., arresting the development or further development of the disease, disorder or condition or clinical symptoms association therewith), and prolonging survival of a subject as compared to expected survival if not receiving treatment or as compared to a published standard of care therapy for a particular disease.

[0049] The term “in need of treatment” as used herein refers to a judgment made by a physician or similar professional that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of the physician’s expertise, which may include a positive diagnosis of a disease, disorder or condition.

[0050] As used herein, the “administration” of or “administering” an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration may be carried out by any suitable route, including but not limited to, orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intratumorally or topically. Administration includes self-administration and the administration by another. 13 299641857

[0051] The term “standard of care” widely accepted and recommended treatment approach, involving for example chemotherapy, radiation therapy, a targeted agent, or combinations thereof, used by medical experts to treat a specific type of cancer. Detailed standard of care guidelines are published, for example, by National Comprehensive Cancer Network (NCCN), which may be adapted to account for geographical differences (e.g., NCCN – Asia Consensus Statements), German Guideline Program in Oncology, and other international societies of medical oncology (e.g., European Society for Medical Oncology, Japanese Society of Medical Oncology, Chinese Society of Medical Oncology, and the like).

[0052] As used herein, “specifically binds” refers to a molecule (e.g., an antibody or antigen binding fragment thereof) which recognizes and binds another molecule (e.g., an antigen), but that does not substantially recognize and bind other molecules. The terms “specific binding,” “specifically binds to,” or is “specific for” a particular molecule (e.g., a polypeptide, or an epitope on a polypeptide), as used herein, can be exhibited, for example, by a molecule having a KD for the molecule to which it binds to of about 10−6M, 10−7M, 10−8M, 10−9M, 10−10M, 10−11M, or 10−12M. The term “specifically binds” may also refer to binding where a molecule (e.g., an antibody or antigen binding fragment thereof) binds to a particular polypeptide, or an epitope on a particular polypeptide, without substantially binding to any other polypeptide, or polypeptide epitope.

[0053] The term “antibody,” as used herein, is used in the broadest sense and encompasses various antibody and antibody-like structures that specifically bind to a single antigen or to multiple antigens (e.g., monospecific antibodies, multispecific antibodies, polyepitopic antibodies, etc.), including but not limited to full-length antibodies, antigen-binding fragments, heavy chain antibodies, single-chain antibodies, and higher order variants of single-chain antibodies. Thus, any reference to an antibody should be understood to refer to the antibody in intact form or an antigen-binding fragment (including an antigen-binding fragment derived from a full-length antibody) unless the context requires otherwise. Preferably, but not necessarily, antibodies useful herein are isolated and may be produced recombinantly. The term “isolated antibody” refers to an antibody that has been separated from a component of its natural environment. In some embodiments, an isolated antibody is purified to greater than 95% or 99% purity as determined by, for example, electrophoresis or chromatography (e.g., ion exchange or reverse phase HPLC). 14 299641857

[0054] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody having a structure substantially similar to a native antibody structure or having heavy chains that contain an Fc region.

[0055] The term “antibody fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antigen-binding fragment include, without limitation, a diabody, a Fab, a Fab′, a F(ab′)2, a F(ab)c, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv′), a disulfide stabilized diabody (ds diabody), a triabody, a tetrabody, a single-chain antibody, an scFv, an scFv dimer, a single domain antibody, a single- domain antibody, and a multivalent domain antibody. Typically, binding fragments compete with the intact antibody from which they were derived for specific binding. Binding fragments may be produced by recombinant DNA techniques, or by enzymatic or chemical separation of intact immunoglobulins.

[0056] As used herein, the term “therapeutic agent” is intended to mean a compound that, when present in an effective amount, produces a desired therapeutic effect on a subject in need thereof.

[0057] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in, a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a therapeutic agent or composition comprising a therapeutic agent administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The therapeutic agent(s) and compositions comprising the therapeutic agent(s) can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic agent(s) or compositions comprising the therapeutic agent(s) may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a “therapeutically effective amount” of a therapeutic agent or composition comprising a therapeutic agent refers to level(s) of the therapeutic agent or composition 15 299641857comprising a therapeutic agent in which the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount may be given in one or more administrations.

[0058] Additionally, amounts, ratios, and other numerical values are sometimes presented herein in a range format. It is to be understood that such range format is used for convenience and brevity and should be understood flexibly to include numerical values explicitly specified as limits of a range, but also to include all individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly specified. For example, a value in the range of about 1 to about 200 should be understood to include the explicitly recited limits of about 1 and about 200, but also to include individual values such as about 2, about 3, and about 4, and sub-ranges such as about 10 to about 50, about 20 to about 100, and so forth.

[0059] Also as used herein, “and / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0060] As used herein, the term “comprising” is intended to mean that the compositions and methods include the recited elements, but not excluding others. “Consisting essentially of” when used to define compositions and methods, shall mean excluding other elements of any essential significance to the composition or method. “Consisting of” shall mean excluding more than trace elements of other ingredients for claimed compositions and substantial method steps. Examples and implementations defined by each of these transition terms are within the scope of this disclosure. Accordingly, it is intended that the methods and compositions can include additional steps and components (comprising) or alternatively including steps and compositions of no significance (consisting essentially of) or alternatively, intending only the stated method steps or compositions (consisting of).

[0061] As used herein, “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0062] “Substantially pure” indicates that a component (e.g., a compound according to this disclosure) makes up greater than about 50% of the total content of the composition, and 16 299641857typically greater than about 60% of the total content. More typically, “substantially pure” refers to compositions in which at least 75%, at least 85%, at least 90% or more of the total composition is the component of interest. In some cases, the component of interest will make up greater than about 90%, or greater than about 95% of the total content of the composition. Biomarkers of the Disclosure

[0063] The present disclosure relates to a biomarker or a combination of biomarkers (e.g., 2, 3, or 4), and use of these biomarkers individually or in combination in clinical and diagnostic settings to affect treatment.

[0064] In various embodiments, a biomarker of the present disclosure may be an expression level of a protein (e.g., NR4A1, NR4A2, NR4A3, TOX1), or a nucleic acid encoding said protein, relative to a reference level. The terms “NR4A1,” “NR4A2,” “NR4A3,” and “TOX1” refer herein to proteins in humans encoded by the NR4A1, NR4A2, NR4A3, and TOX genes, respectively, and naturally occurring variants or fragments of NR4A1, NR4A2, NR4A3, and TOX1 e.g., originating from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g., by proteolysis. The amino acid sequence of exemplary human NR4A1, NR4A2, NR4A3, and TOX1 may be found under UniProt Accession Numbers P22736, P43354, Q92570, and O94900, respectively. For clarity, the term “expression level” when used in regard to a protein (e.g., an NR4A1 expression level, NR4A2 expression level, NR4A3 expression level, or a TOX1 expression level), may refer to a detectable level of a polynucleotide (e.g., mRNA) encoding the protein and in some instances fragments thereof, or to a detectable level of the translated polypeptide and in some instances fragments thereof. The polynucleotide and / or protein may be tumor-associated, e.g., detectable in or on tumor cells or other cells within the tumor microenvironment (e.g., immune cells, stromal cells, fibroblasts, endothelial cells, etc.), or may be in the tumor microenvironment, e.g., detectable in the tumor microenvironment even though it is not in or on the tumor cells or other cells within the tumor microenvironment, or may be circulating, e.g., detectable in or on intact cells or subcellular structures (e.g., exosomes) in blood or lymph or extracts thereof, or detectable as a soluble form in blood, lymph or extracts thereof. Expression levels or amounts equal to or above a reference level are “high” and expression levels or amounts below a reference level are “low”. Accordingly, a biomarker high tumor (e.g., a NR4A1 high tumor, etc.) is a tumor where the expression level of the biomarker is equal to or above a reference level in a sample of the tumor. Similarly, high expression of a peripheral 17 299641857biomarker (e.g., high NR4A1 expression in a blood derived sample, etc.) refers to an expression level of the biomarker, measured in a peripheral sample that is equal to or above a reference level.

[0065] Non-limiting examples of uses contemplated herein for the biomarkers of the present disclosure include identifying subjects with cancer that have a poor prognosis, improving outcomes for subjects with a poor prognosis, determining effectiveness of a therapy comprising a A2R antagonist for treating cancer in subjects, identifying subjects for treatment with an A2R antagonist, identifying subjects for a clinical trial and / or stratifying subjects into treatment arms during a clinical trial (e.g., based on prognosis and / or predicted responsiveness to A2R antagonists), determining whether a cancer is likely to respond to a therapy comprising an A2R antagonist, and the like. As described in further detail herein, the effectiveness of a therapy (e.g., a standard of care therapy) may be improved by administering the therapy in combination with an A2R antagonist to a subject with a cancer characterized by one or more biomarker(s) disclosed herein. Improved effectiveness may be demonstrated in a clinical trial setting, for example by comparing an outcome measured in an experimentally treated cohort of subjects to the same outcome measured in a biomarker-selected subset of that cohort (e.g., prospectively or retrospectively selected based on the biomarker or combination of biomarkers). In another example, improved effectiveness may be demonstrated in a clinical trial setting with an experimentally treated cohort of subjects by comparing an outcome in a biomarker “high” subset of the cohort to the same outcome measured in a biomarker “low” subset of the cohort (e.g., prospectively or retrospectively selected based on the biomarker or combination of biomarkers). Non-limiting examples of suitable outcomes include response rates (e.g., measures of complete responses, partial responses, stable disease, progressive disease, objective response rate, etc.), duration of response, time to response, progression free survival, and overall survival. Improved effectiveness does not require a therapy to always be effective in treating every individual subject of the biomarker selected cohort.

[0066] When a biomarker is a protein expression level (e.g., an expression level of NR4A1, NR4A2, NR4A3, or TOX1), expression level may be measured in a sample by immunohistochemistry (IHC), immunocytochemistry, immunofluorescence microscopy, immunophenotyping by flow cytometry, ELISA, electrochemiluminescence-based immunoassays or other immunoassays, mass spectrometry, proximity extension assays (e.g., Olink assays), aptamer-based assays (e.g., SOMAscan assays), etc. Non-limiting examples of 18 299641857suitable samples include tissue, cells and / or fluid from biopsies (e.g., bone marrow biopsies, lymph node biopsies, skin biopsies, surgical biopsies, tumor biopsies, etc.) or resections, tumor lysates, tissue extracts such as homogenized tissue, lymph, blood, blood-derived cells, plasma, and serum. Protein expression levels may be characterized in a number of different ways using different algorithms. For example, protein expression level measured by IHC may be characterized by percent cell positivity, percent cell positivity relative to an area, an H-score, percent positivity at a given intensity level, and the like. Protein expression level measured, for example, by ELISA, immunoassay, mass spectrometry, proximity extension assays, aptamer- based assays, and the like may be characterized by an absolute amount, a relative amount, or a fold-change. For methods that use an antibody to detect the protein of interest (e.g., IHC), it will be appreciated that any given antibody that specifically binds the protein may be used with a particular assay protocol and / or scoring terminology to derive protein expression level. Sensitivities can vary between different antibodies and therefore thresholds defined by one particular assay may not be applicable to another, even if using the same scoring algorithm. Although there may not be agreement between absolute threshold values, different assays and algorithms – when used at different thresholds – may identify the same population of patients and concordance between different approaches may be empirically determined by a person of skill in the art.

[0067] When a biomarker is a polynucleotide expression level, expression level may be measured by transcriptomic profiling of a tumor sample or specific cells within the tumor sample (e.g., tumor cells, immune cells, stromal cells and other cells present in the tumor sample) using amplification-based, hybridization-based, or sequencing-based methods. Nonlimiting examples of these methods include in situ hybridization approaches (e.g., chromogenic in situ hybridization (CISH), fluorescence in situ hybridization (FISH), RNAscopeTM, BaseScopeTM, and the like), sequencing based approaches (e.g., next-generation sequencing (NGS), RNA sequencing (RNA-Seq), single-cell RNA-seq (scRNA-seq) and the like), PCR based approaches (quantitative PCR, quantitative RT-PCR, and the like), microarray and high-throughput fluorescence-based approaches (e.g., traditional microarray technologies, Nanostring nCounter Analysis System, Nanostring GeoMX Digital Spatial Profiler and the like) or other multiplexed assays for detecting a polynucleotide. Non-limiting examples of suitable samples include tissue, cells and / or fluids from biopsies (e.g., bone marrow biopsies, lymph node biopsies, skin biopsies, surgical biopsies, tumor biopsies, etc.) or resections, tumor lysates, tissue extracts such as homogenized tissue, lymph, blood, blood- 19 299641857derived cells and plasma. Polynucleotide expression levels may be characterized in a number of different ways using different algorithms, which may describe an expression level by an absolute amount, a relative amount (normalized against a housekeeping gene, library size, transcript length (e.g., RPKM), or the like), or a fold-change. For example, the expression of the one or more biomarkers may be characterized by a gene expression score.

[0068] In some embodiments, expression level of one or more biomarkers according to this disclosure (e.g., NR4A1, NR4A2, NR4A3, and / or TOX1) is greater than or equal to a reference level. When NR4A1, NR4A2, NR4A3, and / or TOX1 expression is detected by immunohistochemistry (IHC), for example by an IHC assay staining for NR4A1, NR4A2, NR4A3, or TOX1 using an antibody that specifically binds to NR4A1, NR4A2, NR4A3, or TOX1, positive staining may refer to partial or complete membrane staining (exclusive of cytoplasmic staining) at any intensity or may refer to membranous, cytoplasmic, and punctate staining at any intensity, depending upon the particular IHC assay and tumor type being evaluated.

[0069] When the expression of one or more biomarkers according to the disclosure (e.g., NR4A1, NR4A2, NR4A3, and / or TOX1) is determined using staining techniques (e.g., IHC), the expression level may be characterized as a positive tumor cell fraction (i.e., %TC or tumor proportion score (TPS)), an H-score, a score that captures both intensity and proportion of positive cells (e.g., 2+-3+ H-score), a score that captures the proportion of positive cells over a given area, or a positive immune cell fraction (%IC).

[0070] In the above embodiments, the sample may be obtained from a subject with cancer, optionally a solid tumor.

[0071] In embodiments where a biomarker is used to select a therapy, expression of the biomarker is measured or was measured in a sample obtained prior to administration of the therapy (e.g., a baseline tumor sample). High baseline NR4A1, NR4A2, NR4A3 and / or TOX1 expression compared to low baseline NR4A1, NR4A2, NR4A3 and / or TOX1 expression may be associated with poor clinical outcomes (e.g., poor survival, progressive disease, etc.) in patients receiving a standard of care therapy (e.g., chemotherapy).

[0072] In embodiments where a biomarker or combination of biomarkers is used as a pharmacodynamic marker or to affect clinical decisions after treatment has begun, expression 20 299641857of each biomarker is measured or was measured in two or more samples, e.g., a baseline sample and a second sample obtained after one or more treatments (e.g., about 1, 2, 3, 4, 5, 6, or 7 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks after a prior treatment), or as a first on- treatment sample and second on-treatment sample (e.g., about 1, 2, 3, 4, 5, 6, or 7 days, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks after the first treatment). The second sample or the second on-treatment sample can be obtained or could have been obtained within the same dosing cycle as the baseline sample or the first on-treatment sample, respectively, or from a different dosing cycle. A decrease in NR4A1, NR4A2, and / or NR4A3 expression in the second sample as compared to the first sample, or the second on-treatment sample as compared to the first on-treatment sample, is referred to herein as “a reduction” or “a post-treatment decrease.” A post-treatment (e.g., after administration of a A2R antagonist) decrease (i.e., reduction) in NR4A1, NR4A2, and / or NR4A3 expression may indicate the tumor is responding favorably to treatment and administration of a therapy comprising the A2R antagonist should continue. Post-treatment decrease in NR4A1, NR4A2, and / or NR4A3 expression following treatment with an A2R antagonist may also predict favorable treatment response if treatment is continued. For example, a post-treatment decrease in NR4A1, NR4A2, and / or NR4A3 expression measured about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after start of treatment with a therapy comprising an A2R antagonist may predict favorable treatment response (e.g., greater tumor size reduction, longer progression free survival, longer overall survival, etc.), particularly if the decrease is greater than a suitable reference level. A non-limiting example of a suitable reference level may be a post-treatment change (post-treatment – baseline) in expression of the biomarker for patients with the same type of cancer that have baseline expression less than the median for the type of cancer.

[0073] Optimal biomarker cut-offs (e.g., reference levels) may be analyzed using the median levels described above, or using quartiles or tertiles, as reference points. Statistical methods, including but not limited to receiver operating characteristic (ROC) analysis and those described in the examples, can also be used to determine the cut-off of best fit, whereby the difference in treatment response between biomarker-high and biomarker-low populations is most significant. Additional datasets and descriptive statistics of biomarker distribution can also inform clinically relevant cut-off / threshold selection.

[0074] In some embodiments, a reference level may be a median expression level, or an amount within about 30% above or below a median expression level, derived from samples 21 299641857obtained from a group of subjects known to have the same disease, such as about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below a median expression level. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% above a median expression level derived from samples obtained from a group of subjects known to have the same disease. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% below a median expression level derived from samples obtained from a group of subjects known to have the same disease.

[0075] In some embodiments, a reference level may be a tertile (i.e., the 33rdor 66thpercentile), or an amount within about 30% above or below a tertile, derived from samples obtained from a group of subjects known to have the same disease, such as about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below a tertile. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% above a tertile derived from samples obtained from a group of subjects known to have the same disease. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% below a tertile expression level derived from samples obtained from a group of subjects known to have the same disease.

[0076] In some embodiments, a reference level may be a quartile (i.e., the 25th, 50th, or 75thpercentile), or an amount within about 30% above or below a quartile, derived from samples obtained from a group of subjects known to have the same disease, such as about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below a quartile. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% above a quartile derived from samples obtained from a group of subjects known to have the same disease. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% below a quartile derived from samples obtained from a group of subjects known to have the same disease.

[0077] In some embodiments, a reference level may be a best-cut value or an amount within about 30% above or below a best-cut value, determined based on the overall survival and / or progression free survival for a group of subjects known to have the same disease, such as about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below a best-cut value. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% above a best-cut value determined based on the overall survival and / or progression 22 299641857free survival for a group of subjects known to have the same disease. In some embodiments, a reference level can be an amount within about 30%, 25%, 20%, 15%, 10%, or 5% below a best-cut value determined based on the overall survival and / or progression free survival for a group of subjects known to have the same disease. Methods of Use

[0078] The present disclosure is directed to uses of biomarkers described herein. The biomarkers described herein are useful in a variety of methods described further below, including, but not limited to, methods of identifying a subject for treatment with a therapy, methods of treating a disease in a subject (e.g., a subject in need thereof), methods of determining a prognosis for a subject who has been identified as having or as at risk for a disease or condition (e.g., cancer), and methods for determining a prognosis for subject or a patient, such as a subject or a patient who has received or is yet to receive a therapy (e.g., a therapy comprising an A2R antagonist).

[0079] Particular embodiments of the disclosure are directed to methods of treating a cancer in a subject or methods of improving a therapeutic outcome in a subject, the methods comprising administering an A2R antagonist to a subject having a cancer with high expression of one or more of the biomarkers described herein. Related embodiments of the disclosure also provide methods of identifying a human subject that is responsive to treatment with an A2R antagonist, the methods comprising measuring expression of one or more of the biomarkers described herein from a baseline tumor sample obtained from the subject, wherein responsiveness is characterized by an increased expression of the one or more biomarkers compared to a reference level.

[0080] In certain embodiments, a biomarker is assessed, observed, measured, analyzed, or characterized in a sample obtained from a subject. The sample may be a sample comprising tumor cells. For instance, in some embodiments, method of quantifying an A2R antagonist responsive biomarker in a tumor sample is provided, the method comprising measuring expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 in a tumor sample. In some embodiments, the one or more biomarkers are (a) NR4A1, NR4A2 or NR4A3, or any combination thereof, (b) TOX1, or (c) NR4A1, NR4A2, NR4A3, and TOX1. The sample may be a sample comprising immune cells. The sample may be a sample 23 299641857comprising tumor cells and immune cells. How to obtain such as sample is known in the art. In particular, the sample may be obtained by biopsy or resection.

[0081] The sample to be analyzed in the context of the methods of the present disclosure may be obtained prior, during, or after treatment of a disease (e.g., cancer) as described herein. In embodiments where a biomarker is used to select a therapy, expression of the biomarker is measured or was measured in a “baseline sample,” i.e., a sample obtained prior to treatment (e.g., administration of the therapy for a given line of therapy). A sample obtained prior to treatment may be obtained not more than one year, not more than six, five, four, three or two months, or one month prior to the initiation of said treatment. It is also contemplated to obtain a sample not more than two weeks, or not more than one week prior to said treatment. A sample obtained after treatment may be obtained after the end of the treatment (e.g., once all treatment has stopped), or after an amount of time has elapsed on treatment. A sample obtained after the end of treatment may be obtained not more than three years, obtained not more than one year, not more than six, five, four, three or two months, or one month after said treatment. It is also contemplated to obtain a sample not more than two weeks, or not more than one week after said treatment. For samples obtained after an amount of time has elapsed on treatment, the sample may be obtained not more than four, three, or two weeks, or not more than one week after the most recent administration of the treatment. Additionally, samples obtained after an amount of time has elapsed on treatment may be obtained after 1, 2, 3, or 4 weeks have elapsed on treatment, or after 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, or 24 months have elapsed on treatment.

[0082] Determining the amount of the biomarkers referred to in this disclosure relates to measuring a detectable level in a biological sample, preferably qualitatively or quantitatively. Measuring may be done directly or indirectly. Direct measuring relates to measuring the biomarker based on a signal which is obtained from the biomarker itself, and the intensity of which directly correlates with the number of molecules of the polypeptide or polynucleotide present in the sample. Such a signal - sometimes referred to herein as intensity signal - may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the biomarker. Indirect measuring includes measuring a signal obtained from a secondary component (i.e., a component not being the biomarker itself) or a biological read-out system, e.g., measurable cellular responses, ligands, labels, or enzymatic reaction products. 24 299641857

[0083] In accordance with the present disclosure, determining the amount of a biomarker may be achieved by all known means suitable for determining the amount of a protein or nucleic acid biomarker, respectively, for a given sample. Non-limiting examples of suitable means for detecting protein and nucleic acid biomarkers are described above.

[0084] In some embodiments, an amount or level (e.g., expression level) of a biomarker is compared to a reference level. The term “comparing” as used herein encompasses comparing the amount of the biomarker comprised by the sample to be analyzed with an amount of a suitable reference source specified elsewhere in this description. It is to be understood that comparing as used herein refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount, while a concentration is compared to a reference concentration, or an intensity signal obtained from a test sample is compared to the same type of intensity signal of a reference sample. The comparison may be carried out manually or computer assisted. For a computer assisted comparison, the value of the determined amount may be compared to values corresponding to suitable references which are stored in a database by a computer program. The computer program may further evaluate the result of the comparison, e.g., automatically provide the desired assessment in a suitable output format. Based on the comparison, it is possible to predict one or more of a patient’s likelihood of responding to a therapy described herein, or a patient’s prognosis before or after receiving a therapy described herein. The comparison also allows for selecting or identifying a candidate for a particular therapy described herein.

[0085] The term “reference level” as used herein refers to amounts of the biomarker(s) which allow for predicting whether a patient or subject is likely to respond to a therapy described herein, is a suitable candidate to receive a therapy described herein, and / or has a favorable or poor prognosis before or following a therapy described herein. In some embodiments, the reference level may be derived from a group of subjects known to have a particular disease (e.g., cancer, type of cancer, type of cancer and stage of cancer, etc.) that is the same as the patient in question. For clarity, this group of subjects is “unselected” with respect to said biomarker(s). Preferably, said reference level is derived from samples obtained from the aforementioned subjects (i.e., the reference level is a predetermined value). The reference value is a threshold that distinguishes, and in some instances maximizes treatment benefit between patient high vs patient low groups. Exemplary treatment benefits include progression free survival, overall survival, disease control rate, etc. The reference value can be 25 299641857an expression level within the 10thto 90thpercentile for a group of subjects with the same cancer, or within the 20thto 80thpercentile. In some embodiments, the reference value can be a quartile (i.e., the 25th, 50th, or 75thpercentile). In some embodiments, the reference value can be a tertile (i.e., the 33rdor 66thpercentile). In some embodiments, the reference value can be a median, or a value within about 30% above or below the median expression level. In some embodiments, the reference value can be a best-cut value determined based on the overall survival and / or progression free survival for group of subjects with the same cancer.

[0086] In the context of the methods of the present disclosure, the amount of more than one biomarker may be determined. For clarity, determined amounts shall be compared to various reference amounts, i.e., to a reference amount for the individual biomarker tested.

[0087] Moreover, the reference levels, preferably, define threshold amounts or thresholds. Suitable reference amounts or threshold amounts may be determined by the method of the present disclosure from a reference sample. The reference sample may be analyzed simultaneously with the test sample or may be analyzed before or after a test sample is analyzed. Once a threshold is established for a particular disease, comparison to a reference amount does not require reanalyzing a reference sample with each test sample.

[0088] In some embodiments, the reference level is a median expression level, or an amount that is within about 30%, or about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below a median expression level, derived from samples obtained from a group of subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is an amount within about 30% above or below a median expression level derived from samples obtained from group of subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is an amount within about 25% above or below a median expression level derived from samples obtained from a group of subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is an amount within about 20% above or below a median expression level derived from samples obtained from a group subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some 26 299641857embodiments, the reference level is an amount within about 15% above or below a median expression level derived from samples obtained from a group of subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is an amount within about 10% above or below a median expression level derived from samples obtained from a group of subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is an amount within about 5% above or below a median expression level derived from samples obtained from a group of subjects known to have the same disease (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy).

[0089] In some embodiments, the reference level is an expression level within the 10thto 90thpercentile for a group of subjects with the same cancer, more preferably within the 20thto 80thpercentile for a group of subjects with the same cancer (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is at or above the 20thpercentile for a group of subjects with the same cancer, such as, for example, at or above the 25th, 30th, 35th, 40th, 45th, 50th, 55th, 60th, 65th, 70th, 75th, or 80thpercentile for a group of subjects with the same cancer (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy).

[0090] In some embodiments, the reference level is the best-cut value determined based on the overall survival and / or progression free survival for a group of subjects with the same cancer (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is the best-cut value determined based on the overall survival for a group of subjects with the same cancer (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). In some embodiments, the reference level is the best-cut value determined based on the progression free survival for a group of subjects with the same cancer (e.g., same cancer, optionally same disease stage and in some embodiments same setting / line of therapy). Methods of Treatment

[0091] The present disclosure provides methods for treating a disease (e.g., cancer) in a patient known to have the disease, the method comprising administering an effective amount of a therapy to the patient when the patient is determined to have a particular pattern of 27 299641857expression of one or more biomarkers according to this disclosure. The biomarkers of the below embodiments are further described elsewhere herein, and those disclosures are incorporated by reference into this section. Suitable samples are also described elsewhere herein and incorporated by reference into this section.

[0092] In some embodiments, this disclosure provides a method of treating a human subject having a cancer with a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1, the method comprising administering to the subject a therapeutically effective amount of an A2R antagonist, wherein the high expression of the one or more biomarkers is characterized by a baseline tumor sample obtained from the subject having an increased expression level of the one or more biomarkers, compared to a reference level.

[0093] In various embodiments, a method of treating cancer in a human subject is provided, the method comprising (a) comparing the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 in a tumor sample from the subject to a reference level of the one or more biomarkers; and (b) administering an A2R antagonist to the subject if the tumor sample from the subject has an increased expression of the one or more biomarkers compared to the reference levels. In further embodiments, a method of treating cancer in a human subject is provided, the method comprising a step for measuring one or more biomarkers selected from selected from NR4A1, NR4A2, NR4A3, and TOX1 from a tumor sample from the subject, and administering an A2R antagonist to the subject if the expression of the one or more biomarkers is higher than a reference level.

[0094] In any of these methods, expression of the one or more biomarkers is measured in a baseline tumor sample obtained from the subject and compared to a reference level. In some embodiments, the reference level is a) a median expression level, or an amount that is within about 30%, about 25%, or about 20% or about 15% or about 10% or about 5% above or below a median expression level of the one or more biomarkers measured in a group of subjects with the same cancer; b) an expression level within the 20thto 80thpercentile for a group of subjects with the same cancer; c) an expression level at or about the 20thpercentile for a group of subjects with the same cancer; or d) the best-cut value determined based on the overall survival and / or progression free survival for a group of subjects with the same cancer. 28 299641857

[0095] In any of these or related methods, the one or more biomarkers comprise NR4A1, NR4A2, or NR4A3, or any combination thereof. In some embodiments, the one or more biomarkers comprise NR4A1, NR4A2, and NR4A3. In some embodiments, the one or more biomarkers comprise TOX1. In some embodiments, the one or more biomarkers comprise NR4A1, NR4A2, NR4A3, and TOX1. In some embodiments, the one or more biomarkers consist of NR4A1, NR4A2, or NR4A3, or any combination thereof. In some embodiments, the one or more biomarkers consist of NR4A1, NR4A2, and NR4A3. In some embodiments, the one or more biomarkers consist of TOX1. In some embodiments, the one or more biomarkers consist of NR4A1, NR4A2, NR4A3, and TOX1. In various embodiments, the expression of one or more biomarkers is measured by detecting a polynucleotide encoding the biomarker or fragment thereof. In various embodiments, the expression of one or more biomarkers is measured by detecting an NR4A1, NR4A2, NR4A3, and / or TOX1 polypeptide or fragment thereof. Methods of measuring biomarkers (e.g., as a polynucleotide or a polypeptide) are described further herein and include, but are not limited to: immunohistochemistry (IHC), immunocytochemistry, immunofluorescence microscopy, or immunophenotyping by flow cytometry for detecting a polypeptide and in situ hybridization approaches (e.g., chromogenic in situ hybridization (CISH), fluorescence in situ hybridization (FISH), RNAscopeTM, BaseScopeTM, and the like), sequencing based approaches (e.g., next-generation sequencing (NGS), RNA sequencing (RNA-Seq), single-cell RNA-seq (scRNA-seq) and the like), PCR based approaches (quantitative PCR, quantitative RT-PCR, and the like), microarray and high- throughput fluorescence-based approaches (e.g., traditional microarray technologies, Nanostring nCounter Analysis System, Nanostring GeoMX Digital Spatial Profiler and the like) or other multiplexed assays for detecting a polynucleotide.

[0096] In any of these or related methods, the A2R antagonist is a small molecule A2R antagonist. In some embodiments, the small molecule A2R antagonist is selected from etrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF- 1129, ILB2109, CS3005, and taminadenant. In some embodiments, the small molecule A2R antagonist is etrumadenant.

[0097] In some embodiments, the methods herein further comprise administering an additional therapy to the subject. In various embodiments, the additional therapy is a standard of care therapy for the cancer. In various embodiments, the additional therapy comprises one or more of an immune checkpoint inhibitor, chemotherapy, and / or radiation therapy. In 29 299641857particular embodiments, the methods of treating may comprise administering an immune checkpoint inhibitor that antagonizes PD-1, PD-L1, BTLA, LAG-3, a B7 family member, TIM- 3, TIGIT, or CTLA-4. In some embodiments, the methods of treatment may comprise administering a chemotherapy selected from a platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or a combination thereof. In some embodiments, the methods of treatment may comprise administering a chemotherapy comprising an agent selected from gemcitabine, nab-paclitaxel, paclitaxel, fluorouracil, and oxaliplatin, or a combination thereof.

[0098] In various embodiments, the cancer is pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, or prostate cancer. Other oncology or oncology-related disorders that may be treated by the disclosed methods are described elsewhere and these disclosures are incorporated by reference into this section.

[0099] In any of these embodiments, the treatment (e.g., treating the subject with the A2R antagonist) may result in an improvement in an outcome as compared to subjects who have not been treated with A2R antagonist. Improved outcomes (e.g., prolonged progression free survival, a reduced hazard ratio for disease progression or death, prolonged overall survival, and / or greater tumor size reduction) are further described elsewhere herein, and those disclosures are incorporated by reference into this section. In any of these embodiments, the treatment (e.g. treating the subject with the A2R antagonist) may result in a tumor response in the subject. Exemplary tumor responses (e.g., tumor size reduction, increased immune cell infiltration in the tumor, and / or a reduction in the amount of adenosine in the tumor) are further described elsewhere herein, and those disclosures are incorporated by reference into this section. Methods for identifying a patient for therapy

[0100] The present disclosure provides methods for identifying a patient for treatment with a therapy, such as a therapy comprising an A2R antagonist. In some embodiments, provided herein is a method for identifying a suitable candidate for a particular therapy described herein. In some embodiments, provided is a method for selecting a patient for a particular therapy described herein. In some embodiments, the patient is a patient having cancer, such as a solid tumor. The biomarkers of the below embodiments are further described elsewhere herein, and 30 299641857those disclosures are incorporated by reference into this section. Suitable samples are also described elsewhere herein and incorporated by reference into this section.

[0101] In various embodiments, therefore, a method is provided for identifying a human subject with a cancer that is responsive for treatment with an A2R antagonist, the method comprising measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 from a baseline tumor sample obtained from the subject, wherein responsiveness is characterized by an increased expression of the one or more biomarkers compared to a reference level. As described further above, the reference level herein can be a) a median expression level, or an amount that is within about 30%, or about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below a median expression level of the one or more biomarkers measured in a group of subjects with the same cancer; b) an expression level within the 20thto 80thpercentile for a group of subjects with the same cancer; c) an expression level at or about the 20thpercentile for a group of subjects with the same cancer; or d) the best-cut value determined based on the overall survival and / or progression free survival for a group of subjects with the same cancer. As described further above, the one or more biomarkers may be (a) NR4A1, NR4A2, or NR4A3, or any combination thereof, (b) TOX1, or (c) NR4A1, NR4A2, NR4A3, and TOX1. As described further above, the A2R antagonist may be a small molecule A2R antagonist. Suitable small molecule A2R antagonists are described elsewhere herein, and those disclosures are incorporated by reference into this section. In various embodiments, the small molecule A2R antagonist is selected from etrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF-1129, ILB2109, CS3005, and taminadenant. In various embodiments, the A2R antagonist is etrumadenant. Accordingly, in various embodiments, the methods provided herein identify a human subject with a cancer that is responsive for treatment with etrumadenant.

[0102] In various embodiments, the cancer may be pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, prostate cancer, or any other cancer or oncology- related condition described elsewhere herein and incorporated by reference into this section.

[0103] In various embodiments, treatment with the A2R antagonist may result in increased immune cell infiltration in the tumor and / or reduced adenosine levels in the tumor. These improved outcomes are further described elsewhere herein, and those disclosures are 31 299641857incorporated by reference into this section. In various embodiments, reduced adenosine levels in the tumor may be characterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post-treatment tumor sample obtained from the subject, as compared to the expression of the same biomarker in the baseline tumor sample. Expression of these biomarkers may be measured by detecting a polynucleotide encoding the biomarker or a fragment thereof. Alternatively, expression of these biomarkers may be measured by detecting a NR4A1, NR4A2, NR4A3, and / or TOX1 polypeptide or fragment thereof. Methods of measuring polynucleotides and / or polypeptides and fragments thereof are described elsewhere herein and incorporated by reference into this section. Improved Treatment Response

[0104] In various embodiments, the methods of treating a subject with cancer as described herein results in an improved treatment response for the subject. In various embodiments, methods provided herein that comprise administering an A2R antagonist to a subject (e.g., a subject having a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1) result in an improvement in an outcome (e.g., a treatment benefit) of the subject as compared to subjects who have not been treated with an A2R antagonist. In various embodiments, the improved outcome comprises one or more of (a) a prolonged progression free survival; (b) a reduced hazard ratio for disease progression or death; (c) prolonged overall survival and / or (d) greater tumor size reduction. Each of these outcomes are described further below. In further embodiments, the methods provided herein result in a tumor response in the subject. In various embodiments, the tumor response is (a) tumor size reduction; (b) increased immune cell infiltration in the tumor; and / or (c) a reduction in the amount of adenosine in the tumor as described further below. In an aspect, the reduction in the amount of adenosine is characterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post-treatment tumor sample obtained from the subject as compared to the expression of the same biomarker in the baseline tumor sample.

[0105] In some embodiments, the present disclosure provides a method of improving treatment response to a therapy in a human subject having a cancer. For example, the present disclosure provides a method of improving treatment response to a therapy in a human subject having a cancer with a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1, said method comprising administering an A2R antagonist (e.g., an small molecule A2R antagonist) to the subject. In some embodiments, the therapy is standard 32 299641857of care therapy. For instance, in various embodiments, a method of improving treatment response to a chemotherapy or radiation therapy is provided. In further embodiments, a method of improving treatment response to a chemotherapeutic agent is provided, wherein the chemotherapeutic agent is selected from platinum-based chemotherapeutic agent, a taxoid- based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or any combination thereof. In further embodiments, a method of improving treatment response to a chemotherapeutic agent is provided, wherein the chemotherapeutic agent is selected from gemcitabine, nab-paclitaxel, and oxaliplatin, or any combination thereof. In each of these embodiments, the improved treatment response may be an improved outcome characterized by one or more of (a) a prolonged progression free survival; (b) a reduced hazard ratio for disease progression or death; (c) prolonged overall survival; and / or (d) greater tumor size reduction. In each of these embodiments, administering the A2R antagonist may result in a tumor response in the subject. In various embodiments, the tumor response is (a) tumor size reduction; (b) increased immune cell infiltration in the tumor; and / or (c) a reduction in the amount of adenosine in the tumor.

[0106] Further embodiments of the disclosure provide for methods of increasing efficacy of a therapy (e.g., improving treatment response) in a human subject having cancer, the method comprising the steps of (a) measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX in a tumor sample from the subject; (b) comparing the expression of the one or more biomarkers with the expression of the one or more biomarkers in a reference sample; and (c) for a subject with increased expression of the one or more biomarkers in said tumor sample as compared to said reference sample, treating the subject with an A2R antagonist in combination with the therapy, thereby increasing the efficacy of the therapy.

[0107] In various embodiments, a method of increasing efficacy (e.g., improving treatment response) of chemotherapy or radiation therapy is provided. In further embodiments, a method of increasing efficacy (e.g., improving treatment response) of a chemotherapeutic agent is provided wherein the chemotherapeutic agent is selected from a platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or any combination thereof. In further embodiments, a method of increasing efficacy of a chemotherapy agent is provided, wherein the chemotherapeutic agent is selected from gemcitabine, nab-paclitaxel, and oxaliplatin, or any combination thereof. Each 33 299641857of these methods may comprise the steps of: (a) measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX in a tumor sample from the subject, (b) comparing the expression of the one or more biomarkers with the expression of the one or more biomarkers in a reference sample; and (c) for a subject with increased expression of the one or more biomarkers in said tumor sample as compared to said reference sample, treating the subject with an A2R antagonist in combination with the therapy, thereby increasing the efficacy (e.g., improving treatment response) of the therapy. In each of these methods, the human subject may have a cancer with a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 and the method comprises administering an A2R antagonist (e.g., a small molecule A2R antagonist as described herein).

[0108] In any of these embodiments, expression of one or more biomarkers is measured in a baseline tumor sample obtained from the subject and compared to a reference level. In various embodiments, the reference level is a) the median expression level, or an amount that is within about 30%, or about 25%, or about 20%, or about 15%, or about 10%, or about 5% above or below the median expression level of the same biomarker, or combination of biomarkers, measured in a group of subjects with the same cancer; b) an expression level within the 20thto 80thpercentile for a group of subjects with the same cancer; c) an expression level at or about the 20thpercentile for a group of subjects with the same cancer; or d) the best-cut value determined based on the overall survival and / or progression free survival for a group of subjects with the same cancer. In various embodiments, the one or more biomarkers are (a) NR4A1, NR4A2, or NR4A3, or any combination thereof, (b) TOX1, or (c) NR4A1, NR4A2, NR4A3, and TOX1.

[0109] In various embodiments, the improved treatment response can be identified by comparison to untreated subjects with the same cancer, subjects with the same cancer being treated with a standard therapy but not with an A2R antagonist, or subjects with the same cancer being treated with a therapy comprising an A2R antagonist but not having increased baseline expression of the biomarkers described herein. Treatment response can be assessed using known methods, for example, by following the Response Evaluation Criteria in Solid Tumors (RECIST) guidelines.

[0110] In various embodiments, the improved treatment response to a therapy may be an improved outcome characterized by one or more of (a) prolonged progression free survival as 34 299641857compared to the therapy alone; (b) a reduced hazard ratio for disease progression or death as compared to the therapy alone; (c) prolonged overall survival as compared to the therapy alone; and / or greater tumor size reduction as compared to the therapy alone. In various embodiments, administering the A2R antagonist results in a tumor response in the subject. In embodiments, the tumor response is (a) tumor size reduction; (b) increased immune cell infiltration in the tumor; and / or (c) a reduction in the amount of adenosine in the tumor. As discussed further herein, the reduction in the amount of adenosine may be characterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post-treatment tumor sample obtained from the subject, as compared to the expression of the same biomarker in a baseline tumor sample obtained from the subject.

[0111] In various embodiments, the methods of treating a subject with a therapeutically effective amount of an A2R antagonist, or a therapeutically effective amount of an A2R antagonist and an additional therapy, results in an improvement in progression free survival in the subject. The improved progression free survival can be by comparison to untreated subjects with the same cancer, subjects with the same cancer being treated with a standard therapy but not with an A2R antagonist, or subjects with the same cancer being treated with a therapy comprising an A2R antagonist but not having increased baseline expression of the biomarkers described herein. In various embodiments, treating the subject with a therapeutically effective amount of an A2R antagonist results in an increase in progression free survival by at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or greater to at least about 60%, as compared to a subject that has not received the A2R antagonist. In various embodiments, treating the subject with a therapeutically effective amount of an A2R antagonist can result in progression free survival that is increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, or at least about 100% as compared to a subject that has not received the A2R antagonist. 35 299641857

[0112] In various embodiments, the methods of treating a subject with a therapeutically effective amount of an A2R antagonist or a therapeutically effective amount of an A2R antagonist and an additional therapy results in a reduced hazard ratio for disease progression or death in the subject. The reduced hazard ratio for disease progression or death can be by comparison to untreated subjects with the same cancer, subjects with the same cancer being treated with a standard therapy but not with an A2R antagonist, or subjects with the same cancer being treated with a therapy comprising an A2R antagonist but not having increased baseline expression of the biomarkers described herein. In various embodiments, treating the subject with a therapeutically effective amount of an A2R antagonist, alone or in combination with an additional therapy, reduces the hazard ratio for disease progression or death by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% as compared to a subject that has not received the A2R antagonist. In various embodiments, treating the subject with a therapeutically effective amount of an A2R antagonist, alone or in combination with an additional therapy, reduces the hazard ratio for disease progression or death by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90% or about 90% to about 100% as compared to a subject that has not received the A2R antagonist.

[0113] In various embodiments, the methods of treating a subject with a therapeutically effective amount of an A2R antagonist or a therapeutically effective amount of an A2R antagonist and an additional therapy results in prolonged overall survival (OS) in the subject. The prolonged overall survival can be by comparison to the median overall survival in untreated subjects with the same cancer, subjects with the same cancer being treated with a standard therapy but not with an A2R antagonist, or subjects with the same cancer being treated with a therapy comprising an A2R antagonist but not having increased baseline expression of the biomarkers described herein. In various embodiments, treating the subject with a therapeutically effective amount of an A2R antagonist or a therapeutically effective amount of an A2R antagonist and an additional therapy results in an increase of overall survival by at least about 5% or greater to at least about 100%, at least about 10% or greater to at least about 95% or greater, at least about 20% or greater to at least about 80% or greater, at least about 40% or 36 299641857greater to at least about 60%, as compared to a subject that has not received the A2R antagonist. In various embodiments, treating the subject with a therapeutically effective amount of an A2R antagonist or a therapeutically effective amount of an A2R antagonist and an additional therapy can result in an overall survival that is increased at least about 5% or greater, at least about 10% or greater, at least about 15% or greater, at least about 20% or greater, at least about 25% or greater, at least about 30% or greater, at least about 35% or greater, at least about 40% or greater, at least about 45% or greater, at least about 50% or greater, at least about 55% or greater, at least about 60% or greater, at least about 65% or greater, at least about 70% or greater, at least about 75% or greater, at least about 80% or greater, at least about 85% or greater, at least about 90% or greater, at least about 95% or greater, or at least about 100% as compared to a subject that has not received the A2R antagonist.

[0114] In various embodiments, the methods of treating a subject with a therapeutically effective amount of an A2R antagonist or a therapeutically effective amount of an A2R antagonist and an additional therapy results in greater tumor size reduction in the subject. The greater tumor size reduction can be by comparison to untreated subjects with the same cancer, subjects with the same cancer being treated with a standard therapy but not with an A2R antagonist, or subjects with the same cancer being treated with a therapy comprising an A2R antagonist but not having increased baseline expression of the biomarkers described herein. In various embodiments, treating a subject with an A2R antagonist or a therapeutically effective amount of an A2R antagonist and an additional therapy may reduce tumor size by at least 5% to at least about 10%, at least about 10% to at least about 15%, at least about 15% to at least about 20%, at least about 20% to at least about 25%, at least about 25% to at least about 30%, at least about 30% to at least about 35%, at least about 35% to at least about 40%, at least about 40% to at least about 45%, at least about 45% to at least about 50%, at least about 50% to at least about 55%, at least about 55% to at least about 60%, at least about 60% to at least about 65%, at least about 65% to at least about 70%, at least about 70% to at least about 75%, at least about 75% to at least about 80%, at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 95%, at least about 95% to at least about 100% more as compared to a subject that has not received the A2R antagonist.

[0115] In various embodiments, the methods of treating a subject herein result in a tumor response in a subject. In various embodiments, the tumor response may be a reduction in tumor 37 299641857size (i.e., “tumor size reduction”), increased immune cell infiltration into the tumor and / or a reduction in the amount of adenosine in the tumor.

[0116] In various embodiments, the methods of treating a subject provided herein results in tumor size reduction. For instance, the methods of treating a subject with an A2R antagonist, alone or in combination with an additional therapy, as described herein may reduce tumor size by at least about 5% to at least about 10%, at least about 10% to at least about 15%, at least about 15% to at least about 20%, at least about 20% to at least about 25%, at least about 25% to at least about 30%, at least about 30% to at least about 35%, at least about 35% at least about 40%, at least about 40% to at least about 45%, at least about 45% to at least about 50%, at least about 50% to at least about 55%, at least about 55% to at least about 60%, at least about 60% to at least about 65%, at least about 65% to at least about 70%, at least about 70% to at least about 75%, at least about 75% to at least about 80%, at least about 80% to at least about 85%, at least about 85% to at least about 90%, at least about 90% to at least about 95%, or at least about 95% to at least about 100% as compared to a tumor size prior to administering the A2R antagonist.

[0117] In various embodiments, the methods of treating a subject as provided herein results in increased immune cell infiltration into the tumor. The immune cell infiltration may be measured directly or indirectly using methods known in the art (e.g., comparing the immune cell infiltration of a baseline sample obtained from the subject to the immune cell infiltration of a sample obtained from the same subject after administration of a therapy comprising an A2R antagonist). Immune cell infiltration can be measured using, for example, a surrogate measure of T-cell activity (e.g., the T cell-inflamed gene expression profile (GEP) signature (Cristescu et al., Science, 2018, 362(6411):eaar3593)). In an aspect, the methods of treating a subject with an A2R antagonist, alone or in combination with an additional therapy, as described herein may increase immune cell infiltration into the tumor by at least about 0.5 fold, at least about 1 fold, at least about 2 fold, at least about 5 fold, at least about 10 fold, at least about 15 fold, at least about 20 fold, at least about 25 fold, at least about 30 fold, at least about 35 fold, at least about 40 fold, at least about 45 fold, at least about 50 fold, at least about 55 fold, at least about 60 fold, at least about 65 fold, at least about 70 fold, at least about 75 fold, at least about 80 fold, at least about 85 fold, at least about 90 fold, at least about 95 fold, or at least about 100 fold as compared to levels of immune cell infiltration prior to administering the A2R antagonist. 38 299641857

[0118] Immune cell infiltration may also be measured by enumerating T cells in a given sample, or a representative area of a sample, for example by using IHC or immunofluorescence-based methods with the appropriate antibodies. In certain such embodiments, the methods of treating a subject with an A2R antagonist, alone or in combination with an additional therapy, as described herein may increase immune cell infiltration into the tumor by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% as compared to levels of immune cell infiltration prior to administering the A2R antagonist. In various embodiments, treating a subject with an A2R antagonist, alone or in combination with an additional therapy, as described herein may increase immune cell infiltration into the tumor by about 5% to about 10%, about 10% to about 15%, about 15% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100% as compared to levels of immune cell infiltration prior to administering the A2R antagonist.

[0119] In various embodiments, the methods of treating a subject with an A2R antagonist, alone or in combination with an additional therapy, as described herein result in a reduction in an amount of adenosine in the tumor. In an aspect, treating a subject with an A2R antagonist, alone or in combination with an additional therapy, may reduce adenosine levels by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100% as compared to adenosine levels in the tumor prior to administering the A2R antagonist. In various embodiments, the reduction in the amount of adenosine is characterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post- treatment tumor sample obtained from the subject, as compared to the expression of the same biomarker in the baseline tumor sample. Accordingly, a method for indirectly measuring adenosine levels in a tumor sample is also provided, the method comprising measuring the expression of one or more biomarkers selected from NR4A1, NR4A2 and NR4A3 in the tumor sample. 39 299641857

[0120] Also provided herein are methods of measuring the response of a cancer in a human subject to treatment with an A2R antagonist, the methods comprising (a) measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 from a first tumor sample obtained from the subject prior to administration of the A2R antagonist, (b) administering the A2R antagonist to the subject; and (c) measuring the expression of the one or more biomarkers from a second tumor sample obtained from the subject after administration of the A2R antagonist; wherein a response is characterized by a decrease in the expression of the one or more biomarkers in the second tumor sample compared to the first tumor sample.

[0121] In any of these methods, the A2R antagonist may be a small molecule A2R antagonist. In various embodiments, the small molecule A2R antagonist is selected from etrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF- 1129, ILB2109, CS3005, and taminadenant. In certain embodiments, the small molecule A2R antagonist is etrumadenant.

[0122] In any of these methods, the expression of one or more biomarkers may be measured by detecting a polynucleotide encoding the biomarker or fragment thereof. In any of these methods, the expression of one or more biomarkers may be measured by detecting a NR4A1, NR4A2, NR4A3, and / or TOX1 polypeptide or fragment thereof. Methods of detecting a polynucleotide and / or polypeptide are described elsewhere herein and incorporated by reference into this section.

[0123] In any of these methods, the subject may have a cancer selected from pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer, ovarian cancer, and prostate cancer or any other oncological condition that are described elsewhere herein and incorporated by reference into this section. Therapy

[0124] In various embodiments, the present disclosure contemplates the use of the biomarkers disclosed herein to affect treatment decisions (for example, for treating a human subject with cancer and / or improving treatment response to a therapy in a human subject having cancer).

[0125] In embodiments directed to treating cancer, a treatment may comprise administering an effective amount of an A2R antagonist. As used herein, the term A2R 40 299641857antagonist refers to small molecule A2R antagonists. In some embodiments the A2R antagonist is a dual A2AR / A2BR antagonist. As used herein, the term A2AR / A2BR refers to a dual A2AR and A2BR antagonist. In some embodiments the A2R antagonist is an A2AR antagonist. In other embodiments the A2R antagonist is an A2bR antagonist. In some embodiments, the A2R antagonist is a small molecule A2R antagonist. Exemplary small molecule A2R antagonists include, but are not limited to etrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF-1129, ILB2109, CS3005, and taminadenant. In some embodiments, the A2R antagonist is etrumadenant.

[0126] In some embodiments, the A2R antagonist is etrumadenant. In some embodiments, etrumadenant is orally administered (e.g., as an appropriate pharmaceutical composition) at a total daily dose of about 5 mg to about 250 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg, about 25 mg to about 250 mg, about 25 mg to about 200 mg, about 50 mg to about 250 mg, about 50 mg to about 200 mg, about 75 mg to about 250 mg, about 75 mg to about 200 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg. In some embodiments, etrumadenant is orally administered at a dose of about 5 mg / day, about 10 mg / day, about 15 mg / day, about 20 mg / day, about 25 mg / day, about 30 mg / day, about 35 mg / day, about 40 mg / day, about 45 mg / day, about 50 mg / day, about 55 mg / day, about 60 mg / day, about 65 mg / day, about 70 mg / day, about 75 mg / day, about 80 mg / day, about 85 mg / day, about 90 mg / day, about 95 mg / day, about 100 mg / day, about 110 mg / day, about 120 mg / day, about 130 mg / day, about 140 mg / day, about 150 mg / day, about 160 mg / day, about 170 mg / day, about 180 mg / day, about 190 mg / day, about 200 mg / day, about 210 mg / day, about 220 mg / day, about 230 mg / day, about 240 mg / day, or 250 mg / day). In some embodiments, etrumadenant is orally administered at a dose of about 75 mg / day. In some embodiments, etrumadenant is orally administered at a dose of about 100 mg / day. In some embodiments, etrumadenant is orally administered at a dose of about 150 mg / day.

[0127] Depending upon the disease (e.g., type of cancer), the A2R antagonist may be used as a monotherapy or may be used in combination with one or more additional therapies. In some embodiments, the A2R antagonist may be used as a monotherapy. Thus, the disclosed methods may comprise administering the A2R antagonist to the subject. In further embodiments, the A2R antagonist may be used in combination with one or more additional therapies. For example, in one embodiment, the A2R antagonist may be used in combination 41 299641857with chemotherapy. As such, the disclosed methods may comprise administering one or more than one additional therapy to the subject.

[0128] In certain embodiments, the methods herein are directed to improving treatment response to the one or more additional therapies. When used in combination, each additional therapy may be a therapeutic agent or another treatment modality. Selection of the additional therapies may be informed by the current standard of care for a particular cancer and / or mutational status of a subject’s cancer and / or stage of disease. Detailed standard of care guidelines are published, for example, by National Comprehensive Cancer Network (NCCN). See, for instance NCCN Colon Cancer v4.2024, NCCN Rectal Cancer v3.2024, NCCN Kidney Cancer, v1.2025, NCCN NSCLC v7.2024, NCCN Small Cell Lung Cancer v3.2024, NCCN Pancreatic Adenocarcinoma v2.2024, NCCN Esophageal and Esophagogastric Junction Cancers v3.2024, NCCN Gastric Cancer v2.2024, NCCN Breast Cancer v4. 2024, NCCN Ovarian Cancer / Fallopian Tube Cancer / Primary Peritoneal Cancer v3.2024, and NCCN Prostate Cancer v4.2024.

[0129] In embodiments comprising one or more additional treatment modality, the A2R antagonist may be administered before, after or during treatment with the additional treatment modality. Non-limiting examples of additional treatment modalities include surgical resection of a tumor, bone marrow transplant, radiation therapy, and photodynamic therapy. In embodiments comprising one or more additional therapeutic agents, the therapeutic agents used in such combination therapy may be formulated as a single composition or as separate compositions. If administered separately, each therapeutic agent in the combination may be given at or around the same time, or at different times. Furthermore, the therapeutic agents are administered “in combination” even if they have different forms of administration (e.g., oral capsule and intravenous), they are given at different dosing intervals, one therapeutic agent is given at a constant dosing regimen while another is titrated up, titrated down or discontinued, or each therapeutic agent in the combination is independently titrated up, titrated down, increased or decreased in dosage, or discontinued and / or resumed during a patient’s course of therapy. If the combination is formulated as separate compositions, in some embodiments, the separate compositions are provided together in a kit. 42 299641857

[0130] In some embodiments, one or more of the additional therapies is an additional treatment modality. Exemplary treatment modalities include but are not limited to surgical resection of a tumor, bone marrow transplant, radiation therapy, and photodynamic therapy.

[0131] In some embodiments of the disclosure, the additional therapy is a standard of care therapy for the cancer.

[0132] In some embodiments, one or more of the additional therapies is a therapeutic agent. Exemplary therapeutic agents include chemotherapy, radiopharmaceuticals, hormone therapies, epigenetic modulators, targeted therapies, signal transduction inhibitors, RAS signaling inhibitors, HIF inhibitors, AXL inhibitors, immunotherapeutic agents, cellular therapies, gene therapies, immune checkpoint inhibitors, and agonists of stimulatory or co- stimulatory immune checkpoints.

[0133] In some embodiments, one or more of the additional therapies is an agent that induces immunogenic cell death (e.g., chemotherapy, antitumor peptides (e.g., LTX-315), oncolytic viruses, and certain physiochemical approaches (e.g., radiation therapy, photodynamic therapy, cold plasma irradiation, etc.)). In some embodiments, the one or more additional therapies is chemotherapy and / or radiation therapy.

[0134] In some embodiments, one or more of the additional therapeutic agents is chemotherapy. The chemotherapy may be a chemotherapeutic agent or combination of chemotherapeutic agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, 43 299641857pomalidomide, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pemetrexed, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide; procarbazine; razoxane; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxanes and taxoids, e.g., paclitaxel, nab paclitaxel, and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum and platinum coordination complexes (i.e., “platinum-containing chemotherapeutic agent”) such as cisplatin, carboplatin, oxaliplatin, nedaplatin, and satraplatin; vinca alkaloids such as vinblastine, vincristine, vindesine, vinorelbine; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT11; proteasome inhibitors such as bortezomib, carfilzomib and ixazomib; topoisomerase inhibitors such as irinotecan, SN-38 (including liposomal SN38, antibody drug conjugates comprising SN38, dendrimer-enhanced SN-38, and the like), topotecan, etoposide, mitoxantrone, teniposide; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; anthracyclines and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, combination therapy comprises chemotherapy that includes one or more chemotherapeutic agents. In some embodiments, the chemotherapy comprises an agent selected from a platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or a combination thereof. In some embodiments, the chemotherapeutic agent comprises an agent selected from gemcitabine, nab-paclitaxel, paclitaxel, fluorouracil, and oxaliplatin, or a combination thereof. In one embodiment, 44 299641857combination therapy comprises chemotherapy comprising one or more of FLOX (fluorouracil, leucovorin, and oxaliplatin), FOLFOX (folinic acid, fluorouracil, and oxaliplatin), FOLFIRI (e.g., folinic acid, fluorouracil, and irinotecan), FOLFOXIRI or FOLFIRINOX (e.g., fluorouracil, leucovorin, irinotecan, and oxaliplatin), CAPOX (capecitabine and oxaliplatin), a taxane or taxoid (e.g., docetaxel, paclitaxel, nab-paclitaxel, etc.), NALIRIFOX (fluorouracil, leucovorin, liposomal irinotecan, and oxaliplatin), a fluoropyrimidine-containing chemotherapeutic agent (e.g., fluorouracil, capecitabine, floxuridine), a platinum-containing chemotherapeutic agent, a topoisomerase inhibitor, and / or gemcitabine. In some embodiments, the chemotherapy comprises nab-paclitaxel and gemcitabine.

[0135] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. As used herein, the terms “immune checkpoint inhibitor” and “CPI” may be used interchangeably to refer to an antagonist of an inhibitory or co-inhibitory immune checkpoint. Immune checkpoint inhibitors may antagonize an inhibitory or co-inhibitory immune checkpoint by interfering with receptor -ligand binding and / or altering receptor signaling. Examples of immune checkpoints (ligands and receptors), some of which are selectively upregulated in various types of cancer cells, that can be antagonized include PD-1; PD-L1 (PD- 1 ligand); BTLA (B and T lymphocyte attenuator); CTLA-4 (cytotoxic T-lymphocyte associated antigen 4); TIM-3 (T cell immunoglobulin and mucin domain containing protein 3); LAG-3 (lymphocyte activation gene 3); TIGIT (T cell immunoreceptor with Ig and ITIM domains); CD276 (B7-H3); PD-L2; Galectin 9; CEACAM-1; CD69; Galectin-1; CD113; GPR56; VISTA; 2B4; CD48; GARP; PD1H; LAIR1; TIM-1; and TIM-4; and Killer Inhibitory Receptors.

[0136] In some embodiments, an immune checkpoint inhibitor is a CTLA-4 antagonist. In further embodiments, the CTLA-4 antagonist may be an antagonistic CTLA-4 antibody. Suitable antagonistic CTLA-4 antibodies include, for example, monospecific antibodies such as ipilimumab or tremelimumab, as well as bispecific antibodies such as MEDI5752 and KN046.

[0137] In some embodiments, an immune checkpoint inhibitor is a PD-1 antagonist that blocks an interaction between PD-1 and a ligand (e.g., PD-L1). In further embodiments, the PD-1 antagonist may be an antagonistic PD-1 antibody (“an anti-PD-1 antibody”), small molecule or peptide. Suitable antagonistic PD-1 antibodies include, for example, monospecific 45 299641857antibodies such as balstilimab, budigalimab, camrelizumab, cosibelimab, dostarlimab, cemiplimab, ezabenlimab (BI-754091), MEDI-0680 (AMP-514; WO2012 / 145493), nivolumab, pembrolizumab, pidilizumab (CT-011), pimivalimab, retifanlimab, sasanlimab, spartalizumab, sintilmab, tislelizumab, toripalimab, and zimberelimab; as well as bi-specific antibodies such as LY3434172, cadonilimab, KN046, rilvegostomig, volrustomig and the like. In still further embodiments, the PD-1 antagonist may be a recombinant protein composed of the extracellular domain of PD-L2 (B7-DC) fused to the Fc portion of IgGl (AMP-224).

[0138] In some embodiments, an immune checkpoint inhibitor is zimberelimab. In some embodiments, zimberelimab is intravenously administered at a dose of about 100 mg to about 600 mg or about 200 mg to about 600 mg, about 600 mg to about 800 mg. In some embodiments, zimberelimab is intravenously administered at a dose of about 100 mg, 150 mg, 200 mg, 220 mg, 240 mg, 260 mg, 300 mg, 320 mg, 340 mg, 360 mg, 380 mg, 400 mg, 420 mg, 440 mg, or 460 mg. Doses of zimberelimab may be intravenously administered once a week, or less frequently (e.g., once every two, three, four, five, six weeks, or more). In some embodiments, a dosing cycle comprises intravenously administering zimberelimab at a dose of about 360 mg once every three weeks or at a dose of about 480 mg once every four weeks.

[0139] In some embodiments, an immune checkpoint inhibitor is a PD-L1 antagonist that blocks an interaction between PD-L1 and PD-1. In further embodiments, the PD-L1 antagonist may be an antagonistic PD-L1 antibody (“an anti-PD-L1 antibody”). Suitable antagonistic PD- Ll antibodies include, for example, monospecific antibodies such as avelumab, atezolizumab, durvalumab, BMS-936559, and envafolimab as well as bi-specific antibodies such as LY3434172 and KN046.

[0140] In some embodiments, an immune checkpoint inhibitor is a TIGIT antagonist that blocks an interaction between TIGIT and CD155. In further embodiments, the TIGIT antagonist may be an antagonistic TIGIT antibody (“an anti-TIGIT antibody”). Suitable antagonistic TIGIT antibodies include but are not limited to monospecific antibodies such as AGEN1327, AB308 (WO2021247591), BAT6021, COM902, domvanalimab, belrestotug, etigilimab, IBI-939, JS006, PM1021, dargistotug, ociperlimab, renvistobart, SEA-TGT, tiragolumab, vibostolimab; as well as bi-specific antibodies such as AGEN1777, AZD2936, D3L-002, HB036, HLX301, KA-1874, PM1009, SHR02992, and SIM0348. In certain embodiments, an immune checkpoint inhibitor is an antagonistic TIGIT antibody disclosed in 46 299641857WO2017152088 or WO2021247591. In certain embodiments, an immune checkpoint inhibitor is domvanalimab or AB308.

[0141] In some embodiments, an immune checkpoint inhibitor is domvanalimab. In some embodiments, domvanalimab is intravenously administered at a dose of about 500 mg to about 2000 mg, about 800 mg to about 1600 mg, about 600 mg to about 800 mg, about 900 mg to about 1200 mg, about 1200 mg to about 1600 mg. Doses of domvanalimab may be intravenously administered once a week, or less frequently (e.g., once every two, three, four, five, six weeks, or more). In some embodiments, a dosing cycle comprises intravenously administering domvanalimab at a dose of about 1200 mg once every three weeks or at a dose of about 1600 mg once every four weeks.

[0142] In some embodiments, an immune checkpoint inhibitor is a LAG-3 antagonist. In further embodiments, the LAG-3 antagonist may be an antagonistic LAG-3 antibody (“an anti- LAG-3 antibody”). Suitable antagonistic LAG-3 antibodies include, for example, fianlimab, BMS-986016 (WO10 / 19570, WO14 / 08218), or IMP-731 or IMP-321 (WO08 / 132601, WO09 / 44273).

[0143] In certain embodiments, an immune checkpoint inhibitor is a B7-H3 antagonist. In further embodiments, the B7-H3 antagonist is an antagonistic B7-H3 antibody (“an anti-B7- H3 antibody”). Suitable antagonist B7-H3 antibodies include, for example, enoblituzumab (MGA271; WO11 / 109400), omburtumab, DS-7300a, ABBV-155, and SHR-A1811.

[0144] In some embodiments, an immune checkpoint inhibitor is a TIM-3 antagonist. In further embodiments, the TIM-3 antagonist may be an antagonistic TIM-3 antibody (“an anti- TIM-3 antibody”). Suitable antagonistic TIM-3 antibodies include, for example, dostarlimab, sabatolimab, BMS-986258, and RG7769 / RO7121661.

[0145] In some embodiments, the one or more additional therapies comprise i) an immune checkpoint inhibitor, chemotherapy, and / or radiation therapy; ii) an immune checkpoint inhibitor that antagonizes PD-1, PD-L1, BTLA, LAG-3, a B7 family member, TIM-3, TIGIT, or CTLA-4; iii) a chemotherapy comprising an agent selected from a platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or a combination thereof; or iv) a chemotherapy comprising an agent 47 299641857selected from gemcitabine, nab-paclitaxel, paclitaxel, fluorouracil, and oxaliplatin, or a combination thereof.

[0146] In some embodiments, the additional therapy comprises one or more of an immune checkpoint inhibitor, chemotherapy, and / or radiation therapy. In some embodiments, the additional therapy comprises chemotherapy and / or radiation therapy.

[0147] In some embodiments, the additional therapy comprises an immune checkpoint inhibitor that antagonizes PD-1, PD-L1, BTLA, LAG-3, a B7 family member, TIM-3, TIGIT, or CTLA-4

[0148] In some embodiments, the one or more additional therapies comprise a chemotherapy comprising an agent selected from a platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or a combination thereof.

[0149] In some embodiments, the one or more additional therapies comprise a chemotherapy comprising an agent selected from gemcitabine, nab-paclitaxel, paclitaxel, fluorouracil, and oxaliplatin, or a combination thereof. Oncology and Oncology-related Disorders

[0150] In one or more embodiments of this disclosure, the biomarkers described herein are useful in the treatment or prognosis of a disease, e.g., cancer. In certain embodiments, the cancer may be early stage cancer, e.g., Stage I or Stage II. In other embodiments, the cancer may be locally advanced and / or unresectable, metastatic, or at risk of becoming metastatic. Alternatively, or in addition, the cancer may be recurrent or no longer responding to a treatment, such as a standard of care treatment known to one of skill in the art. Exemplary types of cancer contemplated by this disclosure include cancer of the genitourinary tract (e.g., bladder, kidney, renal cell, penile, prostate, testicular, etc.), uterus, cervix, ovary, breast, gastrointestinal tract (e.g., esophagus, oropharynx, stomach, small or large intestines, colon, or rectum), bone, bone marrow, skin (e.g., melanoma), head and neck, liver, gall bladder, bile ducts, heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain (e.g., gliomas), ganglia, central nervous system (CNS), peripheral nervous system (PNS), the hematopoietic system (i.e., hematological malignancies), and the immune system (e.g., spleen or thymus). In particular embodiments, the cancer is a solid tumor. In particular embodiments, the cancer is 48 299641857pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, or prostate cancer.

[0151] In some embodiments, the biomarkers according to this disclosure are useful in the treatment or prognosis of hematological malignancies. Exemplary types of cancer affecting the hematopoietic system include leukemias, lymphomas and myelomas, including acute myeloid leukemia, adult T-cell leukemia, T-cell large granular lymphocyte leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, acute monocytic leukemia, Hodgkin’s and Non-Hodgkin’s lymphoma, Diffuse large B Cell lymphoma, and multiple myeloma.

[0152] In another embodiment, the biomarkers according to this disclosure are useful in the treatment or prognosis of solid tumors. The solid tumor may be, for example, ovarian cancer, endometrial cancer, breast cancer, lung cancer (small cell or non-small cell), colon cancer, prostate cancer, cervical cancer, biliary cancer, pancreatic cancer, gastric cancer, esophageal cancer, liver cancer (e.g., hepatocellular carcinoma), kidney cancer (e.g., renal cell carcinoma), head-and-neck tumors, mesothelioma, melanoma, sarcomas, central nervous system (CNS) hemangioblastomas, and brain tumors (e.g., gliomas, such as astrocytoma, oligodendroglioma and glioblastomas).

[0153] In some embodiments, the biomarkers according to this disclosure are useful in the treatment or prognosis of CD73 expressing cancers (e.g., cancers with 1 % CD73 expression by tumor cell (TC) staining). Exemplary CD73 expressing cancers include colorectal cancer, pancreatic cancer, gastric cancer, non-small cell lung cancer (adenocarcinoma and squamous call carcinoma), esophageal cancer, renal cell carcinoma (clear cell or papillary), breast cancer (e.g., triple negative breast cancer), ovarian cancer, and prostate cancer. In some embodiments, the cancer has high CD73 expression.

[0154] In some embodiments, the biomarkers according to this disclosure are useful in the treatment or prognosis of colon cancer, rectal cancer, or colorectal cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the colorectal cancer is metastatic colorectal cancer.

[0155] In some embodiments, the biomarkers according to this disclosure are useful in the treatment or prognosis of lung cancer. In further embodiments, the lung cancer is 49 299641857mesothelioma, small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). In some embodiments, the lung cancer is SCLC. In other embodiments, the lung cancer is NSCLC. In still further embodiments, the NSCLC is lung squamous cell carcinoma or lung adenocarcinoma.

[0156] In some embodiments, the biomarkers according to this disclosure are useful in the treatment or prognosis of pancreatic cancer. In further embodiments, the pancreatic cancer is pancreatic neuroendocrine tumor or pancreatic adenocarcinoma.

[0157] In some embodiments, the biomarkers according to this disclosure are useful in the treatment or prognosis of gastrointestinal (GI) cancer. In some embodiments, the GI cancer is an upper GI cancer, such as esophageal or gastric cancer. In further embodiments, the upper GI cancer is an adenocarcinoma, a squamous cell carcinoma, or any combination thereof. In still further embodiments, the upper GI cancer is esophageal adenocarcinoma (EAC), esophageal squamous cell carcinoma (ESCC), gastroesophageal junction adenocarcinoma (GEJ), gastric adenocarcinoma (also referred to herein as “gastric cancer”) or any combination thereof. In some embodiments, the GI cancer is lower GI cancer. In further embodiments, the lower GI cancer is colorectal cancer.

[0158] In some embodiments, biomarkers according to this disclosure are useful in the treatment or prognosis of kidney cancer. In further embodiments, the kidney cancer is renal cell carcinoma. In still further embodiments, the renal cell carcinoma is clear cell renal carcinoma.

[0159] In some embodiments, the biomarkers according to this disclosure are useful in the treatment of breast cancer. In further embodiments, the breast cancer is hormone receptor positive (e.g., ERα-positive breast cancer, PR-positive breast cancer, ERα-positive and PR- positive breast cancer), HER2 positive breast cancer, HER2 over-expressing breast cancer, or any combination thereof. In still further embodiments, the breast cancer is triple negative breast cancer.

[0160] In some embodiments, the biomarkers according to this disclosure are useful in the treatment of ovarian cancer. In further embodiments, the ovarian cancer is metastatic. In further embodiments, the ovarian cancer is epithelial ovarian cancer (EOC). In some embodiments, the ovarian cancer is high grade serous ovarian cancer (HGSOC). In some embodiments, the 50 299641857ovarian cancer is characterized by a mesenchymal (MES) molecular subtype. In some embodiments, the ovarian cancer is resistant to therapy, including but not limited to platinum- or taxane-based therapies (e.g., platinum-resistant ovarian cancer (PROC)).

[0161] In some embodiments, the biomarkers according to this disclosure are useful in the treatment of prostate cancer. In further embodiments, the prostate cancer is prostate adenocarcinoma. In some embodiments, the prostate cancer is transitional cell carcinoma. In some embodiments, the prostate cancer is squamous cell carcinoma. In further embodiments, the prostate cancer is small cell prostate cancer. Kits, in vitro assay, etc.

[0162] The present disclosure provides kits, in vitro assays, etc. The biomarkers of the below embodiments are further described elsewhere herein, and those disclosures are incorporated by reference into this section. Suitable samples are also described elsewhere herein and incorporated by reference into this section.

[0163] In some embodiments, the kits herein can include instructions for use in accordance with any of the methods described herein. The included instructions can have a description of administration of the one or more disclosed therapeutic agents (e.g., an A2R antagonist) to treat cancer. In some embodiments, the kit can further include a description of selecting an individual suitable for treatment based on identifying whether that individual has a cancer responsive to the treatment, e.g., applying the diagnostic method as described herein.

[0164] In some embodiments, kit instructions relating to the use of one or more disclosed therapeutic agents (e.g., an A2R antagonist) can generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers can be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0165] The label or package insert indicates that the therapeutic agent(s) described herein are used for treating, delaying the onset and / or alleviating cancer. In some embodiments, instructions are provided for practicing any of the methods described herein. 51 299641857

[0166] In some embodiments, the kit includes instructions and / or appropriate reagents for measuring one or more biomarkers according to this disclosure. The kit may further comprise instructions for treating a subject based on the biomarkers measured in a sample obtained from the subject.

[0167] The kits of this disclosure are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device such as a minipump. In some embodiments, a kit has a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). In some embodiments, the container also has a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).

[0168] In some embodiments, kits herein can optionally provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides articles of manufacture comprising contents of the kits described above. EXAMPLES Example 1: Discovery of Biomarkers That are Predictive of Treatment Benefit with an Adenosine Pathway Inhibitor or Antagonist

[0169] In this example, transcriptomic data was used to identify biomarkers that are predictive of treatment benefit with an adenosine pathway inhibitor or antagonist (e.g., a CD73 inhibitor or an A2R antagonist) using the CD73 inhibitor quemliclustat.

[0170] Samples for this analysis were obtained from an open-label, dose-escalation and dose-expansion study of quemliclustat (“Q”) in combination with standard doses of gemcitabine / nab-paclitaxel (“G / nP,” 100 mg / m2and 125 mg / m2, of G and nP respectively, intravenous (IV) on days 1, 8, and 15 of each 28-day cycle) with or without zimberelimab (“Z,” a PD-1 inhibitor, 240 mg IV once every two weeks (Q2W)) in participants with advanced pancreatic cancer (“ARC-8”). The dose escalation portion enrolled patients with treatment naïve (i.e., first line (1L)) metastatic pancreatic ductal carcinoma (mPDAC) who were 52 299641857administered escalating doses of quemliclustat (IV Q2W) in combination with Z+G / nP. The study also included an expansion cohort in treatment naïve mPDAC (Cohort A), followed by a portion whereby patients with treatment naïve mPDAC were randomized 2:1 to either QZ+G / nP (Cohort A1) or Q+G / nP (Cohort A2). Based on the dose escalation phase, 100 mg of quemliclustat (Q100) was selected as an initial dose for expansion and was used in Cohort A, A1, and A2 (IV Q2W). The overall study design has been described, as well as safety and efficacy data for a pooled group of 1L mPDAC patients treated with 100 mg of quemliclustat in the dose-escalation portion, dose-expansion portion (i.e., Cohort A), or the randomized portion (i.e., Cohorts A1 and A2). See, for example, Wainberg ZA, et al. ASCO GI, Jan. 19, 2024, data cut off of June 19, 2023. This pooled group of 1L mPDAC patients is referred to in the Examples as the “pooled Q100 Q(±Z)+G / nP cohort”.

[0171] The transcriptomic data were investigated retrospectively in a biomarker evaluable patient (BEP) subset of the pooled Q100 Q(±Z)+G / n cohort. In this example, the BEP subset had a baseline biopsy sample available for sequencing. The baseline samples were formalin- fixed, paraffin embedded (FFPE) archival tumor samples obtained ≤ 24 months prior to screening or non-archival (“fresh”) FFPE biopsy samples obtained prior to the first dose of treatment on cycle 1 day 0. Baseline biopsies were available from 80 patients.

[0172] The transcriptome of the FFPE human tissue was measured using RNA sequencing (RNA-seq). Briefly, serial 4-5 micron sections were mounted on positive charged glass slides and the first slide was stained with hematoxylin and eosin (H&E). The subsequent slides were macrodissected where possible to enrich for greater than 70% tumor content. RNA and DNA were extracted using the MagMAX™ FFPE DNA / RNA Ultra Kit (ThermoFisher). RNA-seq was performed using TruSeq RNA Exome (Illumina). Gene expression was calculated from sequencing reads using a custom RNA-seq Nextflow pipeline. Briefly, reads were aligned using STAR aligner, gene counts were estimated using salmon, and resulting estimated values were transformed using trimmed mean of M values (TMM)-voom normalization.

[0173] The enrichment of specific pathway activities was evaluated using single sample Gene Set Enrichment Analysis (ssGSEA) on gene expression values. For a given gene set, ssGSEA is a non-parametric method that calculates an enrichment score for a given sample as the normalized difference between the empirical cumulative distribution function (eCDF) of gene expression ranks of gene set members compared to non-members in the universe of genes 53 299641857(Barbie D.A., et al. (2009) Nature.5; 462(7269): 108–112). A total of 88 gene signatures were evaluated in an unbiased manner, including: HALLMARK pathways (50), published signatures (36), and internal gene sets (2). See Table 1. Associations between gene signatures and patient survival (overall survival, OS, and progression-free survival, PFS) were assessed using Cox proportional hazards models and Kaplan-Meier curves (KM curves) with log-rank tests. For analysis of KM curves, biomarker cut-off values (e.g., reference values or thresholds) distinguishing “high” from “low” expression were set using a “best-cut” analysis to identify optimal cutoffs by maximizing the logrank statistic (Kassambara A., rdrr.io / cran / survminer / man / surv_cutpoint.html). To prevent outliers driving the difference, a minimal of 25% of samples needed to be in either group. A best-cut point of -0.19 (ssGSEA threshold) was used to distinguish “high” (≥ best-cut point) from “low” (< best-cut point) NR4A / TOX signatures.

[0174] Using this unbiased approach, a gene signature consisting of higher expression of each of NR4A1, NR4A2, NRA43, and TOX (referred to in this example as a “NR4A / TOX signature”) was found to be the most predictive of treatment benefit among the 88 signatures evaluated (FIG.1A and FIG.1B). This contrasts against other immune-related signatures such as a T cell-inflamed gene expression profile (GEP) signature largely associated with interferon gamma, which did not show any association with outcomes. Associations between the NR4A / TOX signature at baseline and patient survival (OS and PFS) assessed using Cox proportional hazards models and KM curves with log-rank tests and a best-cut point of -0.19 (ssGSEA score) distinguishing “high” (≥ best-cut point) from “low” (< best-cut point) NR4A / TOX signatures found that high expression of baseline NR4A / TOX signature was associated with better OS and PFS benefit from a quemliclustat-containing regimen (FIG. 2A and FIG. 2B, high vs. low HR 0.37 (0.19, 0.73) and 0.52 (0.31, 0.89), respectively for OS and PFS).

[0175] A similar analysis was performed with an NR4A family signature (i.e., expression of NR4A1, NR4A2, and NR4A3). High expression of the NR4A family signature showed significant survival benefit by Cox proportional hazards regression in a continuous manner in the ARC-8 biomarker-evaluable cohort (PFS: HR, 0.732 (95% CI: 0.56–0.96); P = 0.0238; OS: HR, 0.678 (95% CI: 0.49–0.95); P = 0.0239) (FIG. 3A and FIG. 3B). In contrast, previously published adenosine signatures (Sidders_ADO and Willingham_ADO, see Table 1) were not associated with OS or PFS in the ARC-8 biomarker evaluable cohort (FIG.2C and 54 299641857FIG. 2D). These findings suggest that NR4A family expression is also predictive of patient benefit on a CD73 inhibitor regimen rather than prognostic of general PDAC outcomes.

[0176] High expression of baseline NR4A family signature, defined using a best-cut point (ssGSEA score of -0.13) as the reference value to discriminate high vs low, was also associated with better OS and PFS (high vs. low HR 0.41 (0.2, 0.86) and 0.42 (0.23, 0.76), respectively for OS and PFS). Similar trends were observed when a median cutoff was applied (high vs. low HR 0.64 (0.36, 1.16) and 0.56 (0.33, 0.94), respectively for OS and PFS). The median PFS for the NR4AHighgroup was 11.0 months compared with 4.9 months for the NR4ALowgroup as defined by best-cut. The median OS was 26.1 months for patients in the NR4AHighgroup compared with 12.4 months for the NR4ALowgroup. Table 1: Gene Signatures

[0177] The ARC-8 trial did not contain a control arm of G / nP (i.e., treatment with G / nP ± Z in the absence of quemliclustat). Therefore to assess the predictive utility of the NR4A / TOX signature in the absence of an adenosine pathway inhibitor or antagonist, associations between 55 299641857the gene signature and patient survival (OS and PFS) were tested using a treatment-naïve mPDAC patient cohort treated with G / nP and a PD-1 inhibitor from another clinical trial (PRINCE trial, G / nP + nivolumab). The RNA-seq data from the PRINCE clinical trial was accessed from a public repository (Padrón, L.J., et al., Nat Med, 2022, 28, 1167–1177). Data from baseline samples were available for 25 patients in the G / nP + nivolumab arm of the PRINCE trial. Associations between the N4RA / TOX gene signature and patient survival (OS and PFS) were again assessed using Cox proportional hazards models.

[0178] Comparative analysis across the ARC-8 pooled Q100 Q(±Z)+G / nP and PRINCE G / nP+nivolumab cohorts revealed contrasting effects of higher NR4A / TOX signatures (FIG. 3A and FIG. 3B). In the PRINCE G / nP + nivolumab cohort, patients with higher NR4A / TOX signatures were associated with poor or neutral overall survival outcomes (hazard ratio, HR, ≥ 1). However, in the ARC-8 pooled Q100 Q(±Z)+G / nP cohort, higher NR4A / TOX signatures were associated with improved overall survival (HR < 1). This contrast in outcomes suggests that addition of an adenosine pathway inhibitor or antagonist (e.g., a CD73 inhibitor, such as quemliclustat or an A2R antagonist, such as etrumadenant) to G / nP (or G / nP + a PD-1 inhibitor) is associated with improved treatment benefit (e.g., overall survival, PFS) in biological contexts that are typically linked to poor clinical performance (e.g., 1L mPDAC treated with G / nP). The data also suggest that higher NR4A / TOX signatures, or NR4A family signatures, may be used to prospectively identify these biological contexts where the addition of an adenosine pathway inhibitor or antagonist (e.g., a CD73 inhibitor or an A2R antagonist) to a standard of care improves treatment outcomes.

[0179] The individual components of the NR4A / TOX gene signature were also assessed independently and in combinations across the ARC-8 pooled Q100 Q(±Z)+G / nP and PRINCE G / nP+nivolumab cohorts for their association with both OS and PFS (FIG. 3A and FIG. 3B). It was found that higher expression of each component individually (e.g., NR4A1, NR4A2, NR4A3, and TOX, each individually), as well as combination of NR4A family members (e.g., NR4A1, NR4A2, and NR4A3), and combination of NR4A family members and TOX (e.g., NR4A1, NR4A2, NR4A3 and TOX) were associated with better outcomes in ARC-8.

[0180] To begin to assess the potential contribution of zimberelimab to these observations, we compared the clinical outcomes of NR4AHighand NR4ALowgroups in the Q+Z+G / nP (n = 65) and Q+G / nP (n = 15) cohorts. We observed similar PFS and OS trends in both clinical 56 299641857cohorts, suggesting that the presence of zimberelimab did not contribute meaningfully to the predictive value of NR4A gene expression and that this signature is likely reflecting the impact of quemliclustat on modulating adenosine biology in the tumor. Example 2: Impact of Adenosine Modulation on Treatment Benefit

[0181] To investigate whether treatment benefit with an adenosine pathway inhibitor or antagonist (e.g., a CD73 inhibitor, an A2R antagonist) is linked to adenosine modulation, changes in adenosine were indirectly measured by assessing changes in expression of NR4A family members using baseline and on-treatment patient biopsies obtained from two different clinical trials; ARC-8 with quemliclustat (CD73 inhibitor) and ARC-9 with etrumadenant (A2AR / A2BR antagonist).

[0182] Transcriptomic data were investigated retrospectively in a BEP subset of the trials. In this example, in order to evaluate potential treatment-related changes in gene expression, the BEP subset included patients that had a baseline biopsy sample and an on-treatment biopsy sample available for sequencing. The baseline samples were FFPE archival tumor samples obtained ≤ 24 months prior to screening or non-archival (“fresh”) FFPE biopsy samples obtained prior to the first dose of treatment on cycle 1 day 0, while on-treatment biopsies were collected at day 29 (i.e., Cycle 2 day 1) ± 7 days.

[0183] In ARC-8, the BEP subset was from the pooled Q100 Q(±Z)+G / nP cohort.37 paired biopsy samples were available. In ARC-9, the BEP subset was from the etrumadenant (E) experimental arm of Cohort B. Cohort B enrolled third-line (3L) metastatic colorectal cancer (mCRC) patients who previously progressed on both oxaliplatin and irinotecan containing regimens. Patients in Cohort B were randomized in a 2:1 ratio to receive EZFB: E (150 mg orally [PO] once daily [QD]) + Z (240 mg IV Q2W) + mFOLFOX-6 + bevacizumab (5 mg / kg IV Q2W), or regorafenib (160 mg PO QD [days 1-21 every 4 weeks]).10 paired biopsy samples from the EZFB arm of Cohort B were available. To evaluate potential treatment-related changes in gene expression, RNA-seq data was obtained and processed as described in Example 1 and ssGSEA scores of the NR4A gene family and the T cell-inflamed GEP signature were calculated.

[0184] Significant downregulation of NR4A1, NR4A2 and NR4A3 expression (“NR4A family expression”) in on-treatment tumor samples was observed at Cycle 2, Day 1 (C2D1) 57 299641857compared to baseline tumor samples for both trials (FIG. 4A and FIG. 5A). Patients in both trials were further stratified by baseline median NR4A family expression. Notably, tumors with higher baseline levels of NR4A family expression exhibited a larger decrease in NR4A family expression compared to those with lower baseline levels (FIG. 4A, FIG. 4B, FIG. 5B, FIG. 5C).

[0185] A significant increase in inflammation, as measured by the T cell-inflamed GEP signature, was observed for both ARC-8 and ARC-9 in the on-treatment biopsies compared to the baseline biopsies (FIG. 6A). This suggested treatment with quemliclustat or etrumadenant led to an increase in tumor inflammation.

[0186] A potential association between the magnitude of NR4A change and quemliclustat mechanism of action was hypothesized. The paired molecular samples (n = 37) from ARC-8 were stratified into "maximal decrease" versus "minimal decrease" subgroups based on the median cut of NR4A change after quemliclustat treatment (post-treatment to pre-treatment). Based on the immunosuppressive role of adenosine and pre-clinical data, several T-cell activation signatures were evaluated in the paired samples (see Table 1). Significant upregulation was observed of multiple signatures in the NR4A maximal decrease but not in the NR4A minimal decrease subgroup. Gene-level analysis revealed significant upregulation of T- cell lineage / activation markers (CD8A, EOMES) and effector molecules (FASLG, GZMA, GZMK, PRF1) exclusively in the NR4A maximal decrease subgroup (FIG.6B). These findings suggest that high magnitude NR4A downregulation following quemliclustat treatment may be associated with the degree of T-cell activation, potentially shedding light on a mechanism of action of treatment in ARC-8.

[0187] We further examined NR4A family expression levels in relation to pharmacodynamic (PD) changes in the ARC-8 BEP subset. Patients were stratified by median change in NR4A family expression (post-treatment – pre-treatment) into patients with the highest decrease (greater than or equal to the median change) and patients with the lowest decrease (less than the median change) of NR4A family expression, visualized using a series of boxplots. Survival analysis using a Kaplan-Meier curve and logrank test was performed to investigate the association of overall survival (OS) against patients with lowest versus highest decrease of NR4A family expression. Patients with the highest decreases in NR4A family expression were associated with longer OS benefit (HR: 0.24 [0.08, 0.67]) (FIG.7A). A similar 58 299641857trend was observed using a Kaplan-Meier analysis investigating progression free survival (FIG. 7B). These results demonstrated that the magnitude of NR4A family expression downregulation after treatment was predictive of clinical outcomes in ARC-8 and further supported a link between clinical benefit of quemliclustat regimens and adenosine modulation in the tumor microenvironment (TME).

[0188] In the ARC-9 BEP subset, the relationship between tumor size changes and NR4A family expression PD changes was assessed using waterfall plot analysis. Patients exhibiting the highest decreases in NR4A family expression experienced the most significant reductions in tumor size (FIG. 8).

[0189] Taken together, these observations suggest that NR4A family expression may be a surrogate measure of adenosine levels and that its downregulation may be associated with an increase in tumor inflammation by IFNγ. The association with outcomes establishes a compelling link between treatment benefit from a CD73 inhibitor (e.g., quemliclustat) and an A2R antagonist (e.g., etrumadenant) with NR4A family expression changes, thereby suggesting that adenosine modulation by either mechanism of action plays a role in the treatment’s efficacy. Example 3: Biomarkers as a Proxy for Tumor Adenosine Levels Example 3A

[0190] In this example, isolated human cells in tissue culture were treated with vehicle, stimulated with the adenosine receptor agonist, NECA, or provided with adenosine monophosphate (AMP) that was converted into adenosine by the endogenously expressed CD73 on the cell surface. Cancer cell lines NCI-H650 (lung), HCT 116 (colorectal), AsPC-1 (pancreatic) and PANC-1 (pancreatic) as well as pancreatic tumor fibroblasts (PDAC CAF), CD8+T cells and CD4+T cells were evaluated for NR4A family gene expression after treatment with vehicle, upon NECA stimulation, or upon accumulation of extracellular adenosine hydrolyzed from AMP by CD73 on the cultured cells. We also examined ex vivo human macrophages in their naïve M0 state and successfully polarized them into M2-like macrophages. After this transformation, we assessed their ability to induce the NR4A gene family when stimulated with NECA. It is important to note that because these cells did not 59 299641857express CD73, we did not evaluate the NR4A gene family in response to the accumulation of extracellular adenosine hydrolyzed from AMP by CD73.

[0191] Gene expression levels measured after vehicle treatment were set as “1.0”. As shown in Table 2 below, all depicted cell types showed increases in the expression of NR4A genes above the 1.0 vehicle control upon NECA or AMP administration to the cells. Notably, the gene expression increases were suppressed below their respective levels in the NECA group through the administration of the A2AR / A2BR adenosine receptor antagonist, etrumadenant (etruma). The results demonstrate that the A2R adenosine receptors are critical for NR4A induction upon extracellular adenosine-mediated signaling. Furthermore, the addition of quemliclustat (quemli), an inhibitor of CD73 enzymatic activity, suppressed the NR4A gene expression changes below the respective levels observed in the AMP groups demonstrating the similar effects can be produced with both etruma and quemli in the appropriate cellular context. Altogether, these results demonstrate that the NR4A gene family comprised of NR4A1, NR4A2, and NR4A3 can be modulated through extracellular adenosine signaling and that those effects can be suppressed through the activity of an adenosine pathway antagonist or inhibitor, such as etruma or quemli, respectively. Table 2. Gene expression fold change over vehicle controls (vehicle = 1.0)60 299641857n.e. – not evaluated Materials and Methods

[0192] Quemliclustat (quemli) and etrumadenant (etruma) were supplied by Arcus Biosciences. 5'-N-Ethylcarboxamidoadenosine (NECA) was obtained from Tocris Bioscience (Cat. 16-911-0) and dissolved in DMSO. Adenosine-5'-monophosphate (AMP) was obtained from ThermoFisher Scientific (Cat. J61643.06) and dissolved in DMSO. Erythro-9-(2- Hydroxy-3-nonyl)adenine (EHNA) was obtained from Sigma-Aldrich (Cat. 324630) and dissolved in water. The adenosine deaminase inhibitor, EHNA, was added to these cultures in order to stabilize the adenosine produced from AMP.

[0193] Panc-1 cancer cell line was obtained from ATCC (Cat. CRL-1469) and cultured in Dulbecco’s Modified Eagle’s Medium from Gibco (Cat. 11966025) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL Penicillin-Streptomycin. AsPc-1 cancer cell line was obtained from ATCC (Cat. CRL-1682) and cultured in RPMI-1640 from Gibco (Cat. 11875085) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL Penicillin- Streptomycin. NCI-H650 cancer cell line was obtained from ATCC (Cat. CRL-5835) and cultured in RPMI-1640 from Gibco (Cat.11875085) supplemented with 5% fetal bovine serum (FBS) and 100 U / mL Penicillin-Streptomycin.

[0194] HCT 116 cancer cell line was obtained from ATCC (Cat. CCL-247) and cultured in McCoy’s 5A (Modified) Medium from Gibco (Cat.16600082) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL Penicillin-Streptomycin. Primary PDAC-derived CAFs were acquired from Neuromics (Cat. ABCTC5543) and were provided in passage 1 (p1) and cultured in a complete human pancreatic growth medium (Cat. ABMTM5543). Human T cells were isolated from healthy donor blood using the EasySepTMHuman T cell isolation kits (Stemcell Technologies, #17592, 17953) and cultured in CTS OpTmizer (Cat. A1048501). All cells were authenticated using short tandem repeat DNA profiling and tested for pathogen 61 299641857contamination, including Mycoplasma spp. (STAT-Myco and IMPACT II testing) via RT-PCR (IDEXX Bioresearch). All cell lines were maintained at 37°C, 21% O2, and 5% CO2.

[0195] In vitro polarized M0 and M2 human monocyte-derived macrophages were prepared from CD14+ monocytes isolated from healthy human donors' peripheral blood mononuclear cells (PBMCs). The CD14+ monocytes were then cultured in a complete growth medium (RPMI supplemented with 10% heat-inactivated FBS, two mM L-glutamine, 25 U / ml penicillin, and 25 μg / ml streptomycin). Subsequently, the human CD14+ monocytes were differentiated into uncommitted macrophages (M0) by exposing them to macrophage colony- stimulating factor (M-CSF) for seven days at 5% CO2and 37 °C. M2 polarization was achieved by culturing cells for the final 24 hours in interleukin-4 (IL-4).

[0196] Applied Biosystems TaqMan Gene Expression assays (FAM or VIC modified) were used for quantitative real-time PCR analysis of NR4A1 (assay ID. Hs00374226), NR4A2 (assay ID. Hs01117527), and NR4A3 (assay ID. Hs00545009) expression levels. HPRT1 (assay ID. Hs02800695) and ACTB (assay ID. Hs01060665) were reference genes.

[0197] Cells were plated in 6-well plates (1 x 105-2 x 105cells per well) 24h before treatment. Cells were serum-starved overnight and then stimulated with NECA (5 μM) + / - etruma (2 μM) or EHNA (2.5 μM) & AMP (50 μM) + / - quemli (2 μM) for 6 hours. The cells were then lysed, and RNAs were extracted from each condition. SuperScript IV Reverse Transcriptase (RT) was used for highly efficient full-length cDNA synthesis. Changes in NR4A gene expression levels were analyzed using 2-ΔΔCT. Gene expression levels were expressed as fold change over the vehicle condition (DMSO). Example 3B

[0198] The effect of chemotherapy on NR4A expression was also assessed by leveraging a public single nucleus RNA-sequencing (snRNA-seq) data of patients who underwent surgical resection with or without neoadjuvant chemotherapy treatment (Hwang et al., Nat Genet 54, 1178-1191 (2022)). Processed snRNA-seq data was downloaded from Gene Expression Omnibus, accession ID GSE202051, and was analyzed using the R programming language using the Seurat package. The author-normalized RNA assay and reductions for PCA, harmony and the uniform manifold approximation and projection (UMAP) were extracted and converted to a Seurat object for further analysis. Author cell type annotations were used for analysis. 62 299641857AUCell (Aibar et al., Nat Methods 14, pages 1083–1086 (2017)) was used to calculate geneset scores for the NR4A family.

[0199] NR4A genes were upregulated in many cell types in the TME, including CAFs, endothelial, pericyte, malignant epithelial and myeloid cells (FIG.9A). Individual members of the NR4A family were upregulated at varying degrees by different cell types (FIG. 9A). Of note, NR4A1 was expressed at high levels across many cell types and showed the most differential expression between treated compared with untreated samples. This may reflect, in part, generation of adenosine as a result of ATP release following cytotoxic killing of cancer cells. Example 3C

[0200] Also investigated was a potential mechanism of NR4A gene regulation through the cyclic adenosine monophosphate (cAMP)–protein kinase A (PKA)–cAMP-responsive element binding protein (CREB) pathway downstream of adenosine receptor activation. PANC-1 cancer cell line was obtained from ATCC (Cat. CRL-1469) and cultured in Dulbecco’s Modified Eagle’s Medium from Gibco (Cat. 11966025) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL Penicillin-Streptomycin. HCT 116 cancer cell line was obtained from ATCC (Cat. CCL-247) and cultured in McCoy’s 5A (Modified) Medium from Gibco (Cat. 16600082) supplemented with 10% fetal bovine serum (FBS) and 100 U / mL Penicillin- Streptomycin. Cultured cells were stimulated with 0.2 or 2.0 μM of forskolin, an adenylyl cyclase activator, for 1 hour to activate the CREB pathway. Alternatively, cells were stimulated with EHNA (2.5 μM) & AMP (50 μM) for 1 hour in the presence of 2.0 μM CREB inhibitor, 666-15, (Cat. HY-101120). Gene expression changes of NR4A family members were then analyzed by real-time PCR.

[0201] Treatment of PANC-1 or HCT 116 cells with forskolin, an adenylyl cyclase activator, resulted in dose-dependent upregulation of all three NR4A family members (FIG. 9B). PANC-1 and HCT 116 cells treated with AMP+EHNA showed significant NR4A upregulation, while co-treatment with a CREB inhibitor inhibited adenosine-mediated induction to near-baseline levels (FIG. 9B). These results confirmed that adenosine can regulate NR4A expression through the cAMP-PKA-CREB signaling axis, establishing NR4A family members as downstream targets of adenosine biology (FIG. 9C). 63 299641857Example 4:

[0202] A mechanism of action of quemliclustat may involve the reversal of an immunosuppressive adenosine environment and a subsequent increase in cytotoxic T-cell abundance and activation in the vicinity of cancer cells expressing high levels of NR4A in the TME. In this example, the spatial expression patterns of NR4A gene family members were evaluated in tissue samples from ARC-8 patients, and the abundance and spatial distribution of NR4A1 expressing cells and activated T cells was assessed.

[0203] The spatial expression patterns of NR4A gene family members were evaluated in tissue samples from ARC-8 patients using dual NR4A1 / IFNγ in situ hybridization (ISH). A total of 71 tumor samples from the dose expansion cohort were assessed.

[0204] ISH protocols for NR4A1, NR4A2, and NR4A3 were developed and tested on cell line controls and procured PDAC samples and showed that NR4A1 was the most abundantly and broadly expressed family member in multiple cell types in the TME, including cancer cells, CAFs and myeloid cells, consistent with the findings from snRNA-seq analyses described in Example 3B. Additionally, a multiplex immunofluorescence assay was deployed on tumor tissues to quantify the numbers and spatial distribution of cytotoxic, regulatory and exhausted T cells in the TME.

[0205] The abundance and spatial distribution of NR4A1 expressing cells and activated T cells was assessed, as indicated by IFNγ expression in 150 different T-cell enriched regions of interest (ROIs), and determined a median T-cell content of 329.8 CD3+cells / mm2and 78.44 CD8+T cells / mm2(means of 597.4 and 280.5 cells / mm2, respectively). Using the 75th percentile of average NR4A1 copies per cell as a threshold, NR4A1 expression was categorized into high and low status. There were significantly fewer IFNγ copies per cell in NR4A1Highversus NR4A1LowROIs (FIGS.3A, 3B, and 3C). To confirm that the IFNγ finding was indeed a measurement of IFNγ-expressing CD8+T cells, fused images were generated, combining the ISH- and multiplex immunofluorescent (mIF)-stained serial sections, and demonstrated that IFNγ in CD8+T cells and NR4A1 were largely co-localized to cancer cells. 64 299641857

[0206] Next, proximity analysis was conducted to quantify the number of T cells in close proximity to cancer cells, which are often the predominant source of NR4A1 expression in the TME. Interestingly, NR4AHightumors (n = 10) showed significantly fewer T cells within 50 μm of PanCK+cancer cells compared with NR4ALowtumors (n = 22), with a mean of 57% of total T cells versus 80%, respectively (P = 0.0055). This difference was driven by T cells in the range of 0–10 μm and 10–20 μm, which were scarce in NR4AHightumors versus NR4ALowtumors, with means of 15% versus 32% within 10 μm (P = 0.0141) and 14% versus 21% within 10–20 μm (P = 0.0170), respectively. This finding was also demonstrated by an average distance of CD3+to PanCK+cells of 66 μm in NR4AHightumors versus 23 μm in NR4ALowtumors (P = 0.0066).

[0207] Expanding on the spatial distribution of T cells in relation to NR4A status and an immune suppressed TME, a nearest neighbor and proximity analysis was performed on the dual ISH slides to evaluate adenosine’s influence on immune activation using NR4A1 expression as an indicator of adenosine levels and IFNγ as a marker of T-cell activation and inflammation. Concentric circles drawn around NR4A1Lowand NR4AHighcopy number regions in the TME were used to quantify the percentage of IFNγ+cells within 10 μm intervals of NR4A1 expressing cells. The percentage of total IFNγ+cells located across 10 μm intervals within a 50 μm distance from NR4A1Highcells was significantly lower than that observed for NR4A1Lowcells. These spatial analyses showed that IFNγ+T cells were distributed significantly further away from cells expressing high levels of NR4A1 (67% of IFNγ+cells located >50 μm away) compared with cells with low NR4A1 expression (37% of IFNγ+cells located >50 μm away) (P < 0.0001). This spatial relationship was also reflected in IFNγ+T cell average distance measurements of 74.6 μm relative to cells with NR4A1Highcopy numbers versus 40.9 μm relative to NR4A1Lowregions (P = 0.0171). These findings support the presence of an adenosine immunosuppressive gradient effect that results in a scarcity of IFNγ+T cells in close proximity to cancer cells expressing high levels of NR4A in the TME. Methods

[0208] Dual in situ hybridization (ISH). Dual ISH was used to detect mRNA transcripts for NR4A1 and IFNγ in FFPE tissue sections. Staining was performed on the Leica Bond Rx automated staining platform using the RNAscope™ 2.5 LS Duplex Reagent Kit (Cat# 322440, 65 299641857Advanced Cell Diagnostics, Newark, California) according to the manufacturer’s recommended protocol.

[0209] Multiplex immunofluorescence (mIF) staining. mIF staining was performed on the Leica Bond Rx automated staining platform using the Opal 6-plex Detection Kit (Cat# NEL871001KT, Akoya Biosciences, Marlborough, Massachusetts) for sequential staining of each marker. Markers were optimized, validated and tested in multiple staining positions subjected to multiple rounds of heat-induced epitope retrieval at pH = 9.0 (BOND Epitope Retrieval Solution 2, Cat# AR9640, Leica Biosystems, Deer Park, Illinois) to determine sequence position and antibody stripping efficiency using single-plex chromogenic detection before incorporation into the mIF panel. The final panel used included LAG3 (clone 12H6, Cat# PA0300, Leica Biosystems, Deer Park, Illinois), TOX (clone E613Q, Cat# 73758S, Cell Signaling Technology, Danvers, Massachusetts), CD3 (clone LN10, Cat# PA0553, Leica Biosystems, Deer Park, Illinois), PanCK (clone AE1 / AE3 + 5D3, Cat# ab86734, Cambridge, United Kingdom), FoxP3 (clone D2W8E, Cat# 98377S, Cell Signaling Technology, Danvers, Massachusetts) and CD8 (clone D8A8Y, Cat# 85336S, Cell Signaling Technology, Danvers, Massachusetts) in corresponding positions one through six. FFPE slides were cut at 4–5 μm thickness on a Leica rotary microtome and baked at 60ºC for 1 hour; deparaffinization, rehydration, peroxidase blocking and antigen retrieval were performed according to the standard Leica Bond IHC protocol. Each marker was detected in the order listed above using MACH2 Universal HRP Polymer (Cat# M2U522L, Biocare Medical, Pacheco, California) followed by OPAL dyes 690, 520, 570, 480, 620, and lastly OPAL TSA DIG followed by OPAL 780. A heat-induced primary antibody stripping step of 20 minutes at 98ºC in pH = 9.0 buffer was performed after detection of each of the first five primary antibodies. Lastly, spectral DAPI was applied, and slides were mounted using Prolong Diamond Antifade Mountant (P36961, Life Technologies, Carlsbad, California).

[0210] Dual ISH Analysis. ISH module v4.2.11.14 was used for RNAScope™ analysis. Green chromogen signal was used for detection of NR4A1 and red chromogen for detection of IFNG. An exclusion stain was designated to exclude any artifacts. RNAscope™ Cell Scoring within the ISH module was used for scoring probe copies according to 1+, 2+, 3+ and 4+ scores based on minimum copies / cell of 1, 4, 10 and 16. Quantification of probes was performed according to tumor and stroma classification previously described. Cells with NR4A1 less than four copies were termed as NR4A1Low, and cells with NR4A1 with more than 16 copies were 66 299641857termed as NR4A1HighCells with no NR4A1 copies were termed as NR4A1-negative. IFNγ+ cells are defined as all cells that are positive for IFNγ irrespective of copy number.

[0211] Image fusing. Chromogenic RNAScope™ images were deconvoluted and converted into pseudofluorescent images using the Deconvolution module (version 2.0.1) in HALO software (Version 4.0, Indica Labs, Albuquerque, New Mexico). The deconvoluted RNAScope™ image was then registered with its corresponding mIF image using serial stain registration. The registered deconvoluted RNAScope™ image and mIF image were fused using serial stain fusing to create a composite image of ISH + mIF. The fused images generated were used for demonstrating the levels and spatial proximity of immune phenotypes, such as CD3+ / CD8+ in relation to NR4A1Highor NR4A1Lowcopy cells and expression of IFNG.

[0212] Spatial analysis. Spatial plots were generated using object data to show spatial location of immune phenotypes (CD8+, CD3+ / CD8+, CD3+ / LAG3+, CD8+ / Lag3+, CD3+ / FoxP3+) in relation to PanCK+cells and NR4A1 high or low copy cells. Nearest neighbor and proximity analyses were performed HALO Spatial Analysis module. Nearest neighbor analysis was performed on RNAscope™ images to quantify the distance between IFNγ+cells and NR4A1Negative, NR4A1Lowcopy and NR4A1Highcopy cells. Proximity analysis was performed on RNAScope™ images to quantify %IFNγ+cells in 10 μm intervals within 50 μm from NR4A1Lowcopy and NR4A1Highcopy cells. For mIF staining, proximity analysis was performed to measure CD3+T cells and T-cell subsets in 10 μm intervals within 50 μm of PanCK+ cancer cells. Example 5: Protein and RNA Transcript Expression are Correlated

[0213] The Clinical Proteomic Tumor Analysis Consortium (CPTAC) is a National Cancer Institute (NCI) program that uses proteogenomics to study tumors and understand the molecular basis of cancer across several indications. CPTAC datasets measure DNA, RNA, and protein molecules in tumors and nearby normal tissues, and include data such as whole- genome sequencing, RNA sequencing, and clinical and protein expression data. Data was accessed through CPTAC’s Google BigQuery platform. RNA transcript level expression was measured using log2(FPKM). Protein expression was quantified using the protein_abundance_log2ratio, a metric derived by calculating the log2 of the ratio between the gene / protein abundance in the sample of interest and that in control samples. 67 299641857

[0214] In this example, tumor samples were evaluated, specifically those with both protein and RNA expression data available. The analysis included samples from all tumor types represented in the CPTAC dataset. The analysis revealed a strong linear correlation between RNA and protein levels for each gene (NR4A1, NR4A2, NR4A3, TOX) within the NR4A / TOX signature that is statistically significant (p-value < 0.05) (FIG. 10A and FIG. 10B). 68 299641857

Claims

CLAIMS What is claimed is:

1. A method of treating a human subject with a cancer having a high expression of one ormore biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1, said method comprising administering to the subject a therapeutically effective amount of an A2R antagonist, wherein the high expression of one or more of the biomarkers is characterized by a baseline tumor sample obtained from the subject having an increased expression level of one or more of the biomarkers, compared to a reference level for each biomarker.

2. A method of claim 1, wherein the A2R antagonist is an A2AR / A2BR antagonist.

3. A method of claim 1, wherein the A2R antagonist is an A2AR or A2BR antagonist.

4. The method of any one of claims 1-3, wherein the reference level is:a. a median expression level, or an amount that is within about 30%, or about 25%,or about 20%, or about 15%, or about 10%, or about 5% above or below a median expression level of the one or more biomarkers measured in a group of subjects with the same cancer; b. an expression level within the 20th to 80th percentile for a group of subjects withthe same cancer; c. an expression level at or above the 20th percentile for a group of subjects withthe same cancer; or d. a best-cut value determined based on overall survival and / or progression freesurvival for a group of subjects with the same cancer.

5. The method of any one of claims 1-4, wherein the one or more biomarkers are:a. NR4A1, NR4A2, or NR4A3, or any combination thereof;b. TOX1; orc. NR4A1, NR4A2, NR4A3, and TOX1.69 2996418576. The method of any one of claims 1-5, wherein the A2R antagonist is a small moleculeA2R antagonist.

7. The method of claim 6, wherein the small molecule A2R antagonist is selected frometrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF-1129, ILB2109, CS3005, and taminadenant.

8. The method of claim 7, wherein the small molecule A2R antagonist is etrumadenant,optionally wherein a totally daily dose of about 75 mg to about 200 mg or about 100 mg to about 150 mg of etrumadenant is orally administered.

9. The method of any one of claims 1-8, wherein the cancer is pancreatic cancer, colorectalcancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, or prostate cancer.

10. The method of any one of claims 1-9, further comprising administering an additionaltherapy to the subject.

11. The method of claim 10, wherein the additional therapy is a standard of care therapyfor the cancer.

12. The method of claim 11, wherein the additional therapy comprises one or more of animmune checkpoint inhibitor, chemotherapy, and / or radiation therapy.

13. The method of claim 12, wherein the immune checkpoint inhibitor antagonizes PD-1,PD-L1, BTLA, LAG-3, a B7 family member, TIM-3, TIGIT, or CTLA-4.

14. The method of claim 12, wherein the chemotherapy comprises an agent selected froma platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or a combination thereof.

15. The method of claim 12, wherein the chemotherapy comprises an agent selected fromgemcitabine, nab-paclitaxel, paclitaxel, fluorouracil, and oxaliplatin, or a combination thereof. 70 29964185716. The method of any one of claims 1-15, wherein the treatment results in an improvementin an outcome as compared to subjects who have not been treated with an A2R antagonist.

17. The method of claim 16, wherein the improved outcome comprises one or more of:a. prolonged progression free survival;b. a reduced hazard ratio for disease progression or death;c. prolonged overall survival; and / ord. greater tumor size reduction.

18. The method of any one of claims 1-17, wherein the treatment results in a tumor responsein the subject.

19. The method of claim 18, wherein the tumor response is:a. tumor size reduction;b. increased immune cell infiltration in the tumor; and / orc. a reduction in the amount of adenosine in the tumor.

20. The method of claim 19, wherein the reduction in the amount of adenosine ischaracterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post-treatment tumor sample obtained from the subject, as compared to expression of the same biomarker in the baseline tumor sample.

21. The method of any one of claims 1-20, wherein the expression of the one or morebiomarkers is measured by detecting a polynucleotide encoding the biomarker or a fragment thereof.

22. The method of any one of claims 1-20, wherein the expression of the one or morebiomarkers is measured by detecting a NR4A1, NR4A2, NR4A3, and / or TOX1 polypeptide or fragment thereof. 71 29964185723. A method of identifying a human subject with a cancer that is responsive to treatmentwith an A2R antagonist, the method comprising measuring the expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1 from a baseline tumor sample obtained from the subject, wherein responsiveness is characterized by an increased expression of the one or more biomarkers compared to a reference level.

24. The method of claim 23, wherein the reference level is:a. a median expression level, or an amount that is within about 30%, or about 25%,or about 20%, or about 15%, or about 10%, or about 5% above or below a median expression level of the one or more biomarkers measured in a group of subjects with the same cancer; b. an expression level within the 20th to 80th percentile for a group of subjects withthe same cancer; c. an expression level at or above the 20th percentile for a group of subjects withthe same cancer; or d. a best-cut value determined based on overall survival and / or progression freesurvival for a group of subjects with the same cancer.

25. The method of claim 23 or 24, wherein the one or more biomarkers are:a. NR4A1, NR4A2, or NR4A3, or any combination thereof;b. TOX1; orc. NR4A1, NR4A2, NR4A3, and TOX1.

26. The method of any one of claims 23-25, wherein the A2R antagonist is a small moleculeA2R antagonist.

27. The method of claim 26, wherein the small molecule A2R antagonist is selected frometrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF-1129, ILB2109, CS3005, and taminadenant. 72 29964185728. The method of claim 27, wherein the small molecule A2R antagonist is etrumadenant,optionally wherein a totally daily dose of about 75 mg to about 200 mg or about 100 mg to about 150 mg of etrumadenant is orally administered.

29. The method of any one of claims 23-28, wherein the cancer is pancreatic cancer,colorectal cancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, or prostate cancer.

30. The method of any one of claims 23-29, wherein treatment with the A2R antagonistresults in increased immune cell infiltration in the tumor and / or reduced adenosine levels in the tumor.

31. The method of claim 30, wherein the reduced adenosine levels in the tumor ischaracterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post-treatment tumor sample obtained from the subject, as compared to expression of the same biomarker in the baseline tumor sample.

32. The method of any one of claims 23-31, wherein the expression of the one or morebiomarkers is measured by detecting a polynucleotide encoding the biomarker or a fragment thereof.

33. The method of claim any one of claims 23-31, wherein the expression of the one ormore biomarkers is measured by detecting a NR4A1, NR4A2, NR4A3, and / or TOX1 polypeptide or fragment thereof.

34. A method of improving treatment response to a therapy in a human subject having acancer with a high expression of one or more biomarkers selected from NR4A1, NR4A2, NR4A3, and TOX1, said method comprising administering an A2R antagonist to the subject.

35. The method of claim 34, wherein the expression of the one or more biomarkers ismeasured in a baseline tumor sample obtained from the subject and compared to a reference level.

36. The method of claim 35, wherein the reference level is:73 299641857a. a median expression level, or an amount that is within about 30%, or about 25%,or about 20%, or about 15%, or about 10%, or about 5% above or below the median expression level of the same biomarker, or combination of biomarkers, measured in a group of subjects with the same cancer; b. an expression level within the 20th to 80th percentile for a group of subjects withthe same cancer; c. an expression level at or above the 20th percentile for a group of subjects withthe same cancer; or d. a best-cut value determined based on overall survival and / or progression freesurvival for a group of subjects with the same cancer.

37. The method of any one of claims 34-36, wherein the one or more biomarkers are:a. NR4A1, NR4A2, or NR4A3, or any combination thereof;b. TOX1; orc. NR4A1, NR4A2, NR4A3, and TOX1.

38. The method of any one of claims 34-37, wherein the therapy is a standard of caretherapy.

39. The method of any one of claims 34-38, wherein the therapy comprises chemotherapyand / or radiation therapy.

40. The method of claim 39, wherein the chemotherapy comprises a chemotherapeuticagent selected from a platinum-based chemotherapeutic agent, a taxoid-based chemotherapeutic agent, and an anthracycline-based chemotherapeutic agent, or any combination thereof.

41. The method of claim 39, wherein the chemotherapy comprises a chemotherapeuticagent selected from gemcitabine, nab-paclitaxel, and oxaliplatin, or any combination thereof. 74 29964185742. The method of any one of claims 34-41, wherein the A2R antagonist is a small moleculeA2R antagonist.

43. The method of claim 42, wherein the small molecule A2R antagonist is selected frometrumadenant, inupadenant, ciforadenant, TT-702, TT-10, TT-4, M-1069, INCB106385, PBF-1129, ILB2109, CS3005, and taminadenant.

44. The method of claim 43, wherein the small molecule A2R antagonist is etrumadenant,optionally wherein a totally daily dose of about 75 mg to about 200 mg or about 100 mg to about 150 mg of etrumadenant is orally administered.

45. The method of any one of claims 34-44, wherein the cancer is pancreatic cancer,colorectal cancer, gastric cancer, lung cancer, esophageal cancer, kidney cancer, breast cancer (e.g., triple negative breast cancer), ovarian cancer, or prostate cancer.

46. The method of any one of claims 34-45, wherein the improved treatment response ischaracterized by one or more of: a. prolonged progression free survival as compared to the therapy alone;b. a reduced hazard ratio for disease progression or death as compared to thetherapy alone; c. prolonged overall survival as compared to the therapy alone; and / ord. greater tumor size reduction as compared to the therapy alone.

47. The method of any one of claims 34-46 wherein administration of the A2R antagonistresults in a tumor response in the subject.

48. The method of claim 47, wherein the tumor response is:a. tumor size reduction;b. increased immune cell infiltration in the tumor; and / orc. a reduction in the amount of adenosine in the tumor.75 29964185749. The method of claim 48, wherein the reduction in the amount of adenosine ischaracterized by a reduced expression of one or more biomarkers selected from NR4A1, NR4A2, and NR4A3 in a post-treatment tumor sample obtained from the subject, as compared to expression of the same biomarker in a baseline tumor sample obtained from the subject.

50. The method of any one of claims 34-49, wherein the expression of the one or morebiomarkers is measured by detecting a polynucleotide encoding the biomarker or fragment thereof.

51. The method of any one of claims 34-49, wherein the expression of the one or morebiomarkers is measured by detecting a NR4A1, NR4A2, NR4A3, and / or TOX polypeptide or fragment thereof. 76 299641857

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