WO2016138182A1 - Méthodes et compositions d'immunomodulation - Google Patents

Méthodes et compositions d'immunomodulation Download PDF

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Publication number
WO2016138182A1
WO2016138182A1 PCT/US2016/019423 US2016019423W WO2016138182A1 WO 2016138182 A1 WO2016138182 A1 WO 2016138182A1 US 2016019423 W US2016019423 W US 2016019423W WO 2016138182 A1 WO2016138182 A1 WO 2016138182A1
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Prior art keywords
cells
cell
sample
treatment
immune
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PCT/US2016/019423
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English (en)
Inventor
Drew Hotson
Andy CONROY
Erwan LE SCOLAN
Rachael HAWTIN
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Nodality, Inc.
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Publication of WO2016138182A1 publication Critical patent/WO2016138182A1/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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • cellular pathways may be influenced by cells associated with the condition, e.g., tumor cells, such that certain cells, e.g., immune cells are inhibited or stimulated.
  • Screening potential therapeutic agents, targeting therapy for the condition, and other activities may require an understanding of the functionality of pathways in one or more cell types, and/or optionally expression levels in the cell types and/or in diseased cells such as tumor cells of various ligands, receptors, or other cell components.
  • the invention provides a method of treating a patient suffering from a pathological condition comprising treating the patient with a treatment for the condition.
  • an aspect of treating the patient with the treatment is based on an outcome of a treatment decision process.
  • the treatment decision process comprises consideration of at least two of a first, second, and/or third quantitative value, or a value or values derived from the at least two quantitative values.
  • the first, second, and third quantitative values are obtained from results of a first, second, and/or third assay, respectively.
  • the first assay comprises determining surface expression levels of a first immunomodulatory receptor (FMR) of a first cell population cell population (CP in a first sample from the patient.
  • FMR immunomodulatory receptor
  • the second assay comprises determining functional status of a second EVIR in single cells of a second CP or a subpopulation thereof in a second sample from the patient.
  • the third assay comprises determining surface expression levels of an FMR ligand (IMRL) for a third FMR in a third cell population in a third sample from the patient.
  • IMRL FMR ligand
  • the condition is cancer.
  • the surface expression levels of the first IMR in the first assay are determined in single cells, or the surface expression levels of the IMRL of the third IMR in the third assay are determined in single cells, or both.
  • the method of determination in the assay comprises cytometry.
  • the cytometry is flow cytometry or mass cytometry.
  • the cytometry is flow cytometry.
  • the cytometry is mass cytometry.
  • the aspect of treating the patient comprises a decision to treat the patient or not treat the patient with the treatment, a choice of the treatment or a component of the treatment, a choice of the timing of the treatment or of a component of the treatment, a choice of a dosage of the treatment or a component of the treatment, or a combination thereof.
  • the outcome of the treatment decision process comprises a first likelihood of the patient responding to the treatment, a second likelihood of prolongation of the patient's life due to receiving the treatment, or a third likelihood of the patient experiencing an adverse treatment effect, or any combination of the first, second, and/or third likelihoods.
  • the assays comprise the first assay and the second assay, the assays are performed on single cells, the first and second samples are the same sample, the first and second EVIRs are the same EVER, and the first and second cell populations are the same population, and the second quantitative value represents a functional status of the IMR for the subpopulation of the population, the process of obtaining the second quantitative value comprises gating the results for functional status of the IMR in the single cells of the cell population on the basis of the results of the
  • the gating comprises establishing a threshold for expression level of the IMR in a single cell and single cells in the cell population having an expression level of the IMR above the threshold are included in the subpopulation and single cells in the cell population having an expression level equal to or below, or below, the threshold are excluded from the subpopulation.
  • the treatment is a combination treatment comprising an immunotherapy treatment.
  • the combination treatment further comprises a targeted treatment, a chemotherapy treatment, a radiation treatment, or a surgical treatment.
  • the first and second cell populations are immune cell populations.
  • the first and second immune cell populations are the same immune cell population. In some embodiments, the first and second immune cell populations are different immune cell populations. In some embodiments, the third cell population is a non-immune cell population. In some embodiments, the third cell population is a tumor cell population.
  • the first sample and the second sample, and optionally the third sample are the same sample.
  • the sample is a blood or blood- derived sample, or bone marrow or bone marrow-derived sample.
  • the first and second samples, and optionally the third sample are solid samples or solid-sample- derived samples.
  • the sample comprise a tumor sample.
  • the tumor sample is a primary tumor sample or a metastatic tumor sample.
  • the first and second samples comprise tumor-infiltrating lymphocytes (TILS) derived from a solid tumor sample and the third sample comprises tumor cells derived from the same solid tumor sample.
  • the sample is a peripheral blood mononuclear cell (PBMC) sample.
  • PBMC peripheral blood mononuclear cell
  • the surface expression levels of a plurality of IMRLs in the third assay are determined in single cells.
  • the plurality of IMRLs comprises a plurality of IMRLS of Figure 15 and the description thereof.
  • the surface expression levels of a plurality of first FMRs in the first assay are determined.
  • the surface expression levels of a plurality of second FMRs in the second assay are determined.
  • the surface expression levels of a plurality of FMRs in the first assay and the second assay are determined.
  • the plurality of FMRs comprises a plurality of FMRs of Figure 15 and the description thereof.
  • the condition is cancer
  • the therapy is a combination therapy comprising immunotherapy
  • in first assay and second assay a plurality of IMRs is assayed
  • the aspect of the treatment comprises choice of the combination therapy.
  • the assay of the functional status of the FMR in the second assay comprises determining the change in an activation level of an intracellular activatable element or change in expression level of an intracellular expression element.
  • the activatable element is an activatable element of TABLE 1, or Figure 20.
  • the activatable element comprises p-ERK or p-AKT.
  • the treatment decision process further comprises consideration of a characteristic of the patient.
  • the characteristic comprises a genetic characteristic, age, gender, race, health status, previous treatment history, or any combination thereof.
  • the first and second cell populations comprise a first and second cell immune cell population of TABLE 1 or Figure 17. In some
  • the first and second cell population are the same cell population.
  • the cell populations are identified by surface expression levels of at least three of the cell surface markers of Table 1 or Figure 17.
  • the F RL corresponds to the first EVER in the first assay or the second EVER in the second assay.
  • the second assay comprises determining the functional status of the EVER in the presence and absence of an
  • the second assay comprises determining the functional status of the EVER in the presence and absence of a plurality of
  • the invention further provides a kit comprising (i) a distinguishably detectable binding element configured for use in binding to and distinguishably detecting a first intracellular element; and (ii) a distinguishably detectable binding element configured for use in binding to and distinguishably detecting a cell surface EVER on the cell or a cell surface EVERL on a cell of a population of cells of a non-immune cell type.
  • a change in the expression level and/or activation level of the first intracellular element in a cell of an immune cell type in response to exposure of the cell to an activator of the immune cell type is indicative of activation of the cell.
  • the kit further comprises the activator of the immune cell type.
  • the kit comprises a plurality of distinguishably detectable binding elements configured for use in binding to and
  • the kit further comprises instructions for use of the kit in an assay for predicting the response of a patient to immunotherapy.
  • the immunotherapy is an immunotherapy that directly or indirectly affects activation of the population of cells of the immune cell type.
  • the kit further comprises a plurality of distinguishably detectable binding elements, each configured for use in binding to and distinguishably detecting a different cell surface marker.
  • the level of at least two of the plurality of different cell surface markers can be used to type the cell as a cell of an immune cell population.
  • the plurality of different surface EVERs and/or the plurality of different cell surface EVERLs is a plurality of different surface IMRs and/or a plurality of different cell surface IMRLs of Figure 15 and the description thereof.
  • the plurality of FMRs comprise PD-1 and CTLA-4 and the plurality of FMRLs comprise at least two of B7-1, B7-2, PDL-1, and PDL-2.
  • the plurality of cell surface markers comprise a plurality of cell surface markers listed in TABLE 1 or Figure 17.
  • the cell surface FMR or the cell surface FMRL comprises an FMR or an FMRL or of FIGURE 15 and the description thereof.
  • the FMR is PD-1 and the FMRL is PDL-1 or PDL-2.
  • the intracellular element is an intracellular activatable element.
  • the activatable element is an activatable element of TABLE 1 or Figure 20.
  • the invention further provides a kit comprising at least three distinguishably detectable binding elements.
  • the at least three distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, or three different cell surface FMRs on single cells of an immune cell population and/or at least one, two, or three cell surface FMRLs on single cells of a nonimmune cell population.
  • the FMR or FMRs, and/or FMRL or FMRLs are an IMR and/or FMRL of Figure 15 and the description thereof.
  • the kit comprises at least five distinguishably detectable binding elements, In some embodiments, the at least five distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, three, four, or five different cell surface FMRs on single cells of an immune cell population and/or at least one, two, three, four cell or five surface FMRLs on single cells of a non-immune cell population. In some embodiments, the kit comprises at least five detectable binding elements. In some embodiments, at least three distinguishably detectable binding elements are each configured for use in binding to and distinguishably detecting a cell surface FMR on the cell of an immune cell population or a cell surface FMRL on a cell of a non-immune cell population.
  • the invention further provides a pharmaceutical package comprising one or more immunotherapeutic agents and (i) instructions and/or an imprint indicating that the one or more immunotherapeutic agents is to be used for treatment of a patient who suffers from a pathological condition; (ii) instructions and/or an imprint indicating that the patient is to be stratified by one or more the methods described herein that produces a result that can be used to determine if condition (i)(a), (b), (c), and/or (d) is satisfied; and/or (iii) one or more necessary materials to carry out the one or more of methods of part (ii).
  • cells e.g., cells associated with the patient's pathological condition, an immune cell population from a sample from the patient
  • non-cell samples e.g., a non-cell liquid from a sample from the patient
  • cells associated with the patient's pathological condition are characterized by surface expression of an IMRL at a level greater than, or greater than or equal to a threshold level of expression or surface expression of a plurality of different IMRLs at levels greater than, or greater than or equal to, a plurality of threshold expression levels.
  • an immune cell population from a sample from the patient is characterized by surface expression level of a first IMR that is greater than, or greater than or equal to a threshold expression level. In some embodiments, an immune cell population from a sample from the patient is characterized by a change in the expression level and/or activation level of an intracellular element that is less than, or less than or equal to a threshold change.
  • the change in the expression level or activation level of the intracellular element in a cell of an immune cell type is in response to contact with an activator of that immune cell type and is indicative of the activation level of the cell, and the change in the level may be measured in the presence and/or absence of an activator and/or inhibitor of an FMR that can be expressed on the cell of the immune cell type.
  • a non-cell liquid from a sample from the patient contains an immune effector molecule at a level greater than, greater than or equal to, less than, or less than or equal to a threshold level.
  • any combination of the above-mentioned cells or non-cell samples can be characterized.
  • the pharmaceutical package further comprises one or more components for use in gathering, treating, and/or transporting one or more samples from the patient for use in the one or more methods of the above-mentioned
  • the pathological condition is cancer.
  • the cells associated with the pathological condition comprise tumor cells.
  • the cancer is characterized by tumor cell surface expression of an FMRL that modulates an inhibitory IMR of Figure 15 and the description thereof.
  • the tumor cell surface expression level of the FMRL is greater than, or greater than or equal to, a threshold level.
  • the cancer is characterized by tumor cell surface expression of an FMRL that activates PD-1. In some embodiments, the cancer is
  • the cancer is characterized by tumor cell surface expression of an IMRL that activates PD-1 and tumor cell surface expression of an EVIRL that activates CTLA4.
  • the intracellular element is an intracellular activatable element and the activation level of the element is indicative of the activation level of the cell.
  • the intracellular activatable element is an activatable element of TABLE 1 or Figure 20.
  • the intracellular activatable element comprises p-ERK, p- AKT, P-ZAP70, PLCg, p-PKC ⁇ , p-p38, or pNFkBp65.
  • the activatable element comprises p-ERK or p-AKT.
  • the intracellular activatable element comprises p-STATl, p-STAT3, p-STAT4, p-STAT5, or p-STAT6, or a combination thereof.
  • the invention further provides a method for screening a first agent at a first screening level.
  • the method comprises (i) contacting a first immune cell population expressing a first IMR on their surfaces with the first agent and activating the cells of the first population by contacting them with an activator; (ii) activating the cells of a second immune cell population expressing the first IMR on their surfaces that have not been contacted with the first agent by contacting them with the activator; (iii) determining (a) expression levels of an intracellular expression element in single cells of the first population or a subpopulation thereof and expression levels of the intracellular element in single cells of the second population or a subpopulation thereof, and/or (b) activation levels of an intracellular activatable element in single cells of the first population or a subpopulation thereof and activation levels of the intracellular activatable element in single cells of the second population or a subpopulation thereof; (iv) making a determination to send or not send the agent to a second screening level based on the results of (i)
  • the intracellular expression element is an element whose expression levels changes upon activation of the cells of the first and second immune cell populations.
  • the intracellular activatable element is an activatable element whose activation level changes upon activation of the cell of the first and second immune cell populations.
  • the determination of step (iv) comprises an evaluation of a result of a comparison of the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element in the single cells of the first population, or a first quantitative value derived therefrom, with the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element in the single cells of the second population, or a second quantitative value derived therefrom.
  • the result is a third quantitative value.
  • the determination of step (iv) comprises comparing the third quantitative value with a threshold value to determine if the third value is greater than, greater than or equal to, less than, or less than or equal to the threshold value.
  • the agent is sent to the second screening level if the third quantitative value is greater than, or greater than or equal to, the threshold value.
  • the agent is sent to the second screening level if the third quantitative value is less than, or less than or equal to, the threshold value.
  • the first and second cell populations are the same immune cell population.
  • the identity of the first and second immune cell populations is determined by determining the levels of at least one cell surface marker in single cells of the first and second immune cell populations.
  • the method further comprises determining the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element in single cells of a third immune cell population type that have not been activated and that have not been contacted with the agent.
  • the first, second, and third immune cell populations are the same immune cell population.
  • the method further comprises determining surface expression levels of the first IMR in single cells of the first and second immune cell populations.
  • the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element are determined in subpopulations of the first and second immune cell populations.
  • a cell is gated into the subpopulation of the first or second population on the basis of its surface expression level of the first IMR.
  • a cell is gated by comparing its surface expression level of the IMR to a threshold expression level value for the first EVER.
  • the cell is gated into the subpopulation if its surface expression level of the first EVER is greater than the threshold value, or greater than or equal to the threshold value.
  • the method further comprises screening a second agent in combination with the first agent and step (i) of the above-mentioned method of screening further comprises contacting the first immune cell population with the second agent; and the cells of the second immune cell population further express the second EVER on their surfaces and in step (ii) of the above-mentioned method of screening the cells of the second population have not been contacted with the second agent.
  • the second agent is different from the first agent.
  • the cells of the first immune cell population further express a second EVER on their surfaces.
  • the method further comprises determining surface expression levels of the second EMR in single cells of the first and second immune cell populations.
  • the expression levels of the intracellular expression element and/or the activation levels of the intracellular activatable element are determined in subpopulations of the first and second populations.
  • a cell is gated into the subpopulation of the first and second population on the basis of its surface expression level of the first IMR and its surface expression level of the second IMR.
  • a cell is gated by comparing its surface expression level of the first IMR to a threshold expression level value for the first IMR and its surface expression level of the second IMR to a threshold expression level value for the second IMR.
  • the cell is gated into the subpopulation if its surface expression level of the first FMR is greater than the threshold value for the surface expression level of the first FMR and its surface expression level of the second FMR is greater than the threshold value for the surface expression level of the second FMR, or greater than or equal to the threshold values for the surface expression of the first and second FMRs.
  • the cells of the first and second immune cell populations expressing the first FMR have been induced to express the first FMR by activation of the cells of the first and second immune cell populations at a time previous to steps (i) and (ii).
  • the cells are derived from a sample from a healthy individual, a plurality of samples from the healthy individual, or a plurality of samples from a plurality of healthy individuals.
  • the cells are from cell lines.
  • the cells are derived from a sample from an individual suffering from a pathological condition, or a plurality of samples from the individual, or a plurality of samples from a plurality of individuals suffering from the pathological condition.
  • the cells are derived from a sample from an individual suffering from a pathological condition, or a plurality of samples from the individual, or a plurality of samples from a plurality of individuals suffering from the pathological condition.
  • pathological condition is cancer.
  • the invention further provides a method of determining a phenotype of a population of cells of an immune cell population in a sample from a patient suffering from a pathological condition, comprising determining in single cells of the immune cell population surface expression levels of at least at least three different FMRs, and determining the phenotype based on the levels of the at least three different IMRs.
  • the method comprises determining the phenotype based on surface expression levels of at least 4 different FMRs on single cells of the population.
  • the invention further provides a method of treating a patient comprising determining an immunotherapy for the patient, based on a phenotype determined by the above-mentioned method.
  • the immunotherapy is a combination immunotherapy.
  • the combination immunotherapy is a combination comprising at least two different immunotherapies.
  • the invention further provides a method of determining a phenotype of a population of cells of an non-immune cell population in a sample from a patient suffering from a pathological condition, comprising determining in single cells of the cell population surface expression levels of at least at least three different IMRLs and determining the phenotype based on the levels of the at least three different IMRLs.
  • the method comprises determining the phenotype based on surface expression levels of at least 4 different EVIRLs on single cells of the population.
  • the invention further provides a method of treating a patient comprising determining an immunotherapy for the patient based on a phenotype determined by the above-mentioned method.
  • the immunotherapy is a combination immunotherapy.
  • the immunotherapy is a combination immunotherapy.
  • the immunotherapy is a combination immunotherapy.
  • the immunotherapy is a combination immunotherapy.
  • the combination immunotherapy is a combination of two different immunotherapies.
  • the invention further provides a method of determining a phenotype of a population of cells of an immune cell population in a sample from a patient suffering from a pathological condition, comprising determining in single cells of the cell population a functional status of an IMR expressed on the surface of the cells and determining the phenotype based on the functional status of the IMR.
  • the method comprises determining the phenotype based on surface expression levels of at least 2 different EVIRs expressed on the surfaces of single cells of the population.
  • the method further comprises determining the surface expression levels of the EVER in the single cells.
  • the cell population is a subpopulation of an immune cell population.
  • each single cell is placed or not placed in the subpopulation based on its surface expression level of the IMR.
  • the invention further provides a method of treating a patient comprising determining an immunotherapy for the patient based on a phenotype determined by the above-mentioned method.
  • the immunotherapy is a combination immunotherapy.
  • the combination immunotherapy is a combination of two different immunotherapies.
  • the invention further provides a method of treating a patient suffering from a pathological condition.
  • the method comprises treating the patient with a treatment for the condition.
  • an aspect of treating the patient with the treatment is based on an outcome of a treatment decision process comprising consideration of a quantitative value, or a value or values derived from the quantitative value.
  • the quantitative value is obtained from results of an assay comprising determining functional status of an IMR in single cells of an immune cell population or a subpopulation thereof in a sample from the patient.
  • the method further comprises determining surface expression levels of the FMR in the single cells.
  • the method is performed using a subpopulation of the immune cell population.
  • single cells of the subpopulation are gated into the subpopulation on the basis of the surface expression level of the IMR of the single cell.
  • the method of determination in the assay comprises cytometry.
  • the cytometry is flow cytometry or mass cytometry.
  • the cytometry is flow cytometry.
  • the cytometry is mass cytometry.
  • the aspect of treating the patient comprises a decision to treat the patient or not treat the patient with the treatment, a choice of the treatment or a component of the treatment, a choice of the timing of the treatment or of a component of the treatment, a choice of a dosage of the treatment or a component of the treatment, or a combination thereof.
  • the outcome of the treatment decision process comprises a first likelihood of the patient responding to the treatment, a second likelihood of prolongation of the patient's life due to receiving the treatment, or a third likelihood of the patient experiencing an adverse treatment effect, or any combination of the first, second, and/or third likelihoods.
  • the pathological condition is cancer.
  • the invention further provides a method of diagnosing, prognosing, predicting, or monitoring an individual suffering from or suspected of suffering from a solid tumor, comprising evaluating single non-tumor cells in a non-tumor sample taken from the individual.
  • the single cells can be immune cells.
  • the sample can be a blood or blood-derived sample, e.g., a PBMC sample.
  • the sample can be a bone marrow mononuclear cell (BMMC) sample.
  • the cells can be immune cells, e.g., immune cells belonging to one or more immune cell populations as shown in Table 1 or Figure 17.
  • the method can comprise measuring cell surface markers to place the cells in an immune cell population or subpopulation and measuring the activation levels of one or more activatable elements in the cells, wherein the measuring is performed in single cells of the sample.
  • the method can further comprise treating the cells with a modulator, such as a cytokine, a TCR activator, a BCR activator, or a TLR receptor activator.
  • the modulator can comprise a modulator, e.g., activator, of Table 1 or Figure 20A or 20B.
  • the activatable element can be an activatable element of Table 1 or Figure 20A or 20B.
  • the cells can be assessed for expression level of one or more FMRs or FMRLs, on a single cell basis, such as one or more FMRs or FMRLs of Figure 15.
  • the cells can be assessed for expression level of two or more FMRs or FMRLs, on a single cell basis.
  • the cells can be assessed for expression level of three or more IMRs or IMRLs, on a single cell basis.
  • the IMR or IMRL can comprise PD1 or PDL1.
  • the cancer is melanoma, breast cancer, lung cancer, e.g., small cell lung carcinoma or non-small cell lung carcinoma, or prostate cancer.
  • the cancer is melanoma or breast cancer.
  • the cancer is melanoma.
  • the cancer is breast cancer.
  • the invention provides a method of diagnosing, prognosing, predicting, or monitoring an individual suffering from or suspected of suffering from breast cancer, comprising evaluating single non-tumor cells in a non-tumor sample taken from the individual.
  • the single cells can be immune cells.
  • the sample can be a blood or blood- derived sample, such as a PBMC sample.
  • the sample can be a bone marrow mononuclear cell (BMMC) sample.
  • the cells can be immune cells belonging to one or more immune cell populations, such as shown in Table 1 or Figure 17.
  • the method can comprise measuring cell surface markers to place the cells in an immune cell population or subpopulation and measuring the activation levels of one or more activatable elements in the cells, wherein the measuring is performed in single cells of the sample.
  • the method can include further treating the cells with a modulator.
  • the modulator can comprise a cytokine, a TCR activator, a BCR activator, or a TLR receptor activator.
  • the modulator can comprise a modulator, e.g., activator, of Table 1 or Figure 20A or 20B.
  • the activatable element is an activatable element of Table 1 or Figure 20A or 20B.
  • the modulator can be a TCR activator.
  • the activatable element can be an activatable element in the TCR pathway.
  • the activatable element can be selected from the group consisting of p-ERK, p-AKT, p-PLCg2, p-CD3z, p-s6, and combinations thereof.
  • the cells can be assessed for expression level of one or more FMRs or FMRLs, on a single cell basis, such as one or more FMRs or FMRLs are those of Figure 15.
  • the cells can be assessed for expression level of two or more FMRs or FMRLs, on a single cell basis.
  • the cells can be assessed for expression level of three or more FMRs or FMRLs, on a single cell basis.
  • the one or more FMRs or IMRLs are selected from the group consisting of PD1, PDL1, OX-40, TFM-3, GITR.
  • the FMR or FMRL comprises PD1 or PDLl .
  • the invention provides a method of diagnosing, prognosing, predicting, or monitoring an individual suffering from or suspected of suffering from melanoma, comprising evaluating single non-tumor cells in a non-tumor sample taken from the individual, the single cells can be immune cells.
  • the sample can be a blood or blood- derived sample, such as a PBMC sample.
  • the sample can be a bone marrow mononuclear cell (BMMC) sample.
  • the cells can be immune cells, such as belonging to one or more immune cell populations as shown in Table 1 or Figure 17.
  • the method can comprise measuring cell surface markers to place the cells in an immune cell population or
  • the cells can further be treated with a modulator.
  • the modulator can comprise a cytokine, a TCR activator, a BCR activator, or a TLR receptor activator.
  • the modulator comprises a cytokine, such as an interleukin, for example IL15.
  • the modulator comprises a modulator, e.g., activator, of Table 1 or Figure 20A or 20B.
  • the activatable element is an activatable element of Table 1 or Figure 20A or 20B, such as an activatable element downstream of cytokine activation as shown in Figure 20A or 20B.
  • the activatable element is selected from the group consisting of p-STAT 1, p-STAT 3, p-STAT 4, p-STAT 5, p-STAT 6, and combinations thereof.
  • the activatable element comprises p-STAT5.
  • the cells are assessed for expression level of one or more FMRs or FMRLs, on a single cell basis.
  • the one or more FMRs or FMRLs are those of Figure 15.
  • the cells are assessed for expression level of two or more FMRs or FMRLs, on a single cell basis. In certain embodiments, the cells are assessed for expression level of three or more FMRs or FMRLs, on a single cell basis. In certain embodiments, the FMR or FMRL comprises PD1 or PDL1.
  • the invention provides a kit for evaluating single non-tumor cells in a non-tumor sample taken from an individual suffering from or suspected of suffering from a solid tumor, comprising (i) one or more distinguishably detectable binding elements specific to cell surface proteins on the non-tumor cells; (ii) one or more distinguishably detectable binding elements specific to one or more activatable elements in the non-tumor cells; and (iii) one or more distinguishably detectable binding elements specific to one or more FMRs and/or FMRLs on the surface of the non-tumor cells.
  • the kit comprises at least two or more distinguishably detectable binding elements specific to cell surface proteins on the non-tumor cells.
  • kit comprises at least two or more distinguishably detectable binding elements specific to two or more activatable elements in the non-tumor cells. In certain embodiments, the kit comprises at least two or more distinguishably detectable binding elements specific to two or more FMRs and/or FMRLs on the surface of the non-tumor cells. In certain embodiments, the kit comprises at least three or more distinguishably detectable binding elements specific to three or more IMRs and/or IMRLs on the surface of the non-tumor cells. In certain embodiments, the kit comprises at least four or more distinguishably detectable binding elements specific to four or more IMRs and/or IMRLs on the surface of the non-tumor cells. In certain embodiments, the kit comprisses at least five or more distinguishably detectable binding elements specific to five or more IMRs and/or IMRLs on the surface of the non-tumor cells. In certain
  • the kit comprises at least six or more distinguishably detectable binding elements specific to six or more IMRs and/or IMRLs on the surface of the non-tumor cells.
  • Cell surface markers are as described herein.
  • Activatable elements are as described herein.
  • IMR and IMRLs are as described herein.
  • the kit can further comprise additional component, such as described in the Section "Kits,” herein.
  • Figure 1 depicts an example of the immune system cell communication network.
  • Figure 2 provides results from Example 10.
  • Figure 3 shows patient demographics for Example 11.
  • Figure 4 shows monocyte hyporesponsiveness in melanoma vs. healthy patients.
  • Figure 5 shows TCR signaling in various patient groups and health individual in Example 11.
  • Figure 6 shows reduced IL-15 signaling in samples from patients that received ipilimumab.
  • Figure 7 shows CTLA-4 defined differential signaling populations in CD4+ T cells.
  • Figure 8 shows ipilimumab promotes in vitro T cell activation.
  • Figure 9 shows co-stimulation effects of anti-CD3, anti-CD28, anti-PDl, and anti- ICOS, in various combinations, in normal T cells.
  • Figure 10 shows signaling pathways interrogated in healthy and CLL T cells.
  • Figure 11 shows basal signaling in CLL vs. healthy T cells.
  • Figure 12 shows modulated signaling in CLL vs. healthy T cells.
  • Figure 13 shows increased TCR signaling in CLL PD1+ CD8 T cells compared to healthy.
  • Figure 14 shows decreased TCR induced proliferation in CLL PD-1+ cells compared to healthy, c311s activated 48 hr with anti-CD3/CD28 +/- PD-1 blockade.
  • FIG. 15 shows exemplary immunomodulator receptors (FMRs), which can be expressed on the surface of cells of one or more immune cell populations and which have a role in immunomodulation in normal immune function as well as immunomodulation in a variety of pathological conditions, for example, immunosuppression in cancer, and which can be inhibitory (decrease the activation of the cells in response to one or more activators) or costimulatory (increase activation of the cells in response to one or more activators).
  • a single cell may have multiple FMRs, which can be of either or both types (inhibitory and/or costimulatory)
  • the inhibitory FMRs shown in this Figure, and their corresponding FMRLs, as well as an additional inhibitory FMR not shown in the Figure, and its FMRL, are examples of the inhibitory FMRs shown in this Figure, and their corresponding FMRLs, as well as an additional inhibitory FMR not shown in the Figure, and its FMRL, are
  • inhib FMR CTLA-4, FMRLs: B7-1 (aka CD80), B7-2 (aka CD86)
  • inhib FMR PD-1
  • FMRLs PD-L1 (aka CD274, B741)
  • PDL-2 aka CD273, B7DC
  • inhib FMR BTLA (aka CD272)
  • FMRL HVEM
  • inhib FMR LAG3 (aka CD223)
  • FMRL MHC class II molecules
  • inhib FMR TFM-3 (HAvcr2), FMRL: Gal9
  • inhib FMR VISTA
  • FMRL unknown (putatively VISTAL, or the ligand for VISTA);
  • inhib FMR A2aR
  • FMRL adenosine.
  • Treg cells express high levels of the exoenzymes CD39 (aka NTPDase 1), which converts extracellular ATP to AMP, and CD73 (aka 5' -NT), which converts AMP to adenosine.
  • CD39 aka NTPDase 1
  • CD73 aka 5' -NT
  • A2aR engagement by adenosine drives T cells to become Treg cells, this can produce a self-amplifying loop within the tumor, and expression levels of one or both.
  • A2aR is an important EVIR, adenosine an important IMRL, and the exoenzymes CD39 and CD73 important immune effector molecules, any of which or any combination of which may be used as markers to determine immunosuppression, e.g., in the tumor microenvironment or in peripheral blood, and/or as target or targets for immunotherapy
  • costim EVIR CD28, FMRL: B7-1 (aka CD80);
  • costim FMR GITR (aka T FRSR18, AITR, CD357, GITR-D), FMRL: GITRL;
  • costim FMR OX-40 (aka CD 134, T FRSF4, ACT35, FMD16, TXGP1L), FMRL: OX-40L,
  • costim FMR 4-lBB (aka CD137, TNFRSF9, ILA, CDwl37), FMRL: 4-1BBL,
  • costim FMR CD40L (aka CD 154, CD40 ligand; it is a costimulatory FMR despite the misleading name), FMRL: CD40,
  • costim FMR CD27, FMRL: CD70
  • costim FMR ICOS (aka CD278)
  • FMRL B7-RP1 (aka CD275, ICOSLG).
  • Fig 15 also shows a group of FMRs designated KIRs (killer cell immunoglobulin-like receptors), some of which are costimulatory FMRs and some of which are inhibitory FMRs, expressed on K cells and certain T cells, FMRLs: MHC class I molecules.
  • KIRs killer cell immunoglobulin-like receptors
  • compositions and methods of the invention involve measuring, in single cells, expression levels of one or more of CTLA-4, PD-1, PD-Ll, TFM- 3, LAG3, GITR, OX40, CD27, 4-lBB, and/or CD40L.
  • compositions and methods of the invention involve measuring, in single cells, expression levels of two or more of CTLA-4, PD-1, PD-Ll, TFM-3, LAG3, GITR, OX40, CD27, 4-lBB, and/or CD40L
  • the compositions and methods of the invention involve measuring, in single cells, expression levels of three or more of CTLA-4, PD-1, PD- Ll, TFM-3, LAG3, GITR, OX40, CD27, 4-lBB, and/or CD40L
  • the compositions and methods of the invention involve measuring, in single cells, expression levels of four or more of CTLA-4, PD-1, PD-Ll, TFM-3, LAG3, GITR, OX40, CD27, 4- 1BB, and/or CD40L
  • the compositions and methods of the invention involve measuring, in single cells, expression levels of five or more of CTLA-4, PD-1, PD- Ll, TFM-3, LAG3,
  • Figure 16 shows selected results from Example 15, in which cells from samples from AML patients and from healthy volunteers were compared for surface expression levels of four costimulatory FMRs (4-lBB, OX-40, CD27, and GITR), three inhibitory FMRs (PD-1, LAG3, and TFM-3), and an FMRL (PD-Ll), which were measured in single cells from different cell populations to determine surface expression levels of the FMRs and FMRLs in the different cell populations.
  • Cell surface expression levels of PD-1, PD-Ll, TFM-3, 4-lBB, and OX-40 in both CD4+ and CD8+ cell populations are shown; both PD-1 and OX-40 showed upregulation in cell populations in the AML patients.
  • Figure 17 shows several populations of immune cells that can be the subject of the methods and compositions of the invention, and a selection of their corresponding cell surface markers.
  • CD3+CD4+ cells are Thelper lineage cell populations
  • CD3+CD8+ cells are Tcytotoxic lineage cell populations
  • EM effector memory cell subpopulations (T helper subpopulation if CD4+CD62Llow,CD45RAlow, Tcyto subpopulation if CD8+CD62LlowCD45RAhigh)
  • CM central memory cell subpopulation (T helper subpopulation if CD4+CD62LhighCD45RAlow, Tcyto subpopulation if
  • CD8+CD62LhighCD45RAlow CD8+CD62LhighCD45RAlow
  • E effector cell subpopulation (Thelper subpopulation if CD4+CD62LlowCD45RAhigh, Tcyto subpopulation if CD8+CD62LlowCD45RAhigh)
  • N naive cell subpopulation (Thelper subpopulation if CD4+CD62LhighCD45RAhigh, Tcyto subpopulation if CD8+CD62LhighCD45RAhigh).
  • Treg subpopulation if CD4+Foxp3+ which can be further subdivided into CD4+CD25+Foxp3+ Treg subpopulation and CD4+CD25-Foxp3+Treg subpopulation.
  • CD3- cells are non-T cell lineage cell populations, of which B cells, NK cell, and monocytes are subpopulations. or Cell surface markers for B cell subpopulations are CD3-CD14-CD20+, which can be further divided into subpopulations on the basis of CD27 (+ or -), IgD (+ or -) (not shown, see Table 1.
  • NK cell subpopulation is CD3-CD19-CD14- CD20-CD56+, further subdivided into subpopulations CD56bright and CD56dim.
  • Figure 18 shows the protocol and results of Example 16.
  • Figure 19 shows the protocol and results of Example 17.
  • Figure 20A shows exemplary pathways interrogated by SCNP, activators for the pathways, intracellular expressed elements in the pathways, and intracellular activatable elements in the pathways.
  • Figure 20B shows exemplary pathways interrogated by SCNP, activators for the pathways, intracellular expressed elements in the pathways, and intracellular activatable elements in the pathways, an alternative view.
  • Figure 21 A shows neutralization of KIR signaling in NK cells enhances degranulation of the NK cells.
  • CD107a is used as a surrogate marker for degranulation. See Example 18.
  • Figure 21B shows potential to associate induced degranulation of NK cells to response to therapy.
  • Figure 22 illustrates heterogeneity of FMR expression in AML compared to healthy cells.
  • Figure 23 shows elevated FMR expression in CD34+ cells from AML donors.
  • Figure 24 shows overall signaling response (Uu metric) in AML samples in the context of PD-1 expression, with reduced signaling in PD1+ subsets.
  • Figure 25 shows selected data from Figure 24, shown as log2 differences. Fewer patients are shown because a cutoff of 50 events per readout was sued.
  • FIG. 26 illustrates that Single Cell Network Profiling (SCNP) enables analysis of signaling in the context of FMR expression.
  • SCNP Single Cell Network Profiling
  • Figure 27 shows FMR profiling in CLL and healthy donors (HD)
  • Figure 28 shows expression of FMRS in CD8+ T cell subsets.
  • Figure 29 shows reduced TCR dependent p-ERK and p-Akt signaling in T cells observed when comparing CLL to healthy donors correlated to reduced TIM3 expression.
  • Figure 30 shows CLL donors show higher IL-2 induced signaling in CD8+ T cell subsets compared to healthy donors.
  • Figure 31 shows CLL donors show higher IL-2 induced signaling in CD8+ T cell subsets compared to healthy donors
  • Figure 32 shows SCNP identifies "association" between signaling and CLL surrogate markers.
  • Figure 33 shows profiling of cell signaling capacity in PD-1+ and PD-1- cell subsets defines functional signaling differences in CLL donor T cells.
  • Figure 34 shows differential cytokines modulated signaling in CD4+ T cells.
  • Figure 35 shows quantification of PI3Ki and BTKi activity in PD1+ vs PD1- T cell subsets.
  • Figure 36 shows SCNP identifies Akt independent phaosphorylation of S6 by measuring BTKi activity in T cells.
  • Figure 37 shows the metrics used in Example 21.
  • Figure 38 shows breast cancer samples display elevated levels of PD-1 and PD-L1 expression as compared to healthy.
  • Figure 39 shows high expression of OX-40 and TFM-3 is also observed in breast cancer donor samples relative to healthy.
  • Figure 40 shows trends observed in PD-L1 expression patterns on K cells in breast cancer patients treated with Fresolimumab.
  • Figure 41 shows slightly elevated FMR expression patterns with higher dose of Fresolimumab in breast cancer patients.
  • Figure 42 shows FMR expression patterns show subtle changes over the course of treatment in breast cancer patients.
  • Figure 43 shows TCR signaling is lower in breast cancer samples compared to healthy donors.
  • Figure 44 shows PD-1+ CD4+ and CD8+ T cells demonstrate reduced TCR signaling as compared to PD-1 T cells.
  • Figure 45 shows in vitro Keytruda increases TCR ⁇ p-ERK/p-AKT in PD-1+ T cells, a basis for an in vitro assay to quantify activity and donor sensitivity.
  • Figure 46A-D show FMR vs FMR associations in health and breast cancer samples.
  • Figure 47A and 47B show correlations observed between FMR expression and basal signaling in T cell subsets of breast cancer patients and healthy donors.
  • Figure 48A-C show FMR correlations with modulated signaling similar between healthy and breast cancer patients.
  • A Heat maps showing correlations;
  • B Correlation between TCR- p-AKT and PD-L1 expression in CD4+ T cells (left) and correlation between TCR- p-AKT and PD-1 expression in CD4+ T cells;
  • C Correlation between TCR- p-AKT and GITR expression in CD4+ T cells (left) and correlation between TCR- p-AKT and TFM- 3 expression in CD4+ T cells.
  • Figure 49A and 49B show correlations between PD-1 expression and in vitro Keytruda activity in healthy and breast cancer samples.
  • Figure 50 shows higher FMR expression on T cell subsets associates with lower progression-free survival (PFS)
  • Figure 51 shows lower TCR mediated signaling in PBMCs associates with lower PFS
  • Figure 52 shows weak in vitro Fresolimumab activity detected in breast cancer samples.
  • Figure 53 shows Keytruda activity over two doses of Fresolimumab and over course of treatment.
  • Figure 54 shows older breast cancer patients correlate with higher PFS and greater survival through week 15 of treatment.
  • Figure 55 shows significant associations between IL-15- pSTAT 5 signaling and PFS in melanoma patients being treated with ipilimumab.
  • Figure 56 shows association between IL-15- pSTAT5 signaling and PFS was observed at baseline.
  • Figure 57 shows linear adjustment (i.e. correcting for batch effect) of melanoma data by the control data indicates there is still a significant association between cytokine ⁇ p Stat and PFS in a solid cancer, e.g., melanoma, being treated with an immunomodulatory agent, e.g., a checkpoint inhibitor such as ipilimumab.
  • an immunomodulatory agent e.g., a checkpoint inhibitor such as ipilimumab.
  • Other exemplary checkpoint inhibitors include novolumab/pembrolizumab (aPD-1) and atezolizumab (aPD-Ll).
  • Figure 58 shows exemplary cell types targeted by cancer immunotherapy, exemplary sample types of use in the methods and compositions of the invention, and exemplary cell types examined by SC P
  • Figure 59 shows exemplary classes of biological modulators and types of readouts of use in the methods and compositions of the invention
  • Figure 60 shows that SCNP nodes (TCR- p-ERK and TCR ⁇ p-AKT, in this example) in PBMC samples from individual donor cancer patients with solid tumors match signaling in TILS samples from the same donors, indicating that a liquid sample, e.g., a blood or blood-derived sample such as a PBMC sample, in different cell populations (CD4+ and CD8+ T cells, in this example) and for different levels of expression of FMR (PD-1+ and PD- 1-, in this example).
  • Each line represents an individual donor, and can be designated by its starting point (PD1-CD4+, p-AKT or p-ERK, PBMC or TILS).
  • Second from top line in PD1-CD4+ p-AKT PBMC cells is same donor as top line in PD1-CD4+ p-ERK PBMC cells, top line in PD1-CD4+ p-AKT TILS cells, and top line in PD1-CD4+ p-ERK TILS cells.
  • Third from top line in PD1-CD4+ p-AKTT PBMC cells is same donor as third from top line in PD1-CD4+ p-ERK PBMC cells, second from top line in PD1-CD4+ p-AKT TILS cells, and second from top line in PDl-CD4+p-ERK TILS cells.
  • PD1-CD4+ p-AKTT PBMC cells Fifth from top line in PD1-CD4+ p-AKTT PBMC cells is same donor as bottom line in PD1-CD4+ p-ERK PBMC cells, third from top line in PD1-CD4+ p-AKT TILS cells, and third from top line in PDl-CD4+p-ERK TILS cells.
  • Bottom line in PD1-CD4+ p-AKTT PBMC cells is same donor as second from top line in PD1-CD4+ p-ERK PBMC cells, bottom line in PD1-CD4+ p-AKT TILS cells, and bottom line in PDl-CD4+p-ERK TILS cells.
  • the data shows that different donors can be differentiated, i.e., stratified, for example, TCR- pattern in TILS is generally similar to that of PBMC, but the magnitude of signal shows a broad range across the 4 donors.
  • Figure 61 shows the results of comparison of SC P readouts and haplotype in different cell populations, in this case mDCs and monocytes.
  • Pathway readout X in this case expressed as log2fold difference between modulated and unmodulated, varies according to haplotype in both monocytes and mDCs, and in response to 2 immunostimulants. This indicates that a direct readout of a therapeutic target pathway activation can serve as a pharmacodynamics marker, and supports the use of haplotype as a selection marker. This allows activity quantification of immunostimulatory therapeutics in the context of genotypes, which can be, e.g., the basis for patient selection biomarkers.
  • FIG. 62 shows that SCNP can identify modulator- and cell subset-specific FMR induction.
  • TLR such as TLR4 (e.g., LPS) modulation
  • PD-L1 expression measured in single cells in NK cells and in monocytes, in three different donors; each line represents a different donor.
  • the right graph shows the effect of cytokine modulation, in this case, for 24 hours, on TFM3 expression measured in single cells in NK cells and in monocytes; in contrast to the TLR modulation, cytokine modulation induces TFM-3 expression in NK cells but not in monocytes.
  • TLR4 e.g., LPS
  • Figure 63 shows the effect of a therapeutic and cytokine, on various FMRs, measured in single cells, using, in this case, a log2fold metric, and log2fold increase of 0.2 as a threshold level indicating induction of an FMR.
  • Therapeutic X induced TIM-3 expression in NK cells, but did not induce expression of other FMRs in NK cells (OX-40, CLTA-4, 4-1BB, GITR).
  • cytokine X induced expression of multiple FMRs (OX-40, CLTA-4, 4- 1BB, GITR, TFM-3), most strongly in NK cells. This can be useful in, e.g., informing clinical combination studies, identifying possible mechanisms of resistance, and the like.
  • Data is shown for NK cells, but other immune cell subsets (populations) can be analyzed, such as T, B, and monocyte cell subsets.
  • patents and applications that are incorporated by reference include U.S. Patent Nos. 7,381,535, 7,393,656, 7,563,584, 7,695,924, 7,695,926, 7,939,278, 8,148,094,
  • One convenient way to class single cells as part of a cell population is to determine the level of a cell surface marker of a given cell population on the single cell.
  • the term "cell surface marker” and “extracellular cell marker” are used interchangeably herein.
  • T cells can be identified and classed based on the presence or absence, or relative abundance, of the CD4+ marker; thus one cell population can be CD4+ T cells, or T helper cells.
  • markers and classifications are well-known in the art and any suitable method of classification may be used.
  • a cell population can also be a subpopulation of another cell population.
  • the Thelper cell population is a subpopulation of the T cell lineage population
  • the Thelper effector population is a subpopulation of the Thelper population.
  • Other examples of cell populations that are subpopulations of another cell population are shown in Table 1.
  • an "immune cell population,” as that term is used herein, encompasses populations of cells of the immune system, for example, the human immune system. Such cell populations are known in the art and any suitable system for classifying cells as cells of an immune cell population may be used.
  • a "non-immune cell population,” as that term is used herein, encompasses any population of cells that is not an immune cell population, for example, any population of human cells, whether normal or abnormal, that is not an immune cell population, such as a population of cancer cells.
  • a population of immune cells may also be an abnormal, e.g., pathological population, such as a cancer cell population.
  • pathological population such as a cancer cell population.
  • immune system cells themselves may be cancer cells. In these cases, the cells are classed as part of a non-immune population, for example, a tumor population, despite their origin as immune cells.
  • modulation of the immune response to the condition by cells associated with the pathological condition is an important aspect of the condition.
  • One such pathological condition is cancer, where the tumor cells themselves develop strategies of immunosuppression to decrease various aspects of the host immune response; modulation of the immune system occurs in other pathological conditions as well, e.g., autoimmunity and HIV infection, and the invention encompasses such conditions.
  • the invention is described in many instances herein in terms of cancer; one of skill in the art will make the necessary alterations for any particular pathological condition. Detailed descriptions of immunosuppression in cancer and cancer immunotherapies are available, see Characiejus et al.
  • IMRs immunomodulatory receptors
  • IMRLs IMR ligands
  • certain immune cell populations e.g., antigen-presenting cells (APCs)
  • APCs antigen-presenting cells
  • MHC I molecules MHC I molecules
  • a pathological condition e.g. cancer cells.
  • the EVIRL is a soluble factor (e.g., adenosine) and the pathological condition can affect its levels in the extracellular environment.
  • Immune cells e.g., T cells and non-T cells such as NK cells, monocytes, B cells, and dendritic cells (DC), and subpopulations thereof, such as Treg and Tcyto, express a variety of receptors that either inhibit the activation of the immune cell, (e.g., in the T cell, stimulation at the T Cell Receptor (TCR), similarly with other receptor or receptors for a particular immune cell population, see Table 1), or activate (costimulate) the activation of the cells. Both inhibitory and activating (costimulatory) receptors are referred to as "immunomodulatory receptors" (IMRs) herein.
  • IMRs immunomodulatory receptors
  • Tumor cells as well as antigen-presenting cells (APCs) and other cells, often express IMR ligands (FMRLs) on their surface that interact with one or more of these receptors, thus blunting the immune response and decreasing effectiveness of the immune system in eradicating the tumor.
  • the ligand is a soluble molecule, e.g., adenosine. See, e.g., Figure 15, which shows various stimulatory (costimulatory) and inhibitory IMRs found on T cells and the corresponding ligands found on, e.g., APCs or tumor cells, and Table 1, which provides exemplary methods of activating various immune cell populations.
  • the description of Figure 15 in the Brief Description of the Drawings provides further details and description, as well as additional FMRs and FMRLs that are encompassed by the methods and compositions of the invention.
  • an "immunotherapy” encompasses any therapy directed at altering modulation of the immune system of a patient, where the patient's immune system has been modulated by the pathological condition from which he or she suffers, e.g., immunotherapies in cancer seek to counteract, by one or preferably more than one, mechanism the immune suppression seen in a particular cancer.
  • Immunotherapy e.g., cancer immunotherapy, may be directed at any aspect of
  • immunosuppression or multiple aspects, for example, at modulating one or more of FMR- FMRL interactions, such as immune checkpoint blockade (blocking an inhibitory FMR with an antagonist, or blocking an inhibitory FMRL) but also including activating a costimulatory FMR); vaccination to bolster the immune response (often used in combination with modulation of FMR- FMRL interaction and generally involving DCs); cytokine therapy, e.g., treating a patient with IL-2, to bolster immune response; and adoptive immunotherapy, e.g., treating a patient with T cells that have been removed from the patient and modulated ex vivo to increase their tumor-killing capacity.
  • FMR- FMRL interactions such as immune checkpoint blockade (blocking an inhibitory FMR with an antagonist, or blocking an inhibitory FMRL) but also including activating a costimulatory FMR)
  • vaccination to bolster the immune response (often used in combination with modulation of FMR- FMRL interaction and generally involving DCs)
  • immunotherapy does not encompass therapies in which an antibody targeting a tumor-associated antigen (TAA) is used, alone or conjugated to a therapeutic agent, to directly attack the tumor, even though it involves a component of the immune system, an antibody or a fragment thereof; such therapies are a type of "targeted therapies,” see, e.g., Vanneman, M., and Dranoff, G., Nature Reviews: Cancer 12:237-251 (2012).
  • TAA tumor-associated antigen
  • one such strategy, or immunotherapy is to modulate the activation of an IMR by its corresponding EVERL or IMRLs; this type of therapy is sometimes called checkpoint therapy if the therapy is aimed at decreasing the interaction between an inhibitory EVER and its ligand or ligands; however, therapies in which costimulatory IMRs are activated are also being developed.
  • the therapy may be aimed at blocking the EVER if it is an inhibitory EVER, or blocking one or more ligands for the inhibitory EVER, or activating a costimulatory EVER with an agonist to its EVERL, or otherwise modulating the EVER-EVERL interaction, and/or modulating the activity of the EVER, so that the activity of the EVER and its EVER pathway is modulated— increased, in the case of a costimulatory EVER, or decreased, in the case of an inhibitory EVER.
  • the ultimate result is thought to be that cells of one or more immune cell populations experience less immunosuppression and ultimately the immune system is able to attack and destroy the tumor cells.
  • ipilimumab therapy for malignant melanoma, which blocks the CTLA-4 (inhibitory) receptor, thus stimulating immune cells, e.g., T cells.
  • Other similar therapies are being developed or tested, such as molecules to block the PD-1 (inhibitory) IMR or one or both of its cognate ligands.
  • Anti-PD-1 therapies are now being tested, and some response is being seen in non-small cell lung cancer (NSCLC) and in renal cancer, but not all patients respond, and current stratification techniques are not effective. For example, patient stratification may be attempted by analyzing the level of expression of an EVERL, e.g., PD1 ligand (PD-L1) on tumor cells.
  • an EVERL e.g., PD1 ligand (PD-L1) on tumor cells.
  • immunotherapy may be used to inhibit an inhibitory EVER pathway or pathways, or stimulate activating (costimulatory) EVER pathway or pathways.
  • Diagnosis, prognosis, monitoring, selection of an aspect of treatment of a patient, and screening candidate agents, e.g., drug candidates, to develop for such therapies are all aided by understanding and using knowledge of the pathways involved in the activity of EVERs (EVER pathways), especially at the single cell level, because the initial effect of an EVER pathway is at the level of the cell expressing the EVER on its surface.
  • methods such as selection of an aspect of therapy for a patient suffering from a particular condition, e.g., cancer may be based at least in part on characterization of one or more of the IMR pathways, for example, the functional status of one or more IMR pathways.
  • the advantage of using functional status over traditional biomarkers such as expression levels, is that it gives a measure of the actual state of single cells of a cell population, e.g., a cell population from a sample from an individual such as a patient, or a cell population used in screening drug candidates.
  • functional status relies on modulating the IMR or EVIRs, preferably in single cells, to determine the level at which the particular EVER or IMRs is functioning, e.g., by activating the IMR or IMRs.
  • the functional status of all the cells of a population may be analyzed, one by one.
  • one bases decisions on how the cells actually respond to a stimulus generally a stimulus that results in activation similar to activation in the body, and how that response is modified by the one or more IMRs.
  • values such as the expression level of the IMR, may also give an indication of, e.g., whether or not, and to what degree, the IMR is affecting a particular cell, it is not a direct indication, and does not take into account the complexity of the cell's actual function.
  • a particular cell may express a particular IMR at a high level, but, when the IMR is activated in conjunction with overall activation of the cell, the IMR may have little or no effect on the cell's response to overall activation.
  • the expression level of the IMR as a marker for its effect in the cell will give an erroneous view of its influence on the cell; by surface expression level, its effect should be high, but by actual interrogation of its effect, it is low.
  • expression levels may be sufficient to provide a marker for cell or cell population function, and/or may be useful in gating cells from a population so that the functional status of an FMR is determined only in cells expressing the FMR above a certain threshold level on their surface is determined,
  • “functional status,” for example used in reference to an FMR pathway or FMR, encompasses the magnitude of effect or potential effect on (e.g., modulation of, or potential modulation of), the immune activity of a particular cell or cell population due to the effects or potential effects of the FMR/EVIR pathway in that cell or cell population. For example, if, when an immune cell is activated in the presence of activation of an FMR/EVIR pathway, and in the absence of such activation, there is no difference in the activation level of the immune cell, the functional status of the EVIR/EVIR pathway is low in that cell, for example, can be expressed as 0.
  • the functional status of the EVIR/IMR pathway is high.
  • the functional status of an FMR/FMR pathway can be expressed in any suitable manner, for example as a quantitative value whose magnitude corresponds with magnitude of the effect of the IMR/EVIRL on a particular cell or cell population.
  • the modulation or potential modulation of the activity of the cell or cell population can be an increase in the immune activity of the cell or cell population or a decrease in the immune activity of the cell or cell population.
  • the activation of the cell or population refers, e.g., to its response when activated by one or more activators for that particular cell or cell population, thus the functional status of an IMR or IMR pathway is generally, assessed in the context of activation of the cell or cell population in which it operates.
  • activation can be achieved by well- known methods, such as those described herein and known in the art, e.g., to specifically activate T cells, one may contact the T cells with one or more activators of T cells, such as ⁇ CD3 and DCD28.
  • the functional status of one or more IMRs/IMR pathways in T cells can be assessed by activating the T cells in the presence and absence of activation of the one or more IMRs/IMR pathways and assessing the difference in the activation of the T cells with and without such activation of the FMR/IMR pathway.
  • Activation of the cell or cell population may be determined by any suitable means.
  • determining the activation of a cell or cell population can include determining a change in the expression level of one or more intracellular expressed elements, and/or the change in the activation level of one or more intracellular activatable elements, as described herein, compared to the level without activation of the cell or cell population.
  • the response of one or more immune cell populations to a modulator may be used as an alternative, e.g., surrogate, for the above measurements, or in addition to such measurements; e.g., as shown in the examples, certain modulator ⁇ activatable element (nodes) combinations are seen to be correlated with certain diagnostic, prognositic, predictive, monitoring, and other characteristics useful in evaluating an individual suffering from, or suspected of suffering from, a pathological condition, such as cancer.
  • Intracellular activatable elements and intracellular expressed elements are collectively referred to herein as "intracellular elements.”
  • Intracellular expressed elements are typically proteins, e.g., intracellular proteins whose expression levels change in response to activation of the cell or cell population, e.g., where the change in expression levels corresponds to the level of activation of the cell or cell population.
  • Intracellular activatable elements are typically proteins, e.g., proteins whose activation levels change in response to the activation of the cell or cell population, e.g., where the change in activation level corresponds to the level of activation of the cell or cell population.
  • the kinetics of change in activation levels of one or more intracellular activatable elements, and/or expression levels of one or more intracellular expressed elements, can also be indicative of the activation of the cell or cell population. See Table 1 for examples of immune cell populations, cell surface markers, in vitro activators, intracellular activatable elements, and intracellular expressed elements.
  • Immune cell population cell surface markers in vitro activator(s), intracellular activatable elements, and intracellular expressed elements
  • Immune cell Exemplary In vitro Exemplary IntraExemplary IntraExemplary population cell surface activatort activators cellular intracellular expressed markers for ype activatable cellular expres-sed intragating element 2 activatable element 3 , cellular population 1 readout, elements type elements type
  • CD14- ligand agonists JAK/STAT, pERK, p- HLA-DR+ and/or NFkB AKT, others
  • Changes in activation level can also be measured in longer time frames, e.g., hours, or days, such as at least 4, 8, 12, 16, 20, or 24 hours after contacting the cells with the activator, allowing intercellular communication events to occur.
  • Change in expression level of intracellular expression element detectable in hours or days, e.g., at least 12, 16, 20, 24, 36 or 48 hours after contact with activator and can correspond to later events in activation.
  • time or days e.g., at least 12, 16, 20, 24, 36 or 48 hours after contact with activator and can correspond to later events in activation.
  • communication can play a role in a manner similar to intercellular communication in vivo, allowing a different view of overall interactions that occur in vivo, that may be different from the short-term activation events seen with activatable elements, which occur in a time frame (minutes), in which the intracellular and extracellular events necessary for cell-cell communication have not yet occurred.
  • Intracellular expression elements useful in the invention also include non-cytokine elements, such as cell cycle elements, e.g., Ki67 and Cyclin A2
  • TCR activators are “specific T cell activators,” as they mainly or exclusively activate T cells, and TLR activators are “nonspecific T cell activators.
  • a “surrogate activator” may be used to determine the functional status of one or more IMRs in a cell population, e.g., T cells, e.g., use of cytokine activator (surrogate activator) such as IL-6, IL-10, IL-15, IL-21, IL-2, IL-4, IL-12, IFNa, or lFNg, or any combination thereof, and measuring the activation level of an activatable element in the JAK/STAT pathway, such as p-STATs (e.g., 1, 3, 4, 5, or 6 or any combination thereof), with or without modulation of the IMR or IMRs of interest, in single cells of the population, e.g., T cell population.
  • cytokine activator such as IL-6, IL-10, IL-15, IL-21, IL-2,
  • basal levels of the activatable element or element may alone be a surrogate for functional status of an IMR or IMRs, e.g., by comparison with a value derived from analysis of samples with known functional status of the IMR or IMRs. See Example 14
  • cytokines includes the subclass of cytokines known as chemokines.
  • BCR activators are “specific B cell activators,” as they activate mainly or exclusively B cells, and TLRs are “nonspecific B cell activators,” as they activate other cell populations besides B cells
  • the success of diagnosis, prognosis, monitoring, prediction, and/or therapy, or even drug development may depend on knowledge of the complex interplay between conditions produced by a pathological condition, such as tumor cells produced in a cancer, and one or more IMRs, in one or more immune cell populations; in addition, or alternatively, knowledge of other aspects of immunosuppression may be required or useful.
  • Certain embodiments of the current invention are based on characterization of one or more of EVER pathways, in one or more immune cell populations, to, e.g., guide diagnosis, prognosis, monitoring, prediction, and/or therapy for pathological conditions, such as cancer, and/or to aid in drug development for these conditions.
  • the characterization may include
  • expression level of an IMR and “surface expression level” of an IMR are used synonymously herein.
  • expression level when referring to an IMR, means surface expression level, unless otherwise indicated.
  • the characterization may include characterizing the functional level of one or more pathways affected by the one or more IMRs/IMR pathways in one or more immune cell populations, indicative of the functional status of the IMR/IMR pathway or FMR pathways/IMR pathways; in some embodiments, this is done by determining levels of one or more intracellular activatable elements in single cells of the populations, such as the levels after modulation of the cells; in some embodiments it may be done by characterizing levels of one or more intracellular expressible elements in the cells.
  • the methods include modulation, e.g., activation, of one or more IMR/IMR pathways.
  • the surface expression levels of one or more IMR ligands (EVIRLs) or other components of cells associated with a pathological condition, such as cancer cells, e.g., tumor cells, may also be characterized.
  • IMR ligands IMR ligands
  • Other embodiments of the methods and compositions of the invention are as described below.
  • the activation levels of intracellular activatable elements can change in a matter of minutes, much faster than a change in expression levels can be detected, and can be measured at a time no greater than 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, or 240 min., but also can be measured at later times, e.g., at least 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours after exposure of the immune cells to activator, or at time that is greater than or equal to 2, 4, 6, 8, 10, 15, 20, 25, 30, 45, 60, 90, 120, 150, or 240 min.
  • Intracellular activatable elements of use in the invention include any suitable activatable element; exemplary activatable elements are shown in Table 1 and described elsewhere herein, see, e.g., Activatable Elements, Signaling Pathways, and Figures 20A and 20B, and can include phosphoproteins and/or proteins activated by cleavage, also as described herein.
  • changes, e.g., increases or decreases, in p-ERK, pZAP70, PLCg, p-pKCtheta, p-p38 and/or p-AKT activation levels can correspond to immune cell, e.g., T-cell activation.
  • intracellular expression levels of intracellular expressed elements change more slowly than activation levels of activation elements in response to activation of an immune cell population, and are measured hours or even days after the activation of the immune cell or cell population.
  • the expression level of one or more intracellular expression elements may be determined at least 1, 2, 4, 8, 12, 16, 20, 24, 30, 36, 42, or 48 hours after activation of the immune cell or cell population.
  • the expression level is compared with an expression level in an cell or population from the same immune cell population that was not activated; the difference is the expression level of the intracellular expressible element.
  • Intracellular expressed elements of use in the invention include any suitable expressed element; exemplary expressed elements are shown in Table 1 and described elsewhere herein.
  • Intracellular expression elements useful in the invention also include non-cytokine elements, such as cell cycle elements, e.g., Ki67 and Cyclin A2.
  • a "surrogate activator” may be used to determine the functional status of one or more FMRs/FMR pathways in a cell or cell population, e.g., T cells, e.g., use of cytokine activator (surrogate activator) such as IL-6, IL-10, IL-15, IL-21, IL-2, IL-4, IL-12, IFNa, or IFNg, or any combination thereof, and measuring the activation level of an activatable element in the JAK/STAT pathway, such as p-STATs (e.g., 1, 3, 4, 5, or 6 or any combination thereof), with or without modulation of the FMR or EVIRs of interest, in single cells of the population, e.g., T cell population.
  • cytokine activator such as IL-6, IL-10, IL-15, IL-21, IL-2, IL-4, IL-12, IFNa, or IFNg, or any combination thereof
  • p-STATs e.g.
  • basal levels of the activatable element or element may alone be a surrogate for functional status of an FMR or EVIRs, e.g., by comparison with a value derived from analysis of samples with known functional status of the FMR or FMRs. See Example 14.
  • such "surrogate activators" and their corresponding readouts may be correlated or otherwise linked to a particular condition or outcome, without necessarily directly measuring the functional status of a particular FMR/FMR pathway, and it is typically assumed, without being bound by theory, that such correlation or other linkage is indicative of a functional change in one or more such pathways, or other changes in the cell that occur as a result of modulation of one or more such pathways (e.g., induction of off-target effects with immunomodulatory treatment, such as a side effect, for example, colitis in treatment of melanoma or other cancer with ipilimumab).
  • induction of off-target effects with immunomodulatory treatment such as a side effect, for example, colitis in treatment of melanoma or other cancer with ipilimumab.
  • determining the functional status of an FMR/FMR pathway can in certain embodiments entail the use of an activator of the immune cell population or populations of interest, an activator of the FMR/FMR pathway of interest, and the determination of either or both of a change in activation level of an intracellular activatable element, e.g.,
  • phosphoprotein or change in the expression level of an intracellular expressed element, in response to contact of the cells of the cell population with the activator, and in the presence and absence of activation of the EVIR/IMR pathway.
  • a surrogate activator of the immune cells of the immune cell population is used.
  • basal activation levels of one or more activatable elements are used as an indicator of EVER/EVER pathway status, alone or in combination with other elements as described herein.
  • EVIRs are typically expressed in response to activation of an immune cell, and in quiescent cells one or more IMRs may be at very low levels, but in activated cells, one or more EVIRs will be expressed on the surface of the cell in order to allow modulation of the now-active immune response.
  • immune cells from a sample from a patient suffering from a condition, e.g. cancer already are activated, and EVIRs are already expressed on their surface; indeed, this is one mechanism by which the cancer suppresses the immune response.
  • immune cells from sample from an individual e.g., a patient, such as a cancer patient, are used "as is", without further significant modulation of the expression of their IMRs.
  • one or more modulations of the cells may be necessary in order to induce measurable or useful surface expression levels of one or more EVIRs in cells, or otherwise prepare the cells for meaningful measurements.
  • cells from healthy individuals are usually quiescent, and expression of one or more EVIRs can be induced, for example if the cells are to be used for screening agents that may affect EVIRs/EVIR pathways.
  • Any suitable method to induce surface expression of one or more EVIRs may be used; a preferred method is to activate the immune cells, which is the normal route by which expression of EVIRs is induced, for a certain period of time, e.g., at least 12, at least 24, at least 36, or at least 48 hours or any other suitable interval, to allow expression of the EVIRs, then use the cells, e.g., to study functional status of the EVIRs/IMR pathways in the cells, often after resting the cells for a period so that the initial activation of the cell can subside but EVER expression levels remain high enough to use. See Example 16 and Figure 18.
  • Surface expression levels of one or more EVIRs on cells of one or more immune cell populations may also be determined in certain embodiments of the invention.
  • surface expression levels of at least 1, at least 2, at least 3, at least 4, or at least 5, 6, 7, 8, 9, or 10 different EVIRs may be determined in single cells of one or more immune populations, for example, determined on the same cell, and such levels for all or a certain portion of the cells in a cell population or subpopulation used.
  • the surface expression levels may be used alone, or in combination with other determinations, e.g., in combination with determination of the functional status of one or more EVIRs, for example, at least 1, at least 2, at least 3, or at least 4, 5, 6, 7, 8, 9, or 10 different IMRs, e.g., determined in single cells of an immune cell population.
  • the expression level of an IMR and the functional status of the same IMR, e.g., PD-1, may be measured on the same cell of an immune cell population. This determination may be used, e.g., to gate the cell into or out of a population.
  • FMR or FMRs when surface expression levels of one or more FMRs are measured in single cells of a population and the functional status of the FMR or FMRs is also measured in the same cells, it may be desirable to determine the functional status of the FMR or FMRs only in cells that are expressing the FMR on their surface at a level greater than, or greater than or equal to, a threshold level, and cells may first be gated into the population with such expression levels before the functional status of the FMR or FMR is determined. Functional status may be determined directly or by use of surrogate activators, as described herein.
  • tumor cell surface markers useful in classifying cells as members of an immune cell population (e.g., T cell, B cell, etc.), or as members of a non-immune cell population (e.g., tumor cells) may also be determined in one or more embodiments of the invention.
  • Tumor cell surface markers are well-known in the art, and any suitable marker or markers may be used.
  • immune cell surface markers are also well-known in the art and any suitable marker or markers may be used to place immune cells into one or more immune cell populations; exemplary cell surface markers and corresponding immune cell populations are shown in Table 1 and Figure 17, and throughout the Description and Examples, but any suitable set of cell surface markers corresponding to any suitable immune cell populations may be used, as will be apparent to those of skill in the art.
  • Cells can be gated into a particular population by well-known techniques based on their surface expression levels of particular cell surface markers.
  • Certain embodiments of the invention are directed to methods and compositions involving a patient suffering from, or suspected of suffering from, a pathological condition.
  • the pathological condition involves modulation of the patient's immune system by, for example, cells associated with the condition, for example, immunosuppression by tumor cells.
  • the pathological condition may be cancer, e.g., any one of the known cancers or cancers described herein. For convenience, description herein often refers to cancer, but non- cancer pathological conditions such as autoimmune disease or HIV infection are included.
  • the pathological condition is classified by a designation that mainly or entirely is derived from the results of one or more measures of the functionality of one or more FMRs in one or more immune cell populations derived from a sample obtained from an individual, optionally including surface expression levels of an FMR or FMRs on immune cells or cell populations, and/or surface expression levels of an FMR or EVIRL on cell of a non-immune cell population, and is not based entirely or mainly on traditional diagnostic criteria. That is, the classification of the condition is condition-agnostic, e.g. cancer-agnostic and is, instead based at least in part on the above criteria.
  • Certain embodiments of the invention are directed to methods and compositions involving treating a patient suffering from a pathological condition, e.g., cancer, with a treatment.
  • the treatment may be any treatment known or devised in the art.
  • the treatment includes immunotherapy.
  • Immunotherapies are as described elsewhere herein, and can include one or more of vaccines, therapies aimed at modulating one or more interactions between IMRs and EVIRLs, or modulating the EVIR pathway, e.g., checkpoint therapies such as anti-PD-1 therapies and/or anti-CTLA-4 therapies (exemplary checkpoint inhibitors include ipilimumab (antiCTLA4), nivolumab/pembrolizumab (anti- PD1), atezolizumab (antiPD-Ll), and the like), adoptive immune cell therapy, cytokine therapy, and the like, as described elsewhere herein.
  • checkpoint therapies such as anti-PD-1 therapies and/or anti-CTLA-4 therapies
  • exemplary checkpoint inhibitors include ipilimumab (antiCTLA4), nivolumab/pembrolizumab (anti- PD1), atezolizumab (antiPD-Ll), and the like
  • adoptive immune cell therapy adoptive immune cell therapy
  • the treatment is a combination treatment, that is, at least two different treatments, such as a combination immunotherapy treatment, such as one or more immunotherapies and one or more non- immunotherapy treatments, or two or more different immunotherapy treatments, such as a vaccine and at least one other immunotherapy, such as modulation of one or more IMRs/IMR pathways.
  • the treatment is a combination immunotherapy of two or more different immunotherapies in which one of the immunotherapies is modulation of an IMR/IMR pathway; or in which 2, 3, 4, or 5 or more of the immunotherapies is modulation of 2, 3, 4, or 5 or more different IMR/IMR pathways, such as the EVIRs IMR pathways shown in Figure 15.
  • one of the pathways is the PD-1 pathway.
  • one of the pathways is the CTLA-4 pathway.
  • two of the pathways are the PD-1 pathway and the CTLA-4 pathway.
  • the treatment comprises an immunotherapy comprising modulation of PD-l/PD-1 pathway, alone or in combination with modulation of one or more other IMR/IMR pathways, such as one or more of the IMRs/IMR pathways shown in Figure 15 and the Description thereof, for example, CTLA-4/CTLA-4 pathway.
  • the combination treatment is a combination of one or more immunotherapies and a non-immunotherapy treatment, such as one or more of a targeted treatment (a treatment specifically targeted at tumor cells such as MAb or conjugated MAb therapy), chemotherapy, radiation treatment, or surgical treatment.
  • the immunotherapy comprises an immunotherapy that comprises modulation of one FMR/FMR pathway, such as one of the IMR/IIVIR pathways shown in Figure 15 and the Description thereof.
  • the treatment comprise modulation of the the PD-1 pathway.
  • the treatment comprises modulation of the CTLA-4 pathway.
  • the treatment comprises modulation of the PD-1 pathway.
  • the treatment comprises modulation of the LAG-3 pathway.
  • the treatment comprises modulation of the TIM-3 pathway.
  • the treatment comprises modulation of the VISTA pathway.
  • the treatment comprises modulation of the GITR pathway. In certain embodiments, the treatment comprises modulation of the OX-40 pathway. In certain embodiments, the treatment comprises modulation of the CD27 pathway. In certain embodiments, the treatment comprises modulation of the 4-1BB pathway.
  • Modulation of an IMR refers to a modulation that specifically alters the IMR-IMRL interaction and its effects on immune cells on which the FMR is expressed, for example, contacting the FMR with an agonist, contacting the FMR with an antagonist (blocking the FMR), contacting the FMRL with a blocking agent, such as an antibody, and any other method of specifically altering the FMR-FMRL interaction and its effects on the immune cell.
  • the immunotherapy is treatment with an immunotherapy that comprises modulation of two FMR/FMR pathways, such as two of the FMR/FMR pathways shown in Figure 15 and the Description thereof.
  • the treatment comprises modulation of the PD-1 pathway and one other FMR pathway.
  • the treatment comprises modulation of the CTLA-4 pathway and one other FMR pathway.
  • the treatment comprises modulation of the LAG-3 pathway and one other IMR pathway.
  • the treatment comprises modulation of the TFM-3 pathway and one other IMR pathway.
  • the treatment comprises modulation of the VISTA pathway and one other FMR pathway.
  • the treatment comprises modulation of the GITR pathway and one other FMR pathway.
  • the treatment comprises modulation of the OX- 40 pathway and one other FMR pathway. In certain embodiments, the treatment comprises modulation of the CD27 pathway and one other IMR pathway. In certain embodiments, the treatment comprises modulation of the 4-1BB pathway and one other FMR pathway. In certain embodiments the treatment comprises modulating the PD-1 pathway and the CTLA-4 pathway. In certain embodiments the treatment comprises modulating the PD-1 pathway and the OX-40 pathway, for example, in an AML patient.
  • Certain embodiments of the invention are directed to methods and compositions involving one or more aspects of treating a patient with a treatment.
  • An aspect of treating a patient with a treatment encompasses any element of the treatment that may affect treatment outcome, where treatment outcome includes the effect of the treatment on the pathological condition, and/or on the overall health and/or comfort of the patient.
  • aspects of treatment include, but are not limited to, a decision to treat the patient or not treat the patient with a treatment or component of the treatment, choice of the treatment or a component of the treatment, a choice of the timing of the treatment or of a component of the treatment, a choice of a dosage of the treatment or a component of the treatment, or a combination thereof.
  • Certain embodiments of the invention are directed to methods and compositions involving a decision process, for example, a treatment decision process, generally engaged in by at least the patient and/or one or more of the patient's healthcare providers.
  • a treatment decision process includes any process by which an outcome, e.g., an outcome regarding an aspect of treatment, is determined.
  • Exemplary outcomes of a treatment decision process include a first likelihood of the patient responding to the treatment, a second likelihood of prolongation of the patient's life due to receiving the treatment, or a third likelihood of the patient experiencing an adverse treatment effect, or any combination of the first, second, and/or third likelihoods.
  • a treatment decision process includes consideration of a quantitative value, or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 quantitative values, or a value or values derived therefrom, or at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 quantitative values derived therefrom. In certain embodiments a treatment decision process includes
  • the quantitative value or values is generally engaged in by at least the patient and/or one or more of the patient's healthcare providers, and can comprise, for example, comparing the one or more quantitative values to a threshold value, e.g., comparing a quantitative value to a threshold value to decide whether or not the patient will respond to a particular treatment or component of a treatment, comparing the quantitative value to a continuous function, e.g., comparing it to a function that indicates probability of response of the patient to a treatment given the quantitative value or values, or a combination thereof.
  • a threshold value e.g., comparing a quantitative value to a threshold value to decide whether or not the patient will respond to a particular treatment or component of a treatment
  • comparing the quantitative value to a continuous function e.g., comparing it to a function that indicates probability of response of the patient to a treatment given the quantitative value or values, or a combination thereof.
  • Comparison can also be done automatically, for example, so as to give a simple yes/no answer for the patient and/or healthcare provider(s) to consider, or to give a probability for the patient and/or healthcare provider(s) to consider, or any combination thereof. Any other comparison involving the quantitative value or values, or value or values derived therefrom, that can influence the outcome of a treatment decision process may also be used.
  • a decision to treat a patient may be made by a patient and his or her healthcare provider, where the decision process includes consideration of a quantitative value, or two quantitative values, or a quantitative value that is derived therefrom, or two quantitative values derived therefrom, where the quantitative value is determined at least in part by interrogating one or more immune cell populations from a sample from the patient, and considering the value using a classifier, e.g., a hierarchical classifier or a continuous function classifier such as a linear classifier, e.g., where the consideration indicates that the patient will respond to the treatment.
  • the interrogating the one or more immune cell populations may comprise determining the functional status of one or more IMRs in the one or more immune cell populations;
  • a treatment decision process for whether or not to treat a patient with an immunotherapy may include comparing a first quantitative value to a first threshold value, such that if the first quantitative value is greater than or equal to the first threshold value, the patient is likely to respond to the immunotherapy (e.g., is a responder). Either the patient and/or their healthcare provider make the comparison, or the comparison may be made automatically, e.g., in the form of a simple yes/no.
  • the first quantitative value can, e.g., correspond to the functional status of an IMR pathway in cells of one or more immune cell populations from a sample from the patient.
  • the first quantitative value is derived from a plurality of initial quantitative values, each of which represents, for a single cell in the one or more immune cell populations, an activation level of an intracellular activatable element in the single cell, for example, a signal magnitude from labels on antibodies that binds to the intracellular activatable elements in the particular cell.
  • the derivation procedure may be complex, and involve one or more intermediate quantitative values between the plurality of initial values and the final first quantitative value, see, e.g., Table 2.
  • a second quantitative value may be obtained, for example, a quantitative value that corresponds to surface expression levels of the IMRL that activates the first IMR, on tumor cells from a sample from the patient.
  • the second quantitative value may also be compared to a threshold, or the first and second quantitative values may be combined, e.g., with weighting of each value to reflect its relative importance in predicting response to the immunotherapy, to obtain a third value, and it is the third value that is used in the treatment decision process.
  • a single quantitative value is used in the treatment decision process, and or a single quantitative value is used for each aspect of the treatment that the treatment decision process addresses.
  • Obtaining the threshold values and/or continuous functions to which a quantitative value is compared may be done, in the case of treatment, diagnosis, prognosis, prediction, or monitoring decision processes, through retrospective and/or prospective studies where, e.g., a training phase establishes putative threshold value(s) and/or continuous function(s), as well as any manipulations of quantitative values necessary to obtain a meaningful quantitative value for comparison, and a validation phase validates them.
  • a training phase establishes putative threshold value(s) and/or continuous function(s), as well as any manipulations of quantitative values necessary to obtain a meaningful quantitative value for comparison, and a validation phase validates them.
  • Such methods are well-known in the art.
  • a decision process e.g., a prognostic, diagnostic, prediction, or monitoring decision process, such as a treatment decision process, may also comprise consideration of a characteristic of the patient, such a genetic characteristic, age, gender, race, health status, previous treatment history, or any combination thereof.
  • a characteristic of the patient such as a genetic characteristic, age, gender, race, health status, previous treatment history, or any combination thereof.
  • certain therapies are only given to patients with a certain genetic characteristic, such as the presence or absence of a gene mutation; e.g., cetuximab is only used in patients with wild-type (unmutated) KRAS genes.
  • cetuximab is only used in patients with wild-type (unmutated) KRAS genes.
  • any suitable characteristic as known in the art or as discovered, related to a particular condition, e.g., pathological condition, from which an individual may suffer or potentially suffer, may be used in the methods and compositions of the invention.
  • a decision process e.g., a prognostic, diagnostic, prediction, or monitoring decision process, such as a treatment decision process, may comprise consideration of the number of cells in one or more immune cell populations, or a ratio of cell numbers in one immune cell population to the number of cells in another immune cell population or other cell population or combination of populations. For example, a low Treg/Tcyto ratio in tumor infiltrating lymphocytes (TILS) is related to better overall prognosis.
  • TILS tumor infiltrating lymphocytes
  • a prognosis decision process includes any process by which an outcome, e.g., an outcome affecting a decision regarding a prognosis, is made.
  • Exemplary outcomes of a prognosis decision process include a likelihood of a healthy individual developing a pathological condition, for example, within a certain period of time; a likelihood of a patient suffering from a pathological condition experiencing a worsening of the condition, e.g. within a certain period of time; and the like.
  • the prognosis decision process is analogous to the treatment decision process, above, and any differences and/or modifications will be readily apparent to one of ordinary skill in the art; for example, the prognosis decision process can be partially or completely automated, can be performed by one or more of the individual's healthcare providers, etc.
  • Certain embodiments of the invention are directed to monitoring, e.g., monitoring a patient with a pathological condition who is or is not receiving treatment.
  • a patient who is receiving treatment for a pathological condition such as for a cancer, is monitored for, e.g., response to the treatment, development of side effects, and the like.
  • the methods and compositions of the invention provide many avenues to obtain useful information and to inform decisions in drug development.
  • disease profiling the methods and compositions as described herein can be used, e.g., in identify pathways, identify potential drug targets, and validate these. This is useful in, e.g., providing phenotypic- and target-based drug discovery information.
  • drug profiling the methods and compositions as described herein can be used, e.g., for lead optimization, to identify and test potential drug combinations, and to ascertain mechanism of action (MO A). This is useful in, e.g., faster go/no-go decisions, and in identifying on- and off-target drug effects.
  • MO A mechanism of action
  • the methods and compositions as described herein can be used, e.g., for indications for use, biomarkers, predictors of response, selection of combination therapies, toxicity, and the like. This is useful in, e.g., reduced trial size and costs, increased likelihood of success.
  • a drug screening decision process includes any process by which one or more candidate therapeutic agents are determined to move or not move to a next level of screening, and can be engaged in by a person or persons, performed automatically, or any combination thereof.
  • Certain embodiments of the invention are directed to methods and compositions involving one or more quantitative values. Any or all of activation level of an intracellular activatable element, intracellular expression level of an intracellular expressed element, surface expression level of a cell surface marker, or other markers or characteristics described herein, and/or a change thereof, is expressed as a quantitative value in certain embodiments herein. Such quantitative values can be used to derive further quantitative values. Examples of quantitative values of use in the invention are given in Table 2, however a quantitative value derived from one or more such values may be used. In certain embodiments, a quantitative value or a derivative thereof (generally another quantitative value) is compared to a threshold value. The result of the comparison varies depending on the embodiment.
  • the quantitative value is compared to a continuous function, such as a linear function, for example, to determine probability of response of a patient to treatment.
  • cells can be gated based on the results of comparison of one or more quantitative values or their derivative, e.g., values reflecting surface expression levels of cell surface markers, or surface expression levels of EVIRs, may be used to gate cells.
  • the selection of the treatment may be based on the outcome of a decision process that includes consideration of one or more quantitative values, that is indicative, e.g. of the probability that the patient will respond to a treatment, such as an immunotherapy, or one or more such treatments.
  • one or more of activation level of an intracellular activatable element, intracellular expression level of an intracellular expressed element, surface expression level of a cell surface marker, or other markers or characteristics described herein is determined in single cells, for example single cells derived from a sample from a patient, such as a blood or a blood-derived sample, e.g., a peripheral blood
  • PBMC mononuclear cell
  • BMMC bone marrow mononuclear sample
  • solid tumor sample may be a primary tumor sample or a metastatic tumor sample, and obtained, e.g., as a biopsy or during a surgical procedure, such as surgical resection of a tumor. Sample and methods of sampling are described in more detail elsewhere herein.
  • any suitable method may be used to determine characteristics of single cells in a sample, as described herein, such as cytometry, for example, flow cytometry or mass cytometry.
  • one or more distinguishably detectable binding elements is used in order to provide a detectable signal corresponding to a characteristic to be measured, e.g., the activation level of an intracellular activatable element in a cell.
  • a "distinguishably detectable binding element” is a binding element, as that term is used herein, for example, an antibody or antibody fragment, that both binds to a component of interest, e.g., an activatable element in a particular activation state, and whose bound form can be detected, e.g., through a label, such as a fluorescent label for flow cytometry or a mass label, also referred to as a mass tag, in mass cytometry, that produces a signal that can be detected, e.g., by a cytometer. Its signal can be distinguished from that of any other detectable binding element used in the particular process in which it is used.
  • the signal that is detected is a quantitative value and it may be manipulated to produce other quantitative values, see, e.g., Table 2.
  • the values may be used to gate cells, as known in the art and as described herein.
  • Gating may include an automatic component.
  • Gating may include a manual component.
  • gating includes both a manual and an automatic component; see, e.g., U.S. Patent Application No. 20130173618.
  • treatment and treatment decisions, and or prognoses, or drug screening may be partially or completely condition- (e.g. cancer-) agnostic; that is, a treatment is selected, or a prognosis formulated, or a candidate agent selected, not solely or mainly based on traditional patient phenotypes, e.g.
  • cancer phenotypes such as colon cancer, prostate cancer, ovarian cancer, renal cancer, cancer stages, cancer histology, etc., but solely or mainly based on a patient phenotype based on the state of immunomodulation, e.g., immunosuppression, in the patient, such as a phenotype based on the one or more of the cell population phenotypes described herein.
  • non-tumor cells can reflect the tumor environment and thus, a non-tumor sample may be used, alone or in conjunction with a tumor or tumor- derived sample, to evaluate an individual suffering from, or suspected of suffering from, a solid tumor.
  • the invention provides methods and compositions for diagnosing, prognosing, predicting, or monitoring an individual suffering from or suspected of suffering from a solid tumor, comprising evaluating single non-tumor cells in a non-tumor sample taken from the individual.
  • the sample can be any suitable non-tumor sample, such as a blood or blood-derived sample, e.g., a PBMC sample, or a bone marrow mononuclear cell (BMMC) sample.
  • a blood or blood-derived sample e.g., a PBMC sample, or a bone marrow mononuclear cell (BMMC) sample.
  • BMMC bone marrow mononuclear cell
  • the cells can be immune cells, e.g., cells belonging to one or more immune cell populations such as those described herein, for example, as shown in Table 1 or Figure 17.
  • the cells can be assessed for cell surface markers to gate them into one or more immune cell populations such as those described herein.
  • the cells can be further assessed.
  • the cells are assessed for levels of one or more activatable elements, such as those described herein, either with or without treatment with a modulator.
  • cells may be exposed to one or more of a cytokine, such as an interleukin, or a TCR activator, or a BCR activator, or a TLR activator, or any activator or combination of activators as described in Table 1 or Figures 20A or 20B herein, then assessed, on a single cell basis, for the activation levels of one or more activatable elements, such as a pSTAT, or other activatable element as shown in Table 1 or Figures 20A and 20B.
  • a cytokine such as an interleukin, or a TCR activator, or a BCR activator, or a TLR activator, or any activator or combination of activators as described in Table 1 or Figures 20A or 20B herein
  • the cells may alternatively or additionally be assessed for expression levels of one or more FMRs or FMRLs, as described herein, such as one, two, three, four, five, or more than five FMRs and/or FMRLs as shown in Figure 15.
  • the cells may be assessed for expression levels of PD-1, and/or PDL1.
  • cells may be gated as positive or negative for the one or more FMRs or FMRLs, e.g., PD1+ or PD1-; the cells in one gated group, e.g., PD1+, may be further assessed, e.g., for levels of one or more activatable elements.
  • cells from a blood or blood-derived sample from an individual suffering from or suspected of suffering from a solid tumor are assessed on a single cell basis for 1) expression levels of one or more surface markers that can be used to classify the cells into one or more immune cell populations or subpopulations; and 2) activation levels of one or more activatable elements, with or without contacting the cells with a modulator.
  • cells from a blood or blood- derived sample from an individual suffering from or suspected of suffering from a solid tumor are assessed on a single cell basis for 1) expression levels of one or more surface markers that can be used to classify the cells into one or more immune cell populations or subpopulations; 2) expression levels of at least one, two, three, four, or five FMRs and/or FMRLs; and 3) activation levels of one or more activatable elements, with or without contacting the cells with a modulator.
  • these methods and compositions are not directed to circulating tumor cells, or to serum markers, though either or both may be used in addition to the methods and compositions to further refine diagnosis, prognosis, prediction, or monitoring.
  • the cancer comprises melanoma, breast cancer, small cell lung carcinoma, non-small cell lung carcinoma, prostate cancer, or bladder cancer.
  • the cancer is melanoma, breast cancer, lung cancer, e.g., small cell lung carcinoma or non-small cell lung carcinoma, or prostate cancer.
  • the cancer is melanoma or breast cancer.
  • the cancer is melanoma.
  • the individual is known to suffer from melanoma; in certain of these embodiments the cells are treated with a cytokine, e.g., IL-15, and the levels of a pSTAT, e.g., pSTAT-5, are measured.
  • the levels may indicate prognosis, e.g., length of progression-free survival.
  • the levels may alternatively, or in addition, indicate, alone or with other measures, likelihood of adverse effects, e.g., in the case of treatment with a checkpoint inhibitor such as ipilimumab, likelihood of development of colitis, or grade of colitis, or both.
  • the information can be used to select or not select treatment, modify treatment, as described herein.
  • levels of FMRs and/or EVIRLS are measured, which can include PDl and/or CTLA4.
  • the levels of IMRs and/or FMRLs, such as, e.g., PDl and/or CTLA4 can be indicative of likely response or non-response, or probability of response, to treatment, e.g., treatment with a checkpoint inhibitor such as ipilimumab.
  • the levels of the IMRs and/or IMRLs can be used to determine combination treatments, such as a combination of ipilimumab with another treatment, e.g., another checkpoint modulator, e.g., another checkpoint inhibitor, such as a PDl or PDLl inhibitor.
  • the cancer is breast cancer. See, e.g., Example 21.
  • the individual is known to suffer from breast cancer; in certain of these embodiments the cells are treated with a TCR activator, e.g., aCD3 and aCD28, and downstream elements of T cell activation measured, e.g., p-ERK, p-AKT, p-PLCg2, p-CD3z, p-s6, and the like, typically in T cells or T cell subpopulations, though after sufficient time, other cell populations may also show an effect.
  • a TCR activator e.g., aCD3 and aCD28
  • downstream elements of T cell activation measured, e.g., p-ERK, p-AKT, p-PLCg2, p-CD3z, p-s6, and the like, typically in T cells or T cell subpopulations, though after sufficient time, other cell populations may also show an effect.
  • IMRs and/or IMRLs can additionally or alternatively be measured in single cells, for example, one or more of the FMRs/FMRLs shown in Figure 15, such as one or more of, for example 2, 3, 4 or more of, PDl, PDLl, OX-40, TFM-3, GITR, CTLA4, or one or more of, for example 2, 3, 4 or more of PDl, PDLl, OX-40, TFM-3, GITR.
  • Levels, either of activatable elements, of IMRs/FMRLs, or both can be measured in any of a number of immune cell populations as described herein. The levels, may indicate prognosis, e.g., length of progression-free survival.
  • the levels of one or more FMRs/FMRLs, comprising GITR can indicate PFS.
  • the levels may alternatively, or in addition, indicate, alone or with other measures, likelihood of adverse effects, e.g., in the case of treatment with a checkpoint inhibitor such as ipilimumab, likelihood of development of colitis, or grade of colitis, or both; this is merely an exemplary treatment and any other suitable treatment and its potential side effects are included.
  • the information can be used to select or not select treatment, modify treatment, as described herein.
  • levels of FMRs and/or EVIRLS are measured, which can include PDl and/or CTLA4.
  • the levels of FMRs and/or FMRLs can be indicative of likely response or non- response, or probability of response, to treatment, e.g., treatment with a checkpoint inhibitor such as a PDl or PDLl modulator.
  • the levels of the FMRs and/or FMRLs can be used to determine combination treatments, such as a combination of an immunomodulator, e.g., anti- PD1 or anti-PDLl, with another treatment, e.g., another treatment to counteract potential side effects of the immunodulator, e.g., a treatment regulate T cell suppression, such as Fresolimumab.
  • the information described above can be gathered before a treatment decision is made, and/or after a treatment decision is made, for example, to monitor a cancer, such as the cancers described above, e.g., melanoma or breast cancer.
  • Treatment can be administered or not administered, or mode or other characteristic of administration modified, based at least in part on the information described above.
  • Combination treatments e.g., of a primary immunotherapeutic, such as a checkpoint modulator, for example a checkpoint inhibitor such as ipilimumab
  • a primary immunotherapeutic such as a checkpoint modulator, for example a checkpoint inhibitor such as ipilimumab
  • Combination treatments can include treatment with another immunomodulatory treatment, such as another checkpoint modulator, e.g., checkpoint inhibitor, and/or treatment to ameliorate potential or existing side effects, and/or other combinations.
  • Combination treatments are described elsewhere herein.
  • the invention provides methods and compositions related to determining a functional status of an EVIR in cells of a cell population, e.g., an immune cell population.
  • the IMR may be any suitable IMR, for example of Figure 15 and the description thereof, for which knowledge of a functional status is desired in a particular immune cell population, such as any of the immune cell populations described herein, e.g., of Table 1 or Figure 17.
  • the cells may be gated into the immune cell population by standard gating methods, e.g., by determining the surface expression levels of one or more cell surface markers of the cells and gating them accordingly. Exemplary cell surface markers are shown in Table 1 and Figure 17, but any suitable cell surface markers may be used.
  • the functional status of an EVIR or EVIRs may be determined in a plurality of cell populations, for example a plurality of immune cell populations.
  • the method can comprise contacting cells of the immune cell population or populations with an activator that activates the cells, in the presence and absence of activation of the EVIR, e.g., in the presence and absence of an EVIRL or an EVIR agonist, and determining a change in the activation level of one or more intracellular activatable elements, as described herein, and/or the change in expression levels of one or more intracellular expressed elements, as described herein, for example, in single cells of the population.
  • Any suitable EVIRL or EVIR agonist may be used to activate the EVIR, such as one or more of those shown in Figure 15 and the description thereof or Table 1, so long as it activates the particular EVIR whose functional status is to be determined.
  • Suitable intracellular activatable elements, e.g., phosphoproteins include those shown in Table 1 and Figure 20, for example, p-ERK and/or p-AKT.
  • the particular activator used to activate the cells can depend on the immune cell population; for example, TCR activators will be specific to T cell populations, BCR activators will be specific to B cell populations; other activators, such as TLR agonists and ligands, may have a broader effect, activating cells of more than one immune cell population, or may be specific, in some cases. Any suitable activator may be used, such as one of those shown in Table 1, or a combination of activators. In certain embodiments, a surrogate activator, e.g., one or more cytokines, may be used, and the appropriate activatable element measured, such as one or more p-STATs (e.g., 1, 3, 4, 5, or 6), assessed.
  • TCR activators will be specific to T cell populations
  • BCR activators will be specific to B cell populations
  • other activators such as TLR agonists and ligands
  • Any suitable activator may be used, such as one of those shown in Table 1, or a combination of activators.
  • the surface expression level of the IMR or IMRs may also be determined for the single cells, for example, in the same cell a surface expression level and a functional status of the same IMR may be determined.
  • Surface expression levels of a different IMR, or a plurality of different EVIRs, including others whose functional status is not determined, may be determined in the single cells in addition to, or alternatively to, the expression level of the IMR whose functional status is determined in the cell.
  • the surface expression of the IMR may be used to gate the cells, so that the functional status of the IMR is determined only in cells expressing the IMR at a level greater than, or equal to or greater than, a threshold value.
  • the determination of functional status of the IMR or IMRs may also be determined when contacting the cells of the cell population with an agent, for example, an agent being screened as a potential therapeutic agent, or a therapeutic agent that can be used to treat a patient from whom the cells were derived, and the effect of the agent on the functional status of the IMR or IMRs determined.
  • an agent for example, an agent being screened as a potential therapeutic agent, or a therapeutic agent that can be used to treat a patient from whom the cells were derived, and the effect of the agent on the functional status of the IMR or IMRs determined.
  • Any suitable method of evaluating single cells may be used, for example, cytometry, such as flow cytometry or mass cytometry.
  • the functional status of a plurality of IMRs may also be determined.
  • the IMRs are evaluated in the same cell, for example, if it is wished to determine if a certain combination therapy or combination agent that affects the plurality of IMR pathways may be effective, for example, in treating a patient from whom the cells have been derived.
  • the functional status of a plurality of IMRs is determined, but at least some or all of the IMRs are evaluated on different cells, so as to determine the functional status of each IMR separately. This eliminates the additive or synergistic effects that may be seen when a plurality of IMRs are evaluated on the same cell, which can be desirable in some applications.
  • Certain sets of embodiments relate to determining a phenotype, such as for a cell population, e.g., an immune cell population or a non-immune cell population.
  • a second phenotype may also be determined, where the second phenotype is based, at least in part, on the first phenotype, for example, determining a phenotype for an individual, where the phenotype is based, at least in part, on a phenotype for an immune cell population in a sample or samples from the individual.
  • a phenotype may be determined based, at least in part, on two or more different phenotypes determined in different sets of embodiments as presented herein, or non-immune cell populations, as presented herein, for example, determining a phenotype for an individual, where the phenotype is based, at least in part, on two or more different phenotypes determined in different sets of embodiments as presented herein for immune cell populations in a sample or samples from the individual, and/or a phenotype for non-immune cell populations, such as tumor cells, in a sample or samples from the individual, as presented herein.
  • an individual may present with a cancer, for example AML, and be phenotyped as PD-1 positive, OX-40 negative, meaning that one or more immune cell populations and/or tumor cell populations in the individual showed high functioning and/or expression for PD-1 but low or no functioning for OX-40.
  • An aspect of treating the patient with an immunotherapy could be based, at least in part, on the phenotype; for example the PD-1+ OX-40- phenotype patient could be a candidate for treatment with a PD-1 pathway modulator but not for treatment with an OX-40 pathway modulator.
  • a patient with a PD- 1+OX-40+ phenotype could be a candidate for a combination treatment with both a PD-1 modulator and an OX-40 modulator.
  • embodiments of the invention embrace different type of phenotyping for use in various situations, so long as the phenotyping involves one or more of the sets of embodiments provided herein.
  • the invention provides methods and compositions related to determining a phenotype of a population of cells of an immune cell population, e.g., an immune cell population derived from a sample from a patient suffering from a pathological condition, such as cancer, comprising determining in single cells of the cell population a functional status of one or more IMRs expressed or potentially expressed on the surface of the immune population cells and determining the phenotype based on the functional status of the IMR or IMRs.
  • an immune cell population derived from a sample from a patient suffering from a pathological condition, such as cancer
  • the functional status of the FMR may be determined by any suitable method, for example, by any of the methods used in the first set of embodiments, optionally including methods in which surface expression levels of one or more IMRs, such as the one or more IMRs whose functional status is being determined, are also determined (for example, where the surface expression level and the functional level of an FMR are determined in the same cell), as well as, optionally, surface expression levels of one or more other IMRs, is determined; the expression levels thus determined can be used, e.g., in gating the cells, as described above, or as separate pieces of information about the cells of the immune cell population, or both.
  • the sample can be any sample as described herein, for example, a PBMC sample, a BMMC sample, or a solid tumor sample; the immune cell population phenotype may be determined in TILS, such as TILS from a solid tumor sample or TILS in a blood or blood-derived sample.
  • the immune cell population may be any immune cell population described herein, for example, an immune cell population of Table 1 or Figure 17.
  • the method comprises determining the phenotype based on functional statuses of 2 different IMRs, where the functional status of each FMR may be determined in separate cells, or the functional status of the 2 different IMRs determined in the same cell, optionally with determination of surface expression levels of one or both of the 2 different IMRs on the cells, e.g., in the same cells in which functional status is determined.
  • the results can be a single functional status that reflects the influence of both the IMRs, when activated, on the activation of the immune cells, whereas when functional status is determined for each FMR in separate cells, the results can be multiple functional statuses, each reflecting a different FMR. Combinations of the two different approaches may be used.
  • the method comprises determining the phenotype based on functional status of at least 3, 4, 5, 6, 7, 8, 9, or 10 different FMRs, where the functional status of each FMR may be determined in separate cells, or the functional status of the FMRs determined on the same cell, in any combination. In certain embodiments, the functional status of each FMR is determined in separate cells so that the functional status of one FMR is determined in any given cell.
  • the FMR or FMRs may be any suitable FMR, such as FMRs shown in Figure 15 and its accompanying description.
  • the invention includes treating a patient based on the phenotype thus determined for one or more immune cell populations derived from a sample obtained from the patient, where the treatment may be any treatment as described herein, such as an immunotherapy, e.g. a combination
  • immunotherapy for example an immunotherapy that is a combination of at least two immunotherapies.
  • exemplary treatments are given herein, and include a combination immunotherapy that includes modulation of the PD-1 pathway, or modulation of the CTLA-4 pathway, or both, or a monotherapy that involves modulation of any of the FMR pathways shown in Figure 15 and its description, such as PD-1, or CTLA-4.
  • the invention includes treating a patient based on the phenotype thus determined, optionally also based on a phenotype as determined by, e.g., any of the methods used in the third set of embodiments, and optionally based on a phenotype as determined by, e.g., any of the methods used in the fourth set of embodiments, or a phenotype derived from the phenotypes (e.g., a phenotype of the patient), where the treatment may be any treatment as described herein, such as an immunotherapy, e.g. a combination immunotherapy, for example an immunotherapy that is a combination of at least two immunotherapies.
  • an immunotherapy e.g. a combination immunotherapy
  • an immunotherapy that is a combination of at least two immunotherapies.
  • Exemplary treatments are given herein, and include a combination immunotherapy that includes modulation of the PD-1 pathway, or modulation of the CTLA-4 pathway, or both, or a monotherapy that involves modulation of any of the IMR pathways shown in Figure 15 and its description, such as PD-1, or CTLA-4.
  • the invention provides methods and compositions related to determining a phenotype of a population of cells of an non-immune cell population in a sample from, e.g., a patient suffering from a pathological condition, such as cancer, comprising determining in single cells of the cell population, e.g., tumor cells, surface expression levels of at least at least one, for example, at least two, such as at least three, different IMRLs and determining the phenotype based on the levels of the at least one, two, or three different IMRLs.
  • the phenotype may be determined based on the surface expression levels of at least 4 different IMRLs on single cells of the population.
  • the phenotype may be determined based on the surface expression levels of at least 5, 6, 7, 8, 9, or 10 different IMRLs on single cells of the population.
  • the surface expression levels for the different IMRLS may be determined on the same cell for the cells of the non-immune cell population, as long as they may be
  • the sample can be any sample as described herein, for example, a PBMC sample, a BMMC sample, or a solid tumor sample, and the non-immune cell population phenotype may be determined in tumor cells.
  • the IMRLs may be any suitable FMRL, such as IMRLs shown in Figure 15 and its accompanying description.
  • the invention includes treating a patient based on the phenotype thus determined, optionally also based on a phenotype as determined by, e.g., any of the methods used in the second set of embodiments, and optionally based on a phenotype as determined by, e.g., any of the methods used in the fourth set of embodiments, or a phenotype derived from the phenotypes (e.g., a phenotype of the patient), where the treatment may be any treatment as described herein, such as an immunotherapy, e.g. a combination immunotherapy, for example an immunotherapy that is a combination of at least two immunotherapies.
  • an immunotherapy e.g. a combination immunotherapy
  • an immunotherapy that is a combination of at least two immunotherapies.
  • Exemplary treatments are given herein, and include a combination immunotherapy that includes modulation of the PD-1 pathway, or modulation of the CTLA-4 pathway, or both, or a monotherapy that involves modulation of any of the IMR pathways shown in Figure 15 and its description, such as PD-1, or CTLA-4.
  • the invention provides methods and compositions related to determining the phenotype of a population of cells of an immune cell population in a sample, for example, a sample from a patient suffering from a pathological condition, comprising determining in single cells of the immune cell population surface expression levels of at least one, for example two, such as at least three different IMRs, and determining the phenotype based on the surface expression levels of the at least one, two, or three different IMRs.
  • the pathological condition can be cancer.
  • the sample can be any sample as described herein, for example, a PBMC sample, a BMMC sample, or a solid tumor sample, and the immune cell population phenotype may be determined in cells that include TILS.
  • the immune cell population may be any immune cell population described herein, for example, an immune cell population of Table 1 or Figure 17.
  • the method comprises determining the phenotype based on surface expression levels of at least 4 different EVIRs on single cells of the population. In certain embodiments, the method comprises determining the phenotype based on surface expression levels of at least 5, 6, 7, 8, 9, or 10 different EVIRs on single cells of the population.
  • the EVIRs may be any suitable IMR, such as EVIRs shown in Figure 15 and its accompanying description.
  • the surface expression levels for the different FMRS may be determined on the same cell for the cells of the non-immune cell population, as long as they may be distinguishably determined on the same cell; alternatively or additionally, the surface expression level of each may each be determined on different cells, or any combination of determination of any number on the same cell or different cells.
  • the invention includes treating a patient based on the phenotype thus determined, optionally also based on a phenotype as determined by, e.g., any of the methods used in the second set of embodiments, and optionally based on a phenotype as determined by, e.g., any of the methods used in the third set of embodiments, or a phenotype derived from the phenotypes (e.g., a phenotype of the patient), where the treatment may be any treatment as described herein, such as an immunotherapy, e.g. a combination immunotherapy, for example an immunotherapy that is a combination of at least two immunotherapies.
  • an immunotherapy e.g. a combination immunotherapy
  • an immunotherapy that is a combination of at least two immunotherapies.
  • Exemplary treatments are given herein, and include a combination immunotherapy that includes modulation of the PD-1 pathway, or modulation of the CTLA-4 pathway, or both, or a monotherapy that involves modulation of any of the IMR pathways shown in Figure 15 and its description, such as PD-1, or CTLA-4.
  • the invention provides methods and compositions related to treating a patient suffering from a pathological condition including treating the patient with a treatment for the condition, wherein an aspect of treating the patient with the treatment is based on an outcome of a treatment decision process comprising consideration of a first quantitative value, or a value or values derived from the first quantitative value, wherein the first quantitative value is obtained from results of an assay comprising determining functional status of one or more IMRs in single cells of a immune cell population or a subpopulation thereof in a sample from the patient.
  • the pathological condition may be any suitable pathological condition as described herein, e.g., cancer.
  • Determining the functional status of the one or more IMRs may be accomplished by any suitable method, such one or more of the methods used in the first set of embodiments.
  • the treatment may be any suitable treatment, such as any suitable treatment described herein, such as treatments described in the second, third, or fourth sets of embodiments, or any other suitable treatment, such as a combination treatment that includes an immunotherapy and also includes one or more of a targeted therapy, radiation therapy, surgical therapy, or
  • chemotherapy or a combination treatment that includes two different immunotherapies, such as vaccine and modulation of one or more IMRs/IMR pathways, and the like.
  • the methods may also comprise determining surface expression levels of the IMR or IMRs in the single cells, for example, by any of the methods used in the first set of embodiments, for example, using cytometry, such as flow cytometry or mass cytometry.
  • the expression levels may be used to gate cells into populations in which functional status is determined, for example to gate cells into a subpopulation of the immune cell population, and wherein single cells of the subpopulation are gated into the subpopulation on the basis of the surface expression levels of the IMR or IMRs of the single cell.
  • the expression levels of the IMR or FMRs may, in addition or alternatively, be used to obtain a second quantitative value or values, which may also be considered in the treatment decision process.
  • the methods may also comprise determining surface expression levels of an FMRL or FMRLs, for example in single cells of a non-immune cell population, for example, a tumor cell population that can be derived from a sample from the patient, for example, by any of the methods used in the third set of embodiments.
  • the expression levels of the FMRL or FMRLs maybe used to obtain a third quantitative value or values, which may also be considered in the treatment decision process, e.g., in a process where the first quantitative value is considered, or the first and second quantitative values are considered.
  • Treatment decision processes, outcomes of treatment decision process, and aspects of treating the patient may be any suitable process or processes, outcome or outcomes, and/or aspect or aspects, for example as described herein.
  • an aspect of treating the patient may comprise a decision to treat the patient or not treat the patient with the treatment, a choice of the treatment or a component of the treatment, a choice of the timing of the treatment or of a component of the treatment, a choice of a dosage of the treatment or a component of the treatment, or a combination thereof.
  • an outcome of the treatment decision process may comprise a first likelihood of the patient responding to the treatment, a second likelihood of prolongation of the patient's life due to receiving the treatment, or a third likelihood of the patient experiencing an adverse treatment effect, or any combination of the first, second, and/or third likelihoods.
  • the invention provides methods and compositions related to treating a patient suffering from a pathological condition, e.g., cancer, comprising treating the patient with a treatment for the condition; wherein an aspect of treating the patient with the treatment is based on an outcome of a treatment decision process, wherein the treatment decision process comprises consideration of at least two of a first, second, and third quantitative value, or a value or values derived from the at least two quantitative values; and
  • first, second, and/or third quantitative values are obtained from results of a first, second, and/or third assay, respectively, wherein
  • the first assay comprises determining surface expression levels of a first immunomodulatory receptor (IMR) of a first cell population cell population (CP in a first sample from the patient;
  • IMR immunomodulatory receptor
  • the second assay comprises determining functional status of a second IMR in single cells of a second CP or a subpopulation thereof in a second sample from the patient;
  • the third assay comprises determining surface expression levels of an IMR ligand (FMRL) for a third IMR in a third cell population in a third sample from the patient.
  • FMRL IMR ligand
  • the expression levels may be determined in single cells, by any suitable method, for example, by any of the methods used the first set of embodiments, using any suitable detection technique, such as cytometry, e.g., flow cytometry or mass cytometry.
  • cytometry e.g., flow cytometry or mass cytometry.
  • functional status of the second IMR as determined in single cells the functional status may be determined in single cells by any suitable method, for example, by any of the methods used the first set of embodiments, using any suitable detection technique, such as cytometry, e.g., flow cytometry or mass cytometry.
  • the levels may be determined in single cells, by any suitable method, for example, by any of the methods used the third set of embodiments, using any suitable detection technique, such as cytometry, e.g., flow cytometry or mass cytometry.
  • cytometry e.g., flow cytometry or mass cytometry.
  • an aspect of treating the patient comprises a decision to treat the patient or not treat the patient with the treatment, a choice of the treatment or a component of the treatment, a choice of the timing of the treatment or of a component of the treatment, a choice of a dosage of the treatment or a component of the treatment, or a combination thereof.
  • an outcome of the treatment decision process comprises a first likelihood of the patient responding to the treatment, a second likelihood of prolongation of the patient's life due to receiving the treatment, or a third likelihood of the patient experiencing an adverse treatment effect, or any combination of the first, second, and/or third likelihoods, etc.
  • assays comprise the first assay and the second assay, wherein the assays are performed on single cells, the first and second samples are the same sample, the first and second FMRs are the same FMR, and the first and second cell populations are the same population, and wherein the second quantitative value represents a functional status of the FMR for the subpopulation of the population, wherein the process of obtaining the second quantitative value comprises gating the results for functional status of the FMR in the single cells of the cell population on the basis of the results of the determination of the expression level of the FMR in the same single cells of the population.
  • the gating comprises establishing a threshold for expression level of the IMR in a single cell and single cells in the cell population having an expression level of the IMR above the threshold are included in the subpopulation and single cells in the cell population having an expression level equal to or below, or below, the threshold are excluded from the subpopulation.
  • the first and second cell populations are immune cell populations, for example, the first and second immune cell populations can be the same immune cell population or the first and second immune cell populations can be different immune cell populations.
  • the third cell population can be a nonimmune cell population, such as a tumor cell population.
  • the first and second cell populations can comprise a first and second cell immune cell population of TABLE 1 or Figure 17.
  • the cell populations can be identified by any suitable method, e.g., by surface expression levels of at least one, two, three of the cell surface markers of Table 1 or Figure 17.
  • the first sample and the second sample, and optionally the third sample are the same sample.
  • the first, second and third samples can be any suitable samples, as described herein, for example, a blood or blood-derived sample, such as a PBMC sample, or bone marrow or bone marrow- derived sample, such as a BMMC, or a solid sample or solid-sample-derived samples, such as a tumor sample, for example a primary tumor sample or a metastatic tumor sample.
  • the first and second samples comprise tumor-infiltrating lymphocytes (TILS) derived from a solid tumor sample and the third sample comprises tumor cells derived from the same solid tumor sample.
  • TILS tumor-infiltrating lymphocytes
  • TILS may also be found in a blood or blood-derived sample, such as a PBMC sample, a bone or bone marrow-derived sample, such as a BMMC.
  • a blood or blood-derived sample such as a PBMC sample, a bone or bone marrow-derived sample, such as a BMMC.
  • tumor cells may be found in a blood or blood-derived sample, such as circulating tumor cells (CTCs)
  • a plurality of FMRLs is determined, e.g., in single cells.
  • the plurality of FMRLs can comprise a plurality of FMRLS of Figure 15 and the description thereof.
  • a plurality of FMRs is assayed.
  • the plurality of FMRs can comprise a plurality of FMRs of Figure 15 and the description thereof.
  • the condition is cancer
  • the therapy is a combination therapy comprising immunotherapy
  • the aspect of the treatment comprises choice of the combination therapy.
  • the treatment decision process further comprises consideration of a
  • the EVIRL corresponds to the IMR for a) or b).
  • the assay of part (b) comprises determining the functional status of the IMR in the presence and absence of an immunotherapeutic agent, or determining the functional status of the EVER in the presence and absence of a plurality of immunotherapeutic agents, such as immunotherapeutic agent or agents that are candidates for use in the treatment, or agents that represent a class of immunotherapeutic agents that are candidates for use in the treatment.
  • the invention provides methods and compositions related to a pharmaceutical package comprising one or more immunotherapeutic agents and
  • immunotherapeutic agents is to be used for treatment of a patient who suffers from a pathological condition, e.g., cancer, wherein either
  • cells associated with the patient's pathological condition e.g., tumor cells
  • an immune cell population from a sample from the patient is characterized by surface expression level of a first IMR that is greater than, or greater than or equal to a threshold expression level;
  • an immune cell population from a sample from the patient is characterized by a change in the expression level and/or activation level of an intracellular element that is less than, or less than or equal to a threshold change, wherein the change in the expression level or activation level of the intracellular element in a cell of an immune cell type is in response to contact with an activator of that immune cell type and is indicative of the activation level of the cell, and wherein the change in the level may be measured in the presence and/or absence of an activator and/or inhibitor of an EVER that can be expressed on the cell of the immune cell type; or
  • a non-cell liquid from a sample from the patient contains an immune effector molecule at a level greater than, greater than or equal to, less than, or less than or equal to a threshold level; or
  • the pharmaceutical package may further comprise one or more components for use in gathering, treating, and/or transporting one or more samples from the patient for use in the one or more methods of part (ii).
  • the cancer in certain embodiments in which the pathological condition is cancer, can be characterized by tumor cell surface expression of an IMRL that modulates an inhibitory IMR of Figure 15, for example, PD-1 and the description thereof, wherein the tumor cell surface expression level of the IMRL is greater than, or greater than or equal to, a threshold level.
  • the cancer can be characterized by tumor cell surface expression of plurality of FMRLs, each of which modulates a different inhibitory IMR of Figure 15, such as an FMRL that activates PD-1 and an FMRL that activates CTLA-4, and the description thereof, wherein the surface expression level of each of the FMRLs is greater than, or greater than or equal to, a threshold level for surface expression for that FMRL.
  • Intracellular activatable elements and their assay are as described in the methods and compositions used in the first set of embodiments.
  • the intracellular activatable element comprises p-ERK, p-AKT, p-ZAP70, PLCg, p-PKC0, p-p38, or p FkBp65, such as p-ERK or p-AKT.
  • the intracellular activatable element comprises p-STATl, p-STAT3, p-STAT4, p-STAT5, or p-STAT6, or a combination thereof.
  • the invention provides methods and compositions related to screening a first agent, for example an agent for potential use in treatment of a pathological condition, such as cancer, at a first screening level comprising
  • the determination of step (iv) can comprise an evaluation of a result of a comparison of the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element in the single cells of the first population, or a first quantitative value derived therefrom, with the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element in the single cells of the second population, or a second quantitative value derived therefrom, the result can be a third quantitative value.
  • the determination of step (iv) can comprise comparing the third quantitative value with a threshold value to determine if the third value is greater than, greater than or equal to, less than, or less than or equal to the threshold value.
  • the agent can be sent to the second screening level if the third quantitative value is greater than, or greater than or equal to, the threshold value.
  • the agent can be sent to the second screening level if the third quantitative value is less than, or less than or equal to, the threshold value.
  • the first and second cell populations can the same immune cell population, or they can be different immune cell populations.
  • the identity of the first and second immune cell populations can be determined by determining the levels of at least one cell surface marker in single cells of the first and second immune cell populations.
  • the method can further comprise determining the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element in single cells of a third immune cell population type that have not been activated and that have not been contacted with the agent.
  • the first, second, and third immune cell populations can be the same immune cell population, or one or more of them can be different from the others.
  • the method can further comprise determining surface expression levels of the first IMR in single cells of the first and second immune cell populations, for example the expression levels of the intracellular element and/or the activation levels of the intracellular activatable element can be determined in subpopulations of the first and second immune cell populations, and a cell is gated into the subpopulation of the first or second population on the basis of its surface expression level of the first IMR.
  • a cell can be gated by comparing its surface expression level of the EVER to a threshold expression level value for the first IMR, wherein the cell is gated into the subpopulation if its surface expression level of the first IMR is greater than the threshold value, or greater than or equal to the threshold value. See, e.g., compositions and methods described for use in the first set of embodiments.
  • the method can further comprising screening a second agent in combination with the first agent wherein the second agent is different from the first agent and wherein the cells of the first immune cell population further express a second IMR on their surfaces and step (i) further comprises contacting the first immune cell population with the second agent; and the cells of the second immune cell population further express the second EVER on their surfaces and in step (ii) the cells of the second population have not been contacted with the second agent.
  • the method can further comprise determining surface expression levels of the second EVER in single cells of the first and second immune cell populations.
  • Expression levels of the intracellular expression element and/or the activation levels of the intracellular activatable element can be determined in subpopulations of the first and second populations, and a cell can be gated into the subpopulation of the first and second population on the basis of its surface expression level of the first EVER and its surface expression level of the second EVIR.
  • a cell can be gated by comparing its surface expression level of the first EVER to a threshold expression level value for the first EVIR and its surface expression level of the second EVIR to a threshold expression level value for the second EVIR, wherein the cell is gated into the subpopulation if its surface expression level of the first EVER is greater than the threshold value for the surface expression level of the first EVIR and its surface expression level of the second EVIR is greater than the threshold value for the surface expression level of the second EVIR, or greater than or equal to the threshold values for the surface expression of the first and second EVIRs.
  • the cells of the first and second immune cell populations expressing the first IMR, and, optionally, second IMR have been induced to express the first IMR, and, optionally, second EVER, by activation of the cells of the first and second immune cell populations at a time previous to steps (i) and (ii).
  • the cells can be, e.g., derived from a sample from a healthy individual, a plurality of samples from the healthy individual, or a plurality of samples from a plurality of healthy individuals.
  • the cells can be from cell lines.
  • the cells can derived from a sample from an individual suffering from a pathological condition, e.g., cancer, or a plurality of samples from the individual, or a plurality of samples from a plurality of individuals suffering from the pathological condition, e.g., cancer.
  • a pathological condition e.g., cancer
  • the cells can derived from a sample from an individual suffering from a pathological condition, e.g., cancer, or a plurality of samples from the individual, or a plurality of samples from a plurality of individuals suffering from the pathological condition, e.g., cancer.
  • kits In these sets of embodiments, the invention provides kits.
  • a kit may comprise:
  • At least 1, 2, 3, or 4 for example in certain embodiments at least 1, such as in certain embodiments at least 2, distinguishably detectable binding elements for determination of activation levels of at least 1, 2, 3, or 4, for example in certain embodiments at least 1, such as in certain embodiments at least 2, intracellular activatable elements whose levels indicate the functional status of one or more IMRs; and/or
  • (iii) at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, for example in certain embodiments at least 1, in certain embodiments at least 2, in certain embodiments at least 3, in certain embodiments at least 4, in certain embodiments at least 5, distinguishably detectable binding elements for determining surface expression levels of one or more different IMRs; and/or
  • activators for ) at least 1, 2, 3, or 4, for example in certain embodiments at least 1, such as in certain embodiments at least 2, further such as at least 3 different immune cell populations; and/or
  • Distinguishably detectable binding elements can be any suitable distinguishably detectable binding elements, such as those described herein.
  • the plurality of elements described herein can be any suitable distinguishably detectable binding elements, such as those described herein.
  • the plurality of elements described herein can be any suitable distinguishably detectable binding elements, such as those described herein.
  • the plurality of elements described herein can be any suitable distinguishably detectable binding elements, such as those described herein.
  • distinguishably detectable binding elements can be antibodies, as that term is defined herein, which can be labeled, for example, directly labeled, with distinguishably detectable labels suitable for detection in a flow cytometer, for example, fluorophores.
  • the distinguishably detectable binding elements can be antibodies, as that term is defined herein, which can be labeled, for example, directly labeled, with distinguishably detectable labels suitable for detection in a mass cytometer, for example, mass labels or mass tags.
  • Exemplary mass labels are those used for detection in the CyToF instruments, available from Fludigm.
  • kits described herein can contain the elements of the kit as described above and can further include, if the kit does not already include it, one or more of the following:
  • kits e.g., packaging suitable for transport from a kit manufacturer or distributor to an end user, where a single kit may be packaged in one or more than one packages, so long as all the packages are for use for a single purpose, which may be indicated, e.g., on a website, or in a set of instructions, or the like;
  • reagents for use in conducting the procedure or procedures for which the elements of the kit are intended such as buffers, permeabilizers, fixatives, and the like, as described elsewhere herein;
  • components for use in for use in conducting the procedure or procedures for which the elements of the kit are intended such as microtiter plates, e.g. 96-well plates, which can be unloaded or preloaded with one or more elements of the kit, buffers, etc.;
  • kit material for interpreting the results of use of the kit, such as scientific papers, where the materials may be written or available electronically, e.g., on a website;
  • kits may be a kit intended for use in a companion diagnostic process for a therapeutic agent, such as an immunotherapeutic agent for use in an immunotherapy comprising modulation of one or more IMR pathways, e.g., an IMR modulator, or IMRL modulator, where the kit includes a distinguishably detectable antibody configured for use in binding to and detecting an activatable element, where the magnitude of change in the activation level of the activatable element in single cells of an immune population derived from a sample from a patient in whom the immunotherapeutic agent may be used corresponds to the functional level of an IMR pathway, and instructions for use, where the antibody is suitably packaged for transport from a manufacturer or a distributor to an end user and the instructions for use are suitably configured to be used by the end user, such as written instructions including with or separate from the kit, instructions on a website, and the like, to accomplish the purpose of the kit, which may be to provide results suitable for determining if a given patient will or will not respond to the therapeutic agent,
  • a therapeutic agent such as an
  • kits can comprise a plurality of components in any combination as described above, so long as they are supplied from a manufacturer or distributor to an end user for a particular use, e.g., in
  • the particular use may be to produce one or more results that is used in a decision process, such as a decision process described herein, e.g., a treatment decision process, a prognosis, diagnosis, or monitoring process, a screening process, etc.
  • the invention provides a kit comprising [00241] a distinguishably detectable binding element configured for use in binding to and distinguishably detecting a first intracellular element, wherein a change in the expression level and/or activation level of the first intracellular element in a cell of an immune cell type in response to exposure of the cell to an activator of the immune cell type is indicative of activation of the cell; and
  • a distinguishably detectable binding element configured for use in binding to and distinguishably detecting a cell surface IMR on the cell or a cell surface EVERL on a cell of a population of cells of a non-immune cell type.
  • the kit can further comprise the activator.
  • the kit includes a plurality of distinguishably detectable binding elements configured for use in binding to and distinguishably detecting a plurality of different cell surface EVERs or a plurality of different cell surface EVERLs.
  • the plurality of different surface IMRs and/or the plurality of different cell surface EVIRLs can be a plurality of different surface EVIRs and/or a plurality of different cell surface EVIRLs of Figure 15 and the description thereof.
  • the plurality of EVIRs can comprise PD-1 and CTLA-4 and the plurality of EVIRLs can comprise at least two of B7-1, B7-2, PDL-1, and PDL-2.
  • the kit further comprises instructions for use of the kit, for example in an assay for predicting the response of a patient to immunotherapy, such as wherein the immunotherapy is an immunotherapy that directly or indirectly affects activation of the population of cells of the immune cell type.
  • the kit further comprises a plurality of distinguishably detectable binding elements, each configured for use in binding to and distinguishably detecting a different cell surface marker, wherein the level of at least two of the plurality of different cell surface markers can be used to type the cell as a cell of an immune cell population.
  • the plurality of cell surface markers can comprise any suitable plurality, as known in the art, for example, a plurality of cell surface markers listed in TABLE 1 or Figure 17.
  • the cell surface EVER or the cell surface EVIRL comprises an EVER, or an EVIRL or of FIGURE 15 and the description thereof.
  • the EVER can be PD-1 and the EVIRL can be PDL-1 or PDL-2.
  • the intracellular element is an intracellular activatable element, such as an activatable element of TABLE 1 or Figure 20.
  • the invention provides a kit comprising at least one, for example in certain embodiments at least two, such as in certain embodiments at least three, distinguishably detectable binding elements, wherein the at least one, two, or three distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, or three different cell surface IMRs on single cells of an immune cell population and/or at least one, two, or three cell surface IMRLs on single cells of a non-immune cell population, e.g., a tumor cell population
  • the kit comprises at least four distinguishably detectable binding elements, wherein the at least four distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, three, or four different cell surface IMRs on single cells of an immune cell population and/or at least one, two, three, or four cell surface IMRLs on single cells of a nonimmune cell population.
  • the kit comprises at least five distinguishably detectable binding elements, wherein the at least five distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, three, four, or five different cell surface IMRs on single cells of an immune cell population and/or at least one, two, three, four or five cell surface IMRLs on single cells of a non-immune cell population.
  • the kit comprises at least six distinguishably detectable binding elements, wherein the at least six distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, three, four, five, or six different cell surface IMRs on single cells of an immune cell population and/or at least one, two, three, four, five, or six surface IMRLs on single cells of a non-immune cell population.
  • the kit comprises at least seven distinguishably detectable binding elements, wherein the at least seven distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, three, four, five, six, or seven different cell surface IMRs on single cells of an immune cell population and/or at least one, two, three, four, five, six, or seven surface IMRLs on single cells of a non-immune cell population.
  • the kit comprises at least eight distinguishably detectable binding elements, wherein the at least eight distinguishably detectable binding elements are configured for use in binding to and distinguishably detecting at least one, two, three, four, five, six, seven, or eight different cell surface IMRs on single cells of an immune cell population and/or at least one, two, three, four, five, six, seven, or eight surface IMRLs on single cells of a non-immune cell population.
  • the kit comprises both one or more distinguishably detectable binding elements configured for use in binding to and distinguishably detecting at least one, two, three, four, five, six, seven, or eight different cell surface IMRs on single cells of an immune cell population and one or more distinguishably detectable binding elements configured for use in binding to and distinguishably detecting at least one, two, three, four, five, six, seven, or eight cell surface IMRLs on single cells of a non-immune cell population, e.g., a tumor cell population.
  • a non-immune cell population e.g., a tumor cell population.
  • the surface IMRs and/or cell surface IMRLs can be surface IMRs and/or cell surface FMRLs of Figure 15 and the description thereof.
  • the FMRs can comprise PD-1 and CTLA-4 and the FMRLs can comprise at least two of B7-1, B7-2, PDL-1, and PDL-2.
  • the invention provides methods and
  • compositions related to a system are provided.
  • the invention provides a system for treating a patient suffering from a pathological condition, e.g., cancer, with a treatment, wherein the system comprises
  • a system for determining a quantitative value, or a value or values derived from the quantitative value wherein the quantitative value is obtained from results of an assay comprising determining functional status of an FMR, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 FMRs, in single cells of an immune cell population in the first sample from the patient, and/or a quantitative value, or a value or values derived from the quantitative value, wherein the quantitative value is obtained from results of an assay comprising determining surface expression levels of one or more FMRLs in single cells of a non-immune cell population in the second sample, where the first and second samples may be the same or different, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 FMRLS;
  • a communication system to communicate the quantitative value, values, or a value or values derived from the quantitative value or values, to the patient and/or the healthcare provider, and/or to a system wherein the value or values may be further analyzed and/or modified;
  • an aspect of treating the patient with the treatment is based on an outcome of a treatment decision process comprising consideration by the patient and/or healthcare provider of the quantitative value, or the value or values derived from the quantitative value, or consideration by the patient and/or healthcare provider of the results of the further analysis and/or modification.
  • the invention provides a system
  • the invention provides a system for screening potential agents for immunotherapy comprising
  • a communication system to communicate the result or results to the person or persons or entity and/or to a system wherein the result or results may be further analyzed and/or modified to produce a further result or results and the result or results communicated to the person or persons or entity.
  • the invention provides methods and
  • compositions related to determining a vaccine therapy for a patient suffering from a pathological condition comprising
  • an immune cell population e.g., DC cells or a population derived therefrom, from a sample obtained from the patient, a functional status of an IMR or EVIRs, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 EVIRs, in single cells of the sample; and
  • SC P Single cell network profiling
  • compositions of the invention as described herein, problem is that to be successful in immunomodulation, such as immune-oncology treatments, it is very useful to have a deep understanding of immune system function, and the highly complex biologic impact of drugs that inhibit (e.g., anti-OX40) or stimulate (e.g., anti-CTLA4 and anti-PDl) an immune response.
  • SC P is one solution in that it uniquely quantifies functional signaling, e.g., across multiple signaling pathways with resolution to rare signals across, e.g., multiple and rare immune cell subsets that other cell-averaging technologies miss ant that brings important dimensionality to immunomodulation, such as immune-oncology, studies.
  • SCNP allows for rational drug development, among other things, by uniquely enabling the evaluation of mechanism of action (MO A) and on-/off-target effects and identifying predictive biomarkers of pharmacodynamics (PD), toxicity, and response in immune cell subsets from patient samples.
  • MO A mechanism of action
  • PD pharmacodynamics
  • SCNP has the advantage over other techniques in that it can quantify functional signaling information simultaneously across multiple immune cells with resolution down to rare cell subsets; analyze cell-cell interactions in mixtures of human primary cells (e.g., innate-adaptive immune cells); has an assay sensitivity that identifies signaling events that other cell-averaging technologies miss; provides dynamic rather than static measurements; allows correlation of short-term signaling with phenotypic changes associated with, e.g., therapeutic response and/or toxicity; can be synergistic with genomics and proteomics; and is a CLIA-validated platform for testing.
  • human primary cells e.g., innate-adaptive immune cells
  • has an assay sensitivity that identifies signaling events that other cell-averaging technologies miss provides dynamic rather than static measurements; allows correlation of short-term signaling with phenotypic changes associated with, e.g., therapeutic response and/or toxicity; can be synergistic with genomics and proteomics; and is a CLIA-validated platform for testing.
  • SCNP is used alone to, e.g., diagnose, prognose, predict, or monitor a condition, such as a cancer, e.g., a solid tumor cancer.
  • a condition such as a cancer, e.g., a solid tumor cancer.
  • blood or blood-derived samples such as PBMC samples, and/or TILS samples, from an individual suffering from or suspected of suffering from a cancer such as a hematological cancer or a solid tumor
  • SCNP can be evaluated by SCNP to diagnose, prognose, predict (e.g., predict response to a treatment such as a drug or combination of drugs, and/or predict toxicity), or monitor the cancer, such as a hematological cancer or a solid tumor.
  • SCNP can be used in combination with any other suitable measurement, such as measurements of one or more IMRs (which also can be done on a single cell basis, as described elsewhere herein), tumor geography (e.g., immunohistochemistry), tumor mutational status (genomics), clinical characteristics as known in the art or as developed for a particular cancer, and/or any other suitable measurement.
  • tumor geography e.g., immunohistochemistry
  • tumor mutational status e.g., cancer, and/or any other suitable measurement.
  • the invention provides methods and compositions directed at breast cancer.
  • Embodiments include various aspects of treatment of breast cancer, including treating the cancer with one or more treatments, including combination treatments, such as treatment with an immunotherapeutic agent and another treatment (which may be a second immunotherapeutic agent); dose selection; making a decision whether or not to treat; making a decision as to what treatment or combination of treatments to use; monitoring treatment, and the like.
  • Embodiments also include aspects of screening agents for potential use in the treatment of breast cancer.
  • Embodiments include kits for use in diagnosis, prognosis, monitoring, and/or drug screening in breast cancer.
  • a sample is used, for example, a sample that is not a tumor sample, such as a liquid sample, e.g., a blood sample or a blood-derived sample such as a PBMC sample.
  • a blood sample can be treated as described herein, e.g., to produce a peripheral blood mononuclear cell (PBMC) sample.
  • PBMC peripheral blood mononuclear cell
  • treatment or other aspect is based on the analysis of one or more non-tumor samples, such as PBMC sample/s, in whole or in part.
  • a breast cancer patient is treated with a treatment where at least one aspect of the treatment is based on an analysis of one or more blood samples or blood-derived samples from the patient, e.g., one or more PBMC samples.
  • blood samples or blood-derived samples e.g., one or more PBMC samples.
  • samples can be used for diagnostic, prognostic, or monitoring purposes, e.g., to determine if a particular treatment is effective in a patient (and potentially adjust dosage, frequency, etc.), or whether or not the treatment is producing or likely to produce side effects that can be monitored not only by clinical aspects but by alterations in the characteristics of the blood or blood-derived samples, or whether one or more additional treatments should be used, and the like.
  • Such samples can also be used for drug screening purposes, using the methods described herein.
  • a blood or blood-derived sample such as a PBMC sample
  • PBMC sample is analyzed, generally in single cells from the sample, for one, two, three, four, or all
  • cell surface markers used to classify cells in the sample into a plurality of populations and/or subpopulations, such as immune cell populations, e.g., one or more surface markers and immune cell populations such as those depicted in Figure 17;
  • response to one or more agents can be based on changes in one or more of the markers of l)-4), for example, by a change in one or more intracellular activatable elements on modulation of the cells, and where the agent can be, e.g., a therapeutic agent or a potential therapeutic agent.
  • the sample is analyzed for expression levels in single cells of at least one IMR or IMRL, e.g., at least one of the EVIRs and IMRLs shown in Figure 15; or at least two EVIRs or IMRLs, e.g., at least two of the IMRs and IMRLs shown in Figure 15; or at least three FMRs or FMRLs, e.g., at least three of the FMRs and FMRLs shown in Figure 15; or at least four FMRs or FMRLs, e.g., at least four of the FMRs and FMRLs shown in Figure 15.
  • IMR or IMRL e.g., at least one of the EVIRs and IMRLs shown in Figure 15
  • at least two EVIRs or IMRLs e.g., at least two of the IMRs and IMRLs shown in Figure 15
  • at least three FMRs or FMRLs e.g., at least three of the FMRs and FMRLs shown
  • the one or more FMRs or FMRLs are selected from the group consisting of PD-1, PD-L1, OX-40, GITR, and TFM-3.
  • the FMR or FMRLs includes at least PD-1.
  • the FMR or FMRLs includes at least PD-L1.
  • the FMR or FMRLs includes at least both PD-1 and PD-L1.
  • the FMR or FMRL includes PD-1 and/or PD-L1 and at least one other, or at least two other, or at least three other FMRs or FMRLs.
  • the levels of one or more activatable elements, basal and/or in response to modulation can be measured, e.g., in single cells of the sample.
  • one or more intracellular activatable elements can be any suitable element, such as phosphoproteins and/or cleavable proteins, as described herein.
  • the intracellular activatable elements are elements in a T cell receptor pathway, such as elements reflecting cell proliferation and/or cell survival.
  • levels are determined on a single cell basis of one or more of those shown in Table 1, or one or more of p-ERK and p-AKT, p- PLCg2, p-CD3z, p-s6, and IkB.
  • basal levels are determined and used to decide, e.g., one or more aspects of treatments.
  • a basal level of p-ERK, and/or p-AKT can be determined in one or more cell populations and used.
  • levels in response to one or modulators may be determined in one or more cell populations.
  • the cell population can be one or more of T cells, or a subpopulation thereof such as CD4+ or CD8+ T cells; K cells; and/or monocytes.
  • the modulator/s can be, e.g., T cell modulators, such as those shown in Table 1, for example, DCD3 and DCD28.
  • the sample is exposed to DCD3 and DCD28 and one or more of the intracellular activatable elements is measured in unexposed and exposed cells, for example, p-ERK and or p-AKT.
  • a blood or blood-derived sample from a breast cancer patient is evaluated, generally in single cells, for both expression levels of one or more FMRs or FMRLs and levels of one or more activatable elements, such as basal levels and/or levels after exposure of cells to a modulator, such as a T cell modulator.
  • levels of one or more intracellular activatable elements, modulated and/or basal are measured without the need to measure surface expression of EVIRs or IMRLs.
  • the cells can be analyzed by any suitable method, as described herein.
  • the cells are analyzed by flow cytometry.
  • the cells are analyzed by mass cytometry.
  • EVIRs, cell surface markers, intracellular activatable elements, and the like can be labeled with distinguishably detectable binding elements, also as described herein, such as antibodies, e.g., labeled antibodies, such as fluorescently labeled or mass labeled antibodies.
  • a blood or blood-derived sample contains a large number of cells of each different cell population.
  • Methods of the invention include gating the data from a sample so that data from only a portion of the cells in the sample is used.
  • the gating can include one or more of 1) gating on living vs. dead cells; 2) gating on healthy cells (as indicated by, e.g., apoptosis markers, such as levels of cPARP); 3) gating on a cell population or subpopulation; 4) gating on IMR or IMRLs; 5) gating on levels of one or more intracellular elements, such as intracellular activatable elements.
  • the order of gating can be important and in certain embodiments a certain order of gating is used.
  • one or more blood or blood-derived sample is obtained from a breast cancer patient and a decision is made regarding treatment, diagnosis, prognosis, drug screening, and the like, based at least in part on information from one or more particular cell population or populations in the sample. See, e.g., Example 21.
  • a patient can be treated with a particular treatment, for example, with a particular immunomodulatory agent or agents, where the decision to treat, and the selection of immunomodulatory agent or agents, and/or other aspects of the treatment, such as dosing, use of other treatments, and the like, is based on one or more characteristics, as described herein, of one or more cell populations or subpopulations in the one or more blood or blood-derived samples.
  • the populations or subpopulations can, e.g., include one or more of T cells or T cell subsets (e.g., CD4+ and/or CD8+ T cells), monocytes, and/or NK cells.
  • the characteristic/s include expression levels of one or more FMRs or FMRLs, as described herein. In certain embodiments, the characteristic/s include levels of intracellular activatable elements, which can be basal levels and/or modulated levels.
  • a primary treatment such as a primary immunomodulatory treatment or a nonimmunomodulatory treatment, has already been decided for a breast cancer patient, and the methods and compositions of the invention are used to determine whether or not to use a combination treatment.
  • the invention includes treating a breast cancer patient with a combination of treatments based, at least in part, on the methods described above.
  • Immunomodulatory treatments for breast cancer include any such treatments as described herein.
  • Specific types of treatment include vaccines and checkpoint blockade; the former includes nelipepimut-S, and the latter include ipilimumab, pembrolizumab
  • compositions of the invention See, e.g., Example 21, in which, in vitro, Keytruda had no effect on TCR signaling (measured by p-ERK and p-AKT readouts), whereas there was a clear trend toward an increase in signaling with Keytruda treatment in PD-1 positive (high expression) samples.
  • TCR signaling in single cells from a sample from a breast cancer patient as measured by levels of one or more intracellular activatable elements, such a p- ERK and p-AKT, in one or more cell populations (e.g., CD4+ T cells) and/or expression levels of one or more IMRs or IMRLs, such as PD-1, in one or more cell populations (e.g., CD4+ T cells), can be used to determine whether or not the patient is likely to respond to an immunomodulatory agent (e.g., an agent that reduces communication through the PD-1 pathway, such as pembrolizumab).
  • an immunomodulatory agent e.g., an agent that reduces communication through the PD-1 pathway, such as pembrolizumab.
  • the invention includes treating a patient based on such a determination.
  • the invention also includes monitoring treatment of a patient based on such a determination.
  • the invention also includes dosing a patient based on such a determination, where the dosage is based at least in part on the determination. In addition, such
  • determinations can be used to screen potential agents, e.g., potential immunomodulatory agents such as checkpoint modulators (stimulators or inhibitors) and to determine whether a particular agent is potentially useful.
  • potential immunomodulatory agents such as checkpoint modulators (stimulators or inhibitors)
  • a diagnosis is made, a treatment is determined or modified, a prognosis is made, or a treatment is monitored; in embodiments regarding a treatment, the embodiment can include in some cases administration of said treatment, for a breast cancer patient, based at least in part on analysis of a sample or samples from the patient, for example, based on analysis of a blood or blood-derived sample, such as a PBMC sample, e.g., analysis of one or more immune cell populations in the sample.
  • the prognosis can be, e.g., likelihood of PMS for a given period of time.
  • the prognosis can be based on one or more of IMR/IMRL expression levels in single cells of one or more immune cell populations; e.g., in Example 21 higher FMR expression in T cell subsets (PD-L1 in CD4+ T cells, NK cells; PD-1 in C4+ cells, GITR in CD4+ and CD8+ T cells) correlated with lower PFS.
  • the prognosis can additionally, or alternatively, be based on basal and/or modulated levels of activatable elements in single cells of one or more immune cell populations; e.g., in Example 21, lower TCR signaling (indicated by p-AKT or P-ERK in CD4+ and CD8+ T cells) correlated with lower PFS.
  • activatable elements reflective of the TCR pathway and/or TCR modulators may be used.
  • kits that are used in conjunction with breast cancer, for example kits for diagnosis, prognosis, treatment selection, treatment monitoring, drug screening, and the like, in breast cancer are provided.
  • the kits include 1) one, two, three, four or more than four distinguishably detectable binding elements, for example, antibodies, to determine immune cell populations, such as those shown in Figure 17; 2) one, two, three, four or more than four distinguishably detectable binding elements, for example, antibodies, to intracellular activatable elements, for example, elements in the TCR pathway, such as those shown in Table 1, or for example selected from the group consisting of p-ERK and p- AKT, p-PLCg2, p-CD3z, p-s6, and IkB; optionally 3) one, two, three, four or more modulators for modulating one or more cell populations in a sample, such as a PBMC sample, e.g., T cell modulator(s) such as TCR activators, such as DCD3 and D CD28;
  • kits optionally 4) one, two, three, four or more than four distinguishably detectable binding elements, e.g., antibodies, to markers of cell health, for example, cPARP; optionally 5) one, two, three, four, or more distinguishably detectable binding elements, e.g., antibodies, to FMRs or FMRLs, such as those shown in Figure 15; optionally 6) instructions for use.
  • Other elements, as described elsewhere herein for kits, may also be included in the kits of the invention.
  • the invention may involve analysis of one or more samples from an individual.
  • An individual or a patient is any multi-cellular organism; in some embodiments, the individual or patient is an animal, e.g., a mammal. In some embodiments, the individual or patient is a human.
  • the sample may be any suitable type that allows for the methods of the invention.
  • a solid sample such as a tumor biopsy, or a liquid sample, such as a blood or blood-derived, e.g. PBMC sample, or both, is used.
  • a blood or blood-derived sample is used.
  • the advantage of a blood or blood- derived sample is that it is easy to obtain and often stored so that retrospective studies can be performed, and it can reflect, e.g., the tumor microenvironment. See Example 23.
  • the sample may be any suitable type that allows for the analysis of single cells. Samples may be obtained once or multiple times from an individual.
  • Multiple samples may be obtained from different locations in the individual (e.g., blood samples, bone marrow samples, lymph node samples, biopsies, and/or resection material), at different times from the individual (e.g., a series of samples taken to monitor response to treatment or to monitor for return of a pathological condition), or any combination thereof.
  • a liquid sample is used, for example a blood or blood- derived (e.g., PBMC) sample, or a bone marrow sample.
  • a solid sample is used, such as a solid tumor sample, from which may be derived, e.g., tumor- infiltrating lymphocytes (TILS).
  • TILS tumor- infiltrating lymphocytes
  • the sample is selected from the group consisting of whole blood, bone marrow, and PBMC.
  • the sample is a TILS sample.
  • a combination of samples is used, e.g., a PBMC sample and a TILS sample from a cancer patient suffering from a solid tumor.
  • samples When samples are obtained as a series, e.g., a series of blood samples obtained after treatment, the samples may be obtained at fixed intervals, at intervals determined by the status of the most recent sample or samples or by other characteristics of the individual, or some combination thereof. For example, samples may be obtained at intervals of
  • an individual who has undergone treatment for a cancer may be sampled (e.g., by blood draw) relatively frequently (e.g., every month or every three months) for the first six months to a year after treatment, then, if no abnormality is found, less frequently (e.g., at times between six months and a year) thereafter. If, however, any abnormalities or other circumstances are found in any of the intervening times, or during the sampling, sampling intervals may be modified.
  • Fluid samples include normal and pathologic bodily fluids and aspirates of those fluids. Fluid samples also comprise rinses of organs and cavities (lavage and perfusions). Bodily fluids include whole blood, peripheral blood mononuclear cells (PBMCs), bone marrow aspirate, synovial fluid, cerebrospinal fluid, saliva, sweat, tears, semen, sputum, mucus, menstrual blood, breast milk, urine, lymphatic fluid, amniotic fluid, placental fluid and effusions such as cardiac effusion, joint effusion, pleural effusion, and peritoneal cavity effusion (ascites).
  • PBMCs peripheral blood mononuclear cells
  • Rinses can be obtained from numerous organs, body cavities, passageways, ducts and glands. Sites that can be rinsed include lungs (bronchial lavage), stomach (gastric lavage), gastrointestinal track (gastrointestinal lavage), colon (colonic lavage), vagina, bladder (bladder irrigation), breast duct (ductal lavage), oral, nasal, sinus cavities, and peritoneal cavity (peritoneal cavity perfusion).
  • the sample or samples is blood.
  • a solid tissue sample is used.
  • Solid tissue samples may also be used, either alone or in conjunction with fluid samples.
  • a solid tissue sample is a tumor sample.
  • Tumor samples contain tumor cells and, generally, immune cells such as tumor infiltrating lymphocytes, and it is of interest to determine characteristics of one or both of these cell types.
  • Solid samples may be derived from individuals by any method known in the art including surgical specimens, biopsies, and tissue scrapings, including cheek scrapings.
  • Surgical specimens include samples obtained during exploratory, cosmetic, reconstructive, or therapeutic surgery. Biopsy specimens can be obtained through numerous methods including bite, brush, cone, core, cytological, aspiration, endoscopic, excisional, exploratory, fine needle aspiration, incisional, percutaneous, punch, stereotactic, and surface biopsy.
  • Samples may include circulating tumor cells (CTC).
  • CTC circulating tumor cells
  • Methods for isolating CTC are known in the art. See for example: Toner M et al. Nature 450, 1235-1239 (20 December 2007); Lustenberger P et al. Int J Cancer. 1997 Oct 21;74(5):540-4; Reviews in Clinical Laboratory Sciences, Volume 42, Issue 2 March 2005 , pages 155 - 196; and Biotechno, pp.109-113, 2008 International Conference on Biocomputation, Bioinformatics, and
  • the sample is a blood or PMBC sample.
  • Blood and PBMC samples are particularly suited to analysis of hematopoietic cancers such as leukemia and lymphoma; however, PBMC samples can also be used with solid tumors if the status of the circulating immune cells can be correlated, e.g. with a treatment outcome.
  • the sample is a bone marrow sample.
  • the sample is a lymph node sample.
  • the sample is cerebrospinal fluid.
  • combinations of one or more of a blood, bone marrow, cerebrospinal fluid, and lymph node sample are used.
  • a lavage or perfusion may be used, e.g., for lung, bladder, stomach, colon and other cancer sites may provide sufficient immune and/or tumor cells for an analysis.
  • a sample may be obtained from an apparently healthy individual during a routine checkup and analyzed so as to provide an assessment of the individual's general health status.
  • a sample may be taken to screen for commonly occurring diseases. Such screening may encompass testing for a single disease, a family of related diseases or a general screening for multiple, unrelated diseases. Screening can be performed once, weekly, bi-weekly, monthly, bi-monthly, every several months, annually, or in several year intervals and may replace or complement existing screening modalities.
  • an individual with a known increased probability of disease occurrence may be monitored regularly to detect for the appearance of a particular disease or class of diseases.
  • An increased probability of disease occurrence can be based on familial association, age, previous genetic testing results, or occupational, environmental or therapeutic exposure to disease causing agents.
  • Breast and ovarian cancer related to inherited mutations in the genes BRCA1 and BRCA2 are examples of diseases with a familial association wherein susceptible individuals can be identified through genetic testing.
  • Another example is the presence of inherited mutations in the adenomatous polyposis coli gene predisposing individuals to colorectal cancer.
  • environmental or therapeutic exposure include individuals occupationally exposed to benzene that have increased risk for the development of various forms of leukemia, and individuals therapeutically exposed to alkylating agents for the treatment of earlier malignancies.
  • Individuals with increased risk for specific diseases can be monitored regularly for the first signs of an appearance of an abnormal discrete cell population. Monitoring can be performed once, weekly, bi-weekly, monthly, bi-monthly, every several months, annually, or in several year intervals, or any combination thereof. Monitoring may replace or complement existing screening modalities. Through routine monitoring, early detection of the presence of disease causative or associated cells may result in increased treatment options including treatments with lower toxicity and increased chance of disease control or cure.
  • testing can be performed to confirm or refute the presence of a suspected genetic or physiologic abnormality associated with increased risk of disease.
  • Such testing methodologies can replace other confirmatory techniques like cytogenetic analysis or fluorescent in situ histochemistry (FISH). See U.S. S.N. 12/688,851.
  • FISH fluorescent in situ histochemistry
  • testing can be performed to confirm or refute a diagnosis of a pre- pathological or pathological condition.
  • one or more samples may be obtained and analyzed to predict the response of the individual to available treatment options, or to determine the optimal treatment.
  • an individual treated with the intent to reduce in number or ablate cells that are causative or associated with a pre-pathological or pathological condition can be monitored to assess the decrease in such cells over time.
  • a reduction in causative or associated cells may or may not be associated with the disappearance or lessening of disease symptoms. If the anticipated decrease in cell number does not occur, further treatment with the same or a different treatment regiment may be warranted.
  • the immunological profile of the individual may be monitored, for example, during and after immunotherapy, to determine the effectiveness of the treatment in terms of immune system function, as well as to monitor for any changes that indicate that the treatment effect is declining.
  • an individual treated to reverse or arrest the progression of a pre-pathological condition can be monitored to assess the reversion rate or percentage of cells arrested at the pre-pathological status point. If the anticipated reversion rate is not seen or cells do not arrest at the desired pre-pathological status point further treatment with the same or a different treatment regiment can be considered.
  • Individuals may also be monitored for the appearance or increase in cell number of another discrete cell population(s) that are associated with a good prognosis. If a beneficial, discrete cell population is observed, measures can be taken to further increase their numbers, such as the administration of growth factors. Alternatively, individuals may be monitored for the appearance or increase in cell number of another discrete cell population(s) associated with a poor prognosis. In such a situation, renewed therapy can be considered including continuing, modifying the present therapy or initiating another type of therapy.
  • fluid samples can be analyzed in their native state with or without the addition of a diluent or buffer.
  • fluid samples may be further processed to obtain enriched or purified cell populations prior to analysis.
  • Numerous enrichment and purification methodologies for bodily fluids are known in the art.
  • a common method to separate cells from plasma in whole blood is through centrifugation using heparinized tubes. By incorporating a density gradient, further separation of the lymphocytes from the red blood cells can be achieved.
  • a variety of density gradient media are known in the art including sucrose, dextran, bovine serum albumin (BSA), FICOLL diatrizoate (Pharmacia), FICOLL metrizoate (Nycomed), PERCOLL (Pharmacia), metrizamide, and heavy salts such as cesium chloride.
  • BSA bovine serum albumin
  • FICOLL diatrizoate Pharmacia
  • FICOLL metrizoate Nycomed
  • PERCOLL Pharmacia
  • metrizamide metrizamide
  • heavy salts such as cesium chloride.
  • red blood cells can be removed through lysis with an agent such as ammonium chloride prior to centrifugation.
  • Whole blood can also be applied to filters that are engineered to contain pore sizes that select for the desired cell type or class.
  • filters that are engineered to contain pore sizes that select for the desired cell type or class.
  • rare pathogenic cells can be filtered out of diluted, whole blood following the lysis of red blood cells by using filters with pore sizes between 5 to 10 ⁇ , as disclosed in U.S. Patent Application No. 09/790,673.
  • whole blood can be separated into its constituent cells based on size, shape, deformability or surface receptors or surface antigens by the use of a microfluidic device as disclosed in U.S. Patent Application No. 10/529,453.
  • Select cell populations can also be enriched for or isolated from whole blood through positive or negative selection based on the binding of antibodies or other entities that recognize cell surface or cytoplasmic constituents.
  • U.S. Patent No. 6,190,870 to Schmitz et al. discloses the enrichment of tumor cells from peripheral blood by magnetic sorting of tumor cells that are magnetically labeled with antibodies directed to tissue specific antigens.
  • Solid tissue samples may require the disruption of the extracellular matrix or tissue stroma and the release of single cells for analysis.
  • Various techniques are known in the art including enzymatic and mechanical degradation employed separately or in combination.
  • An example of enzymatic dissociation using collagenase and protease can be found in Wolters GHJ et al.
  • Examples of mechanical dissociation can be found in Singh, NP. Technical Note: A rapid method for the preparation of single-cell suspensions from solid tissues. Cytometry 31 :229-232 (1998).
  • single cells may be removed from solid tissue through microdissection including laser capture microdissection as disclosed in Laser Capture Microdissection, Emmert-Buck, M. R. et al. Science, 274(8):998-1001, 1996. See also U.S. Patent Publication Number 20130129681, for descriptions of method for release of single cells from solid tissue samples.
  • single cells can be analyzed within a tissue sample, such as a tissue section or slice, without requiring the release of individual cells before determining step is performed.
  • the cells can be separated from body samples by centrifugation, elutriation, density gradient separation, apheresis, affinity selection, panning, FACS, centrifugation with
  • antibodies specific for markers identified with particular cell types By using antibodies specific for markers identified with particular cell types, a relatively homogeneous population of cells may be obtained.
  • a relatively homogeneous population of cells may be obtained.
  • a relatively homogeneous population of cells may be obtained.
  • heterogeneous cell population can be used.
  • Cells can also be separated by using filters. Once a sample is obtained, it can be used directly, frozen, or maintained in appropriate culture medium for short periods of time.
  • Methods to isolate one or more cells for use according to the methods of this invention are performed according to standard techniques and protocols well-established in the art. See also U.S.S. Nos. 12/432,720 and 13/493,857 and U.S. Patent No. 8,227,202. See also, the commercial products from companies such as BD and BCI.
  • the cells are cultured post collection in a media suitable for revealing the activation level of an activatable element (e.g. RPMI, DMEM) in the presence, or absence, of serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
  • an activatable element e.g. RPMI, DMEM
  • serum such as fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, or goat serum.
  • serum is present in the media it could be present at a level ranging from 0.0001 % to 30%.
  • an "activatable element” is an element, e.g, a protein, that can exist in two or more states. In general, activation can result in a change in the activatable element, e.g., protein, that results in a conformation that is detectably different from the non-activated form.
  • An example is a phosphoprotein, which can exist in one or, in some cases, more than one phosphorylated forms, and a nonphosphorylated form.
  • Another example is a protein that is activated by cleavage, where the cleaved protein can be considered an activated form. For convenience, one form can be designated the "activated" form, and another an "unactivated” form; though there can be several forms, similar principles apply.
  • a cell possesses a plurality of a particular activatable element, some of which are in the activated form and some of which are in the unactivated form.
  • One form or both forms can be distinguishably detectable, for example, the activated form may be distinguishably detectable, for example through binding of a binding element that is specific to the activated form.
  • the binding element representing some fraction of the total number of activatable elements, and generate a measurable signal.
  • the measurable signal corresponding to the summation of individual activated elements of a particular type that are activated in a single cell can be the "activation level" for that activatable element in that cell.
  • an activatable element may be referred to in its unactivated form, and in certain instances in its activated form; in general, the two are synonymous, and either may be considered the "activatable element.”
  • Activation levels for a particular activatable element may vary among individual cells so that when a plurality of cells is analyzed, the activation levels follow a
  • the distribution may be a normal distribution, also known as a Gaussian distribution, or it may be of another type. Different populations of cells may have different distributions of activation levels that can then serve to distinguish between the populations.
  • Cellular constituents that may include activatable elements include without limitation proteins, carbohydrates, lipids, nucleic acids and metabolites.
  • a change occurs to the activatable element, such as covalent modification of the activatable element (e.g., binding of a molecule or group to the activatable element, such as
  • Such changes generally contribute to changes in particular biological, biochemical, or physical properties of the cellular constituent that contains the activatable element.
  • Activation states of activatable elements may result from chemical additions or modifications of biomolecules and include biochemical processes such as glycosylation, phosphorylation, acetylation, methylation, biotinylation, glutamyl ati on, glycylation, hydroxylation, isomerization, prenylation, myristoylation, lipoylation,
  • biomolecules include the formation of protein carbonyls, direct modifications of protein side chains, such as o- tyrosine, chloro-, nitrotyrosine, and dityrosine, and protein adducts derived from reactions with carbohydrate and lipid derivatives.
  • modifications may be non-covalent, such as binding of a ligand or binding of an allosteric modulator.
  • a covalent modification is the substitution of a phosphate group for a hydroxyl group in the side chain of an amino acid (phosphorylation).
  • phosphorylation A wide variety of proteins are known that recognize specific protein substrates and catalyze the
  • kinases phosphorylation of serine, threonine, or tyrosine residues on their protein substrates. Such proteins are generally termed "kinases.” Substrate proteins that are capable of being phosphorylated are often referred to as phosphoproteins (after phosphorylation). Once phosphorylated, a substrate phosphoprotein may have its phosphorylated residue converted back to a hydroxyl one by the action of a protein phosphatase that specifically recognizes the substrate protein. Protein phosphatases catalyze the replacement of phosphate groups by hydroxyl groups on serine, threonine, or tyrosine residues.
  • a protein may be reversibly phosphorylated on a multiplicity of residues and its activity may be regulated thereby.
  • the presence or absence of one or more phosphate groups in an activatable protein is a preferred readout in the present invention.
  • Another example of a covalent modification of an activatable protein is the acetylation of histones.
  • histone acetylation and histone deactelyation have been linked with malignant progression. See Nature, 2004 May 27; 429(6990): 457-63.
  • Another form of activation involves cleavage of the activatable element.
  • one form of protein regulation involves proteolytic cleavage of a peptide bond. While random or misdirected proteolytic cleavage may be detrimental to the activity of a protein, many proteins are activated by the action of proteases that recognize and cleave specific peptide bonds. Many proteins derive from precursor proteins, or pro-proteins, which give rise to a mature isoform of the protein following proteolytic cleavage of specific peptide bonds. Many growth factors are synthesized and processed in this manner, with a mature isoform of the protein typically possessing a biological activity not exhibited by the precursor form.
  • proteolytically activated proteins are relatively short-lived proteins, and their turnover effectively results in deactivation of the signal. Inhibitors may also be used.
  • enzymes that are proteolytically activated are serine and cysteine proteases, including cathepsins and caspases respectively.
  • Activation of an activatable element can involve prenylation of the element.
  • prenyl ati on is meant the addition of any lipid group to the element.
  • prenylation include the addition of farnesyl groups, geranylgeranyl groups, myristoylation and palmitoylation. In general these groups are attached via thioether linkages to the activatable element, although other attachments may be used.
  • the activatable element can be a protein.
  • proteins that can be activatable elements include, but are not limited to kinases, phosphatases, lipid signaling molecules, adaptor/scaffold proteins, cytokines, cytokine regulators, ubiquitination enzymes, adhesion molecules, cytoskeletal/contractile proteins, heterotrimeric G proteins, small molecular weight GTPases, guanine nucleotide exchange factors, GTPase activating proteins, caspases, proteins involved in apoptosis, cell cycle regulators, molecular chaperones, metabolic enzymes, vesicular transport proteins, hydroxylases, isomerases, deacetylases, methylases, demethylases, tumor suppressor genes, proteases, ion channels, molecular transporters, transcription factors/DNA binding factors, regulators of transcription, and regulators of translation.
  • Exemplary proteins that may be activated include HER receptors, PDGF receptors, FLT3 receptor, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, erythropoetin receptor, thromobopoetin receptor, CD114, CD116, ⁇ 1, ⁇ 2, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGF ⁇ receptors, BMP receptors, MEKKs, ASK, MLKs, DLK, PAKs, Mek 1, Mek 2, MKK3/6, MKK4/7, ASK1, Cot, NIK, Bub, Myt
  • An activatable element can be a nucleic acid. Activation and deactivation of nucleic acids can occur in numerous ways including, but not limited to, cleavage of an inactivating leader sequence as well as covalent or non-covalent modifications that induce structural or functional changes. For example, many catalytic RNAs, e.g. hammerhead ribozymes, can be designed to have an inactivating leader sequence that deactivates the catalytic activity of the ribozyme until cleavage occurs.
  • An example of a covalent modification is methylation of DNA. Deactivation by methylation has been shown to be a factor in the silencing of certain genes, e.g. STAT regulating SOCS genes in lymphomas. See Leukemia.
  • An activatable element can be a small molecule, carbohydrate, lipid or other naturally occurring or synthetic compound capable of having an activated isoform.
  • the activation level of an activatable element in a cellular pathway, or signaling pathway can be determined.
  • the methods of the invention are employed to determine the status of an activatable element in a signaling pathway.
  • Signaling pathways for F Rs are the main pathways of interest in the invention; however, one or more pathways may be related to an F R pathway or otherwise affected by it, for example, a pathway for activation of a cell, e.g., an immune cell. Signaling pathways and their members have been described.
  • Exemplary signaling pathways include the following pathways and their members: the JAK- STAT pathway including JAKs, STATs 1, 2,3 4, 5, and 6 the FLT3L signaling pathway, the MAP kinase pathway including Ras, Raf, MEK, ERK and Elk; the PI3K/Akt pathway including PI3-kinase, PDK1, Akt and Bad; the NF-DB pathway including IKKs, IkB and NF- ⁇ B, and the Wnt pathway including frizzled receptors, beta-catenin, APC and other co- factors and TCF.
  • the JAK- STAT pathway including JAKs, STATs 1, 2,3 4, 5, and 6 the FLT3L signaling pathway
  • the MAP kinase pathway including Ras, Raf, MEK, ERK and Elk
  • the PI3K/Akt pathway including PI3-kinase, PDK1, Akt and Bad
  • the NF-DB pathway including IKKs, IkB and NF-
  • Nuclear Factor-kappaB (NF- ⁇ ) Pathway Nuclear factor-kappaB (NF-kappaB) transcription factors and the signaling pathways that activate them are central coordinators of innate and adaptive immune responses. More recently, it has become clear that NF-kappaB signaling also has a critical role in cancer development and progression. NF-kappaB provides a mechanistic link between inflammation and cancer, and is a major factor controlling the ability of both pre-neoplastic and malignant cells to resist apoptosis-based tumor-surveillance mechanisms.
  • NF- ⁇ complexes In mammalian cells, there are five NF- ⁇ family members, RelA (p65), RelB, c-Rel, p50/pl05 (NF-KB1) and p52/pl00 (NF-KB2) and different NF- ⁇ complexes are formed from their homo and heterodimers. In most cell types, NF- ⁇ complexes are retained in the cytoplasm by a family of inhibitory proteins known as inhibitors of NF- ⁇ (IKBS). Activation of NF- ⁇ typically involves the phosphorylation of ⁇ by the ⁇ kinase (IKK) complex, which results in ⁇ ubiquitination with subsequent degradation.
  • IKK ⁇ kinase
  • NF-KB non-phosphorylated IkB can serves as an activatable element in this pathway.
  • the genes regulated by NF- KB include those controlling programmed cell death, cell adhesion, proliferation, the innate- and adaptive-immune responses, inflammation, the cellular-stress response and tissue remodeling.
  • the expression of these genes is tightly coordinated with the activity of many other signaling and transcription-factor pathways. Therefore, the outcome of NF-KB activation depends on the nature and the cellular context of its induction. For example, it has become apparent that NF- ⁇ activity can be regulated by both oncogenes and tumor suppressors, resulting in either stimulation or inhibition of apoptosis and proliferation.
  • Phosphatidylinositol 3-kinase (PI3-K)/AKT Pathway PI3-Ks are activated by a wide range of cell surface receptors to generate the lipid second messengers
  • PIP2 phosphatidylinositol 3,4-biphosphate
  • PIP3 phosphatidylinositol 3,4,5-trisphosphate
  • receptor tyrosine kinases include but are not limited to FLT3 LIGAND, EGFR, IGF-1R, HER2/neu, VEGFR, and PDGFR.
  • the lipid second messengers generated by PDKs regulate a diverse array of cellular functions.
  • the specific binding of PI3,4P2 and PI3,4,5P3 to target proteins is mediated through the pleckstrin homology (PH) domain present in these target proteins.
  • PH pleckstrin homology
  • One key downstream effector of PI3-K is Akt, a
  • Akt serine/threonine kinase, which is activated when its PH domain interacts with PI3, 4P2 and PI3,4,5P3 resulting in recruitment of Akt to the plasma membrane.
  • Akt is phosphorylated at threonine 308 by 3-phosphoinositide-dependent protein kinase- 1 (PDK-1) and at serine 473 by several PDK2 kinases.
  • Akt then acts downstream of PI3K to regulate the phosphorylation of a number of substrates, including but not limited to forkhead box O transcription factors, Bad, GSK-3P, ⁇ - ⁇ , mTOR, MDM-2, and S6 ribosomal subunit.
  • Deregulation of the PI3K pathway occurs by activating mutations in growth factor receptors, activating mutations in a PI3-K gene (e.g. PIK3CA), loss of function mutations in a lipid phosphatase (e.g. PTEN), up-regulation of Akt, or the impairment of the tuberous sclerosis complex (TSCl/2). All these events are linked to increased survival and proliferation.
  • a PI3-K gene e.g. PIK3CA
  • loss of function mutations in a lipid phosphatase e.g. PTEN
  • up-regulation of Akt Akt
  • TSCl/2 tuberous sclerosis complex
  • Wnt Pathway The Wnt signaling pathway describes a complex network of proteins well known for their roles in embryogenesis, normal physiological processes in adult animals, such as tissue homeostasis, and cancer. Further, a role for the Wnt pathway has been shown in self-renewal of hematopoietic stem cells (Reya T et al., Nature. 2003 May
  • Cytoplasmic levels of ⁇ -catenin are normally kept low through the continuous proteosomal degradation of ⁇ -catenin controlled by a complex of glycogen synthase kinase 3 ⁇ (GSK-3 ⁇ ), axin, and adenomatous polyposis coli (APC).
  • GSK-3 ⁇ glycogen synthase kinase 3 ⁇
  • APC adenomatous polyposis coli
  • ⁇ -catenin Upon Wnt signaling and inhibition of the ⁇ -catenin degradation pathway, ⁇ -catenin accumulates in the cytoplasm and nucleus. Nuclear ⁇ -catenin interacts with transcription factors such as lymphoid enhanced-binding factor 1 (LEF) and T cell-specific transcription factor (TCF) to affect transcription of target genes
  • LEF lymphoid enhanced-binding factor 1
  • TCF T cell-specific transcription factor
  • PKC Protein Kinase C
  • PKC isoforms have distinct and overlapping roles in cellular functions.
  • PKC was originally identified as a phospholipid and calcium-dependent protein kinase.
  • the mammalian PKC superfamily consists of 13 different isoforms that are divided into four subgroups on the basis of their structural differences and related cofactor requirements cPKC (classical PKC) isoforms ( ⁇ , ⁇ , ⁇ and ⁇ ), which respond both to Ca2+ and DAG (diacylglycerol), nPKC (novel PKC) isoforms ( ⁇ , ⁇ , ⁇ and ⁇ ), which are insensitive to Ca2+, but dependent on DAG, atypical PKCs (aPKCs, ⁇ / ⁇ , ⁇ ), which are responsive to neither co-factor, but may be activated by other lipids and through protein-protein
  • PKC protein kinase N family
  • PKN1, PKN2 and PKN3 proteins kinase N family
  • PKN1 protein kinase N
  • PKN2 protein kinase N2
  • PKN3 protein kinase N 3
  • PKC isoforms differ in their structure, tissue distribution, subcellular localization, mode of activation and substrate specificity.
  • PDK-1 phosphoinositide-dependent kinase 1
  • the phospholipid DAG has a central role in the activation of PKC by causing an increase in the affinity of classical PKCs for cell membranes accompanied by PKC activation and the release of an inhibitory substrate (a pseudo-substrate) to which the inactive enzyme binds. Activated PKC then phosphorylates and activates a range of kinases.
  • the downstream events following PKC activation are poorly understood, although the MEK-ERK (mitogen activated protein kinase kinase-extracellular signal-regulated kinase) pathway is thought to have an important role. There is also evidence to support the involvement of PKC in the PI3K-Akt pathway.
  • PKC isoforms probably form part of the multi-protein complexes that facilitate cellular signal transduction. Many reports describe dysregulation of several family members. For example alterations in PKCs have been detected in thyroid cancer, and have been correlated with aggressive, metastatic breast cancer and PKCi was shown to be associated with poor outcome in ovarian cancer.
  • MAPK Mitogen Activated Protein
  • MAPKs are activated by protein kinase cascades consisting of three or more protein kinases in series: MAPK kinase kinases
  • MAP3Ks activate MAPK kinases (MAP2Ks) by dual phosphorylation on S/T residues; MAP2Ks then activate MAPKs by dual phosphorylation on Y and T residues MAPKs then phosphorylate target substrates on select S/T residues typically followed by a proline residue.
  • MAP3K is usually a member of the Raf family.
  • Many diverse MAP3Ks reside upstream of the p38 and the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/ SAPK) MAPK groups, which have generally been associated with responses to cellular stress.
  • the kinase cascades may themselves be stimulated by combinations of small G proteins, MAP4Ks, scaffolds, or oligomerization of the MAP3K in a pathway.
  • Ras family members In the ERK1/2 pathway, Ras family members usually bind to Raf proteins leading to their activation as well as to the subsequent activation of other
  • the MAPK pathway has been a focus of intense investigation for therapeutic targeting.
  • Many receptor tyrosine kinases are capable of initiating MAPK signaling. They do so after activating phosphorylation events within their cytoplasmic domains provide docking sites for src-homology 2 (SH2) domain-containing signaling molecules.
  • SH2 src-homology 2
  • adaptor proteins such as Grb2 recruit guanine nucleotide exchange factors such as SOS-1 or CDC25 to the cell membrane.
  • the guanine nucleotide exchange factor is now capable of interacting with Ras proteins at the cell membrane to promote a conformational change and the exchange of GDP for GTP bound to Ras.
  • Ras isoforms have been described, including K-Ras, N-Ras, and H-Ras. Termination of Ras activation occurs upon hydrolysis of RasGTP to RasGDP. Ras proteins have intrinsically low GTPase activity. Thus, the GTPase activity is stimulated by GTPase-activating proteins such as NF-1 GTPase-activating protein/neurofibromin and pi 20 GTPase activating protein thereby preventing prolonged Ras stimulated signaling. Ras activation is the first step in activation of the MAPK cascade.
  • Raf (A-Raf, B-Raf, or Raf-1) is recruited to the cell membrane through binding to Ras and activated in a complex process involving phosphorylation and multiple cofactors that is not completely understood.
  • Raf proteins directly activate MEKl and MEK2 via phosphorylation of multiple serine residues.
  • MEKl and MEK2 are themselves tyrosine and threonine/serine dual-specificity kinases that subsequently phosphorylate threonine and tyrosine residues in Erkl and Erk2 resulting in activation.
  • Erk has multiple targets including Elk-1, c-Etsl, c-Ets2, p90RSKl, MNK1, M K2, and TOB .
  • the cellular functions of Erk are diverse and include regulation of cell proliferation, survival, mitosis, and migration. McCubrey, J. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochimica et Biophysica Acta. 2007; 1773 : 1263-1284, hereby fully incorporated by reference in its entirety for all purposes, Friday and Adjei, Clinical Cancer Research (2008) 14, p342-346.
  • JNK c-Jun N-terminal kinase
  • SAPK stress-activated protein kinase
  • JNKs c-Jun N-terminal kinases
  • JNK1, JNK2 and JNK3 phosphorylate the N-terminal transactivation domain of the c- Jun transcription factor. This phosphorylation enhances c-Jun dependent transcriptional events in mammalian cells.
  • JNK1, JNK2 and JNK3 are ubiquitous, whereas JNK3 is relatively restricted to brain.
  • the predominant MAP2Ks upstream of JNK are MEK4 (MKK4) and MEK7 (MKK7).
  • MAP3Ks with the capacity to activate JNK/SAPKs include MEKKs (MEKK1, -2, -3 and -4), mixed lineage kinases (MLKs, including MLK1-3 and DLK), Tpl2, ASKs, TAOs and TAKl .
  • MEKKs MEKK1, -2, -3 and -4
  • MLKs mixed lineage kinases
  • Tpl2 ASKs
  • TAOs TAKl
  • Knockout studies in several organisms indicate that different MAP3Ks predominate in JNK/SAPK activation in response to different upstream stimuli.
  • the wiring may be comparable to, but perhaps even more complex than, MAP3K selection and control of the ERKl/2 pathway.
  • JNK/SAPKs are activated in response to inflammatory cytokines; environmental stresses, such as heat shock, ionizing radiation, oxidant stress and DNA damage; DNA and protein synthesis inhibition; and growth factors.
  • JNKs phosphorylate transcription factors c-Jun, ATF-2, p53, Elk-1, and nuclear factor of activated T cells (NFAT), which in turn regulate the expression of specific sets of genes to mediate cell proliferation, differentiation or apoptosis. JNK proteins are involved in cytokine production, the inflammatory response, stress-induced and
  • p38 Map kinases Several independent groups identified the p38 Map kinases, and four p38 family members have been described ( ⁇ , ⁇ , ⁇ , ⁇ ). Although the p38 isoforms share about 40% sequence identity with other MAPKs, they share only about 60% identity among themselves, suggesting highly diverse functions. p38 MAPKs respond to a wide range of extracellular cues particularly cellular stressors such as UV radiation, osmotic shock, hypoxia, proinflammatory cytokines and less often growth factors. Responding to osmotic shock might be viewed as one of the oldest functions of this pathway, because yeast p38 activates both short and long-term homeostatic mechanisms to osmotic stress.
  • cellular stressors such as UV radiation, osmotic shock, hypoxia, proinflammatory cytokines and less often growth factors.
  • osmotic shock might be viewed as one of the oldest functions of this pathway, because yeast p38 activates both short and long-term homeostatic mechanisms to o
  • p38 is activated via dual phosphorylation on the TGY motif within its activation loop by its upstream protein kinases MEK3 and MEK6.
  • MEK3/6 are activated by numerous MAP3Ks including MEKK1-4, TAOs, TAK and ASK.
  • p38 MAPK is generally considered to be the most promising MAPK therapeutic target for rheumatoid arthritis as p38 MAPK isoforms have been implicated in the regulation of many of the processes, such as migration and accumulation of leucocytes, production of cytokines and pro-inflammatory mediators and angiogenesis, that promote disease pathogenesis. Further, the p38 MAPK pathway plays a role in cancer, heart and neurodegenerative diseases and may serve as promising therapeutic target. Cuenda, A.
  • Src Family Kinases Src is the most widely studied member of the largest family of nonreceptor protein tyrosine kinases, known as the Src family kinases (SFKs). Other SFK members include Lyn, Fyn, Lck, Hck, Fgr, Blk, Yrk, and Yes.
  • Src kinases can be grouped into two sub-categories, those that are ubiquitously expressed (Src, Fyn, and Yes), and those which are found primarily in hematopoietic cells (Lyn, Lck, Hck, Blk, Fgr).
  • SFKs are key messengers in many cellular pathways, including those involved in regulating
  • SFKs The activity of SFKs is highly regulated intramolecularly by interactions between the SH2 and SH3 domains and intermolecularly by association with cytoplasmic molecules. This latter activation may be mediated by focal adhesion kinase (FAK) or its molecular partner Crk-associated substrate (CAS), which plays a prominent role in integrin signaling, and by ligand activation of cell surface receptors, e.g. epidermal growth factor receptor (EGFR).
  • FAK focal adhesion kinase
  • CAS Crk-associated substrate
  • EGFR epidermal growth factor receptor
  • Src can also be activated by dephosphorylation of tyrosine residue Y530. Maximal Src activation requires the autophosphorylation of tyrosine residue Y419 (in the human protein) present within the catalytic domain. Elevated Src activity may be caused by increased transcription or by deregulation due to overexpression of upstream growth factor receptors such as EGFR, FIER2, platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor, ephrins, integrin, or FAK. Alternatively, some human tumors show reduced expression of the negative Src regulator, Csk.
  • Src kinases Increased levels, increased activity, and genetic abnormalities of Src kinases have been implicated in both solid tumor development and leukemias. Ingley, E. Src family kinases: Regulation of their activities, levels and identification of new pathways. Biochimica et Biophysica Acta. 2008; 1784 56-65, hereby fully incorporated by reference in its entirety for all purposes. Benati and Baldari., Curr Med Chem. 2008; 15(12): 1154-65, Finn (2008) Ann Oncol. May 16, hereby fully incorporated by reference in its entirety for all purposes.
  • Janus kinase (JAK)/ Signal transducers and activators of transcription (STAT) pathway The JAK/STAT pathway plays a crucial role in mediating the signals from a diverse spectrum of cytokine receptors, growth factor receptors, and G-protein-coupled receptors.
  • Signal transducers and activators of transcription (STAT) proteins play a crucial role in mediating the signals from a diverse spectrum of cytokine receptors growth factor receptors, and G-protein-coupled receptors.
  • STAT directly links cytokine receptor stimulation to gene transcription by acting as both a cytosolic messenger and nuclear transcription factor.
  • JAK Janus Kinase
  • STAT Janus Kinase
  • JFK JAK family kinase
  • Tyrosine phosphorylated STAT forms a dimer, translocates to the nucleus, and binds to specific DNA elements to activate target gene transcription, which leads to the regulation of cellular proliferation, differentiation, and apoptosis.
  • the entire process is tightly regulated at multiple levels by protein tyrosine phosphatases, suppressors of cytokine signaling and protein inhibitors of activated STAT.
  • JAKs contain two symmetrical kinase-like domains; the C-terminal JAK homology 1 (JH1) domain possesses tyrosine kinase function while the immediately adjacent JH2 domain is enzymatically inert but is believed to regulate the activity of JH1.
  • JAK family members JAK1, JAK2, JAK3 and tyrosine kinase 2 (Tyk2). Expression is ubiquitous for JAK1, JAK2 and TYK2 but restricted to hematopoietic cells for JAK3.
  • JAK3 e.g. JAK3A572V, JAK3V722I, JAK3P132T
  • fusion JAK2 e.g. ETV6-JAK2, PCM1- JAK2, BCR-JAK2 mutations
  • JAK2 V617F, JAK2 exon 12 mutations
  • MPL MPLW515L/K/S MPLS505N
  • JAK2 mutations primarily JAK2V617F, are invariably associated with polycythemia vera (PV). This mutation also occurs in the majority of patients with essential thrombocythemia (ET) or primary myelofibrosis (PMF) (Tefferi n., Leukemia & Lymphoma, March 2008; 49(3): 388 - 397). STATs can be activated in a JAK-independent manner by sre family kinase members and by oncogenic FLt3 ligand-ITD (Hayakawa and Naoe, Ann N Y Acad Sci. 2006 Nov; 1086:213-22; Choudhary et al.
  • STAT5 Activation mechanisms of STAT5 by oncogenic FLt3 ligand-ITD. Blood (2007) vol. 110 (1) pp. 370-4).
  • mutations of STATs have not been described in human tumors, the activity of several members of the family, such as STAT1, STAT3 and STAT5, is dysregulated in a variety of human tumors and leukemias.
  • STAT3 and STAT5 acquire oncogenic potential through constitutive phosphorylation on tyrosine, and their activity has been shown to be required to sustain a transformed phenotype. This was shown in lung cancer where tyrosine phosphorylation of STAT3 was JAK-independent and mediated by EGF receptor activated through mutation and Src.
  • STAT1 In contrast to STAT3 and STAT5, STAT1 negatively regulates cell proliferation and angiogenesis and thereby inhibits tumor formation. Consistent with its tumor suppressive properties, STAT1 and its
  • the response to DNA damage is a protective measure taken by cells to prevent or delay genetic instability and tumorigenesis. It allows cells to undergo cell cycle arrest and gives them an opportunity to either: repair the broken DNA and resume passage through the cell cycle or, if the breakage is irreparable, trigger senescence or an apoptotic program leading to cell death.
  • DNA damage sensor protein complexes are positioned at strategic points within the DNA damage response pathway and act as sensors, transducers or effectors of DNA damage.
  • double stranded breaks, single strand breaks, single base alterations due to alkylation, oxidation etc there is an assembly of specific DNA damage sensor protein complexes in which activated ataxia telangiectasia mutated (ATM) and ATM- and Rad3 related (ATR) kinases phosphorylate and subsequently activate the checkpoint kinases Chkl and Chk2.
  • ATM telangiectasia mutated
  • ATR ATM- and Rad3 related
  • Chk2 Maximal kinase activation of Chk2 involves phosphorylation and homo- dimerization with ATM-mediated phosphorylation of T68 on Chk2 as a preliminary event. This in turn activates the DNA repair. As mentioned above, in order for DNA repair to proceed, there can be a delay in the cell cycle. Chk2 seems to have a role at the Gl/S and G2/M junctures and may have overlapping functions with Chkl . There are multiple ways in which Chkl and Chk2 mediate cell cycle suspension. In one mechanism Chk2
  • CDC25A and CDC25C phosphatases phosphorylates the CDC25A and CDC25C phosphatases resulting in their removal from the nucleus either by proteosomal degradation or by sequestration in the cytoplasm by 14-3-3. These phosphatases are no longer able to act on their nuclear CDK substrates. If DNA repair is successful cell cycle progression is resumed.
  • Chk2 substrates that operate in a p53-independent manner include the E2F1 transcription factor, the tumor suppressor promyelocytic leukemia (PML) and the polo-like kinases 1 and 3 (PLK1 and PLK3).
  • E2F1 drives the expression of a number of apoptotic genes including caspases 3, 7, 8 and 9 as well as the pro-apoptotic Bcl-2 related proteins (Bim, Noxa, PUMA).
  • the p53 In its response to DNA damage, the p53 activates the transcription of a program of genes that regulate DNA repair, cell cycle arrest, senescence and apoptosis.
  • the overall functions of p53 are to preserve fidelity in DNA replication such that when cell division occurs tumorigenic potential can be avoided. In such a role, p53 is described as "The
  • Bcl-2 family Key regulators of apoptosis are proteins of the Bcl-2 family.
  • the founding member, the Bcl-2 proto-oncogene was first identified at the chromosomal breakpoint of t(14: 18) bearing human follicular B cell lymphoma. Unexpectedly, expression of Bcl-2 was proved to block rather than promote cell death following multiple pathological and physiological stimuli
  • the Bcl-2 family has at least 20 members which are key regulators of apoptosis, functioning to control mitochondrial permeability as well as the release of proteins important in the apoptotic program.
  • the ratio of anti- to pro-apoptotic molecules constitutes a rheostat that sets the threshold of susceptibility to apoptosis for the intrinsic pathway, which utilizes organelles such as the mitochondrion to amplify death signals.
  • the family can be divided into 3 subclasses based on structure and impact on apoptosis.
  • Family members of subclass 1 including Bcl-2, Bcl-XL and Mcl-1 are characterized by the presence of 4 Bcl-2 homology domains (BH1, BH2, BH3 and BH4) and are anti -apoptotic.
  • the structure of the second subclass members is marked for containing 3 BH domains and family members such as Bax and Bak possess pro-apoptotic activities.
  • the third subclass termed the BH3-only proteins include Noxa, Puma, Bid, Bad and Bim. They function to promote apoptosis either by activating the pro-apoptotic members of group 2 or by inhibiting the anti- apoptotic members of subclass 1.
  • Activated caspase 9 classified as an intiator caspase, then cleaves procaspase 3 which cleaves more downstream procaspases, classified as executioner caspases, resulting in an amplification cascade that promotes cleavage of death substrates including poly(ADP-ribose) polymerase 1 (PARP).
  • PARP poly(ADP-ribose) polymerase 1
  • IAPs inhibitors of apoptosis
  • caspase proteases with aspartate specificity play significant roles in both inflammation and apoptosis.
  • Caspases exhibit catalytic and substrate recognition motifs that have been highly conserved. These characteristic amino acid sequences allow caspases to interact with both positive and negative regulators of their activity. The substrate preferences or specificities of individual caspases have been exploited for the development of peptides that successfully compete for caspase binding.
  • the catalytic domains of the caspases require at least four amino acids to the left of the cleavage site with P4 as the prominent specificity-determining residue.
  • WEHD, VDVAD, and DEVD are examples of peptides that preferentially bind caspase-1, caspase-2 and caspase-3, respectively. It is possible to generate reversible or irreversible inhibitors of caspase activation by coupling caspase-specific peptides to certain aldehyde, nitrile or ketone compounds. These caspase inhibitors can successfully inhibit the induction of apoptosis in various tumor cell lines as well as normal cells. Fluoromethyl ketone (FMK)-derivatized peptides act as effective irreversible inhibitors with no added cytotoxic effects.
  • FMK Fluoromethyl ketone
  • Inhibitors synthesized with a benzyl oxycarbonyl group also known as BOC or Z
  • BOC or Z Benzyloxycarbonyl group
  • ZVAD Benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone
  • the status of an activatable element within an apoptosis pathway is determined.
  • the activatable element within the apoptosis pathway is selected from the group consisting of Cleaved PARP (PARP+), Cleaved Caspase 8, and Cytoplasmic Cytochrome C.
  • the status of an activatable element within a DNA damage pathway is determined.
  • the activatable element within a DNA damage pathway is selected from the group consisting of p-CHkl, p-Chk-2, p-ATM, p-p53, p-ATR, p-21, and p-H2AX.
  • the cell cycle is the series of events that take place in a cell leading to its division and duplication (replication).
  • the cell cycle consists of five distinct phases: Gl phase, S phase (synthesis), G2 phase (collectively known as interphase) and M phase (mitosis).
  • M phase is itself composed of two tightly coupled processes: mitosis, in which the cell's chromosomes are divided between the two daughter cells, and cytokinesis, in which the cell's cytoplasm divides forming distinct cells. Activation of each phase is dependent on the proper progression and completion of the previous one. Cells that have temporarily or reversibly stopped dividing are said to have entered a state of quiescence called GO phase.
  • Regulation of the cell cycle involves processes crucial to the survival of a cell, including the detection and repair of genetic damage as well as the prevention of uncontrolled cell division.
  • the molecular events that control the cell cycle are ordered and directional; that is, each process occurs in a sequential fashion and it is impossible to "reverse" the cycle.
  • cyclins and cyclin-dependent kinases determine a cell's progress through the cell cycle.
  • Many of the genes encoding cyclins and CDKs are conserved among all eukaryotes, but in general more complex organisms have more elaborate cell cycle control systems that incorporate more individual components.
  • Many of the relevant genes were first identified by studying yeast, especially Saccharomyces cerevisiae genetic nomenclature in yeast dubs many these genes cdc (for "cell division cycle") followed by an identifying number, e.g., cdc25.
  • Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin.
  • CDKs When activated by a bound cyclin, CDKs perform a common biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle.
  • Different cyclin-CDK combinations determine the downstream proteins targeted.
  • CDKs are constitutively expressed in cells whereas cyclins are synthesised at specific stages of the cell cycle, in response to various molecular signals.
  • Gl cyclin-CDK complexes Upon receiving a pro-mitotic extracellular signal, Gl cyclin-CDK complexes become active to prepare the cell for S phase, promoting the expression of transcription factors that in turn promote the expression of S cyclins and of enzymes required for DNA replication.
  • the Gl cyclin-CDK complexes also promote the degradation of molecules that function as S phase inhibitors by targeting them for ubiquitination. Once a protein has been ubiquitinated, it is targeted for proteolytic degradation by the proteasome. Active S cyclin- CDK complexes phosphorylate proteins that make up the pre-replication complexes assembled during Gl phase on DNA replication origins.
  • the phosphorylation serves two purposes: to activate each already-assembled pre-replication complex, and to prevent new complexes from forming. This ensures that every portion of the cell's genome will be replicated once and only once. The reason for prevention of gaps in replication is fairly clear, because daughter cells that are missing all or part of crucial genes will die. However, for reasons related to gene copy number effects, possession of extra copies of certain genes would also prove deleterious to the daughter cells.
  • Mitotic cyclin-CDK complexes which are synthesized but inactivated during S and G2 phases, promote the initiation of mitosis by stimulating downstream proteins involved in chromosome condensation and mitotic spindle assembly.
  • a critical complex activated during this process is an ubiquitin ligase known as the anaphase-promoting complex (APC), which promotes degradation of structural proteins associated with the chromosomal kinetochore.
  • APC also targets the mitotic cyclins for degradation, ensuring that telophase and cytokinesis can proceed.
  • Interphase Interphase generally lasts at least 12 to 24 hours in mammalian tissue. During this period, the cell is constantly synthesizing RNA, producing protein and growing in size. By studying molecular events in cells, scientists have determined that interphase can be divided into 4 steps: Gap 0 (GO), Gap 1 (Gl), S (synthesis) phase, Gap 2 (G2).
  • Cyclin D is the first cyclin produced in the cell cycle, in response to extracellular signals (e.g. growth factors). Cyclin D binds to existing CDK4, forming the active cyclin D- CDK4 complex. Cyclin D-CDK4 complex in turn phosphorylates the retinoblastoma susceptibility protein (Rb). The hyperphosphorylated Rb dissociates from the E2F/DPl/Rb complex (which was bound to the E2F responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like cyclin E, cyclin A, DNA polymerase, thymidine kinase, etc.
  • Rb retinoblastoma susceptibility protein
  • Cyclin E thus produced binds to CDK2, forming the cyclin E-CDK2 complex, which pushes the cell from Gl to S phase (Gl/S transition).
  • Cyclin B along with cdc2 (cdc2 - fission yeasts (CDK1 - mammalia)) forms the cyclin B-cdc2 complex, which initiates the G2/M transition.
  • Cyclin B-cdc2 complex activation causes breakdown of nuclear envelope and initiation of prophase, and subsequently, its deactivation causes the cell to exit mitosis.
  • the Cip/Kip family includes the genes p21, p27 and p57. They halt cell cycle in Gl phase, by binding to, and inactivating, cyclin-CDK complexes.
  • p21 is a p53 response gene (which, in turn, is triggered by DNA damage eg. due to radiation).
  • p27 is activated by Transforming Growth Factor ⁇ (TGF ⁇ ), a growth inhibitor.
  • TGF ⁇ Transforming Growth Factor ⁇
  • the INK4a/ARF family includes pl6INK4a, which binds to CDK4 and arrests the cell cycle in Gl phase, and pl4arf which prevents p53 degradation.
  • Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. The cell cannot proceed to the next phase until checkpoint requirements have been met.
  • Gl/S checkpoint is a rate-limiting step in the cell cycle and is also known as restriction point.
  • An alternative model of the cell cycle response to DNA damage has also been proposed, known as the postreplication checkpoint.
  • p53 plays an important role in triggering the control mechanisms at both Gl/S and G2/M checkpoints.
  • binding element includes any molecule, e.g., peptide, nucleic acid, small organic molecule which is capable of detecting form of an activatable element over another form of the activatable element.
  • a "detectable binding element” as that term is used herein, encompasses a binding element, that both preferentially binds to one form of an activatable element, and whose bound form can be detected, e.g., through a label, such as a fluorescent label for flow cytometry or a mass label, also referred to as a mass tag, in mass cytometry, that produces a signal that can be detected, e.g., by a cytometer.
  • a "detectable binding element” as that term is used herein, encompasses a detectable binding element signal whose signal can be distinguished from that of any other detectable binding element in the particular process or composition in which it is used.
  • the signal that is detected can a quantitative value and it may be manipulated to produce other quantitative values.
  • the values may be used to gate cells, as known in the art and as described herein.
  • Gating may include an automatic component.
  • Gating may include a manual component. In certain embodiments, gating includes both a manual and an automatic component; see, e.g., U.S. Patent Application No. 2013/01763618.
  • the binding element is a peptide, polypeptide, oligopeptide or a protein.
  • the peptide, polypeptide, oligopeptide or protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
  • amino acid or “peptide residue”, as used herein include both naturally occurring and synthetic amino acids.
  • homo-phenylalanine, citrulline and noreleucine are considered amino acids.
  • the side chains may be in either the (R) or the (S) configuration.
  • the amino acids are in the (S) or L-configuration.
  • non-amino acid substituents may be used, for example to prevent or retard in vivo degradation.
  • Proteins including non-naturally occurring amino acids may be synthesized or in some cases, made recombinantly.
  • the binding element is an antibody. In some embodiment, the binding element is an activation state-specific antibody.
  • antibody includes full length antibodies and antibody fragments, and can refer to a natural antibody from any organism, an engineered antibody, or an antibody generated recombinantly for experimental, therapeutic, or other purposes as further defined below.
  • antibody fragments as are known in the art, such as Fab, Fab', F(ab')2, Fv, scFv, or other antigen-binding subsequences of antibodies, either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
  • antibody comprises monoclonal and polyclonal antibodies.
  • Antibodies can be antagonists, agonists, neutralizing, inhibitory, or stimulatory. They can be humanized, glycosylated, bound to solid supports, and posses other variations. See U.S.S. Nos U.S.S.N. 12/432,720, 12/229,476, 12/460,029, and 12/910,769 for more information about antibodies as binding elements.
  • the antigenicity of an activated form of an activatable element can be any antigenicity.
  • an activated isoform of an element possesses an epitope that is absent in a non-activated isoform of an element, or vice versa.
  • this difference is due to covalent addition of a moiety to an element, such as a phosphate moiety, or due to a structural change in an element, as through protein cleavage, or due to an otherwise induced conformational change in an element which causes the element to present the same sequence in an
  • Such a conformational change can cause an activated isoform of an element to present at least one epitope that is not present in a non-activated isoform, or to not present at least one epitope that is presented by a non-activated isoform of the element.
  • proteins that can be analyzed with the methods described herein include, but are not limited to, kinases, HER receptors, PDGF receptors, FLT3 receptor, Kit receptor, FGF receptors, Eph receptors, Trk receptors, IGF receptors, Insulin receptor, Met receptor, Ret, VEGF receptors, TIE1, TIE2, erythropoetin receptor, thromobopoetin receptor, CD114, CD116, FAK, Jakl, Jak2, Jak3, Tyk2, Src, Lyn, Fyn, Lck, Fgr, Yes, Csk, Abl, Btk, ZAP70, Syk, IRAKs, cRaf, ARaf, BRAF, Mos, Lim kinase, ILK, Tpl, ALK, TGFp receptors, BMP receptors, MEKKs
  • phosphatases PTEN, SHTPs, myotubularins, lipid signaling, phosphoinositide kinases, phopsholipases, prostaglandin synthases, 5 -lipoxygenase, sphingosine kinases,
  • sphingomyelinases adaptor/scaffold proteins, She, Grb2, BLNK, LAT, B cell adaptor for PI3-kinase (BCAP), SLAP, Dok, KSR, MyD88, Crk, CrkL, GAD, Nek, Grb2 associated binder (GAB), Fas associated death domain (FADD), TRADD, TRAF2, RIP, T-Cell leukemia family, cytokines, IL-2, IL-4, IL-8, IL-6, interferon ⁇ , interferon a, cytokine regulators, suppressors of cytokine signaling (SOCs), ubiquitination enzymes, Cbl, SCF ubiquitination ligase complex, APC/C, adhesion molecules, integrins, Immunoglobulin-like adhesion molecules, selectins, cadherins, catenins, focal adhesion kinase, pl30CAS, cytoskeletal/contractile proteins,
  • a binding element can be a peptide comprising a recognition structure that binds to a target structure on an activatable protein.
  • recognition structures are well known in the art and can be made using methods known in the art, including by phage display.
  • a binding element can be a nucleic acid.
  • nucleic acid includes nucleic acid analogs, for example, phosphoramide, phosphorothioate, phosphorodithioate, O- methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones.
  • Cells can be contacted with one or more modulators.
  • a modulator can be, e.g., an activator, a therapeutic compound, an inhibitor or a compound capable of impacting a cellular pathway. Modulators can also take the form of environmental cues and inputs.
  • Modulation can be performed in a variety of environments. Cells can be exposed to a modulator immediately after collection. In some embodiments where there is a mixed population of cells, purification of cells is performed after modulation. In some embodiments where there is a mixed population of cells, purification of cells is performed after modulation. In some embodiments where there is a mixed population of cells, purification of cells is performed after modulation. In some embodiments where there is a mixed population of cells, purification of cells is performed after modulation. In some
  • whole blood is collected to which a modulator is added.
  • cells are modulated after processing for single cells or purified fractions of single cells.
  • whole blood can be collected and processed for an enriched fraction of lymphocytes that is then exposed to a modulator.
  • Modulation can include exposing cells to more than one modulator. For instance, a sample of cells can be exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more modulators. See U.S. Patent
  • Cells can be cultured post collection in a suitable media before exposure to a modulator.
  • the media is a growth media.
  • the growth media is a complex media that may include serum.
  • the growth media comprises serum.
  • the serum is selected from the group consisting of fetal bovine serum, bovine serum, human serum, porcine serum, horse serum, and goat serum.
  • the serum level ranges from 0.0001% to 30%, about 0.001% to 30%, about 0.01% to 30%, about 0.1% to 30% or 1% to 30%.
  • the growth media is a chemically defined minimal media and is without serum.
  • cells are cultured in a differentiating media.
  • Modulators include chemical and biological entities, and physical or environmental stimuli. Modulators can act extracellularly or intracellularly. Chemical and biological modulators include growth factors, mitogens, cytokines, drugs, immune modulators, ions, neurotransmitters, adhesion molecules, hormones, small molecules, inorganic compounds, polynucleotides, antibodies, natural compounds, lectins, lactones, chemotherapeutic agents, biological response modifiers, carbohydrate, proteases and free radicals. Modulators include complex and undefined biologic compositions that may comprise cellular or botanical extracts, cellular or glandular secretions, physiologic fluids such as serum, amniotic fluid, or venom.
  • Physical and environmental stimuli include electromagnetic, ultraviolet, infrared or particulate radiation, redox potential and pH, the presence or absences of nutrients, changes in temperature, changes in oxygen partial pressure, changes in ion concentrations and the application of oxidative stress.
  • Modulators can be endogenous or exogenous and may produce different effects depending on the concentration and duration of exposure to the single cells or whether they are used in combination or sequentially with other modulators. Modulators can act directly on the activatable elements or indirectly through the interaction with one or more intermediary biomolecule. Indirect modulation includes alterations of gene expression wherein the expressed gene product is the activatable element or is a modulator of the activatable element.
  • the modulator can be selected from the group consisting of growth factors, mitogens, cytokines, adhesion molecules, drugs, hormones, small molecules, polynucleotides, antibodies, natural compounds, lactones, chemotherapeutic agents, immune modulators, carbohydrates, proteases, ions, reactive oxygen species, peptides, and protein fragments, either alone or in the context of cells, cells themselves, viruses, and biological and non- biological complexes (e.g. beads, plates, viral envelopes, antigen presentation molecules such as major histocompatibility complex).
  • the modulator is a physical stimuli such as heat, cold, UV radiation, and radiation.
  • modulators include but are not limited to Growth factors, such as Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bone morphogenetic proteins (BMPs),Brain-derived neurotrophic factor (BD F), Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cell line-derived neurotrophic factor (GD F), Granulocyte colony- stimulating factor (G-CSF), Granulocyte macrophage colony-stimulating factor (GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growth factor (IGF), Migration-stimulating factor, Myostatin (GDF-8), Nerve growth factor (NGF) and other neurotrophins, Platelet-derived growth factor (PDGF), Stromal Derived Growth Factor, (SDGF), Thrombopoietin (TPO), TPO
  • T cells Activates T cells, IL-2- T-cell growth factor. Stimulates IL-1 synthesis. Activates B-cells and K cells, IL-3- Stimulates production of all non-lymphoid cells, IL-4- Growth factor for activated B cells, resting T cells, and mast cells,IL-5- Induces differentiation of activated B cells and eosinophils, IL-6- Stimulates Ig synthesis. Growth factor for plasma cells, and IL-7- Growth factor for pre-B cells.
  • Cell motility factors such as peptide growth factors, (e.g., EGF, PDGF, TGF-beta), substrate-adhesion molecules (e.g., fibronectin, laminin), cell adhesion molecules (CAMs), and metalloproteinases, hepatocyte growth factor (HGF) or scatter factor (SF), autocrine motility factor (AMF), and migration-stimulating factor (MSF).
  • peptide growth factors e.g., EGF, PDGF, TGF-beta
  • substrate-adhesion molecules e.g., fibronectin, laminin
  • CAMs cell adhesion molecules
  • metalloproteinases e.g., hepatocyte growth factor (HGF) or scatter factor (SF), autocrine motility factor (AMF), and migration-stimulating factor (MSF).
  • HGF hepatocyte growth factor
  • SF scatter factor
  • AMF autocrine motility factor
  • MSF migration-stimulating factor
  • modulators include SDF- ⁇ , IFN-a, IFN- ⁇ , IL-10, IL-6, IL-27, G-CSF, FLT-3L, IGF-1, M- CSF, SCF, PMA, Thapsigargin, H202, Etoposide, Mylotarg, AraC, daunorubicin, staurosporine, benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD), lenalidomide, EPO, azacitadine, decitabine, IL-3, IL-4, GM-CSF, EPO, LPS, TNF-a, and CD40L, and combinations thereof.
  • the modulator is an activator. In some embodiments the modulator is an inhibitor. In some embodiments, cells are exposed to one or more modulators. In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators. In some embodiments, cells are exposed to at least two modulators, wherein one modulator is an activator and one modulator is an inhibitor. In some embodiments, cells are exposed to at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 modulators, where at least one of the modulators is an inhibitor.
  • the modulator can be a cross-linker.
  • the cross-linker can be a molecular binding entity.
  • the molecular binding entity is a monovalent, bivalent, or multivalent is made more multivalent by attachment to a solid surface or tethered on a nanoparticle surface to increase the local valency of the epitope binding domain.
  • the modulator can be an inhibitor.
  • the inhibitor can be an inhibitor of a cellular factor or a plurality of factors that participates in a cellular pathway (e.g. signaling cascade) in the cell.
  • the inhibitor is a phosphataseor a tyrosine kinase inhibitor.
  • phosphatase inhibitors include, but are not limited to H202, siRNA, miRNA, Cantharidin, (-)-p-Bromotetramisole, Microcystin LR, Sodium Orthovanadate, Sodium Pervanadate, Vanadyl sulfate, Sodium oxodiperoxo(l, 10-phenanthroline)vanadate, bis(maltolato)oxovanadium(IV), Sodium Molybdate, Sodium Perm olybdate, Sodium
  • the activation level of an activatable element in a cell can be determined by contacting the cell with an inhibitor and a modulator, where the modulator can be an inhibitor or an activator. In some embodiments, the activation level of an activatable element in a cell is determined by contacting the cell with an inhibitor and an activator. In some
  • the activation level of an activatable element in a cell is determined by contacting the cell with two or more modulators.
  • a phenotypic profile of a population of cells can be determined by measuring the activation level of an activatable element when the population of cells is exposed to a plurality of modulators in separate cultures.
  • the modulators include PMA, SDF1 a, CD40L, IGF-1, IL-7, IL-6, IL-10, IL-27, IL-4, IL-2, IL-3, and/or a combination thereof.
  • a population of cells can be exposed to one or more, all or a combination of the following combination of modulators:; PMA; SDFla; CD40L; IGF-1; IL-7; IL-6; IL-10; IL-27; IL-4; IL-2; IL-3;;.
  • the phenotypic profile of the population of cells is used to classify the population as described herein.
  • One or more activatable elements can be detected and/or quantified by any method that detects and/or quantitates the presence of the activatable element of interest.
  • Such methods may include flow cytometry, mass cytometry, radioimmunoassay (RIA) or enzyme linked immunoabsorbance assay (ELISA), immunohistochemistry, immunofluorescent histochemistry with or without confocal microscopy, reversed phase assays, homogeneous enzyme immunoassays, and related non-enzymatic techniques, Western, Northern, and Southern blots, PCR, nucleic acid sequencing, whole cell staining ,
  • a cytometer can be used, for example a flow cytometer or a mass cytometer, e.g., a CyToF, may be used.
  • Commercial instruments are available through Becton Dickinson, Beckman Coulter, and Fluidigm, among others.
  • fluorescence can be measured using a fluorimeter.
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics.
  • the excitation optics deliver the excitation radiation to excite the sample.
  • fluorescent proteins in the sample emit radiation that has a wavelength that is different from the excitation wavelength.
  • Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned.
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed.
  • the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer.
  • the computer also can transform the data collected during the assay into another format for presentation.
  • known robotic systems and components can be used.
  • detecting fluorescence may also be used, e.g., Quantum dot methods as well as confocal microscopy.
  • flow cytometry involves the passage of individual cells through the path of a laser beam. The scattering the beam and excitation of any fluorescent molecules attached to, or found within, the cell is detected by photomultiplier tubes to create a readable output, e.g. size, granularity, or fluorescent intensity.
  • the binding element may be detected by a signal from a label that is detectable by a mass spectrometer, e.g., a mass tag.
  • a mass spectrometer e.g., a mass tag.
  • An example is an Inductively Coupled Plasma
  • ICP-MS Spectrometer
  • mass cytometry when coupled with cytometric techniques similar to those used in flow cytometry, is referred to as "mass cytometry" herein.
  • the technique is very similar to flow cytometry, and sample preparation can be carried out using essentially identical techniques, e.g., 96-well plates, modulation, fixing, permeabilizing, the use of antibodies as binding agents. The difference is that the antibodies are tagged with mass labels rather than fluorescent labels, and that detection is carried out by mass spectrometry.
  • the signals from the mass spectrometer are analogous to those of a flow cytometer, i.e., a signal is generated that is proportional to the level of a particular element of the cell being investigated, such as the expression level of a surface marker, or the activation level of an activatable element.
  • Mass cytometry presents the potential advantage of being capable of detecting a larger number of signals that flow cytometry, e.g., the CyTof instrument (Fluidigm), can detect up to 34 parameters for a single cell, as opposed to the current maximum of 12 parameters for flow cytometers (in addition to scatter characteristics, e.g., SSC and FSC).
  • the cell population or subpopulation the cell belongs to which usually requires the determination of levels of 1-4 or even more surface markers.
  • APCs and/or tumor cells it may be desirable to determine the levels of a plurality of ligands for FMRs, i.e., the same number or even more than the number of the FMRs themselves, and/or surface markers for identification of tumor or of APC type and/or markers for intracellular pathways in the cells to determine functional status of the pathways. This presents a challenge for even the most advanced of presently available flow cytometers, but a mass cytometer can give reliable readings for a sufficient number of different channels to allow such measurements.
  • the number of channels required for the methods and compositions of the invention is such that a flow cytometer can be used as the detection instrument.
  • detection instruments and techniques are described herein for flow cytometers using, typically, fluorescent detection.
  • mass cytometry the same or similar techniques can be used for mass cytometry, and one of skill in the art understands the necessary adjustments required to apply the flow cytometric techniques to mass cytometry.
  • the detecting, sorting, or isolating step of the methods of the present invention can entail fluorescence-activated cell sorting (FACS) techniques, where FACS is used to select cells from the population containing a particular surface marker, or the selection step can entail the use of magnetically responsive particles as retrievable supports for target cell capture and/or background removal.
  • FACS fluorescence-activated cell sorting
  • a variety of FACS systems are known in the art and can be used in the methods described herein (see e.g., W099/54494, filed Apr. 16, 1999; U.S. Ser. No. 20010006787, filed Jul. 5, 2001, each expressly incorporated herein by reference).
  • a FACS cell sorter e.g. a FACSVantageTM Cell Sorter, Becton Dickinson Immunocytometry Systems, San Jose, Calif.
  • FACSVantageTM Cell Sorter Becton Dickinson Immunocytometry Systems, San Jose, Calif.
  • Other flow cytometers that are commercially available include the LSR II and the Canto II both available from Becton Dickinson others are available from Attune Acoustic Cytometer (Life
  • the cells are first contacted with fluorescent-labeled activation state-specific binding elements (e.g. antibodies) directed against specific activation state of specific activatable elements.
  • the amount of bound binding element on each cell can be measured by passing droplets containing the cells through the cell sorter. By imparting an electromagnetic charge to droplets containing the positive cells, the cells can be separated from other cells. The positively selected cells can then be harvested in sterile collection vessels.
  • Fluorescent compounds such as Daunorubicin and Enzastaurin are problematic for flow cytometry based biological assays due to their broad fluorescence emission spectra. These compounds get trapped inside cells after fixation with agents like paraformaldehyde, and are excited by one or more of the lasers found on flow cytometers. The fluorescence emission of these compounds is often detected in multiple PMT detectors which complicates their use in multiparametric flow cytometry. A way to get around this problem is to compensate out the fluorescence emission of the compound from the PMT detectors used to measure the relevant biological markers.
  • positive cells can be sorted using magnetic separation of cells based on the presence of an isoform of an activatable element.
  • cells to be positively selected are first contacted with specific binding element (e.g., an antibody or reagent that binds an isoform of an activatable element).
  • the cells are then contacted with retrievable particles (e.g., magnetically responsive particles) that are coupled with a reagent that binds the specific element.
  • the cell-binding element-particle complex can then be physically separated from non-positive or non-labeled cells, for example, using a magnetic field.
  • the positive or labeled cells can be retained in a container using a magnetic field while the negative cells are removed.
  • cell analysis by flow cytometry on the basis of the activation level of at least one element is combined with a determination of other flow cytometry readable outputs, such as the presence of surface markers, granularity and cell size to provide a further information on other cell qualities measurable by flow cytometry for single cells.
  • methods described herein also provide for the ordering of element clustering events in signal transduction.
  • the methods described herein allow the artisan to construct an element clustering and activation hierarchy based on the correlation of levels of clustering and activation of a multiplicity of elements within single cells. Ordering can be accomplished by comparing the activation level of a cell or cell population with a control at a single time point, or by comparing cells at multiple time points to observe subpopulations arising out of the others.
  • the methods described herein provide a valuable method of determining the presence of cellular subsets within cellular populations.
  • signal transduction pathways are evaluated in homogeneous cell populations to ensure that variances in signaling between cells do not qualitatively nor quantitatively mask signal transduction events and alterations therein.
  • the ultimate homogeneous system is the single cell, the present invention allows the individual evaluation of cells to allow true differences to be identified in a significant way.
  • a suitable protease e.g. collagenase, dispase, etc; and the like.
  • An appropriate solution is used for dispersion or suspension.
  • Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hanks balanced salt solution, etc., conveniently
  • Convenient buffers include HEPES1 phosphate buffers, lactate buffers, etc.
  • the cells may be fixed, e.g. with 3% paraformaldehyde, and are usually permeabilized, e.g. with ice cold methanol; HEPES- buffered PBS containing 0.1% saponin, 3% BSA; covering for 2 min in acetone at -2000C; and the like as known in the art and according to the methods described herein.
  • a permeabilizing agent for example, a methanol dispensing instrument can used to permeabilize the cells. It is important to ensure that the correct volume of methanol is being dispensed into the wells, otherwise the labeling reagents will not have access to their targets. To ensure that the appropriate amount of methanol is dispensed, the dispenser is charged beforehand with methanol or is charged with methanol either manually or automatically.
  • the methanol dispensing heads in the instrument can be stored with methanol or air in the dispensing channels. Air can be drawn through the dispensing heads, then an alcohol solution and then stored air dried or with methanol. Upon reuse of the instrument or any restart of the process, the dispensing heads are recharged with methanol. A bleeder valve can be used to fill up the head with the correct amount of methanol. In one embodiment, the instrument dispenser is charged by flushing several methanol washes through the dispenser head. In one embodiment, 2, 3, 4, 5, 6, washes are used to fill and clean the head.
  • the present invention uses platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • One embodiment uses microtiter plates and reference will be made to this embodiment as a representative of those articles that can contain samples to be analyzed.
  • one or more cells are contained in a well of a 96 well plate or other commercially available multiwell plate.
  • the reaction mixture or cells are in a cytometric measurement device.
  • Other multiwell plates useful in the present invention include, but are not limited to 384 well plates and 1536 well plates. Still other vessels for containing the reaction mixture or cells and useful for the present invention will be apparent to the skilled artisan. Methods to automate the analysis are shown in U.S. Ser. No. 12/606,869 which is hereby incorporated by reference in its entirety.
  • the activation level of an activatable element is measured using a mass spectrometer, e.g., an Inductively Coupled Plasma Mass Spectrometer (ICP- MS).
  • ICP- MS Inductively Coupled Plasma Mass Spectrometer
  • a binding element that has been labeled with a specific element binds to the activatable element.
  • the cell is introduced into the ICP, it is atomized and ionized.
  • the elemental composition of the cell, including the labeled binding element that is bound to the activatable element is measured. The presence and intensity of the signals corresponding to the labels on the binding element indicates the level of the activatable element on that cell.
  • compositions find use in a variety of other assay formats in addition to cytometry analysis.
  • Confocal microscopy can be used for detection. Confocal microscopy relies on the serial collection of light from spatially filtered individual specimen points, which is then electronically processed to render a magnified image of the specimen. The signal processing involved confocal microscopy has the additional capability of detecting labeled binding elements within single cells, the cells can be labeled with one or more binding elements. In some embodiments the binding elements used in connection with confocal microscopy are antibodies conjugated to fluorescent labels, however other binding elements, such as other proteins or nucleic acids are also possible. [00412] Another detection method is an "In-Cell Western Assay.” In such an assay, cells are initially grown in standard tissue culture flasks using standard tissue culture techniques.
  • the growth media is removed and cells are washed and trypsinized.
  • the cells can then be counted and volumes sufficient to transfer the appropriate number of cells are aliquoted into microwell plates (e.g., Nunc TM 96 Microwell TM plates).
  • the individual wells are then grown to optimum confluency in complete media whereupon the media is replaced with serum-free media.
  • controls are untouched, but experimental wells are incubated with a modulator, e.g. EGF. After incubation with the modulator cells are fixed and stained with labeled antibodies to the activation elements being investigated.
  • a modulator e.g. EGF
  • the plates can be scanned using an imager such as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniques described in the Odyssey Operator's Manual vl .2., which is hereby incorporated in its entirety. Data obtained by scanning of the multiwell plate can be analyzed and activation profiles determined as described below.
  • an imager such as the Odyssey Imager (LiCor, Lincoln Nebr.) using techniques described in the Odyssey Operator's Manual vl .2., which is hereby incorporated in its entirety.
  • Data obtained by scanning of the multiwell plate can be analyzed and activation profiles determined as described below.
  • the detecting is by high pressure liquid chromatography (HPLC), for example, reverse phase HPLC, and in a further aspect, the detecting is by mass spectrometry.
  • HPLC high pressure liquid chromatography
  • These instruments can fit in a sterile laminar flow or fume hood, or are enclosed, self-contained systems, for cell culture growth and transformation in multi-well plates or tubes and for hazardous operations.
  • the living cells may be grown under controlled growth conditions, with controls for temperature, humidity, and gas for time series of the live cell assays. Automated transformation of cells and automated colony pickers may facilitate rapid screening of desired cells.
  • the methods described herein include the use of liquid handling components.
  • the liquid handling systems can include robotic systems comprising any number of components.
  • any or all of the steps outlined herein may be automated; thus, for example, the systems may be completely or partially automated. See U.S. Ser. Nos. 12/606,869 and 12/432,239.
  • Fully robotic or microfluidic systems include automated liquid-, particle-, cell- and organism-handling including high throughput pipetting to perform all steps of screening applications.
  • This includes liquid, particle, cell, and organism manipulations such as aspiration, dispensing, mixing, diluting, washing, accurate volumetric transfers; retrieving, and discarding of pipet tips; and repetitive pipetting of identical volumes for multiple deliveries from a single sample aspiration.
  • These manipulations are cross-contamination-free liquid, particle, cell, and organism transfers.
  • This instrument performs automated replication of microplate samples to filters, membranes, and/or daughter plates, high-density transfers, full-plate serial dilutions, and high capacity operation.
  • chemically derivatized particles, plates, cartridges, tubes, magnetic particles, or other solid phase matrix with specificity to the assay components are used.
  • the binding surfaces of microplates, tubes or any solid phase matrices include non- polar surfaces, highly polar surfaces, modified dextran coating to promote covalent binding, antibody coating, affinity media to bind fusion proteins or peptides, surface-fixed proteins such as recombinant protein A or G, nucleotide resins or coatings, and other affinity matrix are useful in this invention.
  • platforms for multi-well plates, multi-tubes, holders, cartridges, minitubes, deep-well plates, microfuge tubes, cryovials, square well plates, filters, chips, optic fibers, beads, and other solid-phase matrices or platform with various volumes are accommodated on an upgradable modular platform for additional capacity.
  • This modular platform includes a variable speed orbital shaker, and multi-position work decks for source samples, sample and reagent dilution, assay plates, sample and reagent reservoirs, pipette tips, and an active wash station.
  • the methods described herein include the use of a plate reader.
  • thermocycler and thermoregulating systems are used for stabilizing the temperature of heat exchangers such as controlled blocks or platforms to provide accurate temperature control of incubating samples from Oo C to 100 ⁇ C.
  • interchangeable pipet heads with single or multiple magnetic probes, affinity probes, or pipetters robotically manipulate the liquid, particles, cells, and organisms.
  • Multi-well or multi-tube magnetic separators or platforms manipulate liquid, particles, cells, and organisms in single or multiple sample formats.
  • the instrumentation will include a detector, which can be a wide variety of different detectors, depending on the labels and assay.
  • useful detectors include a microscope(s) with multiple channels of fluorescence; plate readers to provide fluorescent, ultraviolet and visible spectrophotometric detection with single and dual wavelength endpoint and kinetics capability, fluorescence resonance energy transfer (FRET), luminescence, quenching, two-photon excitation, and intensity redistribution; CCD cameras to capture and transform data and images into quantifiable formats; and a computer workstation.
  • the robotic apparatus includes a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus. Again, as outlined below, this may be in addition to or in place of the CPU for the multiplexing devices described herein.
  • a central processing unit which communicates with a memory and a set of input/output devices (e.g., keyboard, mouse, monitor, printer, etc.) through a bus.
  • input/output devices e.g., keyboard, mouse, monitor, printer, etc.
  • this may be in addition to or in place of the CPU for the multiplexing devices described herein.
  • the general interaction between a central processing unit, a memory, input/output devices, and a bus is known in the art. Thus, a variety of different procedures, depending on the experiments to be run, are stored in the CPU memory.
  • robotic fluid handling systems can utilize any number of different reagents, including buffers, reagents, samples, washes, assay components such as label probes, etc. See U.S. Serial No. 12/606,869 for automated systems.
  • any of the steps above can be performed by a computer program product that comprises a computer executable logic that is recorded on a computer readable medium.
  • the computer program can execute some or all of the following functions: (i) exposing reference population of cells to one or more modulators, (ii) exposing reference population of cells to one or more binding elements, (iii) detecting the activation levels of one or more activatable elements, (iv) characterizing one or more cellular pathways and/or , (v) classifying one or more cells into one or more classes based on the activation level (vi) determining cell health status of a cell, (vii) determining the percentage of viable cells in a sample; (viii) determining the percentage of healthy cells in a sample; (ix) determining a cell signaling profile; (x) adjusting a cell signaling profile based on the percentage of healthy cells in a sample; (xi) adjusting a cell signaling profile for an individual cell based on the health of the cell; (xii)
  • the computer executable logic can work in any computer that may be any of a variety of types of general-purpose computers such as a personal computer, network server, workstation, or other computer platform now or later developed.
  • a computer program product is described comprising a computer usable medium having the computer executable logic (computer software program, including program code) stored therein.
  • the computer executable logic can be executed by a processor, causing the processor to perform functions described herein.
  • some functions are implemented primarily in hardware using, for example, a hardware state machine.
  • the program can provide a method of determining the status of an individual by accessing data that reflects the activation level of one or more activatable elements in the reference population of cells.
  • the data e.g., fluorescent intensity raw data
  • the detector such as a flow cytometer
  • the data is subject to processing using metrics outlined below.
  • the data can be fed to a model, such as machine learning, data mining, classification, or regression to provide a model for an outcome.
  • a model such as machine learning, data mining, classification, or regression to provide a model for an outcome.
  • models There is also a selection of models to produce an outcome, which can be a prediction or a prognosis.
  • the data can also be processed by using characteristics of cell health and cell maturity. Information on how to use cell health to analyze cells is shown in U.S.S.No.
  • a method is provided to analyze cells comprising obtaining cells, determining if the cell is undergoing apoptosis and then excluding cells from a final analysis that are undergoing apoptosis.
  • One way to determine if a cell is undergoing apoptosis is by measuring the intracellular level of one or more activatable elements related to cell health such as cleaved PARP, MCL-1, or other compounds whose activation state or activation level correlate to a level of apoptosis within single cells.
  • Indicators for cell health can include molecules and activatable elements within molecules associated with apoptosis, necrosis, and/or autophagy, including but not limited to caspases, caspase cleavage products such as dye substrates, cleaved PARP, cleaved cytokeratin 18, cleaved caspase, cleaved caspase 3, cytochrome C, apoptosis inducing factor (AIF), Inhibitor of Apoptosis (IAP) family members, as well as other molecules such as Bcl-2 family members including anti-apoptotic proteins (MCL-1, BCL-2, BCL-XL), BH3-only apoptotic sensitizers (PUMA, NOXA, Bim, Bad), and pro-apoptotic proteins (Bad, Bax) (see below), p53, c-myc proto-oncogene, APO-l/Fas/CD95, growth stimulating genes, or
  • Another general method for analyzing cells takes into account the maturity level of the cells.
  • cells that are immature are included in the analysis and mature cells are not included.
  • the analysis can include all the patient's cells if they go above a certain threshold for the entire sample, for example, a call will be made on the basis of the entire sample. For example, samples having greater than 50, 60, 65, 70, 75, 80, 85, 90, or 95% immature cells can be classified as immature as a whole.
  • only those specific cells which are classified as immature are included in the analysis, irrespective of the total number of immature cells, for example, only those cells that are classified as immature will be part of the analysis for each sample.
  • the metrics that are employed can relate to absolute cell counts, signal, e.g., fluorescence, intensity, frequencies of cellular populations (univariate and bivariate), relative signal, e.g., fluorescence, readouts (such as signal above background, etc.), and
  • raw intensity data is corrected for variances in the instrument. Then the biological effect can be measured, such as measuring how much signaling is going on using the basal, fold, total and delta metrics. Also, a user can look at the number of cells that show signaling using the Mann Whitney model below.
  • flow cytometry experiments are performed and the results are expressed as fold changes using graphical tools and analyses, including, but not limited to a heat map or a histogram to facilitate evaluation.
  • graphical tools and analyses including, but not limited to a heat map or a histogram to facilitate evaluation.
  • One common way of comparing changes in a set of flow cytometry samples is to overlay histograms of one parameter on the same plot.
  • Flow cytometry experiments ideally include a reference sample against which experimental samples are compared. Reference samples can include normal and/or cells associated with a condition (e.g. tumor cells). See also U.S. Ser. No. 12/501,295 for visualization tools.
  • the "basal" metric is calculated by measuring the autofluorescence of a cell that has not been stimulated with a modulator or stained with a labeled antibody.
  • the “total phospho” metric is calculated by measuring the autofluorescence of a cell that has been stimulated with a modulator and stained with a labeled antibody.
  • the "fold change” metric is the measurement of the total phospho metric divided by the basal metric.
  • the quadrant frequency metric is the frequency of cells in each quadrant of the contour plot
  • a user may also analyze multimodal distributions to separate cell populations.
  • metrics can be used for analyzing bimodal and spread distribution.
  • a Mann-Whitney U Metric is used.
  • metrics that calculate the percent of positive above unstained and metrics that calculate MFI of positive over untreated stained can be used.
  • a user can create other metrics for measuring the negative signal. For example, a user may analyze a "gated unstained” or ungated unstained autofluorescence population as the negative signal for calculations such as "basal” and “total". This is a population that has been stained with surface markers such as CD33 and CD45 to gate the desired population, but is unstained for the fluorescent parameters to be quantitatively evaluated for node
  • every antibody has some degree of nonspecific association or “stickyness” which is not taken into account by just comparing fluorescent antibody binding to the autofluorescence.
  • the user may stain cells with isotype-matched control antibodies.
  • (phospho) or non phosphopeptides which the antibodies should recognize will take away the antibody's epitope specific signal by blocking its antigen binding site allowing this "bound" antibody to be used for evaluation of non-specific binding.
  • a user may block with unlabeled antibodies. This method uses the same antibody clones of interest, but uses a version that lacks the conjugated fluorophore.
  • a user may block other high protein concentration solutions including, but not limited to fetal bovine serum, and normal serum of the species in which the antibodies were made, i.e. using normal mouse serum in a stain with mouse antibodies. (It is preferred to work with primary conjugated antibodies and not with stains requiring secondary antibodies because the secondary antibody will recognize the blocking serum).
  • a user may treat fixed cells with phosphatases to enzymatically remove phosphates, then stain.
  • One embodiment of the present invention is software to examine the correlations among phosphorylation or expression levels of pairs of proteins in response to stimulus or modulation.
  • the software examines all pairs of proteins for which phosphorylation and/or expression was measured in an experiment.
  • the total phosho metric (sometimes called "FoldAF") is used to represent the phosphorylation or expression data for each protein; this data is used either on linear scale or log2 scale.
  • Delta CRNR stim the difference between Pearson correlation coefficients for each protein pair for the responding patients and for the non-responding patients in the stimulated or treated state.
  • DeltaDelta CRNR the difference between Delta CRNRstim and Delta
  • Protein-protein pairs are identified for closer analysis by the following criteria:
  • All pair data is plotted as a scatter plot with axes representing phosphorylation or expression level of a protein.
  • Data for each sample (or patient) is plotted with color indicating whether the sample represents a responder (generally blue) or non-responder (generally red).
  • Each graph is annotated with the Pearson correlation coefficient and linear regression parameters for the individual classes and for the data as a whole.
  • the resulting plots are saved in PNG format to a single directory for browsing using Picassa. Other visualization software can also be used.
  • a Mann Whitney statistical model is used for describing relative shifts in cellular populations.
  • a Mann Whitney U test or Mann Whitney Wilcoxon (MWW) test is a non parametric statistical hypothesis test for assessing whether two independent samples of observations have equally large values. See Wikipedia at http(colon)(slashslash)en.wikipedia.org(slash)wiki/Mann%E2%80%93Whitney_U .
  • the U metric may be more reproducible in some situations than Fold Change in some applications.
  • Uu is a measure of the proportion of cells that have an increase (or decrease) in protein levels upon modulation from the basal state. It is computed by dividing the scaled Mann-Whitney U statistic
  • Modulated (m) and modulated (u) populations are being compared
  • n m number of cells in the modulated population
  • n u number of cells in the unmodulated population
  • Ui is another value that is the same as U u except that the isotype control is used as the reference instead of the unmodulated well.
  • This metric is always calculated relative to the unmodulated level of
  • unmodulated and modulated states are typically measured on the same plate several factors such as autofluorescense, batch effects, etc. are implicitly corrected for in this calculation.
  • the metric is "Rel l0g2 F I R xr Fisotype control
  • n u number of cells in the single cell events are unmodulated population used in the calculation.
  • n a number of cells in the It is formally a scaled autofluorescence population Mann-Whitney U metric
  • Modulated (m) and unmodulated (u) proportion of cells in a populations are being compared. modulated state relative to the population seen in
  • n u number of cells in the Mann-Whitney U metric unmodulated population (AUC).
  • n m number of cells in the modulated
  • a given measure e.g. MFI, ERF, etc.
  • Each protein pair can be further annotated by whether the proteins comprising the pair are connected in a "canonical" pathway.
  • canonical pathways are defined as the pathways curated by the NCI and Nature Publishing Group. This distinction is important; however, it is likely not an exclusive way to delineate which protein pairs to examine.
  • High correlation among proteins in a canonical pathway in a sample may indicate the pathway in that sample is "intact" or consistent with the known literature.
  • One embodiment of the present invention identifies protein pairs that are not part of a canonical pathway with high correlation in a sample as these may indicate the non-normal or
  • This method will be used to identify stimulator/modulator-stain-stain combinations that distinguish classes of patients.
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using classification algorithms.
  • Any suitable classification algorithm known in the art can be used. Examples of classification algorithms that can be used include, but are not limited to, multivariate classification algorithms such as decision tree techniques:
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using random forest algorithm.
  • Random forest or random forests is an
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using lasso algorithm.
  • the method of least squares is a standard approach to the approximate solution of overdetermined systems, i.e. sets of equations in which there are more equations than unknowns. "Least squares" means that the overall solution minimizes the sum of the squares of the errors made in solving every single equation. The best fit in the least-squares sense minimizes the sum of squared residuals, a residual being the difference between an observed value and the fitted value provided by a model.
  • nodes and/or nodes/metric combinations can be analyzed and compared across sample for their ability to distinguish among different groups (e.g., CR vs. NR patients) using BBLRS model building methodology.
  • Best subsets selection of main effects is used to identify the combination of predictors that yields the largest score statistic among models of a given size in each bootstrap sample. Models having from 1 to 2> ⁇ N/10 are typically entertained at this stage, where N is the number of observations. This is much larger than the number of predictors generally recommended when building a generalized linear prediction model (Harrell, 2001) but subsequent model building rules are applied to reduce the likelihood of over-fitting. At the conclusion of this step, there will be a "best" main effects model of each size for each bootstrap sample, though the number of unique models of each size may be considerably fewer.
  • each of the unique "best" models of each size, identified in the previous step are fit to each of a subset of the bootstrap samples, where the number of bootstrap samples in the subset is under the control of the user (i.e. a tuning parameter) so that the processing time required at this step can be controlled.
  • the median SBC of the "best" models of the same size is calculated and the model size yielding the lowest median SBC in that bootstrap sample is identified.
  • the optimal model size is then determined as the size for which the median SBC is smallest most often over the subset of bootstrap samples.
  • the procedure described here results in the selection of the effects (main effects and possibly two-way interactions) to be included in the final model, but not specification of the model itself.
  • the latter includes the effects and the specific regression coefficients associated with the intercept and each of the model effects.
  • Another method of the present invention relates to display of information using scatter plots.
  • Scatter plots are known in the art and are used to visually convey data for visual analysis of correlations. See U. S. Patent No. 6,520, 108.
  • the scatter plots illustrating protein pair correlations can be annotated to convey additional information, such as one, two, or more additional parameters of data visually on a scatter plot.
  • the diameter of the circles representing the phosphorylation or expression levels of the pair of proteins may be scaled according to another parameter. For example they may be scaled according to expression level of one or more other proteins such as transporters (if more than one protein, scaling is additive, concentric rings may be used to show individual
  • additional shapes may be used to indicate subclasses of patients. For example they could be used to denote patients who responded to a second drug regimen or where CRp status. Another example is to show how samples or patients are stratified by another parameter (such as a different stim-stain-stain combination). Many other shapes, sizes, colors, outlines, or other distinguishing glyphs may be used to convey visual information in the scatter plot.
  • the size of the dots is relative to the measured expression and the box around a dot indicates a NRCR patient that is a patient that became CR (Responsive) after more aggressive treatment but was initially NR (Non-Responsive). Patients without the box indicate a NR patient that stayed NR.
  • the Total Phospho metric for p-Akt and p-Statl are correlated in response to peroxide ("H202") treatment. On log2 scale the Pearson correlation coefficient for p-Akt and p-Statl in response to HOOH for samples from patients who responded to first treatment is 0.89 and the p-value for linear regression line fit is 0.0075.
  • Table 3(a) below shows nodes identified by a fold change metric.
  • Table 3(b) below shows node identified by a variety of methods. In some embodiments, the nodes depicted in Tables 3(a) and 3(b) are used according to the methods described herein for classification, diagnosis, prognosis of AML or for the selection of treatment and/or predict outcome after administering a therapeutic.
  • analyses are performed on healthy cells.
  • the health of the cells is determined by using cell markers that indicate cell health.
  • cells that are dead or undergoing apoptosis will be removed from the analysis.
  • cells are stained with apoptosis and/or cell death markers such as PARP or Aqua dyes.
  • Cells undergoing apoptosis and/or cells that are dead can be gated out of the analysis.
  • apoptosis is monitored over time before and after treatment. For example, in some embodiments, the percentage of healthy cells can be measured at time zero and then at later time points and conditions.
  • the measurements of activatable elements are adjusted by measurements of sample quality for the individual sample, such as the percent of healthy cells present.
  • a regression equation will be used to adjust raw node readout scores for the percentage of healthy cells at 24 hours post-thaw.
  • means and standard deviations will be used to standardize the adjusted node readout scores.
  • 0 1 are the coefficients from the regression equation used to adjust for the percentage of healthy cells (P ct healthy ⁇ anc j residual mean and residual _sd are the mean and stanc j arc i deviation, respectively, for the adjusted signal readouts in the training set data.
  • “parameter” is equal to "percenthealthy24Hrs”.
  • the SCNP classifier will be applied to the z values for the node-metrics to calculate the continuous SCNP classifier score and the binary induction response assignment (pNR or pCR) for each sample.
  • the measurements of activatable elements are adjusted by measurements of sample quality for the individual cell populations or individual cells, based on markers of cell health in the cell populations or individual cells. Examples of analysis of healthy cells can be found in U.S. application serial number 61/374,613 filed August 18, 2010, the content of which is incorporated herein by reference in its entirety for all purposes. Conditions
  • the methods of the invention are applicable to any condition in an individual involving, indicated by, and/or arising from, in whole or in part, altered physiological status in cells.
  • a condition involving or characterized by altered physiological status may be readily identified, for example, by determining the state of one or more activatable elements in cells from different populations, as taught herein.
  • the condition is cancer.
  • the cancer may produce solid tumors or hematological tumors.
  • Cancers that produce solid tumors include adrenal cortical cancer, anal cancer, bile duct cancer (e.g. peripheral cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma,
  • osteochrondroma hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g.
  • ductal carcinoma in situ infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia
  • Castleman disease e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia
  • cervical cancer colorectal cancer
  • endometrial cancer e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma, clear cell
  • esophagus cancer gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g.
  • kidney cancer e.g. renal cell cancer
  • laryngeal and hypopharyngeal cancer e.g.
  • hemangioma hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma
  • lung cancer e.g. small cell lung cancer, non-small cell lung cancer
  • mesothelioma mesothelioma
  • plasmacytoma, nasal cavity and paranasal sinus cancer e.g. esthesioneuroblastoma, midline granuloma
  • nasopharyngeal cancer e.g. neuroblastoma, oral cavity and oropharyngeal cancer
  • ovarian cancer pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g.
  • thyroid cancer e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma
  • vaginal cancer e.g. vulvar cancer
  • uterine cancer e.g. uterine leiomyosarcoma
  • Primary cancers and metastases as well as cancers of unknown primary are included.
  • Cancers that produce hematological tumors include but are not limited to Non- Hodgkin Lymphoma, Hodgkin or other lymphomas, acute or chronic leukemias, and multiple myeloma.
  • the cancer is non-B lineage derived, such as Acute myeloid leukemia (AML), Chronic Myeloid Leukemia (CML), non-B cell Acute lymphocytic leukemia (ALL), or non-B cell lymphomas.
  • the cancer is a B-Cell or B cell lineage derived cancer.
  • B-Cell or B cell lineage cancers include but are not limited to Chronic Lymphocytic Leukemia (CLL), B lymphocyte lineage leukemia, B lymphocyte lineage lymphoma, and Multiple Myeloma.
  • CLL Chronic Lymphocytic Leukemia
  • B lymphocyte lineage leukemia B lymphocyte lineage lymphoma
  • Multiple Myeloma Other conditions within the scope of the present invention include, but are not limited to, cancers such as gliomas, lung cancer, colon cancer and prostate cancer.
  • kits provided by the invention may comprise one or more of the state-specific binding elements described herein, such as phospho-specific antibodies.
  • a kit may also include other reagents that are useful in the invention, such as modulators, fixatives, containers, plates, buffers, therapeutic agents, instructions, and the like.
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of PI3-Kinase (p85, pi 10a, pi 10b, pl lOd), Jakl, Jak2, SOCs, Rac, Rho, Cdc42, Ras-GAP, Vav, Tiam, Sos, Dbl, Nek, Gab, PRK, SHP1, and SHP2, SHIP1, SHIP2, sSHIP, PTEN, She, Grb2, PDK1, SGK, Aktl, Akt2, Akt3, TSC1,2, Rheb, mTor, 4EBP-1, p70S6Kinase, S6, LKB-1, AMPK, PFK, Acetyl-CoAa Carboxylase, DokS, Rafs, Mos, Tpl2, MEK1/2, MLK3, TAK, DLK, MKK3/6, MEKK1,4, MLK3, ASK1, MKK4/7, SAPK
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Erk, Syk, Zap70, Lck, Btk, BLNK, Cbl, PLCy2, Akt, RelA, p38, S6.
  • the kit comprises one or more of the phospho-specific antibodies specific for the proteins selected from the group consisting of Aktl, Akt2, Akt3, SAPK/JNK 1,2,3, p38s, Erkl/2, Syk, ZAP70, Btk, BL K, Lck, PLCy, PLCy 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, F-kB, GSK3p, CARMA/BcllO and Tcl-1.
  • the proteins selected from the group consisting of Aktl, Akt2, Akt3, SAPK/JNK 1,2,3, p38s, Erkl/2, Syk, ZAP70, Btk, BL K, Lck, PLCy, PLCy 2, STAT1, STAT 3, STAT 4, STAT 5, STAT 6, CREB, Lyn, p-S6, Cbl, F-kB, GSK3p, CARMA/BcllO and Tcl-1.
  • the state-specific binding element of the invention can be conjugated to a solid support and to detectable groups directly or indirectly.
  • the reagents may also include ancillary agents such as buffering agents and stabilizing agents, e.g., polysaccharides and the like.
  • the kit may further include, where necessary, other members of the signal-producing system of which system the detectable group is a member (e.g., enzyme substrates), agents for reducing background interference in a test, control reagents, apparatus for conducting a test, and the like.
  • the kit may be packaged in any suitable manner, typically with all elements in a single container along with a sheet of printed instructions for carrying out the test. .
  • kits may additionally comprise one or more therapeutic agents.
  • the kit may further comprise a software package for data analysis of the physiological status, which may include reference profiles for comparison with the test profile.
  • kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials. Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in some embodiments, be marketed directly to the consumer.
  • PBMC peripheral blood mononuclear cell
  • BMMC bone marrow mononuclear cell
  • Sample inclusion criteria can require collection at a time point prior to initiation of induction chemotherapy, AML classification by the French-American-British (FAB) criteria as M0 through M7 (excluding M3), and availability of appropriate clinical annotations (e.g., disease response after one or two cycles of induction chemotherapy).
  • Induction chemotherapy can consist of at least one cycle of standard cytarabine-based induction therapy (i.e., daunorubicin 60 mg/m2 x 3 days, cytarabine 100-200 mg/m2 continuous infusion x 7 days); responses are measured after one cycle of induction therapy.
  • Standard clinical and laboratory criteria can be used for defining complete responders (CR) in the patient samples. Leukemia samples obtained from patients who do not meet the criteria for CR or samples obtained from those who died during induction therapy are considered non-complete response ( R) for the primary analyses.
  • Cell network profiling assays involved measuring the expression of protein levels and their post-translational modification by phosphorylation in different populations of cells at baseline and after perturbation with various modulators.
  • the populations that can be analyzed include myeloid leukemic cells, B cells, T cells, dendritic cells, monocytes, macrophages, neutrophils, eosinophils, and basophils.
  • Other cells such as epithelial cells can also be analyzed.
  • a pathway "node” is defined as a combination of a specific proteomic readout in the presence or absence of a specific modulator.
  • Levels of signaling proteins, as well as expression of cell surface markers are detected by multiparameter flow cytometry using fluorochrome- conjugated antibodies to the target proteins. Multiple nodes (including surface receptors and transporters), using multiple modulators can be assessed in the two studies.
  • a minimum yield of 100,000 viable cells and 500 cells per gated sample in gate of interest can be used for each patient sample to be classified as evaluable.
  • phenotypic markers e.g., CD45, CD33
  • intracellular stains e.g., CD45, CD33
  • Data acquisition and cytometry analysis Data is acquired using FACS DIVA software on both LSR II and CANTO II Flow Cytometers (BD). For all analyses, dead cells and debris are excluded by FSC (forward scatter), SSC (side scatter), and Amine Aqua Viability Dye measurement. Leukemic cells are identified as cells that lacked the
  • CD45++, CD33- characteristics of mature lymphocytes (CD45++, CD33-), and that fit the CD45 and CD33 versus right-angle light-scatter characteristics consistent with myeloid leukemia cells.
  • Other cell populations are identified using markers known in the art.
  • MFI median fluorescence intensity
  • Basal MFI log2(MFIUnmodulated Stained) - log2(MFIGated Unstained (Autofluoresence)), designed to measure the basal levels of a certain protein under unmodulated conditions
  • Fold Change MFI log2(MFFModulated Stained) - log2(MFIUnmodulated Stained), a measure of the change in the activation state of a protein under modulated conditions
  • Total Phospho MFI log2(MFIModulated Stained) - log2(MFIGated Unstained (Autofluorescence)), a measure of the total levels of a protein under modulated conditions.
  • Fold over Control MFI log2(MFIStain) - log2(MFIControl), a measure of the levels of surface marker staining relative to control antibody staining
  • Percent Cell Positivity a measure of the frequency of cells that have surface markers staining at an intensity level greater than the 95th percentile for control antibody staining
  • a low signaling node is defined as a node having a fold change metric or total phosphoprotein signal equal to I log2(Fold) I >.15. However, it is not necessary to use this as an exclusion criterion in this study.
  • a random result would produce an AUC value of 0.5.
  • a (bio)marker with 100% specificity and selectivity would result in an AUC of 1.0.
  • the cell population/node/metric combinations are independently tested for differences between patient samples whose response to standard induction therapy was CR vs NR. No corrections are applied to the p-values to correct for multiple testing. Instead, simulations are performed by randomly permuting the clinical variable to estimate the number of cell population/node/metric combinations that might appear to be significant by chance. For each permutation, nine donors are randomly chosen (without replacement) and assigned to the CR category and the remaining are assigned to the NR category. By comparing each cell population/node/metric combination to the permuted clinical variable, the student t-test p-values are computed. This process is repeated. The results from these simulations are then used to estimate the number of cell
  • the corners classifier is a rules-based algorithm for dividing subjects into two classes (in this case the dichotomized response to induction therapy) using one or more numeric variables (defined in our study as a node/metric combination).
  • This method works by setting a threshold on each variable, and then combining the resulting intervals (e.g., X ⁇ 10, or Y > 50) with the conjunction (and) operator (reference).
  • Threshold values are chosen by minimizing an error criterion based on the logit-transformed misclassification rate within each class.
  • the method assumes only that the two classes (i.e. response or lack of response to induction therapy) tend to have different locations along the variables used, and is invariant under monotone transformations of those variables.
  • a bagging also known as bootstrapped aggregation, is used i to internally cross- validate the results of the above statistical model.
  • Bootstrap re-samples are drawn from the original data.
  • Each classifier i.e. combination of cell population/node/metric, is fit to the resample, and then used to predict the class membership of those patients who were excluded from the resample.
  • each patient acquires a list of predicted class memberships based on classifiers that are fit using other patients.
  • Each patient's list is reduced to the fraction of target class predictions; members of the target class should have fractions near 1, unlike members of the other class.
  • the set of such fractions, along with the patient's true class membership, is used to create an ROC curve and to calculate its AUC.
  • Patient samples Sets of fresh or cryopreserved samples from patients can be analyzed.
  • the sets can consist of cells samples derived from the lymph nodes, synovium and/or synovial fluid of rheumatoid patients. All patients will be asked for consent for the collection and use of their samples for institutional review board (IRB)-approved research purposes. All clinical data is de-identified in compliance with Health Insurance Portability and Accountability Act (HIPAA) regulations.
  • HIPAA Health Insurance Portability and Accountability Act
  • Sample inclusion criteria can include: (i) A diagnosis of rheumatoid arthritis by the 1987 ACR criteria, (ii) Definite bony erosions, (iii) Age of disease onset greater than 18 years, (iv) Patient does not have psoriasis, inflammatory bowel disease, or systemic lupus erythematosus.
  • Standard clinical and laboratory criteria can be used for defining RA patients that are able to respond to a treatment in the patient samples.
  • RA samples obtained from patients who do not meet the criteria for patients that are able to respond are considered non-complete responders for the primary analyses.
  • possible treatments include nonsteroidal antiinflammatory drugs (NSAIDs) such as Acetyl salicylate (aspirin), naproxen (Naprosyn), ibuprofen (Advil, Medipren, Motrin), and etodolac (Lodine); Corticosteroid;
  • NSAIDs nonsteroidal antiinflammatory drugs
  • Example 1 Populations of cells that can be analyzed using the methods described in Example 1 include B cells, T cells, dendritic cells, monocytes, macrophages, neutrophils, eosinophils, and basophils. Other cells such as mesechymal cells and epithelial cells can also be analyzed.
  • EXAMPLE 3 CELLULAR AND INTRACELLULAR NETWORK
  • JAK/STAT signaling in hematopoietic cells has shown to be involved in certain hematological and immune diseases; thus, the regulation of JAK/STAT signaling is an important research area.
  • Signaling pathway- and cell type-specific responses to various cytokines in the immune system signaling network can elicit a wide range of biological outcomes due to the combinatorial use of a limited set of kinases and STAT proteins.
  • SCNP Single Cell Network Profiling
  • the SC P assay was performed using a fluorophore-labeled antibody cocktail to simultaneously measure signaling in six distinct cell populations, including: neutrophils, CD20+ B cells, CD3+CD4+T cells, CD3+CD4- T cells (CD 8 enriched), CD3-CD20- lymphocytes (NK cell enriched), and CD14+ monocytes.
  • neutrophils CD20+ B cells
  • CD3+CD4+T cells CD3+CD4- T cells
  • CD3+CD4- T cells CD3+CD4- T cells (CD 8 enriched)
  • CD3-CD20- lymphocytes NK cell enriched
  • CD14+ monocytes CD14+ monocytes.
  • the median fluorescent intensity of phospho (p)- STAT1(Y701), p-STAT3(Y705), and p-STAT5(Y694) were measured in each defined cell population for each experimental condition.
  • IL-6 induced signaling was only observed in CD4+ T cells and monocytes with peak p-STAT3 levels at 3 minutes followed by p-STATl and p-STAT5 at 10-15 minutes.
  • signal resolution fell to baseline levels at 45 minutes in monocytes, while the CD4+ T cells showed sustained elevated signaling, suggesting a cell- type specific regulation.
  • IFN-D ⁇ stimulation activated all 3 STAT proteins, peaking at 10 minutes with similar kinetics in all cell subsets.
  • IFN- ⁇ signaling resolution was faster and almost complete at 45 minutes in monocytes, while in the all other subsets the signal was sustained. This efficient signal termination in monocytes was also observed with GM-CSF - p-STAT5, while neutrophils maintained persistent p- STAT5 levels.
  • IL-27 induced p-STAT 1 and p-STAT3 in T cell subsets, B cells, and monocytes with peak activation at 30 minutes. In general, signaling characteristics were remarkably uniform across the healthy donors.
  • IL-6- p-STAT3 was particularly consistent across time points and ligand concentrations, while p-STATl and p-STAT5 showed more variation. More results are provided in Example 5.
  • PBMCs peripheral blood mononuclear cells
  • This study was designed to apply SCNP to generate a map of IFN-D -mediated signaling responses, with emphasis on Stat proteins, in PBMCs from healthy donors.
  • the data provides a reference for future studies using PBMCs from patient samples in which IFND ⁇ - mediated signaling is aberrantly regulated.
  • IFN-a-mediated signaling responses were measured by SCNP in PBMC samples from 12 healthy donors.
  • PBMCs were processed for flow cytometry by fixation and permeabilization followed by incubation with fluorochrome-conjugated antibodies that recognize extracellular lineage markers and intracellular signaling molecules.
  • the levels of several phospho-proteins (p-Statl, p-Stat3, p-Stat4, p-Stat5, p-Stat6, and p-p38) were measured in multiple cell populations (CD14+ monocytes, CD20+ B cells, CD4+ CD3+ T cells, and CD4- CD3+ T cells) at 15 minutes, 1, 2 and 4 hours post IFN-a exposure as described in Example 6.
  • Results The data revealed distinct phospho-protein activation patterns in different cell subsets within PBMCs in response to IFN-a exposure. For example, activation of p- Stat4 was detected in T cell subsets (both CD4+ and CD4- T cells), but not in monocytes or B cells. Such cell-type specific activation patterns likely play a key role in mediating specific functions within different cell types in response to IFN-a. Differences in the kinetics of activation by IFN-a for different phospho-proteins were also observed.
  • the peak response for activation of p-Statl, p-Stat3, and p-Stat5 was at 15 minutes in most of the cell types interrogated in this study, whereas for the activation of p-Stat4, p-Stat6, and p-p38 it was at 1 hr in the majority of cell types tested.
  • the relationships between phospho-protein readouts in each cell subset were determined by calculating the Pearson correlation coefficients. For example, the activation of p-Statl and p-Stat5 at 15 minutes was positively correlated in both B cells and T cells. More results are provided in Example 6.
  • Activation levels of pStatl, pStat3 and pStat5 were measured using flow cytometry at 15 minutes after treatment with the modulators.
  • Activation levels of pStatl, pStat3 and pStat5 were measured using flow cytometry at 15 minutes after treatment with the modulators.
  • several other elements were measured in order to separate the cells into discrete populations according to cell type. These markers included CD45, CD34, CD71 and CD235ab.
  • CD45 was used to segregate Lymphocytes, Myeloid(pl) cells and nRBCs. The nRBCs were further segregated into 4 distinct cell populations based on expression of CD71 and CD235ab: ml, m2, m3 and m4. These cell populations correspond to RBC maturity and are illustrated in FIG. 2.
  • FIG. 2 of U.S. S.N. 12/877,998 illustrates the different activation levels of pStatl, pStat3 and pStat5 observed in EPO, G-CSF and EPO + G-CSF treated Lymphocytes, nRBCl cells, Myeloid(pl) cells and stem cells.
  • Activation levels observed in different samples from the normal and low risk populations are plotted as dots.
  • different cell discrete populations demonstrated different induced activation levels. Although this was true in both the healthy and the low risk patients, the different discrete cell populations exhibited a narrower range of induced activation levels in then normal samples than in the low risk samples.
  • cell differentiation in disease may be inhibited or stunted, causing cells to exhibit characteristics that are different from other cells of the same type.
  • EXAMPLE 6 NORMAL CELL RESPONSE TO VARYING CONCENTRATIONS OF GM-CSF, IL-27, IFNa AND IL-6
  • Activation levels of different cell surface markers were also profiled using single cell network profiling and used in conjunction with gating to segregate the cells into discrete cell populations.
  • SSC-A and FSC-A were first used to segregate lymphocytes from non-lymphocytes.
  • CD 14 and CD4 were then used to segregate the non- lymphocytes into populations of neutrophils and CD14+ cells (monocytes).
  • CD3 and CD20 were then used to segregate the lymphocytes into populations of CD20+ (B Cells), CD3+(T Cells) and CD20-CD3- cells.
  • CD4 was used to segregate the CD3+ T cells into populations of CD3+CD4- and CD3+CD4+ T cells.
  • FIG. 3 of U.S. S.N. 12/877,998 illustrates the kinetic responses of different discrete cell populations in the normal samples.
  • the line graphs contained in FIG. 3 of U.S. S.N. 12/877,998 plot the activation levels observed in all of the donors over the time intervals at which they were measured.
  • the different concentrations of IL-6 tabulated above are represented by solid and dashed lines.
  • the normal samples demonstrated similar activation profiles over time according to the concentration of sample given.
  • Different concentrations of the modulator IL-6 yielded dramatically different activation profiles for some of the Stat phosphoproteins measured.
  • IL-6-induced pStat3 response varied at early time points (5-15 minutes) for the different concentrations of IL-6 but became more uniform at later time points. This uniformity of response supports the idea that normal cells exhibit a narrow range of activation.
  • EXAMPLE 7 STUDY EXAMINING MODULATED PROTEOMIC READOUTS IN PRE-TREATMENT PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC) FROM PATIENTS WITH METASTATIC MELANOMA WHO RECEIVED IPILIMUMAB
  • the current example identified signaling differences in PBMC samples from patients with metastatic melanoma vs. healthy donors.

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  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
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Abstract

L'invention concerne l'immunomodulation de cellules et la détection et l'utilisation associées, par exemple, dans le criblage de médicaments, notamment des méthodes, des compositions et des systèmes s'y rapportant, ou dans un aspect relatif aux soins de santé, tel que le pronostic, le diagnostic, un aspect de traitement, de surveillance, et analogue, ainsi que des méthodes, des compositions, et des systèmes à cet effet.
PCT/US2016/019423 2015-02-24 2016-02-24 Méthodes et compositions d'immunomodulation WO2016138182A1 (fr)

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US201562120217P 2015-02-24 2015-02-24
US62/120,217 2015-02-24
US201562192956P 2015-07-15 2015-07-15
US62/192,956 2015-07-15
US201562242901P 2015-10-16 2015-10-16
US62/242,901 2015-10-16
US201662295999P 2016-02-16 2016-02-16
US62/295,999 2016-02-16

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CN113201481A (zh) * 2021-04-19 2021-08-03 清华大学深圳国际研究生院 皮肤微球及其制备方法和应用
WO2023081243A1 (fr) * 2021-11-03 2023-05-11 Toreador Therapeutics, Inc. Procédés et systèmes pour l'étude à super-résolution de thérapies

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WO2019023624A1 (fr) * 2017-07-28 2019-01-31 Bristol-Myers Squibb Company Biomarqueur sanguin périphérique prédictif pour inhibiteurs de points de contrôle
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CN113201481A (zh) * 2021-04-19 2021-08-03 清华大学深圳国际研究生院 皮肤微球及其制备方法和应用
CN113201481B (zh) * 2021-04-19 2023-10-13 清华大学深圳国际研究生院 皮肤微球及其制备方法和应用
WO2023081243A1 (fr) * 2021-11-03 2023-05-11 Toreador Therapeutics, Inc. Procédés et systèmes pour l'étude à super-résolution de thérapies

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