WO2013192339A1 - Biomarkers for the immunoediting-escape phase - Google Patents

Biomarkers for the immunoediting-escape phase Download PDF

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WO2013192339A1
WO2013192339A1 PCT/US2013/046638 US2013046638W WO2013192339A1 WO 2013192339 A1 WO2013192339 A1 WO 2013192339A1 US 2013046638 W US2013046638 W US 2013046638W WO 2013192339 A1 WO2013192339 A1 WO 2013192339A1
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biomarker
cancer
sample
fold
subject
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French (fr)
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Arie S. Belldegrun
Joseph Riss
Frederic D. BIRKHAUSER
Allan PANTUCK
Antoni Ribas
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The Regents Of The University Of California
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/5743—Specifically defined cancers of skin, e.g. melanoma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57438—Specifically defined cancers of liver, pancreas or kidney
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Metastatic RCC and melanoma are members of a restricted class of solid tumors that are significantly responsive to, and in a small percentage of cases, curable by systemic treatment with high-dose interleukin-2.
  • the identification of tumor- associated antigens (TA s) overexpressed in RCC has led to the development of humoral and cell-mediated immunotherapies.
  • TAAs carbonic anhydrase IX (CAIX / G250).
  • CAIX belongs to the carbonic anhydrase group of enzymes that regulate reversible proton exchange to buffer intracellular H, a function crucial for cell survival, especially in the harsh hypoxic tumor m icroenvironraent.
  • CALX expression has been described in many normal and cancerous tissues and is directly regulated by the hypoxia-inducible factor la- von Hippel-Lindau (VHL) pathway.
  • VHL hypoxia-inducible factor la- von Hippel-Lindau pathway.
  • stabilization of HIF- I a occurs leading to the overexpression of CAIX, rendering it useful as a diagnostic and prognostic biomarker, but also as a target for humoral and ce!l-rnediaied immunotherapies.
  • a peptide-based dendritic cell (DC) vaccination in HLA-A24-postive metastatic RCC patients was shown to be safe, with a median survival time of 21 months.
  • the method comprises: (a) measuring at least one biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of the bioraarkers listed in Table 5 and their human orthologs; and
  • the immunoediting-escape status indicates the subject's risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, the presence or absence of disease, stage of disease, or effectiveness of treatment for a disease.
  • the immunoediting-escape status indicates the presence, absence, or stage of a cancer or the effectiveness of treatment for a cancer in the subject.
  • the cancer is renal cell carcinoma.
  • the method further comprises: (c) managing treatment of the subject based on the immunoediting-escape status.
  • managing treatment comprises ordering a further diagnostic test, performing surgery, administering a therapy, or taking no further action, in some embodiments, the therapy is immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment.
  • the method comprises measuring at least two (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more) biomarkers in the sample from the subject and correlating the measurement of the biomarkers with the subject's immune response, in some embodiments, the correlating step is performed by a software classification system.
  • the method comprises: determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, is indicative of a status of cancer, immune-escape, or immune disease,
  • methods of monitoring the treatment of a subject for a disease or disorder are provided.
  • the method comprises:
  • an expression level of the at least one biomarker in the first expression profile that is above a threshold level indicates the presence of the disease or disorder
  • an expression level of the at least one biomarker in the second expression profile that is below a threshold level indicates efficacy of treatment.
  • the determining of an expression profile comprises:
  • nucleic acid sample is labeled.
  • the determining of an expression profile comprises:
  • the determining of an expression profile comprises: fi) providing a tissue sample from the subject; and
  • the substrate is a microti ter plate comprising one or more biospecific affinity reagents that bind to the biomarker, and wherein the biomarker is detected using a fluorescent label.
  • the expression profile is determined using a nucleic acid array.
  • the determining of an expression profile comprises detecting the presence or absence of the at least one biomarker, quantifying the amount of the at least one biomarker, and qualifying the type of the at least one biomarker.
  • the at least one biomarker is measured using a biochip array.
  • the biochip array is an antibody chip array, tissue chip array, protein chip array, nucleic acid array, or a peptide chip array.
  • one or more capture reagents that bind the at least one biomarker are immobilized on the biochip array.
  • the at least one biomarker is a protein translated from an mRNA listed in Table 5 and the protein bioraarker is measured by immunoassay.
  • methods for identifying a candidate agent to treat a disease or disorder in a subject comprises:
  • the one or more target biomarkers is an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human orthoiog thereof, and wherein differential expression of the one or more target biomarkers in the expression profile as compared to a control identifies the candidate agent as useful for treating the disease or disorder in the subject.
  • the candidate agent is a small molecule, a peptide, an immunotherapeutic, a vaccine, or a nucleic acid.
  • the candidate agent is identified in silica using pharmacogenomics.
  • the method comprises: detecting in a biological sample from a subject having the cancer an altered level of at least one biomarker listed in Table 5 or a human orthoiog thereof. In some embodiments, the method comprises detecting an elevated level of at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample. In some embodiments, the method comprises detecting a decreased level of at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample.
  • the level of the at least one biomarker in the sample from the subject having the cancer is altered by at least 1.2-fold as compared to the level of the at least one biomarker in a control sample, in some embodiments, a level of the at least one bioraarker in the sample from the subject having the cancer that is increased by at least 1.2-fold as compared to the control indicates that the cancer is not likely to be responsive to
  • the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA)-cytokine fusion protein. In some embodiments, the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
  • TAA tumor-associated antigen
  • the method comprises: detecting in a biological sample from the individual an altered level of at least one biomarker listed in Table 5 or a human oriholog thereof, in some embodiments, the method comprises detecting an elevated level of the at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample.
  • an elevated level of the biomarker in the sample from the individual indicates thai the individual has a cancer that is not likely to be responsive to the immunotherapy.
  • the method comprises detecting a decreased level of the at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample.
  • the level of the at least one biomarker in the sample from the subject having the cancer is altered by at least 1.2-fold as compared to the level of the at least one biomarker in a control sample.
  • the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA)--cytokine fusion protein.
  • TAA tumor-associated antigen
  • the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
  • methods of monitoring the efficacy of treatment for an individual having a cancer are provided.
  • the method comprises:
  • the treatment is an immunotherapy.
  • the immunotherapy comprises immune stimulation with a tumor-associated antigen
  • the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
  • an elevated level of the at least one biomarker relative to a control indicates that the cancer is not responsive to the treatment, In some embodiments, wherein the altered level of the at least one biomarker indicates that tire cancer is not responsive to the treatment, the method further comprising administering to the individual an alternative treatment.
  • the at least one biomarker is a protein translated from an mRNA listed in Table 5 or its human ortholog, In some embodiments, the at least one biomarker is an mRNA. in some embodiments, the at least one biomarker is an miRNA.
  • the at least one biomarker is an alternative isoform mRNA. In some embodiments, the at least one biomarker is a LincK A. In some embodiments, the at least one biomarker is a gene. In some embodiments, the at least one biomarker is a biomarker listed in Table 2 or its human ortholog. in some embodiments, the at least one biomarker is a biomarker listed in Table 4A and/or 4B or its human ortholog.
  • the at least one biomarker is Cel l, Cxcl9, Hmgbl, FgI2, Cd209a, Foxj l, Klra2, miR- 1 186, iniR-98, miR-5097, miR-1942, or miR-708.
  • the biomarker is selected from Cell, Cxcl9, Hmgbl , Cd209a, Fgl2, Kira2, and Foxj l .
  • the biomarker is selected from miR- 1 186, raiR-98, miR-5097, mill- 1942, and miR-708.
  • the sample is from blood, serum, plasma, urine, saliva, feces, kidney, lung, liver, prostate, bladder, intestine, colon, pancreas, or a tumor tissue.
  • the disease or disorder is a a cancer, e.g., a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is renal cell carcinoma.
  • the cancer is a metastatic cancer.
  • the disease or disorder is an immune system disease or disorder.
  • the disease or disorder is ischemia.
  • kits for detecting immunoediting-escape phase biomarkers comprise: at least one capture reagent that binds a biomarker selected from the group consisting of an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, and human orthologs thereof.
  • the kit comprises a plurality of capture reagents, wherein each capture reagent binds a biomarker selected from the group consisting of the biomarkers listed in Table 5 and their human orthologs.
  • the capture reagent is an antibody that binds to a protein translated from an mRNA listed in Table 5, In some embodiments, the capture reagent is a nucleic acid that is substantially complementary to an mRNA, miRNA, or LincRNA listed in Table 5. in some embodiments, the kit further comprises a wash solution that selectively retains the bound biomarker to the capture reagent after washing. In some embodiments, the kit further comprises instructions for using the capture reagent to detect the biomarker. In some embodiments, the instructions provide for contacting a test sample with the capture reagent and detecting the biomarker that is present in the sample, if any, that is retained by the capture reagent. In some embodiments, the kit further comprises instructions for using the kit for the detection of a disease or disorder.
  • FIG. 1 Bone marrow cells are harvested from syngeneic Balb/c mice, ex vivo differentiated to DCs and transduced with either Ad-GM-CAIX (DC-Ad-GM-CAIX) or with sham Ad-Nu!l (DC-Ad-Nuli).
  • mice are vaccinated by two rounds of subcutaneous transplantation of DC-Ad-GM-CAIX (groups A, B) or DC-Ad-Null (groups C, D) or not vaccinated (groups E, F), Twelve days after the second vaccination, mice are challenged by subcutaneous transplantation with either NPR-IX (groups A, C, E) or RENCA (groups B, D, F) tumor cells.
  • NPR-IX tumor cells are first subcutaneously transplanted, and only after tumors are established the mice are vaccinated with DC-Ad-GM-CAIX (group G), DC-Null (group H) or not vaccinated (group ⁇ ).
  • FIG. 1 Tumor volume, survival, and body weight Prevention model: (A) Tumor growth over time, (B) time to observable tumor growth (Kaplan-Meier curve), and (C, D) repeated study, (G, I) Body weight over time and (H, J) time to 15% weight loss (Kaplan- Meier curve). Treatment model: (E) Tumor growth over time and (F) time to tumor growth >600 mm 3 (Kaplan-Meier curve). ( ) Body weight ove time and L) time to 1 5% weight loss (Kaplan-Meier curve).
  • Prevention model (A) DC-Ad-GM-CAIX-treated mouse (group A, 10% hCAIX positive); (B) DC-Ad-Null-treated mouse (group C, 70% hCAIX positive); (C) Non- treated mouse (group E, 70% hCAIX positive).
  • FIG. 4 ELISA of mterferon-gamma (IFN- ⁇ ).
  • IFN- ⁇ mterferon-gamma
  • group A co-incubated with NPR-IX relative to co-incubation with RENCA.
  • group E co-incubated with NPR- ⁇ or RENCA did not induce !FN- ⁇ .
  • FIG. 1 Clusters of differential (A) niRNA and (B) miRNA expression between tumors of group A and C. Multidimensional scaling using 1 -correlation as distance metric. Validation of (C) gene expression and (D) miRNA by qPCR. Error bars shown are standard error of the mean. Tumor expression levels relative to control are shown. Gene and miRNA expressions were normalized to 188 rR A and to snoRNA234 (except for mir-708, which was normalized to snoRNA202), respectively. [0034] Figure 6.
  • FIG. 7 Dot plots of FACS for hCAIX in NPR-IX and RENCA cells.
  • A Unstained NPR-IX.
  • B NPR-IX stained with anti-hCAIX-FlTC, showing 95.30% of cells positive for hCAIX expression.
  • C Unstained RENCA.
  • D RENCA stained with anti- hCAIX-FITC, showing 2.15% of cells positive for hCAIX expression, which is assumed to be an artifact of nonspecific binding,
  • FIG. 8 Western Mot of the fusion protein hGM-CAIX.
  • the 70 kDa weight seen in both blots shows that the hGM-CAIX fusion gene is primarily expressed as a fused protein.
  • Lane i 293T control; Jane 2: 293 T infected with Ad-Null; lane 3; 293T infected with Ad-GM-CAIX.
  • A Gel exposed for actin loading control and hCAIX.
  • B Gel exposed for actin loading control and liGM-CSF.
  • Ad-GM-CAIX stained with anti-hCAIX-FITC showing 70.1% of cells positive for hCAIX expression, indicating successful infection of DCs with Ad-GM-CAIX.
  • C DC-Ad- GM'CAIX stained with anti-rnCd86-PE, showing 96.3% of cells positive for mCd86 expression, indicating successful differentiation from BMCs into DCs.
  • D DC-Ad- GM-CAIX double-stained with anti-hCAIX-FITC and with anti-mCd86-PE, showing 28.4% of cells positive for both hCAIX and mCd86 expression, representing the percentage of hCAiX-expressing DCs.
  • FIG. 10 Dot plots of FACS for hCAIX and hGM-CSF expression in DC-Ad- GM-CAIX.
  • A Unstained DC-Ad-GM-CAIX.
  • B DC-Ad-GM-CAIX stained with anti- hCAIX-FlTC, showing 49.51 % of ceils positive for hCAIX expression.
  • C DC-Ad- GM-CAIX stained with anti-hGM-CSF-PerCP, showing 30.35% of cells positive for hGM- CSF expression.
  • FIG. 12 Mean standardized metastatic foci area is um * per 10 um " total Sun area.
  • FIG 13 Immunohistochemical analysis of NPR-IX lung metastatic tumors with human CAIX (hCAIX).
  • the panels show staining of membranous hCAIX in brown, Originai magnification, 40x.
  • Non-treated mice group A, 90% hCAIX positive
  • DC- Ad-GM-CAIX-treated mice group B, 10% hCAIX positive.
  • GM-CAIX fusion protein GM-CSF-CAIX
  • SCID severe combined immunodeficiency
  • GM-CAIX was capable of inducing human dendritic cell (DC) maturation.
  • Adenoviral vectors are more potent in eliciting CD4+ and CD8+ TAA- specific polyclonal CTLs compared with peptide-pulsed.
  • dendritic cells expressing GM-CAIX referred to herein as "DC-Ad- GM-CAIX” or “DC-Ad-GM-CAIX" were found to generate hCAIX-specific immune response attributed to CTLs in Balb/c immunocompetent mouse models, which are capable of both preventing and intervening in RCC growth.
  • the results revealed significant hCAIX- specific immunotherapeutic efficacy with no signs of systemic toxicity,
  • Ad-GM-CAIX / Ad-GM-CAIX Adeno-virus vector expressing GM-CAIX
  • AP Alkaline phosphatase
  • APC Antigen-presenting cell
  • CAIX / CA9 / G250 Carbonic anhydrase IX
  • CD4 Cluster of differentiation 4
  • CD8 Cluster of differentiation 8
  • CD86 Cluster of diiXerentiation 86
  • cDNA Complementary DNA
  • CL Chemiluminescence assays: CTL: Cytotoxic T lymphocyte; DAPI: 4',6-diamidino-2-phenylindole;
  • DC Dendritic ceil;
  • DC-Ad-GM-CAIX / DC-Ad-GM-CAIX Dendritic cells transduced with Ad-GM-CAIX: DC-Ad-Null: Dendritic ceils transduced with an empty Adeno-virus vector;
  • EIA Enzyme immunoas
  • the term "immunoediting-escape” refers to the ability of cancer cells (e.g., tumors) in a subject to evade or escape elimination by the immune system of the subject, in some embodiments, a cancer ceil or tumor that evades or escape the immune system progresses to a more ad vanced form of cancer and/or metastasizes.
  • immunoediting-escape status refers to the extent to which a cancer cell or tumor has evaded or is predicted to evade elimination by the immune system of a subject.
  • immunoediting-escape status is determined by measuring the level or amount of expression of one or more (e.g., I, 2, 3, 4, 5, 6, 7. 8, 9, 10, 15, 20, 25, 30, or more) biomarkers selected from the group of biomarkers listed in Table 5 and their human orthoiogs; and correlating the level or amount of expression with an immune response.
  • the level or amount of expression of the one or more biomarkers is compared to a threshold level or a control (e.g., a control sample from a non-diseased sample, a control sample from a subject having an effective immune response to the cancer cell or tumor, or a control sample from a subject known not to have an effective immune response to the cancer cell or tumor).
  • a threshold level or a control e.g., a control sample from a non-diseased sample, a control sample from a subject having an effective immune response to the cancer cell or tumor, or a control sample from a subject known not to have an effective immune response to the cancer cell or tumor.
  • biomarker refers to a molecule (e.g., a nucleic acid, e.g., mRNA, miRNA, or LincRNA, or a protein) thai is differentially expressed (overexpressea or underexpressed) in a ceil, on the surface of a ceil, or secreted by a cell (e.g., a cancer cell) in comparison to a normal control cell, and which is useful for the qualification of
  • a biomarker useful for the qualification of immunoediting-escape status or diagnosis or prognosis of a disease or condition in a subject as disclosed herein exhibits over 1-fold over/under expression, e.g., at least 1 ,2-fold over/under expression, 1.5-fold over/under expression, 2-fold over/under expression, 3-fold over/under expression, 4-fold over/under expression, 5-fold over/under expression, 6-fold over/under expression, 7-fold over/under expression, 8-fold over/under expression, 9-fold over/under expression, 10-fold over/underexpression, 15-fold
  • a biomarker useful for the qualification of immunoediting-escape status or diagnosis or prognosis of a disease or condition in a subject as disclosed herein is an mRNA, miRNA, or LincR A listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof.
  • the biomarker is a biomarker listed in Table 2 or a human ortholog thereof.
  • the biomarker is biomarker listed in Table 4A and/or 4B or a human ortholog thereof.
  • a biomarker useful for the qualification of immunoediting-escape status or diagnosis or prognosis of a disease or condition in a subject as disclosed herein may also function in other cellular or biological processes over than immunoediting-escape phase function.
  • a biomarker of the present invention is at least a biomarker of immunoediting-escape phase function.
  • markers may be used singly or in combination with other markers for any of the uses, e.g., qualification of immunoediting- escape status or diagnosis or prognosis of a disease or condition (e.g., a cancer such as renal cell carcinoma) as disclosed herein.
  • a disease or condition e.g., a cancer such as renal cell carcinoma
  • agent refers to any molecule, either naturally occurring or synthetic, e.g., peptide, protein, oligopeptide (e.g., from about 5 to about 25 am ino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule (e.g., an organic molecule having a molecular weight of less than about 2500 daltons, e.g..
  • peptide e.g., siRNA or shRNA
  • polypeptide e.g., aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-linked glycol-like molecules
  • peptide polypeptide
  • protein are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturaliy occurring amino acid polymer.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid rmmetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, ⁇ - earboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g.
  • amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof.
  • the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
  • Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonat.es, chira!-methyl phosphonates, 2-O-methyl ribonucleotides, and pepiide-nuc!eic acids (PNAs).
  • nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), complementary sequences, splice variants, and nucleic acid sequences encoding truncated forms of proteins, as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 ( 1991 ); Ohtsuka et ah, J. Biol.
  • nucleic acid is used interchangeably with gene, cDNA, mRNA, miRNA, LincRNA, shRNA, siR A, oligonucleotide, and polynucleotide.
  • nucleic acids refers to a first nucleic acid that has at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the complement of a second nucleic acid.
  • a first nucleic acid that is substantially complementary to a. second nucleic acid is able to bind to the second nucleic acid under moderately stringent hybridization conditions.
  • overexpress overexpression
  • upregulate upreguiation
  • nucleic acid or protein expression of a biomarker of interest in a sample e.g., a tumor sample
  • control e.g., non-cancer
  • Upreguiation can be due to increased transcription, post transcriptional processing, translation, post transiational processing, altered stability, or altered protein degradation, as well as local upreguiation due to altered protein traffic patterns (increased nuclear localization), and augmented functional activity, e.g., as a transcription factor.
  • Upreguiation can be detected usiiig conventional techniques for detecting nucleic acid (e.g. , RT-PCR, PCR, microarray) or proteins (e.g., EL1SA, Western blots, flow cytometry, immunofluorescence,
  • nucleic acid e.g. , RT-PCR, PCR, microarray
  • proteins e.g., EL1SA, Western blots, flow cytometry, immunofluorescence,
  • Upreguiation can be at least 10%, 20%, 30%, 40%, 50%», 60%, 70%, 80%, 90% or more for the biomarker of interest in the test sample (e.g., a tumor sample) in comparison to a control (e.g., non-cancer) sample, in certain instances, upreguiation is at least 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3, 5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, i 1-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16-fold, 17-fold, i 8-fold, 19-fold, 20-fold or more higher levels of UNA or protein levels for the biomarker of interest in the test sample (e.g., tumor sample) in comparison to a control (e.g., non-cancer) sample.
  • a control e.g., non-cancer
  • downstream interchangeably refer to nucleic acid or protein expression of a biomarker of interest in a sample (e.g., a tumor sample) that is detectabiy lower than the nucleic acid or protein expression of the biomarker of interest in a control (e.g., non-cancer) sample.
  • a sample e.g., a tumor sample
  • a control e.g., non-cancer
  • Downregulation can be due to decreased transcription, post transcriptional processing, translation, post translational processing, altered stability, or altered protein degradation, as well as local downregulation due to altered protein traffic patterns (decreased nuclear localization), and augmented functional activity, e.g., as a transcription factor.
  • Downregulation can be detected using conventional techniques for detecting nucleic acid (e.g., RT-PCR, PCR, microarray) or proteins (e.g. , ELISA, Western blots, flow cytometry, immunofluorescence, imniunobistochemistry, DNA binding assay techniques).
  • nucleic acid e.g., RT-PCR, PCR, microarray
  • proteins e.g. , ELISA, Western blots, flow cytometry, immunofluorescence, imniunobistochemistry, DNA binding assay techniques.
  • Downregulation can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less for tiie biomarker of interest in the test sample (e.g., a tumor sample) in comparison to a control (e.g., non-cancer) sample, in certain instances, downregulation is at least 1.2-fold, 1.
  • RNA or protein levels for the biomarker of interest in the test sample e.g., tumor sample
  • a control e.g., non-cancer
  • an expression profile refers to the amount of nucleic acid and/or protein that is expressed for a given biomarker or set of biomarkers in a biological sample.
  • an expression profile comprises the levels of nucleic acid and/or protein expression for a plurality of biomarkers as described herein, e.g., for 2. 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more biomarkers selected from an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof.
  • Cancer is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells.
  • the term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers.
  • Examples of different types of cancer include, but are not limited to, digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and esophageal cancer; breast cancer; lung cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer, ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer (e.g., melanoma); lymphomas; gliomas; choriocarcinomas; head and neck cancers; osteogenic sarcomas; and blood cancers.
  • a "tumor” comprises one or more cancerous cells.
  • the cancer is renal cell carcinoma
  • the cancer is a metastatic cancer.
  • sample or “biological sample” includes blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like); sputum or saliva; kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue; cultured cells, e.g., primary cultures, explants, and transformed cells, stent cells, stool, urine, etc.
  • biological samples also include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes, in some embodiments, a sample is a tumor tissue sample.
  • a biological sample is typically obtained from a "subject," i.e., a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, or mouse; rabbit: or a bird; reptile; or fish.
  • a "subject" i.e., a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, or mouse; rabbit: or a bird; reptile; or fish.
  • administer refers to methods of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical deliver)', parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, colonical delivery, rectal delivery, or intraperitoneal delivery. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Oilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's,
  • the present invention relates to methods of qualifying the iram unoediting-escape status of a subject, in some embodiments, the method comprises:
  • biomarker is selected from the group consisting of the biomarkers listed in Table 5 and their human orthologs;
  • the method comprises measuring at least two biomarkers (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers) and correlating the measurement of the biomarkers with the .subject's immune response.
  • the immunoediting-escape status indicates the subject's risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, the presence or absence of a disease ⁇ e.g., cancer), the stage of a disease, or effectiveness of treatment for a disease
  • the immunoediting-escape status indicates the presence, absence, or stage of a cancer or the effectiveness of treatment for a cancer in the subject
  • the immunoediting-escape status indicates the presence, absence, or stage of a renal ceil carcinoma or the effectiveness of treatment for a. renal ceil carcinoma in the subject.
  • the at least one biomarker is an mRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human orihoiog thereof, in some embodiments, the mRNA or protein is an mRNA or protein that is involved in inflammatory response, cell proliferation, cell growth, cell movement, or cell-to-cell signaling, in some embodiments, the mRNA or protein is an inflammatory response gene or protein listed in Table 4A. in some embodiments, the mRNA or protein is a cellular growth and/or proliferation gene or protein listed in Table 4B. In some embodiments, the at least one biomarker is an miRNA or LincRNA that is involved in cell (e.g., tumor) growth or suppression of tumor growth.
  • the at least one biomarker is an miRNA or LincRNA listed in Table 5 or a human ortholog thereof. In some embodiments, the at least one biomarker is an miRNA or LincRNA listed in Table 2 or a human orihoiog thereof.
  • the at least one biomarker is a cytokine or a regulator and/or marker of myeloid cells, dendritic cells, natural killer cells, and/or T-cells.
  • the at least one biomarker is Cel l , Cxcl9, Hmgbi, Fgl2, Cd209a, Foxj i , Klra2, miR-1 186, miR-98, miR-5097, miR-1942, or miR-708.
  • the at least one biomarker is selected from Cell , Cxcl9, Hmgbl, FgI2, Cd209a, Foxj 1, and Klra2.
  • the at least one biomarker is selected from miR-1 186, miR-98, miR- 5097, miR-1 42, and miR-708.
  • upregulation of at least one biomarker selected from Cell, Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l, KIra2, miR-1 186, miR- 98, miR-5097, and miR-1942 is indicative of immune escape, cancer (e.g., renal ceil carcinoma), or immune disease.
  • downregulation of miR-708 is indicative of immune escape, cancer (e.g., renal cell carcinoma), or immune disease.
  • the at least one biomarker e.g., an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof
  • the at least one biomarker is upregulated or more highly expressed in the sample from the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to the level of expression of the biomarker in the control (e.g., non-diseased) sample.
  • the at least one biomarker is upregulated or more highly expressed in the sample from the subject by at least 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16- fold, 17-fold, 18- fold, 19-fold, 20-fold or more as compared to the level of expression of the biomarker in the control (e.g., non-diseased) sample.
  • the at least one biomarker e.g., an mRNA. rniRNA, or LincRNA listed in Table 5.
  • a protein translated from an mRNA listed in Table 5, or a human ortholog thereof is downregulated in the sample from the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to the level of expression of the biomarker in the control (e.g., non- diseased) sample.
  • the at least one biomarker is downregulated in the sample from the subject by at least 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-foid, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more as compared to the level of expression of the biomarker in the control (e.g., non-diseased) sample.
  • Samples e.g., non-diseased
  • a biological sample comprises a ceil.
  • the cell is derived from a tissue or organ (e.g., prostate, breast, kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, keletal muscle, cartilage, skin, or bone tissue).
  • the cell is derived from a biological fluid, e.g., blood (e.g., an erythrocyte), lymph (e.g., a monocyte, macrophage, neutrophil, eosinophil, basophil, mast cell, T cell, B cell, and/or NK cell), serum, urine, sweat, tears, or saliva.
  • the ceil is derived from a biopsy (e.g., a skin biopsy, a muscle biopsy, a bone marrow biopsy, a liver biopsy, a gastrointestinal biopsy, a lung biopsy, a nervous system biopsy, or a lymph node biopsy), in some embodiments, the cell is derived from a cultured cell (e.g., a primary cell culture) or a ceil line (e.g., PC3, HEK293T, NIH3T3, Jurkat, or Ramos).
  • a biopsy e.g., a skin biopsy, a muscle biopsy, a bone marrow biopsy, a liver biopsy, a gastrointestinal biopsy, a lung biopsy, a nervous system biopsy, or a lymph node biopsy
  • the cell is derived from a cultured cell (e.g., a primary cell culture) or a ceil line (e.g., PC3, HEK293T, NIH3T3, Jurkat, or Ramos).
  • the sample comprises a cancer cell (e.g., a cell obtained or derived from a tumor).
  • the cancer is prostate cancer, breast cancer, bladder cancer, urogenital cancer, lung cancer, renal ceil carcinoma, endometrial cancer, melanoma, ovarian cancer, thyroid cancer, or brain cancer, in some embodiments, the cancer is a metastatic cancer.
  • the sample is from blood, serum, plasma, urine, saliva, feces, kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, rectum, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus.
  • the sample is from a renal tumor, renal cyst, renal metastasis, kidney cell or cells, or kidney tissue.
  • the sample is from a diseased tissue, in some embodiments, the sample is from a hypoxic tumor, in some embodiments, the sample is from a tumor tissue (e.g., from a cancer type as described herein), in some embodiments, the sample is from ischemic tissue.
  • the sample is from a human subject.
  • the biological sample is from a non-human mammal (e.g., chimpanzee, dog, cat, pig, mouse, rat, sheep, goat or horse), avian (e.g., pigeon, penguin, eagle, chicken, duck, or goose), reptile (e.g., snake, lizard, alligator, or turtle), amphibian (e.g., frog, toad, salamander, caecilian, or newt), or fish (e.g., shark, salmon, trout, or sturgeon).
  • a non-human mammal e.g., chimpanzee, dog, cat, pig, mouse, rat, sheep, goat or horse
  • avian e.g., pigeon, penguin, eagle, chicken, duck, or goose
  • reptile e.g., snake, lizard, alligator, or turtle
  • amphibian e.
  • the detection methods for measuring and/or quantifying one or more biomarkers as described herein can be carried out, for example, using standard nucleic acid and/or polypeptide detection techniques known in the art. Detection can be accomplished by labeling a nucleic acid probe or a primary antibody or secondary antibody with, for example, a radioactive isotope, a fluorescent label, an enzyme or any other detectable label known in the art.
  • Antibody reagents can be used in assays to detect protein expression levels for the at least one hiomarker of interest in patient samples using any of a number of imm noassays known to those skilled in the art.
  • immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay,” 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach," Oxford University Press, 2000.
  • a variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al, Ciirr. Opin. Biotechnol, 7:60-65 (1996)).
  • immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EI A) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), lgM antibody capture EL1SA (MAC ELISA), and microparticle enzyme immunoassay (MELA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (R1A); itnmunoradiometric assays (I MA); immunofluorescence (IF); fluorescence polarization immunoassays (FPIA); and
  • chemi!uminescence assays can be automated.
  • Immunoassays can aiso be used in conjunction with laser induced fluorescence ⁇ see, e.g., Schmalzing et al, Electrophoresis, 18:2184-93 (1997); Bao, J, Chromatogr. B. Biomed. Set, 699:463-80 (1997)).
  • Specific immunological binding of an antibody to a protein can be detected directly or indirectly.
  • Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody.
  • An antibody labeled with iodine- 125 ( r25 I) can be used.
  • a chemiluminescence assay using a cherniluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels.
  • An antibody labeled with fluorochrome is aiso suitable.
  • fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoeiythrin, rhodamine, Texas red, and lissamine.
  • Indirect labels include various enzymes welt known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), ⁇ - galactosidase, urease, and the like.
  • a horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • TMB tetramethylbenzidine
  • An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm.
  • a ⁇ -galactosidase detection system can be used with the chromogenic substrate o-niirophenyi- -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm
  • a urease detection system can be used with a substrate such as urea- bromocresoi purple (Sigma immunochemicals; St. Louis, MO).
  • a signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 1 ⁇ 5 ⁇ ; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength.
  • a quantitative analysis can be made using a spectrophotometer such as an EMAX Microptate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions.
  • the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected
  • the amount of signal can be quantified using an automated high-content imaging system.
  • High-content imaging systems are commercially available (e.g., ImageXpress, Molecular Devices Inc., Sunnyvale, CA).
  • Antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate materia! or membrane (e.g., plastic, nylon, paper), and the like.
  • An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
  • nucleic, acid binding molecules such as probes, oligonucleotides, oligonucleotide arrays, and primers can be used in assays to detect differential nucleic acid expression of a biomarker of interest in ubject samples, e.g., RT-PCR.
  • RT-PCR is used according to standard methods known in the art.
  • PGR assays such as Taqman® assays available from, e.g. , Applied Biosystems, can be used to detect nucleic acids and variants thereof.
  • qPCR and nucleic acid microarrays can be used to detect nucleic acids.
  • Reagents that bind to selected markers of interest can be prepared according to methods known to those of skill in the art or purchased commercially.
  • nucleic acids can be achieved using routine techniques such as Southern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), or any other methods based on hybridization to a nucleic acid sequence that is complementary to a portion of the marker coding sequence (e.g. , slot blot hybridization) are also within the scope of the present invention.
  • Applicable PCR amplification techniques are described in, e.g., Ausubel et al. and innis et al, supra.
  • General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999.
  • Amplification or hybridization of a plurality of nucleic acid sequences can also be performed from mRNA or cDNA sequences arranged in a microarray.
  • Microarray methods are generally described in Hardiman, "Microarrays Methods and
  • Analysis of nucleic acid markers can also be performed using techniques known in the art including, without limitation, microarrays, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis.
  • PCR polymerase chain reaction
  • a non-limiting example of a PCR- based analysis includes a Taqraan® allelic discrimination assay available from Applied Biosy stems.
  • sequence analysis include Maxam-Giibert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman er a/, . . Methods Mol.
  • sequencing with mass spectrometry such as matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et cel., Nat. BiotechnoL, 16:381 -384 (1998)), and sequencing by hybridization.
  • MALDI-TOF/MS matrix- assisted laser desorption/ionization time-of-flight mass spectrometry
  • Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis.
  • Other methods for detecting nucleic acid variants include, e.g., the INVADER® assay from Third Wave Technologies, nc.. restriction fragment length polymorphism (RFLP) analysis, alleie-specific oligonucleotide hybridization, a heterodupiex mobility assay, single strand conformational polymorphism (SSCP) analysis, single-nucleotide primer extension (SNUPE), and pyrosequencing.
  • RFLP restriction fragment length polymorphism
  • SSCP single strand conformational polymorphism
  • SNUPE single-nucleotide primer extension
  • a detectable moiety can be used in the assays described herein.
  • a wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions.
  • Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes ⁇ e.g., fluorescein, fluorescein isothioeyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodirnine isothiocynate (TRJTC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like.
  • fluorescent dyes ⁇ e.g., fluorescein, fluorescein isothioeyanate (FITC), Oregon GreenTM, rhodamine, Texas red, tetrarhodirnine isothi
  • Useful physical formats comprise surfaces having a. plurality of discrete, addressable locations for the detection of a plurality of different markers.
  • Such formats include microarrays and certain capillary devices. See, e.g., Ng et al, J. Cell Mol. Med., 6:329-340 (2002); U.S. Pat. No, 6,019,944,
  • each discrete surface location may comprise antibodies or oligonucleotides to immobilize one or more markers for detection at each location.
  • Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticies) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more markers for detection.
  • Other useful physical formats include sticks, wells, sponges, and the like.
  • the antibodies or nucleic acid probes of the invention can be applied to subject samples immobilized on microscope slides.
  • the resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art,
  • Analysis of the protein or nucleic acid can also be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALD1/MS, MALDI-TOF MS, tandem MS, etc.). Correlating Biomarker Measurements to Immune Response
  • the level or amount of expression of the at least one biomarker is correlated with an immune response in the subject
  • the correlating step comprises correlating expression of the at least one biomarker with an immune response by the subject that is above or below a threshold level (e.g., an immune response level in a. healthy control subject or population of subjects).
  • a level of expression of the at least one biomarker that is over 1 -fold over the threshold or control level correlates the biomarker with an immune response
  • a level of expression of the at least one biomarker that is over 1-fold over the threshold or control level indicates that a diseased cell or tissue (e.g., a cancer cell or tumor) has evaded or escaped elimination by the immune system (i.e., has an imrnunoediting-escape status)
  • the correlating step is performed by a software classification system.
  • the immune response is the secretion of iramunosuppressor factors (e.g., IL-IO, TGFp).
  • the immune response is a change in the expression and/or activity of immune system cells (e.g., regulatory T cells or effector cells).
  • immune system cells e.g., regulatory T cells or effector cells.
  • the methods of qualifying the imrnunoediting-escape status of a subject further comprise managing treatment of the subject based on the imrnunoediting- escape status.
  • the method comprises: measuring at least one biomarker (e.g., an mRNA, miR A, or LincR.N A listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) in a sample from the subject;
  • the at least one biomarker is differentially expressed (upregulated or downregulated) in the sample from the subject as compared to a control subject;
  • the correlating step comprises correlating a level of expression of the at least one biomarker that is altered by more than 1 -fold relative to a threshold or control level (e.g., that is upregulated or downregulated by at least 1.2-fold, 1 ,5- fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-foid, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more relative to the threshold or control level) with a diseased cell or tissue in the subject (e.g., a cancer cell or tumor) evading or escaping elimination by the immune system (i.e., having an immunoediting-escape status), in some embodiments, a level of expression of the at least one biomarker that is altered by more than 1-fold relative to a threshold or control level
  • managing treatment comprises ordering a further diagnostic test, performing surgery, administering a therapy, or taking no further action, in some embodiments, wherein managing treatment comprises administering a therapy, the therapy comprises immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment.
  • the present invention relates to methods of diagnosing or prognosing a disease, disorder, or condition in a subject using a biomarker of immunoediting- escape status as described herein.
  • diagnosis or “diagnosis” refers to detecting a disease, disorder, or condition (e.g., a cancer). In any method of diagnosis exists false positives and false negatives. Any one method of diagnosis does not provide 100% accuracy.
  • providing a prognosis refers to to providing a prediction of the probable course and outcome of a disease, disorder, or condition such as a cancer, including prediction of metastasis, disease free survival, overall survival, etc.
  • the methods can also be used to devise a suitable therapy for treatment, e.g., by indicating whether or not a disease (e.g., a cancer) is still at an early stage or if the disease has advanced to a stage where aggressive therapy would be ineffective.
  • a disease e.g., a cancer
  • methods of diagnosing the status of a tissue or organ comprises: determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, is indicative of a status of cancer, immune-escape, or immune disease in the tissue or organ of the subject.
  • a differential pattern or level of expression of the at least one biomarker in the sample from the subject is indicative of the presence, absence, or stage of a disease (e.g., a cancer) in the tissue or organ or the effectiveness of treatment for the disease (e.g., cancer) in the subject.
  • a disease e.g., a cancer
  • an increased or upreguiated level of expression of the at least one biomarker in the sample from the subject is indicative of the presence, absence, or stage of a disease or the effectiveness of treatment for the disease in the subject.
  • the level of the at least one biomarker is increased by at least 1.2-fold, 1 .5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fo!d, 19- fold, 20-fold or more, in some embodiments, a level of expression of the at least one biomarker in the sample from the subject that is increased by at least 1.2- fold, 1 , 5-fold, 2- fold.
  • the method comprises diagnosing renal status. Irs sorne embodiments, a differential pattern or level of expression of the at least one biomarker in the sample from the subject is indicati ve of the presence, absence, or stage of renal cell carcinoma or the effectiveness of treatment for renal ceil carcinoma in the subject. In some embodiments, an increased or upreguiated level of expression of the at least one biomarker in the sample from the subject is indicative of the presence, absence, or stage of renal cell carcinoma or the effectiveness of treatment for renal ceil carcinoma in the subject. [0095] In some embodiments, methods of diagnosing whether or not an individual has a disease or disorder that is likely to be responsive to a therapy (e.g., immunotherapy) are provided.
  • a therapy e.g., immunotherapy
  • the method comprises: detecting in a biological sample from the individual an altered level of at least one biomarker listed in Table 5 or a human ortho!og thereof, in some embodiments, the method comprises detecting an elevated or upregulated level of expression of the least one biomarker, wherein the elevated or upregulated level of expression indicates that individual has a disease or disorder that is not likely to be responsive to the therapy. In some embodiments, the method comprises detecting a decreased or downregulated level of expression of the least one biomarker, wherein the decreased or downregulated level of expression indicates that the individual has a disease or disorder that is likely to be responsive to the therapy.
  • the level of the at least one biomarker is altered (e.g., elevated or decreased) by at least 1 ,2-fold, 1.5-fold, 2- foid, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more.
  • the disease or disorder is a cancer, e.g...
  • the disease or disorder is an immune system disease or disorder.
  • the disease or disorder is ischemia.
  • the method further comprises administering the therapy to the individual.
  • methods of determining whether or not a disease or disorder is likely to be responsive to a therapy comprise: detecting in a biologicai sample from a subject having the disease or disorder an altered level of at least one biomarker listed in Table 5 or a human ortholog thereof.
  • the method comprises detecting an elevated or upregulated level of expression of the least one biomarker, wherein the elevated or upregulated level of expression indicates that the disease or disorder is not likely to be responsive to the therapy.
  • the method comprises detecting a decreased or downregulated level of expression of the least one biomarker, wherein the decreased or downregulated le vel of expression indicates that the disease or disorder is likely to be responsive to the therapy.
  • the level of the at least one biomarker is altered (e.g., elevated or decreased) by at least 1.2-foki, 1.5-fold, 2-fold, 2,5-fold, 3-fold, 3 ,5-fold, 4-fold, 4 ,5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13- fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more.
  • the disease or disorder is a cancer, e.g., a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is an immune system disease or disorder, in some embodiments, the disease or disorder is ischemia. In some embodiments, wherein the altered level of the biomarker indicates that the disease or disorder is likely to be responsive to the therapy (e.g., immunotherapy), the method further comprises administering the therapy to the individual.
  • the therapy e.g., immunotherapy
  • the present invention provides therapeutic methods for identifying subjects for treatment and treating su jects in need thereof.
  • treatment include: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mamma! that may be exposed to the organism but does not yet experience or display symptoms of the disease: (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms.
  • the method comprises: administering a therapy to a subject that has been identified as having an immunoediting-escape status, wherein identifying the subject as having an immunoediting- escape status comprises determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, and wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, identities the subject as having an immunoediting-escape status.
  • an increased or upregulated level of expression of the at least one biomarker in the sample from the subject identifies the subject as having an
  • the disease or disorder is a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is an immune system disease or disorder. In some embodiments, the disease or disorder is ischemia.
  • the method comprises: detecting in a biological sample from the individual a level of at least one biomarker selected from an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in ' fable 5, or a human orthoJog thereof; and
  • disease or disorder is a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate. bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is a metastatic cancer or renal cell carcinoma, in some embodiments, the disease or disorder is ischemia, in some
  • an elevated level of the at least one biomarker relative to a control indicates that the disease or disorder is not responsive to the treatment. In some embodiments, wherein an elevated level of the at least one biomarker relative to a control indicates that the disease or disorder is not responsive to the treatment, the method further comprises administering to the individual an alternative treatment.
  • the method comprises detecting the level of the at least one biomarker in two or more samples from the individual taken at different timepoints (e.g., before treatment and during the course of treatment; at different times during the course of treatment; before treatment and after the completion of a round of treatment; or during the course of treatment and after the completion of a round of treatment) and determining whether the level of the biornarker is altered in each sample is altered relative to a control.
  • timepoints e.g., before treatment and during the course of treatment; at different times during the course of treatment; before treatment and after the completion of a round of treatment; or during the course of treatment and after the completion of a round of treatment
  • the method comprises: (a) determining a first expression profile for at least one biomarker selected from an mRNA, miRNA, or LincR A listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof in a first sample from the subject, wherein the first expression profile is determined prior to the administration of a therapeutic- agent;
  • the disease or disorder is a cancer (e.g., a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus).
  • the disease or disorder is an immune system disease or disorder, in some embodiments, the disease or disorder is ischemia.
  • an expression level of the at least one biomarker in the first expression profile that is above a threshold level indicates the presence of the disease or disorder.
  • an expression levei of the at least one biomarker in the second expression profile that is below a threshold level indicates efficacy of treatment.
  • the determining of an expression profile comprises:
  • a sample e.g., a nucleic acid sample, protein sample, or a tissue sample
  • an expression profile is determined using a biochip array (e.g., an antibody chip array, a tissue chip array, a protein chip array, a nucleic acid array, or a peptide chip array).
  • the determining of an expression profile comprises detecting the presence or absence of the at least one biomarker, quantifying the amount of the at least one biomarker, and qualifying the type of the at least one biomarker (e.g., categorizing at least one function of the biomarker, e.g., a function as described in Table 2, Table 4A, or Table 4B).
  • the substrate is a microliter plate comprising one or more biospecific affinity reagents that bind to the biomarker, and wherein the biomarker is detected using a fluorescent label.
  • the one or more capture reagents are immobilized on a biochip array, in some embodiments, the biochip array is an antibody chip array, a tissue chip array, a protein chip array, a nucleic acid array, or a peptide chip array.
  • the at least one biomarker is a protein translated from an mRNA listed in Table 5 and wherein the protein biomarker is measured by immunoassay.
  • the at least one biomarker for use according to the diagnostic, prognostic, and therapeutic methods described herein is an mRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof.
  • the mRNA or protein is an mRN A or protein that is involved in inflammatory response, cell proliferation, cell growth, cell movement, or cell-to-cell signaling.
  • the mRNA or protein is an inflammatory response gene or protein listed in Table 4A.
  • the mRNA or protein is a cellular growth and/or proliferation gene or protein listed in Table 4B.
  • the at least one biomarker is an miRNA or LincR A that is involved in cell (e.g., tumor) growth or suppression of tumor growth.
  • the at least one biomarker is an miRNA or LincRNA listed in Table 5 or a human ortholog thereof.
  • the at least one biomarker is a cytokine or a regulator and/or marker of myeloid cells, dendritic cells, natural killer ceils, and/or T-celis.
  • the at least one biomarker is Cell , Cxcl9, Hrngbl , Fgl2, Cd209a, Foxj l , Klra2, miR-1 186, miR-98, miR-5097, miR- 1942, or miR-708.
  • the at least one biomarker is selected from Cell , Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l, and Klra2.
  • the at least one biomarker is selected from miR-1 186, miR-98, miR-5097, miR-1942, and miR-708,
  • the disease or disorder is cancer.
  • the cancer is anal carcinoma, bladder carcinoma, breast carcinoma, cervix carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukem ia, head and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non- Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung (non-small ceil carcinoma), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, soft tissue sarcoma, brain tumor, or rectal tumor, in some embodiments, the cancer is renal carcinoma, skin cancer, bladder cancer, prostate cancer, or breast cancer.
  • the cancer is a metastatic cancer. In some embodiments, the cancer is an invasive cancer. In some embodiments, the cancer is a solid tumor, in some embodiments, the cancer is a carcinoma of the kidney, lung, liver, spleen., pancreas, intestine, colon, mammary g!and, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, brain, or esophagus, in some embodiments, the cancer is a hypoxic tumor, e.g., a hypoxic solid tumor. In some embodiments, the tumor (e.g., hypoxic tumor) overexpresses CALX.
  • the disease or disorder is an immune disease or disorder
  • the immune disease or disorder is an autoimmune disease, asthma, ataxia telangiectasia, arthritis (e.g., rheumatoid arthritis), autoimmune polyglandular syndrome, Burkitt lymphoma, diabetes (e.g., type 1 diabetes), DiGeorge syndrome, familiar
  • Mediterranean fever Guillain-Barre syndrome, inflammatory bowel disease, leukemia, lupus, multiple sclerosis, common variable immunodeficiency, severe combined immunodeficiency, human immunodeficiency virus/acquired immune deficiency syndrome, or drug-induced immunodeficiency.
  • the disease or disorder is ischemia.
  • the ischemia is cardiac ischemia, brain ischemia, kidney ischemia, ischemia! colitis, mesenteric ischemia, or an ischemia-reperfusion injury.
  • the disease or disorder is wound healing, in some embodiments, the disease or disorder is acute renal failure.
  • a therapeutic agent for use according to any of the methods of the present invention can be any composition that has or may have a pharmacological activity.
  • Agents include compounds that are known drugs, compounds for which pharmacological activity has been identified but which, are undergoing further therapeutic evaluation, and compounds that are members of collections and libraries that are screened for a pharmacological activity.
  • the therapeutic agent is a cell, e.g., a stem cell; an anti-cancer, e.g., an anti- signaling agent (e.g., a cytostatic drug) such as a monoclonal antibody or a tyrosine kinase inhibitor; an anti-pro!iferative agent; a chemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeutic agent; and/or a radioiherapeutic agent.
  • an anti-cancer e.g., an anti- signaling agent (e.g., a cytostatic drug) such as a monoclonal antibody or a tyrosine kinase inhibitor
  • an anti-pro!iferative agent e.g., a chemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeutic agent; and/or a radioiherapeutic agent.
  • an anti-cancer e.g., an anti- signaling agent (
  • the therapeutic agent is administered at a therapeutically effective amount or dose.
  • a therapeutically effective amount or dose will vary according to several factors, including the chosen route of administration, the forrnuiation of the composition, patient response, the severity- of the condition, the subject's weight and the judgment of the prescribing physician.
  • the dosage can be increased or decreased over time, as required by an individual patient. In certain instances, a patient initially is given a low dose, which is then increased to an efficacious dosage tolerable to the patient. Determination of an effective amount is well within the capability of those skilled in the art.
  • the route of administration of a therapeutic agent can be oral, intraperitoneal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, infusion; iiposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
  • a therapeutic agent is formulated as a pharmaceutical composition
  • a pharmaceutical composition incorporates particulate forms, protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method/mode of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, etc.
  • a pharmaceutical composition comprises an acceptable carrier and/or excipients.
  • a pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the therapeutic agent.
  • the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration.
  • Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s).
  • Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipienis or other stabilizers and/or buffers.
  • carbohydrates such as glucose, sucrose, or dextrans
  • antioxidants such as ascorbic acid or glutathione
  • chelating agents such ascorbic acid or glutathione
  • low molecular weight proteins compositions that reduce the clearance or hydrolysis of the active agents, or excipienis or other stabilizers and/or buffers.
  • Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005.
  • Various pharmaceutically acceptable excipienis are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipienis (5
  • the therapeutic treatment comprises immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment.
  • the therapeutic treatment comprises immune stimulation with an immunogenic composition, e.g., a tumor- associated antigen (TAA)-cytokine fusion protein.
  • TAA tumor-associated antigen
  • a subject is administered a TAA-cytokine fusion protein to induce a cell-mediated immune response
  • the TAA-cytokine fusion protein can be formulated according to methods known in the art.
  • the TAA-cytokine fusion protein is administered as a polypeptide comprising a fusion protein, as dendritic cells or other cells pulsed with the fusion protein, as a nucleic acid encoding a fusion protein (e.g., as a gene therapy vector, e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, etc.), as dendritic cells or other cells transfected with a nucleic acid encoding a fusion protein (e.g., via a recombinant virus, plasmid DMA transfection, etc.), or as a nucleic acid encoding a fusion protein complexed with a transfection agent.
  • a gene therapy vector e.g., adenovirus,
  • the TAA-cytokine fusion protein comprises the TAA carbonic anhydrase IX (CAIX), or a portion thereof, attached to a granulocyte macrophage colony stimulating factor (GM-CSF).
  • the fusion protein comprises human CAIX, or a portion thereof, attached to human GM-CSF.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • Suitable CAIX-GM-CSF fusion proteins, as well as methods of making, formulating, and administering such fusion proteins, are described in US 7,572,891 and US 8,378,084, incorporated by reference herein.
  • the CAIX protein in the fusion protein is a "CAIX variant" protein as described in US 8,378,084.
  • an imrauno-effector is co-administered with the TAA- cytokine fusion protein
  • immuno-effectors include, but are not limited to, interferoii-ot, interferon- ⁇ , interferon- ⁇ , interferon-t, interferon- ⁇ , tumor necrosis factor- , tumor necrosis factor- ⁇ , interleukin-2, interieukin-6, interleukin-7, interleukin-12, interleukin-15, B7- 1 T cell co-stursuiatory molecule, B7-2 T cell co-stimulatory molecule, immune cell adhesion molecule (ICAM)-l , granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, and combinations thereof,
  • IAM immune cell adhesion molecule
  • adoptive immunotherapy is administered.
  • adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) tumor cells.
  • the immune response results in inhibition of tumor and/or metastatic cell growth and/or proliferation and most preferably results in neoplastic cell death and/or resorption,
  • the immune ceils can be derived from a different organism/host
  • dendritic cells e.g., isolated from the patient or autologous dendritic cells
  • the dentritic cells can be transfected with nucleic acids encoding the TAA- cytokine fusion protein and then re-introduced into a patient
  • the present invention provides methods of identifying a drug candidate agent, e.g., for treating a disease or disorder, using biomarkers of immunoeditmg- escape status.
  • the method comprises:
  • the one or more target biomarkers is an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof, and wherein differential expression of the one or more target biomarkers in the expression profile of the eel! contacted with the candidate agent relative to a control identifies the candidate agent as useful for treating the disease or disorder in the subject.
  • the method of identifying a drug candidate agent comprises detecting the expression profile of at least one biomarker (e.g., I, 2, 3, 4, 5, 6, 7, S, 9, 10 or more target biomarkers) selected from an rnRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof, in some embodiments, the rnRNA or protein biomarker is an rnRNA or protein that is involved in inflammatory response. In some embodiments, the mRNA or protein biomarker is an inflammatory response gene or protein listed in Table 4A.
  • at least one biomarker e.g., I, 2, 3, 4, 5, 6, 7, S, 9, 10 or more target biomarkers
  • the mRNA or protein biomarker is an mRNA or protein that is involved in cellular growth and/or proliferation, in some embodiments, the mRNA or protein biomarker is a cellular growth and/or proliferation gene or protein listed in Table 4B. In some embodiments, the at least one biomarker is an miRNA or LincRNA listed in Table 5 or a human ortholog thereof.
  • the at least one biomarker is selected from Ccl 1 , Cxc!9, Hmgb l, I- -12, Cd209a, Foxj l, Klra2, miR-1 186, miR-98, miR-5097, miR-1942, and miR- 708.
  • the at least one biomarker is Cell, Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l, or Klra2.
  • the at least one biomarker is miR-1 186, miR-98, miR-5097, miR-1942, or miR-708.
  • differential expression of at least one biomarker selected from Cell , Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l , lra2, miR-1 186, miR-98, miR-5097, and miR-1942, and miR-708 in the ceil contacted with the candidate agent e.g., upregulation or downregulation of the biomarker in the cell contacted with the candidate agent relative to a control cell not contacted with the candidate agent
  • upregulation or downregulation of the biomarker in the cell contacted with the candidate agent relative to a control cell not contacted with the candidate agent identifies the candidate agent as useful for treating the cancer or immune system disease or disorder.
  • disease or disorder is a cancer.
  • the cancer is ana! carcinoma, bladder carcinoma, breast carcinoma, cervix carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukemia, head and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non- Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung (non-small cell carcinoma), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, or soft tissue sarcoma.
  • the cancer is renal carcinoma, skin cancer, bladder cancer, prostate cancer, or breast cancer, in some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is an invasive cancer.
  • the disease or disorder is an immune system disease or disorder.
  • the immune disease or disorder is an autoimmune disease, asthma, ataxia telangiectasia, arthritis (e.g., rheumatoid arthritis), autoimmune polyglandular syndrome, Burkitt lymphoma, diabetes (e.g., type 1 diabetes), DiGeorge syndrome, familiar Mediterranean fever, Guiilain-Barre syndrome, inflammatory bowel disease, leukemia, lupus, multiple sclerosis, common variable immunodeficiency, severe combined immunodeficiency, human immunodeficiency virus/acquired immune deficiency syndrome, or drug-induced immunodeficiency.
  • the disease or disorder is ischemia.
  • the ischemia is cardiac ischemia, brain ischemia, kidney ischemia, ischemial colitis, mesenteric ischemia, or an ischemia-reperfusion injury, in some embodiments, the disease or disorder is wound healing. In some embodiments, the disease or disorder is acute renal failure.
  • a drug candidate agent is identified as suitable for use in treating the disease or disorder when the one or more biomarkers is differentially expressed (upregulated or downregulated) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or by at least 1.2-fold, 1 .5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold in the ceil as compared to a threshold level or a control cell (e.g., a non-diseased cell).
  • a control cell e.g., a non-diseased cell
  • Drug candidate agents that can be evaluated according to the methods described herein include, but are not limited to, peptides, proteins, oligopeptides, circular peptides, pepticiomimeties, antibodies, polysaccharides, lipids, fatty acids, inhibitory RNAs (e.g., siRNA, miRNA, or shRNA), immunotherapeutics, vaccines, polynucleotides,
  • the agent is a small organic molecule. In some embodiments, the agent is a peptide or protein. In some embodiments, the agent is an immunotherapeutic.
  • Drug candidate agents are not limited by therapeutic category, and can include, for example, analgesics, anti- inflammatory agents, antihelminthics, anti-arrhythmic agents, antibacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti- epileptics, anti-fungal agent, anti -gout agents, anti-hypertensive agents, anti-ma lariais, antimigraine agents, anti-muscarinic agents, anti-neoplastic agents, erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, ⁇ -blockers, cardiac inotropic agents, corticosteroids, diuretics, anti-park insonian agents, gastro-iniestinal agents, histamine receptor antagonists, keratolyses, lipid regulating agents, anti-anginal agents, Cox-2
  • the candidate agent is identified in siiico using
  • a candidate agent is identified from a library of agents.
  • the library of agents comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000 agents or more, it will be appreciated that there are many suppliers of chemical compounds, including Sigma (St.
  • the library is a combinatorial chemical or peptide library.
  • a combinatorial chem ical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length ⁇ i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • a candidate agent can be identified by screening a library containing a large number of potential therapeutic compounds.
  • the library can be screened in one or more assays, as described herein, to identify those library members that display a desired characteristic activity,
  • the compounds thus identified can serve as conventional "lead compounds” (e.g., for identifying other potential therapeutic compounds) or can themselves be used as potential or actual therapeutics.
  • Libraries of use in the present invention can be composed of amino acid compounds, nucleic acid compounds,
  • Carbohydrate libraries have been described in, for example, Liang et al, Science, 274: 1520-1522 (1996); and U.S. Patent No. 5,593,853.
  • Representative amino acid compound libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent Nos. 5,010,175; 6,828,422; and 6,844,161; Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991); Houghton et al. , Nature, 354:84-88 (1991 ); and Eichier, Comb Chern High Throughput Screen., 8:135 (2005)), peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication No. WO 93/20242), random bio- oligomers (PCT Publication No.
  • peptide libraries see, e.g., U.S. Patent Nos. 5,010,175; 6,828,422; and 6,844,161; Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991); Houghton et
  • WO 92/00091 vinyiogous polypeptides (Hagihara et al., ,1. Amer. Chern. Soc, 1 14:6568 ( 1992)), nonpeptidal peptidomimetics with ⁇ -D-glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc., 1 14:9217-9218 (1992)), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., U.S. Patent Nos. 6,635,424 and 6,555,310; PCT Application No.
  • nucleic acid compound libraries include, but are not limited to, genomic DNA, cDNA, mRNA, inhibitory R A (e.g., RNAi, siRNA), and antisense RNA libraries. See, e.g., Ausubel, Current Protocols in Molecular Biology, eds. 1987-2005, Wiley Interscience; and Sambrook and Russell, Molecular Cloning: A Laboratory Manual , 2000, Cold Spring Harbor Laboratory Press. Nucleic acid libraries are described in, for example, U.S. Patent Nos . 6,706,477: 6,582,914; and 6,573,098. cDNA libraries are described in, for example, U.S. Patent Nos. 6,846,655: 6,841,347; 6,828,098; 6,808,906; 6,623,965; and
  • RNA libraries for example, ribozyme, RNA interference, or siRNA libraries, are described in, for example, Downward, Cell, 121 : S 13 (2005) and Akashi et al., Nat. Rev. Mol Cell Biol, 6:413 (2005).
  • Antisense RNA libraries are described in, for example, U.S. Patent Nos. 6,586, 180 and 6,518,017. f0137j
  • Representative small organic molecule libraries include, but are not limited to, diversomers such as hydantoins, benzodiazepines, and dipeptides (Hobbs et at, Proc. Nat. Acad. Sci.
  • thiazolidinones and metathiazanones e.g., U.S. Patent No. 5,549,974; pyrrolidines (e.g., U.S. Patent Nos. 5,525,735 and 5,519,134); morpholino compounds (e.g., U.S. Patent. No. 5,506,337); tetracyclic benzimidazoles (e.g., U.S. Patent No, 6,515,122): dihydrobenzpyrans (e.g. , U.S. Patent No. 6/790,965); amines (e.g., U.S. Patent No, 6,750,344); phenyl compounds (e.g., U.S. Patent No. 6,740,712); azoles (e.g., U.S. Patent No. 6,683,191);
  • pyridine carboxarnides or sulfonamides e.g. , U.S. Patent No. 6,677,452
  • 2- aminobenzoxazoles e.g., U.S. Patent No. 6,660,858
  • isoindoles, isoox indoles, or isooxyquinolines e.g., U.S. Patent No. 6,667,406
  • oxazolidinones e.g., U.S. Patent No. 6,562,844
  • hydroxylamines e.g., U.S. Patent No, 6,541,276).
  • the invention further provides compositions, kits, and arrays for practicing the methods described herein.
  • the invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more capture reagents (e.g., polynucleotides or polypeptides) that bind a biomarker as described herein (e.g., an niRN A, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) immobilized on a solid support, and a labeling reagent.
  • the assay compositions can also include additional reagents that are desirable for hybridization.
  • kits for carrying out the methods described herein can include one or more capture reagents (e.g., polynucleotides or polypeptides) that bind a biomarker as described herein (e.g., an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human orthoiog thereof) as well as other components, in some embodiments, the kit comprises a capture reagent that is an antibody that binds to a protein translated from an mRNA listed in Table 5.
  • a biomarker e.g., an mRNA, miRNA, or LincRNA listed in Table 5
  • the kit comprises a capture reagent that is an antibody that binds to a protein translated from an mRNA listed in Table 5.
  • the kit comprises a capture reagent that is a nucleic acid that is substantially complementary to an mRNA, miRNA, or LincRNA listed in Table 5.
  • the kit comprises a a plurality of capture reagen ts, wherein each capture reagent binds a biomarker selected from the group consisting of the biomarkers listed in Table 5 and their human ortho!ogs,
  • a detectable label is conjugated to the capture reagent for indicating the presence of the capture reagent and therefore the biomarker.
  • the kit comprises one or more capture reagents and one or more detection reagents (e.g., a primary antibody that is a capture reagent for a biomarker of interest, and a secondary antibody having a binding specificity for the primary antibody and having a detectable label or moiet)').
  • the kit further comprises additional reagents for the detection reaction, e.g., buffers, drugs, cytokines, or wash solutions for selectively retaining the bound biomarker to the capture reagent after washing.
  • the kit includes a container containing the capture reagent(s).
  • kits of the present invention can include instructions to the kit user (e.g., instructions for using the capture reagent to detect the biomarker, instructions that provide for contacting a test sample with the capture reagent and detecting the biomarker that is present in the sample, if any, that is retained by the capture reagent; and/or instructions for using the kit for detecting a disease or disorder as described herein. VII. Examples
  • Example ⁇ Dendritic Cell-Based Immunotherapy HI Prevention and Treatment of Renal Cell Carcinoma: Efficacy, Safety, and Anti-Tumor Activity of Ad-GM-CAIX in Immunocompetent Mouse Models
  • the dendritic cell vaccine DC- Ad-GM-CAIX is an active, specific immunotherapy with the potential of providing a safe and effective therapy against renai cell carcinoma (RCC), Using immunocompetent Balb/c mouse models we tested the efficacy and mechanism of the vaccine to prevent and treat the growth of a syngeneic RCC (RE CA) engineered to overexpress the human TAA carbonic anhydrase IX ( PR-IX).
  • RE CA syngeneic RCC
  • PR-IX human TAA carbonic anhydrase IX
  • NPR- IX tumor development was specifically and significantly delayed by 13 days in DC-Ad- GM-CAIX- treated mice ( ⁇ G.001), and tumor volumes were 79% smaller at study termination compared to the controls (day 24, P ⁇ Q, 07), Six of these mice remained tumor- free for >1 year, in a treatment model, NPR-TX tumors remained smaller in DC-Ad- GM-CAIX-treated mice for 8 days (P ⁇ 0.002), achieving a 60% growth inhibition at termination, No vaccine-related toxicity was observed.
  • the critical mechanistic parameter separating responsive from non-responsive tumors was hCAIX protein expression, demonstrated by aggressive growth of tumors that did not express hCATX protein and in sham-treated mice (DC-Ad-Null), and by in vitro anti-hCAIX cytotoxic T-cell IFN- ⁇ response (Cd8+). No murine serum anti-hCAIX antibodies were detected.
  • RCC renal cell carcinoma
  • TAAs tumor-associated antigens
  • CAIX carbon ic anhydrase IX
  • CAIX regulates proton exchange to buffer intracellular H, a function crucial for cell survival (4), CAIX expression has been described in many normal and cancerous tissues (4, 5) and is directly regulated by the hypoxia-inducible factor la-von Hippel-Lindau (VHL) pathway. Upon loss of pVHL function in clear cell RCC, stabilization of HIF- ⁇ occurs, leading to the overexpression of CAIX and rendering it useful as a diagnostic and therapeutic TAA (6-10).
  • VHL hypoxia-inducible factor la-von Hippel-Lindau
  • GM-CAIX to induce a C AIX- targeted, T-cell mediated, and MHC-restricted anti-tumor activity (13-1 5).
  • hCAIX human CAIX
  • RENCA cell line 16
  • the cells were then sorted for hCAIX expression (Newly Purified RENCA-CAIX; NPR-IX) and transplanted in syngeneic Balb/c mice.
  • mice in group A maintained their body weight throughout the experiment, while mice in each of the control groups B-F suffered significant weight loss ( ⁇ 0.001; Fig. 2G-J).
  • group G showed a significantly smaller median weight loss compared to the control groups H-I combined from day 15 to 21 ( ⁇ 0.01 1 ; Fig. 2K-L).
  • none of the organs in groups A-I showed evidence of metastases or systemic toxicity caused by the immunotherapy, such as histopathologic inflammatory lesions or atrophy.
  • IFN- ⁇ levels were consistently and predominately increased in splenocytes of DC-Ad-GM-CAlX treated mice (group A) co-incubated with NPR-IX. However, lower IFN- ⁇ levels were also released after co-incubation with RENCA . Splenocytes of non-treated mice (group E) co-incubated with NPR-IX or RENCA did not release IFN- ⁇ (Fig. 4).
  • MHC class ⁇ (MHC-I) expression is crucial for CTL targeting of tumor cell
  • tumors that evaded the DC-Ad-GM-CAIX therapy exhibited differential expression of MHC-L The qPCR analysis suggested that the Balb/c MHC-I markers H-2K d and H-2D d were expressed and at similar levels in tumors that evaded the DC-Ad-GM-CAIX therapy and in tumors of mice treated with DC-Ad-Null (Table 2, Fig.
  • the differentially expressed genes were associated with significantly enriched gene ontology categories (IPA, ingenuity, CA; P ⁇ 0.GS) including immune response, proliferation, cell growth, cell movement, and cell-to-cell signaling (Table 4A-B).
  • cancer immunoediting is a complex but orderly continuum that can be separated into a series of three overlapping phases: elimination (tumor destruction), equilibrium (dormancy), and evasion (escape) (22).
  • Hmgbl, Cd209a (DC-SIGN) and Fgl2 all of which were found to be up-regulated in the evading tumors (group A), have been suggested to function as recruiters of immunosuppressive myeloid-derived suppressor cells and Treg (27-29), leading to evasion from immune surveillance (30, 31) and to inhibition of DC maturation and T-cell proliferation (Table 2, Fig. 5C) (32).
  • Another characteristic of RCC are the presence and role of NK cells (33), One of the negative regulators of murine myeloid cells is Klra2 (Ly49B).
  • Klra2 is not normally expressed at detectable levels on NK cells, it appears to be up- regulated on myeloid cells and NK cells following encounter with inflammatory cytokines or bacterial products, decreasing their reactivity and preventing non-specific- inflammatory responses (34, 35), Now for the first time we report that Kira2 is also up-regulated in tumors that have evaded the DC-Ad-GM-CAIX immunotherapy, suggesting an interesting new role In inhibiting NK cell-mediated target RCC lysis (Table 2, Fig, SC, Table 5).
  • miR-98 is suggested to have tumor suppressive function in breast tumors, tumor promoting function in other types of tumors (40-42).
  • miR-708 expression which we found to be down- regulated in tumors that evaded immunotherapy, was recently described to be attenuated in RCC and was suggested to have a role as a pro-apoptoic tumor suppressor (43).
  • RENCA is a spontaneous murine renal cortical adenocarcinoma arising in BaJb/c mice and is a model of human adult RCC in
  • RENCA cells were a gift from Dr. R. Wiltrout (NCi/NIH) (44).
  • RENCA-CAIX cells were generated by stably transducing the human CAIX gene in RENCA cells (16), NPR-IX cells are hCATX FACS-purified RENCA-CAIX ceils, which repeatedly had >90% hCAIX-positive cells in flow cytometry analysis (Fig. 7).
  • RCC in vivo models had 6 groups of 8 immunocompetent Balb/c mice each. Groups A-D were immunized by subcutaneous transplantation of 2x10 6 DCs in 150 ul of PBS I into the right flank. Two groups (A, B) were transplanted with DC- Ad-GM-CAIX and two groups (C, D) with DC-Ad-Null. Groups E and F had no treatment. The DC transplantation was repeated after 6 days. Twelve days (day 0) after the first DC transplantation, 5xI0 5 CC cells in 100 ⁇ of PBS lx were subcutaneous! ⁇ ' transplanted into the left flank. Three groups (A. C, E) were transplanted with NPR-IX, and three groups (B, D, F) with RENCA cells. The treatment model had three groups (G, H, 1) of 16
  • V ab 2 :*:Pi/6, where "a” is the longest diameter of the tumor and 'b" is the longest diameter perpendicular to "a".
  • the slides were then stained with hCAIX (rabbit, 1 : 1000 dilution, Novus Biological, CO), rnCAiX (goat, 1 :200, R&D System, MN), Kt67 (rat, 1 : 100, DAKO, CA), Pecaml (Cd31) (goat, 1 : 100, Santa Cruz, CA), Cdl l b (rat, 1 :50, AbDSerotec, NC), and F4/80 (rat, 1 :50, AbDSerotec), and the sections were incubated with secondary rabbit and rat (Cdl l b, F4/S0, K167) or rabbit anti-goat (Cd31, mCAIX) immunoglobulin for 30 min at 1 :200 dilution (DakoCytomation).
  • hCAIX rabbit, 1 : 1000 dilution, Novus Biological, CO
  • rnCAiX goat, 1 :200, R&D System
  • the signal was detected using the Dakocvtoniation Envision System Labelled Polymer HRP anti rabbit (DakoCytomation) for all.
  • an ApoptagPlus Peroxidase In Situ Apoptosis kit (Miliipore, MA) was used. Staining was performed according to the manufacturer's instructions. All sections were visualized with the diaminobenzidine reaction and
  • mice splenocytes (5x10 5 ) from groups A and E were separately co-incubated with RENCA and NPR-IX cells (2.5x10 ), respectively. After 48 hours, supernatants were analyzed for mouse lFN- ⁇ by ELISA (eBiosctence, CA).
  • RNA was isolated using Trizoi Reagent (Invitrogen, CA). Total RNA (1 g) was reverse transcribed in a volume of 50 ⁇ . Five ⁇ of the resulting solution was then used for PGR according to the manufacturer's instructions (Applied Biosystems, Inc., Foster City, CA). Gene expression signatures were quantified relative to the expression level of ribosomal 18s. and for iniRNA relative to snoRNA 234 or snoRjNA202. All probes were purchased from Applied Biosystems or Integrated DNA Technologies (IDT, CA). Normalized data are presented as fold difference in log? gene expression. Primers are listed below in Table 3.
  • icroRNA array v6, Exiqon, Denmark having more than 1,891 capture probes covering all human, mouse, rat, and viral miRNAs and 66 proprietary miRNA s spotted in quadruplicate were used for measuring genome miRNA expression, intensities were determined by GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA) and were normalized by median centering of the ratios to a reference control array.
  • Global miRNA expression profiles of samples were examined by multidimensional scaling of about 4,200 features that were flagged as found in >50% of the samples. The features found in ⁇ 60% of samples in both groups A and C were eliminated from further analysis. There were 104 miRNAs
  • Tso CL Zisman A, Pantuck A, et al. Induction of G250-targeted and T-cell-mediated antitumor activity against renal ceil carcinoma using a chimeric fusion protein consisting of G250 and gran ulocy te/monocyte-colony stimulating factor. Cancer Res 2001 ; 61 : 7925-33. 14. Mukouyama H, Janzen NK, Hernandez JM, et al. Generation of kidney cancer- specific antitumor immune responses using peripheral blood monocytes transduced with a recombinant adenovirus encoding carbonic anhydrase 9. Clin Cancer Res 2004; 10: 1421-9.
  • GM-CSF granulocyte/macrophage colony-stimulating factor
  • RCC in vivo metastasis model The renal orthotopic RCC metastasis model had 2 groups of 10 immunocompetent Balb/e mice each. Mice were orthotopicaJ!y transplanted in the left kidney with IxlO 5 NPR-IX cells in 100 ⁇ ! of 90% High Concentration Matrigel (354262, Becton, Dickinson, CA) balanced with PBS Ix (Salumbides et al., Current Protocols in Pharmacology: Pre -Clinical Models of Renal Carcinoma and Their Utility in Drag Development, Vol. Unit 14.13 DOT. 2009). Tumor bearing animals were randomly assigned into the 2 groups receiving monotherapy of DC-Ad-GM- CAIX, or no therapy.
  • group B was subcutaneously transplanted in the right flank with 2xl0 6 DC-Ad-GM-CAIX in 150 ⁇ of PBS Ix and group A had no treatment.
  • Mouse performance status was assessed daily, and body weight was measured twice a week over the first 2 weeks and then daily.
  • At termination partial necropsy was performed in all mice and lung metastatic foci were counted and area size measured. The whole study was repeated for the second time using an identical protocol (rounds A and B).
  • DC-Ad-GM-CAIX monotherapy significantly reduced the amount and area size of lung NPR-IX metastatic foci in syngeneic Balb/c mice (group B; Figs. 11 and 12), In these groups, mean amounts of lung metastatic foci were 2.5-fold smaller (p ⁇ 0.05) and with a mean 3.4 smaller area size (p ⁇ 0,05) than the untreated mice (group A),
  • Table 4A Gene ontology category - Inflammatory Response
  • Table 4B Gene ontology category - Cellular Growth and/or Proliferation
  • IGFBP3 JL 1 RL 3 ⁇ ⁇ JRGM, J " UN,LA MA4, LAMB l .LOX RPl (includes EG: 16971), MCC,Ms4a4b (includes others),NOD2,Prl2c2 (includes others), RCA 1 ,REG 1 A.RG Sl 6,
  • ILIRL IJRGM, LOX, Ms4a4b (includes
  • CD209,CHRNA1,FGL2 ( FOXJ 1 ,FYB,HM GB 1 ,HPGDS,ICAM 1 ,IL 1 RL 1 ,
  • IRGM,Ms4a4b (includes
  • chr5 120 0.646 0.006315277 A .

Abstract

The present invention provides methods of qualifying immunoediting-escape status in a subject, comprising measuring at least one biomarker in a sample from the subject and correlating the measurement with the subject's immune response. The present invention also provides diagnostic and therapeutic methods using the biomarkers of immunoediting-escape status as described herein.

Description

BIOMARKERS FOR THE IMMUNOEDITING-ESCAPE PHASE
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Application No. 61/661,786, filed June 19, 2012, and to U.S. Provisional Application No. 61/669,633 filed July 9, 2012, the contents of each of which are incorporated by reference herein in its entirety.
BACKGROUND OF THE INVENTION
[0002] The treatment of advanced renal cell carcinoma (RCC) represents a serious medical challenge, with approximately 13.000 deaths in the United States in 2010. In spite of new targeted therapies, including the FDA-approved agents sorafenib, sunitmib, temsirolimus, everolimus, bevacizumab, and pazopanib, complete remission of metastatic disease is often not achieved. In addition, these cytostatic therapies require chronic administration and are associated with adverse side effects and treatment resistance typically develops within year.
[0003] Metastatic RCC and melanoma are members of a restricted class of solid tumors that are significantly responsive to, and in a small percentage of cases, curable by systemic treatment with high-dose interleukin-2. The efficacy seen with high-dose interleukin-2 therapy in patients with metastatic RCC tumors, although limited, has led to great interest in the development of novel immunotherapy approaches that can be both effective in larger patient cohorts while also having fewer toxic side effects. The identification of tumor- associated antigens (TA s) overexpressed in RCC has led to the development of humoral and cell-mediated immunotherapies. Among the few TAAs discovered for RCC is carbonic anhydrase IX (CAIX / G250).
[0004] CAIX belongs to the carbonic anhydrase group of enzymes that regulate reversible proton exchange to buffer intracellular H, a function crucial for cell survival, especially in the harsh hypoxic tumor m icroenvironraent. CALX expression has been described in many normal and cancerous tissues and is directly regulated by the hypoxia-inducible factor la- von Hippel-Lindau (VHL) pathway. Upon loss of pVTIL in clear cell RCC, stabilization of HIF- I a occurs leading to the overexpression of CAIX, rendering it useful as a diagnostic and prognostic biomarker, but also as a target for humoral and ce!l-rnediaied immunotherapies. Several strategies to target CA1X therapeutically have been tested clinically. A peptide-based dendritic cell (DC) vaccination in HLA-A24-postive metastatic RCC patients was shown to be safe, with a median survival time of 21 months. Another study of adoptive cell transfer of CAIX, chimeric immune receptor-transduced T-cells, was aborted due to liver toxicity, thought to be a result of autoimmune action against CAIX-expressing bile duct cells.
[0005] The recent FDA approval of sipuieucel-T (Dendreon Corporation) and its associated body of research have clearly shown the immunotherapeutic benefit of fusing a potent cytokine such as granulocyte-macrophage colony stimulating factor (GM-CSF) with a TAA, and transducing the fused protein into APCs, leading to an effective GM-CSF-mediated immune-stimulation at the site of the targeted tumor. While the precise mechanism of action needs to be further verified, it is designed to activate antigen-presenting cells ex vivo (APCs; e.g., DCs) and then prime T-celis to recognize and kill TAA-positive cancer cells. However, tumor-induced immunosuppression remains a significant obstacle that limits the efficacy of this kind of biological therapy. The present invention addresses this need and others.
BRIEF SUMMARY OF THE INVENTION
[0006] In one aspect methods of qualifying the immunoediting-escape status of a subject are provided. In some embodiments, the method comprises: (a) measuring at least one biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of the bioraarkers listed in Table 5 and their human orthologs; and
(b) correlating the measurement with the subject's immune response; thereby qualifying the immunoediting-escape status of the sub ject. [0007] In some embodiments, the immunoediting-escape status indicates the subject's risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, the presence or absence of disease, stage of disease, or effectiveness of treatment for a disease. In some embodiments, the immunoediting-escape status indicates the presence, absence, or stage of a cancer or the effectiveness of treatment for a cancer in the subject. In some embodiments, the cancer is renal cell carcinoma.
[0008] In some embodiments, the method further comprises: (c) managing treatment of the subject based on the immunoediting-escape status.
[0009J in some embodiments, managing treatment comprises ordering a further diagnostic test, performing surgery, administering a therapy, or taking no further action, in some embodiments, the therapy is immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment.
[0010] In some embodiments, the method comprises measuring at least two (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more) biomarkers in the sample from the subject and correlating the measurement of the biomarkers with the subject's immune response, in some embodiments, the correlating step is performed by a software classification system.
[0011] In another aspect, methods of diagnosing tissue or organ status (e.g., renal status) are provided, in some embodiments, the method comprises: determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, is indicative of a status of cancer, immune-escape, or immune disease,
10012] In yet another aspect, methods of monitoring the treatment of a subject for a disease or disorder are provided. In some embodiments, the method comprises:
(a) determining a first expression profile for at least one biomarker selected from an mRNA, miR A, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof in a first sample from the subject, wherein the first expression profile is determined prior to the administration of a therapeutic agent;
(b) administering a therapeutically effective amount of the therapeutic agent to the subject; and
(c) determining a second expression profile for at least one biomarker listed in Table 5 in a second sample from the subject, wherein the second expression profile is determined subsequent to the administration of the therapeutic agent; wherein modulation of the expression profile indicates efficacy of treatment with the therapeutic agent. [00ί3] in some embodiments, an expression level of the at least one biomarker in the first expression profile that is above a threshold level indicates the presence of the disease or disorder, in some embodiments, an expression level of the at least one biomarker in the second expression profile that is below a threshold level indicates efficacy of treatment. |0014] In some embodiments, the determining of an expression profile comprises:
(i) pro viding a nucleic acid sample from the subject; and (is) capturing the at least one biomarker that is present in the nucleic acid sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at least one biomarker. In some embodiments, the nucleic acid sample is labeled.
[0015] in some embodiments, the determining of an expression profile comprises:
(i) providing a protein sample from the subject; and
(ii) capturing the at least one biomarker that is present in the protein sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at least one biomarker.
[0026] In some embodiments, the determining of an expression profile comprises: fi) providing a tissue sample from the subject; and
(ii) capturing the at least one biomarker that is present in the tissue sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at least one biomarker.
[0017] In some embodiments, the substrate is a microti ter plate comprising one or more biospecific affinity reagents that bind to the biomarker, and wherein the biomarker is detected using a fluorescent label. In some embodiments, the expression profile is determined using a nucleic acid array. In some embodiments, the determining of an expression profile comprises detecting the presence or absence of the at least one biomarker, quantifying the amount of the at least one biomarker, and qualifying the type of the at least one biomarker. In some embodiments, the at least one biomarker is measured using a biochip array. In some embodiments, the biochip array is an antibody chip array, tissue chip array, protein chip array, nucleic acid array, or a peptide chip array. In some embodiments, one or more capture reagents that bind the at least one biomarker are immobilized on the biochip array. [0018] In some embodiments, the at least one biomarker is a protein translated from an mRNA listed in Table 5 and the protein bioraarker is measured by immunoassay.
[0019] in still another aspect, methods for identifying a candidate agent to treat a disease or disorder in a subject are provided, in some embodiments, the method comprises:
(a) contacting a cell with the candidate agent; and
(b) detecting the expression profile of one or more target biomarkers in the ceil, wherein the one or more target biomarkers is an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human orthoiog thereof, and wherein differential expression of the one or more target biomarkers in the expression profile as compared to a control identifies the candidate agent as useful for treating the disease or disorder in the subject.
[0020] In some embodiments, the candidate agent is a small molecule, a peptide, an immunotherapeutic, a vaccine, or a nucleic acid. In some embodiments, the candidate agent is identified in silica using pharmacogenomics.
[Θ021] In still another aspect, methods of determining whether or not a cancer is likely to be responsive to immunotherapy are provided, "in some embodiments, the method comprises: detecting in a biological sample from a subject having the cancer an altered level of at least one biomarker listed in Table 5 or a human orthoiog thereof. In some embodiments, the method comprises detecting an elevated level of at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample. In some embodiments, the method comprises detecting a decreased level of at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample. In some embodiments, the level of the at least one biomarker in the sample from the subject having the cancer is altered by at least 1.2-fold as compared to the level of the at least one biomarker in a control sample, in some embodiments, a level of the at least one bioraarker in the sample from the subject having the cancer that is increased by at least 1.2-fold as compared to the control indicates that the cancer is not likely to be responsive to
immunotherapy. In some embodiments, the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA)-cytokine fusion protein. In some embodiments, the immunotherapy comprises immune stimulation with CAIX-GM-CSF. [0022] In yet another aspect, methods of diagnosing whether or not an individual has a cancer that is likely to be responsive to immunotherapy are provided. In some embodiments, the method comprises: detecting in a biological sample from the individual an altered level of at least one biomarker listed in Table 5 or a human oriholog thereof, in some embodiments, the method comprises detecting an elevated level of the at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample. In some embodiments, an elevated level of the biomarker in the sample from the individual indicates thai the individual has a cancer that is not likely to be responsive to the immunotherapy. In some embodiments, the method comprises detecting a decreased level of the at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample. In some embodiments, the level of the at least one biomarker in the sample from the subject having the cancer is altered by at least 1.2-fold as compared to the level of the at least one biomarker in a control sample. In some embodiments, the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA)--cytokine fusion protein. In some embodiments, the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
[0023] in still another aspect, methods of monitoring the efficacy of treatment for an individual having a cancer are provided. In some embodiments, the method comprises:
(a) detecting in a biological sample from the individual a level of at least one biomarker listed in Table 5 or a human oriholog thereof; and
(b) determining whether or not the level of the at least one biomarker in the sample is altered compared to a control; thereby determining whether the cancer is responsive to the treatment.
[0024] In some embodiments, the treatment is an immunotherapy. In some embodiments, the immunotherapy comprises immune stimulation with a tumor-associated antigen
(TA A) -cytokine fusion protein. In some embodiments, the immunotherapy comprises immune stimulation with CAIX-GM-CSF. In some embodiments, an elevated level of the at least one biomarker relative to a control indicates that the cancer is not responsive to the treatment, In some embodiments, wherein the altered level of the at least one biomarker indicates that tire cancer is not responsive to the treatment, the method further comprising administering to the individual an alternative treatment. [0025] In some embodiments, the at least one biomarker is a protein translated from an mRNA listed in Table 5 or its human ortholog, In some embodiments, the at least one biomarker is an mRNA. in some embodiments, the at least one biomarker is an miRNA. In some embodiments, the at least one biomarker is an alternative isoform mRNA. In some embodiments, the at least one biomarker is a LincK A. In some embodiments, the at least one biomarker is a gene. In some embodiments, the at least one biomarker is a biomarker listed in Table 2 or its human ortholog. in some embodiments, the at least one biomarker is a biomarker listed in Table 4A and/or 4B or its human ortholog. In some embodiments, the at least one biomarker is Cel l, Cxcl9, Hmgbl, FgI2, Cd209a, Foxj l, Klra2, miR- 1 186, iniR-98, miR-5097, miR-1942, or miR-708. in some embodiments, the biomarker is selected from Cell, Cxcl9, Hmgbl , Cd209a, Fgl2, Kira2, and Foxj l . In some embodiments, the biomarker is selected from miR- 1 186, raiR-98, miR-5097, mill- 1942, and miR-708.
[0026] In some embodiments, the sample is from blood, serum, plasma, urine, saliva, feces, kidney, lung, liver, prostate, bladder, intestine, colon, pancreas, or a tumor tissue. [0027] in some embodiments, the disease or disorder is a a cancer, e.g., a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is renal cell carcinoma. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the disease or disorder is an immune system disease or disorder. In some embodiments, the disease or disorder is ischemia.
[0028] In yet another aspect, kits for detecting immunoediting-escape phase biomarkers are provided. In some embodiments, the kit comprises: at least one capture reagent that binds a biomarker selected from the group consisting of an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, and human orthologs thereof. In some embodiments, the kit comprises a plurality of capture reagents, wherein each capture reagent binds a biomarker selected from the group consisting of the biomarkers listed in Table 5 and their human orthologs. In some embodiments, the capture reagent is an antibody that binds to a protein translated from an mRNA listed in Table 5, In some embodiments, the capture reagent is a nucleic acid that is substantially complementary to an mRNA, miRNA, or LincRNA listed in Table 5. in some embodiments, the kit further comprises a wash solution that selectively retains the bound biomarker to the capture reagent after washing. In some embodiments, the kit further comprises instructions for using the capture reagent to detect the biomarker. In some embodiments, the instructions provide for contacting a test sample with the capture reagent and detecting the biomarker that is present in the sample, if any, that is retained by the capture reagent. In some embodiments, the kit further comprises instructions for using the kit for the detection of a disease or disorder.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] Figure 1. Experimental outlines. (A) Prevention Model: Bone marrow cells are harvested from syngeneic Balb/c mice, ex vivo differentiated to DCs and transduced with either Ad-GM-CAIX (DC-Ad-GM-CAIX) or with sham Ad-Nu!l (DC-Ad-Nuli). First, mice are vaccinated by two rounds of subcutaneous transplantation of DC-Ad-GM-CAIX (groups A, B) or DC-Ad-Null (groups C, D) or not vaccinated (groups E, F), Twelve days after the second vaccination, mice are challenged by subcutaneous transplantation with either NPR-IX (groups A, C, E) or RENCA (groups B, D, F) tumor cells. (B) Treatment Model: NPR-IX tumor cells are first subcutaneously transplanted, and only after tumors are established the mice are vaccinated with DC-Ad-GM-CAIX (group G), DC-Null (group H) or not vaccinated (group Ϊ).
[0030] Figure 2. Tumor volume, survival, and body weight Prevention model: (A) Tumor growth over time, (B) time to observable tumor growth (Kaplan-Meier curve), and (C, D) repeated study, (G, I) Body weight over time and (H, J) time to 15% weight loss (Kaplan- Meier curve). Treatment model: (E) Tumor growth over time and (F) time to tumor growth >600 mm3 (Kaplan-Meier curve). ( ) Body weight ove time and L) time to 1 5% weight loss (Kaplan-Meier curve).
[0031] Figure 3. ImmMnobistochcmical analysis of NPR-IX tumors with human CAIX (hCAfX), The panels show staining of membranous hCAIX in brown. Original
magnification, x200. Prevention model: (A) DC-Ad-GM-CAIX-treated mouse (group A, 10% hCAIX positive); (B) DC-Ad-Null-treated mouse (group C, 70% hCAIX positive); (C) Non- treated mouse (group E, 70% hCAIX positive). Treatment model: D) DC-Ad-GM-CAIX- treated mouse (group G, 10% hCAIX positive); (E) DC-Ad-Null-treated mouse (group H, 80% hCAIX positive); (F) Non-treated mouse (group I, 50% hCAIX positive).
[0032] Figure 4. ELISA of mterferon-gamma (IFN-γ). The trend of IFN-γ was consistently up-regulated (123- 151 % ) in 3 replicas of splenocytes of DC-Ad-GM-CAIX- treated mice (group A) co-incubated with NPR-IX relative to co-incubation with RENCA. Three replicas of splenocytes of non-treated mice (group E) co-incubated with NPR-ίΧ or RENCA did not induce !FN-γ.
[0033] Figure 5. Clusters of differential (A) niRNA and (B) miRNA expression between tumors of group A and C. Multidimensional scaling using 1 -correlation as distance metric. Validation of (C) gene expression and (D) miRNA by qPCR. Error bars shown are standard error of the mean. Tumor expression levels relative to control are shown. Gene and miRNA expressions were normalized to 188 rR A and to snoRNA234 (except for mir-708, which was normalized to snoRNA202), respectively. [0034] Figure 6. Dot plots of indirect FACS for anti-hCAIX antibodies using (A) NPR-IX and (B) RENCA stained by mouse serum from group A, and (C) NPR-IX and (D) RENCA stained by mouse serum from group E, followed by staining with FITC-conjugated anti- mouse IgG. Comparable rates of positive staining for mouse IgG within group A and group E suggest that sera of mice of group A and E do not contain measurable amounts of mouse anti- hCAIX antibodies.
]003S] Figure 7. Dot plots of FACS for hCAIX in NPR-IX and RENCA cells. (A) Unstained NPR-IX. (B) NPR-IX stained with anti-hCAIX-FlTC, showing 95.30% of cells positive for hCAIX expression. (C) Unstained RENCA. (D) RENCA stained with anti- hCAIX-FITC, showing 2.15% of cells positive for hCAIX expression, which is assumed to be an artifact of nonspecific binding,
[0036] Figure 8. Western Mot of the fusion protein hGM-CAIX. The 70 kDa weight seen in both blots shows that the hGM-CAIX fusion gene is primarily expressed as a fused protein. Lane i : 293T control; Jane 2: 293 T infected with Ad-Null; lane 3; 293T infected with Ad-GM-CAIX. (A) Gel exposed for actin loading control and hCAIX. (B) Gel exposed for actin loading control and liGM-CSF.
[0037] Figure 9. Dot plots of FACS for hCAIX (representative of hGM-CAIX expression) and mCd86 in DC-Ad-GM-CAIX. (A) Unstained DC-Ad-GM-CAIX. (B) DC-
Ad-GM-CAIX stained with anti-hCAIX-FITC, showing 70.1% of cells positive for hCAIX expression, indicating successful infection of DCs with Ad-GM-CAIX. (C) DC-Ad- GM'CAIX stained with anti-rnCd86-PE, showing 96.3% of cells positive for mCd86 expression, indicating successful differentiation from BMCs into DCs. (D) DC-Ad- GM-CAIX double-stained with anti-hCAIX-FITC and with anti-mCd86-PE, showing 28.4% of cells positive for both hCAIX and mCd86 expression, representing the percentage of hCAiX-expressing DCs.
[00381 Figure 10. Dot plots of FACS for hCAIX and hGM-CSF expression in DC-Ad- GM-CAIX. (A) Unstained DC-Ad-GM-CAIX. (B) DC-Ad-GM-CAIX stained with anti- hCAIX-FlTC, showing 49.51 % of ceils positive for hCAIX expression. (C) DC-Ad- GM-CAIX stained with anti-hGM-CSF-PerCP, showing 30.35% of cells positive for hGM- CSF expression. (D) DC-Ad-GM-CAIX double-stained with anti-hCAIX-FITC and anti- hGM-CSF-PerCP, showing 12.70% of cells positive for both hCAIX and hGM-CSF expression. [0039] Figure 11. Mean number of metastatic foci per 1Θ8 urn2 total lung area. Non- treated mice (group A) and the DC-Ad-GM-CAIX-treated mice (group B). * - significantly lower (group B vs. group A).
[0040] Figure 12. Mean standardized metastatic foci area is um* per 10 um" total Sun area. Non-treated mice (group A) and the DC-Ad-GM-CAIX-treated mice (group B). * ::: significantly lower (group B vs. group A).
[0041] Figure 13, Immunohistochemical analysis of NPR-IX lung metastatic tumors with human CAIX (hCAIX). The panels show staining of membranous hCAIX in brown, Originai magnification, 40x. Non-treated mice (group A, 90% hCAIX positive) and the DC- Ad-GM-CAIX-treated mice (group B, 10% hCAIX positive).
DETAILED DESCRIPTION OF THE INVENTION I, Introduction
[0042J To overcome the immune impairment in patients with metastatic renal cell carcinoma (RCC) and benefit from the expected RCC responsiveness to immunotherapies, the fusion protein GM-CSF-CAIX (referred to herein as "GM-CAIX" or "GM-C AIX") has previously been shown to induce a specific cytotoxic lymphocytes immune response in vitro and natural kill (NK) cells in a severe combined immunodeficiency (SCID) mouse model. Furthermore, GM-CAIX was capable of inducing human dendritic cell (DC) maturation. See US 7,572,891; see also US 12/479,415 (published as US 2010/0129313 Al) and US 12/520,084 (published as US 2010/0183637 Al), the contents of each of which are incorporated by reference herein in its entirety. [0Θ43] To model clear cell RCC and the immunotherapy in immunocompetent mice, the human CAIX (hCAlX) tumor associated antigen (TAA) was constitutively and stably overexpressed in RENCA cells (RENCA-CAIX), and the cells were transplanted into a syngeneic Balb/c mouse. To transduce the GM-CAIX fusion protein in DCs, an adenoviral vector was utilized. Adenoviral vectors are more potent in eliciting CD4+ and CD8+ TAA- specific polyclonal CTLs compared with peptide-pulsed. As demonstrated in US 09/783,708 and US 12/520,084, dendritic cells expressing GM-CAIX (referred to herein as "DC-Ad- GM-CAIX" or "DC-Ad-GM-CAIX") were found to generate hCAIX-specific immune response attributed to CTLs in Balb/c immunocompetent mouse models, which are capable of both preventing and intervening in RCC growth. The results revealed significant hCAIX- specific immunotherapeutic efficacy with no signs of systemic toxicity,
[0044] Here, we have identified new gene and miRNA signatures in tumors that evade immunotherapy. As described herein in the Examples section, differential gene and miRNA expression profiles were compared between tumor tissue that escaped the therapy and tumor responsive tissues (Table 5). The identification and characterization of these "escape" genes and msRNAs provide information that can be used for the development of more effective immunotherapies, including but not limited to: (1) early identification of immunotherapy escape phase; (2) inhibition of the immune-escape processes that may result in a beneficial combination therapy (e.g., GM-CAIX immunotherapy); (3) information regarding mRNAs and miRNAs that have not been previously published or well-characterized; and (4) new- molecular targets for therapies.
II. Definitions
[0045] Abbreviation index: Ad-GM-CAIX / Ad-GM-CAIX: Adeno-virus vector expressing GM-CAIX; AP: Alkaline phosphatase; APC: Antigen-presenting cell; CAIX / CA9 / G250: Carbonic anhydrase IX; CD4: Cluster of differentiation 4; CD8: Cluster of differentiation 8; CD86: Cluster of diiXerentiation 86; cDNA: Complementary DNA; CL: Chemiluminescence assays: CTL: Cytotoxic T lymphocyte; DAPI: 4',6-diamidino-2-phenylindole; DC: Dendritic ceil; DC-Ad-GM-CAIX / DC-Ad-GM-CAIX: Dendritic cells transduced with Ad-GM-CAIX: DC-Ad-Null: Dendritic ceils transduced with an empty Adeno-virus vector; EIA: Enzyme immunoassays; ELISA: Enzyme-linked immunosorbent assay: FACS: Fluorescence-activated cell sorting; FITC: Fluorescein isothiocyanate; GM-CSF: Granulocyte-macrophage colony stimulating factor; GM-CSF-CAIX / GM-CAIX / GM-CAIX: Fusion protein of human GM- CSF with CA1X; hCAIX: human carbonic anhydrase IX; HIF-Ι : Hypoxia inducible factor la; HLA: Human leukocyte antigen; HRP: Horseradish peroxidase; IF: immunofluorescence; JFN-γ: interferon gamma; ig: Immunoglobulin; IgG; Immunoglobulin G; lincRNA: long intergenic non-coding RNA; mCAIX; mouse carbonic anhydrase IX; MHC: Major histocompatibility complex; miR A: microRNA; mRNA: messenger RNA; NK ceils:
Natural killer ceils; NPR-IX / RENCA-CAIX: RENCA ceil line transduced with hCAIX; PBC: Phosphate-buffered saline; PGR: Polymerase chain reaction; qPCR: quantitative realtime reverse transcription PGR; RCC: Renal cell carcinoma: RENCA: Balb/c mouse renal cancer ceil line; RNA: Ribonucleic acid; rRNA: Ribosomal RNA ; RT-PCR: Reverse transcription PGR; SCID mice: Severe combined immunodeficiency mice; shRNA: Short hairpin RNA; siRNA: Small interfering RNA; TAA: Tumor-associated antigen; VHL: von Hippel-Lindau.
[0046] As used herein, the term "immunoediting-escape" refers to the ability of cancer cells (e.g., tumors) in a subject to evade or escape elimination by the immune system of the subject, in some embodiments, a cancer ceil or tumor that evades or escape the immune system progresses to a more ad vanced form of cancer and/or metastasizes.
[0047] As used herein, the term, "immunoediting-escape status" refers to the extent to which a cancer cell or tumor has evaded or is predicted to evade elimination by the immune system of a subject. In some embodiments, immunoediting-escape status is determined by measuring the level or amount of expression of one or more (e.g., I, 2, 3, 4, 5, 6, 7. 8, 9, 10, 15, 20, 25, 30, or more) biomarkers selected from the group of biomarkers listed in Table 5 and their human orthoiogs; and correlating the level or amount of expression with an immune response. In some embodiments, for determining an immunoediting-escape status the level or amount of expression of the one or more biomarkers is compared to a threshold level or a control (e.g., a control sample from a non-diseased sample, a control sample from a subject having an effective immune response to the cancer cell or tumor, or a control sample from a subject known not to have an effective immune response to the cancer cell or tumor).
[0048] As used herein, the term "biomarker" refers to a molecule (e.g., a nucleic acid, e.g., mRNA, miRNA, or LincRNA, or a protein) thai is differentially expressed (overexpressea or underexpressed) in a ceil, on the surface of a ceil, or secreted by a cell (e.g., a cancer cell) in comparison to a normal control cell, and which is useful for the qualification of
immunoediting-escape status or diagnosis or prognosis of a disease or condition (e.g., renal ceil carcinoma) as disclosed herein, For example, in some embodiments, a biomarker useful for the qualification of immunoediting-escape status or diagnosis or prognosis of a disease or condition in a subject as disclosed herein exhibits over 1-fold over/under expression, e.g., at least 1 ,2-fold over/under expression, 1.5-fold over/under expression, 2-fold over/under expression, 3-fold over/under expression, 4-fold over/under expression, 5-fold over/under expression, 6-fold over/under expression, 7-fold over/under expression, 8-fold over/under expression, 9-fold over/under expression, 10-fold over/underexpression, 15-fold
over/underexpression, or 20-fold over/under expression or more in a sample from the subject in comparison to a normal tissue, ϊη some embodiments, a biomarker useful for the qualification of immunoediting-escape status or diagnosis or prognosis of a disease or condition in a subject as disclosed herein is an mRNA, miRNA, or LincR A listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof. In some embodiments, the biomarker is a biomarker listed in Table 2 or a human ortholog thereof. In some embodiments, the biomarker is biomarker listed in Table 4A and/or 4B or a human ortholog thereof. In some embodiments, a biomarker useful for the qualification of immunoediting-escape status or diagnosis or prognosis of a disease or condition in a subject as disclosed herein may also function in other cellular or biological processes over than immunoediting-escape phase function. Thus, in some embodiments, a biomarker of the present invention is at least a biomarker of immunoediting-escape phase function.
|0049] It will be understood by the skilled artisan that markers may be used singly or in combination with other markers for any of the uses, e.g., qualification of immunoediting- escape status or diagnosis or prognosis of a disease or condition (e.g., a cancer such as renal cell carcinoma) as disclosed herein.
[OOSOj As used herein, the term "agent" refers to any molecule, either naturally occurring or synthetic, e.g., peptide, protein, oligopeptide (e.g., from about 5 to about 25 am ino acids in length, preferably from about 10 to 20 or 12 to 18 amino acids in length, preferably 12, 15, or 18 amino acids in length), small organic molecule (e.g., an organic molecule having a molecular weight of less than about 2500 daltons, e.g.. less than 2000, less than 1000, or less than 500 daltons), circular peptide, peptidomimetic, antibody, polysaccharide, lipid, fatty acid, inhibitory RNA (e.g., siRNA or shRNA), polynucleotide, oligonucleotide, aptamer, drug compound, or other compound. [0051] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturaliy occurring amino acid polymer.
[0Θ52] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid rmmetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g. , hydroxyproline, γ- earboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g. , homoserine, norieucine, methionine sulfoxide, methionine methyl sulfoniurn. Such analogs have modified R groups (e.g. , norieucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0053] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, and complements thereof. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonat.es, chira!-methyl phosphonates, 2-O-methyl ribonucleotides, and pepiide-nuc!eic acids (PNAs).
[0054] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), complementary sequences, splice variants, and nucleic acid sequences encoding truncated forms of proteins, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 ( 1991 ); Ohtsuka et ah, J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al, Mol Cell. Probes, 8:91-98 (1994)), The term nucleic acid is used interchangeably with gene, cDNA, mRNA, miRNA, LincRNA, shRNA, siR A, oligonucleotide, and polynucleotide.
[0055] The term ''substantially complementary," as used herein with reference to nucleic acids, refers to a first nucleic acid that has at least about 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the complement of a second nucleic acid. In some embodiments, a first nucleic acid that is substantially complementary to a. second nucleic acid is able to bind to the second nucleic acid under moderately stringent hybridization conditions. [0056] The terms "overexpress," "overexpression," "upregulate," and "upreguiation" interchangeably refer to nucleic acid or protein expression of a biomarker of interest in a sample (e.g., a tumor sample) that is detectabiy higher than the nucleic acid or protein expression of the biomarker of interest in a control (e.g., non-cancer) sample. Upreguiation can be due to increased transcription, post transcriptional processing, translation, post transiational processing, altered stability, or altered protein degradation, as well as local upreguiation due to altered protein traffic patterns (increased nuclear localization), and augmented functional activity, e.g., as a transcription factor. Upreguiation can be detected usiiig conventional techniques for detecting nucleic acid (e.g. , RT-PCR, PCR, microarray) or proteins (e.g., EL1SA, Western blots, flow cytometry, immunofluorescence,
iminunohistochemistry, DNA binding assay techniques). Upreguiation can be at least 10%, 20%, 30%, 40%, 50%», 60%, 70%, 80%, 90% or more for the biomarker of interest in the test sample (e.g., a tumor sample) in comparison to a control (e.g., non-cancer) sample, in certain instances, upreguiation is at least 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3, 5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, i 1-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16-fold, 17-fold, i 8-fold, 19-fold, 20-fold or more higher levels of UNA or protein levels for the biomarker of interest in the test sample (e.g., tumor sample) in comparison to a control (e.g., non-cancer) sample.
[0057] The terms "underexpress," "underexpression," "downregulate," and
"downregulation" interchangeably refer to nucleic acid or protein expression of a biomarker of interest in a sample (e.g., a tumor sample) that is detectabiy lower than the nucleic acid or protein expression of the biomarker of interest in a control (e.g., non-cancer) sample.
Downregulation can be due to decreased transcription, post transcriptional processing, translation, post translational processing, altered stability, or altered protein degradation, as well as local downregulation due to altered protein traffic patterns (decreased nuclear localization), and augmented functional activity, e.g., as a transcription factor.
Downregulation can be detected using conventional techniques for detecting nucleic acid (e.g., RT-PCR, PCR, microarray) or proteins (e.g. , ELISA, Western blots, flow cytometry, immunofluorescence, imniunobistochemistry, DNA binding assay techniques).
Downregulation can be at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or less for tiie biomarker of interest in the test sample (e.g., a tumor sample) in comparison to a control (e.g., non-cancer) sample, in certain instances, downregulation is at least 1.2-fold, 1. -fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, i 8-fold, 19-fold, 20-fold or more lower levels of RNA or protein levels for the biomarker of interest in the test sample (e.g., tumor sample) in comparison to a control (e.g., non-cancer) sample.
[0058] As used herein, the term "expression profile" refers to the amount of nucleic acid and/or protein that is expressed for a given biomarker or set of biomarkers in a biological sample. In some embodiments, an expression profile comprises the levels of nucleic acid and/or protein expression for a plurality of biomarkers as described herein, e.g., for 2. 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more biomarkers selected from an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof.
[0059] "Cancer" is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, soft tissue, or solid, and cancers of all stages and grades including pre- and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and esophageal cancer; breast cancer; lung cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer, ovarian cancer; renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system; skin cancer (e.g., melanoma); lymphomas; gliomas; choriocarcinomas; head and neck cancers; osteogenic sarcomas; and blood cancers. As used herein, a "tumor" comprises one or more cancerous cells. In some embodiments, the cancer is renal cell carcinoma, in some embodiments, the cancer is a metastatic cancer.
[0060] The term "sample" or "biological sample" includes blood and blood fractions or products (e.g., serum, plasma, platelets, red blood cells, and the like); sputum or saliva; kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, or bone tissue; cultured cells, e.g., primary cultures, explants, and transformed cells, stent cells, stool, urine, etc. Such biological samples also include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes, in some embodiments, a sample is a tumor tissue sample. A biological sample is typically obtained from a "subject," i.e., a eukaryotic organism, most preferably a mammal such as a primate, e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, or mouse; rabbit: or a bird; reptile; or fish.
[0061] The terms "administer," "administered," or "administering" refer to methods of delivering agents, compounds, or compositions to the desired site of biological action. These methods include, but are not limited to, topical deliver)', parenteral delivery, intravenous delivery, intradermal delivery, intramuscular delivery, colonical delivery, rectal delivery, or intraperitoneal delivery. Administration techniques that are optionally employed with the agents and methods described herein, include e.g., as discussed in Goodman and Oilman, The Pharmacological Basis of Therapeutics, current ed.; Pergamon; and Remington's,
Pharmaceutical Sciences (current edition), Mack Publishing Co., Eastern, PA.
ΪΠ. Methods of Qualifying Immunoediting-Escape Status
[0062] In one aspect, the present invention relates to methods of qualifying the iram unoediting-escape status of a subject, in some embodiments, the method comprises:
(a) measuring at least one biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of the biomarkers listed in Table 5 and their human orthologs; and
(b) correlating the measurement with the subject's immune response; thereby qualifying the iramunoediting-escape status of the subject,
[00631 In some embodiments, the method comprises measuring at least two biomarkers (e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more biomarkers) and correlating the measurement of the biomarkers with the .subject's immune response. [Θ064] In some embodiments, the immunoediting-escape status indicates the subject's risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, the presence or absence of a disease {e.g., cancer), the stage of a disease, or effectiveness of treatment for a disease, in some embodiments, the immunoediting-escape status indicates the presence, absence, or stage of a cancer or the effectiveness of treatment for a cancer in the subject, in some embodiments, the immunoediting-escape status indicates the presence, absence, or stage of a renal ceil carcinoma or the effectiveness of treatment for a. renal ceil carcinoma in the subject.
Biomarkers
[0065] In some embodiments, the at least one biomarker is an mRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human orihoiog thereof, in some embodiments, the mRNA or protein is an mRNA or protein that is involved in inflammatory response, cell proliferation, cell growth, cell movement, or cell-to-cell signaling, in some embodiments, the mRNA or protein is an inflammatory response gene or protein listed in Table 4A. in some embodiments, the mRNA or protein is a cellular growth and/or proliferation gene or protein listed in Table 4B. In some embodiments, the at least one biomarker is an miRNA or LincRNA that is involved in cell (e.g., tumor) growth or suppression of tumor growth. In some embodiments, the at least one biomarker is an miRNA or LincRNA listed in Table 5 or a human ortholog thereof. In some embodiments, the at least one biomarker is an miRNA or LincRNA listed in Table 2 or a human orihoiog thereof.
{0066] In some embodiments, the at least one biomarker is a cytokine or a regulator and/or marker of myeloid cells, dendritic cells, natural killer cells, and/or T-cells. In some embodiments, the at least one biomarker is Cel l , Cxcl9, Hmgbi, Fgl2, Cd209a, Foxj i , Klra2, miR-1 186, miR-98, miR-5097, miR-1942, or miR-708. In some embodiments, the at least one biomarker is selected from Cell , Cxcl9, Hmgbl, FgI2, Cd209a, Foxj 1, and Klra2. In some embodiments, the at least one biomarker is selected from miR-1 186, miR-98, miR- 5097, miR-1 42, and miR-708. in some embodiments, upregulation of at least one biomarker selected from Cell, Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l, KIra2, miR-1 186, miR- 98, miR-5097, and miR-1942 is indicative of immune escape, cancer (e.g., renal ceil carcinoma), or immune disease. In some embodiments, downregulation of miR-708 is indicative of immune escape, cancer (e.g., renal cell carcinoma), or immune disease. [0067] in some embodiments, the at least one biomarker (e.g., an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) is upregulated or more highly expressed in the sample from the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to the level of expression of the biomarker in the control (e.g., non-diseased) sample. In some embodiments, the at least one biomarker is upregulated or more highly expressed in the sample from the subject by at least 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16- fold, 17-fold, 18- fold, 19-fold, 20-fold or more as compared to the level of expression of the biomarker in the control (e.g., non-diseased) sample. In some embodiments, the at least one biomarker (e.g., an mRNA. rniRNA, or LincRNA listed in Table 5. a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) is downregulated in the sample from the subject by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more as compared to the level of expression of the biomarker in the control (e.g., non- diseased) sample. In some embodiments, the at least one biomarker is downregulated in the sample from the subject by at least 1.2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-foid, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15- fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more as compared to the level of expression of the biomarker in the control (e.g., non-diseased) sample. Samples
[0068] In some embodiments, a biological sample comprises a ceil. In some embodiments, the cell is derived from a tissue or organ (e.g., prostate, breast, kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, keletal muscle, cartilage, skin, or bone tissue). In some embodiments, the cell is derived from a biological fluid, e.g., blood (e.g., an erythrocyte), lymph (e.g., a monocyte, macrophage, neutrophil, eosinophil, basophil, mast cell, T cell, B cell, and/or NK cell), serum, urine, sweat, tears, or saliva. In some embodiments, the ceil is derived from a biopsy (e.g., a skin biopsy, a muscle biopsy, a bone marrow biopsy, a liver biopsy, a gastrointestinal biopsy, a lung biopsy, a nervous system biopsy, or a lymph node biopsy), in some embodiments, the cell is derived from a cultured cell (e.g., a primary cell culture) or a ceil line (e.g., PC3, HEK293T, NIH3T3, Jurkat, or Ramos).
[0069] In some embodiments, the sample comprises a cancer cell (e.g., a cell obtained or derived from a tumor). In some embodiments, the cancer is prostate cancer, breast cancer, bladder cancer, urogenital cancer, lung cancer, renal ceil carcinoma, endometrial cancer, melanoma, ovarian cancer, thyroid cancer, or brain cancer, in some embodiments, the cancer is a metastatic cancer.
[0070] in some embodiments, the sample is from blood, serum, plasma, urine, saliva, feces, kidney, lung, liver, heart, brain, nervous tissue, thyroid, eye, skeletal muscle, cartilage, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, rectum, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus. In some embodiments, the sample is from a renal tumor, renal cyst, renal metastasis, kidney cell or cells, or kidney tissue.
[0071] in some embodiments, the sample is from a diseased tissue, in some embodiments, the sample is from a hypoxic tumor, in some embodiments, the sample is from a tumor tissue (e.g., from a cancer type as described herein), in some embodiments, the sample is from ischemic tissue.
[0072] in some embodiments, the sample is from a human subject. In some embodiments, the biological sample is from a non-human mammal (e.g., chimpanzee, dog, cat, pig, mouse, rat, sheep, goat or horse), avian (e.g., pigeon, penguin, eagle, chicken, duck, or goose), reptile (e.g., snake, lizard, alligator, or turtle), amphibian (e.g., frog, toad, salamander, caecilian, or newt), or fish (e.g., shark, salmon, trout, or sturgeon).
Measurement of Biomarikgll
[0073] 'The detection methods for measuring and/or quantifying one or more biomarkers as described herein can be carried out, for example, using standard nucleic acid and/or polypeptide detection techniques known in the art. Detection can be accomplished by labeling a nucleic acid probe or a primary antibody or secondary antibody with, for example, a radioactive isotope, a fluorescent label, an enzyme or any other detectable label known in the art.
[0074] Antibody reagents can be used in assays to detect protein expression levels for the at least one hiomarker of interest in patient samples using any of a number of imm noassays known to those skilled in the art. immunoassay techniques and protocols are generally described in Price and Newman, "Principles and Practice of Immunoassay," 2nd Edition, Grove's Dictionaries, 1997; and Gosling, "Immunoassays: A Practical Approach," Oxford University Press, 2000. A variety of immunoassay techniques, including competitive and non-competitive immunoassays, can be used (see, e.g., Self et al, Ciirr. Opin. Biotechnol, 7:60-65 (1996)). The term immunoassay encompasses techniques including, without limitation, enzyme immunoassays (EI A) such as enzyme multiplied immunoassay technique (EMIT), enzyme-linked immunosorbent assay (ELISA), lgM antibody capture EL1SA (MAC ELISA), and microparticle enzyme immunoassay (MELA); capillary electrophoresis immunoassays (CEIA); radioimmunoassays (R1A); itnmunoradiometric assays (I MA); immunofluorescence (IF); fluorescence polarization immunoassays (FPIA); and
chemi!uminescence assays (CL). If desired, such immunoassays can be automated.
Immunoassays can aiso be used in conjunction with laser induced fluorescence {see, e.g., Schmalzing et al, Electrophoresis, 18:2184-93 (1997); Bao, J, Chromatogr. B. Biomed. Set, 699:463-80 (1997)). [0075] Specific immunological binding of an antibody to a protein can be detected directly or indirectly. Direct labels include fluorescent or luminescent tags, metals, dyes, radionuclides, and the like, attached to the antibody. An antibody labeled with iodine- 125 (r25I) can be used. A chemiluminescence assay using a cherniluminescent antibody specific for the protein marker is suitable for sensitive, non-radioactive detection of protein levels. An antibody labeled with fluorochrome is aiso suitable. Examples of fluorochromes include, without limitation, DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R- phycoeiythrin, rhodamine, Texas red, and lissamine. Indirect labels include various enzymes welt known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), β- galactosidase, urease, and the like. A horseradish-peroxidase detection system can be used, for example, with the chromogenic substrate tetramethylbenzidine (TMB), which yields a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm. An alkaline phosphatase detection system can be used with the chromogenic substrate p- nitrophenyl phosphate, for example, which yields a soluble product readily detectable at 405 nm. Similarly, a β-galactosidase detection system can be used with the chromogenic substrate o-niirophenyi- -D-galactopyranoside (ONPG), which yields a soluble product detectable at 410 nm, A urease detection system can be used with a substrate such as urea- bromocresoi purple (Sigma immunochemicals; St. Louis, MO).
[0076] A signal from the direct or indirect label can be analyzed, for example, using a spectrophotometer to detect color from a chromogenic substrate; a radiation counter to detect radiation such as a gamma counter for detection of 1κ5Ι; or a fluorometer to detect fluorescence in the presence of light of a certain wavelength. For detection of enzyme-linked antibodies, a quantitative analysis can be made using a spectrophotometer such as an EMAX Microptate Reader (Molecular Devices; Menlo Park, CA) in accordance with the manufacturer's instructions. If desired, the assays of the present invention can be automated or performed robotically, and the signal from multiple samples can be detected
simultaneously. In some embodiments, the amount of signal can be quantified using an automated high-content imaging system. High-content imaging systems are commercially available (e.g., ImageXpress, Molecular Devices Inc., Sunnyvale, CA).
[0077] Antibodies can be immobilized onto a variety of solid supports, such as magnetic or chromatographic matrix particles, the surface of an assay plate (e.g., microtiter wells), pieces of a solid substrate materia! or membrane (e.g., plastic, nylon, paper), and the like. An assay strip can be prepared by coating the antibody or a plurality of antibodies in an array on a solid support. This strip can then be dipped into the test sample and processed quickly through washes and detection steps to generate a measurable signal, such as a colored spot.
[0078] Alternatively, nucleic, acid binding molecules such as probes, oligonucleotides, oligonucleotide arrays, and primers can be used in assays to detect differential nucleic acid expression of a biomarker of interest in ubject samples, e.g., RT-PCR. In one embodiment, RT-PCR is used according to standard methods known in the art. in another embodiment, PGR assays such as Taqman® assays available from, e.g. , Applied Biosystems, can be used to detect nucleic acids and variants thereof. In other embodiments, qPCR and nucleic acid microarrays can be used to detect nucleic acids. Reagents that bind to selected markers of interest can be prepared according to methods known to those of skill in the art or purchased commercially.
[0079] Analysis of nucleic acids can be achieved using routine techniques such as Southern analysis, reverse-transcriptase polymerase chain reaction (RT-PCR), or any other methods based on hybridization to a nucleic acid sequence that is complementary to a portion of the marker coding sequence (e.g. , slot blot hybridization) are also within the scope of the present invention. Applicable PCR amplification techniques are described in, e.g., Ausubel et al. and innis et al, supra. General nucleic acid hybridization methods are described in Anderson, "Nucleic Acid Hybridization," BIOS Scientific Publishers, 1999. Amplification or hybridization of a plurality of nucleic acid sequences (e.g., genomic DNA, mRNA or cDNA) can also be performed from mRNA or cDNA sequences arranged in a microarray.
Microarray methods are generally described in Hardiman, "Microarrays Methods and
Applications: Nuts & Bolts," DNA Press, 2003; and Baldi et al, "DNA Microarrays and Gene Expression: From Experiments to Data Analysis and Modeling," Cambridge
University Press, 2002.
[0080] Analysis of nucleic acid markers can also be performed using techniques known in the art including, without limitation, microarrays, polymerase chain reaction (PCR)-based analysis, sequence analysis, and electrophoretic analysis. A non-limiting example of a PCR- based analysis includes a Taqraan® allelic discrimination assay available from Applied Biosy stems. Non-limiting examples of sequence analysis include Maxam-Giibert sequencing, Sanger sequencing, capillary array DNA sequencing, thermal cycle sequencing (Sears et al, Biotechniques, 13:626-633 (1992)), solid-phase sequencing (Zimmerman er a/,.. Methods Mol. Cell Biol,, 3:39-42 (1992)), sequencing with mass spectrometry such as matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS; Fu et cel., Nat. BiotechnoL, 16:381 -384 (1998)), and sequencing by hybridization. Chee et al, Science, 274:610-614 (1996); Drmanac et al, Science, 260: 1649-1652 (1993); Drmanac el al, Nat. BiotechnoL, 16:54-58 (1 98). Non-limiting examples of electrophoretic analysis include slab gel electrophoresis such as agarose or polyacrylamide gel electrophoresis, capillary electrophoresis, and denaturing gradient gel electrophoresis. Other methods for detecting nucleic acid variants include, e.g., the INVADER® assay from Third Wave Technologies, nc.. restriction fragment length polymorphism (RFLP) analysis, alleie-specific oligonucleotide hybridization, a heterodupiex mobility assay, single strand conformational polymorphism (SSCP) analysis, single-nucleotide primer extension (SNUPE), and pyrosequencing.
[0081 j A detectable moiety can be used in the assays described herein. A wide variety of detectable moieties can be used, with the choice of label depending on the sensitivity required, ease of conjugation with the antibody, stability requirements, and available instrumentation and disposal provisions. Suitable detectable moieties include, but are not limited to, radionuclides, fluorescent dyes {e.g., fluorescein, fluorescein isothioeyanate (FITC), Oregon Green™, rhodamine, Texas red, tetrarhodirnine isothiocynate (TRJTC), Cy3, Cy5, etc.), fluorescent markers (e.g., green fluorescent protein (GFP), phycoerythrin, etc.), autoquenched fluorescent compounds that are activated by tumor-associated proteases, enzymes (e.g., luciferase, horseradish peroxidase, alkaline phosphatase, etc.), nanoparticles, biotin, digoxigenin, and the like. [©082] Useful physical formats comprise surfaces having a. plurality of discrete, addressable locations for the detection of a plurality of different markers. Such formats include microarrays and certain capillary devices. See, e.g., Ng et al, J. Cell Mol. Med., 6:329-340 (2002); U.S. Pat. No, 6,019,944, In these embodiments, each discrete surface location may comprise antibodies or oligonucleotides to immobilize one or more markers for detection at each location. Surfaces may alternatively comprise one or more discrete particles (e.g., microparticles or nanoparticies) immobilized at discrete locations of a surface, where the microparticles comprise antibodies to immobilize one or more markers for detection. Other useful physical formats include sticks, wells, sponges, and the like. [0083] The analysis can be carried out in a variety of physical formats. For example, the use of microliter plates or automation could be used to facilitate the processing of large numbers of test samples.
[0084] Alternatively, the antibodies or nucleic acid probes of the invention can be applied to subject samples immobilized on microscope slides. The resulting antibody staining or in situ hybridization pattern can be visualized using any one of a variety of light or fluorescent microscopic methods known in the art,
[0085] Analysis of the protein or nucleic acid can also be achieved, for example, by high pressure liquid chromatography (HPLC), alone or in combination with mass spectrometry (e.g., MALD1/MS, MALDI-TOF MS, tandem MS, etc.). Correlating Biomarker Measurements to Immune Response
[0086] In some embodiments, the level or amount of expression of the at least one biomarker (e.g., an mRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) is correlated with an immune response in the subject, in some embodiments, the correlating step comprises correlating expression of the at least one biomarker with an immune response by the subject that is above or below a threshold level (e.g., an immune response level in a. healthy control subject or population of subjects). In some embodiments, a level of expression of the at least one biomarker that is over 1 -fold over the threshold or control level (e.g., at least 1 .2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13- fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more over the threshold or control level) correlates the biomarker with an immune response, in some embodiments, a level of expression of the at least one biomarker that is over 1-fold over the threshold or control level indicates that a diseased cell or tissue (e.g., a cancer cell or tumor) has evaded or escaped elimination by the immune system (i.e., has an imrnunoediting-escape status), in some embodiments, the correlating step is performed by a software classification system.
[0087] In some embodiments, the immune response is the secretion of iramunosuppressor factors (e.g., IL-IO, TGFp). In some embodiments, the immune response is a change in the expression and/or activity of immune system cells (e.g., regulatory T cells or effector cells). Methods for detecting and/or quantifying an immune response in a subject are well known. For example, immunoassays as described herein can be used to detect and/or quantify the expression of proteins. Flow cytometry can also be used to determine the presence or level of one or more markers in a sample or to measure and/or characterize cells such as T-ceSis,
■Mgjrcsgj.ng Treatment Based on Immanoediting-Escape Status
[0088] in some embodiments, the methods of qualifying the imrnunoediting-escape status of a subject further comprise managing treatment of the subject based on the imrnunoediting- escape status. For example, in some embodiments, the method comprises: measuring at least one biomarker (e.g., an mRNA, miR A, or LincR.N A listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) in a sample from the subject;
determining whether the at least one biomarker is differentially expressed (upregulated or downregulated) in the sample from the subject as compared to a control subject;
correlating the differential expression of the at least one biomarker to the subject's risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, the presence or absence of a disease (e.g., cancer), the stage of a disease, or effectiveness of treatment for a disease; and
managing treatment of the subject based on the risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, presence or absence of disease, stage of disease, or effectiveness of treatment for disease identified for the subject.
[0089] In some embodiments, the correlating step comprises correlating a level of expression of the at least one biomarker that is altered by more than 1 -fold relative to a threshold or control level (e.g., that is upregulated or downregulated by at least 1.2-fold, 1 ,5- fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-foid, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more relative to the threshold or control level) with a diseased cell or tissue in the subject (e.g., a cancer cell or tumor) evading or escaping elimination by the immune system (i.e., having an immunoediting-escape status), in some embodiments, a level of expression of the at least one biomarker that is altered by more than 1-fold relative to a threshold or control level (e.g., that is upreguiated or downregulated by at least 1 ,2-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-foid, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12- fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more relative to the threshold or control level) correlates the biomarker with an increased risk of cancer, an increased risk of an immune disease, decreased immune response, decreased likelihood of responding therapy, decreased likelihood of tissue repair, increased likelihood of acute organ failure, increased risk for organ transplantation, increased likelihood of the presence of a disease (e.g., cancer), increased likelihood of an advanced stage of a disease, or decreased likelihood of effectiveness of treatment for a disease.
[ΘΘ90] In some embodiments, managing treatment comprises ordering a further diagnostic test, performing surgery, administering a therapy, or taking no further action, in some embodiments, wherein managing treatment comprises administering a therapy, the therapy comprises immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment.
Therapeutic methods suitable for managing treatment of the subject are described herein, e.g., in section IV below,
IV. Diagnostic, Prognostic, and Therapeutic Methods
"Diagnostic and Prognostic Methods
[0091] In another aspect, the present invention relates to methods of diagnosing or prognosing a disease, disorder, or condition in a subject using a biomarker of immunoediting- escape status as described herein. As used herein, the term "diagnosing" or "diagnosis" refers to detecting a disease, disorder, or condition (e.g., a cancer). In any method of diagnosis exists false positives and false negatives. Any one method of diagnosis does not provide 100% accuracy. The term "providing a prognosis," as used herein, refers to to providing a prediction of the probable course and outcome of a disease, disorder, or condition such as a cancer, including prediction of metastasis, disease free survival, overall survival, etc. The methods can also be used to devise a suitable therapy for treatment, e.g., by indicating whether or not a disease (e.g., a cancer) is still at an early stage or if the disease has advanced to a stage where aggressive therapy would be ineffective.
[0092] In some embodiments, methods of diagnosing the status of a tissue or organ are provided, in some embodiments, the method comprises: determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, is indicative of a status of cancer, immune-escape, or immune disease in the tissue or organ of the subject.
[0093] In some embodiments, a differential pattern or level of expression of the at least one biomarker in the sample from the subject is indicative of the presence, absence, or stage of a disease (e.g., a cancer) in the tissue or organ or the effectiveness of treatment for the disease (e.g., cancer) in the subject. In sorne embodiments, an increased or upreguiated level of expression of the at least one biomarker in the sample from the subject is indicative of the presence, absence, or stage of a disease or the effectiveness of treatment for the disease in the subject. In some embodiments, the level of the at least one biomarker is increased by at least 1.2-fold, 1 .5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8- fold, 9-fold, 10-fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fo!d, 19- fold, 20-fold or more, in some embodiments, a level of expression of the at least one biomarker in the sample from the subject that is increased by at least 1.2- fold, 1 , 5-fold, 2- fold. 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 3 7-fold, 18-fold, 39-fold, 20-fold or more indicates the presence of a disease (e.g., cancer), an advanced stage of a disease (e.g., cancer), or a decreased likelihood of effectiveness of treatment for the disease in the subject.
[0094] in some embodiments, the method comprises diagnosing renal status. Irs sorne embodiments, a differential pattern or level of expression of the at least one biomarker in the sample from the subject is indicati ve of the presence, absence, or stage of renal cell carcinoma or the effectiveness of treatment for renal ceil carcinoma in the subject. In some embodiments, an increased or upreguiated level of expression of the at least one biomarker in the sample from the subject is indicative of the presence, absence, or stage of renal cell carcinoma or the effectiveness of treatment for renal ceil carcinoma in the subject. [0095] In some embodiments, methods of diagnosing whether or not an individual has a disease or disorder that is likely to be responsive to a therapy (e.g., immunotherapy) are provided. In some embodiments, the method comprises: detecting in a biological sample from the individual an altered level of at least one biomarker listed in Table 5 or a human ortho!og thereof, in some embodiments, the method comprises detecting an elevated or upregulated level of expression of the least one biomarker, wherein the elevated or upregulated level of expression indicates that individual has a disease or disorder that is not likely to be responsive to the therapy. In some embodiments, the method comprises detecting a decreased or downregulated level of expression of the least one biomarker, wherein the decreased or downregulated level of expression indicates that the individual has a disease or disorder that is likely to be responsive to the therapy. In some embodiments, the level of the at least one biomarker is altered (e.g., elevated or decreased) by at least 1 ,2-fold, 1.5-fold, 2- foid, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more. in some embodiments, the disease or disorder is a cancer, e.g.. a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus. In some embodiments, the disease or disorder is an immune system disease or disorder. In some embodiments, the disease or disorder is ischemia. In some embodiments, wherein the altered level of the biomarker indicates that the individual has a cancer is likely to be responsive to the therapy (e.g., immunotherapy), the method further comprises administering the therapy to the individual.
[00961 In some embodiments, methods of determining whether or not a disease or disorder is likely to be responsive to a therapy (e.g., immunotherapy) are provided. In some embodiments, the method comprises: detecting in a biologicai sample from a subject having the disease or disorder an altered level of at least one biomarker listed in Table 5 or a human ortholog thereof. In some embodiments, the method comprises detecting an elevated or upregulated level of expression of the least one biomarker, wherein the elevated or upregulated level of expression indicates that the disease or disorder is not likely to be responsive to the therapy. In some embodiments, the method comprises detecting a decreased or downregulated level of expression of the least one biomarker, wherein the decreased or downregulated le vel of expression indicates that the disease or disorder is likely to be responsive to the therapy. In some embodiments, the level of the at least one biomarker is altered (e.g., elevated or decreased) by at least 1.2-foki, 1.5-fold, 2-fold, 2,5-fold, 3-fold, 3 ,5-fold, 4-fold, 4 ,5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 1 1-fold, 12-fold, 13- fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold or more. In some embodiments, the disease or disorder is a cancer, e.g., a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is an immune system disease or disorder, in some embodiments, the disease or disorder is ischemia. In some embodiments, wherein the altered level of the biomarker indicates that the disease or disorder is likely to be responsive to the therapy (e.g., immunotherapy), the method further comprises administering the therapy to the individual.
Therapeutic Methods
[0097] In another aspect, the present invention provides therapeutic methods for identifying subjects for treatment and treating su jects in need thereof. The terms "treating" or
"treatment" include: (1) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a mamma! that may be exposed to the organism but does not yet experience or display symptoms of the disease: (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; and (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms. [0098] In another aspect, methods of treating a disease or disorder are provided, in some embodiments, the method comprises: administering a therapy to a subject that has been identified as having an immunoediting-escape status, wherein identifying the subject as having an immunoediting- escape status comprises determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, and wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, identities the subject as having an immunoediting-escape status.
[0099] In some embodiments, an increased or upregulated level of expression of the at least one biomarker in the sample from the subject identifies the subject as having an
immunoediting-escape status. In some embodiments, the disease or disorder is a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is an immune system disease or disorder. In some embodiments, the disease or disorder is ischemia.
[0100] In still another aspect, methods of monitoring the efficacy of treatment for an individual having a disease or disorder are provided, in some embodiments, the method comprises: detecting in a biological sample from the individual a level of at least one biomarker selected from an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in 'fable 5, or a human orthoJog thereof; and
determining whether or not the level of at the least one biomarker in the sample is altered relative to a control: thereby determining whether the disease or disorder is responsive to the treatment.
[0101] in some embodiments, disease or disorder is a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate. bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus, in some embodiments, the disease or disorder is a metastatic cancer or renal cell carcinoma, in some embodiments, the disease or disorder is ischemia, in some
embodiments, an elevated level of the at least one biomarker relative to a control indicates that the disease or disorder is not responsive to the treatment. In some embodiments, wherein an elevated level of the at least one biomarker relative to a control indicates that the disease or disorder is not responsive to the treatment, the method further comprises administering to the individual an alternative treatment.
[0102] In some embodiments, the method comprises detecting the level of the at least one biomarker in two or more samples from the individual taken at different timepoints (e.g., before treatment and during the course of treatment; at different times during the course of treatment; before treatment and after the completion of a round of treatment; or during the course of treatment and after the completion of a round of treatment) and determining whether the level of the biornarker is altered in each sample is altered relative to a control.
[0103] In still another aspect, methods of monitoring treatment of a subject for a disease or disorder, e.g., for monitoring treatment of a subject for a cancer, are provided. In some embodiments, the method comprises: (a) determining a first expression profile for at least one biomarker selected from an mRNA, miRNA, or LincR A listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof in a first sample from the subject, wherein the first expression profile is determined prior to the administration of a therapeutic- agent;
(b) administering a therapeutically effective amount of the therapeutic agent to the subject; and
(c) determining a second expression profile for at least one biomarker listed in Table 5 in a second sample from the subject, wherein the second expression profile is determined subsequent to the administration of the therapeutic agent; wherein modulation of the expression profile indicates efficacy of treatment with the therapeutic agent.
[0104] In .some embodiments, the disease or disorder is a cancer (e.g., a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus). In some embodiments, the disease or disorder is an immune system disease or disorder, in some embodiments, the disease or disorder is ischemia.
[0105] In some embodiments, an expression level of the at least one biomarker in the first expression profile that is above a threshold level indicates the presence of the disease or disorder. In some embodiments, an expression levei of the at least one biomarker in the second expression profile that is below a threshold level indicates efficacy of treatment.
[0106] In some embodiments, the determining of an expression profile comprises:
(i) providing a sample (e.g., a nucleic acid sample, protein sample, or a tissue sample) from the subject; and
(if) capturing the at least one biomarker that is present in the sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at least one biomarker.
[0107] in some embodiments, an expression profile is determined using a biochip array (e.g., an antibody chip array, a tissue chip array, a protein chip array, a nucleic acid array, or a peptide chip array). In some embodiments, the determining of an expression profile comprises detecting the presence or absence of the at least one biomarker, quantifying the amount of the at least one biomarker, and qualifying the type of the at least one biomarker (e.g., categorizing at least one function of the biomarker, e.g., a function as described in Table 2, Table 4A, or Table 4B).
[0108] in some embodiments, the substrate is a microliter plate comprising one or more biospecific affinity reagents that bind to the biomarker, and wherein the biomarker is detected using a fluorescent label. In some embodiments, the one or more capture reagents are immobilized on a biochip array, in some embodiments, the biochip array is an antibody chip array, a tissue chip array, a protein chip array, a nucleic acid array, or a peptide chip array. In some embodiments, the at least one biomarker is a protein translated from an mRNA listed in Table 5 and wherein the protein biomarker is measured by immunoassay. [0109] In some embodiments, the at least one biomarker for use according to the diagnostic, prognostic, and therapeutic methods described herein is an mRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof. In some embodiments, the mRNA or protein is an mRN A or protein that is involved in inflammatory response, cell proliferation, cell growth, cell movement, or cell-to-cell signaling. In some embodiments, the mRNA or protein is an inflammatory response gene or protein listed in Table 4A. In some embodiments, the mRNA or protein is a cellular growth and/or proliferation gene or protein listed in Table 4B. In some embodiments, the at least one biomarker is an miRNA or LincR A that is involved in cell (e.g., tumor) growth or suppression of tumor growth. In some embodiments, the at least one biomarker is an miRNA or LincRNA listed in Table 5 or a human ortholog thereof. In some embodiments, the at least one biomarker is a cytokine or a regulator and/or marker of myeloid cells, dendritic cells, natural killer ceils, and/or T-celis. In some embodiments, the at least one biomarker is Cell , Cxcl9, Hrngbl , Fgl2, Cd209a, Foxj l , Klra2, miR-1 186, miR-98, miR-5097, miR- 1942, or miR-708. In some embodiments, the at least one biomarker is selected from Cell , Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l, and Klra2. In some embodiments, the at least one biomarker is selected from miR-1 186, miR-98, miR-5097, miR-1942, and miR-708,
Diseases
[0110] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is anal carcinoma, bladder carcinoma, breast carcinoma, cervix carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukem ia, head and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non- Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung (non-small ceil carcinoma), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, soft tissue sarcoma, brain tumor, or rectal tumor, in some embodiments, the cancer is renal carcinoma, skin cancer, bladder cancer, prostate cancer, or breast cancer. In some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is an invasive cancer. In some embodiments, the cancer is a solid tumor, in some embodiments, the cancer is a carcinoma of the kidney, lung, liver, spleen., pancreas, intestine, colon, mammary g!and, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, brain, or esophagus, in some embodiments, the cancer is a hypoxic tumor, e.g., a hypoxic solid tumor. In some embodiments, the tumor (e.g., hypoxic tumor) overexpresses CALX.
[0111] in some embodiments, the disease or disorder is an immune disease or disorder, in some embodiments, the immune disease or disorder is an autoimmune disease, asthma, ataxia telangiectasia, arthritis (e.g., rheumatoid arthritis), autoimmune polyglandular syndrome, Burkitt lymphoma, diabetes (e.g., type 1 diabetes), DiGeorge syndrome, familiar
Mediterranean fever, Guillain-Barre syndrome, inflammatory bowel disease, leukemia, lupus, multiple sclerosis, common variable immunodeficiency, severe combined immunodeficiency, human immunodeficiency virus/acquired immune deficiency syndrome, or drug-induced immunodeficiency.
[0112] In some embodiments, the disease or disorder is ischemia. In some embodiments, the ischemia is cardiac ischemia, brain ischemia, kidney ischemia, ischemia! colitis, mesenteric ischemia, or an ischemia-reperfusion injury. In some embodiments, the disease or disorder is wound healing, in some embodiments, the disease or disorder is acute renal failure.
Therapies and Therapeutic Agents
[0113] A therapeutic agent for use according to any of the methods of the present invention can be any composition that has or may have a pharmacological activity. Agents include compounds that are known drugs, compounds for which pharmacological activity has been identified but which, are undergoing further therapeutic evaluation, and compounds that are members of collections and libraries that are screened for a pharmacological activity. In some embodiments, the therapeutic agent is a cell, e.g., a stem cell; an anti-cancer, e.g., an anti- signaling agent (e.g., a cytostatic drug) such as a monoclonal antibody or a tyrosine kinase inhibitor; an anti-pro!iferative agent; a chemotherapeutic agent (i.e., a cytotoxic drug); a hormonal therapeutic agent; and/or a radioiherapeutic agent.
[0114] Generally, the therapeutic agent is administered at a therapeutically effective amount or dose. A therapeutically effective amount or dose will vary according to several factors, including the chosen route of administration, the forrnuiation of the composition, patient response, the severity- of the condition, the subject's weight and the judgment of the prescribing physician. The dosage can be increased or decreased over time, as required by an individual patient. In certain instances, a patient initially is given a low dose, which is then increased to an efficacious dosage tolerable to the patient. Determination of an effective amount is well within the capability of those skilled in the art.
[0115] The route of administration of a therapeutic agent can be oral, intraperitoneal, transdermal, subcutaneous, by intravenous or intramuscular injection, by inhalation, topical, intralesional, infusion; iiposome-mediated delivery; topical, intrathecal, gingival pocket, rectal, intrabronchial, nasal, transmucosal, intestinal, ocular or otic delivery, or any other methods known in the art.
[0116] in some embodiments, a therapeutic agent is formulated as a pharmaceutical composition, in some embodiments, a pharmaceutical composition incorporates particulate forms, protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method/mode of administration. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectibles, implantable sustained-release formulations, etc.
[0117] In some embodiments, a pharmaceutical composition comprises an acceptable carrier and/or excipients. A pharmaceutically acceptable carrier includes any solvents, dispersion media, or coatings that are physiologically compatible and that preferably does not interfere with or otherwise inhibit the activity of the therapeutic agent. Preferably, the carrier is suitable for intravenous, intramuscular, oral, intraperitoneal, transdermal, topical, or subcutaneous administration. Pharmaceutically acceptable carriers can contain one or more physiologically acceptable compound(s) that act, for example, to stabilize the composition or to increase or decrease the absorption of the active agent(s). Physiologically acceptable compounds can include, for example, carbohydrates, such as glucose, sucrose, or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins, compositions that reduce the clearance or hydrolysis of the active agents, or excipienis or other stabilizers and/or buffers. Other pharmaceutically acceptable carriers and their formulations are well-known and generally described in, for example, Remington: The Science and Practice of Pharmacy, 21st Edition, Philadelphia, PA. Lippincott Williams & Wilkins, 2005. Various pharmaceutically acceptable excipienis are well-known in the art and can be found in, for example, Handbook of Pharmaceutical Excipienis (5th ed., Ed. Rowe et a!. , Pharmaceutical Press, Washington, D.C.).
[0118] In some embodiments, the therapeutic treatment comprises immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment. In some embodiments, the therapeutic treatment comprises immune stimulation with an immunogenic composition, e.g., a tumor- associated antigen (TAA)-cytokine fusion protein.
TAA-Cytokine Fusion Proteins for Immunotherapy
[0119] in some embodiments, a subject is administered a TAA-cytokine fusion protein to induce a cell-mediated immune response, The TAA-cytokine fusion protein can be formulated according to methods known in the art. In some embodiments, the TAA-cytokine fusion protein is administered as a polypeptide comprising a fusion protein, as dendritic cells or other cells pulsed with the fusion protein, as a nucleic acid encoding a fusion protein (e.g., as a gene therapy vector, e.g., adenovirus, adeno-associated virus, retrovirus, lentivirus, etc.), as dendritic cells or other cells transfected with a nucleic acid encoding a fusion protein (e.g., via a recombinant virus, plasmid DMA transfection, etc.), or as a nucleic acid encoding a fusion protein complexed with a transfection agent.
[0120] In some embodiments, the TAA-cytokine fusion protein comprises the TAA carbonic anhydrase IX (CAIX), or a portion thereof, attached to a granulocyte macrophage colony stimulating factor (GM-CSF). In some embodiments, the fusion protein comprises human CAIX, or a portion thereof, attached to human GM-CSF. Suitable CAIX-GM-CSF fusion proteins, as well as methods of making, formulating, and administering such fusion proteins, are described in US 7,572,891 and US 8,378,084, incorporated by reference herein. In some embodiments, the CAIX protein in the fusion protein is a "CAIX variant" protein as described in US 8,378,084. [012 J ] In some embodiments, an imrauno-effector is co-administered with the TAA- cytokine fusion protein, Examples of immuno-effectors include, but are not limited to, interferoii-ot, interferon-β, interferon-γ, interferon-t, interferon-θ, tumor necrosis factor- , tumor necrosis factor-β, interleukin-2, interieukin-6, interleukin-7, interleukin-12, interleukin-15, B7- 1 T cell co-stursuiatory molecule, B7-2 T cell co-stimulatory molecule, immune cell adhesion molecule (ICAM)-l , granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, and combinations thereof,
[0122] In some embodiments, adoptive immunotherapy is administered. Adoptive immunotherapy refers to a therapeutic approach for treating cancer or infectious diseases in which immune cells are administered to a host with the aim that the cells mediate either directly or indirectly specific immunity to (i.e., mount an immune response directed against) tumor cells. In some embodiments, the immune response results in inhibition of tumor and/or metastatic cell growth and/or proliferation and most preferably results in neoplastic cell death and/or resorption, The immune ceils can be derived from a different organism/host
(exogenous immune cells) or can be cells obtained from the subject organism (autologous immune cells), in some embodiments, dendritic cells (e.g., isolated from the patient or autologous dendritic cells) are pulsed with the TAA-cytokine fusion protein and then injected back into the subject where they present and activate immune cells in vivo, in addition, or alternatively, the dentritic cells can be transfected with nucleic acids encoding the TAA- cytokine fusion protein and then re-introduced into a patient, Methods of performing adoptive immunotherapy are well known to those of skill in the art, See, e.g., U.S. Pat. Nos,
5,081,029, 5,985,270, 5,830,464, 5,776,451, 5,229, 1 15, 690,915, and the like.
V. Methods of identifying Drug Candidate Agents
[0123] In yet another aspect, the present invention provides methods of identifying a drug candidate agent, e.g., for treating a disease or disorder, using biomarkers of immunoeditmg- escape status. In some embodiments, the method comprises:
(a) contacting a ceil with the candidate agent; and
(b) detecting the expression profile of one or more target biomarkers in the cell, wherein the one or more target biomarkers is an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof, and wherein differential expression of the one or more target biomarkers in the expression profile of the eel! contacted with the candidate agent relative to a control identifies the candidate agent as useful for treating the disease or disorder in the subject.
[0124] In some embodiments, the method of identifying a drug candidate agent comprises detecting the expression profile of at least one biomarker (e.g., I, 2, 3, 4, 5, 6, 7, S, 9, 10 or more target biomarkers) selected from an rnRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof, in some embodiments, the rnRNA or protein biomarker is an rnRNA or protein that is involved in inflammatory response. In some embodiments, the mRNA or protein biomarker is an inflammatory response gene or protein listed in Table 4A. In some embodiments, the mRNA or protein biomarker is an mRNA or protein that is involved in cellular growth and/or proliferation, in some embodiments, the mRNA or protein biomarker is a cellular growth and/or proliferation gene or protein listed in Table 4B. In some embodiments, the at least one biomarker is an miRNA or LincRNA listed in Table 5 or a human ortholog thereof.
[0125] In some embodiments, the at least one biomarker is selected from Ccl 1 , Cxc!9, Hmgb l, I- -12, Cd209a, Foxj l, Klra2, miR-1 186, miR-98, miR-5097, miR-1942, and miR- 708. In some embodiments, the at least one biomarker is Cell, Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l, or Klra2. in some embodiments, the at least one biomarker is miR-1 186, miR-98, miR-5097, miR-1942, or miR-708. in some embodiments, differential expression of at least one biomarker selected from Cell , Cxcl9, Hmgbl, Fgl2, Cd209a, Foxj l , lra2, miR-1 186, miR-98, miR-5097, and miR-1942, and miR-708 in the ceil contacted with the candidate agent e.g., upregulation or downregulation of the biomarker in the cell contacted with the candidate agent relative to a control cell not contacted with the candidate agent) identifies the candidate agent as useful for treating the cancer or immune system disease or disorder.
[0126] In some embodiments, disease or disorder is a cancer. In some embodiments, the cancer is ana! carcinoma, bladder carcinoma, breast carcinoma, cervix carcinoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, endometrial carcinoma, hairy cell leukemia, head and neck carcinoma, lung (small cell) carcinoma, multiple myeloma, non- Hodgkin's lymphoma, follicular lymphoma, ovarian carcinoma, brain tumors, colorectal carcinoma, hepatocellular carcinoma, Kaposi's sarcoma, lung (non-small cell carcinoma), melanoma, pancreatic carcinoma, prostate carcinoma, renal cell carcinoma, or soft tissue sarcoma. In some embodiments, the cancer is renal carcinoma, skin cancer, bladder cancer, prostate cancer, or breast cancer, in some embodiments, the cancer is a metastatic cancer. In some embodiments, the cancer is an invasive cancer.
[0127] In some embodiments, the disease or disorder is an immune system disease or disorder. In some embodiments, the immune disease or disorder is an autoimmune disease, asthma, ataxia telangiectasia, arthritis (e.g., rheumatoid arthritis), autoimmune polyglandular syndrome, Burkitt lymphoma, diabetes (e.g., type 1 diabetes), DiGeorge syndrome, familiar Mediterranean fever, Guiilain-Barre syndrome, inflammatory bowel disease, leukemia, lupus, multiple sclerosis, common variable immunodeficiency, severe combined immunodeficiency, human immunodeficiency virus/acquired immune deficiency syndrome, or drug-induced immunodeficiency.
[0128] In some embodiments, the disease or disorder is ischemia. In some embodiments, the ischemia is cardiac ischemia, brain ischemia, kidney ischemia, ischemial colitis, mesenteric ischemia, or an ischemia-reperfusion injury, in some embodiments, the disease or disorder is wound healing. In some embodiments, the disease or disorder is acute renal failure.
[0129] In some embodiments, a drug candidate agent is identified as suitable for use in treating the disease or disorder when the one or more biomarkers is differentially expressed (upregulated or downregulated) by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or by at least 1.2-fold, 1 .5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold in the ceil as compared to a threshold level or a control cell (e.g., a non-diseased cell).
[0130] Drug candidate agents that can be evaluated according to the methods described herein include, but are not limited to, peptides, proteins, oligopeptides, circular peptides, pepticiomimeties, antibodies, polysaccharides, lipids, fatty acids, inhibitory RNAs (e.g., siRNA, miRNA, or shRNA), immunotherapeutics, vaccines, polynucleotides,
oligonucleotides, aptamers, small organic molecules, or drug compounds. In some embodiments, the agent is a small organic molecule. In some embodiments, the agent is a peptide or protein. In some embodiments, the agent is an immunotherapeutic. [01.31] Drug candidate agents are not limited by therapeutic category, and can include, for example, analgesics, anti- inflammatory agents, antihelminthics, anti-arrhythmic agents, antibacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti- epileptics, anti-fungal agent, anti -gout agents, anti-hypertensive agents, anti-ma lariais, antimigraine agents, anti-muscarinic agents, anti-neoplastic agents, erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, β-blockers, cardiac inotropic agents, corticosteroids, diuretics, anti-park insonian agents, gastro-iniestinal agents, histamine receptor antagonists, keratolyses, lipid regulating agents, anti-anginal agents, Cox-2 inhibitors, leukotriene inhibitors, macroiides, muscle relaxants, anti-osteoporosis agents, anti- obesity agents, cognition enhancers, anti-urinary incontinence agents, nutritional oils, anti- benign prostate hypertrophy agents, essentia! fatly acids, non-essential fatty acids, and the like, as well as mixtures thereof.
[0132] In some embodiments, the candidate agent is identified in siiico using
pharmacogenomics. In siiico methods for identifying and validating drug candidate agents are known in the ait. See, e.g., In Siiico Technologies in Drug Target Identification and Validation, Leon and Markei, eds., CRC Press, 2006. [0133] In some embodiments, a candidate agent is identified from a library of agents. In some embodiments, the library of agents comprises at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 5000, 10,000, 20,000, 30,000, 40,000, 50,000 agents or more, it will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigraa-Aldrich (St. Louis, MO), Fluka Chem tka-Biochemica Analytika (Bucfas Switzerland), as well as providers of small organic molecule and peptide libraries ready for screening, including Chembridge Corp. (San Diego, CA), Discovery Partners International (San Diego, CA), Triad Therapeutics (San Diego, CA), Nanosyn (Menlo Park, CA), Affyraax (Palo Alto, CA), ConiGenex (South San Francisco, CA), and Tripos, Inc. (St. Louis, MO). In some embodiments, the library is a combinatorial chemical or peptide library. A combinatorial chem ical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length {i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks. The preparation and screening of chemical libraries is well known to those of skill in the art (see, e.g., Beeler el al, Curr Opin Che Biol,, 9:277 (2005); and Shang et al, Curr Opin Chern Biol, 9:248 (2005)).
[0134] in some embodiments, a candidate agent can be identified by screening a library containing a large number of potential therapeutic compounds. The library can be screened in one or more assays, as described herein, to identify those library members that display a desired characteristic activity, The compounds thus identified can serve as conventional "lead compounds" (e.g., for identifying other potential therapeutic compounds) or can themselves be used as potential or actual therapeutics. Libraries of use in the present invention can be composed of amino acid compounds, nucleic acid compounds,
carbohydrates, or small organic compounds. Carbohydrate libraries have been described in, for example, Liang et al, Science, 274: 1520-1522 (1996); and U.S. Patent No. 5,593,853.
[0135] Representative amino acid compound libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent Nos. 5,010,175; 6,828,422; and 6,844,161; Furka, Int. J. Pept. Prot. Res., 37:487-493 (1991); Houghton et al. , Nature, 354:84-88 (1991 ); and Eichier, Comb Chern High Throughput Screen., 8:135 (2005)), peptoids (PCT Publication No. WO 91/19735), encoded peptides (PCT Publication No. WO 93/20242), random bio- oligomers (PCT Publication No. WO 92/00091), vinyiogous polypeptides (Hagihara et al., ,1. Amer. Chern. Soc, 1 14:6568 ( 1992)), nonpeptidal peptidomimetics with β-D-glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc., 1 14:9217-9218 (1992)), peptide nucleic acid libraries (see, e.g., U.S. Patent No. 5,539,083), antibody libraries (see, e.g., U.S. Patent Nos. 6,635,424 and 6,555,310; PCT Application No. PCTYUS96/10287; and Vaughn et al , Nature Biotechnology, 14:309-314 (1996)), and peptidyl phosphonates (Campbell et l, J. Org Chem., 59:658 (1994)).
[0136] Representative nucleic acid compound libraries include, but are not limited to, genomic DNA, cDNA, mRNA, inhibitory R A (e.g., RNAi, siRNA), and antisense RNA libraries. See, e.g., Ausubel, Current Protocols in Molecular Biology, eds. 1987-2005, Wiley Interscience; and Sambrook and Russell, Molecular Cloning: A Laboratory Manual , 2000, Cold Spring Harbor Laboratory Press. Nucleic acid libraries are described in, for example, U.S. Patent Nos . 6,706,477: 6,582,914; and 6,573,098. cDNA libraries are described in, for example, U.S. Patent Nos. 6,846,655: 6,841,347; 6,828,098; 6,808,906; 6,623,965; and
6,509,175. RNA libraries, for example, ribozyme, RNA interference, or siRNA libraries, are described in, for example, Downward, Cell, 121 : S 13 (2005) and Akashi et al., Nat. Rev. Mol Cell Biol, 6:413 (2005). Antisense RNA libraries are described in, for example, U.S. Patent Nos. 6,586, 180 and 6,518,017. f0137j Representative small organic molecule libraries include, but are not limited to, diversomers such as hydantoins, benzodiazepines, and dipeptides (Hobbs et at, Proc. Nat. Acad. Sci. USA, 90:6909-6913 (1993)); analogous organic syntheses of small compound libraries (Chen et a!., J. Amer. Chem. Soc, 1 16:2661 (1994)); oligocarbamates (Cho et al, Science, 261 : 1303 (1993)); benzodiazepines (e.g., U.S. Patent No. 5,288,514; and Baura, C&EN, Jan 18, page 33 (1993)); isoprenoids (e.g., U.S. Patent No. 5,569,588);
thiazolidinones and metathiazanones (e.g., U.S. Patent No. 5,549,974); pyrrolidines (e.g., U.S. Patent Nos. 5,525,735 and 5,519,134); morpholino compounds (e.g., U.S. Patent. No. 5,506,337); tetracyclic benzimidazoles (e.g., U.S. Patent No, 6,515,122): dihydrobenzpyrans (e.g. , U.S. Patent No. 6/790,965); amines (e.g., U.S. Patent No, 6,750,344); phenyl compounds (e.g., U.S. Patent No. 6,740,712); azoles (e.g., U.S. Patent No. 6,683,191);
pyridine carboxarnides or sulfonamides (e.g. , U.S. Patent No. 6,677,452); 2- aminobenzoxazoles (e.g., U.S. Patent No. 6,660,858); isoindoles, isoox indoles, or isooxyquinolines (e.g., U.S. Patent No. 6,667,406); oxazolidinones (e.g., U.S. Patent No. 6,562,844): and hydroxylamines (e.g., U.S. Patent No, 6,541,276).
[0138] Devices for the preparation of libraries are commercially available. See, e.g., 357 MPS and 390 MPS from Advanced Chem. Tech (Louisville, KY), Symphony from. Ratnin Instruments (Woburn, MA), 433 A from Applied Biosystems (Foster City, CA), and 9050 Plus from Miiiipore (Bedford, MA).
VI. Compositions, Kits, and Arrays
[0139] The invention further provides compositions, kits, and arrays for practicing the methods described herein. [01.40] The invention provides assay compositions for use in solid phase assays; such compositions can include, for example, one or more capture reagents (e.g., polynucleotides or polypeptides) that bind a biomarker as described herein (e.g., an niRN A, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof) immobilized on a solid support, and a labeling reagent. In each case, the assay compositions can also include additional reagents that are desirable for hybridization. Appropriate controls for carrying out the screen (e.g., known positive and/or negative controls for the capture reagents) can also be included in the assay compositions. [0141] The invention also provides kits for carrying out the methods described herein, The kits can include one or more capture reagents (e.g., polynucleotides or polypeptides) that bind a biomarker as described herein (e.g., an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human orthoiog thereof) as well as other components, in some embodiments, the kit comprises a capture reagent that is an antibody that binds to a protein translated from an mRNA listed in Table 5. In some embodiments, the kit comprises a capture reagent that is a nucleic acid that is substantially complementary to an mRNA, miRNA, or LincRNA listed in Table 5. In some embodiments, the kit comprises a a plurality of capture reagen ts, wherein each capture reagent binds a biomarker selected from the group consisting of the biomarkers listed in Table 5 and their human ortho!ogs, Optionally, a detectable label is conjugated to the capture reagent for indicating the presence of the capture reagent and therefore the biomarker. In some embodiments, the kit comprises one or more capture reagents and one or more detection reagents (e.g., a primary antibody that is a capture reagent for a biomarker of interest, and a secondary antibody having a binding specificity for the primary antibody and having a detectable label or moiet)'). In some embodiments, the kit further comprises additional reagents for the detection reaction, e.g., buffers, drugs, cytokines, or wash solutions for selectively retaining the bound biomarker to the capture reagent after washing. In some embodiments, the kit includes a container containing the capture reagent(s). [0142] In addition, the kits of the present invention can include instructions to the kit user (e.g., instructions for using the capture reagent to detect the biomarker, instructions that provide for contacting a test sample with the capture reagent and detecting the biomarker that is present in the sample, if any, that is retained by the capture reagent; and/or instructions for using the kit for detecting a disease or disorder as described herein. VII. Examples
[0143] The following examples are offered to illustrate, but not to limit, the claimed invention, Example ί : Dendritic Cell-Based Immunotherapy HI Prevention and Treatment of Renal Cell Carcinoma: Efficacy, Safety, and Anti-Tumor Activity of Ad-GM-CAIX in Immunocompetent Mouse Models
ABSTRACT
[0144] The dendritic cell vaccine DC- Ad-GM-CAIX is an active, specific immunotherapy with the potential of providing a safe and effective therapy against renai cell carcinoma (RCC), Using immunocompetent Balb/c mouse models we tested the efficacy and mechanism of the vaccine to prevent and treat the growth of a syngeneic RCC (RE CA) engineered to overexpress the human TAA carbonic anhydrase IX ( PR-IX). In a prevention model, NPR- IX tumor development was specifically and significantly delayed by 13 days in DC-Ad- GM-CAIX- treated mice ( <G.001), and tumor volumes were 79% smaller at study termination compared to the controls (day 24, P<Q, 07), Six of these mice remained tumor- free for >1 year, in a treatment model, NPR-TX tumors remained smaller in DC-Ad- GM-CAIX-treated mice for 8 days (P<0.002), achieving a 60% growth inhibition at termination, No vaccine-related toxicity was observed. The critical mechanistic parameter separating responsive from non-responsive tumors was hCAIX protein expression, demonstrated by aggressive growth of tumors that did not express hCATX protein and in sham-treated mice (DC-Ad-Null), and by in vitro anti-hCAIX cytotoxic T-cell IFN-γ response (Cd8+). No murine serum anti-hCAIX antibodies were detected. Moreover, altered mechanisms of immunoediting as a means for immune escape were suggested by differential gene expression (Cell, Hmgbl , Fgl2, Cd209a, K3ra2) and miRNAs (miR-1 1 86, miR-98, miR-5097, miR-1942, miR-708) in tumors that evaded DC-Ad-GM-CAlX immunotherapy. This is the first study in immunocompetent mice that provides a proof of concept for the specificity, efficacy, safety-', and mechanisms of the DC-Ad-GM-CAIX immunotherapy, forming the basis for a first-in-human phase 1 trial in RCC patients.
INTRODUCTION
[0145] Despite the FDA approval of six targeted therapies for advanced renal cell carcinoma (RCC) in the past five years, complete and durable remissions of metastatic disease are rarely if ever achieved. Moreover, these agents are mainly cytostatic, are associated with significant side effects, require chronic administration, and treatment resistance typically develops within one year. On the other hand, RCC belongs with melanoma to a class of solid tumors that are significantly responsive to and are, in a small percentage of cases, curable by systemic, treatment with high-dose interleukin-2 (1). The identification of tumor- associated antigens (TAAs) overexpressed in RCC, such as carbon ic anhydrase IX (CAIX), has led to the development of humoral and cell-mediated
immunotherapies (2, 3).
[0146] CAIX regulates proton exchange to buffer intracellular H, a function crucial for cell survival (4), CAIX expression has been described in many normal and cancerous tissues (4, 5) and is directly regulated by the hypoxia-inducible factor la-von Hippel-Lindau (VHL) pathway. Upon loss of pVHL function in clear cell RCC, stabilization of HIF- Ια occurs, leading to the overexpression of CAIX and rendering it useful as a diagnostic and therapeutic TAA (6-10). [0147] The recent FDA approval of sipuleucel-T and its associated body of research have clearly shown the immunotherapeutic benefit of fusing a potent cytokine such as granulocyte- macrophage colony stimulating factor (GM-CSF) with a TA A. Transduction of such a fusion protein ex vivo into antigen presenting cells is capable of leading to an effective immune stimulation leading to the improved survival of patients with advanced solid cancer (11, 12). We have previously shown the in vitro ability of the fusion protein GM-CSF-CAIX
(GM-CAIX) to induce a C AIX- targeted, T-cell mediated, and MHC-restricted anti-tumor activity (13-1 5). To model the role of T-cell anti-tumor activity against clear cell RCC in mice with an intact immune system, we constitutively and stably overexpressed human CAIX (hCAIX) in a CAIX-negative parental RENCA cell line (16). The cells were then sorted for hCAIX expression (Newly Purified RENCA-CAIX; NPR-IX) and transplanted in syngeneic Balb/c mice. An adenoviral vector was used to transduce the GM-CAIX fusion protein in syngeneic DCs that were then transplanted in the same mice. Our study shows for the first time that murine DCs expressing hGM-CAIX can generate a potent T-cell mediated hCAIX- specific inhibition of tumor growth in immunocompetent mice. Differential gene and rniRNA expression characterization revealed candidate markers for immune evasion mechanisms. Collectively, these encouraging pre-clinical results provide the basis for the initiation of clinical trials using DC-Ad-GM-CAIX immunotherapy.
RESULTS
Prevention model
[0148] DC-Ad-GM-CAIX immunotherapy significantly and specifically prevented the growth of the NPR-IX tumors in syngeneic Baib/c mice (group A in Fig. 1A). In this group the mean time to development of a palpable tumor (~50 mm") was 13 days longer than in the control groups (23 days in group A vs. 10 days in the sham and RENCA tumors control groups B-F, <0.001). At study termination, median tumor volume in group A was 79% smaller than in the control groups B-F (day 24, all PO.007; Fig. 2A), and 50% of the mice were tumor- free (Fig, 2B). Repetition of this study yielded similar results, with 7 of 8 (88%) mice in group A remaining tumor-free at termination (Fig. 2C-D). Tumor growth was prevented for >1 year in 6 of 16 mice.
[0149] Although the growth rate of NPR-IX tumors (group E; 45.3 mm day) was significantly faster than that of RENCA tumors (group F: 27.6 mnrVday; P<0.001), the growth rate of tumors that eventually de veloped in group A showed no significant difference compared to groups B-F (all .P>0.03).
Treatment model
[0150] To test whether the DC-Ad-GM-CAIX immunotherapy would have an inhibitory effect on established RCC tumors (Fig. IB), three groups of mice bearing syngeneic NPR-IX tumors were treated with DC- Ad-GM- CALX, DC-Ad-Null, or no DCs (groups G, H, and I, respectively). Tumor growth was inhibited in group G relative to the control groups H-I, achieving a 60% median growth inhibition at day 26 (Fig. 2E-F). Group G had a smaller median tumor volume than the control groups from day 19 to 26 ( <0.002). Additionally, median tumor growth rate was significantly lower in group G (27.7 mm day) compared to the control groups (group H 62.3 mmVday; group 1 66.0 mra'Vday; both <0.001). Tumor growth rates between group H and i were statistically not different (P= .6Q2).
Histopathology, toxicity, and in vitro immune-monitoring
[0151] In the prevention model, mice in group A maintained their body weight throughout the experiment, while mice in each of the control groups B-F suffered significant weight loss (Ρ<0.001; Fig. 2G-J). In the treatment model, group G showed a significantly smaller median weight loss compared to the control groups H-I combined from day 15 to 21 (Ρ<0.01 1 ; Fig. 2K-L). At study termination, none of the organs in groups A-I showed evidence of metastases or systemic toxicity caused by the immunotherapy, such as histopathologic inflammatory lesions or atrophy.
[0152] Immunohistoiogical analyses showed absent or minimal hCAIX expression in tumors that evaded DC-Ad-GM-CAIX immunotherapy (groups A and G), but strong expression in the control groups C, E, H-I (Fig. 3A-F), suggesting that DC-Ad-GM-CAIX immunotherapy is specifically targeting and killing hCAIX-expressing tumor cells. Murine CAIX (mCAIX) staining, however, was minimal in all groups (Table 1). In addition, tumor necrosis levels and staining for Ki-67, murine macrophages (F4/80), and murine granulocytes (Cd l lb) were similar in groups A, C and E (Table 1).
Table 1. Prevention Model: Histopathologic analysis
Figure imgf000048_0001
- negative 4 1-33% pos. / low ++ 34-66% pos. / middle 4 67-100% pos. / high
[0153] Next we tested the sera for the presence of murine antibodies against the hCAIX protein expressed in NPR-IX and DC-Ad-GM-CAlX. FACS analysis of sera from groups A and E did not show evidence for the presence of murine anti-hCAIX antibodies (Fig, 6). [0154] To validate that the tumor growth inhibition in group A was related to an anti- hCAlX cytotoxic T-cell response (Cd84), an in vitro interferon gamma (IFN-γ) test was conducted. Splenocytes harvested from group A and group E were co-incubated with NPR- IX or RENCA. IFN-γ levels were consistently and predominately increased in splenocytes of DC-Ad-GM-CAlX treated mice (group A) co-incubated with NPR-IX. However, lower IFN-γ levels were also released after co-incubation with RENCA . Splenocytes of non-treated mice (group E) co-incubated with NPR-IX or RENCA did not release IFN-γ (Fig. 4).
Differential gene and miRNA expression in tumors that evaded DC~Ad~GM-CAiX therapy and control
[0155] To test if the down-regulation in hCAl'X protein expression originated at the transcription or protein level (Fig. 3), qPCR analysis was performed and revealed similar hCAIX expression levels in tumors that evaded DC- Ad-GM-CAIX therapy (group A) and in tumors of mice treated with DC-Ad-Null (groups C), suggesting hCAIX protein regulation as the cause of its reduced expression. A similar transcription signature was obtained for the endogenous mouse CAIX gene (Car9; Table 2, Fig. 5C). [0156] Since MHC class ί (MHC-I) expression is crucial for CTL targeting of tumor cell we next evaluated whether tumors that evaded the DC-Ad-GM-CAIX therapy exhibited differential expression of MHC-L The qPCR analysis suggested that the Balb/c MHC-I markers H-2Kd and H-2Dd were expressed and at similar levels in tumors that evaded the DC-Ad-GM-CAIX therapy and in tumors of mice treated with DC-Ad-Null (Table 2, Fig.
Table 2. qPCR validated MHC-I, CAIX, and differential expression of genes and miRNA in tumors that evaded DC~Ad-GM;CAIX immunotherapy (Group A) relative to DC-Ad-Null (Group C).
Figure imgf000049_0001
Expression group
Marker A vs. C interest Ref.
miR-1 186 up Short term expression: (37-39) promotion of tumor growth
through Cycli B l (Ccnbl )
activation
miR-98 up Promotion and suppression of (40-42) tumor growth
miR-5097 up Novel description
raiR-1942 p Novel description in RCC /
immunoediting
miR-708 down Tumor suppressor of RCC (43)
[0157] To characterize and evaluate the molecular divergence between NPR-IX tumors that evaded DC-Ad-GM-CAIX therapy (group A; 5 tumors) and NPR-IX tumors of mice treated with DC-Ad-Null (group C; 4 tumors), we preformed genome wide mRNA and miRNA expressions analysis. We found 367 differentialiy expressed gene features between groups A and C altered at feast by i .5-fold at P<0.0\ 5 representing 314 coding genes that are listed at NCBI EntrezGene database (Table 5). Hierarchical clustering of mean centered logarithmic expressions of these genes by average linkage algorithm using 1 -correlation as distance metric clustered the tumor samples into two groups (groups A and C; Fig, 5A). Similar clustering was observed for the miRNA expression microarray spots (n-28; P<0.015) of which 1 1 miRNAs clustered the samples into groups A and C (Fig. SB). The differentially expressed genes were associated with significantly enriched gene ontology categories (IPA, ingenuity, CA; P<0.GS) including immune response, proliferation, cell growth, cell movement, and cell-to-cell signaling (Table 4A-B). Then, using an extensive literature survey (CoreMine and PubMed) we identified which of the differentially expressed genes and miRNAs are known to play a predominant role in mechanisms of immune evasion. Of the evasion genes we observed and validated by qPCR were the differential expression of chemokines (Cel l, Cxcl9), regulators and markers of the myeloid cells, DCs, natural killer (NK) cells and T-cells (Hmgbl, Cd209a, Fgl¾ Klra2, Foxjl), and of 5 miRNAs (miR-1 186, miR-98, miR-5097, miR-1942, miR-708; Table 2, Fig. 5C-D).
DISCUSSION
[0158] Designing targeted and effective immunotherapy strategies for the treatment of cancer requires not only identifying specific TAAs, but also utilizing an innovative approach which re-engages the immune system to specifically recognize and kill tumor cells (17). Our current in vivo results, coupled with our pre viously published in vitro data, support the hypothesis that DC-Ad-GM-CAIX can generate an in vivo T-cell mediated anti-hCAIX- specific immune response resulting in both prevention and treatment of established RCC tumors. Moreover, this therapy appears to be capable of inducing significant inhibition of RCC tumor growth in immunocompetent mice without obvious systemic toxicity. [0J59] As previously established, murine and human GM-CSF act in a species-specific manner (18, 19), and therefore the therapeutic efficacy demonstrated here can likely be attributed to only the hCAIX part of the fusion molecule. Indeed, overexpression of hCAIX appeared to be the critical mechanistic determinant of turner response to DC-Ad-GM-CAIX immunotherapy. Only tumor cells overexpressing hCAIX protein (NPR-IX) were inhibited and only in groups immunized with DC-Ad-GM-CAIX (groups A, G), Moreover, in both the prevention and the treatment models, tumor tissue that subsequently evaded the therapy- appeared to have done so only in tumors with little or no hCAIX protein expression. A significant finding is that, while both mouse and human CATX proteins were absent in the immunotherapy-evading tumors, the CA1X transcripts were expressed without significant change from the controls, suggesting that CAIX expression may also be regulated at the protein level (Table 2; Fig, 3). Similarly, although HLA class I gene down-regulation or loss often occurs in RCC, presumably enabling the tumor to evade cytotoxic T-eeJls (20), in our system both the evading tumors and controls expressed the same transcription levels of the Balb/c MHC class 1 markers H-2Kd and H-2D" (Table 2). Our pharmacodynamics data, the detection of hCAlX-targeted cytotoxic T-cell activity (TFN-γ), the lack of murine serum antibodies against hCAIX, and the uncontrolled growth ofNPR-ΙΧ tumors in the control groups C and E all suggest a hCAlX-specific cytotoxic T-cell response and not a humoral one,
[0160] While the absence of the hCAIX protein appears to have been the critical determinant of evasion, the evasion phase may depend also on other genes and can be regarded as part of the concept of "cancer immunoediting" (21). Clinically and biologically, cancer immunoediting is a complex but orderly continuum that can be separated into a series of three overlapping phases: elimination (tumor destruction), equilibrium (dormancy), and evasion (escape) (22). To gain further molecular insight into the immunoediting processes acti e in our models, we compared the gene and miRNA expression in tumors that evaded the DC-Ad-GM-CAIX immunotherapy with the expression in tumors subject to DC-Ad-Null therapy (groups A and C, respectively). The genes and miRNA expression datasets of tumors that evaded the immunotherapy significantly clustered the tumors apart from the tumors in group C. Our gene ontology analysis of the differential gene expression suggests a significant enrichment of categories such as activation of leukocytes, inflammatory response, and chemotaxis of myeloid cells (Table 4), Consistent with this idea is the differential up- regulation we observed for the chemokines Cell and Cxcl9 in tumors that evaded immunotherapy. Cei l has been suggested to induce Treg mediated immunosuppressive functions (23, 24), and Cxcl9 has been correlated with both T-cell infiltration and promotion of immune surveillance evasion (25, 26), There is also compelling evidence for the role of myeloid cells in tumor evasion. Hmgbl, Cd209a (DC-SIGN) and Fgl2, all of which were found to be up-regulated in the evading tumors (group A), have been suggested to function as recruiters of immunosuppressive myeloid-derived suppressor cells and Treg (27-29), leading to evasion from immune surveillance (30, 31) and to inhibition of DC maturation and T-cell proliferation (Table 2, Fig. 5C) (32). Another characteristic of RCC are the presence and role of NK cells (33), One of the negative regulators of murine myeloid cells is Klra2 (Ly49B). While Klra2 is not normally expressed at detectable levels on NK cells, it appears to be up- regulated on myeloid cells and NK cells following encounter with inflammatory cytokines or bacterial products, decreasing their reactivity and preventing non-specific- inflammatory responses (34, 35), Now for the first time we report that Kira2 is also up-regulated in tumors that have evaded the DC-Ad-GM-CAIX immunotherapy, suggesting an intriguing new role In inhibiting NK cell-mediated target RCC lysis (Table 2, Fig, SC, Table 5). [01613 Tumors that e aded the immunotherapy up-regulated four miR As (miR- 1186, miR-98, miR-5097, miR- 1942) and down-regulated one (miR-708) (Table 2, Fig. 5D, Table 5), Our discovery of the up-regulation of miR-5097 in tumors that evaded immunotherapy is. to the best of our knowledge, the first publication on this raiRNA. The function of miR- 1942 in tumors that evaded therapy is yet to be identified (36). The short-term up-regulation of miR-1 186 was recently suggested to promote tumor growth through Cyclin B 1 (Ccnbl) activation, which is substantially involved in RCC carcinogenesis and progression (37-39). miR-98 is suggested to have tumor suppressive function in breast tumors, tumor promoting function in other types of tumors (40-42). miR-708 expression, which we found to be down- regulated in tumors that evaded immunotherapy, was recently described to be attenuated in RCC and was suggested to have a role as a pro-apoptoic tumor suppressor (43).
[0162] Collectively, the identification and molecular characterization of tumors that evaded immunotherapy may provide information on additional immune evasion processes and may ultimately be useful for the development of immune evasion diagnostic markers and for the rational development of next generation immunotherapies.
METHODS
[0163] Cells and cell culture. RENCA is a spontaneous murine renal cortical adenocarcinoma arising in BaJb/c mice and is a model of human adult RCC in
immunocompetent syngeneic mice (Balb/c) (Murphy et al,, J Natl Cancer Inst 1973; 50: 1013-25). RENCA cells were a gift from Dr. R. Wiltrout (NCi/NIH) (44). RENCA-CAIX cells were generated by stably transducing the human CAIX gene in RENCA cells (16), NPR-IX cells are hCATX FACS-purified RENCA-CAIX ceils, which repeatedly had >90% hCAIX-positive cells in flow cytometry analysis (Fig. 7). All ceils were cultured in RPMI- 1640 medium (Life Technologies, NY) supplemented with 10% FBS (Life Technologies), 1% Pen-Strep (Omega Scientific, CA), 1 % MEM non-essential amino acids solution (Life Technologies), and 1% sodium pyruvate (Life Technologies).
[0164] In vitro DC generation and gese transduction. Bone marrow cells of 8-week-o!d female Balb/c mice were induced to differentiate into DCs as described previously by Koya et al. (45). The differentiated DCs (2.5 10° cells/ml) were cultured in RPMI supplemented with 10% FBS, 1% Pen-Strep, 50 ng/m! triGM-CSF, and 50 ng/ml mIL-4. Half were infected with Ad-GM'CAIX (15) (Fig. 8) and half with an empty adenoviral capsid identical to Ad- GM-CAIX's (Ad-Null; Welgen, MA), each at a multiplicity of infection of 200. After an overnight incubation, the infected DCs were washed with and re-suspended in PBS Ix (I .33xl07 cells/ml). Using intracellular and extracellular FACS analysis, the expression levels ofCdl lc (not shown), Cd86, hCAIX, and hGM-CSF were used to test for DC differentiation and GM-CAIX transduction (Figs. 9 and 10).
[0165] Animals. Eight-week-old Balb/cAnNCr female mice were obtained from the Radiation Oncology Defined Flora Animal Research Facility, UCLA. All animals had free access to water and food. All procedures were approved by the UCLA Animal Research Committee and followed the UCLA institutional Animal Care and Use Committee guidelines according to the American Association for Laboratory Animal Science.
[0166] RCC in vivo models. The prevention model had 6 groups of 8 immunocompetent Balb/c mice each. Groups A-D were immunized by subcutaneous transplantation of 2x106 DCs in 150 ul of PBS I into the right flank. Two groups (A, B) were transplanted with DC- Ad-GM-CAIX and two groups (C, D) with DC-Ad-Null. Groups E and F had no treatment. The DC transplantation was repeated after 6 days. Twelve days (day 0) after the first DC transplantation, 5xI05 CC cells in 100 μί of PBS lx were subcutaneous!}' transplanted into the left flank. Three groups (A. C, E) were transplanted with NPR-IX, and three groups (B, D, F) with RENCA cells. The treatment model had three groups (G, H, 1) of 16
immunocompetent Balb/c mice each, which were subcutaneously transplanted in the left flank with 5x10 ' NPR-IX. On days 8 and 14, group G was subcutaneously transplanted in the right flank with 2x1 if DC-Ad-GM-CAIX and group H with 2xl06 DC- Ad-Null, while group 1 had no treatment. Mouse performance status was assessed daily, body weight and tumors measured twice a week. Tumor volume "V'was calculated according to the formula
V=ab2:*:Pi/6, where "a" is the longest diameter of the tumor and 'b" is the longest diameter perpendicular to "a".
[01671 Western blot. Rabbit a-hCATX (Pierce, IL), mouse a-hGM-CSF (Pierce, IL), rabbit a-actin cell signaling, goat a-rabbit IgG-HRP (Santa Cruz, CA), and goat ct-mouse IgG-BRP (Santa Cruz, CA) antibodies were used. Chem (luminescence was elicited using the
Arners am ECL-Plus (GE Healthcare, J) western blotting detection system.
[01683 Immuaohistochemistry. In three mice of each group of the prevention model, pathology-relevant organs, blood, and tumors were collected. Paraffin-embedded sections were cut at 4 μ.πτ thickness and rehydrated through graded ethanol, Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 min. Heat-induced antigen retrieval and proteolytic induced epitope retrieval were used. The slides were then stained with hCAIX (rabbit, 1 : 1000 dilution, Novus Biological, CO), rnCAiX (goat, 1 :200, R&D System, MN), Kt67 (rat, 1 : 100, DAKO, CA), Pecaml (Cd31) (goat, 1 : 100, Santa Cruz, CA), Cdl l b (rat, 1 :50, AbDSerotec, NC), and F4/80 (rat, 1 :50, AbDSerotec), and the sections were incubated with secondary rabbit and rat (Cdl l b, F4/S0, K167) or rabbit anti-goat (Cd31, mCAIX) immunoglobulin for 30 min at 1 :200 dilution (DakoCytomation). The signal was detected using the Dakocvtoniation Envision System Labelled Polymer HRP anti rabbit (DakoCytomation) for all. For Tunei assays, an ApoptagPlus Peroxidase In Situ Apoptosis kit (Miliipore, MA) was used. Staining was performed according to the manufacturer's instructions. All sections were visualized with the diaminobenzidine reaction and
counterstained with hematoxylin.
[0169] In vitro IFN-γ ELISA. To assess the in vitro immune efficacy of the therapy, mouse splenocytes (5x105) from groups A and E were separately co-incubated with RENCA and NPR-IX cells (2.5x10 ), respectively. After 48 hours, supernatants were analyzed for mouse lFN-γ by ELISA (eBiosctence, CA).
[0170] Flow cytometry. FITC-conjugated anti-hCAIX (U.S. Biologicals, MA) and PE- conjugated anti-mouse Cd86 (BD, MA) were used. Fixation and permeabilization were performed using BD Fix/Wash and BD Perm/Wash (BD, NJ). To detect anti-hCAIX antibodies, 1 : 10 diluted mouse sera from groups A and E and FITC-conjugated goat anti- mouse IgG (Abeam, MA) were used as primary and secondary antibodies, respectively.
[0171] Quantitative real-time reverse transcription-PCR. RNA was isolated using Trizoi Reagent (Invitrogen, CA). Total RNA (1 g) was reverse transcribed in a volume of 50 μΐ. Five μί of the resulting solution was then used for PGR according to the manufacturer's instructions (Applied Biosystems, Inc., Foster City, CA). Gene expression signatures were quantified relative to the expression level of ribosomal 18s. and for iniRNA relative to snoRNA 234 or snoRjNA202. All probes were purchased from Applied Biosystems or Integrated DNA Technologies (IDT, CA). Normalized data are presented as fold difference in log? gene expression. Primers are listed below in Table 3.
Table 3. List of qPCR primers.
Figure imgf000055_0001
Gene Species Vendor
18s Mouse Applied Biosystems (AB, CA)
snoRNA23 Mouse Applied Biosystems (AB, CA)
snoRNA202 Mouse Applied Biosystems (AB, CA)
[0172] Gene expression analysis. The gene expression was investigated by Agilent 60- mer oligonucleotide probe arrays (SurePrint G3 Mouse GE 8x60K, Agilent, CA), using the highest absolute value/score among multiple probe sets for the gene-based interpretation. Background corrected signal values determined by Agilent feature extraction software were normalized by median centering of the ratios to a reference, which is the median of all control arrays. The features called found by extraction software were considered as present or detected. Global expression profiles of samples were examined by multidimensional scaling of about 36,000 features that were found at least in 50% of the arrays. Euclidean distance was used as the dissimilarity measure. The features having presence calls in <80% of samples and a geometric mean signal <50 in both groups A and C were eliminated before further analyses. In those remaining, there were 36? mR A features differentially expressed by 1.5-foJd (two tailed p-value < 0.015 by T-test). Ingenuity pathway analyses were done on the datasets of differentially expressed genes (P<0.05). Publicly available literature from 1966 to 2012 was surveyed using CoreMine and PubMed.
[0173] miRNA expression analysis. Exiqon miRNA arrays (miRCURY L A™
icroRNA array, v6, Exiqon, Denmark) having more than 1,891 capture probes covering all human, mouse, rat, and viral miRNAs and 66 proprietary miRNA s spotted in quadruplicate were used for measuring genome miRNA expression, intensities were determined by GenePix Pro 6.0 software (Molecular Devices, Sunnyvale, CA) and were normalized by median centering of the ratios to a reference control array. Global miRNA expression profiles of samples were examined by multidimensional scaling of about 4,200 features that were flagged as found in >50% of the samples. The features found in <60% of samples in both groups A and C were eliminated from further analysis. There were 104 miRNAs
differentially expressed by 1.5-fold (two tailed p-value < 0.01 5 by T-test), and 11 mouse miRNA replicates appeared 50- 100% of the time at signal levels >200.
[0174] Statistical analysis of tismor volume, weights and survival data. ruskal-Wa!lis one-way AN OVA were used to examine mouse weight and tumor volume. Wilcoxon's rank- sum tests were used to compare differences in weight and tumor volume between pairs of the six groups. The rates of weight change and tumor growth were analyzed using a linear mixed effects model with a random intercept and a random slope (46), Seven primary pairwise comparisons and three primary pairwise comparisons were performed in the prevention and the treatment model, respectively, with Bonferroni's adjustment. Time to tumor development (prevention model: tumor volume >0 mm"'; treatment model: >600 mm3) and to 15% weight loss from baseline were compared using the Kaplan-Meier curves and the log-rank test (47). Missing values of weight and tumor volume up to day 24 were imputed using the Last Observation Carried Forward method. Berger and Boos' method was used to estimate the time intervals of treatment effect in weight loss and tumor volume of the groups A and G in their respective experiments (48). P-values <0.05 were considered statistically significant. S AS Version 9.2 (SAS institute inc., Gary, NC) was used.
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50. Srivatsan S, Peng SL. Cutting edge: Foxj l protects against autoimmunity and inhibits thymocyte egress. J Immunol 2005; 175: 7805-9. Example 2; In vivo testing of DC-Ad-GM«CAIX monotherapies in treating In?ig metastasis
METHODS
[0175] RCC in vivo metastasis model The renal orthotopic RCC metastasis model had 2 groups of 10 immunocompetent Balb/e mice each. Mice were orthotopicaJ!y transplanted in the left kidney with IxlO5 NPR-IX cells in 100 μ! of 90% High Concentration Matrigel (354262, Becton, Dickinson, CA) balanced with PBS Ix (Salumbides et al., Current Protocols in Pharmacology: Pre -Clinical Models of Renal Carcinoma and Their Utility in Drag Development, Vol. Unit 14.13 DOT. 2009). Tumor bearing animals were randomly assigned into the 2 groups receiving monotherapy of DC-Ad-GM- CAIX, or no therapy. On days 8 and 14 following tumor transplantation, group B was subcutaneously transplanted in the right flank with 2xl06 DC-Ad-GM-CAIX in 150 μΐ of PBS Ix and group A had no treatment. Mouse performance status was assessed daily, and body weight was measured twice a week over the first 2 weeks and then daily. At termination partial necropsy was performed in all mice and lung metastatic foci were counted and area size measured. The whole study was repeated for the second time using an identical protocol (rounds A and B).
[0176] Statistical methods. Metastasis areas were first added across all metastases within an animal to compute a total for a given animal (and round). Thus, animal is the unit of analysis. The resulting animal level value was then standardized by the corresponding total lung section area. If "Y" is the area for a given animal and round, the standardized value is computed as Standardized Y - Ys (Y/total lung area) x 10s. That, is, Ys is the value of Y standardized per 10s square urn of lung section area. An examinaton of normal quantile plots (not shown) showed that log(Ys), not Yf„ follows the normal distribution. The marginal means averaging round A and round B were compared across groups under this model. If "M" is the log scale mean, then exp(M)= is the corresponding original scale value. This original scale value is called the geometric mean and is also the model based original scale median. Values of p<0.05 were considered significant in all studies, and all p values were two-tailed.
[0177] Other methods. All other methods {e.g., cell culture, in vivo DC generation and gene transplantation, immunohistocheroistry, and flow cytometry), were conducted as described above for Example 1 and as previously reported (Birkhauser et al., J. Immunoiher. 36(2): 102-1 1 1 (2012)). RESULTS
[0178] DC-Ad-GM-CAIX monotherapy significantly reduced the amount and area size of lung NPR-IX metastatic foci in syngeneic Balb/c mice (group B; Figs. 11 and 12), In these groups, mean amounts of lung metastatic foci were 2.5-fold smaller (p< 0.05) and with a mean 3.4 smaller area size (p< 0,05) than the untreated mice (group A),
[0179] Immunohistologic analyses showed absent or minimal hCAIX expression in metastatic tumors that evaded DC-Ad-GM-CAIX immunotherapy (groups A and Gj, but strong expression in the control group A (Fig. 13), suggesting that DC-Ad-GM-CAIX immunotherapy is specifically targeting and killing hCAIX-expressing metastatic tumor cells. DISCUSSION
[0180] We found that only metastatic tumor cells overexpressing hCAIX protein (NPR-IX) were inhibited and only in the treatment group immunized with DC-Ad-GM-CAIX (group B). Moreover, metastatic tumor tissue that subsequently evaded the therapy appeared to done so only in tumors with little or no hCAIX protein expression. [0181 ] This data suggests that identification of the immunotherapy evasion biomarkers may be useful for the development of metastatic tumor evasion diagnostic markers and for the rational development of next generation immunotherapies to treat lung RCC metastatic tumors.
[0182] it is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. Ail publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Table 4. Differentially expressed genes in tumors that evaded DC-Ad-GM-CAIX inimsinotherapy versus DC-Ad-Null. (A) Geoes in the ontology category "Inflammatory Response." (B) Genes in the ontology category "Cellular Growth and/or Proliferation,"
Table 4A: Gene ontology category - Inflammatory Response
Figure imgf000062_0001
Aggregation of antigen presenting
cei!s 1.39E-02 CCL 1 ,CD209
Migration of Langerhans ceils ! .39E-02 CD207JCAM1
Binding of macrophages L40E-02 CD209,HMGB LICAM1
ANGPT2,CASP4,CCL i ,CTF 1 ,CXCL9,EL
N,
F7.HMGB 1 JCAM 1 ,IL 1 RL 1 ,NOD2 ,PDE4
Inflamm a ory response 1.4IE-02 B, SCGB1 A SOCS 1
ANG PT2,CCL 1 ,CXCL9,EL ,HMGB I ,N
Chernotaxis of myeloid cells I .60E--02 OD2 PDE4B
Inflammation of cells 1 .63E-02 HMGB 1 ,HMGB 1 L 1 K A M i ,IL ! RL 1
Acute inflammatory response 1.71 E-02 CTFl jHMGBl
Aggregation of myeloid ceils 3 .71E-02 CCLIJCAM I
Infiltration by monocytes 1.71E-02 ANGPT2,SOCS l
ANGPT2,CCL 3 ,CXCL9,ELN,HMGB I ,N
Chernotaxis of phagocytes 3 .86E-G2 OD2.PDE4B
Activation of peripheral blood
leukocytes 1.88E-02 F7,SOCSl
Ceil movement of bone marrow- derived macrophages 1.88E-02 HMGBl .lRGM
Quantity of neutrophils 1.92E-02 GJA 1.HMGB 3 JCAMl ,PDE4B
Experimentally induced airway
hyperresponsiveness of organism 2.01E-02 IL3 L3
Homing of endothelial progenitor ceils 2.01E-02 IGFBP3
Table 4B: Gene ontology category - Cellular Growth and/or Proliferation
Function annotation p-Value Molecules
ADAMTS l,AlCDA,ANGPT2,APLN,C8or f4,
CAMK2AiCD209,CDH16,CHRNA l ,CllT A,
CREG1 ,CTF1 ,CXCL9,DLK1 ,ELN,EMP2, ESM1 ,F7,FGL2,FOXJ 1 ,FYB,GJA 1 ,Gm46 17/Ptma,HMGB 1.HMGB 3 L 1 ,HPGDS,ICA Ml ,
IGFBP3 JL 1 RL 3 ,Γ ΗΒΒ JRGM, J "UN,LA MA4, LAMB l .LOX RPl (includes EG: 16971), MCC,Ms4a4b (includes others),NOD2,Prl2c2 (includes others), RCA 1 ,REG 1 A.RG Sl 6,
SCGB 1 A 1 ,SERPI E2,SOCS 1 ,SOCS2,
Proliferation of cells 7.39E-05 TGFBLTP53 Ϊ 3 1 ,ΤΧΙ,Ν A, W WOX.ZIC 1
CTF1 ,ELN5F7,GJA 1 JIMGBl JGFBP3 ,JU
Proliferation of smooth muscle cells 2.S0E-03 N, LRPl (includes EG : 16971 )
CTF 1 ,IG FBP3 , j (J ,SOC S 1 ,ΤΡΜ 3
Growth of fibroblasts 5.1 SE-03 (includes EG:22003)
CD209,CHRNA ! ,CTF1,FGL2,F0XJ1 ,FY B, HMGB 1 ,HPGDS,JCA ί ,
ILIRL IJRGM, LOX, Ms4a4b (includes
Proliferation of blood cells 8.32E-03 others),SCGB iA ) , SOCS 1 , TXLNA
Formation of osteoclast-!ike cells 9.76E-03 }CAM1 ,TM7SF4
Generation of gamma-delta T 1.01E-02 JUN lymphocytes
Growth of cerebellar external
germinal layer cells i .O l E-02 ZIC1
Inhibition of tumor-associated
macrophages 1 .01E-02 HMGB1
Proliferation of proepieardial ceils i .O l E-02 GJAi
Stimulation of chronic lymphocytic
leukemia cells 1.03E-02 T'XLNA
Stimulation of prostate cancer cell
lines i .01E-02 IGFBP3
Proliferation of Th2 cells 1. I 3E-02 ILIRL LSOCS !
CD209 ,CHRNA 1 ,F GL2 ,FOX J I ,F YB ,HM GB l ,ICAM1 ,IL1RL 1 ,IRGM, Ms4a4b
Proliferation of T lymphocytes 1.24E-02 {includes others),SCGB 3 A 1 ,SOCS 1
ANGPT2,CD209,CHRNA 1 ,FGD3 ,HMGB
Stimulation of cells 1.25E-02 1, HMGB 1 L 1 JGFBP3 XLNA
CD209,CHRNA1,FGL2(FOXJ 1 ,FYB,HM GB 1 ,HPGDS,ICAM 1 ,IL 1 RL 1 ,
IRGM,Ms4a4b (includes
Proliferation of lymphocytes L26E-02 others),SCGB lA 1 ,SOCS 1.TXLNA
Growth of endocrine cells 1 .71 E-02 C8orf4,SOCSl
Proliferation of Schwann cells 1.71E-02 LAMA4,SERPJNE2
Growth of thyroid cells 2.01E-02 C8orf4
Proliferation of adrenal glomerulosa
cells 2.01E-G2 ANGPT2
Proliferation of naive helper T cells 2.0 1 ί -02 KAMI
Stimulation of adrenal glomerulosa
cells 2.01.E-02 ANGPT2
Stimulation of peripheral blood
lymphocytes 2.01E-G2 CHRNA1
Figure imgf000065_0001
Figure imgf000065_0002
Figure imgf000066_0001
Figure imgf000067_0001
Figure imgf000068_0001
Figure imgf000069_0001
Figure imgf000070_0001
Figure imgf000071_0001
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
miRNA
ID Treated/ p-vaiue Harvester ProbeName GeneName (Mousse) Description GenelD Gene Name
Control (Human)
Ratio Vehicle
46292 1.881 0 001089679 mmu-mi -5097 hsa-miR-5097
46292 1.737 0.004646469 rnrnu-miR-5097 hsa-miR-5097
46292 2.219 0.001399158 mmu-miR-5097 hsa-miR-5097
Figure imgf000090_0001
Figure imgf000091_0001
Line RN At
LincRN Tumor / p-vaiue Harvester ProbeName Gene ame (Mouse) Description GenelD Gene Name A Namet Vehicle Tiimor vs (Human) (Human)
Ratio Vehicle
chrl:100 0.545 0.002804975 A .30. P010330I6 lincRNAxhrl : 100501706-100608933
.501706- forward strand
1006089
33 F
c!srl:449 0.145 0.002574824 A_30_P01027052 lincRNA:chrl:44915250-44944125 forward
15250- strand
4494412
5 F
ehrl:719 0.566 0.014244125 1 A_30_P01018133 lincRNAxhrl. 1939741-71947891 forward I
39741- strand
7)94789
1 F
chr!0:66 0.296 0.002465136 | A 30 P01021427 | lincRNA : chrlO : 66559716-66647841 1
Figure imgf000092_0001
LincRNAt
Linc N Tumor / p-vaiue Harvester ProbcNam* GeneNamc (Mouse) Description GeneiD Gene Name A Naiuet Vehicle Tumor vs (Human) (Human)
Ratio Vehicle
5 R
chi 15:58 0.551 0.01198452 A. O PO 1021952 !incRNAxhr 15:58327703-58329443
327703- reverse strand
5832944
3 R
chr!5:73 1.646 0.005150095 A..30..P01019822 I incRNA: ctar 15 : 73455424-73477275
455424- reverse strand
7347727
5 R
chrl 5:79 0.615 0.012290236 A. 30JP01020778 lincRN A: chr 15 : 79764481 -79809909
764481- fonvard strand
7980990
9 F
chrl5:90 0.6 1 0.007963963 A 30.P01025795 iincRNA:chrl5:90074814-90087S14
074814- re erse strand
900878 J
4 R
chrl6:32 0.545 0.00484516 A..30..PO 1021750 UncR A:chrl6:32388843-32395264
38SS43- reverse strand
3239526
4 R
chr!6:43 1.526 0.002345.58 A..30. P01025670 tiricRNA : chr 16 :43 23583 -A 3642715
623581 - forward strand
436427 ;
5 F
chrl6:50 0.573 0.005555517 Α_30„Ρ01025300 lincRNA:chrl6:50715231-50841124
715231- reverse strand
50841 12
4 R
chil 7: 12 1.535 0.005885534 A..30. PO 1017675 lincRNA-chr 7: 12965849- 12968519
965849- forward strand
12968 1
9 F
chr l7: 15 0.619 0.005032592 1 A.30..P0J020263 ]incR A:chrl7: 15100982-15115630
100982- reverse strand
151 J 563
0 R
chr 17: 15 0.590 0.002966594 j A 30 Ρ0 Ι 028 Π 6 HncRNAxhrl 7: 15100982- 151 15630
LiiicR At
Figure imgf000094_0001
LincRNAt
LincR Tumor / p-value Harvester ProbeName GeneName (Mouse) Description GeneiD Gene Name A Namet Vehicle Tsirnor vs (Human) (Human)
Ratio Vehicle
86 R
chr4:533 0.656 0.010850314 A 30.P01018969 lmcRNA:chr4:533i5503-53316050 forward
15503- strand
5331605
0 F
chr4:592 0.647 0.0M192S64 A_30_P0102170i IincRNA:cbr4:59264275-59287850 reverse
64275- strand
592S785
0 R
chr5:120 0.646 0.006315277 A .30JP01017756 iincRNA: chr5 : 120269342- i 20279542
269342- reverse strand
1202795
42 R
chr5:149 0.653 0.012420696 A .30JP01019442 lincR A:chr5:14943275-14944205 forward
43275- strand
1494420
5 F
chr5:l49 0.631 0.005543851 A_30_P0101809S IincRNA : chr5 : 1 9751979-149808240
751979- reverse strand
1498082
40 R
chr5:150 0.636 0.012377619 A.30. P01028171 lincRNAxhrS:! 5033568-15033987 forward
33568- strand
1503398
7
chr5:i51 0.650 0.01163016 I A..._30,_P01018533 IincRNA: chr5 : 15146475 -15170100 forward
46475- strand
1537010
0 F
chr5:151 0.629 0.008846335 A_30_P0I024761 IincRNA: chr5 : 15146475- 15170100 forward
46475- strand
1517010
0 F
chr5:15i 0.618 0.0034261 Ϊ Ϊ A_30_P01017834 | lincRNA:chr5:15146475-15170100 forward
46475- ί strand
1517010
0 F
chr6:909 0.626 l_0. i 2306336 1 T A 30 P01033052 [ [ lincRNA:c r6:90937606-9093S335 reverse
Figure imgf000096_0001
Figure imgf000097_0001

Claims

¥HAT IS CLAIMED IS: ί . A method of qualifying the immunoediting-escape status of a subject, the method comprising:
(a) measuring at least one biomarker in a sample from the subject, wherein the biomarker is selected from the group consisting of the biomarkers listed in Table 5 and their human orthoiogs; and
(b) correlating the measurement with the subject's immune response; thereby qualifying the immunoediting-escape status of the subject.
2. The method of claim 1, wherein the immunoediting-escape status indicates the subject's risk of cancer, risk of an immune disease, immune response, response to therapy, regeneration, tissue repair, acute organ failure, organ transplantation, the presence or absence of disease, stage of disease, or effectiveness of treatment for a disease.
3. The method of claim 2, wherein the immunoediting-escape status indicates the presence, absence, or stage of a cancer or the effectiveness of treatment for a cancer in the subject.
4. The method of claim 3, wherein the cancer is renal cell carcinoma.
5. The method of any of claims 1-4, further comprising:
(c) managing treatment of the subject based on the immunoediting-escape status.
6. The method of claim 5, wherein managing treatment comprises ordering a further diagnostic test, performing surgery, administering a therapy, or taking no further action.
7. The method of claim 6, wherein the therapy is immunotherapy, chemotherapy, cell-therapy dialysis, treatment of acute organ failure, organ transplantation, wound healing treatment, or ischemic tissue treatment.
8. The method of any of claims 1 -7, comprising measuring at least two biomarkers in the sample from the subject and con-elating the measurement of the biomarkers with the subject's immune response.
9. The method of any of claims 1-8, wherein the at least one biomarker is a protein translated from an rnRNA listed in Table 5 or its human ortholog.
10. The method of any of claims 1-8, wherein the at least one biomarker is an rnRNA.
1 1. The method of any of claims 1 -8, wherein the at least one biomarker is an miR A.
12. The method of any of claims 1-8, wherein the at least one biomarker is an alternative isoform rnRNA.
13. The method of any of claims 1-8, wherein the at least one biomarker is a LincR A.
14. The method of any of claims 1-8, wherein the at least one biomarker is a gene.
15. The method of any of claims 1-14. wherein the sample is from blood, serum, plasma, urine, saliva, feces, kidney, lung, liver, prostate, bladder, intestine, colon, pancreas, or a tumor tissue.
16. The method of claim 1 , wherein the correlating step is performed by a software classification system.
17. A method of diagnosing renal status in a subject the method comprising: determining the pattern or level of expression of at least one biomarker listed in Table 5 or a human ortholog thereof in a sample from the subject, wherein a differential expression pattern of the at least one biomarker in the subject, relative to the pattern or level of expression of the at least one biomarker in a sample from a control, is indicative of a renal status of cancer, immune-escape, or im une disease.
18. The method of claim 17, wherein the at least one biomarker is a protein translated irons an rnRNA listed in Table 5 or its human ortholog.
19. The method of claim 17, wherein the at least one biomarker is an mRNA.
20. The method of claim 17, wherein the at least one biornarker is an rniRNA,
21. The method of claim 17, wherein the at least one biornarker is a
Linc NA.
22. The method of claim 17, wherein the at least one biornarker is a gene.
23. The method of any of claims 17-22, wherein the sample is from blood, serum, plasma, urine, saliva, feces, kidney, lung, liver, prostate, bladder, intestine, colon, pancreas, or a tumor tissue.
24. A method of monitoring the treatment of a subject for a disease or disorder, the method comprising;
(a) determining a first expression profile for at least one biornarker selected from an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof in a first sample from the subject, wherein the first expression profile is determined prior to the administration of a therapeutic agent;
(b) administering a therapeutically effective amount of the therapeutic agent to the subject; and
(c) determining a second expression profile for at least one biornarker listed in Tabie 5 in a second sample from the subject, wherein the second expression profile is determined subsequent to the administration of the therapeutic agent; wherein modulation of the expression profile indicates efficacy of treatment with the therapeutic agent.
25. The method of claim 24, wherein the disease or disorder is a hypoxic tumor or a carcinoma of kidney, lung, liver, spleen, pancreas, intestine, colon, mammary gland, stomach, prostate, bladder, placenta, uterus, ovary, endometrium, testicle, lymph node, skin, head, neck, or esophagus.
26. The method of claim 24, wherein the disease or disorder is an immune system disease or disorder.
27. The method of claim 24, wherein the disease or disorder is ischemia.
28. The method of any of claims 24-27, wherein an expression level of the at least one biomarker in the first expression profile that is above a threshold level indicates the presence of the disease or disorder.
29. The method of any of claims 24-27, wherein an expression level of the at least one bioinarker in the second expression profile that is below a threshold level indicates efficacy of treatment.
30. The method of any of claims 24-29, wherein the determining of an expression profile comprises:
(i) providing a nucleic acid sample from the subject; and
(ii) capturing the at least one biomarker that is present in the nucleic acid sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at least one biomarker.
31. The method of claim 30, wherein the nucleic acid sample is labeled.
32. The method of any of claims 24-29, wherein the determining of an expression profile comprises:
(i) providing a protein sample from the subject; and
(ii) capturing the at least one biomarker that is present in the protein sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at ieast one biomarker.
33. The method of any of claims 24-29, wherein the determining of an expression profile comprises:
(i) providing a tissue sample from the subject; and
(ii) capturing the at ieast one biomarker that is present in the tissue sample on a surface of a substrate, wherein the surface of the substrate comprises a capture reagent that binds the at least one biomarker.
34. The method of any of claims 30-33, wherein the substrate is a niicrotiier plate comprising one or more biospeeiiic affinity reagents that bind to the biomarker, and wherein the biomarker is detected using a fluorescent label.
35. The method of any of claims 24-29, wherein the expression profile is determined using a nucleic acid array.
36. The method of any of claims 24-35, wherein the determining of an expression profile comprises detecting the presence or absence of the at least one biomarker, quantifying the amount of the at least one biomarker, and qualifying the type of the at least one biomarker.
37. The method of any of claims 1 -36, wherein the at least one biomarker is measured using a biochip array.
.
38. The method of claim 37, wherein the biochip array is an antibody chip array, tissue chip array, protein chip array, nucleic acid array, or a peptide chip array.
39. The method of claim 38, wherein one or more capture reagents that bind the at least one biomarker are immobilized on the biochip array.
40. The method of any of claims 1, 17, or 24, wherein the at least one biomarker is a protein translated from an mRNA listed in Table 5 and wherein the protein biomarker is measured by immunoassay.
41. A method for identifying a candidate agent to treat a disease or disorder in a subject, the method comprising:
(a) contacting a cell with the candidate agent; and
(b) detecting the expression profile of one or more target biomarkers in the cell, wherein the one or more target biomarkers is an mRNA, miRNA, or LincRNA listed in Table 5, a protein translated from an mRNA listed in Table 5, or a human ortholog thereof, and wherein differential expression of the one or more target biomarkers in the expression profile as compared to a control identifies the candidate agent as useful for treating the disease or disorder in the subject.
42. The method of claim 41, wherein the disease or disorder is a hypoxic tumor, renal carcinoma, skin cancer, bladder cancer, prostate cancer, or breast cancer.
43. The method of claim 41, wherein the disease or disorder is an immune system disease or disorder.
44. The method of claim 41, wherein the disease or disorder is ischemia.
45. The method of any of claims 1 -44, wherein the candidate agent is a small molecule, a peptide, an immunotherapeutic, a vaccine, or a nucleic acid.
46. The method of any of claims 41-45, wherein the candidate agent is identified in silica using pharmacogenetics.
47. A method of determining whether or not a cancer is likely to be responsive to immunotherapy, the method comprising: detecting in a biological sample from a subject having the cancer an altered level of at least one biomarker listed in Table 5 or a human ortholog thereof.
48. The method of claim 47, wherein the method comprises detecting an elevated level of at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample.
49. The method of claim 47, wherein the method comprises detecting a decreased level of at least one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a control sample.
50. The method of any of claims 47-49, wherein the level of the at least one biomarker in the sample from the subject having the cancer is altered by at least 1.2-fold as compared to the level of the at least one biomarker in a control sample.
51. The method of any of claims 47-50, wherein the biomarker is an mRNA.
52. The method of claim 51 , wherein the biomarker is selected from Cell,
Cxcl9, Hmgbl , Cd209a, Fg32, Klra2, and Foxj l .
53. The method of any of claims 47-50, wherein the biomarker is an miRNA.
54. The method of claim 53, wherein the biomarker is selected from rniR-
1 186, miR-98, miR-5097, miR- 1942, and miR-708.
55. The method of any of claims 47-50, wherein the biomarker is a iincRNA.
56. The method of any of claims 47-55, wherein the biological sample is a tumor tissue sample,
57. The method of any of claims 47-56, wherein the cancel' is metastatic cancer.
58, The method of any of claims 47 -56, wherein the cancer is renal cell carcinoma.
59, The method of claim 47, wherein the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA)-cytokine fusion protein.
60, The method of claim 59, wherein the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
61. A method of diagnosing whether or not an individual has a cancer that is likely to be responsive to immunotherapy, the method comprising: detecting in a biological sample frorn the individual an altered level of at least one biomarker listed in Table 5 or a human ortholog thereof.
62. The method of claim 61 , wherein the method comprises detecting an elevated level of the at lea st one biomarker listed in Table 5 in the sample from the subject having the cancer as compared to the level of the at least one biomarker in a con trol sample,
63. The method of claim 62, wherein an elevated level of the biomarker in the sample from the individual indicates that the individual has a cancer that is not likely to be responsive to the immunotherapy.
64. The method of claim 61, wherein the method comprises detecting a decreased level of the a t least one biomarker listed in Table 5 in the sample frorn the subject having the cancer as compared to the level of the at least one biomarker in a control sample,
65. The method of any of claims 61-64, wherein the level of the at least one biomarker in the sample from the individual is altered by at least 1.2-fold as compared to the level of the biomarker in a control sample.
66, The method of any of claims 61-65, wherein the biomarker is an mRNA.
67. The method of claim 66, wherein the biomarker is selected from Cel l
Cxcl9, HmgbL Cd209a, Fgl2, Klra2, and Foxj l .
68, The method of any of claims 61-65, wherein the biomarker is an miRNA.
69. The method of claim 68, wherein the biomarker is selected from miR-
1 186, miR-98, miR-5097, miR-1942, and miR-708.
70. The method of any of claims 61-65, wherein the biomarker is a lincRNA.
71. The method of any of claims 61 -70, wherein the biological sample is a tumor tissue sample
The method of any of claims 61-71, wherein the cancer is a metastatic cancer.
73. The method of any of claims 61-71 , wherein the cancer is renal cell carcinoma.
74. The method of any of claims 61 -73, wherein the altered level of the biomarker indicates that the cancer is likely to be responsive to the immunotherapy, further comprising administering the immunotherapy to the individual.
75. The method of claim 61 , wherein the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA) - cytokine fusion protein.
76. The method of claim 75, wherein the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
77. A method of monitoring the efficacy of treatment for an individual having a cancer, the method comprising:
(a) detecting in a biological sample from the individual a level of at least one biomarker listed in Table 5 or a human ortho!og thereof; and
(b) determining whether or not the level of the at least one biomarker in the sample is altered compared to a control; thereby determining whether the cancer is responsive to the treatment.
78. The method of claim 77, wherein the biomarker is an mRNA selected from Cell, Cxcl9, HmgbL Cd209a, Fgl2, Klra2, and Foxj l .
79. The method of claim 77, wherein the biomarker is an m iRNA selected from miR-1 186, miR-98s miR-5097, miR-1942, and miR-708.
SO. The method of any of claims 77-79, wherein the cancer is a metastatic cancer.
81. The method of any of claim s 77-79, wherein the cancer is renal cell carcinoma.
82. The method of any of claims 77-81, wherein the treatment is an immunotherapy.
83. The method of claim 82, wherein the immunotherapy comprises immune stimulation with a tumor-associated antigen (TAA)-cytokine fusion protein.
84. The method of claim 83, wherein the immunotherapy comprises immune stimulation with CAIX-GM-CSF.
85. The method of any of claims 77-84, wherein an elevated level of the at least one biomarker relative to a control indicates that the cancer is not responsive to the treatment.
86. The method of any of claims 77-84, wherein the altered level of the at least one biomarker indicates that the cancer is not responsive to the treatment, the method further comprising administering to the indi vidual an alternative treatment.
87. A kit comprising:
at least one capture reagent that binds a biomarker selected from the group consisting of an mRNA, miRNA, or LincRNA listed in Table 5. a protein translated from an mRNA listed in Table 5, and human orthologs thereof.
88. The kit of claim 87, comprising a plurality of capture reagents, wherein each capture reagent binds a biomarker selected from the group consisting of the biomarkers listed in Table 5 and their human orthologs.
89. The kit of claim 87 or 88, wherein the capture reagent is an antibody that binds to a protein translated from an mRNA listed in Table 5.
90. The kit of claim 87 or 88, wherein the capture reagent is a nucleic acid that is substantially complementary to an mRNA, miR A, or LincRNA !isied in Table 5.
91 . The kit of any of claims 87-90, further comprising a wash solution that selectively retains the bound biomarker to the capture reagent after washing.
92. The kit of any of claims 87-91, further comprising instructions for using the capture reagent to detect the biomarker.
93. The kit of claim 92, wherein the instructions provide for contacting a test sample with the capture reagent and detecting the biomarker that is present in the sample, if any, that is retained by the capture reagent.
94. The kit of any of claims 87-93, further comprising instructions for using the kit for the detection of a disease or disorder.
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