Methods for treating renal cell carcinoma

Abstract

Provided herein, inter alia, are methods for treating renal cell carcinoma using an adenosine A2A receptor antagonist and a CTLA-4 inhibitor, which may optionally further comprise administering a PD-1 inhibitor.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Application No. 63 / 477,256, filed December 27, 2022, the disclosure of which is incorporated herein by reference. [Background technology]

[0002] Renal cell carcinoma (also known as kidney cancer) is a cancer originating in the kidney. Renal cell carcinoma can gradually develop into widespread metastatic disease. Small, localized primary tumors originating on the surface of the renal cortex rarely cause noticeable symptoms during the early stages of the disease (e.g., stages I and II). Early-stage renal cell carcinoma is often identified incidentally by diagnostic readings (e.g., MRI scans) performed during the evaluation of an unrelated condition. As the disease progresses, symptoms may present as the classic triad of hematuria, a palpable mass in the flank or abdomen, and pain. In stages I to III, partial surgical resection of the kidney (e.g., stage I) or total surgical resection (stages II and III) remains the only known effective treatment for localized renal cell carcinoma. In 30% to 50% of patients, progression to metastatic disease (stage IV) occurs before initial diagnosis. However, traditional treatment modalities, including chemotherapy and radiation, are largely ineffective in stage IV patients. There is a need in the art for novel and effective treatments for renal cell carcinoma. The present disclosure is directed to this, as well as other, important ends. Summary of the Invention

[0003] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting, in a biological sample obtained from the patient, an increased level of gene expression of a biomarker gene relative to a control, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof; and (b) administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor. The combination of the CTLA-4 inhibitor and the adenosine A2A receptor antagonist acts in concert to eliminate and block immunosuppressive Tregs and myeloid suppressor cells.

[0004] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of gene expression of a biomarker gene compared to a control, and the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof.

[0005] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising: (a) detecting an increased level of myelosuppressive cells in a biological sample obtained from the patient compared to a control; and (b) administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor. The combination of the CTLA-4 inhibitor and the adenosine A2A receptor antagonist acts in concert to eliminate and block immunosuppressive Tregs and myelosuppressive cells.

[0006] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, the method comprising administering to the patient effective amounts of an adenosine A2A receptor antagonist and a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of myelosuppressor cells compared to a control.

[0007] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist, an effective amount of a CTLA-4 inhibitor, and an effective amount of a PD-1 inhibitor, wherein the effective amounts of the CTLA-4 inhibitor and / or the PD-1 inhibitor are reduced effective amounts.

[0008] These and other embodiments of the present disclosure are described herein. [Brief explanation of the drawings]

[0009] [Figure 1A] We show that the combination of ciforadenant with a CTLA-4 inhibitor (anti-CTLA-4 antibody) promotes superior production of pro-inflammatory cytokines compared to either agent alone, including IFN-gamma (Figure 1A), TNF-alpha (Figure 1B), IL-6 (Figure 1C), IL-12 / IL-23 p40 (Figure 1D), and CXCL10 (Figure 1E). Mouse tumors from the different treatment groups were lysed, and proteins were then extracted for cytokine measurement. [Figure 1B] We show that the combination of ciforadenant with a CTLA-4 inhibitor (anti-CTLA-4 antibody) promotes superior production of pro-inflammatory cytokines compared to either agent alone, including IFN-gamma (Figure 1A), TNF-alpha (Figure 1B), IL-6 (Figure 1C), IL-12 / IL-23 p40 (Figure 1D), and CXCL10 (Figure 1E). Mouse tumors from the different treatment groups were lysed, and proteins were then extracted for cytokine measurement. [Figure 1C]We show that the combination of ciforadenant with a CTLA-4 inhibitor (anti-CTLA-4 antibody) promotes superior production of pro-inflammatory cytokines compared to either agent alone, including IFN-gamma (Figure 1A), TNF-alpha (Figure 1B), IL-6 (Figure 1C), IL-12 / IL-23 p40 (Figure 1D), and CXCL10 (Figure 1E). Mouse tumors from the different treatment groups were lysed, and proteins were then extracted for cytokine measurement. [Figure 1D] We show that the combination of ciforadenant with a CTLA-4 inhibitor (anti-CTLA-4 antibody) promotes superior production of pro-inflammatory cytokines compared to either agent alone, including IFN-gamma (Figure 1A), TNF-alpha (Figure 1B), IL-6 (Figure 1C), IL-12 / IL-23 p40 (Figure 1D), and CXCL10 (Figure 1E). Mouse tumors from the different treatment groups were lysed, and proteins were then extracted for cytokine measurement. [Figure 1E] We show that the combination of ciforadenant with a CTLA-4 inhibitor (anti-CTLA-4 antibody) promotes superior production of pro-inflammatory cytokines compared to either agent alone, including IFN-gamma (Figure 1A), TNF-alpha (Figure 1B), IL-6 (Figure 1C), IL-12 / IL-23 p40 (Figure 1D), and CXCL10 (Figure 1E). Mouse tumors from the different treatment groups were lysed, and proteins were then extracted for cytokine measurement. [Figure 2A]The triple combination of ciforadenant, a CTLA-4 inhibitor (anti-CTLA-4 antibody), and a PD-1 inhibitor (anti-PD-1 antibody) induces activation of the IL-12 / STAT4 signaling axis (Figure 2A), resulting in skewing toward Th1 T cells (Figure 2B), and reducing exhaustion in CD8 T cells (Figure 2C). Transcription levels of the indicated genes in the IL-12 / STAT4 axis were identified using the Nanostring™ mouse bone marrow panel and analyzed with the nCounter® MAX Analysis System. Production of Th1 effector cytokines, marked by IFNγ and TNFα, from CD4 T cells and exhausted CD8 T cells, marked by Eomes (eomesodermin) and LAG3 (lymphocyte activation gene 3), from mouse tumors were detected by flow cytometry. [Figure 2B] The triple combination of ciforadenant, a CTLA-4 inhibitor (anti-CTLA-4 antibody), and a PD-1 inhibitor (anti-PD-1 antibody) induces activation of the IL-12 / STAT4 signaling axis (Figure 2A), resulting in skewing toward Th1 T cells (Figure 2B), and reducing exhaustion in CD8 T cells (Figure 2C). Transcription levels of the indicated genes in the IL-12 / STAT4 axis were identified using the Nanostring™ mouse bone marrow panel and analyzed with the nCounter® MAX Analysis System. Production of Th1 effector cytokines, marked by IFNγ and TNFα, from CD4 T cells and exhausted CD8 T cells, marked by Eomes (eomesodermin) and LAG3 (lymphocyte activation gene 3), from mouse tumors were detected by flow cytometry. [Figure 2C]The triple combination of ciforadenant, a CTLA-4 inhibitor (anti-CTLA-4 antibody), and a PD-1 inhibitor (anti-PD-1 antibody) induces activation of the IL-12 / STAT4 signaling axis (Figure 2A), resulting in skewing toward Th1 T cells (Figure 2B), and reducing exhaustion in CD8 T cells (Figure 2C). Transcription levels of the indicated genes in the IL-12 / STAT4 axis were identified using the Nanostring™ mouse bone marrow panel and analyzed with the nCounter® MAX Analysis System. Production of Th1 effector cytokines, marked by IFNγ and TNFα, from CD4 T cells and exhausted CD8 T cells, marked by Eomes (eomesodermin) and LAG3 (lymphocyte activation gene 3), from mouse tumors were detected by flow cytometry. [Figure 3-1] A high adenosine signature indicates a high ciforadenant response (e.g., increased expression levels of genes for CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2). Note that CXCL8 is equivalent to IL-8. [Figure 3-2] A high adenosine signature indicates a high ciforadenant response (e.g., increased expression levels of genes for CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2). Note that CXCL8 is equivalent to IL-8. DETAILED DESCRIPTION OF THE INVENTION

[0010] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, devices, and materials similar or equivalent to those described herein can be used in the practice of the present disclosure. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not intended to limit the scope of the present disclosure.

[0011] As defined herein, terms such as "inhibition," "inhibit," "inhibiting," and the like, with respect to a protein-inhibitor interaction (e.g., pathway inhibition), refer to adversely affecting (e.g., decreasing) the activity or function of a protein compared to the activity or function of the protein in the absence of the inhibitor. In embodiments, inhibition refers to adversely affecting (e.g., decreasing) the concentration or level of a protein compared to the concentration or level of the protein in the absence of the inhibitor. In embodiments, inhibition refers to a reduction in a disease or disease symptom. In embodiments, inhibition refers to a reduction in the activity of a specific protein target. Thus, inhibition includes at least partially or completely blocking a stimulus, reducing, preventing, or delaying activation, or inactivating, desensitizing, or downregulating signaling or enzymatic activity or the amount of a protein. In embodiments, inhibition refers to a reduction in the activity of a target protein due to a direct interaction (e.g., an inhibitor binds to the target protein). In embodiments, inhibition refers to a reduction in the activity of a target protein due to an indirect interaction (e.g., an inhibitor binds to a protein that activates the target protein, thereby preventing activation of the target protein).

[0012] The terms "inhibitor" or "antagonist" refer interchangeably to a substance that can detectably reduce the expression or activity of a given gene or protein. Antagonists can reduce expression or activity by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control in the absence of the antagonist. In embodiments, expression or activity is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more less than the expression or activity in the absence of the antagonist.

[0013] The term "adenosine A2A receptor" or "A2A adenosine receptor" as provided herein includes any recombinant or naturally occurring form of adenosine A2A receptor (ADORA2A), or a variant or homolog thereof, that maintains ADORA2A protein activity (e.g., at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% activity compared to ADORA2A). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring ADORA2A polypeptide. In an embodiment, ADORA2A is the protein identified by NCBI sequence reference GI:5921992, a homologue or a functional fragment thereof.

[0014] "Adenosine pathway inhibitor" refers to a molecule that inhibits the activity of the adenosine pathway. The adenosine pathway inhibitor may be, but is not limited to, an adenosine receptor (e.g., adenosine A2A receptor or adenosine A2B receptor) antagonist, a CD73 antagonist, a CD38 antagonist, a CD39 antagonist, or an adenosine deaminase. Examples of CD73 antagonists can be found in International Publication Nos. 2017 / 100670, 2018 / 013611, and 2018 / 187512, each of which is incorporated herein by reference in its entirety.

[0015] "Adenosine receptor antagonist" refers to a molecule that inhibits the activity of an adenosine receptor (e.g., an A2A or A2B receptor), typically by direct action. Adenosine receptor antagonists can be small molecule or polymeric antagonists. In embodiments, the adenosine A2A receptor antagonist is a ciforadenant.

[0016] The term "CTLA-4" or "cytotoxic T-lymphocyte protein 4" as provided herein includes any recombinant or naturally occurring form of the CTLA-4 protein receptor, or a variant or homolog thereof, that maintains CTLA-4 protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to CTLA-4). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring CTLA-4 protein. In embodiments, the CTLA-4 is the protein identified as UniProt number P16410, a homolog thereof, or a functional fragment thereof.

[0017] CTLA-4 is expressed in immunosuppressive T regulatory cells and, together with myeloid suppressor cells, dampens the immune response to tumors. Anti-CTLA-4 antibodies bind to CTLA-4-positive Tregs and cause their elimination.

[0018] As provided herein, a "CTLA-4 inhibitor" refers to a substance capable of detectably reducing the expression or activity level of CTLA-4 compared to a control. Inhibited expression or activity of CTLA-4 can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less of the expression or activity in a control. In embodiments, inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to a control. An "inhibitor" is a compound or small molecule that inhibits CTLA-4, for example, by binding to the CTLA-4 receptor, partially or completely blocking stimulation of the CTLA-4 receptor, reducing, preventing, or delaying the activity of the CTLA-4 protein, or inactivating, desensitizing, or downregulating the signaling, gene expression, or enzymatic activity of the CTLA-4 protein. In embodiments, a CTLA-4 inhibitor inhibits the activity or expression of CTLA-4. In embodiments, the CTLA-4 inhibitor is a chemical compound or a small molecule. In embodiments, the CTLA-4 inhibitor is an antibody. In embodiments, the CTLA-4 inhibitor is ipilimumab.

[0019] As referred to herein, "PD-1 protein" or "PD-1" includes any recombinant or naturally occurring form of programmed cell death protein 1 (PD-1), also known as cluster of differentiation 279 (CD279), or a variant or homolog thereof, that maintains PD-1 protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the PD-1 protein). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring PD-1 protein. In embodiments, the PD-1 protein is substantially identical to the protein identified by UniProt reference number Q15116, or a variant or homologue having substantial identity thereto. In embodiments, the PD-1 protein is substantially identical to the protein identified by UniProt reference number Q02242, or a variant or homologue having substantial identity thereto.

[0020] As referred to herein, "PD-L1 protein" or "PD-L1" includes any recombinant or naturally occurring form of programmed death-ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274), or a variant or homologue thereof, that maintains PD-L1 protein activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the PD-L1 protein). In embodiments, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or over a portion of the sequence (e.g., a 50, 100, 150, or 200 contiguous amino acid portion) compared to a naturally occurring PD-L1 protein. In embodiments, the PD-L1 protein is substantially identical to the protein identified by UniProt reference number Q9NZQ7, or a variant or homologue with substantial identity thereto. In embodiments, the PD-L1 protein is substantially identical to the protein identified by UniProt reference number Q9EP73, or a variant or homologue with substantial identity thereto.

[0021] As provided herein, a "PD-1 inhibitor" refers to a substance that can detectably reduce the expression or activity level of the PD-1 signaling pathway compared to a control. Inhibited expression or activity of the PD-1 signaling pathway can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less of the expression or activity of the control. In embodiments, inhibition is 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, or more compared to the control. An "inhibitor" is a compound or small molecule that inhibits the PD-1 signaling pathway, for example, by binding to the PD-1 pathway, partially or completely blocking stimulation of the PD-1 pathway, reducing, preventing, or delaying activation of the PD-1 pathway, or inactivating, desensitizing, or downregulating signal transduction, gene expression, or enzymatic activity of the PD-1 pathway. In embodiments, the PD-1 inhibitor inhibits the activity or expression of PD-1. In embodiments, the PD-1 inhibitor is a compound or small molecule. In embodiments, the PD-1 inhibitor is an antibody. In embodiments, the PD-1 inhibitor is nivolumab.

[0022] The term "myeloid cells" refers to granulocytes and monocytes, which are differentiated progeny of a common progenitor cell derived from hematopoietic stem cells in the bone marrow. Commitment to either granulocytes or monocytes is controlled by transcription factors, followed by terminal differentiation and release into the circulation in response to specific colony-stimulating factors.

[0023] The term "myelosuppressor cells" or "myeloid-derived suppressor cells" refers to a heterogeneous population of cells of myeloid origin that expands during various pathological conditions (including cancer, inflammation, and trauma) and is characterized by increased production of reactive oxygen and nitrogen species and arginase 1 activity. Myelosuppressor cells suppress T cell responses to pathological conditions such as cancer. Myelosuppressor cells can be measured by methods known in the art, such as those described in Florcken et al., Immunol Lett, 168(2):260-267 (2015).

[0024] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof, or their complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides), in either single-, double-, or multi-stranded form. In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," "oligo," and the like refer, in their normal and customary sense, to a linear sequence of nucleotides. The term "nucleoside" refers, in its normal and customary sense, to a glycosylamine containing a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide," in its normal and customary sense, refers to a single polynucleotide unit, i.e., monomer. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a modified form thereof. Examples of polynucleotides contemplated herein include single- and double-stranded DNA, single- and double-stranded RNA, and hybrid molecules containing a mixture of single- and double-stranded DNA and RNA. Examples of nucleic acids, e.g., polynucleotides, contemplated herein include all types of RNA, such as mRNA, siRNA, miRNA, and guide RNA, as well as all types of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "double-stranded" in the context of a polynucleotide refers to double-strandedness in the usual and customary sense. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides, or they can be branched, e.g., the nucleic acid contains one or more arms or branches of nucleotides. Optionally, branched nucleic acids are repeatedly branched to form higher-order structures such as dendrimers. Nucleic acids can include non-specific sequences. As used herein, the term "non-specific sequence" refers to a nucleic acid sequence containing a series of residues that are not designed to be complementary to, or are only partially complementary to, any other nucleic acid sequence.By way of example, a non-specific nucleic acid sequence is a sequence of nucleic acid residues that does not function as an inhibitory nucleic acid when contacted with a cell or organism.

[0025] A polynucleotide generally consists of a specific sequence of the four nucleotide bases: adenine (A), cytosine (C), guanine (G), and thymine (T) (if the polynucleotide is RNA, uracil (U) is substituted for thymine (T)). Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be used for the polynucleotide molecule itself. This alphabetical representation may be input into a database on a computer having a central processing unit and used in bioinformatics applications such as functional genomics and homology searching. A polynucleotide may optionally contain one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0026] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that encode the same or essentially the same amino acid sequence. Due to the degeneracy of the genetic code, many nucleic acid sequences encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," which are one type of conservatively modified variation. Every nucleic acid sequence herein that encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those of skill in the art will understand that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to result in a functionally identical molecule. Thus, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0027] The term "messenger RNA" or "mRNA" refers to a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene and is read by ribosomes in the process of synthesizing proteins.

[0028] As used herein, the term "complement" refers to a nucleotide (e.g., RNA or DNA) or sequence of nucleotides that can base-pair with a complementary nucleotide or sequence of nucleotides. As described herein and generally known in the art, the complementary (matching) nucleotide of adenosine is thymidine, and the complementary (matching) nucleotide of guanidine is cytosine. Thus, a complement may comprise a sequence of nucleotides that base-pair with the corresponding complementary nucleotides of a second nucleic acid sequence. The complementary nucleotides may partially or perfectly match the nucleotides of the second nucleic acid sequence. When the complementary nucleotides perfectly match each nucleotide of the second nucleic acid sequence, the complement forms base pairs with each nucleotide of the second nucleic acid sequence. When the complementary nucleotides partially match the nucleotides of the second nucleic acid sequence, only a portion of the complementary nucleotides form base pairs with the nucleotides of the second nucleic acid sequence. Examples of complementary sequences include coding sequences and non-coding sequences, where the non-coding sequence contains complementary nucleotides to the coding sequence and thus forms the complement of the coding sequence. Further examples of complementary sequences are sense and antisense sequences, where the sense sequence comprises complementary nucleotides to the antisense sequence, thus forming the complement of the antisense sequence.

[0029] The term "gene" refers to a segment of DNA involved in producing a protein. This includes regions preceding and following the coding region (leader and trailer), as well as intervening sequences (introns) between individual coding segments (exons). The leader, trailer, and introns contain regulatory elements required during transcription and translation of a gene. Furthermore, a "protein gene product" is a protein expressed from a particular gene.

[0030] The term "recombinant," when used with reference to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses a gene that is not found in the native (non-recombinant) form of the cell, or expresses a native gene that is otherwise abnormally expressed, under-expressed, or not expressed at all. Transgenic cells and plants are typically those that express heterologous genes or coding sequences as a result of recombinant methods.

[0031] The term "heterologous," when used with reference to portions of a nucleic acid, indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For example, nucleic acids are typically produced recombinantly, having two or more sequences from unrelated genes, e.g., a promoter from one source and a coding region from another source, arranged to create a new functional nucleic acid. Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).

[0032] The phrases "specifically (or selectively) bind" or "specifically (or selectively) immunoreact" with an antibody, when referring to a protein or peptide, often refer to a binding reaction that determines the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under specified immunoassay conditions, a particular antibody will bind to a particular protein at least twice background, and more typically 10-100 times background. Specific binding to an antibody under such conditions requires that the antibody be selected for its specificity for a particular protein. For example, polyclonal antibodies can be selected to obtain only a subset of antibodies that are specifically immunoreactive with a selected antigen and not with other proteins. This selection may be achieved by subtracting out antibodies that cross-react with other molecules. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0033] The terms "isolate" and "isolated," when applied to nucleic acids, viruses, or proteins, mean that the nucleic acid, virus, or protein is essentially free from other cellular components with which it is naturally associated. This can be, for example, a homogeneous state, either dry or in aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. RNA present as the predominant species in a preparation is substantially purified.

[0034] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, which may be conjugated to moieties not consisting of amino acids. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, as well as to naturally occurring and non-naturally occurring amino acid polymers.

[0035] "Percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) to optimally align the two sequences. This percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0036] The terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity over a specified region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with the default parameters described below, or by manual alignment and visual inspection (e.g., www.ncbi.nlm.nih.gov / BLAST / ). Such sequences are therefore "substantially identical." This definition may also refer to or be applied to the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As explained below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 25 amino acids or nucleotides in length, and more preferably over a region that is 50-100 amino acids or nucleotides in length.

[0037] The "position" of an amino acid or nucleotide base is indicated by a number that sequentially identifies each amino acid (or nucleotide base) in a reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, truncations, fusions, etc., which must be considered when determining optimal alignment, the number of amino acid residues in a test sequence, determined simply by counting from the N-terminus, is generally not necessarily the same as the number of corresponding positions in the reference sequence. For example, if a variant has a deletion compared to an aligned reference sequence, there will be no amino acid in the variant that corresponds to the position in the reference sequence at the site of the deletion. If there is an insertion in an aligned reference sequence, the insertion will not correspond to a numbered amino acid position in the reference sequence. In the case of a truncation or fusion, there may be a stretch of amino acids in either the reference sequence or the aligned sequence that does not correspond to any amino acid in the corresponding sequence. When used in the context of the numbering of a given amino acid or polynucleotide sequence, the term "numbered with reference to" or "corresponding to" refers to the numbering of residues in a particular reference sequence when a given amino acid or polynucleotide sequence is compared to the reference sequence.

[0038] The term "about" refers to a range of values ​​that includes the particular value and that one of ordinary skill in the art would consider to be reasonably similar to the particular value. In embodiments, "about" refers to within a standard deviation using measurements generally accepted in the art. In embodiments, "about" refers to a range that covers + / - 10% of the particular value. In embodiments, "about" includes the particular value.

[0039] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0040] As used herein, a "therapeutic agent" refers to an agent (e.g., a compound, biological composition, pharmaceutical composition) that, when administered to a subject, will have an intended preventative effect, e.g., treatment of renal cell carcinoma or symptoms thereof, or an intended therapeutic effect, including any objective or subjective parameter of treatment, i.e., relief, remission, reduction of symptoms, slowing the rate of degeneration or decline, causing less debilitating end points of degeneration, or improving the physical or mental health of the patient. Adenosine A2A inhibitors, CTLA-4 inhibitors, and PD-1 inhibitors are examples of "therapeutic agents."

[0041] As used herein and as well understood in the art, "treating" or "treatment" includes any approach to obtaining a beneficial clinical outcome for a patient. Beneficial clinical outcomes may include, but are not limited to, alleviation or amelioration of one or more symptoms of a disease (e.g., renal cell carcinoma), whether partial or total, and whether detectable or undetectable, reduction in the extent of the disease, stabilization (i.e., not worsening) of the disease, delay or slowing of disease progression, improvement or palliation of the disease, and remission. Treatment may alleviate symptoms of the disease, completely or partially eliminate the underlying cause of the disease, shorten the duration of the disease, or a combination thereof. A method of treatment includes administering a therapeutically effective amount of a therapeutic agent to a subject. The administering step may include a single administration or a series of administrations. The length of the treatment period will depend on various factors, such as the severity of the condition, the age of the patient, the concentration of the therapeutic agent, the activity of the composition used in the treatment, or a combination thereof. It will also be understood that the effective dosage of the therapeutic agent used for treatment may increase or decrease during a particular treatment regimen. Variations in dosage may be effected and made evident by standard diagnostic assays known in the art. Treating does not include preventing.

[0042] In embodiments, "treating renal cell carcinoma" refers to preventing an increase in size or volume of a cancerous tumor. In embodiments, treating renal cell carcinoma includes reducing the size of the cancerous tumor volume. In embodiments, treating renal cell carcinoma includes completely eliminating the cancerous tumor. In embodiments, a cancerous tumor is eliminated when it cannot be detected by imaging tests such as magnetic resonance imaging (MRI), positron emission tomography (PET) scan, X-ray computed tomography (CT), ultrasound, or single-photon emission computed tomography (SPECT). In embodiments, treating renal cell carcinoma refers to reducing or preventing metastasis of a cancerous tumor.

[0043] "Patient" or "subject in need" refers to an organism suffering from renal cell carcinoma that can be treated by administration of a therapeutic agent as provided herein. Non-limiting examples include humans and other mammals, such as dogs and cats. In an embodiment, the patient is a human.

[0044] As used herein, the terms "therapeutically effective amount" and "effective amount" refer to an amount of a therapeutic agent sufficient to treat renal cell carcinoma. For any therapeutic agent described herein, the therapeutically effective amount can be initially determined from cell culture assays. The target concentration is the concentration of the therapeutic agent that can be measured using methods described herein or known in the art to achieve the effects of the methods described herein. As is known in the art, therapeutically effective amounts for use in humans can also be determined from animal models. For example, a human dose can be formulated to achieve a concentration found to be effective in animals. A human dosage can be adjusted by monitoring the efficacy of the compound and adjusting the dosage upward or downward, as described above. Adjusting dosages to achieve maximum efficacy in humans based on the above and other methods is well within the capabilities of one of ordinary skill in the art. Dosages can vary depending on the patient's requirements and the therapeutic agent used. The dose administered to a patient should be sufficient to produce a beneficial therapeutic response in the patient over time. The size of the dose will also be determined by the existence, nature, and extent of any adverse side effects. Determining the appropriate dosage for a particular situation is within the capabilities of one of ordinary skill in the art. Generally, treatment is initiated with lower dosages that are less than the optimal dose of the compound. The dosage is then increased by small increments until the optimal effect under the circumstances is reached. Dosage amounts and intervals can be individually adjusted to provide a level of the administered compound that is effective for the particular clinical indication being treated. This will provide a treatment regimen appropriate to the severity of the individual's condition. "Therapeutically effective amounts" can also be found on the label or prescribing information of commercially available therapeutic agents. In embodiments, an "effective amount" of ciforadenant is about 200 mg per day. In embodiments, an "effective amount" of ciforadenant is about 100 mg twice per day. In embodiments, an "effective amount" of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, an "effective amount" of nivolumab is about 3 mg / kg about once every three weeks.

[0045] The term "reduced effective amount" refers to an amount that is less than the effective amount when compared to the amount normally administered for treating renal cell carcinoma. A reduced effective amount is the same dose administered less frequently, a lower dose administered at the same frequency as the normal dose, or a lower dose administered less frequently. In embodiments, a reduced effective amount is the same dose administered less frequently. In embodiments, a reduced effective amount is a lower dose administered at the same frequency as the normal dose. In embodiments, a reduced effective amount is a lower dose administered less frequently. As described herein, when a CTLA-4 inhibitor (ipilimumab), a PD-1 inhibitor (nivolumab), and an adenosine A2A receptor antagonist (ciforadenant) are administered as a combination therapy to treat renal cell carcinoma, a "reduced effective amount" of the CTLA-4 inhibitor (ipilimumab) and / or the PD-1 inhibitor (nivolumab) is effective to treat renal cell carcinoma.

[0046] The term "administering" is used according to its plain and ordinary meaning and includes oral, topical, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, or implantation of a sustained-release device, e.g., a mini-osmotic pump, to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, or transdermal). Parenteral administration includes, for example, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like. In embodiments, administration does not include administration of any therapeutic agents other than those listed.

[0047] "Biological sample" or "sample" refers to a material obtained or derived from a subject or patient. Biological samples include tissue sections, such as biopsies. Such samples include bodily fluids, such as blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, etc.), sputum, tissues, cultured cells (e.g., primary cultures, explants, and transformed cells), stool, urine, synovial fluid, articular tissue, synovial tissue, synoviocytes, fibroblast-like synoviocytes, macrophage-like synoviocytes, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, and the like. In embodiments, the biological sample is blood. In embodiments, the biological sample is a peripheral blood sample. In embodiments, the biological sample is a serum sample (e.g., the fluid and solute components of blood without clotting factors). In embodiments, the biological sample is a plasma sample (e.g., the liquid portion of blood). In embodiments, the biological sample is a tumor sample. In embodiments, the biological sample is a primary tumor sample. In embodiments, the biological sample is a metastatic tumor sample. In embodiments, the biological sample is a resected tumor sample. In embodiments, the biological sample is a tumor biopsy sample. In embodiments, the biological sample is a tumor sample resected from a primary tumor. In embodiments, the biological sample is a tumor sample resected from a metastatic tumor. In embodiments, the biological sample is a tumor biopsy sample from a primary tumor. In embodiments, the biological sample is a tumor biopsy sample from a metastatic tumor. In embodiments, the tumor sample is tumor cells. Biological samples may be obtained from a subject by methods known in the art and may be analyzed by methods known in the art.

[0048] The term "biomarker" refers to an indicator, e.g., a predictive, prognostic, and / or pharmacodynamic indicator, that can be detected in a biological sample. A biomarker can serve as an indicator of the likelihood that a patient will respond to a particular therapeutic treatment or a particular subtype of a disease or disorder characterized by certain molecular, pathological, histological, and / or clinical features. In embodiments, a biomarker is a gene or set of genes (i.e., biomarker genes). Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number changes (e.g., DNA copy number), polypeptides, or polypeptide and polynucleotide modifications (e.g., post-translational modifications). In embodiments, the biomarker genes are CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof.

[0049] The terms "expression level," "amount," or "level" of a biomarker refer to a detectable level in a biological sample. "Expression" generally refers to the process by which information (e.g., genetic code and / or epigenetic) is converted into structures present and operating in a cell. Thus, "expression" can refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide). Fragments of a transcribed polynucleotide, a translated polypeptide, or a polynucleotide and / or polypeptide modification (e.g., post-translational modification of a polypeptide) are also considered expressed, regardless of whether they are derived from a transcript generated by alternative splicing or a degraded transcript, or from post-translational processing of a polypeptide, for example, by proteolysis. "Expressed genes" include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA but not translated into a polypeptide (e.g., transfer RNA, ribosomal RNA, non-coding RNA). Expression levels can be measured by methods known to those skilled in the art and also disclosed herein. The expression level or amount of a biomarker can be used to identify / characterize subjects who are likely to respond to or benefit from a particular therapeutic agent (e.g., an adenosine A2A receptor antagonist, a CTLA-4 inhibitor, a PD-1 inhibitor).

[0050] The term "elevated expression level" or "elevated level" of gene expression is an expression level of a gene that is higher than the expression level of the gene in a control. The control can be any control known in the art, such as those described herein. In embodiments, an "elevated expression level" of a biomarker gene compared to a control (where the expression level of the biomarker is higher than the corresponding control) is, for example, an increase in expression level of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% or more compared to the control. In embodiments, an "elevated level" of a biomarker gene is an amount that is statistically significantly higher than the expression level of the control.

[0051] Biomarker levels can be detected at either the protein or gene expression level. Proteins expressed by biomarkers can be quantified by immunohistochemistry (IHC) or flow cytometry using antibodies to detect the protein. Biomarker expression can be quantified by multiple platforms known in the art. Quantifying biomarker (gene) expression can alternatively be referred to as detecting the level of biomarker (gene) expression. Platforms that can be used to quantify biomarker (gene) expression or detect biomarker (gene) expression levels include quantitative polymerase chain reaction (qPCR), multiplex quantitative polymerase chain reaction (multiplex qPCR), real-time polymerase chain reaction (rtPCR), Nanostring (e.g., an amplification-free technique that measures nucleic acid content by directly counting molecules), RNA sequencing (using next-generation sequencing (NGS) to reveal the presence and quantity of RNA in a biological sample), or in situ hybridization. Biomarker expression, as measured by Nanostring, varies widely. In embodiments, quantitative rtPCR, Nanostring, RNA sequencing (RNAseq), and in situ hybridization are used to quantify biomarker gene expression. In embodiments, biomarker expression is quantified by RNAseq. In embodiments, biomarker expression is quantified by multiplex qPCR. In embodiments, biomarker expression is quantified by NanoString. With Nanostring, RNA is extracted from a biological sample, and a known amount of RNA is placed in a Nanostring instrument to detect gene expression using gene-specific probes.The number of counts of biomarkers in the sample is determined and normalized to a set of housekeeping genes. To determine the threshold for elevated or decreased biomarker levels, one skilled in the art can evaluate biomarker levels in a control group of samples and select the 10th, 20th, 25th, 30th, 40th, 50th, 60th, 70th, 75th, 80th, or 90th percentile of biomarker gene expression. In embodiments, increased or decreased biomarker expression can be determined by calculating an H-score for biomarker expression. Thus, increased or decreased biomarker expression can have an H-score. As used herein, "H-score" or "histoscore" is a numerical value determined by commonly known semi-quantitative methods for immunohistochemically evaluating protein expression in tumor samples.

[0052] "Control" is used according to its plain and ordinary meaning to refer to an assay, comparison, or experiment in which the experimental subject or reagent is treated identically to a parallel experiment except for the omission of the experimental procedure, reagent, or variable. In embodiments, a control is used as a standard of comparison in assessing experimental efficacy. In embodiments, a control is a gene expression level that is compared against another gene expression level (e.g., a gene expression level of a biomarker gene disclosed herein) to make (e.g., a diagnostic distinction (e.g., prediction and / or prognosis) and / or treatment decision. In embodiments, the control is a healthy patient or a healthy patient population. In embodiments, a healthy patient is a patient who does not have renal cell carcinoma. In embodiments, the control is an average value from a healthy patient population. In embodiments, the control is a level of expression of a biomarker gene that correlates with responsiveness / non-responsiveness to a particular therapeutic agent. In embodiments, the control is a pre-assigned value, e.g., a cut-off value previously determined to significantly distinguish between a first group of patients (e.g., patients with renal cell carcinoma) and a second group of patients (e.g., healthy patients). In embodiments, the cut-off value is the median or average (preferably the median) of gene expression levels in a reference population. A control can also be a reference population of genes obtained, e.g., initially, from the same individual. The control can be obtained from a sample collected before disease or before treatment. Those skilled in the art will recognize that controls can be designed for any number of parameter assessments. In embodiments, the control is a negative control. In embodiments, such as some embodiments directed to detecting the expression level of a gene / protein or a subset of genes / proteins, the control comprises the average expression level (e.g., protein or mRNA) in a population of subjects (e.g., with renal cell carcinoma) or in a healthy or general population. In embodiments, the control comprises the average amount (e.g., expression level) in a population where the number of subjects (n) is 5 or more, 20 or more, 50 or more, 100 or more, 1,000 or more, etc. Those skilled in the art will understand which controls are useful in a given situation and will be able to analyze data based on comparison to the control value. Controls are also useful for determining the significance of data. For example, if the value of a given parameter varies widely in the control, the variation in the test sample is not considered significant.Other controls may be used in the methods described herein to confirm the accuracy of the results and to confirm the absence of impurities or contamination of the reagents.

[0053] method Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting an increased gene expression level of a biomarker gene in a biological sample obtained from the patient compared to a control, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof; and (b) administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor. In embodiments, the adenosine A2A receptor antagonist is ciforadenant. In embodiments, the CTLA-4 inhibitor is ipilimumab. In embodiments, the method further comprises administering to the patient an effective amount of a PD-1 inhibitor. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is tumor tissue or tumor cells. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is tumor cells. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0054] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting an increased gene expression level of a biomarker gene in a biological sample obtained from the patient compared to a control, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof; and (b) administering to the patient an effective amount of ciforadenant and an effective amount of ipilimumab. In embodiments, the effective amount of ciforadenant is about 200 mg per day, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is tumor tissue or tumor cells. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is tumor cells. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0055] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting increased gene expression levels of biomarker genes in a biological sample obtained from the patient compared to a control, wherein the biomarker genes are CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof; and (b) administering to the patient an effective amount of ciforadenant, an effective amount of ipilimumab, and an effective amount of nivolumab. In embodiments, the effective amount of ciforadenant is about 200 mg per day, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is tumor tissue or tumor cells. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is tumor cells. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma). In embodiments, the effective amount of ipilimumab and / or the effective amount of nivolumab are reduced effective amounts.

[0056] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, the method comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of gene expression of a biomarker gene compared to a control, the biomarker gene being CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof. In embodiments, the adenosine A2A receptor antagonist is ciforadenant. In embodiments, the CTLA-4 inhibitor is ipilimumab. In embodiments, the method further comprises administering to the patient an effective amount of a PD-1 inhibitor. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is tumor tissue or tumor cells. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is a tumor cell. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0057] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of ciforadenant and an effective amount of ipilimumab, wherein a biological sample obtained from the patient has an increased level of gene expression of a biomarker gene compared to a control, the biomarker gene being CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof. In embodiments, the effective amount of ciforadenant is about 200 mg per day, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is tumor tissue or tumor cells. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is a tumor cell. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0058] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of ciforadenant, an effective amount of ipilimumab, and an effective amount of nivolumab, wherein a biological sample obtained from the patient has an increased level of gene expression of a biomarker gene compared to a control, the biomarker gene being CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof. In embodiments, the effective amount of ciforadenant is about 200 mg per day, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is tumor tissue or tumor cells. In embodiments, the biological sample is tumor tissue. In embodiments, the biological sample is tumor cells. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma). In embodiments, the effective amount of ipilimumab and / or the effective amount of nivolumab are reduced effective amounts.

[0059] In an embodiment of the method described herein, the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof. These genes are biomarkers for cells that are immunosuppressive and inhibit various T cell-mediated immune responses against tumors. Immunosuppressive myeloid cells are formed in the adenosine-rich tumor microenvironment. The formation of these cells is blocked by inhibiting the adenosine A2A receptor using an adenosine A2A receptor antagonist.

[0060] In embodiments of the methods described herein, the biomarker genes comprise one biomarker gene selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise at least one biomarker gene selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise two biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise at least two biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise three biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise at least three biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise four biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise at least four biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise five biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.In embodiments, the biomarker genes comprise at least five biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise six biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise at least six biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes comprise seven biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In embodiments, the biomarker genes include at least seven biomarker genes selected from the group consisting of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.

[0061] In embodiments, the biomarker genes include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.

[0062] In embodiments, the biomarker genes consist of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2. In relation to this embodiment, "consisting of" means that only the eight specified biomarker genes (i.e., no other biomarker genes) are used in the methods described herein.

[0063] In embodiments of the methods described herein, the elevated level of gene expression is an elevated level of nucleic acid (e.g., RNA) expression or an elevated level of protein expression. The level of gene expression can be determined by methods known in the art, such as those described herein. In embodiments, the elevated level of gene expression is an elevated level of protein expression. In embodiments, the level of a biomarker is determined by immunoassay, liquid chromatography-mass spectrometry (LC-MS), or a combination thereof. In embodiments, the elevated level of gene expression is an elevated level of nucleic acid expression. In embodiments, the elevated level of gene expression is an elevated level of RNA expression. In embodiments, the RNA is mRNA. In embodiments, RNA expression is detected by direct digital counting of nucleic acids, RNA sequencing (RNA-seq), quantitative reverse transcriptase polymerase chain reaction (RT-qPCR), quantitative polymerase chain reaction (qPCR), multiplex qPCR, microarray analysis, or a combination thereof. In embodiments, RNA expression is detected by RNA sequencing. RNA sequencing is a sequencing technique that uses next-generation sequencing (NGS) to reveal the presence and amount of RNA in a biological sample. In embodiments, the gene expression level is the mean gene expression level of the biomarker genes. In embodiments, the mean gene expression level of the biomarker genes is the mean normalized gene expression level of the biomarker genes. In embodiments, the gene expression level of the biomarker genes is the median gene expression level of the biomarker genes. In embodiments, the median gene expression level of the biomarker genes is the median normalized gene expression level of the biomarker genes.

[0064] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting an increased level of myelosuppressive cells in a biological sample obtained from the patient compared to a control; and (b) administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor. In embodiments, the adenosine A2A receptor antagonist is ciforadenant. In embodiments, the CTLA-4 inhibitor is ipilimumab. In embodiments, the method further comprises administering to the patient an effective amount of a PD-1 inhibitor. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is blood. In embodiments, the biological sample is peripheral blood. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0065] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting an increased level of myelosuppressive cells in a biological sample obtained from the patient compared to a control; and (b) administering to the patient an effective amount of ciforadenant and an effective amount of ipilimumab. In embodiments, the effective amount of ciforadenant is about 200 mg per day, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is blood. In embodiments, the biological sample is peripheral blood. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0066] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, the method comprising: (a) detecting an increased level of myelosuppressive cells in a biological sample obtained from the patient compared to a control; and (b) administering to the patient an effective amount of ciforadenant, an effective amount of ipilimumab, and an effective amount of nivolumab. In embodiments, the effective amount of ciforadenant is about 200 mg per day, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is blood. In embodiments, the biological sample is peripheral blood. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma). In embodiments, the effective amount of ipilimumab and / or the effective amount of nivolumab is a reduced effective amount.

[0067] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of myelosuppressor cells. In embodiments, the adenosine A2A receptor antagonist is ciforadenant. In embodiments, the CTLA-4 inhibitor is ipilimumab. In embodiments, the method further comprises administering to the patient an effective amount of a PD-1 inhibitor. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is blood. In embodiments, the biological sample is peripheral blood. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0068] Provided herein is a method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of ciforadenant and an effective amount of ipilimumab, wherein a biological sample obtained from the patient has an increased level of myelosuppressive cells compared to a control. In embodiments, the effective amount of ciforadenant is about 200 mg per day, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, and the effective amount of ipilimumab is about 1 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is blood. In embodiments, the biological sample is peripheral blood. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma).

[0069] Provided herein are methods for treating renal cell carcinoma in a subject in need thereof, comprising administering to the subject an effective amount of ciforadenant, an effective amount of ipilimumab, and an effective amount of nivolumab, wherein a biological sample obtained from the subject has an increased level of myelosuppressor cells compared to a control. In embodiments, the effective amount of ciforadenant is about 200 mg per day, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the effective amount of ciforadenant is about 100 mg twice daily, the effective amount of ipilimumab is about 1 mg / kg about once every three weeks, and the effective amount of nivolumab is about 3 mg / kg about once every three weeks. In embodiments, the biological sample is blood, tumor tissue, or tumor cells. In embodiments, the biological sample is blood. In embodiments, the biological sample is peripheral blood. In embodiments, the control is a population of healthy subjects (e.g., without renal cell carcinoma). In embodiments, the effective amount of ipilimumab and / or the effective amount of nivolumab is a reduced effective amount.

[0070] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist, an effective amount of a CTLA-4 inhibitor, and an effective amount of a PD-1 inhibitor, wherein the effective amounts of the CTLA-4 inhibitor and / or the PD-1 inhibitor are reduced effective amounts. In embodiments, the effective amount of the CTLA-4 inhibitor is a reduced effective amount. In embodiments, the effective amount of the PD-1 inhibitor is a reduced effective amount. In embodiments, the effective amount of the CTLA-4 inhibitor is a reduced effective amount and the effective amount of the PD-1 inhibitor is a reduced effective amount. In embodiments, the adenosine A2A receptor antagonist is ciforadenant. In embodiments, the CTLA-4 inhibitor is ipilimumab. In embodiments, the PD-1 inhibitor is nivolumab. In embodiments, the biological sample obtained from the patient has an increased expression level of a biomarker gene compared to a control, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof. In embodiments, the biological sample obtained from the patient has an increased level of myelosuppressor cells compared to a control.

[0071] Provided herein are methods for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of ciforadenant, an effective amount of ipilimumab, and an effective amount of nivolumab, wherein the effective amounts of ipilimumab and / or nivolumab are reduced effective amounts. In embodiments, the effective amount of ipilimumab is the reduced effective amount. In embodiments, the effective amount of nivolumab is the reduced effective amount. In embodiments, the effective amount of ipilimumab is the reduced effective amount and the effective amount of nivolumab is the reduced effective amount. In embodiments, a biological sample obtained from the patient has increased expression levels of biomarker genes compared to a control, the biomarker genes being CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof. In embodiments, the biological sample obtained from the patient has increased levels of myelosuppressive cells compared to a control.

[0072] In embodiments, the reduced effective amount of ipilimumab is less than 1 mg / kg every three weeks.

[0073] In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 4 weeks or more. In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 4 weeks. In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 5 weeks. In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 6 weeks. In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 7 weeks. In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 8 weeks. In embodiments, the reduced effective amount of ipilimumab is about 1 mg / kg once every 4 weeks to once every 8 weeks.

[0074] In embodiments, the reduced effective amount of ipilimumab is about 0.9 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.8 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.7 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.6 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.5 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.4 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.3 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.2 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg once every three weeks.

[0075] In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.9 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.8 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.7 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.6 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.5 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.4 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.3 mg / kg once every three weeks. In embodiments, the reduced effective amount of ipilimumab is about 0.1 mg / kg to about 0.2 mg / kg once every three weeks.

[0076] In embodiments, the reduced effective amount of nivolumab is less than 3 mg / kg about once every three weeks.

[0077] In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every four or more weeks. In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every four weeks. In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every five weeks. In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every six weeks. In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every seven weeks. In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every eight weeks. In embodiments, the reduced effective amount of nivolumab is about 3 mg / kg once every four to eight weeks.

[0078] In embodiments, the reduced effective amount of nivolumab is about 2.9 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.8 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.7 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.6 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.5 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.4 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.3 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.2 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.1 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 2.0 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.9 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.8 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.7 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.6 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.5 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.4 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.3 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.2 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.1 mg / kg about once every three weeks. In embodiments, the reduced effective amount of nivolumab is about 1.0 mg / kg about once every three weeks.

[0079] In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.9 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.8 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.7 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.6 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.5 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.4 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.3 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.2 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.1 mg / kg about once every three weeks. In an embodiment, the reduced effective amount of nivolumab is about 1.0 mg / kg to about 2.0 mg / kg about once every three weeks.

[0080] Kits and Assays Provided herein are kits containing components, such as reagents and reaction mixtures, for performing assays to detect gene expression described herein. Materials and instructions for storing and using the kit components, for example, are provided as part of the kit. In embodiments, the kit includes one or more of the following: an RNA probe capable of hybridizing to an RNA biomarker; a primer pair capable of priming amplification (e.g., by PCR) of at least a portion of an RNA encoding an RNA marker or polypeptide marker under appropriate reaction conditions; instructions on how to use the kit; and a label or package insert indicating regulatory approval for diagnostic or therapeutic use. In embodiments, the kit further includes an RNA microarray containing molecules that specifically bind to the RNA of the present disclosure or described herein. In embodiments, standard techniques in microarray technology are used to assess RNA expression. Polynucleotide arrays, particularly arrays that bind to the RNA described herein, can also be used for diagnostic purposes, such as to identify subjects with a condition characterized by expression of a polypeptide biomarker.

[0081] "Assaying" or "detecting" means using an analytical procedure to qualitatively assess or quantitatively measure the presence or amount or functional activity of a target entity (e.g., miRNA, mRNA). For example, detecting the level of an RNA (such as miRNA or mRNA) means using an analytical procedure (such as an in vitro procedure) to qualitatively assess or quantitatively measure the presence or amount of the RNA. In embodiments, raw expression values ​​are normalized by quantile normalization to a reference distribution and subsequent log10 transformation. In embodiments, when detecting RNA expression using the nCounter® analytical system commercially available from Nanostring Technologies, a reference distribution is generated by pooling the reported (i.e., raw) counts for a test sample and one or more control samples (preferably at least two samples, more preferably at least 4, 8, or 16 samples) after excluding technical (both positive and negative control) probe values ​​and without intermediate normalization dependent on negative (background adjusted) or positive (synthetic sequences spiked at known dose settings) normalization.

[0082] The term "probe" or "primer" refers to one or more nucleic acid fragments capable of detecting specific hybridization to a sample. Probes or primers can be of any length, depending on the particular technique used. For example, PCR primers are generally 10-40 nucleotides in length, while nucleic acid probes for, e.g., Southern blots, can be over 100 nucleotides in length. Probes or primers can be unlabeled or labeled (e.g., with FRET donor or acceptor labels) as described below so that their binding to the target sequence can be detected. Probes or primers can be designed based on one or more specific (preselected) portions of a chromosome, such as one or more clones, isolated whole chromosomes or chromosomal fragments, or a collection of polymerase chain reaction (PCR) amplification products. One skilled in the art can adjust these factors to provide optimal hybridization and signal generation for a given hybridization and detection procedure, providing the required resolution among different genes or genomic locations.

[0083] Probes and primers can also be immobilized on a solid surface (e.g., nitrocellulose, glass, quartz, fused silica slides), such as in an array. Techniques for producing high-density arrays can also be used for this purpose. Those skilled in the art will recognize that the exact sequences of particular probes and primers can be modified to some extent from the target sequence to produce probes that are "substantially identical" or "substantially complementary" to the target sequence but retain the ability to specifically bind (i.e., specifically hybridize) to the same target from which they were derived.

[0084] The term "capable of hybridizing" refers to a polynucleotide sequence that forms Watson-Crick bonds with a complementary sequence. Those skilled in the art will understand that the percent complementarity need not be 100% for hybridization to occur, depending on the length of the polynucleotide, the length of the complementary region (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, or more bases in length), and the stringency of the conditions. For example, a polynucleotide (e.g., a primer or probe) can bind to a polynucleotide with 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity over the stretch of complementary region.

[0085] In embodiments, the method includes detecting the level of the biomarker using a specific binding agent (e.g., an agent that binds to a protein or nucleic acid molecule). Exemplary binding agents include an antibody or fragment thereof, a detectable protein or fragment thereof, a nucleic acid molecule such as an oligonucleotide / polynucleotide comprising a sequence complementary to the patient's genomic DNA, mRNA, or cDNA generated from the patient's mRNA, or any combination thereof. In embodiments, the antibody is labeled with a detectable moiety, such as a fluorescent compound, an enzyme or functional fragment thereof, or a radioactive agent. In embodiments, the antibody is detectably labeled by conjugating the antibody to a chemiluminescent compound. In embodiments, the presence of the chemiluminescent-labeled antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical response. Non-limiting examples of useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt, and oxalate ester.

[0086] In embodiments, a specific binding agent is an agent that has more than 10-fold, preferably more than 100-fold, and most preferably more than 1000-fold greater affinity for a target molecule compared to another molecule. Those skilled in the art will understand that the term specific is used to indicate that other biomarkers present in a sample do not significantly bind to a binding agent specific for a target molecule. In embodiments, the level of binding to biomolecules other than the target biomarker results in a binding affinity that is at most no more than 10%, no more than 5%, no more than 2%, or no more than 1%, respectively, of the affinity of the target molecule. Preferred specific binding agents meet both the above minimum criteria for affinity and the above minimum criteria for specificity. For example, in embodiments, antibodies have low micromolar (10 -6 ), nanomolar (10 -7 ~10 -9 ) and high affinity antibodies have binding affinities (e.g., Kd) of low nanomolar (10 -9 ) or picomole (10 -12 ) range.

[0087] In embodiments, the subject matter provides compositions comprising a binding agent, the binding agent being bound to a solid support (e.g., a strip, a polymer, a bead, a nanoparticle, a plate such as a multiwell plate, or an array such as a microarray). In embodiments involving the use of nucleic acid probes bound to a solid support (such as a microarray), the nucleic acid in the test sample may be amplified (e.g., using PCR) before or after hybridizing the nucleic acid to be measured with the probe. In embodiments, reverse transcription polymerase chain reaction (RT-PCR) is used to detect mRNA levels. In embodiments, a probe on a solid support is used to convert mRNA (or a portion thereof) in a biological sample to cDNA or partial cDNA, which is then hybridized to the probe (e.g., on a microarray), amplified after hybridization to the probe, or amplified and then hybridized to the probe. In embodiments, the strip may be a porous or non-porous solid support strip coated with a nucleic acid probe, which comprises linking the nucleic acid probe to a carrier to prepare a conjugate and immobilizing the conjugate on a porous solid support. In embodiments, the support or carrier may include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. In embodiments, the nature of the carrier may be soluble to some extent or insoluble for purposes of the present subject matter. In embodiments, the support material may have any structural configuration so long as the bound molecule is capable of binding to a binding agent (e.g., an antibody). In embodiments, the support configuration may be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. In embodiments, the surface may be flat, such as a plate (or well in a multiwell plate), a sheet, a test strip, polystyrene beads, etc. Those of skill in the art will know of many other suitable carriers for binding antibodies or antigens, or will be able to ascertain them using routine experimentation.

[0088] In embodiments, the solid support comprises a polymer to which an agent is chemically bound, immobilized, dispersed, or associated. In embodiments, the polymer support can be, for example, a polymer network and can be prepared in bead form (e.g., by suspension polymerization). In embodiments, the location of active sites incorporated into the polymer support depends on the type of polymer support. In embodiments, in swollen gel bead polymer supports, the active sites are uniformly distributed throughout the beads, while in macroporous bead polymer supports, they are primarily on the inner surface of the macropores. In embodiments, the solid support, e.g., device, can comprise a biomarker binding agent alone or in combination with binding agents for at least one, two, three, or more other biomarkers.

[0089] In embodiments, detection is accomplished using an ELISA or Western blot format. In embodiments, the binding agent comprises a nucleic acid (e.g., a probe or primer complementary to mRNA or cDNA), and the detection step is accomplished using a polymerase chain reaction (PCR) or Northern blot format, or other detection means. In embodiments, the probe or primer is about 10-20, 15-25, 15-35, 15-25, 20-80, 50-100, or 10-100 nucleotides in length, e.g., about 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 nucleotides in length, or less than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, or 100 nucleotides in length.

[0090] In embodiments, cells in a biological sample are lysed to release proteins or nucleic acids. Numerous methods for lysing cells and assessing protein and nucleic acid levels are known in the art. In embodiments, cells are physically lysed by, for example, mechanical disruption, liquid homogenization, high-frequency sonication, freeze / thaw cycles, or manual grinding using detergents. Non-limiting examples of detergents include Tween 20, Triton X-100, and sodium dodecyl sulfate (SDS). Non-limiting examples of assays for determining protein levels include HPLC, LC / MS, ELISA, immunoelectrophoresis, Western blot, immunohistochemistry, and radioimmunoassay. Non-limiting examples of assays for determining mRNA levels include Northern blotting, RT-PCR, RNA sequencing, and qRT-PCR.

[0091] In embodiments, once a suitable biological sample has been obtained, it is analyzed to quantify the expression level of each of the biomarker genes. In embodiments, determining the expression level of a gene comprises detecting and quantifying RNA transcribed from the gene or protein translated from such RNA. In embodiments, the RNA comprises mRNA transcribed from the gene and / or specific spliced ​​variants thereof, and / or fragments of such mRNA and spliced ​​variants.

[0092] In embodiments, raw expression values ​​are normalized by quantile normalization against a reference distribution and subsequent log10 transformation. In embodiments, when detecting gene expression using the nCounter® analysis system commercially available from NanoString® Technologies, a reference distribution is generated by pooling the reported (i.e., raw) counts for the test sample and one or more control samples (preferably at least two samples, more preferably at least 4, 8, or 16 samples) after excluding values ​​for technical (both positive and negative control) probes, without intermediate normalization dependent on negative (background adjusted) or positive (synthetic sequences spiked at known dose settings). In embodiments, a T effector signature score is then calculated as the arithmetic mean of the normalized values ​​for each of the genes in the gene signature.

[0093] In embodiments, the oligonucleotides in the kit can specifically hybridize to a target region of a polynucleotide, such as an RNA transcript or a cDNA generated therefrom. As used herein, specific hybridization means that the oligonucleotide forms an antiparallel duplex structure with the target region under specific hybridization conditions and is unable to form such a structure with a non-target region when incubated with a polynucleotide under the same hybridization conditions. The composition and length of each oligonucleotide in the kit depend on the nature of the transcript containing the target region and the type of assay to be performed using the oligonucleotide, and can be easily determined by one skilled in the art.

[0094] A "detectable agent" or "detectable moiety" is a compound or composition that can be detected by appropriate means, such as spectroscopic, photochemical, biochemical, immunochemical, chemical, magnetic resonance imaging, or other physical means. Detection of the RNAs described herein and the expression levels of the RNAs described herein can be achieved through the use of a detectable moiety in an assay or kit. A detectable moiety is a monovalent detectable agent or a detectable agent attached (e.g., covalently and directly or via a linking group) to another compound, e.g., a nucleic acid. Exemplary detectable agents / moieties for use in the present disclosure include antibody ligands, peptides, nucleic acids, radioisotopes, paramagnetic metal ions, fluorophores (e.g., fluorescent dyes), electron-dense reagents, enzymes (e.g., as commonly used in ELISAs), biotin, biotin-avidin complexes, biotin-streptavidin complexes, magnetic beads, paramagnetic molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticles, ultrasmall superparamagnetic iron oxide nanoparticle aggregates, superparamagnetic iron oxide nanoparticles, superparamagnetic iron oxide nanoparticle aggregates, single-crystalline iron oxide nanoparticles, single-crystalline iron oxide, nanoparticle contrast agents, liposomes, or gadolinium chelates. The radiolabel may be a radioactive molecule, gadolinium, a radionuclide, fluorodeoxyglucose (e.g., fluorine-18 labeled), any gamma-emitting radionuclide, a positron-emitting radionuclide, radiolabeled glucose, radiolabeled water, radiolabeled ammonia, biocolloids, microbubbles, iodinated contrast agents, barium sulfate, thorium dioxide, gold, gold nanoparticles, gold nanoparticle aggregates, fluorophores, two-photon fluorophores, or other delivery vehicles containing haptens and proteins, or other entities that can be made detectable by, for example, incorporating a radiolabel into a peptide or antibody that specifically reacts with a target peptide.

[0095] Embodiments 1 to 24 Embodiment 1. A method for treating renal cell carcinoma in a patient in need thereof, comprising: (a) detecting an increased gene expression level of a biomarker gene in a biological sample obtained from the patient compared to a control, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof; and (b) administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor.

[0096] Embodiment 2. A method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of gene expression compared to a control of a biomarker gene, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof.

[0097] Embodiment 3. The method of embodiment 1 or 2, wherein the biomarker genes comprise CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.

[0098] Embodiment 4. The method of embodiment 1 or 2, wherein the biomarker genes consist of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.

[0099] Embodiment 5. The method of any one of embodiments 1 to 4, wherein the biological sample is a tumor sample.

[0100] Embodiment 6. The method of any one of embodiments 1 to 4, wherein the biological sample is a tumor cell.

[0101] Embodiment 7. A method for treating renal cell carcinoma in a patient in need thereof, comprising: (a) detecting an increased level of myelosuppressor cells in a biological sample obtained from the patient compared to a control; and (b) administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor.

[0102] Embodiment 8. A method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient effective amounts of an adenosine A2A receptor antagonist and a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of myelosuppressive cells compared to a control.

[0103] Embodiment 9. The method of embodiment 7 or 8, wherein the biological sample is blood.

[0104] Embodiment 10. The method of embodiment 7 or 8, wherein the biological sample is peripheral blood.

[0105] Embodiment 11 The method of any one of embodiments 1 to 10, wherein the adenosine A2A receptor antagonist is ciforadenant.

[0106] Embodiment 12. The method of embodiment 11, wherein the effective amount of ciforadenant is 100 mg twice per day.

[0107] Embodiment 13. The method of any one of embodiments 1 to 12, wherein the CTLA-4 inhibitor is ipilimumab.

[0108] Embodiment 14 The method of embodiment 13, wherein the effective amount of ipilimumab is about 1 mg / kg about once every three weeks.

[0109] Embodiment 15. The method of any one of embodiments 1 to 14, further comprising administering to the patient an effective amount of a PD-1 inhibitor.

[0110] Embodiment 16. The method of embodiment 15, wherein the PD-1 inhibitor is nivolumab.

[0111] Embodiment 17. The method of embodiment 16, wherein the effective amount of nivolumab is about 3 mg / kg about once every three weeks.

[0112] Embodiment 18. A method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist, an effective amount of a CTLA-4 inhibitor, and an effective amount of a PD-1 inhibitor, wherein the effective amounts of the CTLA-4 inhibitor and / or the PD-1 inhibitor are reduced effective amounts.

[0113] Embodiment 19. The method of embodiment 18, wherein the adenosine A2A receptor antagonist is ciforadenant, the CTLA-4 inhibitor is ipilimumab, and the PD-1 inhibitor is nivolumab.

[0114] Embodiment 20 The method of embodiment 19, wherein the reduced effective amount of ipilimumab is less than 1 mg / kg every 3 weeks.

[0115] Embodiment 21. The method of embodiment 19 or 20, wherein the reduced effective amount of nivolumab is less than 3 mg / kg every 3 weeks.

[0116] Embodiment 22. The method of any one of embodiments 18 to 21, wherein the effective amount of ciforadenant is 100 mg twice daily.

[0117] Embodiment 23. The method of any one of embodiments 18-22, wherein the biological sample obtained from the patient has an increased expression level of a biomarker gene compared to a control, and the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof.

[0118] Embodiment 24 The method of any one of embodiments 18 to 23, wherein the biological sample obtained from the patient has an increased level of myelosuppressor cells compared to a control.

Claims

1. 1. A method for treating renal cell carcinoma in a patient in need thereof, comprising: (a) detecting an increase in gene expression level of a biomarker gene in a biological sample obtained from the patient compared to a control, wherein the biomarker gene is CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof; (b) administering to said patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor.

2. 1. A method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of gene expression compared to a control of biomarker genes, wherein the biomarker genes are CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof.

3. 2. The method of claim 1, wherein the biomarker genes include CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.

4. 2. The method of claim 1, wherein the biomarker genes consist of CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, and PTGS2.

5. The method of claim 1 , wherein the biological sample is a tumor sample.

6. The method of claim 1 , wherein the biological sample is a tumor cell.

7. 1. A method for treating renal cell carcinoma in a patient in need thereof, comprising: (a) detecting an increased level of myelosuppressor cells in a biological sample obtained from said patient compared to a control; (b) administering to said patient an effective amount of an adenosine A2A receptor antagonist and an effective amount of a CTLA-4 inhibitor.

8. 1. A method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient effective amounts of an adenosine A2A receptor antagonist and a CTLA-4 inhibitor, wherein a biological sample obtained from the patient has an increased level of myelosuppressive cells compared to a control.

9. The method of claim 7 , wherein the biological sample is blood.

10. The method of claim 7 , wherein the biological sample is peripheral blood.

11. 2. The method of claim 1, wherein the adenosine A2A receptor antagonist is ciforadenant.

12. 12. The method of claim 11, wherein the effective amount of ciforadenant is 100 mg twice per day.

13. 2. The method of claim 1, wherein the CTLA-4 inhibitor is ipilimumab.

14. 14. The method of claim 13, wherein the effective amount of ipilimumab is about 1 mg / kg about once every three weeks.

15. 10. The method of claim 1, further comprising administering to the patient an effective amount of a PD-1 inhibitor.

16. 16. The method of claim 15, wherein the PD-1 inhibitor is nivolumab.

17. 17. The method of claim 16, wherein the effective amount of nivolumab is about 3 mg / kg about once every three weeks.

18. 1. A method for treating renal cell carcinoma in a patient in need thereof, comprising administering to the patient an effective amount of an adenosine A2A receptor antagonist, an effective amount of a CTLA-4 inhibitor, and an effective amount of a PD-1 inhibitor, wherein the effective amounts of the CTLA-4 inhibitor and / or the PD-1 inhibitor are reduced effective amounts.

19. 19. The method of claim 18, wherein the adenosine A2A receptor antagonist is ciforadenant, the CTLA-4 inhibitor is ipilimumab, and the PD-1 inhibitor is nivolumab.

20. 20. The method of claim 19, wherein the reduced effective amount of ipilimumab is less than 1 mg / kg every three weeks.

21. 20. The method of claim 19, wherein the reduced effective amount of nivolumab is less than 3 mg / kg every three weeks.

22. 19. The method of claim 18, wherein the effective amount of ciforadenant is 100 mg twice daily.

23. 19. The method of claim 18, wherein the biological sample obtained from the patient has increased expression levels of biomarker genes compared to a control, and the biomarker genes are CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL8, IL-1β, PTGS2, or a combination of two or more thereof.

24. 20. The method of claim 18, wherein the biological sample obtained from the patient has an increased level of myelosuppressor cells compared to a control.

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