WO2019089740A1 - Biomarqueurs de réponse clinique et de bienfait d'une thérapie par inhibiteur de point de contrôle immunitaire - Google Patents

Biomarqueurs de réponse clinique et de bienfait d'une thérapie par inhibiteur de point de contrôle immunitaire Download PDF

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WO2019089740A1
WO2019089740A1 PCT/US2018/058430 US2018058430W WO2019089740A1 WO 2019089740 A1 WO2019089740 A1 WO 2019089740A1 US 2018058430 W US2018058430 W US 2018058430W WO 2019089740 A1 WO2019089740 A1 WO 2019089740A1
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cancer
subject
immune checkpoint
therapy
sample
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PCT/US2018/058430
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English (en)
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Marios GIANNAKIS
Toni K. CHOUEIRI
Haoxin LI
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Dana-Farber Cancer Institute, Inc.
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Publication of WO2019089740A1 publication Critical patent/WO2019089740A1/fr

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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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152

Definitions

  • immune checkpoint therapies can yield durable responses and long-lasting survival benefit across some cancer types (Topalian et al. (2015) Cancer Cell 27:450-461). Indeed, checkpoint therapies have been approved for use in metastatic melanoma, non-small cell lung cancer, bladder cancer, and renal cell carcinoma, including as a first-line therapy for non-small cell lung cancer. However, many subjects among a population of subjects having the same cancer type do not exhibit a therapeutic benefit or relapse despite being treated with the same immune checkpoint therapy.
  • PD1 programmed cell death protein 1
  • RRC renal cell cancer
  • NSCLC non-small-cell lung cancer
  • the present invention is based, at least in part, on the discovery that certain serum metabolites can be specific biomarkers (e.g., kynurenine and/or adenosine, as well as precursors and metabolites thereof) for predicted clinical outcome in a wide variety of cancers afflicting patients who have received anti -immune checkpoint-based therapy (e.g., PD-1 inhibitors).
  • the present invention relates, in part, to methods for stratifying patients and predicting response of a cancer in a subject to immune checkpoint therapy based upon a determination and analysis of amounts of such biomarkers, compared to a control.
  • such analyses can be used in order to provide useful anti -immune checkpoint treatment regimens (e.g. , based on predictions of clinical response, subject survival or relapse, timing of adjuvant or neoadjuvant treatment, etc.).
  • a method of identifying the likelihood of a cancer in a subject to be responsive to an immune checkpoint therapy comprising a) obtaining or providing a subject sample from a subject having cancer; b) measuring the amount of at least one biomarker listed in Table 1 in the subject sample; and c) comparing said amount of the at least one biomarker listed in Table 1 in a control, wherein the absence of or a significantly decreased amount of the at least one biomarker listed in Table 1 in the subject sample, relative to the control, identifies the cancer as being more likely to be responsive to the immune checkpoint therapy; and wherein the presence of or a
  • the method further comprises recommending, prescribing, or administering the immune checkpoint therapy if the cancer is determined likely to be responsive to the immune checkpoint therapy or administering an anti-cancer therapy other than the immune checkpoint therapy if the cancer is determined be less likely to be responsive to the immune checkpoint therapy.
  • the anti-cancer therapy is selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and/or hormonal therapy.
  • the control is determined from a cancerous or non-cancerous sample from either the subject or a member of the same species to which the subject belongs.
  • control is a cancerous or non-cancerous sample from the subject obtained from an earlier point in time than the subject sample, optionally wherein the control sample is obtained before the subject has received immune checkpoint therapy and the subject sample is obtained after the subject has received immune checkpoint therapy.
  • a method of assessing the efficacy of an agent for treating a cancer in a subject comprising a) detecting the amount of at least one biomarker listed in Table 1 in a first subject sample from a subject having the cancer and not being treated with the agent; b) detecting the amount of the at least one biomarker listed in Table 1 in a subsequent subject sample from the subject after the subject has been treated with the agent; and
  • step c) comparing the amount of the at least one biomarker listed in Table 1 from steps a) and b), wherein the absence of or a significantly decreased amount of the at least one biomarker listed in Table 1 detected in step b), relative to the amount detected in step a), indicates that the agent treats the cancer in the subject.
  • a method of assessing the efficacy of an agent for treating a cancer in a subject or prognosing progression of a cancer in a subject comprising a) detecting in a first subject sample at a first point in time the amount of at least one biomarker listed in Table 1 ; b) repeating step a) during at least one subsequent point in time after administration of the agent; and c) comparing the amount detected in steps a) and b), wherein the absence of or a significantly decreased amount of the at least one biomarker listed in Table 1 detected in the at least one subsequent subject sample, relative to the first subject sample, indicates that the cancer is unlikely to progress or that the agent treats the cancer in the subj ect.
  • the subject between the first point in time and the subsequent point in time, has undergone treatment, completed treatment, and/or is in remission for the cancer, optionally wherein the time interval between the first point and the subsequent point in time is 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, or 9 weeks.
  • the subject is unlikely to be responsive to an immune checkpoint therapy.
  • the subject is treated with an immune checkpoint therapy before obtaining the first subject sample and/or between the first point in time and the subsequent point in time.
  • a cell-based assay for screening for agents that have a cytotoxic or cytostatic effect on a cancer cell comprising, contacting the cancer cell with a test agent, and determining the ability of the test agent to decrease the amount of at least one biomarker listed in Table 1 in the subject sample.
  • the step of contacting occurs in vivo, ex vivo, or in vitro.
  • the subject sample and/or the control is selected from the group consisting of ex vivo and in vivo samples.
  • the subject sample and/or the control is a portion of a single sample or pooled samples obtained from the subject.
  • the subject sample and/or the control has not been contacted with any anti-cancer treatment or inhibitor of an immune checkpoint.
  • the subject has not been administered any anti-cancer treatment or inhibitor of an immune checkpoint.
  • the method or assay further comprises recommending, prescribing, or administering at least one additional anti-cancer therapeutic agent, optionally wherein the at least one additional anti-cancer therapeutic agent comprises an immune checkpoint therapy.
  • the subject sample is selected from the group consisting of serum, whole blood, plasma, urine, cells, cell lines, and biopsies.
  • the amount of the at least one biomarker listed in Table 1 is detected using a reagent which specifically binds with the biomarker.
  • the reagent has a label group attached thereto, optionally wherein the label group comprising: a radioisotope, a fluorescent compound, an enzyme, or an enzyme co- factor.
  • the at least one biomarker listed in Table 1 is assessed by liquid chromatography tandem mass spectrometry (LC-MS), HPLC, and/or mass spectrometry.
  • the step of detecting further comprises purifying and/or concentrating the at least one biomarker listed in Table 1.
  • the at least one biomarker listed in Table 1 is adenosine or an adenosine metabolite.
  • the immune checkpoint therapy comprises an inhibitor of at least one selected from the group consisting of PD-1, PD-L1, PD-L2, TIM-3, LAG-3, CTLA-4, and combinations thereof.
  • the inhibitor comprisies at least one antibody selected from the group consisting of anti-PD-1 antibodies, anti-CTLA4 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, and combinations thereof.
  • the immune checkpoint therapy comprises an anti- PD-1 antibody.
  • the immune checkpoint therapy comprises an agent that modulates amounts or activity of adenosine or an adenosine metabolite.
  • the agent is an adenosine receptor antagonist.
  • the likelihood of the cancer in the subject to be responsive to immune checkpoint therapy or the efficacy of the agent to treat the cancer is measured by at least one criteria selected from the group consisting of cellular proliferation, tumor burden, m- stage, metastasis, progressive disease, clinical benefit rate, survival until mortality, pathological complete response, semi -quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria.
  • the cancer is selected from the group consisting of melanoma, bladder cancer, and renal cell cancer (RCC).
  • the subject is a mammal.
  • the mammal is an animal model of cancer.
  • the mammal is a human or a rodent.
  • FIG. 1A - FIG. IE show lineage-associated serum metabolic landscape and metabolite changes in response to PD1 blockade.
  • FIG. 1A shows a schematic of cohort design and specimen collection. Serum samples from 87 melanoma patients and 92 RCC patients were profiled for metabolomic features. Thirty-four of the RCC patients' tumors specimens sampled prior to treatment also underwent whole-transcriptome (RNASeq) analysis.
  • FIG. IB shows that hierarchical clustering based on 80 measured serum polar metabolite levels at baseline displayed cancer-type associated patient classifications.
  • CV co-efficient of variance
  • FIG. 3A - FIG. 3E show that changes in serum kynurenine levels correlate with response to nivolumab in melanoma.
  • FIG. 3C shows that kynurenine level differences between week 6 and baseline according to nivolumab response (mean ⁇ SD), p values were calculated as in (FIG. 3B).
  • FIG. 3A and FIG. 3E show that changes in serum kynurenine levels correlate with response to nivolumab in melanoma.
  • FIG. 3A and FIG. 3B depict volcano plots showing the change in metabolite levels between week 4 (
  • FIG. 3D shows PFS for melanoma patients with outlier kynurenine level changes between week 6 and baseline (bottom 5% changes and top 5% changes).
  • FIG. 4A - FIG.4G show that serum adenosine levels in RCC patients correlate with response to nivolumab.
  • FIG. 4C and FIG. 4D show that RCC patients who failed to respond to nivolumab had significantly higher adenosine levels at baseline (FIG.
  • FIG. 4C shows that high serum adenosine levels measured at baseline (FIG. 4E), at week 4 (FIG. 4F), or at baseline/week 4 average (FIG. 4G) are associated with lack of response to nivolumab (p values were calculated using Fisher' s exact test). The number of patients in each class is labeled.
  • FIG. 5 shows that serum adenosine levels at baseline correlate with adenosine levels measured at week 4 in RCC patients, p, Pearson correlation.
  • FIG. 6A - FIG. 6E show that higher serum adenosine levels at baseline are significantly associated with worse PFS in RCC patients treated with nivolumab.
  • the p value for the binary adenosine feature based survival analysis was calculated by log-rank test and the p value for the continuous adenosine feature was calculated by likelihood ratio test in a Cox proportional hazards regression model.
  • FIG. 6C shows that adenosine levels measured at week 4 were not significantly associated with PFS in RCC patients treated with nivolumab.
  • the p value for the binary adenosine feature based survival analysis was calculated by log-rank test and the p value for the continuous adenosine feature was calculated by likelihood ratio test in a Cox proportional hazards regression model.
  • FIG. 6D and FIG. 6E show that adenosine levels measured at baseline (FIG. 6D) or week 4 (FIG. 6E) did not have significant associations with overall survival (OS) in RCC patients treated with nivolumab. NR, not reached.
  • the p value for the binary adenosine feature based survival analysis was calculated by log-rank test and the p value for the continuous adenosine feature was calculated by likelihood ratio test in a Cox proportional hazards regression model.
  • FIG. 7A - FIG. 7C which show the identification and characterization of high adenosine-associated gene expression signatures in RCC.
  • FIG. 7A shows pathways that are suppressed in the tumors of patients with higher serum adenosine levels at baseline.
  • the GSEA analysis results based on hallmark and gene ontology gene sets are ordered by normalized enrichment scores (NES). Top 20 negatively correlated pathways are shown.
  • certain serum metabolites can be specific biomarkers (e.g., kynurenine and/or adenosine, as well as precursors and metabolites thereof) for predicted clinical outcome in a wide variety of cancers afflicting patients who have received anti-immune checkpoint-based therapy (e.g. , PD-1 inhibitors).
  • the present invention relates, in part, to methods for stratifying patients and predicting response of a cancer in a subject to immune checkpoint therapy based upon a determination and analysis of amounts of such biomarkers, compared to a control.
  • analyses can be used in order to provide useful anti -immune checkpoint treatment regimens (e.g., based on predictions of clinical response, subject survival or relapse, timing of adjuvant or neoadjuvant treatment, etc.).
  • an element means one element or more than one element.
  • altered amount refers to increased or decreased level or concentration (measurable in either absolute or relatively modulated ways) of a biomarker compound, e.g., increased or decreased level or concentration in a cancer sample, as compared to the level or concentration of the biomarker compound in a control sample.
  • altered amount also includes an increased or decreased level or concentration of a biomarker compound in a sample, e.g., a cancer sample, as compared to the corresponding level or concentration in a normal, control sample.
  • the altered amount or level is greater or less than the standard error of the assay employed to assess the level or concentration, and is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more times the level or concentration of the biomarker in a control sample (e.g., sample from a healthy subjects not having the associated disease) and preferably, the average level or concentration of the biomarker in several control samples.
  • a control sample e.g., sample from a healthy subjects not having the associated disease
  • the amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
  • such "significance" can be assessed from any desired or known point of comparison, such as a particular post-treatment versus pre- treatment biomarker measurement measurement, comparison to a control measurement, comparison to a normal measurement, comparison to a group of samples or subjects, and the like.
  • a "significantly" higher or lower amount can be measured as a ratio, such as 1-, 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-. 1.8-, 1.9-, 2.0-, 2.1-, 2.2-, 2.3-,
  • the fold-change can be linear or logarithmic, such as a fold change measured as log2 or loglO units.
  • the measurement is an absolute amount (e.g., 100 nM, 200 nM, 300 nM, 500 nM, 1000 nM, and the like).
  • the measurement can be made over different points of time and/or interventions, such as at baseline, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, and the like.
  • Such "significance" can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.
  • antibody broadly encompass naturally-occurring forms of antibodies (e.g. IgG, IgA, IgM, IgE) and recombinant antibodies such as single-chain antibodies, chimeric and humanized antibodies and multi-specific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives have at least an antigenic binding site.
  • Antibody derivatives may comprise a protein or chemical moiety conjugated to an antibody.
  • antibody as used herein also includes an "antigen-binding portion" of an antibody (or simply “antibody portion”).
  • antigen-binding portion refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody.
  • binding fragments encompassed within the term "antigen- binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al.
  • VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent polypeptides (known as single chain Fv (scFv); see e.g. , Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; and Osbourn et al. 1998, Nature
  • Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
  • Any VH and VL sequences of specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG polypeptides or other isotypes.
  • VH and VL can also be used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g. , Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J , et al. (1994) Structure 2: 1121-1123).
  • an antibody or antigen-binding portion thereof may be part of larger immunoadhesion polypeptides, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion polypeptides include use of the streptavidin core region to make a tetrameric scFv polypeptide (Kipriyanov, S.M., et al.
  • Antibody portions such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion, respectively, of whole antibodies.
  • antibodies, antibody portions and immunoadhesion polypeptides can be obtained using standard recombinant DNA techniques, as described herein.
  • Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof ⁇ e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the present invention bind specifically or substantially specifically to a biomarker polypeptide or fragment thereof.
  • monoclonal antibodies and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen
  • polyclonal antibodies and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen.
  • a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
  • Antibodies may also be "humanized”, which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the non-human antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
  • the humanized antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences ⁇ e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
  • the term "humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • the term "assigned score” refers to the numerical value designated for each of the biomarkers after being measured in a patient sample.
  • the assigned score correlates to the absence, presence or inferred amount of the biomarker in the sample.
  • the assigned score can be generated manually ⁇ e.g., by visual inspection) or with the aid of instrumentation for image acquisition and analysis.
  • the assigned score is determined by a qualitative assessment, for example, detection of a fluorescent readout on a graded scale, or quantitative assessment.
  • an "aggregate score” which refers to the combination of assigned scores from a plurality of measured biomarkers, is determined.
  • the aggregate score is a summation of assigned scores.
  • combination of assigned scores involves performing mathematical operations on the assigned scores before combining them into an aggregate score.
  • the aggregate score is also referred to herein as the "predictive score.”
  • biomarker refers to a measurable entity of the present invention that has been determined to be predictive of immune checkpoint therapy effects on a cancer.
  • Biomarkers can include, without limitation, metabolite compounds, including those shown in Table 1 below, the Examples, and the Figures. Examples of biomarkers include kynurenine and adenosine, as well as precursors and metabolites thereof.
  • AMP adenosine monophosphate
  • ADP adenosine diphosphate
  • ATP adenosine triphosphate
  • PIC picolinic acid
  • ACMS 2-amino-3-carboxymuconate-semialdehyde
  • blocking antibody or an antibody “antagonist” is one which inhibits or reduces at least one biological activity of the antigen(s) it binds.
  • the blocking antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen(s).
  • body fluid refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle,
  • cancer or “tumor” or “hyperproliferative” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. In some embodiments, such cells exhibit such characteristics in part or in full due to the expression and activity of immune checkpoint proteins, such as PD-1, PD-Ll, PD-L2, TEVI, LAG, and/or CTLA-4. Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non- tumorigenic cancer cell, such as a leukemia cell. As used herein, the term "cancer” includes premalignant as well as malignant cancers.
  • Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like.
  • the heavy chain diseases such as, for
  • human sarcomas and carcinomas e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,
  • craniopharyngioma ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease.
  • leukemias e.g., acute lymphocytic le
  • cancers are epithlelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma ⁇ e.g. , serous ovarian carcinoma), or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
  • the cancer or tumor is melanoma and/or renal cell cancer (RCC).
  • joint therapy and “combination therapy,” as used herein, refer to the administration of two or more therapeutic substances, e.g., combinations of anti-immune checkpoint therapies, multiple inhibitors of an immune checkpoint of interest, combinations of immune checkpoint therapy with an inhibitor of PD-1, PD-L1, PD-L2, TIM, LAG, CTLA-4, and the like), and combinations thereof.
  • the different agents comprising the combination therapy may be administered concomitant with, prior to, or following the administration of one or more therapeutic agents.
  • control refers to any reference standard suitable to provide a comparison to the expression products in the test sample.
  • the control comprises obtaining a "control sample” from which expression product levels are detected and compared to the expression product levels from the test sample.
  • a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository.
  • control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy).
  • a certain outcome for example, survival for one, two, three, four years, etc.
  • a certain treatment for example, standard of care cancer therapy
  • control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention.
  • control may comprise normal or non-cancerous cell/tissue sample.
  • control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome.
  • the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level.
  • control may comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer.
  • control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population.
  • control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard;
  • control comprises a control sample which is of the same lineage and/or type as the test sample.
  • control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer.
  • a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome.
  • a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome.
  • the methods of the present invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.
  • the "copy number" of a biomarker nucleic acid refers to the number of DNA sequences in a cell (e.g. , germline and/or somatic) encoding a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. The copy number can be increased, however, by gene amplification or duplication, or reduced by deletion. For example, germline copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in the normal complement of germline copies in a control (e.g., the normal copy number in germline DNA for the same species as that from which the specific germline DNA and corresponding copy number were determined).
  • Somatic copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in germline DNA of a control (e.g., copy number in germline DNA for the same subject as that from which the somatic DNA and corresponding copy number were determined).
  • the "normal" copy number (e.g., germline and/or somatic) of a biomarker nucleic acid or "normal” level of expression of a biomarker nucleic acid, protein, or metabolite is the activity /level of expression or copy number in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow, from a subject, e.g., a human, not afflicted with cancer, or from a corresponding non-cancerous tissue in the same subject who has cancer.
  • a biological sample e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow
  • costimulate with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non-activating receptor mediated signal (a "costimulatory signal") that induces proliferation or effector function.
  • a costimulatory signal can result in cytokine secretion, e.g., in a T cell that has received a T cell-receptor-mediated signal.
  • Immune cells that have received a cell-receptor mediated signal, e.g., via an activating receptor are referred to herein as "activated immune cells.”
  • determining a suitable treatment regimen for the subject is taken to mean the determination of a treatment regimen (i.e., a single therapy or a combination of different therapies that are used for the prevention and/or treatment of the cancer in the subject) for a subject that is started, modified and/or ended based or essentially based or at least partially based on the results of the analysis according to the present invention.
  • a treatment regimen i.e., a single therapy or a combination of different therapies that are used for the prevention and/or treatment of the cancer in the subject
  • Another example is starting an adjuvant therapy after surgery whose purpose is to decrease the risk of recurrence, another would be to modify the dosage of a particular chemotherapy.
  • the determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most cases, the actual determination of the suitable treatment regimen
  • diagnosing cancer includes the use of the methods, systems, and code of the present invention to determine the presence or absence of a cancer or subtype thereof in an individual.
  • the term also includes methods, systems, and code for assessing the level of disease activity in an individual.
  • a molecule is "fixed” or "affixed” to a substrate if it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
  • a fluid e.g. standard saline citrate, pH 7.4
  • expression signature refers to a group of two or more coordinately expressed biomarkers.
  • the genes, proteins, metabolites, and the like making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response.
  • the biomarkers can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer.
  • Expression data and gene expression levels can be stored on computer readable media, e.g., the computer readable medium used in
  • Such expression data can be manipulated to generate expression signatures.
  • “Homologous” as used herein refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue.
  • a region having the nucleotide sequence 5'- ATTGCC-3 1 and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
  • the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%>, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
  • Immune cell refers to cells that play a role in the immune response.
  • Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
  • Immune checkpoint refers to a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
  • Immune checkpoint proteins are well known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7- H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD 160, gp49B, PIR-B, KIR family receptors, TEVl-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO 2012/177624).
  • the term further encompasses biologically active protein fragment, as well as nucleic acids encoding full-length immune checkpoint proteins and biological
  • Immuno checkpoint therapy refers to the use of agents that inhibit immune checkpoint nucleic acids and/or proteins. Inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
  • agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof.
  • Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint proteins ⁇ e.g., a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint nucleic acid transcription or translation; and the like.
  • fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin
  • agents can directly block the interaction between the one or more immune checkpoints and its natural receptor(s) (e.g., antibodies) to prevent inhibitory signaling and upregulate an immune response.
  • agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response.
  • a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can binding to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
  • anti-PD-1 antibodies e.g., Opdivo ® (nivolumab) and Keytruda ® (pembrolizumab)
  • anti-PD-Ll antibodies e.g., Tecentriq ® (atezolizumab)
  • anti-PD-L2 antibodies e.g., Tecentriq ® (atezolizumab)
  • anti-CTLA-4 antibodies either alone or in combination, are used to inhibit immune checkpoints.
  • Immunolimumab is a representative example of an immune checkpoint therapy.
  • Ipilimumab (previously MDX-010; Medarex Inc., marketed by Bristol-Myers Squibb as YERVOYTM) is a fully human anti-human CTLA-4 monoclonal antibody that blocks the binding of CTLA-4 to CD80 and CD86 expressed on antigen presenting cells, thereby, blocking the negative down-regulation of the immune responses elicited by the interaction of these molecules (see, for example, WO 2013/169971, U. S. Pat. Publ. 2002/0086014, and U.S. Pat. Publ. 2003/0086930.
  • Nivolumab is another representative example of an immune checkpoint therapy.
  • Nivolumab (discovered by Medarex, developed by Medarex and Ono Pharmaceutical, and marketed by Bristol-Myers Squibb and Ono as Opdivo ® ) is a human IgG4 anti-PD-1 monoclonal antibody and works as a checkpoint inhibitor, blocking signals that would have prevented activated T cells from attacking the cancer, thus allowing the immune system to clear the cancer.
  • nivolumab for primary or metastatic urothelial carcinoma, the most common form of bladder cancer. It can be prescribed for locally advanced or metastatic form of the condition that experience disease progression during or following platinum-containing chemotherapy or have progression within 12 months of neoadjuvant or adjuvant treatment with platinum-containing chemotherapy.
  • immune response includes T cell mediated and/or B cell mediated immune responses.
  • exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity.
  • immune response includes immune responses that are indirectly effected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g., macrophages.
  • immunotherapeutic agent can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject.
  • Various immunotherapeutic agents are useful in the compositions and methods described herein.
  • cancer includes the decrease, limitation, or blockage, of, for example a particular action, function, or interaction.
  • cancer is "inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented.
  • cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
  • interaction when referring to an interaction between two molecules, refers to the physical contact (e.g., binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules.
  • isolated protein refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • isolated or purified protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of a biomarker polypeptide or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of a biomarker protein or fragment thereof, having less than about 30% (by dry weight) of non-biomarker protein (also referred to herein as a "contaminating protein”), more preferably less than about 20% of non-biomarker protein, still more preferably less than about 10%) of non-biomarker protein, and most preferably less than about 5% non- biomarker protein.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • kits is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe or small molecule, for specifically detecting and/or affecting the expression of a marker of the present invention.
  • the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
  • the kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention.
  • the kit may further comprise a reference standard, e.g., a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival or apoptosis.
  • control proteins including, but not limited to, common molecular tags (e.g. , green fluorescent protein and beta-galactosidase), proteins not classified in any of pathway encompassing cell growth, division, migration, survival or apoptosis by GeneOntology reference, or ubiquitous housekeeping proteins.
  • Reagents in the kit may be provided in individual containers or as mixtures of two or more reagents in a single container.
  • instructional materials which describe the use of the compositions within the kit can be included.
  • neoadjuvant therapy refers to a treatment given before the primary treatment.
  • neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.
  • chemotherapy for example, in treating breast cancer, neoadjuvant therapy can allows patients with large breast cancer to undergo breast-conserving surgery.
  • the "normal" level of expression of a biomarker is the level of expression of the biomarker in cells of a subject, e.g., a human patient, not afflicted with a cancer.
  • An "over- expression” or “significantly higher level or amount” of a biomarker refers to a level or amount in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more higher than the level or amount of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and preferably
  • a "significantly lower level or amount" of a biomarker refers to a level or amount in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20 times or more lower than the level or amount of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease or from a sample from a subject prior to or during therapy) and preferably, the average level or amount of the biomarker in several control samples.
  • a control sample e.g., sample from a healthy subject not having the biomarker associated disease or from a sample from a subject prior to or during therapy
  • pre-determined biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement s) used to, by way of example only, evaluate a subject that may be selected for a particular treatment, evaluate a response to a treatment such as an anti-immune checkpoint inhibitor therapy, and/or evaluate the disease state.
  • a pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without cancer.
  • the pre-determined biomarker amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre-determined biomarker amount and/or activity measurement(s) can vary according to specific subpopulations of patients.
  • Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual.
  • the pre-determined biomarker amount and/or activity can be determined for each subject individually.
  • the amounts determined and/or compared in a method described herein are based on absolute measurements.
  • the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios (e.g., serum biomarker normalized to the expression of a housekeeping or otherwise generally constant biomarker).
  • the pre-determined biomarker amount and/or activity measurement(s) can be any suitable standard.
  • the pre-determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed.
  • the pre-determined biomarker amount and/or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time.
  • the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human.
  • the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group.
  • predictive includes the use of a biomarker compound status, e.g., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to anti -immune checkpoint treatment (e.g., therapeutic antibodies against CTLA-4, PD-1, PD-L1, and the like).
  • a biomarker compound status e.g., over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to anti -immune checkpoint treatment (e.g., therapeutic antibodies against CTLA-4, PD-1, PD-L1, and the like).
  • anti -immune checkpoint treatment e.g., therapeutic antibodies against CTLA-4, PD-1, PD-L1, and the like.
  • Such predictive use of the biomarker may be confirmed by, e.
  • Biotechnol., 86:289-301, or qPCR overexpression or underexpression of a biomarker nucleic acid (e.g., by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g., by IHC), or increased or decreased activity, e.g., in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence in clinical subset of patients with
  • pre-malignant lesions refers to a lesion that, while not cancerous, has potential for becoming cancerous. It also includes the term “pre- malignant disorders” or “potentially malignant disorders.” In particular this refers to a benign, morphologically and/or histologically altered tissue that has a greater than normal risk of malignant transformation, and a disease or a patient's habit that does not necessarily alter the clinical appearance of local tissue but is associated with a greater than normal risk of precancerous lesion or cancer development in that tissue (leukoplakia, erythroplakia, erytroleukoplakia lichen planus (lichenoid reaction) and any lesion or an area which histological examination showed atypia of cells or dysplasia.
  • prevent refers to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
  • probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a biomarker compound. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
  • prognosis includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease.
  • use of statistical algorithms provides a prognosis of cancer in an individual.
  • the prognosis can be surgery, development of a clinical subtype of cancer (e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma), development of one or more clinical factors, development of intestinal cancer, or recovery from the disease.
  • a clinical subtype of cancer e.g., solid tumors, such as lung cancer, melanoma, and renal cell carcinoma
  • response to immune checkpoint therapy or “response to therapy” relates to any response of the hyperproliferative disorder (e.g., cancer) to a therapy, such as an immune checkpoint therapy like immune checkpoint therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • a therapy such as an immune checkpoint therapy like immune checkpoint therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • Hyperproliferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like "pathological complete response" (pCR), "clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
  • pCR pathological complete response
  • cCR clinical complete remission
  • cPR clinical partial remission
  • cSD clinical stable disease
  • cPD clinical progressive disease
  • Assessment of hyperproliferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months.
  • a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed. This is typically three months after initiation of neoadjuvant therapy.
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
  • CR complete remission
  • PR partial remission
  • SD stable disease
  • the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more. Additional criteria for evaluating the response to cancer therapies are related to "survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival" (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g. , death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy.
  • the outcome measurement may be pathologic response to therapy given in the neoadjuvant setting.
  • outcome measures such as overall survival and disease-free survival can be monitored over a period of time for subjects following cancer therapy for whom biomarker measurement values are known.
  • the doses administered are standard doses known in the art for cancer therapeutic agents.
  • the period of time for which subjects are monitored can vary. For example, subjects may be monitored for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or longer, such as 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, or 60 months.
  • Biomarker measurement threshold values that correlate to outcome of a cancer therapy can be determined using well-known methods in the art, such as those described in the Examples section.
  • resistance refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy (i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more.
  • the reduction in response can be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal who is known to have no resistance to the therapeutic treatment.
  • multidrug resistance A typical acquired resistance to chemotherapy is called "multidrug resistance.”
  • the multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi-drug-resistant microorganism or a combination of microorganisms.
  • the term "reverses resistance” means that the use of a second agent in combination with a primary cancer therapy (e.g., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p ⁇ 0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy (e.g., chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.
  • a primary cancer therapy e.g., chemotherapeutic or radiation therapy
  • response refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth.
  • the terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause.
  • To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (i.e. , will exhibit a lack of response or be non-responsive).
  • RNA interfering agent is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi).
  • RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the present invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi).
  • RNA interference is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn, G. and Cullen, B. (2002) J of Virology 76(18):9225), thereby inhibiting expression of the target biomarker nucleic acid.
  • mRNA messenger RNA
  • dsRNA double stranded RNA
  • RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs.
  • siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs.
  • RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target biomarker nucleic acids.
  • inhibiting target biomarker nucleic acid expression includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid.
  • the decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted by an RNA interfering agent.
  • sample used for detecting or determining the presence or level of at least one biomarker is typically whole blood, plasma, serum, saliva, urine, stool (e.g., feces), tears, and any other bodily fluid (e.g., as described above under the definition of "body fluids"), or a tissue sample (e.g., biopsy) such as a small intestine, colon sample, or surgical resection tissue.
  • the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample.
  • cancer means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy (e.g. , ti- immune checkpoint, chemotherapeutic, and/or radiation therapy).
  • a cancer therapy e.g. , ti- immune checkpoint, chemotherapeutic, and/or radiation therapy.
  • normal cells are not affected to an extent that causes the normal cells to be unduly injured by the immune checkpoint therapy.
  • An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res 1982; 42: 2159-2164), cell death assays (Weisenthal L M, Shoemaker R H, Marsden J A, Dill P L, Baker J A, Moran E M, Cancer Res 1984; 94: 161-173; Weisenthal L M, Lippman M E, Cancer Treat Rep 1985; 69: 615-632; Weisenthal L M, In: Kaspers G J L, Pieters R, Twentyman P R, Weisenthal L M, Veerman A J P, eds.
  • the sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 6 month for human and 4-6 weeks for mouse.
  • a composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method.
  • sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.
  • the term "synergistic effect” refers to the combined effect of two or more anti- immune checkpoint agents can be greater than the sum of the separate effects of the anticancer agents alone.
  • siRNA Short interfering RNA
  • small interfering RNA is defined as an agent which functions to inhibit expression of a target biomarker nucleic acid, e.g., by RNAi.
  • An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell.
  • siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3 ' and/or 5' overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides.
  • the length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
  • the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
  • PTGS post-transcriptional gene silencing
  • an siRNA is a small hairpin (also called stem loop) RNA (shRNA).
  • shRNAs are composed of a short ⁇ e.g., 1 -25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand.
  • the sense strand may precede the nucleotide loop structure and the antisense strand may follow.
  • shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, et al. (2003) RNA 9:493-501, incorporated by reference herein).
  • RNA interfering agents e.g., siRNA molecules
  • RNA interfering agents may be administered to a patient having or at risk for having cancer, to inhibit expression of a biomarker gene which is overexpressed in cancer and thereby treat, prevent, or inhibit cancer in the subject.
  • subject refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer, e.g. , lung, ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, as well as melanoma and multiple myeloma.
  • a cancer e.g. , lung, ovarian, pancreatic, liver, breast, prostate, and colon carcinomas, as well as melanoma and multiple myeloma.
  • subject is interchangeable with "patient.”
  • survival includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • therapeutic effect refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
  • the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
  • therapeutically- effective amount means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
  • a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like.
  • certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
  • terapéuticaally-effective amount and “effective amount” as used herein means that amount of a compound, material, or composition comprising a compound of the present invention which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 and the ED50. Compositions that exhibit large therapeutic indices are preferred.
  • the LD50 lethal dosage
  • the LD50 can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more reduced for the agent relative to no administration of the agent.
  • the ED50 i.e., the concentration which achieves a half-maximal inhibition of symptoms
  • the ED50 can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent.
  • the IC50 i.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells
  • the IC50 can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent.
  • cancer cell growth in an assay can be inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100%.
  • a "transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a biomarker nucleic acid and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
  • a polynucleotide e.g. an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA
  • the term "unresponsiveness” includes refractivity of immune cells to stimulation, e.g., stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, e.g., because of exposure to immunosuppressants or exposure to high doses of antigen.
  • the term “anergy” or “tolerance” includes refractivity to activating receptor-mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by lack of cytokine production, e.g., IL-2.
  • T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD-3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and, thus, failure to proliferate.
  • Anergic T cells can, however, proliferate if cultured with cytokines (e.g., IL-2).
  • cytokines e.g., IL-2
  • T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line.
  • a reporter gene construct can be used.
  • anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5' IL-2 gene enhancer or by a mul timer of the API sequence that can be found within the enhancer (Kang et al. (1992) Science 257: 1 134).
  • Arginine AGA, ACG, CGA, CGC, CGG, CGT
  • Aspartic acid Aspartic acid (Asp, D) GAC, GAT Cysteine (Cys, C) TGC, TGT
  • Glycine Gly, G
  • GGC GGG, GGT
  • Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT
  • nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid
  • nucleotide sequence of a DNA or RNA encoding a biomarker nucleic acid can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence.
  • polypeptide amino acid sequence corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
  • description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
  • description and/or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
  • nucleic acid and amino acid sequence information for the loci and biomarkers useful for the present invention are well-known in the art and readily available on publicly available databases, such as the National Center for Biotechnology Information (NCBI).
  • the subject for whom predicted likelihood of efficacy of an immune checkpoint therapy is determined is a mammal (e.g., mouse, rat, primate, non- human mammal, domestic animal, such as a dog, cat, cow, horse, and the like), and is preferably a human.
  • a mammal e.g., mouse, rat, primate, non- human mammal, domestic animal, such as a dog, cat, cow, horse, and the like
  • domestic animal such as a dog, cat, cow, horse, and the like
  • the subject has not undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immune checkpoint therapy.
  • the subject has undergone treatment, such as chemotherapy, radiation therapy, targeted therapy, and/or immune checkpoint therapy.
  • the subject has had surgery to remove cancerous or precancerous tissue.
  • the cancerous tissue has not been removed, e.g., the cancerous tissue may be located in an inoperable region of the body, such as in a tissue that is essential for life, or in a region where a surgical procedure would cause considerable risk of harm to the patient.
  • the methods of the present invention can be used to determine the responsiveness to anti-immune checkpoint therapies of a cancer.
  • the cancer is one for which an immune checkpoint therapy (e.g., anti-PD-1 blocking antibody, anti-PD-Ll blocking antibody, CTLA-4 blocking antibody, and the like) is FDA-approved for treatment, such as those described in the Examples.
  • the cancers are solid tumors, such as lung cancer such as non-small cell lung cancer, bladder cancer, melanoma such as metastatic melanoma, and/or renal cell carcinoma.
  • the cancer is an epithelial cancer such as, but not limited to, brain cancer (e.g., glioblastomas) bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
  • the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
  • the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g., serous ovarian carcinoma), or breast carcinoma.
  • the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, brenner, or undifferentiated.
  • the cancer is a mesenchymal cancer, such as sarcoma.
  • biomarker amount and/or activity measurement(s) in a sample from a subject is compared to a predetermined control (standard) sample.
  • the sample from the subject is typically from a diseased tissue, such as cancer cells or tissues.
  • the control sample can be from the same subject or from a different subject.
  • the control sample is typically a normal, non-diseased sample.
  • the control sample can be from a diseased tissue.
  • the control sample can be a combination of samples from several different subjects.
  • the biomarker amount and/or activity measurement(s) from a subject is compared to a pre-determined level. This pre-determined level is typically obtained from normal samples.
  • a "pre-determined" biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for treatment, evaluate a response to an immune checkpoint therapy, and/or evaluate a response to a combination immune checkpoint therapy.
  • a pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without cancer.
  • the pre-determined biomarker amount and/or activity measurement(s) can be a single number, equally applicable to every patient, or the pre- determined biomarker amount and/or activity measurement(s) can vary according to specific subpopulations of patients.
  • Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual. Furthermore, the pre-determined biomarker amount and/or activity can be determined for each subject individually. In one embodiment, the amounts determined and/or compared in a method described herein are based on absolute measurements.
  • the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios (e.g., biomarker amount/level before a treatment vs. after a treatment, such biomarker measurements relative to a spiked or man-made control, such biomarker measurements relative to the expression of a housekeeping gene, and the like).
  • the relative analysis can be based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.
  • Pre-treatment biomarker measurement can be made at any time prior to initiation of anti-cancer therapy.
  • Post-treatment biomarker measurement can be made at any time after initiation of anti-cancer therapy.
  • post-treatment biomarker measurements are made 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 weeks or more after initiation of anti-cancer therapy, and even longer toward indefinitely for continued monitoring.
  • Treatment can comprise anti-cancer therapy, such as a therapeutic regimen comprising an anti-PDl monoclonal antibody (e.g., nivolumab) alone or in combination with other anti-cancer agents, such as anti-PD-Ll/PD-L2 antibodies, anti- VEGF agents (e.g., bevacizumab), agents described in the Examples, Figures, and Tables.
  • the pre-determined biomarker amount and/or activity measurement(s) can be any suitable standard.
  • the pre-determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed.
  • the pre-determined biomarker amount and/or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time.
  • the control can be obtained from an assessment of another human or multiple humans, e.g., selected groups of humans, if the subject is a human.
  • the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g., other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group.
  • the change of biomarker amount and/or activity measurement(s) from the pre-determined level is about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0 fold or greater, or any range in between, inclusive.
  • Such cutoff values apply equally when the measurement is based on relative changes, such as based on the ratio of pre-treatment biomarker measurement as compared to post-treatment biomarker measurement.
  • Body fluids refer to fluids that are excreted or secreted from the body as well as fluids that are normally not ⁇ e.g.
  • amniotic fluid aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper's fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
  • the subject and/or control sample is selected from the group consisting of cells, cell lines, histological slides, paraffin embedded tissues, biopsies, whole blood, nipple aspirate, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow.
  • the sample is serum, plasma, or urine.
  • the sample is serum.
  • the samples can be collected from individuals repeatedly over a longitudinal period of time (e.g., once or more on the order of days, weeks, months, annually, biannually, etc.). Obtaining numerous samples from an individual over a period of time can be used to verify results from earlier detections and/or to identify an alteration in biological pattern as a result of, for example, disease progression, drug treatment, etc. For example, subject samples can be taken and monitored every month, every two months, or combinations of one, two, or three month intervals according to the present invention.
  • biomarker amount and/or activity measurements of the subject obtained over time can be conveniently compared with each other, as well as with those of normal controls during the monitoring period, thereby providing the subject's own values, as an internal, or personal, control for long-term monitoring.
  • Sample preparation and separation can involve any of the procedures, depending on the type of sample collected and/or analysis of biomarker measurement(s).
  • Such procedures include, by way of example only, concentration, dilution, adjustment of pH, removal of high abundance polypeptides (e.g., albumin, gamma globulin, and transferrin, etc.), addition of preservatives and calibrants, addition of protease inhibitors, addition of denaturants, desalting of samples, concentration of sample proteins, extraction and purification of lipids.
  • the sample preparation can also isolate molecules that are bound in non-covalent complexes to other protein (e.g., carrier proteins).
  • carrier proteins e.g., albumin
  • This process may isolate those molecules bound to a specific carrier protein (e.g., albumin), or use a more general process, such as the release of bound molecules from all carrier proteins via protein denaturation, for example using an acid, followed by removal of the carrier proteins.
  • undetectable proteins from a sample can be achieved using high affinity reagents, high molecular weight filters, ultracentrifugation and/or electrodialysis.
  • High affinity reagents include antibodies or other reagents (e.g., aptamers) that selectively bind to high abundance proteins.
  • Sample preparation could also include ion exchange chromatography, metal ion affinity chromatography, gel filtration, hydrophobic chromatography, chromatofocusing, adsorption chromatography, isoelectric focusing and related techniques.
  • Molecular weight filters include membranes that separate molecules on the basis of size and molecular weight. Such filters may further employ reverse osmosis, nanofiltration, ultrafiltration and microfiltration.
  • Ultracentrifugation is a method for removing undesired polypeptides from a sample. Ultracentrifugation is the centrifugation of a sample at about 15,000-60,000 rpm while monitoring with an optical system the sedimentation (or lack thereof) of particles.
  • Electrodialysis is a procedure which uses an electromembrane or semipermable membrane in a process in which ions are transported through semi-permeable membranes from one solution to another under the influence of a potential gradient. Since the membranes used in electrodialysis may have the ability to selectively transport ions having positive or negative charge, reject ions of the opposite charge, or to allow species to migrate through a semipermable membrane based on size and charge, it renders electrodialysis useful for concentration, removal, or separation of electrolytes.
  • Separation and purification in the present invention may include any procedure known in the art, such as capillary electrophoresis (e.g., in capillary or on-chip) or chromatography (e.g., in capillary, column or on a chip).
  • Electrophoresis is a method which can be used to separate ionic molecules under the influence of an electric field. Electrophoresis can be conducted in a gel, capillary, or in a microchannel on a chip.
  • gels used for electrophoresis include starch, acrylamide, polyethylene oxides, agarose, or combinations thereof.
  • a gel can be modified by its cross-linking, addition of detergents, or denaturants, immobilization of enzymes or antibodies (affinity
  • capillaries used for electrophoresis include capillaries that interface with an electrospray.
  • CE Capillary electrophoresis
  • CZE capillary zone electrophoresis
  • CIEF capillary isoelectric focusing
  • cITP capillary isotachophoresis
  • CEC capillary electrochromatography
  • Capillary isotachophoresis is a technique in which the analytes move through the capillary at a constant speed but are nevertheless separated by their respective mobilities.
  • Capillary zone electrophoresis also known as free-solution CE (FSCE)
  • FSCE free-solution CE
  • CIEF Capillary isoelectric focusing
  • CEC is a hybrid technique between traditional high performance liquid chromatography (UPLC) and CE.
  • Chromatography can be based on the differential adsorption and elution of certain analytes or partitioning of analytes between mobile and stationary phases.
  • Different examples of chromatography include, but not limited to, liquid chromatography (LC), gas chromatography (GC), high performance liquid chromatography (HPLC), etc.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • An "isolated” compound molecule is one which is separated from other molecules which are present in the natural source of the compound molecule.
  • a biomarker metabolite compound molecule of the present invention can be isolated and/or purified using standard molecular biology techniques described herein.
  • Measurement for metabolite compounds are also well known in the art. For example, see Howard et al. (1998) Invest Ophthalmol Vis Sci. 39: 1942-1946 for methods of measuring adenosine concentration in aqueous and vitreous and Ramakers et al. (2008) Curr DrugMetab. 9:679-685 for methods of measuring endogenous adenosine concentration in human in vivo. For similar methods for detecting kynurenine, see Kawai et al. (2007) Int. Congr. Ser. 1304:415-419 and Widner et al. (1999) Adv Exp Med Biol. 467:827-832.
  • such metabolite compounds ⁇ e.g., kynurenine and adenosine, as well as precursors and metabolites thereof) are measured by high-performance liquid chromatography (HPLC) and/or Mass Spectrometry.
  • HPLC high-performance liquid chromatography
  • mass Spectrometry mass Spectrometry.
  • chromatography refers to a process in which a chemical mixture carried by a liquid or gas is separated into components as a result of differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.
  • liquid chromatography means a process of selective retardation of one or more components of a fluid solution as the fluid uniformly percolates through a column of a finely divided substance, or through capillary passageways. The retardation results from the distribution of the components of the mixture between one or more stationary phases and the bulk fluid, (i.e., mobile phase), as this fluid moves relative to the stationary phase(s).
  • Liquid chromatography includes reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC) and high turbulence liquid chromatography (HTLC).
  • HPLC high performance liquid chromatography
  • mass spectrometry refers to an analytical technique to identify compounds by their mass. MS technology generally includes (1) ionizing the compounds to form charged compounds; and (2) detecting the molecular weight of the charged compound and calculating a mass-to-charge ratio (m/z). The compound may be ionized and detected by any suitable means.
  • a “mass spectrometer” generally includes an ionizer and an ion detector. See, e.g., U.S. Pat. Nos. 6,204,500, entitled "Mass
  • Biomarker metabolites such as those shown in Table 1, examples, and figures, can be detected in numerous ways according to well-known techniques.
  • such metabolites, as well as biomarker proteins can be detected using mass spectrometry methods, such as MALDF/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (FIPLC-MS), capillary
  • mass spectrometry methods such as MALDF/TOF (time-of-flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (FIPLC-MS), capillary
  • electrophoresis-mass spectrometry nuclear magnetic resonance spectrometry, or tandem mass spectrometry (e.g., MS/MS, MS/MS/MS, ESI-MS/MS, etc.). See for example, U.S. Patent Application Nos: 20030199001, 20030134304, 20030077616, which are herein incorporated by reference.
  • Mass spectrometry methods are well known in the art and have been used to quantify and/or identify biomolecules, such as chemical metabolites and proteins (see, e.g., Li et al. (2000) Tibtech 18, 151-160; Rowley et al. (2000) Methods 20, 383-397; Kuster and Mann (1998) Curr. Opin. Structural Biol. 8, 393-400). Further, mass spectrometric techniques have been developed that permit at least partial de novo sequencing of isolated proteins (see, e.g., Chait et al. (1993) Science 262, 89-92; Keough et al. (1999) Proc. Natl. Acad. Sci. USA. 96, 7131-7136; reviewed in Bergman (2000) EXS 88, 133-44).
  • a gas phase ion spectrophotometer is used.
  • laser-desorption/ionization mass spectrometry is used to analyze the sample.
  • Modem laser desorption/ionization mass spectrometry (“LDI-MS”) can be practiced in two main variations: matrix assisted laser desorption/ionization (“MALDI”) mass spectrometry and surface-enhanced laser desorption/ionization (“SELDI”).
  • MALDI matrix assisted laser desorption/ionization
  • SELDI surface-enhanced laser desorption/ionization
  • MALDI Metal-organic laser desorption ionization
  • the substrate surface is modified so that it is an active participant in the desorption process.
  • the surface is derivatized with adsorbent and/or capture reagents that selectively bind the protein of interest.
  • the surface is derivatized with energy absorbing molecules that are not desorbed when struck with the laser.
  • the surface is derivatized with molecules that bind the protein of interest and that contain a photolytic bond that is broken upon application of the laser.
  • the derivatizing agent generally is localized to a specific location on the substrate surface where the sample is applied (see, e.g., Hutchens and Yip, U.S. Pat. No.
  • the two methods can be combined by, for example, using a SELDI affinity surface to capture an analyte and adding matrix- containing liquid to the captured analyte to provide the energy absorbing material.
  • Detection of the presence of a marker or other substances will typically involve detection of signal intensity.
  • the signal strength of peak values from spectra of a first sample and a second sample can be compared (e.g., visually or by computer analysis) to determine the relative amounts of particular biomolecules.
  • Software programs such as the Biomarker Wizard program (Ciphergen Biosy stems, Inc., Fremont, Calif.) can be used to aid in analyzing mass spectra.
  • the mass spectrometers and their techniques are well known to those of skill in the art.
  • a control sample may contain heavy atoms (e.g. 13 C) thereby permitting the test sample to be mixed with the known control sample in the same mass spectrometry run.
  • internal controls such as phenylalanine ⁇ and/or valine-d8 can be run with the samples.
  • a laser desorption time-of-flight (TOF) mass spectrometer is used.
  • TOF time-of-flight
  • a substrate with a bound marker is introduced into an inlet system.
  • the marker is desorbed and ionized into the gas phase by laser from the ionization source.
  • the ions generated are collected by an ion optic assembly, and then in a time-of-flight mass analyzer, ions are accelerated through a short high voltage field and let drift into a high vacuum chamber. At the far end of the high vacuum chamber, the accelerated ions strike a sensitive detector surface at a different time. Since the time-of- flight is a function of the mass of the ions, the elapsed time between ion formation and ion detector impact can be used to identify the presence or absence of molecules of specific mass to charge ratio.
  • the relative amounts of one or more biomolecules present in a first or second sample is determined, in part, by executing an algorithm with a
  • the algorithm identifies at least one peak value in the first mass spectrum and the second mass spectrum.
  • the algorithm compares the signal strength of the peak value of the first mass spectrum to the signal strength of the peak value of the second mass spectrum of the mass spectrum.
  • the relative signal strengths are an indication of the amount of the biomolecule that is present in the first and second samples.
  • a standard containing a known amount of a biomolecule can be analyzed as the second sample to provide better quantification of the amount of the biomolecule present in the first sample.
  • the identity of the biomolecules in the first and second sample can also be determined,
  • Adenosine is a naturally occurring nucleoside which exhibits diverse and potent physiological actions in the cardiovascular, nervous, pulmonary, renal and immune systems. Adenosine has been shown to terminate superventricular tachycardia through blockage of atrioventricular nodal conduction (DiMarco et al. (1985) J. Am. Col. Cardiol. 6:417-425; Munoz, et al. (1984) Eur. Heart J. 5 :735-738). Adenosine is a potent vasodilator except in the kidney and placenta (Olsson ( ⁇ 9% ⁇ ) Ann. Rev. Physiol. 43 :385- 395).
  • Adenosine has been implicated as a preventive agent and in treatment of ventricular dysfunction following episodes of regional or global ischemia (Forman and Velasco (1991) Cardiovasc Drugs Ther 5 :901-908) and in cerebral ischemia (Evans et al. (1987) Neurosci. Lett. 83 :287; Von Lubitz et al. ⁇ 9%%) Stroke 19: 1 133).
  • adenosine acts as a signal molecule, is produced by the body, and found in almost every cell. Additional examples of signal molecules include hormones and neurotransmitters. These endogenous molecules are naturally occurring chemicals used by the body to communicate between cells and to coordinate physiological functions. The actions of individual signal molecules are controlled or mediated through specific recognition sites on the surface of cells, called receptors. For adenosine, four receptor subtypes are known (discussed in greater detail below), Al, A2a, A2b, and A3. Each of these is believed to be responsible for a different function. For example, Al-receptors regulate the rhythm of the heart, A2a-receptors dilate blood vessels (vasodilation), and there is growing evidence that stimulation of A3-receptors may impart cardioprotection.
  • Extracelluar adenosine concentration plays an important role in multiple diseases and disorders.
  • Extracellular adenosine mostly comes from two sources. First, it may be derived from the external transport of intracellularly generated adenosine. Second, extracellular adenosine may also be formed from the extracellular hydrolysis of adenine nucleotides. Adenosine used as a second messenger can be the result of de novo purine biosynthesis via adenosine monophosphate (AMP), though it is possible other pathways exist (Miller-Patrick et al. (1993) Chin J Physiol. 36:245-252).
  • AMP adenosine monophosphate
  • extracellular adenosine is derived from the breakdown of extracellular nucleotides, particularly ATP and ADP.
  • the generation of extracellular adenosine from ATP is predominantly controlled through a two-step enzymatic reaction: first, the conversion of ATP or ADP to AMP by ectonucleoside triphosphate diphosphohydrolase 1 (ENTPDl ; also known as CD39), which is followed by AMP hydrolysis to adenosine by ecto-5'- nucleotidase (NT5E; also known as CD73).
  • ENTPDl ectonucleoside triphosphate diphosphohydrolase 1
  • N5E ecto-5'- nucleotidase
  • the concentration of adenosine in the extracellular compartment is the consequence of many biological processes, including extracellular adenosine production, adenosine transport, adenosine formation from intracellular adenosine sources (for example, via the S-adenosylhomocysteine pathway) and adenosine metabolism to inosine or AMP.
  • adenosine deaminase which is present in red blood cells and the vessel wall.
  • ADA irreversibly deaminates adenosine, converting it to the related nucleoside inosine by the substitution of the amino group for a keto group.
  • Inosine can then be deribosylated (removed from ribose) by another enzyme called purine nucleoside phosphorylase (P P), converting it to hypoxanthine. Mutations may cause overexpression of ADA, as one cause of hemolytic anemia (Chottiner et al. (1987) J. Clin. Invest. 79: 1001-1005).
  • Adenosine is known to modulate diverse physiological functions including induction of sedation, vasodilatation, suppression of cardiac rate and contractility, inhibition of platelet aggregability, stimulation of gluconeogenesis and inhibition of lipolysis (see, Stiles (1986) Trends Pharmacol Sci. 7:486-490; Williams ( ⁇ 987) Ann. Rev. Pharmacol. Toxicol. 27:315-345; Ramkumar et al. (1988) Prog. Drug. Res. 32: 195-247).
  • Individual subtypes of adenosine receptor inhibit or stimulate adenylate cyclase (Stiles, ibid.; Williams, ibid). Substantial progress has been made concerning the biochemical and pharmacological properties of these adenosine receptors such as ligand binding
  • adenosine has been shown to open potassium channels, reduce flux through calcium channels, and inhibit or stimulate phosphoinositide turnover through receptor-mediated mechanisms (see, Fredholm and Dunwiddie (1988) Trends Pharmacol. Sci. 9: 130-134; Sebastiao et al. (1990) Br. J. Pharmacol. 100:55-62; Stiles (1990) Clin. Res. 38: 10-18; Nakahata et al. (1991) J. Neurochem. 57:963-969).
  • adenosine The actions of adenosine are mediated through G-protein coupled receptors; the Al,
  • A2a, A2b and A3 adenosine receptors mentioned above.
  • the adenosine receptors were initially classified into Al and A2 subtypes on the basis of pharmacological criteria and coupling to adenylate cyclase (Van Caulker et al. (1979) J. Neurochem. 33 :999-1003). Further pharmacological classification of adenosine receptors prompted subdivision of the A2 class into A2a and A2b subtypes on the basis of high and low affinity, respectively, for adenosine and the agonists NECA and CGS-21680 (Bruns et al. (1986) Mol. Pharmacol. 29:331-346; Wan et al. (1990) J.
  • adenosine receptor subtypes are G-protein-coupled receptors.
  • adenosine receptors have recently shown adenosine receptors to be key in opening the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • Mice dosed with adenosine have shown increased transport across the BBB of amyloid plaque antibodies and prodrugs associated with Parkinson's disease, Alzheimer's, multiple sclerosis, and cancers of the central nervous system (Carman et al. (2011) J. Neurosci. 31 : 13272-13280).
  • the stimulation of Al adenosine receptors also inhibits adenylcyclase and decreases intracellular cyclic AMP.
  • IL- ⁇ interleukin- 1 ⁇
  • TNT tumor necrosis factor
  • the adenosine levels and/or activities are detected and/or measured by the levels of adenosine ⁇ e.g., extracellular adenosine levels, such as serum concentration), adenosine precursors ⁇ e.g., AMP, ADP, ATP, as described herein), and/or adenosine metabolites ⁇ e.g., inosine and hypoxanthine, as described herein), and/or activities of adenosine-related downstream pathway activations, alone or in any
  • Such activities of adenosine-related downstream pathway activations include, at least, induction of sedation, vasodilatation, suppression of cardiac rate and contractility, inhibition of platelet aggregability, stimulation of gluconeogenesis and inhibition of lipolysis, opening the BBB, inhibiting adenylcyclase and decreasing intracellular cyclic AMP, increasing local inflammatory cytokines and chemokines ⁇ e.g., IL- ⁇ , TNF, and others disclosed herein, including CD86, CCL20, IL21R, and IL18), dendritic and natural- killer cell activation markers, reducing expression of interferon receptors ⁇ e.g., IFNAR2, IFNGR2, IL10RB), decreasing transcription of lymphocyte activation molecules
  • adenosine can affect a variety of physiological functions, particular attention has been directed over the years toward actions which might lead to clinical applications. Preeminent has been the cardiovascular effects of adenosine which lead to vasodilation and hypotension but which also lead to cardiac depression. The antilipolytic, antithrombotic and antispasmodic actions of adenosine have also received some attention. Adenosine stimulates steroidogenesis in adrenal cells, again probably via activation of adenylate cyclase. Adenosine has inhibitory effects on neurotransmission and on spontaneous activity of central neurons. Finally, adenosine is a known bronchoconstrictor.
  • Adenosine receptor agonists, antagonists and binding enhancers have been identified and implicated for usage in the treatment of physiological complications resulting from cardiovascular, pulmonary, renal and neurological disorders.
  • the pharmaceutical industry is actively working to develop new drug candidates that bind at one or more adenosine receptor.
  • Adenosine receptor agonists have been identified for use as vasodilators, antihypertensive agents (Taylor et al. (1988) FASEB J. 2: 1799), and anti-psychotic agents (Heffner et al. (1989)
  • Adenosine receptor agonists have been identified for use in improving renal function (Murray Churchill (1985) J. Pharmacol. Exp. Therap. 232: 189- 193).
  • Adenosine receptor allosteric or binding enhancers have shown utility in the treatment of ischemia, seizures or hypoxia of the brain (Bruns et al. (1990) Mol.
  • adenosine inhibitors are designed to antagonize adenosine production, concentration, and/or activities (e.g., through adenosine receptor- mediated pathways), either alone or in any combination.
  • adenosine inhibitors inhibit the de novo purine biosynthesis and/or reduce the levels and/or activities of the adenosine precursors described herein.
  • adenosine inhibitors reduce the levels and/or activies of CD39 and/or CD73.
  • Adenosine inhibitors may also reduce the extracelluar concentration of adenosine, by promoting adenosine degradation (e.g., by increasing levels and/or activity of adenosine deaminase (ADA)) and/or adenosine transportation and absorption (e.g., into cells).
  • adenosine degradation e.g., by increasing levels and/or activity of adenosine deaminase (ADA)
  • adenosine transportation and absorption e.g., into cells.
  • its inhibitors may antagonize the interaction between adenosine and its receptors (e.g., by a ligand trap and/or dominant negative recombinant receptors for adenosine), and/or inhibiting the receptor-induced downstream pathways.
  • Adenosine receptor antagonists have been reported and tested in multiple clinical trials (Chen et al. (2013) Nat Rev Drug Discov. 12:265-286).
  • Exemplary antagonists include xanthine derivatives (Miiller and Jacobson (201 1) Handbook of experimental pharmacology (200): 151-99), such as methylxanthines (e.g., caffeine (Rivera-Oliver and Diaz-Rios (2014) Life Sci. 101 : 1-9), theophylline (Hauber and Muenkle (1996) Naunyn Schmiede bergs Arch. Pharmacol. 354: 179-186), and theobromine).
  • adenosine receptor antagonists tested in clinical trials include, e.g., tonapofylline (clinicaltrials.gov identifier NCT00709865), rolofylline (NCT00328692), istradefylline (KW-6002)
  • NCT00955526 and NCT0057203 preladenant ( CT01155479 and NCT01155466), SYN115 (NCT00783276 and NCT01283594).
  • kynurenine pathway refers to a series of biodegradative enzymatic reactions formed by L-tryptophan metabolic catabolism. The specific enzymatic steps, inputs, metabolic intermediates, and end-products, are shown in Figure 1.
  • "Kynurenines” are a family of tryptophan metabolites formed within the kynurenine pathway.
  • Such metabolites include formylkynurenine (form-KYN), L-kynurenine (L-KYN), kynurenic acid (KYNA), anthranilic acid (AA), 3 -hydrocy kynurenine (3-HK), 3 -hydroxy anthranilic acid (3-HAA), 2-amino-3-carboxymuconate-semialdehyde (ACMS), quinolinic acid (QUIN), 2-aminomuconic acid semialdehyde (AMS), and picolinic acid (PIC), as well as substantially homologous analogs and variations.
  • downstream products of the kynurenine pathway such as NAD+, glutaryl-CoA, and acetyl-CoA are also included.
  • "Kynurenein pathway nucleic acids and proteins” refer to nucleic acids and proteins, respectively of the kynurenein pathway enzymes, which are provided in Figure 1 and include, for example, kynurenine formamidase, kynureninase (KYNase), kynurenine aminotransferase (KAT1/KAT2), kynurenine hydroxylase (KYN-OHase), kynureninase (KYNase), 3 -hydroxy anthralinate-3,4-di oxygenase (3-HAO) and 2-amino 3- carboxymuconate 6-semialdehyde decarboxylase (ACMSD).
  • kynurenine formamidase kynureninase (KYNase), kynurenine amino
  • L-kynurenine is a metabolite of the amino acid L-tryptophan used in the production of niacin.
  • Kynurenine is synthesized by the enzyme tryptophan dioxygenase, which is made primarily but not exclusively in the liver, and indoleamine 2,3-dioxygenase, which is made in many tissues in response to immune activation (Opitz et al. (2011) Nature 478: 197-203).
  • Kynurenine and its further breakdown products carry out diverse biological functions, including dilating blood vessels during inflammation (Wang et al. (2010) Nat Med. 16:279-285) and regulating the immune response (Nguyen et al.
  • Alzheimer's disease (Guillemin et al. (2005) Neuropathol. Appl. Neurobiol. 31 :395-404) and cardiovascular disease (Wirleitner et al. (2003) Eur. J. Clin. Invest. 33 :550-554), where its metabolites are associated with cognitive deficits (Gulaj et al. (2010) Adv. Med. Sci. 55 :204-211) and depressive symptoms (Swardfager et al. (2009)
  • immune checkpoint therapy is predicted according to biomarker amount and/or activity associated with a cancer in a subject according to the methods described herein.
  • immune checkpoint therapy or combinations of therapies ⁇ e.g. , anti-PD-1 antibodies
  • immune checkpoint therapy can be avoided once a subject is indicated as not being a likely responder to immune checkpoint therapy and an alternative treatment regimen, such as targeted and/or untargeted anti-cancer therapies can be administered.
  • Combination therapies are also contemplated and can comprise, for example, one or more
  • chemotherapeutic agents and radiation one or more chemotherapeutic agents and immunotherapy, or one or more chemotherapeutic agents, radiation and chemotherapy, each combination of which can be with immune checkpoint therapy.
  • targeted therapy refers to administration of agents that selectively interact with a chosen biomolecule to thereby treat cancer.
  • Immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells.
  • an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site.
  • the immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen).
  • a cancer antigen or disease antigen e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen.
  • Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.
  • antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.
  • agents that modulate adenosine, adenosine precursors, and/or adenosine metabolites are useful immunotherapeutic agents according to the present invention and are well-known in the art, as described below, such as adenosine receptor antagonists, agents that impair adenosine production, agents that metabolize adenosine, and the like.
  • untargeted therapy referes to administration of agents that do not selectively interact with a chosen biomolecule yet treat cancer.
  • ReRepresentative examples of untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy. In one embodiment, chemotherapy is used.
  • Chemotherapy includes the
  • chemotherapeutic agent may be, but is not limited to, those selected from among the following groups of compounds: platinum compounds, cytotoxic antibiotics, antimetabolities, anti-mitotic agents, alkylating agents, arsenic compounds, DNA topoisomerase inhibitors, taxanes, nucleoside analogues, plant alkaloids, and toxins; and synthetic derivatives thereof.
  • Exemplary compounds include, but are not limited to, alkylating agents: cisplatin, treosulfan, and trofosfamide; plant alkaloids: vinblastine, paclitaxel, docetaxol; DNA topoisomerase inhibitors: teniposide, crisnatol, and mitomycin; anti -folates: methotrexate, mycophenolic acid, and hydroxyurea; pyrimidine analogs: 5-fluorouracil, doxifluridine, and cytosine arabinoside; purine analogs:
  • compositions comprising one or more chemotherapeutic agents (e.g., FLAG, CHOP) may also be used.
  • FLAG comprises fludarabine, cytosine arabinoside (Ara-C) and G-CSF.
  • CHOP comprises cyclophosphamide, vincristine, doxorubicin, and prednisone.
  • PARP e.g.
  • PARP-1 and/or PARP-2) inhibitors are used and such inhibitors are well known in the art (e.g. , Olaparib, ABT-888, BSI-201, BGP-15 (N-Gene Research Laboratories, Inc.); ⁇ -1001 (Inotek Pharmaceuticals Inc.); PI34 (Soriano et al , 2001 ; Pacher et al , 2002b); 3-aminobenzamide (Trevigen); 4-amino- 1,8-naphthalimide; (Trevigen); 6(5H)-phenanthridinone (Trevigen); benzamide (U.S. Pat. Re. 36,397); and NU1025 (Bowman et al.).
  • the mechanism of action is generally related to the ability of PARP inhibitors to bind PARP and decrease its activity.
  • PARP catalyzes the conversion of .beta. -nicotinamide adenine dinucleotide (NAD+) into nicotinamide and poly-ADP-ribose (PAR). Both poly (ADP-ribose) and PARP have been linked to regulation of transcription, cell proliferation, genomic stability, and carcinogenesis
  • Poly(ADP-ribose) polymerase 1 (PARP1) is a key molecule in the repair of DNA single- strand breaks (SSBs) (de Murcia J. et al. 1997. Proc Natl Acad Sci USA 94:7303-7307; Schreiber V, Dantzer F, Ame J C, de Murcia G (2006) Nat Rev Mol Cell Biol 7:517-528; Wang Z Q, et al. (1997) Genes Dev 11 :2347-2358). Knockout of SSB repair by inhibition of PARP1 function induces DNA double-strand breaks (DSBs) that can trigger synthetic lethality in cancer cells with defective homology-directed DSB repair (Bryant H E, et al. (2005) Nature 434:913-917; Farmer H, et al. (2005) Nature 434:917-921).
  • chemotherapeutic agents are illustrative, and are not intended to be limiting.
  • radiation therapy is used.
  • the radiation used in radiation therapy can be ionizing radiation.
  • Radiation therapy can also be gamma rays, X-rays, or proton beams.
  • Examples of radiation therapy include, but are not limited to, external-beam radiation therapy, interstitial implantation of radioisotopes (1-125, palladium, iridium), radioisotopes such as strontium-89, thoracic radiation therapy, intraperitoneal P-32 radiation therapy, and/or total abdominal and pelvic radiation therapy.
  • the radiation therapy can be administered as external beam radiation or teletherapy wherein the radiation is directed from a remote source.
  • the radiation treatment can also be administered as internal therapy or brachytherapy wherein a radioactive source is placed inside the body close to cancer cells or a tumor mass.
  • photodynamic therapy comprising the administration of photosensitizers, such as hematoporphyrin and its derivatives, Vertoporfin (BPD-MA), phthalocyanine,
  • photosensitizer Pc4 demethoxy-hypocrellin A; and 2BA-2-DMHA.
  • hormone therapy is used.
  • Hormonal therapeutic treatments can comprise, for example, hormonal agonists, hormonal antagonists (e.g., flutamide, bicalutamide, tamoxifen, raloxifene, leuprolide acetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesis and processing, and steroids (e.g., dexamethasone, retinoids, deltoids, betamethasone, Cortisol, cortisone, prednisone, dehydrotestosterone, glucocorticoids, mineralocorticoids, estrogen, testosterone, progestins), vitamin A derivatives (e.g., all-trans retinoic acid (ATRA)); vitamin D3 analogs; antigestagens (e.g., mifepristone, onapristone), or antiandrogens (e.g., cyproterone acetate).
  • hormonal antagonists e.g., flutamide, bicalu
  • hyperthermia a procedure in which body tissue is exposed to high temperatures (up to 106°F.) is used. Heat may help shrink tumors by damaging cells or depriving them of substances they need to live. Hyperthermia therapy can be local, regional, and whole-body hyperthermia, using external and internal heating devices.
  • Hyperthermia is almost always used with other forms of therapy (e.g., radiation therapy, chemotherapy, and biological therapy) to try to increase their effectiveness.
  • Local hyperthermia refers to heat that is applied to a very small area, such as a tumor.
  • the area may be heated externally with high-frequency waves aimed at a tumor from a device outside the body.
  • one of several types of sterile probes may be used, including thin, heated wires or hollow tubes filled with warm water; implanted microwave antennae; and radiofrequency electrodes.
  • an organ or a limb is heated. Magnets and devices that produce high energy are placed over the region to be heated.
  • perfusion In another approach, called perfusion, some of the patient's blood is removed, heated, and then pumped (perfused) into the region that is to be heated internally.
  • Whole- body heating is used to treat metastatic cancer that has spread throughout the body. It can be accomplished using warm-water blankets, hot wax, inductive coils (like those in electric blankets), or thermal chambers (similar to large incubators). Hyperthermia does not cause any marked increase in radiation side effects or complications. Heat applied directly to the skin, however, can cause discomfort or even significant local pain in about half the patients treated. It can also cause blisters, which generally heal rapidly.
  • photodynamic therapy also called PDT, photoradiation therapy, phototherapy, or photochemotherapy
  • PDT photoradiation therapy
  • phototherapy phototherapy
  • photochemotherapy is used for the treatment of some types of cancer. It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organisms when the organisms are exposed to a particular type of light.
  • PDT destroys cancer cells through the use of a fixed-frequency laser light in combination with a photosensitizing agent.
  • the photosensitizing agent is injected into the bloodstream and absorbed by cells all over the body. The agent remains in cancer cells for a longer time than it does in normal cells.
  • the photosensitizing agent absorbs the light and produces an active form of oxygen that destroys the treated cancer cells.
  • the laser light used in PDT can be directed through a fiberoptic (a very thin glass strand).
  • the fiber-optic is placed close to the cancer to deliver the proper amount of light.
  • the fiber-optic can be directed through a bronchoscope into the lungs for the treatment of lung cancer or through an endoscope into the esophagus for the treatment of esophageal cancer.
  • An advantage of PDT is that it causes minimal damage to healthy tissue.
  • PDT is mainly used to treat tumors on or just under the skin or on the lining of internal organs.
  • Photodynamic therapy makes the skin and eyes sensitive to light for 6 weeks or more after treatment. Patients are advised to avoid direct sunlight and bright indoor light for at least 6 weeks. If patients must go outdoors, they need to wear protective clothing, including sunglasses. Other temporary side effects of PDT are related to the treatment of specific areas and can include coughing, trouble swallowing, abdominal pain, and painful breathing or shortness of breath.
  • FDA U.S. Food and Drug Administration
  • a photosensitizing agent called porfimer sodium, or Photofrin®, to relieve symptoms of esophageal cancer that is causing an obstruction and for esophageal cancer that cannot be satisfactorily treated with lasers alone.
  • laser therapy is used to harness high-intensity light to destroy cancer cells.
  • This technique is often used to relieve symptoms of cancer such as bleeding or obstruction, especially when the cancer cannot be cured by other treatments. It may also be used to treat cancer by shrinking or destroying tumors.
  • the term "laser” stands for light amplification by stimulated emission of radiation. Ordinary light, such as that from a light bulb, has many wavelengths and spreads in all directions. Laser light, on the other hand, has a specific wavelength and is focused in a narrow beam. This type of high- intensity light contains a lot of energy. Lasers are very powerful and may be used to cut through steel or to shape diamonds.
  • CO2 laser Carbon dioxide
  • This type of laser can remove thin layers from the skin's surface without penetrating the deeper layers. This technique is particularly useful in treating tumors that have not spread deep into the skin and certain precancerous conditions.
  • the CO2 laser is also able to cut the skin. The laser is used in this way to remove skin cancers.
  • Lasers sterilizes the surgery site, thus reducing the risk of infection. Less operating time may be needed because the precision of the laser allows for a smaller incision. Healing time is often shortened; since laser heat seals blood vessels, there is less bleeding, swelling, or scarring. Laser surgery may be less complicated. For example, with fiber optics, laser light can be directed to parts of the body without making a large incision. More procedures may be done on an outpatient basis. Lasers can be used in two ways to treat cancer: by shrinking or destroying a tumor with heat, or by activating a chemical—known as a photosensitizing agent—that destroys cancer cells.
  • a photosensitizing agent that destroys cancer cells.
  • CO2 and Nd:YAG lasers are used to shrink or destroy tumors. They may be used with endoscopes, tubes that allow physicians to see into certain areas of the body, such as the bladder. The light from some lasers can be transmitted through a flexible endoscope fitted with fiber optics. This allows physicians to see and work in parts of the body that could not otherwise be reached except by surgery and therefore allows very precise aiming of the laser beam. Lasers also may be used with low-power microscopes, giving the doctor a clear view of the site being treated.
  • Lasers Used with other instruments, laser systems can produce a cutting area as small as 200 microns in diameter— less than the width of a very fine thread.
  • Lasers are used to treat many types of cancer.
  • Laser surgery is a standard treatment for certain stages of glottis (vocal cord), cervical, skin, lung, vaginal, vulvar, and penile cancers.
  • laser surgery is also used to help relieve symptoms caused by cancer (palliative care).
  • lasers may be used to shrink or destroy a tumor that is blocking a patient's trachea (windpipe), making it easier to breathe. It is also sometimes used for palliation in colorectal and anal cancer.
  • LITT Laser- induced interstitial thermotherapy
  • hyperthermia a cancer treatment
  • heat may help shrink tumors by damaging cells or depriving them of substances they need to live.
  • lasers are directed to interstitial areas (areas between organs) in the body. The laser light then raises the temperature of the tumor, which damages or destroys cancer cells.
  • the duration and/or dose of treatment with anti-immune checkpoint therapies may vary according to the particular anti-immune checkpoint agent or combination thereof.
  • An appropriate treatment time for a particular cancer therapeutic agent will be appreciated by the skilled artisan.
  • the present invention contemplates the continued assessment of optimal treatment schedules for each cancer therapeutic agent, where the phenotype of the cancer of the subject as determined by the methods of the present invention is a factor in determining optimal treatment doses and schedules.
  • any means for the introduction of a polynucleotide into mammals, human or non- human, or cells thereof may be adapted to the practice of this invention for the delivery of the various constructs of the present invention into the intended recipient.
  • the DNA constructs are delivered to cells by transfection, i.e., by delivery of "naked" DNA or in a complex with a colloidal dispersion system.
  • a colloidal system includes macromolecule complexes, nanocapsules,
  • a plasmid containing a transgene bearing the desired DNA constructs may first be experimentally optimized for expression (e.g., inclusion of an intron in the 5' untranslated region and elimination of unnecessary sequences (Feigner, et al., Ann NY Acad Sci 126-139, 1995). Formulation of DNA, e.g.
  • lipid or liposome materials may then be effected using known methods and materials and delivered to the recipient mammal. See, e.g., Canonico et al, Am J Respir Cell Mol Biol 10:24-29, 1994; Tsan et al, Am J Physiol 268; Alton et al., Nat Genet. 5: 135-142, 1993 and U. S. patent No. 5,679,647 by Carson et al.
  • the targeting of liposomes can be classified based on anatomical and mechanistic factors.
  • Anatomical classification is based on the level of selectivity, for example, organ- specific, cell-specific, and organelle-specific.
  • Mechanistic targeting can be distinguished based upon whether it is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs, which contain sinusoidal capillaries.
  • RES reticulo-endothelial system
  • Active targeting involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization.
  • a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting ligand. Naked DNA or DNA associated with a delivery vehicle, e.g. , liposomes, can be administered to several sites in a subject (see below).
  • Nucleic acids can be delivered in any desired vector. These include viral or non- viral vectors, including adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and plasmid vectors. Exemplary types of viruses include HSV (herpes simplex virus), AAV (adeno associated virus), HIV (human immunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV (murine leukemia virus). Nucleic acids can be administered in any desired format that provides sufficiently efficient delivery levels, including in virus particles, in liposomes, in nanoparticles, and complexed to polymers.
  • the nucleic acids encoding a protein or nucleic acid of interest may be in a plasmid or viral vector, or other vector as is known in the art. Such vectors are well known and any can be selected for a particular application.
  • the gene delivery vehicle comprises a promoter and a demethylase coding sequence.
  • Preferred promoters are tissue-specific promoters and promoters which are activated by cellular proliferation, such as the thymidine kinase and thymidylate synthase promoters.
  • Other preferred promoters include promoters which are activatable by infection with a virus, such as the a- and ⁇ -interferon promoters, and promoters which are activatable by a hormone, such as estrogen.
  • Other promoters which can be used include the Moloney virus LTR, the CMV promoter, and the mouse albumin promoter.
  • a promoter may be constitutive or inducible.
  • naked polynucleotide molecules are used as gene delivery vehicles, as described in WO 90/11092 and U.S. Patent 5,580,859.
  • gene delivery vehicles can be either growth factor DNA or RNA and, in certain embodiments, are linked to killed adenovirus. Curiel et al., Hum. Gene. Ther. 3 : 147-154, 1992.
  • Other vehicles which can optionally be used include DNA-ligand (Wu et al., J. Biol. Chem. 264: 16985-16987, 1989), lipid-DNA combinations (Feigner et al., Proc. Natl. Acad. Sci.
  • a gene delivery vehicle can optionally comprise viral sequences such as a viral origin of replication or packaging signal. These viral sequences can be selected from viruses such as astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, retrovirus, togavirus or adenovirus.
  • the growth factor gene delivery vehicle is a recombinant retroviral vector. Recombinant retroviruses and various uses thereof have been described in numerous references including, for example, Mann et al., Cell 33 : 153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.
  • Herpes virus e.g., Herpes Simplex Virus (U.S. Patent No. 5,631,236 by Woo et al., issued May 20, 1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988) Ridgeway, "Mammalian expression vectors," In: Rodriguez R L, Denhardt D T, ed.
  • Vectors A survey of molecular cloning vectors and their uses.
  • viruses include an alphavirus, a poxivirus, an arena virus, a vaccinia virus, a polio virus, and the like. They offer several attractive features for various mammalian cells (Friedmann (1989) Science, 244: 1275-1281 ;
  • target DNA in the genome can be manipulated using well- known methods in the art.
  • the target DNA in the genome can be manipulated by deletion, insertion, and/or mutation are retroviral insertion, artificial chromosome techniques, gene insertion, random insertion with tissue specific promoters, gene targeting, transposable elements and/or any other method for introducing foreign DNA or producing modified DNA/modified nuclear DNA.
  • Other modification techniques include deleting DNA sequences from a genome and/or altering nuclear DNA sequences. Nuclear DNA sequences, for example, may be altered by site-directed mutagenesis.
  • biomarker polypeptides, and fragments thereof can be administered to subjects.
  • fusion proteins can be constructed and administered which have enhanced biological properties.
  • biomarker polypeptides, and fragment thereof can be modified according to well-known
  • pharmacological methods in the art e.g., pegylation, glycosylation, oligomerization, etc.
  • desirable biological activities such as increased bioavailability and decreased proteolytic degradation.
  • the response to a therapy relates to any response of the cancer, e.g., a tumor, to the therapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant chemotherapy.
  • Tumor response may be assessed in a neoadjuvant or adjuvant situation where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation and the cellularity of a tumor can be estimated histologically and compared to the cellularity of a tumor biopsy taken before initiation of treatment.
  • Response may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection.
  • Response may be recorded in a quantitative fashion like percentage change in tumor volume or cellularity or using a semiquantitative scoring system such as residual cancer burden (Symmans et al, J. Clin. Oncol. (2007) 25 :4414-4422) or Miller-Payne score (Ogston et al. , (2003) Breast (Edinburgh, Scotland) 12:320-327) in a qualitative fashion like "pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
  • pCR pathological complete response
  • cCR clinical complete remission
  • cPR clinical partial remission
  • cSD clinical stable disease
  • cPD clinical progressive disease
  • Assessment of tumor response may be performed early after the onset of neoadjuvant or adjuvant therapy, e.g., after a few hours, days, weeks or preferably after a few months.
  • a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed.
  • clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
  • CBR clinical benefit rate
  • the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
  • the CBR for a particular anti-immune checkpoint therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.
  • Additional criteria for evaluating the response to anti-immune checkpoint therapies are related to "survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); "recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
  • the length of said survival may be calculated by reference to a defined start point (e.g., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
  • criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
  • a particular anti- immune checkpoint therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any immune checkpoint therapy.
  • the outcome measurement may be pathologic response to therapy given in the neoadjuvant setting.
  • outcome measures such as overall survival and disease-free survival can be monitored over a period of time for subjects following immune checkpoint therapy for whom biomarker measurement values are known.
  • the same doses of anti-immune checkpoint agents are administered to each subject.
  • the doses administered are standard doses known in the art for anti-immune checkpoint agents. The period of time for which subjects are monitored can vary.
  • Biomarker measurement threshold values that correlate to outcome of an immune checkpoint therapy can be determined using methods such as those described in the Examples section.
  • the methods described herein can be used in a variety of diagnostic, prognostic, and therapeutic applications.
  • any method described herein such as a diagnostic method, prognostic method, therapeutic method, or combination thereof, all steps of the method can be performed by a single actor or, alternatively, by more than one actor.
  • diagnosis can be performed directly by the actor providing therapeutic treatment.
  • a person providing a therapeutic agent can request that a diagnostic assay be performed.
  • the diagnostician and/or the therapeutic interventionist can interpret the diagnostic assay results to determine a therapeutic strategy.
  • such alternative processes can apply to other assays, such as prognostic assays.
  • the compositions described herein can also be used in a variety of diagnostic, prognostic, and therapeutic applications regarding biomarkers described herein, such as those listed in Table 1.
  • any method of diagnosis, prognosis, prevention, and the like described herein can be be applied to a therapy or test agent of interest, such as immune checkpoint therapies, anti-adenosine therapies, anti-cancer therapies, and the like,
  • One aspect of the present invention relates to screening assays, including non-cell based assays.
  • the assays provide a method for identifying whether a cancer is likely to respond to immune checkpoint therapy and/or whether an agent can inhibit the growth of or kill a cancer cell that is unlikely to respond to immune checkpoint therapy.
  • the present invention relates to assays for screening test agents which modulates the amount of at least one biomarker listed in Table 1.
  • a method for identifying such an agent entails determining the ability of the agent to modulate, e.g. reduce, the amount of the at least one biomarker listed in Table 1.
  • an assay is a cell-free or cell-based assay, comprising contacting at least one biomarker listed in Table 1, with a test agent, and determining the ability of the test agent to modulate (e.g. reduce) the amount of the biomarker, such as by measuring direct binding of substrates or by measuring indirect parameters as described below.
  • biomarker molecules can be coupled with a radioisotope or enzymatic label such that binding can be determined by detecting the labeled protein or molecule in a complex.
  • the targets can be labeled with 125 I, 35 S, 14 C, or 3 ⁇ 4, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • the targets can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • Determining the interaction between biomarker and substrate can also be accomplished using standard binding or enzymatic analysis assays.
  • Binding of a test agent to a target can be accomplished in any vessel suitable for containing the reactants.
  • vessels include microtiter plates, test tubes, and micro-centrifuge tubes.
  • Immobilized forms of the antibodies of the present invention can also include antibodies bound to a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of more than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers; a bead, such as that made of agarose or polyacrylamide or latex; or a surface of a dish, plate, or well, such as one made of polystyrene.
  • a solid phase like a porous, microporous (with an average pore diameter less than about one micron) or macroporous (with an average pore diameter of more than about 10 microns) material, such as a membrane, cellulose, nitrocellulose, or glass fibers
  • determining the ability of the agent to modulate the amount of the biomarker can be accomplished by determining the ability of the test agent to modulate the activity of a polypeptide or other product that functions downstream or upstream of its position within the signaling pathway (e.g., feedback loops).
  • feedback loops are well-known in the art (see, for example, Chen and Guillemin (2009) Int. J.
  • the present invention further pertains to novel agents identified by the above- described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent.
  • an antibody identified as described herein can be used in an animal model to determine the mechanism of action of such an agent,
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining the amount and/or activity level of a biomarker listed in Table 1 in the context of a biological sample (e.g., blood, serum, cells, or tissue) to thereby determine whether an individual afflicted with a cancer is likely to respond to immune checkpoint therapy, whether in an original or recurrent cancer.
  • a biological sample e.g., blood, serum, cells, or tissue
  • Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset or after recurrence of a disorder characterized by or associated with biomarker polypeptide, nucleic acid expression or activity.
  • biomarker polypeptide nucleic acid expression or activity.
  • any method can use one or more (e.g., combinations) of biomarkers listed in Table 1.
  • Another aspect of the present invention pertains to monitoring the influence of agents (e.g., drugs, compounds, and small nucleic acid-based molecules) on the expression or activity of a biomarker listed in Table 1.
  • agents e.g., drugs, compounds, and small nucleic acid-based molecules
  • the methods of the present invention implement a computer program and computer system.
  • a computer program can be used to perform the algorithms described herein.
  • a computer system can also store and manipulate data generated by the methods of the present invention which comprises a plurality of biomarker signal changes/profiles which can be used by a computer system in implementing the methods of this invention.
  • a computer system receives biomarker expression data; (ii) stores the data; and (iii) compares the data in any number of ways described herein (e.g., analysis relative to appropriate controls) to determine the state of informative biomarkers from cancerous or pre-cancerous tissue.
  • a computer system (i) compares the determined expression biomarker level to a threshold value; and (ii) outputs an indication of whether said biomarker level is significantly modulated (e.g., above or below) the threshold value, or a phenotype based on said indication.
  • such computer systems are also considered part of the present invention.
  • Numerous types of computer systems can be used to implement the analytic methods of this invention according to knowledge possessed by a skilled artisan in the bioinformatics and/or computer arts.
  • Several software components can be loaded into memory during operation of such a computer system.
  • the software components can comprise both software components that are standard in the art and components that are special to the present invention (e.g., dCHIP software described in Lin et al. (2004) Bioinformatics 20, 1233-1240; radial basis machine learning algorithms (RBM) known in the art).
  • dCHIP software described in Lin et al. (2004) Bioinformatics 20, 1233-1240
  • RBM radial basis machine learning algorithms
  • the methods of the present invention can also be programmed or modeled in mathematical software packages that allow symbolic entry of equations and high-level specification of processing, including specific algorithms to be used, thereby freeing a user of the need to procedurally program individual equations and algorithms.
  • Such packages include, e.g., Matlab from Mathworks (Natick, Mass.), Mathematica from Wolfram Research (Champaign, 111.) or S-Plus from MathSoft (Seattle, Wash.).
  • the computer comprises a database for storage of biomarker data.
  • biomarker production profiles of a sample derived from the non-cancerous tissue of a subject and/or profiles generated from population-based distributions of informative loci of interest in relevant populations of the same species can be stored and later compared to that of a sample derived from the cancerous tissue of the subject or tissue suspected of being cancerous of the subject.
  • the present invention provides, in part, methods, systems, and code for accurately classifying whether a biological sample is associated with a cancer that is likely to respond to immune checkpoint therapy.
  • the present invention is useful for classifying a sample (e.g., from a subject) as associated with or at risk for responding to or not responding to immune checkpoint therapy using a statistical algorithm and/or empirical data (e.g., the amount or activity of a biomarker listed in Table 1).
  • An exemplary method for detecting the amount or activity of a biomarker listed in Table 1, and thus useful for classifying whether a sample is likely or unlikely to respond to immune checkpoint therapy involves obtaining a biological sample from a test subject and contacting the biological sample with an agent, such as a protein-binding agent like an antibody or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of detecting the amount or activity of the biomarker in the biological sample.
  • an agent such as a protein-binding agent like an antibody or antigen-binding fragment thereof, or a nucleic acid-binding agent like an oligonucleotide, capable of detecting the amount or activity of the biomarker in the biological sample.
  • the statistical algorithm is a single learning statistical classifier system.
  • a single learning statistical classifier system can be used to classify a sample as a based upon a prediction or probability value and the presence or level of the biomarker.
  • a single learning statistical classifier system typically classifies the sample as, for example, a likely immune checkpoint therapy responder or progressor sample with a sensitivity, specificity, positive predictive value, negative predictive value, and/or overall accuracy of at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • learning statistical classifier systems include a machine learning algorithmic technique capable of adapting to complex data sets (e.g., panel of markers of interest) and making decisions based upon such data sets.
  • a single learning statistical classifier system such as a classification tree (e.g., random forest) is used.
  • a combination of 2, 3, 4, 5, 6, 7, 8, 9, 10, or more learning statistical classifier systems are used, preferably in tandem.
  • Examples of learning statistical classifier systems include, but are not limited to, those using inductive learning (e.g.,
  • decision/classification trees such as random forests, classification and regression trees (C&RT), boosted trees, etc.), Probably Approximately Correct (PAC) learning, connectionist learning (e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.), reinforcement learning (e.g., passive learning in a known environment such as naive learning, adaptive dynamic learning, and temporal difference learning, passive learning in an unknown environment, active learning in an unknown environment, learning action-value functions, applications of reinforcement learning, etc.), and genetic algorithms and evolutionary programming.
  • connectionist learning e.g., neural networks (NN), artificial neural networks (ANN), neuro fuzzy networks (NFN), network structures, perceptrons such as multi-layer perceptrons, multi-layer feed-forward networks, applications of neural networks, Bayesian learning in belief networks, etc.
  • reinforcement learning e.g., passive learning in a known environment
  • the method of the present invention further comprises sending the sample classification results to a clinician, e.g., an oncologist.
  • a clinician e.g., an oncologist.
  • diagnosis of a subject is followed by administering to the individual a therapeutically effective amount of a defined treatment based upon the diagnosis.
  • the methods further involve obtaining a control biological sample (e.g., biological sample from a subject who does not have a cancer or whose cancer is susceptible to immune checkpoint therapy), a biological sample from the subject during remission, or a biological sample from the subject during treatment for developing a cancer progressing despite immune checkpoint therapy.
  • a control biological sample e.g., biological sample from a subject who does not have a cancer or whose cancer is susceptible to immune checkpoint therapy
  • a biological sample from the subject during remission e.g., a biological sample from the subject during remission
  • a biological sample from the subject during treatment for developing a cancer progressing despite immune checkpoint therapy e.g., a control biological sample
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a cancer that is likely or unlikely to be responsive to immune checkpoint therapy.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation of the amount or activity of at least one biomarker described in Table 1, such as in cancer.
  • the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation of the at least one biomarker described in Table 1, such as in cancer.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with the aberrant biomarker expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, polypeptide, peptide, nucleic acid, small molecule, or other drug candidate
  • compositions described herein can be used in a variety of in vitro and in vivo therapeutic applications using the formulations and/or combinations described herein.
  • anti-immune checkpoint agents can be used to treat cancers determined to be responsive thereto.
  • antibodies that block the interaction between PD-Ll, PD-L2, and/or CTLA-4 and their receptors e.g. , PD-Ll binding to PD-1, PD-L2 binding to PD-1, and the like
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically-effective amount of an agent that modulates (e.g., decreases) biomarker expression and/or activity, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; or (5) aerosol, for example, as an aqueous aerosol, liposomal preparation or solid particles containing the compound.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes
  • parenteral administration for example, by subcutaneous, intramuscular or intravenous injection
  • therapeutically-effective amount means that amount of an agent that modulates (e.g. , inhibits) biomarker expression and/or activity, or expression and/or activity of the complex, or composition comprising an agent that modulates (e.g. , inhibits) biomarker expression and/or activity, or expression and/or activity of the complex, which is effective for producing some desired therapeutic effect, e.g., cancer treatment, at a reasonable benefit/risk ratio.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those agents, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically-acceptable carrier means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject chemical from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and
  • polyethylene glycol polyethylene glycol
  • esters such as ethyl oleate and ethyl laurate
  • agar agar
  • buffering agents such as magnesium hydroxide and aluminum hydroxide
  • alginic acid (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
  • pharmaceutically-acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of the agents that modulates (e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex encompassed by the present invention. These salts can be prepared in situ during the final isolation and purification of the agents, or by separately reacting a purified agent in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • ReRepresentative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like (See, for example, Berge et al. (1977) "Pharmaceutical Salts", J. Pharm. Sci. 66: 1-19).
  • the agents useful in the methods of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically- acceptable salts with pharmaceutically-acceptable bases.
  • pharmaceutically- acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of agents that modulates ⁇ e.g., inhibits) biomarker expression and/or activity, or expression and/or activity of the complex.
  • salts can likewise be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically-acceptable metal cation, with ammonia, or with a pharmaceutically-acceptable organic primary, secondary or tertiary amine.
  • ReRepresentative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • ReRepresentative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like (see, for example, Berge et al , supra).
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabi sulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (
  • Formulations useful in the methods of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, aerosol and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient, which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
  • Methods of preparing these formulations or compositions include the step of bringing into association an agent that modulates (e.g., inhibits) biomarker expression and/or activity, with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association a agent with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or nonaqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a agent as an active ingredient.
  • a compound may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acet
  • compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered peptide or peptidomimetic moistened with an inert liquid diluent.
  • Tablets, and other solid dosage forms may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions, which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding
  • compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active agent may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more agents with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active agent.
  • Formulations which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of an agent that modulates (e.g., inhibits) biomarker expression and/or activity include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active component may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to a agent, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to an agent that modulates (e.g., inhibits) biomarker expression and/or activity, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as
  • the agent that modulates (e.g., inhibits) biomarker expression and/or activity can be alternatively administered by aerosol. This is accomplished by preparing an aqueous aerosol, liposomal preparation or solid particles containing the compound. A nonaqueous (e.g., fluorocarbon propellant) suspension could be used. Sonic nebulizers are preferred because they minimize exposing the agent to shear, which can result in degradation of the compound.
  • an aqueous aerosol is made by formulating an aqueous solution or suspension of the agent together with conventional pharmaceutically acceptable carriers and stabilizers.
  • the carriers and stabilizers vary with the requirements of the particular compound, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), innocuous proteins like serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.
  • Aerosols generally are prepared from isotonic solutions.
  • Transdermal patches have the added advantage of providing controlled delivery of a agent to the body.
  • dosage forms can be made by dissolving or dispersing the agent in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the peptidomimetic across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the peptidomimetic in a polymer matrix or gel.
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions of this invention suitable for parenteral administration comprise one or more agents in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • various antibacterial and antifungal agents for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
  • isotonic agents such as sugars, sodium chloride, and the like into the compositions.
  • prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution, which, in turn, may depend upon crystal size and crystalline form.
  • delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of an agent that modulates (e.g., inhibits) biomarker expression and/or activity, in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
  • agents of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • compositions of this invention may be determined by the methods of the present invention so as to obtain an amount of the active ingredient, which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject.
  • the nucleic acid molecules of the present invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U. S. Pat. No. 5,328,470) or by stereotactic injection (see e.g. , Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91 :3054 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • kits for detecting and/or modulating biomarkers described herein may also include instructional materials disclosing or describing the use of the kit or an antibody of the disclosed invention in a method of the disclosed invention as provided herein.
  • a kit may also include additional components to facilitate the particular application for which the kit is designed.
  • a kit may additionally contain means of detecting the label ⁇ e.g., enzyme substrates for enzymatic labels, filter sets to detect fluorescent labels, appropriate secondary labels such as a sheep anti-mouse-HRP, etc.) and reagents necessary for controls ⁇ e.g. , control biological samples or standards).
  • a kit may additionally include buffers and other reagents recognized for use in a method of the disclosed invention. Non-limiting examples include agents to reduce non-specific binding, such as a carrier protein or a detergent.
  • Example 1 Materials and Methods for Example 2
  • Serum was collected at the specified time-points by centrifugation at 4000g for 4 minutes at 25°C within 2 hours of collection. Samples were frozen immediately and stored at or below -20°C for up to two months followed by storage at -80°C.
  • Polar metabolites were profiled using liquid chromatography tandem mass spectrometry (LC-MS). Briefly, positive ionization mode data were acquired using a 6495 triple quadrupole mass spectrometer coupled to a 1290 Infinity II U-HPLC system (Agilent, Santa Clara, CA). Serum samples (10 ⁇ .) were extracted using 90 of 74.9:24.9:0.2 (v/v/v)
  • acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards 0.2 ng/ ⁇ valine-d8, Isotec.
  • the samples were centrifuged (10 min, 9,000g, 4°C) and the supernatants (10 ⁇ ,) were injected onto a 150 x 2.1 mm Atlantis HILIC column (Waters).
  • the column was eluted isocratically at a flow rate of 250 ⁇ 7 ⁇ with 5% mobile phase A (10 mM ammonium formate and 0.1% formic acid in water) for 1 minute followed by a linear gradient to 40% mobile phase B (acetonitrile with 0.1% formic acid) over 10 minutes.
  • MS data were acquired using multiple reaction monitoring and retention times, mass transitions, and collision energies were determined using authentic reference standards. Other MS parameters were: ion spray voltage, 3.0 kV; source temperature, 200°C; nozzle voltage, 500 V; gas flow, 14 L/min; nebulizer, 40 psi; sheath gas, 250°C; sheath gas flow, 1 L/min; iFunnel high pressure RF, 90; and low pressure RF, 90.
  • Raw data were processed using MassHunter software (Agilent, Santa Clara, CA) for automated peak integration. Metabolite peaks were manually reviewed for quality of integration and compared against standard reference standards to confirm identities.
  • RNA sequencing and immunohistochemistry of RCC tumor biopsies RNA was extracted using standard protocols: 5-10 ⁇ slices from formalin-fixed, paraffin-embedded (FFPE) tumor blocks were obtained, and tumor-enriched tissue was macrodissected. RNA extraction was performed using Qiagen AllPrep DNA/RNA Mini Kit (#51306, Hilden, Germany). Whole transcriptome sequencing from FFPE tissues was aligned using STAR (A. Dobin et al. (2012) Bioinformatics 29: 15-21) and then quantified with RSEM (Li and Dewey (201 1) BMC Bioinformatics 12:323) to yield gene-level expression in transcripts per million (TPM). The RNAseq data in transcripts per million (TPM) were log2 transformed and quantile normalized before further analysis.
  • STAR A. Dobin et al. (2012) Bioinformatics 29: 15-21
  • RSEM Li and Dewey (201 1) BMC Bioinformatics 12:323
  • the more differentially expressed metabolites among patients were pre-selected. Specifically, the variance of each of the 80 metabolites measured in either melanoma or RCC patients was calculated. For each metabolite, this variance was scaled by the corresponding population median. The metabolites with scaled variances above the median value calculated for melanoma and RCC were chose separately for further association analysis with treatment responses. The scaled variances and selected metabolites are included in Tables 4A and 4B.
  • the complete serum metabolomic dataset with all the 80 measured polar metabolites was scaled to have mean 0 and standard deviation 1 to enable relative comparisons.
  • Clustering was done in R with the function hclust.
  • the dissimilarity was calculated as 1 minus the Spearman's correlation between pairwise complete data vectors.
  • the resulting distance matrix was processed by the "ward.D2" method in the hclust function.
  • the heatmap.2 function in the R package gplots was used, g. Gene Set Enrichment Analysis
  • Example 2 Metabolomic correlates of response to PD1 blockade in renal cell carcinoma and melanoma patients
  • PD-L1 as a biomarker is challenged due to the variability among different assays and tumor areas for biopsy, lacking of consensus regarding the cutoff for positivity and responses among PD-L 1 -negative patients (Topalian et al. (2016) Nat. Rev. Cancer 16:275-287). Circulating biomarkers offer the advantages of easy procurement, less sampling bias and monitoring of dynamic changes over time. Recently, pre-treatment serum angiopoietin 2 (ANGPT2) levels and ANGPT2 increases with treatment were associated with reduced response and overall survival in melanoma patients treated with checkpoint inhibitors (Wu et al. (2017) Cancer Immunol. Res. 5: 17-28).
  • ANGPT2 serum angiopoietin 2
  • LC-MS liquid chromatography-mass spectrometry
  • nivolumab therapy was tested during the course of treatment.
  • the metabolite abundance just prior to anti-PD-1 treatment was used as the baseline for each patient, and was compared with the corresponding metabolite levels sampled at later time points.
  • kynurenine was the most significantly changed at week 4 and at week 6 compared to pre- treatment levels (37% increase, q ⁇ l > ⁇ 10 '10 and 34% increase, q ⁇ l l0 "8 respectively, Welch's unequal variances t-test with Benjamini-Hochberg multiple testing correction) ( Figure 1C and ID).
  • microenvironment counterbalance to checkpoint blockade.
  • biomarkers can be selected from Tables 4A-4B for use according to any aspect and/or embodiment of the present invention.
  • Extracellular adenosine which can be produced by the successive actions of CD39 and CD73 (Deaglio et al. (2007) J. Exp. Med. 204: 1257-1265; Mandapathil et al. (2010) J. Biol. Chem. 285:7176-7186) and catabolized by adenosine deaminase (ADA), is known to be functionally important in the tumor microenvironment (Hasko et al. (2008) Nat. Rev.
  • PD-1 resistance in patients with high serum adenosine are due to the maintenance of an immunosuppressive microenvironment with extracellular adenosine, which can be overcome in combination clinical trials with adenosine receptor inhibitors (e.g., direct inhibition of CD39 or CD73, both of which are involved in generating extracellular adenosine (Deaglio et al. (2007), supra; Mandapathil et al. (2010), supra; Borsellino et al. (2007) Blood 1 10: 1225-1232).
  • adenosine receptor inhibitors e.g., direct inhibition of CD39 or CD73, both of which are involved in generating extracellular adenosine
  • IDO/TDO inhibitors are being tested in clinical trials of immune-checkpoint inhibition (Brochez et al. (2017) Eur. J. Cancer 76: 167-182).
  • the observation of significant increases in serum kynurenine levels upon nivolumab treatment indicates that use of IDO/TDO inhibitors in combination with PD 1 checkpoint inhibitors has synergistic effects in the treatment of melanoma, RCC and possibly other malignancies.
  • Adenosine plays an important immune-modulatory role in the tumor environment and pharmacologic targeting of adenosine receptors has been shown to increase anti-tumor T-cell immunity (Ohta et al.
  • immunosuppressive microenvironment state would further justify clinical trials of current immune checkpoint inhibitors in combination with adenosine receptor blockade or inhibition of CD39/CD73, which can generate extracellular adenosine (Deaglio et al.
  • any polynucleotide and polypeptide sequences which reference an accession number correlating to an entry in a public database, such as those maintained by The Institute for Genomic Research (TIGR) on the world wide web and/or the National Center for Biotechnology Information (NCBI) on the worl d wi de web .
  • TIGR The Institute for Genomic Research
  • NCBI National Center for Biotechnology Information

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Abstract

La présente invention concerne, en partie, des méthodes servant à stratifier des patients et à prédire la réponse d'un cancer chez un patient à une thérapie de points de contrôle immunitaire sur la base d'une détermination et d'une analyse des quantités de tels biomarqueurs, par comparaison avec un témoin. De plus, de telles analyses peuvent être utilisées afin de fournir des schémas thérapeutiques de points de contrôle anti-immunitaires utiles (par exemple, sur la base de prédictions de la réponse clinique, de la survie ou de la rechute du patient, de la synchronisation de traitement par adjuvant ou néoadjuvant, etc.). Selon un aspect, l'invention concerne un procédé d'identification de la probabilité qu'un cancer, chez un patient, réponde à une thérapie de point de contrôle immunitaire. Selon un autre aspect, l'invention concerne un procédé d'évaluation de l'efficacité d'un agent pour traiter un cancer chez un patient. La présente invention concerne également des kits de détection et/ou de modulation de biomarqueurs décrits dans la description.
PCT/US2018/058430 2017-11-03 2018-10-31 Biomarqueurs de réponse clinique et de bienfait d'une thérapie par inhibiteur de point de contrôle immunitaire WO2019089740A1 (fr)

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CN113488123A (zh) * 2021-04-21 2021-10-08 广州医科大学附属第一医院 建立基于诊断时效的covid-19分诊系统的方法、该系统及分诊方法
WO2021213292A1 (fr) * 2020-04-20 2021-10-28 山东第一医科大学(山东省医学科学院) Kit de test d'immunofluorescence permettant de mesurer l'expression de pd-l1 dans des cellules tumorales circulantes dans le sang périphérique chez un patient atteint d'un cancer de la prostate, et méthode de mesure
WO2021213261A1 (fr) * 2020-04-20 2021-10-28 山东第一医科大学(山东省医学科学院) Kit et méthode de détection permettant de détecter des mutations du gène pd-l1 dans des cellules tumorales circulantes du sang périphérique d'un patient atteint d'un cancer gastrique
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CN111562375A (zh) * 2020-04-20 2020-08-21 山东第一医科大学(山东省医学科学院) 一种检测胃癌患者外周血循环肿瘤细胞pd-l1表达的免疫荧光试剂盒及检测方法
WO2021213292A1 (fr) * 2020-04-20 2021-10-28 山东第一医科大学(山东省医学科学院) Kit de test d'immunofluorescence permettant de mesurer l'expression de pd-l1 dans des cellules tumorales circulantes dans le sang périphérique chez un patient atteint d'un cancer de la prostate, et méthode de mesure
WO2021213262A1 (fr) * 2020-04-20 2021-10-28 山东第一医科大学(山东省医学科学院) Kit de test d'immunofluorescence pour mesurer l'expression de pd-l1 dans des cellules tumorales circulantes dans le sang périphérique d'un patient atteint d'un cancer de l'estomac et procédé de mesure
WO2021213261A1 (fr) * 2020-04-20 2021-10-28 山东第一医科大学(山东省医学科学院) Kit et méthode de détection permettant de détecter des mutations du gène pd-l1 dans des cellules tumorales circulantes du sang périphérique d'un patient atteint d'un cancer gastrique
CN111562375B (zh) * 2020-04-20 2022-05-06 山东第一医科大学(山东省医学科学院) 一种检测胃癌患者外周血循环肿瘤细胞pd-l1表达的免疫荧光试剂盒及检测方法
CN112735513A (zh) * 2021-01-04 2021-04-30 江苏先声医疗器械有限公司 基于dna甲基化谱的肿瘤免疫检查点抑制剂治疗有效性评估模型的构建方法
CN112735513B (zh) * 2021-01-04 2021-11-19 江苏先声医疗器械有限公司 基于dna甲基化谱的肿瘤免疫检查点抑制剂治疗有效性评估模型的构建方法
CN113488123A (zh) * 2021-04-21 2021-10-08 广州医科大学附属第一医院 建立基于诊断时效的covid-19分诊系统的方法、该系统及分诊方法
CN113488123B (zh) * 2021-04-21 2023-07-18 广州医科大学附属第一医院 建立基于诊断时效的covid-19分诊系统的方法、该系统及分诊方法
WO2024023317A1 (fr) * 2022-07-29 2024-02-01 Astrazeneca Ab Procédés pour détecter des métabolites à l'aide d'un système ce-ms microfluidique

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