Methods and compositions for determining tumor microenvironment composition

Abstract

The present disclosure relates to the field of immunooncology. More specifically, the present disclosure relates to methods and compositions for performing single cell phenotypic and functional analysis of immune cells and cancer cells present within a tumor microenvironment. The methods and compositions allow for determining whether a subject having a solid tumor is likely to respond to a particular immunomodulator, and also allow for treating a solid tumor in a subject by helping select an immunomodulator suitable for treating a solid tumor in a subject.

Description

[0001] This application is a divisional application of Chinese patent application 201980088416.4 (International Application No. PCT / US2019 / 060521), whose application date is November 8, 2019 and whose invention name is “Methods and compositions for determining tumor microenvironment composition”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 758,393, filed on November 9, 2018, the entire disclosure of which is incorporated herein by reference. Field of the Invention

[0004] The present disclosure relates to the field of immuno-oncology. More specifically, the present disclosure relates to methods and compositions for isolating and analyzing tumor samples to determine the phenotypic composition of the tumor microenvironment, assessing the functional status of infiltrating immune cells, quantifying the expression of immunomodulatory receptors and ligands, and predicting response to immunotherapy. background

[0005] The discovery of checkpoint inhibitors in cancer treatment provides a new therapy for treating cancer, which, before this discovery, had escaped successful treatment because these cancers had evolved mechanisms to escape the subject's immune system. However, despite significant progress in the use of checkpoint inhibitors to treat various cancers to date, currently available biomarkers and diagnostic methods have poor predictions of response, and therefore a large number of patients remain unresponsive to immunotherapy treatment. In addition, given the complexity of the tumor microenvironment (TME) of solid tumors (which contains epithelial cells, endothelial cells, mesenchymal cells, stromal cells, cancer cells, and immune cells), it may be more difficult to evaluate potential solid tumor treatment options. In some solid tumors, cancer cells affect the activity or inactivity of immune cells in TME.

[0006] Therefore, despite the progress made to date, there remains a need for methods and compositions that can determine the TME composition of solid tumors, which can then be used to determine which immunomodulatory agents, alone or in combination with other cancer drugs, may be effective in treating a given subject's tumor. SUMMARY OF THE INVENTION

[0007] The present invention is based in part on the following discovery: it is possible to determine the composition of the solid tumor microenvironment TME and to perform single-cell phenotypic and functional analysis of immune cells and cancer cells located within the TME. More specifically, the methods and compositions described herein contribute to the quantitative characterization of immunosuppressive phenotypes and targetable immunomodulatory (e.g., checkpoint) receptors and their ligands in the cell expression within the TME. The methods and compositions can be used to determine whether a subject with a solid tumor is likely to respond to a specific immunomodulator. The methods and compositions also promote the treatment of solid tumors in a given subject by helping to select an immunomodulator suitable for treating a subject's solid tumor.

[0008] Furthermore, the methods and compositions of the present invention provide drug discovery / evaluation tools for (a) early immunotherapy exploratory studies; (b) providing mechanism-based proof of principle in preclinical studies; (c) stratifying patients in clinical trials; and (d) selecting immunomodulatory agents most likely to be effective in treating cancer in a subject.

[0009] The methods described herein also facilitate phenotypic analysis of tumor infiltrating leukocytes (TILs), thereby providing accurate and highly reproducible data sets under validated conditions. Thus, the breadth and quality of the data collected according to the methods described herein provide advantages over conventional cell assessment methods (e.g., conventional immunohistochemistry).

[0010] In one aspect, the present disclosure relates to a method for determining the composition of a solid tumor microenvironment. The method includes combining a single cell suspension of cells derived from a solid tumor with a plurality of labeling reagents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs) and IMR ligand markers (IMR-Ls), and allowing the reagent to simultaneously bind to the cancer cells, immune cells, or both cancer cells and immune cells present in the single cell suspension to produce labeled cells. The method further includes determining the presence and / or content of labeled cells by cytometry, thereby (i) determining the presence and / or content of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor microenvironment and (ii) determining whether cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L, thereby determining the solid tumor microenvironment.

[0011] In another aspect, the present disclosure relates to a method for determining whether a subject with a solid tumor is likely to respond to an immunomodulator. The method includes combining a single-cell suspension of cells derived from a solid tumor with a plurality of labeling reagents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs) and IMR-ligands (IMR-Ls), and allowing the reagents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single-cell suspension to produce labeled cells. The method further includes combining at least a portion of the single-cell suspension of cells with an immunomodulator and determining (i) by cytometry the presence and / or content of labeled cells, thereby determining the presence and / or content of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and whether cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L and (ii) the effect of immunomodulators on cell markers on or in cancer cells, immune cells, or both cancer cells and immune cells, thereby determining whether the subject is likely to respond to an immunomodulator.

[0012] In another aspect, the present disclosure relates to a method for treating a solid tumor in a subject in need. The method includes administering an effective amount of an immunomodulator to the subject, thereby treating the solid tumor. The immunomodulator is selected by a method comprising combining a single cell suspension of cells derived from a solid tumor with a plurality of labeling reagents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs) and IMR-ligands (IMR-Ls), and allowing the reagent to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single cell suspension to produce labeled cells. The method further comprises combining at least a portion of the single-cell suspension of cells with an immunomodulatory agent and determining (i) the presence and / or amount of labeled cells by cytometry to determine the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L and (ii) the effect of the immunomodulatory agent on cellular markers on or in the cancer cells, immune cells, or both cancer cells and immune cells to determine whether the subject is likely to respond to the immunomodulatory agent.

[0013] In certain embodiments of the above aspects, the cytometry is flow cytometry, mass cytometry, image cytometry and / or single cell technology (SCT).

[0014] In certain embodiments of the above aspects, the labeling agent is selected from the group consisting of an oligonucleotide, a fluorophore, an infrared label, and a heavy metal label.

[0015] In certain embodiments of the above aspects, wherein the immune cells include lymphocytes (e.g., T cells (e.g., CD4 + T cells, CD8 + T cells, Tregs), B cells and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages and myeloid-derived suppressor cells), or a combination thereof.

[0016] In certain embodiments, the cell surface markers further include cell activation markers.

[0017] In certain embodiments, cell type markers include lymphocytes (e.g., T cells (e.g., CD4 + T cells, CD8 + In some embodiments, the cell type marker is a cancer cell marker including CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

[0018] In certain embodiments, cell activation markers include CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197) and / or functional markers, e.g., IFNγ, TNFα and / or other cytokines and / or granzyme B.

[0019] In certain embodiments, IMR or IMR-L markers include PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, CD39, CD172a (SIRPα), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and / or CD115 (CSF-1R).

[0020] In certain embodiments, the method further comprises the step of combining the cells with an immunomodulator. In certain embodiments, the method further comprises determining the effect of the immunomodulator on at least a portion of cell marker expression on cancer cells and / or immune cells. The immunomodulator can be combined with the single cell suspension before, during, or after the step of combining the single cell suspension of cells derived from a solid tumor with a plurality of labeling agents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells.

[0021] In certain embodiments, multiple different labeled cells are detected simultaneously during the cytometry process. In certain embodiments, multiple different cell surface markers are detected simultaneously during the cytometry process. In certain embodiments, at least 14 different cell surface markers are detected simultaneously.

[0022] In certain embodiments, receptor-ligand interactions between cells of the marker can be detected and optionally quantified. In certain embodiments, receptor-ligand interactions include interactions between checkpoint inhibitors and their cognate ligands. In certain embodiments, receptor-ligand interactions can be selected from the interaction between PD-1 and PD-L1, CTLA-4 and B7-1 and / or B7-2, TIM3- and Gal9, GITR and GITRL, OX-40 and OX40L, CD-27 and CD70, 4-1BB and 4-1BBL, and / or CD-40L and CD40.

[0023] In certain embodiments, the presence and / or amount of a cell activation marker, an IMR marker, an IMR-L marker, or a combination of an activation marker and an IMR and / or IMR-L marker expressed on cancer cells and / or immune cells is determined.

[0024] The above description describes various aspects and embodiments of the present invention. This patent application specifically contemplates all combinations and permutations of the various aspects and embodiments. These and other aspects and features of the present invention are described in the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The foregoing and other objects, features and advantages of the present invention will become apparent from the following description of the preferred embodiments illustrated in the accompanying drawings. Like reference elements identify common features in the corresponding drawings, in which:

[0026] Figure 1Depicted is a gating tree used to automatically extract metrics from a predefined gating program for granzyme B or cytokine expression in a Treg staining panel and for Treg identification. Flow cytometry data were analyzed using a combination of the Qognit package, Ryvett, and the Verity Software House package, Winlist. The gating tree represents the Boolean logic used to identify cell populations, capturing all cell populations and separating them into more discrete groups as branches.

[0027] Figure 2 Depicted is a gating tree for automatically extracting metrics from a predefined gating program for identifying T cells, B cells, NK cells, and monocytes / macrophages, as well as the expression of IMR and IMR-L on identified cell subsets. Flow cytometry data were analyzed using a combination of the Qognit package Ryvett and the Verity Software House package Winlist. The gating tree represents Boolean logic for identifying cell populations, capturing all cell populations and separating them into more discrete populations as branches of the gating tree.

[0028] Figure 3 Depicted are immunomodulatory receptor (IMR) / immunomodulatory ligand (IMR-L) pairs detectable according to the methods of the present invention.

[0029] Figure 4A Flow cytometric profiles of dissociated tumor samples depicting the surface and intracellular phenotypes of CD3+, CD4+, CD8+, and CD4+CD25 hi The presence of FoxP3+Treg cells. Figure 4B Depicted are the relevant digital plots (expressed as percent positive) showing CD3+, CD4+, CD8+ and CD4+CD25 hi The occupancy rate of FoxP3+Treg cells.CD4+CD25 hi Elevated levels of FoxP3+ Tregs indicate (a) immunosuppressive features associated with tumor dissemination and (b) poor prognosis and objective response.

[0030] (RD-PBMC = PBMC reference donor used in all assays.)

[0031] Figure 5 Depicted are flow cytometry histograms and associated digital plots of gated histograms for one or more basal or induced intracellular detectable antigens (expressed as percent positive). Intracellular cytokine expression (IFNγ and TNFα) was measured in CD4+ and CD8+ T cells isolated from the tumor microenvironment of breast, lung, and kidney tumors. Triangles represent breast tumors, squares represent lung tumors, diamonds represent kidney tumors, and circles represent data from a reference PBMC donor (RD-PBMC).

[0032] Figure 6 Flow cytometry histograms and associated digital plots of gated histograms (expressed as percent positive) are depicted, showing the expression of granzyme B CD8+ cytotoxic T cells in breast, lung, and kidney tumors compared to controls (FMO) and healthy donor PBMCs. Triangles represent breast tumors, squares represent lung tumors, diamonds represent kidney tumors, and circles represent data from a PBMC reference donor. MFI = mean fluorescence intensity.

[0033] Figure 7 Surface phenotype flow cytometric profiles of dissociated tumor samples are depicted, showing the presence of CD45+ leukocytes, CD326+ epithelial tumor cells, CD14+ monocytes / macrophages, and CD4+ and CD8+ T cells.

[0034] Figure 8 Depicted are flow cytometry histograms showing expression of the immune inhibitory checkpoint TIGIT (T cell immunoreceptor with Ig and ITIM domains) on CD4+, CD8+, and CD14+ cells, but not on CD326+ epithelial tumor cells. FMO = control.

[0035] Figure 9 Depicted are numerical representations (expressed as percentages) of several IMRs or IMR-Ls expressed on CD326+ epithelial tumor cells obtained from a panel of isolated breast, lung, and kidney tumors. Medium gray squares represent expression levels in breast tumors, light gray squares represent expression levels in lung tumors, and dark gray squares represent expression levels in kidney tumors.

[0036] Figure 10 Depicted are numerical representations (expressed as percentages) of several IMRs or IMR-Ls expressed on CD14+ myeloid cells obtained from a panel of isolated breast, lung, and kidney tumors. Medium gray squares represent expression levels in breast tumors, light gray squares represent expression levels in lung tumors, and dark gray squares represent expression levels in kidney tumors.

[0037] Figure 11 Depicted are numerical representations (expressed as percentages) of several IMRs or IMR-Ls expressed on CD8+ T cells obtained from a panel of isolated breast, lung, and kidney tumors. Medium gray squares represent expression levels in breast tumors, light gray squares represent expression levels in lung tumors, and dark gray squares represent expression levels in kidney tumors.

[0038] Details

[0039] The present invention is based in part on the following discovery: it is possible to determine the composition of the solid tumor microenvironment and perform single-cell phenotyping and functional analysis of immune cells and cancer cells located within the tumor microenvironment (TME). More specifically, the methods and compositions described herein contribute to the quantitative characterization of immunosuppressive phenotypes and targetable immunomodulatory (e.g., checkpoint) receptors and their ligands in the TME. The methods and compositions can be used to determine whether a given subject with a solid tumor is likely to respond to treatment with a specific immunomodulator, so that the subject is not exposed to a substance that is unlikely to provide a positive treatment result, but is treated with one of a plurality of substances selected to obtain a positive treatment result in the subject.

[0040] Using the methods and compositions described herein, the TME of solid tumors can be quickly and quantitatively analyzed to provide clinically relevant information based on the phenotypic and functional analysis of tumor infiltrating lymphocytes (TILs) within the TME, thereby determining the immune status within the TME. Characterizing TILs in the context of the TME may be important for targeted immunotherapy. For example, phenotypic subset analysis of TILs can be associated with clinical outcomes and used as a treatment guidance analysis for immunotherapy. For example, an increase in the density of CD8+ and CD56+ TILs indicates a positive response to immuno-oncology therapy.

[0041] Similarly, the methods and compositions described herein facilitate the assessment of various subsets of tumor-infiltrating NK, T cells (Treg, CD8+ cytotoxic T cells, exhausted T cells), myeloid-derived suppressor cells (MDSC), and dendritic cells (DC) and macrophages (Mf) in the TME. These methods further facilitate the assessment of the ratio of immune regulatory receptor (IMR) / immune regulatory receptor ligand (IMR-L)-positive immune cells and tumor cells. The method also allows for the analysis of activation / exhaustion markers such as cytokine and granzyme expression.

[0042] In addition, the methods and compositions described herein can be used to determine the functional state of TIL, which can contribute to predicting the clinical response of a given subject with a tumor. For example, using the methods described herein, it can be determined whether immune cells are activated or exhausted for a given solid tumor sample, and whether immune cells are suppressed by Treg, M2 macrophages and MDSC in TME. In addition, the methods and compositions also contribute to the quantification of TIL and tumor cell (total) expression profiles that can target IMR and its cognate ligands, which provides information on whether a specific immunomodulator may have a positive effect on TME and promote the use of an immunomodulator (alone or in combination with another cancer drug) for a positive treatment outcome for a healthcare provider. For example, the selection of a specific immunomodulator may reduce or eliminate the resistance that a specific cancer cell may have to a specific cancer drug treatment.

[0043] Generally, the methods described herein include combining a single cell suspension of cells derived from a solid tumor with a plurality of labeling reagents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, and allowing the reagents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single cell suspension to produce labeled cells. The method includes determining the presence and / or content of labeled cells by cytometry, thereby determining the presence and / or content of cancer cells, immune cells, or both cancer cells and immune cells in the solid tumor microenvironment, while also providing phenotypic information about whether the cells express specific phenotypic markers, such as IMR, IMR-L, and cell activation markers.

[0044] In certain embodiments, the method further comprises the step of combining the cells with an immunomodulatory agent (alone or in combination with a cancer drug) and then determining whether the immunomodulatory agent affects the expression of cell markers on cancer cells and / or immune cells.

[0045] Methods and compositions described herein can be used to determine whether solid tumor patients may respond to immunomodulators.For example, the single cell suspension of cells derived from solid tumors is combined with a variety of labeling reagents that can be combined with the corresponding multiple cell surface markers expressed on cancer cells and / or immune cells, and the reagent is allowed to be combined with the cancer cells, immune cells or cancer cells and immune cells present in the single cell suspension to produce labeled cells. In addition, at least a portion of the single cell suspension of cells is contacted with immunomodulators (alone or in combination with cancer drugs), which can then be analyzed by cytometry to determine the presence and / or content of cancer cells, immune cells or cancer cells and immune cells present in solid tumors. In addition, the effect of immunomodulators on or among cell markers of cancer cells, immune cells or cancer cells and immune cells can be determined. This information can be used to determine whether a subject may actively respond to immunomodulators.

[0046] The methods and compositions described herein can be used in the treatment of solid tumors in subjects in need thereof, whereby an effective amount of an immunomodulator selected by the methods described herein is administered to the subject, thereby treating the solid tumor. An immunomodulator is selected by using a method comprising the following steps: (a) combining a single cell suspension of cells derived from a solid tumor with a plurality of labeling agents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, and allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single cell suspension to produce labeled cells; (b) combining at least a portion of the single cell suspension of cells with an immunomodulator; and (c) determining (i) the presence and / or amount of labeled cells by cytometry, thereby determining the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and (ii) the effect of the immunomodulator on cellular markers on or in the cancer cells, immune cells, or both cancer cells and immune cells, thereby determining whether the subject is likely to respond to the immunomodulator.

[0047] The following sections describe in more detail how to use the methods and compositions described herein to analyze the TME of solid tumors and how to potentially determine whether a subject can respond favorably to a given immunomodulatory agent.

[0048] I. Cell Type

[0049] It is understood that the TME includes tumor cells and immune cells as described herein.

[0050] (a) Tumor cells

[0051] The term "tumor cell" is used herein interchangeably with "cancer cell." Tumor cell types that can be probed using the methods and compositions described herein include epithelial-derived cells, endothelial-derived cells, and mesenchymal-derived cells.

[0052] Tumors that can be detected using the methods described herein include, but are not limited to, anal cancer, bladder cancer, intestinal cancer (large and small intestine), brain cancer, breast cancer, oral cancer, cervical cancer, esophageal cancer, fallopian tube cancer, head and neck cancer, colon cancer, colorectal cancer, lung cancer, ovarian cancer, pancreatic cancer, peritoneal cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, urinary tract cancer, uterine cancer, and vulvar cancer.

[0053] Exemplary tumors include, for example, ovarian cancer (serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid carcinoma), ovarian granulosa cell tumor, fallopian tube adenocarcinoma, peritoneal cancer, uterine (endometrial) adenocarcinoma, sarcomatoid carcinoma, cervical cell carcinoma, cervical adenocarcinoma, vulvar cancer, breast cancer, primary and metastatic (ductal carcinoma, mucinous carcinoma, lobular carcinoma, malignant phyllodes tumor), head and neck cancer, oral cancer (including tongue), primary and metastatic esophageal cancer, adenocarcinoma, gastric adenocarcinoma, primary small intestine cancer, colon adenocarcinoma, primary and metastatic (adenocarcinoma, mucinous carcinoma, large cell neuroendocrine carcinoma, colloid carcinoma), appendiceal adenocarcinoma, colorectal cancer, rectal cancer, anal cancer (squamous, basaloid), carcinoid tumors, primary and metastatic (appendix, small intestine, colon), pancreatic cancer, liver cancer (hepatocellular carcinoma, bile duct cancer), cancer that metastasizes to the liver, Lung cancer, primary and metastatic (squamous cell carcinoma, adenocarcinoma, adenosquamous carcinoma, giant cell carcinoma, non-small cell carcinoma, non-small cell lung cancer (NSCLC), small cell carcinoma, neuroendocrine carcinoma, large cell carcinoma, bronchoalveolar carcinoma), renal cell (kidney) carcinoma, primary and metastatic bladder cancer, primary and metastatic prostate adenocarcinoma, primary and metastatic brain tumors, primary and metastatic (glioblastoma, multiforme, neuroectodermal tumor, neuroectodermal tumor, oligodendroglioma, malignant astrocytoma), skin tumors (malignant melanoma, sebaceous cell carcinoma), thyroid cancer (papillary and follicular), thymic carcinoma, shenoidal carcinoma, cancer of unknown primary, neuroendocrine carcinoma, testicular malignancies (seminoma, embryonal carcinoma, malignant mixed tumor), etc.

[0054] (b) Immune cells

[0055] Immune cells that can be detected and analyzed according to the present invention include lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells (derived granulocytes and monocytes)).

[0056] II. Sample Processing and Cell Counting

[0057] A solid tumor sample can be obtained from a subject and processed into a single-cell suspension of cells by any means known in the art. As used herein, a "single-cell suspension" is a suspension of one or more cells in a liquid sample, wherein the cells are primarily in the form of single cells rather than in the form of cell clusters or aggregates. In certain embodiments, single cells represent 60%, 70%, 80%, 90%, or 95% of the cells in the cell suspension.

[0058] In certain embodiments, by artificial and / or enzymatic digestion of solid tumors.For example, solid tumor samples can be cut into smaller pieces and use trypsin, collagenase, DNAse, dispase and / or hyaluronidase to carry out enzymatic digestion.In certain embodiments, the tissue of digestion is filtered, to remove larger, undigested pieces.

[0059] In addition or in lieu of the above, an automated tissue homogenizer such as the gentleMACS Tissue dissociation was performed using an OctoDissociator (Miltenyi Biotec GmbH, Bergish Gladbach, Germany). Cells can be filtered to remove undigested tissue, for example, using a 70 μM filter.

[0060] The resulting single-cell suspension can contain all cell types present in the tumor microenvironment, including epithelial cells, endothelial cells, mesenchymal cells, stromal cells, tumor / cancer cells, and immune cells.

[0061] After dissociation, the cells can be washed, precipitated, resuspended and / or counted. Cells can be counted using any method known in the art, such as using a manual cell counter or an automated cell counter. For example, for solid tumors, nucleated cell counts can be obtained using an automated cell counter that uses, for example, bright field imaging and / or fluorescence imaging (e.g., dual fluorescence imaging). Exemplary automated cell counters include the Nexcelom Cellometer 2000 (Nexcelom, Lawrence, MA), the Countess II FL Automated Cell Counter (ThermoFisher, Waltham, MA), and the TC20 Automated cell counter (BioRad, Hercules, CA). For whole blood, bone marrow, PBMCs, or BMMCs, an automated cell counter such as a Coulter counter, for example, the Beckman Coulter Act2 Diff (Beckman Coulter, Inc., Brea, CA) can be used.

[0062] After determining the approximate number of cells in the sample, the cells can be pelleted based on the cell count and resuspended in a buffer at the desired concentration. Suitable buffers include RPMI1640 + 10% FBS + 1% penicillin-streptomycin and / or RPMI1640 + 10% FBS or 1X PBS + 0.5% BSA. Cells can be grown at a concentration of about 0.5 to about 5 × 10 6 cells / mL, for example, about 0.5 to about 1×10 6 cells / mL, about 0.5 to about 2×10 6cells / mL, about 0.5 to about 3×10 6 cells / mL, about 0.5 to about 4×10 6 cells / mL, about 1 to about 2×10 6 cells / mL, about 1 to about 3×10 6 cells / mL, about 1 to about 4×10 6 cells / mL, about 1 to about 5×10 6 cells / mL, about 2 to about 3×10 6 cells / mL, about 2 to about 4×10 6 cells / mL, about 2 to about 5×10 6 cells / mL, about 3 to about 4×10 6 cells / mL, about 3 to about 5×10 6 cells / mL, about 4 to about 5×10 6 In certain embodiments, the cells are resuspended at a concentration of about 1.2 to about 2.4×10 6 cells / mL, about 1.2 to about 2×10 6 cells / mL, about 1.2 to about 1.5×10 6 cells / mL, about 1.5 to about 2.4×10 6 cells / mL, about 1.5 to about 2×10 6 Resuspend at a concentration of 10 cells / mL.

[0063] III. Cell Analysis

[0064] To analyze cells initially treated with the TME, the cells, once converted to a single cell suspension, are combined with multiple (e.g., 5, 6, 7, 8, 9, 10, 11, 20, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more) labeling reagents to select cell surface markers to determine whether the cell is a cancer cell or an immune cell or subtype, and / or whether such cell or subtype expresses, for example, IMR, IMR-L, or a cell activation marker.

[0065] Specifically, vital dyes (e.g., amine reactive dyes Alexa750) can be used to coat and stain cells to distinguish between live and dead cells. Cells can be washed in staining buffer (e.g., 1X PBS+0.5% BSA). For example, a Biotek ELx450 deep well plate washer can be used to automate the cleaning step. Standard methods in the art can be used to fix and infiltrate cells, for example, using the FOXP3 / Transcription Staining Buffer Kit from eBioscience (ThermoFisher, Waltham, MA). Cells can then be washed in staining buffer (e.g., 1X PBS+0.5% BSA) and stained with one or more labeling reagents to detect one or more cell markers. The method for staining cells depends on the labeling reagent used.

[0066] (a) Cell surface markers

[0067] The methods disclosed herein can be used to detect any cell surface marker, such as a cancer cell marker, an activation marker, or an IMR or IMR-L marker.

[0068] Cell type markers are proteins or other molecules present in or on a specific cell type (e.g., cancer cells or immune cells). In certain embodiments, the presence of a specific cancer cell marker can indicate the type of cancer. In certain embodiments, the presence of a specific cancer cell marker provides a target for cancer treatment. Exemplary cancer cell markers include CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin and / or vimentin. In certain embodiments, the presence of a specific immune cell marker identifies immune cells as specific immune cell types or subtypes. Exemplary immune cell markers are provided in Table 1 below.

[0069] Table 1

[0070]

[0071]

[0072] Activation markers can be proteins or other molecules. The presence of activation markers in or on cancer cells or immune cells indicates that certain pathways (e.g., immune pathways) are active. Exemplary activation markers include CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197) and / or functional markers (e.g., IFNγ, TNFα and / or other cytokines and / or granzyme B.

[0073] Exemplary IMR or IMR-L markers include PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, CD39, CD172a (SIRPα), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA) and / or CD115 (CSF-1R).

[0074] In certain embodiments, other signaling markers can be detected according to the methods herein. In certain embodiments, the methods include detecting receptors or transporters (e.g., CD3, CD4, CD5, CD8, CD11b, CD11c, CD14, CD15, CD16, CD19, CD20, CD25, CD27, CD28, CD31, CD34, CD38, CD45, CD45RA, CD45RO, CD56, CD69, CD71, CD80, CD86, CD90, CD117, CD123, CD133, CD135, CD235, cytokeratin, EPCAM, FOXP3, HLA-DR, IgD, IgG, IgM, MDR1, ABCG2), DNA damage or apoptosis signaling molecules (e.g., Bcl-2, Bcl-xL, cytochrome C, Caspase 3 or 8, cPARP, Annexin V, DNMT1, 3a, 3b, p-H2AX, p-53BP1, p-ATM, p-DNA-PKcs, p-p53, P53, p21, p-Chk2, p-RPA2, p-BRCA1), immune signaling molecules (e.g., p-Akt, p-Blnk, p-Erk, p-Gsk3b, p-Lyn, p-NFkB, p-Plcg2, p-S6, p-Stat5, p-Syk, p-SLP-76, p-ZAP-70, p-Lck, p-CD3z, p-Vav, p-Lat, p-Pyk2), differentiation, maturation and / or cytokine / chemokine response signaling molecules (e.g., p-Stat1, p-Stat3, p-Stat4, p-Stat5, p-Stat6, p-Erk, p-p38, p-NFkB, IkB, pRelB), intracellular cytokines (e.g., IL-2, IL-4, IL-6, IL-8, IL-10, IL17A, IFNa, IFNg, TNFa), measures of cytotoxic effector function (e.g., CD107a, granzymes, perforin, annexin V), PKC, CA ++ Signaling molecules (e.g., p-Akt, p-Erk, p-PLCg2, p-PKCa, p-S6, p-p38), survival, proliferation, cell cycle, and pattern recognition receptor signaling molecules (e.g., p-Akt, p-NFkB, p-S6, IkB, p-Erk, p-p38, cyclin A2, cyclin B1, p-CDK1, p-HH3, p-MK2, p21, p-CREB, pc-JUN)

[0075] In certain embodiments, a panel of cell markers is measured to identify cell type, IMR and / or IMR-L and / or exhaustion markers. An exemplary panel of cell markers is shown in Table 2.

[0076] Table 2

[0077]

[0078]

[0079]

[0080] For example, in certain embodiments, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more different cell surface markers are detected simultaneously. In certain embodiments, 5 to 19, 6 to 18, 7 to 17, 8 to 16, 9 to 15, 10 to 14, 10 to 13, 11 to 12, 5 to 14, 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 11 to 14, 12 to 14, 13 to 14, 5 to 16, 6 to 16, 7 to 16, 8 to 16, 9 to 16, 10 to 16, 11 to 16, 12 to 16, 13 to 16, 14 to 16, or 15 to 16 different cell surface markers are detected simultaneously. In certain embodiments of the methods described herein, at least 14 different cell surface markers are detected simultaneously.

[0081] In certain embodiments, receptor-ligand interactions between labeled D cells can be detected, such as interactions between a checkpoint inhibitor and its cognate ligand. Exemplary receptor-ligand pairs are depicted in Figure 3 For example, the receptor-ligand interaction can be selected from the interaction between PD-1 and PD-L1, CTLA-4 and B7-1 and / or B7-2, TIM-3 and Gal9, TIGIT / CD112-CD155, GITR and GITRL, OX-40 and OX40L, CD-27 and CD70, 4-1BB and 4-1BBL, LAG3 / MHC, KIR / MHC and / or CD-40L and CD40.

[0082] (b) Labeling reagents for use in combination with single-cell suspensions

[0083] It is contemplated that the single cell suspension, once prepared, can be distributed among a plurality of receiving vessels, such as wells in a multi-well plate, such as a 96 or 364 well plate, and prepared for cytometric analysis.

[0084] Cell plating and subsequent processing steps can be automated, for example, using a Hamilton robotic liquid handler (Hamilton Company, Reno, NV). Cells can be fixed and permeabilized prior to exposure to the labeling agent using any method known in the art. In certain embodiments, if the labeling agent is sensitive to fixation, the cells are exposed to the labeling agent without fixation and permeabilization or before fixation and permeabilization. In certain embodiments, the labeling agent comprises a binding agent (e.g., a member of a binding complex, such as an antibody, protein, aptamer, avimer, Adnectin, or In some embodiments, the binding agent is a ligand, a member of a ligand receptor pair, a small molecule inhibitor), wherein the binding agent binds to a cell surface marker (e.g., a cancer cell marker, an activation marker, or an immunomodulatory receptor (IMR) or an IMR-ligand (IMR-L) marker). In certain embodiments, the cell is exposed to a binding agent (e.g., an antibody) that binds to the cell surface marker to form a cell surface marker / binding agent complex, and the cell surface marker / binding agent complex is exposed to a labeling agent that binds to the cell surface marker / binding agent complex. In certain embodiments, an oligonucleotide conjugate (i.e., an oligonucleotide label) is used, wherein the binding agent (e.g., an antibody) is conjugated to a first oligonucleotide, and a second oligonucleotide that is complementary to the first oligonucleotide (i.e., capable of binding (hybridization)) is directly or indirectly conjugated to one or more labels (e.g., one or more fluorophores). Annealing of the first and second oligonucleotides connects the binding agent to the one or more labels. The annealed oligonucleotide conjugate comprising the binding agent-label conjugate can then be used in cell counting applications (e.g., flow cytometry).

[0085] (c) Binder

[0086] Suitable binding agents for use in accordance with the methods herein include any substance that can preferentially bind to a cell surface marker described herein (e.g., a cancer cell marker, an activation marker, or an immunomodulatory receptor (IMR) or IMR-ligand (IMR-L) marker). For example, binding agents may include antibodies (e.g., monoclonal antibodies), proteins, peptide aptamers, avimers, Adnectins, and Ligands, members of ligand-receptor pairs, and small molecule inhibitors.

[0087] Exemplary binding agents may include CD44 binding agents, CD47 binding agents, CD49f binding agents, CD271 binding agents, CD326 binding agents, cytokeratin binding agents, E-cadherin binding agents, vimentin binding agents, CD25 binding agents, CD26 binding agents, CD27 binding agents, CD28 binding agents, CD38 binding agents, CD40 binding agents, CD44 binding agents, CD62L binding agents, CD69 binding agents, CD80 binding agents, CD86 binding agents, CD95 binding agents, CD95L binding agents, CD127 binding agents, CCR7 (CD197) binding agents, IFNγ binding agents, TNFα binding agents, granzyme B binding agents, PD-1 (CD279) binding agents, PD-L1 (CD274) binding agents, CTLA-4 (CD152) binding agents, LAG3 (CD223) binding agents, OX40 ( binders, CD134) binders, TIM3 (CD366) binders, GITR (CD357) binders, 4-1BB (CD137) binders, KIR (CD158B) binders, 2B4 (CD244) binders, ICOS (CD278) binders, IDO binders, TIGIT binders, CD73 binders, CD39 binders, CD172a (SIRPα) binders, B7H4 (B7S1) binders, VISTA (B7-H5) binders, CD355 (CRTAM) binders, KLRG1 binders, CD160 (BY55, NK1, NK28) binders, CD30 (TNFRSF8) binders, CD224 (GGT1) binders, CD226 binders, CD272 (BTLA) binders, and / or CD115 (CSF-1R) binders.

[0088] Suitable antibodies for use in accordance with the methods herein include anti-CD44 antibodies, anti-CD47 antibodies, anti-CD49f antibodies, anti-CD271 antibodies, anti-CD326 antibodies, anti-cytokeratin antibodies, anti-E-cadherin antibodies, anti-vimentin antibodies, anti-CD25 antibodies, anti-CD26 antibodies, anti-CD27 antibodies, anti-CD28 antibodies, anti-CD38 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD62L antibodies, anti-CD69 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-CD95 antibodies, anti-CD95L antibodies, anti-CD127 antibodies, anti-CCR7 (CD197) antibodies, anti-IFNγ antibodies, anti-TNFα antibodies, anti-granzyme B antibodies, anti-PD-1 (CD279) antibodies, anti-PD-L1 (CD274) antibodies, anti-CTLA-4 (CD152) antibodies, anti-LAG3 (CD223) antibodies, anti-OX 40 (CD134) antibody, anti-TIM3 (CD366) antibody, anti-GITR (CD357) antibody, anti-4-1BB (CD137) antibody, anti-KIR (CD158B) antibody, anti-2B4 (CD244) antibody, anti-ICOS (CD278) antibody, anti-IDO antibody, anti-TIGIT antibody, anti-CD73 antibody, anti-CD39 antibody, anti-CD172a (SIRPa) antibody, anti-B7H4 (B7S1) antibody, anti-VISTA (B7-H5) antibody, anti-CD355 (CRTAM) antibody, anti-KLRG1 antibody, anti-CD160 (BY55, NK1, NK28) antibody, anti-CD30 (TNFRSF8) antibody, anti-CD224 (GGT1) antibody, anti-CD226 antibody, anti-CD272 (BTLA) antibody, and / or anti-CD115 (CSF-1R) antibody.

[0089] (d) Labeling reagent

[0090] (i) Fluorophores (including visible light fluorophore labels and infrared fluorophore labels)

[0091] Fluorophores suitable for use according to the methods herein include, but are not limited to, Cy5.5, Cy5, and Cy7 (GE Healthcare); Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 750, and Alexa Fluor 790 (Invitrogen); VivoTag 680, VivoTag-S 680, and VivoTag-S 750 (VisEn Medical); Dy 677, Dy 682, Dy 752, and Dy 780 (Dyomics); Dy Light 547, Dy Light 647 (Pierce); HiLyte Fluor 647, HiLyte Fluor 680, and HiLyte Fluor 750 (AnaSpec); IRDye 800CW, IRDye 800RS, and IRDye 700DX (Li-Cor); and ADS780WS, ADS830WS and ADS832WS (American Dye Source) and Kodak X-SIGHT650, Kodak X-SIGHT691, Kodak X-SIGHT751 (Carestream Health), PE, PE-Cy7, PerCP, PerCP-Cy5.5, FITC, BV421, BV510, BV605.

[0092] (ii) Heavy Metal Labeling

[0093] Heavy metal labels, such as lanthanides, are used in certain embodiments of the methods described herein, such as mass cytometry. Lanthanides include, but are not limited to, lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), and lutetium (Lu). In certain embodiments, lanthanide isotopes are used, including, for example, 139 La, 141 Pr, 142 Nd, 144 Nd, 145 Nd, 146 Nd, 147 Nd, 147 Sm, 152 Sm, 151 Eu, 153 Eu, 156 Gd, 159 Tb, 164 Dy,165 Ho, 166 Second, 169 Tm, 171 Yb, 174 Yb and 176 Yb.

[0094] (e) Cytometry

[0095] (i) Flow cytometry

[0096] In certain embodiments, the cell of flow cytometry analysis labeling is used.Once using staining procedure as described herein to obtain the cell of labeling, by flow cytometry analysis labeling cell.The exemplary flow cytometer that can be used for this paper method includes for example Attune NxT flow cytometer (ThermoFisher, Waltham, MA), CytoFLEX flow cytometer (Beckman Coulter, Indianapolis, IN), FACSVerse or FACSCanto II flow cytometer (Becton Dickinson, San Jose, CA) or Aurora flow cytometer (Cytek, Oakland, CA) that can measure multiple (for example, 16 kinds) fluorescent dye simultaneously.The standard setting of such as flow cytometer can be used to analyze the data capture from flow cytometer.

[0097] In certain embodiments, prior to acquiring flow cytometry data, information such as sample ID, labeling reagents, and flow cytometer instrument settings and acquisition parameters is incorporated into a plate layout file using Ryvett software from Qognit, Inc. (Qognit, San Carlos, CA). This plate layout file can be imported into the flow cytometry software prior to acquisition. After flow cytometry data is generated, the flow cytometry standard (FCS) file can be analyzed and gated in the Ryvett software using the desired gating program. After manual review of the automated gates, the raw data and calculated metrics can be exported into a CSV file using predefined criteria and data extraction programs.

[0098] In certain embodiments, flow cytometry data can be analyzed using gating in WinList (Software House, Topsham, ME), Ryvett (Qognit, Redwood City, CA), FlowJo (FloJo LLC, Ashland, OR), or Kaluza (Beckman Coulter, Indianapolis, IN).

[0099] Using this software, cell populations in the TME can be interrogated to determine what cancer cells and immune cells are present in the TME, their status (e.g., non-activated, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls.

[0100] As understood in the art, gating trees can be used to analyze and separate cell populations from a flow cytometry experiment into more discrete populations as branches of the gating tree. Figure 1 and Figure 2 Depicted is a gating tree used to automatically extract metrics from a pre-defined gating procedure for identifying Tregs and granzyme B or cytokine expression in a Treg staining panel. Each gate (in Figure 1 and Figure 2 The gates (defined as G4, G9, G10, G17, etc.) consist of multiple regions that are combined to form a gating scheme using Boolean logic (e.g., "and," "or," and "not" statements) to include, exclude, or combine cell populations represented in the gate.

[0101] In addition to determining the presence of a given cell surface marker (e.g., a cell type marker, an immunomodulatory receptor (IMR) or an IMR-ligand (IMR-L) on a cell, the methods described herein can include quantitatively measuring the amount of such a marker, e.g., the amount of the marker can be directly measured as an equivalent amount of a reference fluorophore (EFR). See, e.g., Gaigalas et al. (2016) JOURNAL OF RESEARCH OF THE NATIONAL INSTITUTE OF STANDARDS AND TECHNOLOGY 121:264-281. In certain embodiments, a subject is identified as likely to respond to an immunomodulatory agent if the amount of the measured marker (e.g., an IMR or IMR-L, an activation and / or exhaustion marker) exceeds a particular threshold.

[0102] (ii) Mass cytometry

[0103] In certain embodiments, the labeled cells are analyzed using mass cytometry. Mass cytometry combines flow cytometry and mass spectrometry. Compared to flow cytometry, which distinguishes signals by measuring the fluorescence spectra of different reporter molecules, mass cytometry uses probes (e.g., antibodies) coupled to stable heavy metal isotopes, using a mass cytometer, such as by time-of-flight flow cytometry. ) system (Fluidigm, San Francisco, CA). (Bandura et al. (2009) ANALCHEM. 81:6813-6822; Bjornson et al. (2013) CURRENT OPINION IN IMMUNOLOGY 25:484-494.) Once the labeled cells are obtained using methods known in the art, the labeled cells are analyzed by mass cytometry. Exemplary mass cytometers that can be used in the methods herein include, for example, (Fluidigm, San Francisco, CA). Mass cytometer software, such as Data captured from the mass cytometer were analyzed using Fluidigm® software v.7.0 (Fluidigm, San Francisco, CA).

[0104] In certain embodiments, mass cytometry data can be analyzed using conditional density resampled estimation of mutual information (DREMI), which can optionally be used in conjunction with conditional density rescaled visualization (DREVI) (Krishnaswamy et al. (2014) SCIENCE 346(6213):1250689).

[0105] Thus, like flow cytometry, mass cytometry can be used to interrogate cell populations within the TME to determine which cancer cells and immune cells are present in the TME, their status (e.g., non-activated, activated, or exhausted), and whether the cells express one or more IMRs or IMR-L. In addition, mass cytometry can be used to perform single-cell genomic sequencing to reveal, for example, mutations (e.g., somatic mutations) in single cells (e.g., immune cells or cancer cells).

[0106] (iii) Image cytometry

[0107] In certain embodiments, image cytometry is used to analyze labeled cells. Image cytometry can be used to measure many of the same parameters as flow cytometry, but also includes three-dimensional imaging using automated microscopy and computational image processing and analysis, which allows the collection and identification of tens of thousands of cell events based on fluorescence and / or morphological parameters. Therefore, compared to flow cytometry, image cytometry can also assess cell events through real images. Barteneva et al. (2012) J HISTOCHEM CYTOCHEM 60 (10): 723-733 provides an overview of imaging cytometry.

[0108] Exemplary imaging cytometers include Imaging Flow Cytometer and Mk II Imaging Flow Cytometer (Luminex Corp., Austin, TX).

[0109] In certain embodiments, image cytometry is used to explore cell populations in the TME to determine which cancer cells and immune cells are present in the TME, their status (e.g., non-activated, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls. In addition, in certain embodiments, image cytometry is used to assess the morphological characteristics of cells (e.g., cancer cells and / or immune cells), the co-localization of two proteins, the binding of two cells (e.g., cancer cells and / or immune cells), the visualization of immune synapse formation, and nuclear translocation. In addition, image cytometry can be used to perform single-cell genome sequencing to reveal, for example, mutations (e.g., somatic mutations) in single cells (e.g., immune cells or cancer cells). Image cytometry can also be used in combination with laser capture microdissection (LCM) for laser ablation mass spectrometry as well as proteomics and genomic or mRNA analysis.

[0110] (iv) Single cell technology (SCT)

[0111] Single cell technology (SCT) can also be used to assess the individual cells in TME and their interactions with other cells under different conditions (e.g., in the presence of immunomodulatory and / or cancer drugs) according to the methods disclosed herein. Single cells from TME can be separated and manipulated using a variety of different methods, including fluid-based, physical-based, electric field-driven (e.g., dielectrophoresis (DEP), photoelectric tweezers (OET) and optical techniques such as optical tweezers. (See, e.g., Skelley et al. (2009) NAT. METHODS 6: 147-152; Thieleche et al. (1999) IEEE ENG. MED. BIOL. MAG. 18: 48-52; Taff et al. (2005) ANAL CHEM. 77: 7976-7983; Juan et al. (2011) NAT. PHOTONICS 5: 349-356; and Mirsaidov et al. (2008) LAB CHIP 8: 2174-2181.

[0112] In some embodiments, SCT is performed using a microfluidic chip. For example, SCT can be performed using a fluid dynamics-based microfluidic chip. In other embodiments, dielectrophoretic digital sorting methods use semiconductor control electrode arrays in a microfluidic chip to capture single cells in dielectrophoretic (DEP) cages.

[0113] In certain embodiments, SCT uses a microfluidic slide (e.g., NanoString Technologies, Inc., Seattle, WA). Genesis System in Ann Arbor, MI).

[0114] Single cells can be analyzed using a variety of techniques, including assessment of growth rate (Cermak et al. (2016) NAT. BIOTECH 34: 1052-1059), measurement of cell membrane potential (Liu et al. (2017) NANO LETT. 17: 2757-2764), assessment of the cell genome and / or transcriptome (Horgan (2011) OBSTET. GYNAECOL. 13: 189-195), proteomics (Horgan (2011) supra), and mass spectrometry (Li et al. (2000) TRENDS BIOTECHNOL. 18: 151-160). In certain embodiments, cells are assessed using one or more of the previous SCT techniques to determine their cell type, their state (e.g., non-activated, activated, or exhausted), and whether the cells express one or more IMRs or IMR-Ls.

[0115] V. Immunomodulators

[0116] The above system can also be used to determine whether cells isolated from the TME respond to the addition of immunomodulatory agents alone or in combination with anti-cancer drugs.

[0117] Immunomodulators suitable for use herein include any substance capable of modulating immune cells, for example, by activating the immune system to kill tumor cells or by removing immune suppressor cells from tumor cells. In certain embodiments, cells are exposed to an immunomodulator prior to combining with a labeling agent.

[0118] In certain embodiments, the immunomodulator is a checkpoint inhibitor. The checkpoint inhibitor can be, for example, selected from a PD-1 antagonist, a PD-L1 antagonist, a CTLA-4 antagonist, an adenosine A2A receptor antagonist, a B7-H3 antagonist, a B7-H4 antagonist, a BTLA antagonist, a KIR antagonist, a LAG3 antagonist, a TIM-3 antagonist, a VISTA antagonist, or a TIGIT antagonist.

[0119] In certain embodiments, the checkpoint inhibitor is a PD-1 or PD-L1 inhibitor. PD-1 is a receptor present on the surface of T cells that acts as an immune system checkpoint, inhibiting or regulating T cell activity at the appropriate time to prevent an overactive immune response. However, cancer cells can exploit this checkpoint by expressing a ligand (such as PD-L1), which interacts with PD-1 on the surface of T cells to shut down or regulate T cell activity. Exemplary PD-1 / PD-L1-based immune checkpoint inhibitors include antibody-based therapies. Exemplary treatments using PD-1 / PD-L1-based immune checkpoint inhibition are described in U.S. Patent Nos. 8,728,474 and 9,073,994 and EP Patent No. 1537878B1, and include, for example, the use of anti-PD-1 antibodies. Exemplary anti-PD-1 antibodies are described, for example, in U.S. Patent Nos. 8,952,136, 8,779,105, 8,008,449, 8,741,295, 9,205,148, 9,181,342, 9,102,728, 9,102,727, 8,952,136, 8,927,697, 8,900,587, 8,735,553, and 7,488,802. Exemplary anti-PD-1 antibodies include, for example, nivolumab ( Bristol-Myers Squibb Co.), pembrolizumab ( Merck Sharp & Dohme Corp.), PDR001 (Novartis Pharmaceuticals), and pilivizumab (CT-011, Cure Tech). Exemplary anti-PD-L1 antibodies are described, for example, in U.S. Patent Nos. 9,273,135, 7,943,743, 9,175,082, 8,741,295, 8,552,154, and 8,217,149. Exemplary anti-PD-L1 antibodies include, for example, atuzumab ( Genentech), durvalumab (AstraZeneca), MEDI4736, avelumab, and BMS 936559 (Bristol Myers Squibb Co.).

[0120] In certain embodiments, the immunomodulator is a CTLA-4 inhibitor. In the CTLA-4 pathway, CTLA-4 on T cells interacts with its ligands (e.g., CD80, also known as B7-1, and CD86) on the surface of antigen-presenting cells (rather than cancer cells), leading to T-cell inhibition. Exemplary CTLA-4-based immune checkpoint inhibitor methods are described in U.S. Patent Nos. 5,811,097, 5,855,887, and 6,051,227. Exemplary CTLA-4 antibodies are described in U.S. Patent Nos. 6,984,720, 6,682,736, 7,311,910, 7,307,064, 7,109,003, 7,132,281, 6,207,156, 7,807,797, 7,824,679, 8,143,379, 8,263,073, 8,318,916, 8,017,114, 8,784,815, and 8,883,984, International (PCT) Publication Nos. WO98 / 42752, WO00 / 37504, and WO01 / 14424, and European Patent No. EP 1212422 B1. Exemplary CTLA-4 antibodies include ipilimumab or tremelimumab.

[0121] In certain embodiments, an immunomodulator is administered in combination with an IDO inhibitor. Exemplary IDO inhibitors include 1-methyl-D-tryptophan (known as indomod), epacadostat (INCB24360), navoximod (GDC-0919), and BMS-986205.

[0122] Other immunomodulators include, for example, anti-CD20 antibodies, such as (ofatumumab, GlaxoSmithKine), (rituximab, Genentech, Biogen) and (rituximab, Roche); and anti-CD52 antibodies, such as (Alemtuzumab, Genzyme).

[0123] Other antibody-based immunomodulators include those listed in Table 3. For certain antibodies in Table 3, the cancer type targeted by the antibody or antibody-drug conjugate is also indicated.

[0124] Table 3

[0125]

[0126]

[0127]

[0128]

[0129]

[0130] Once one or more immunomodulators (alone or in combination with an anticancer drug (such as the compounds discussed below)) are identified that provide a positive outcome on cancer cells and / or immune cells in the TME, the immunomodulators (alone or in combination with the anticancer drug) can be administered to a subject.

[0131] IX. Administration of Pharmaceutical Compositions and Immunomodulators

[0132] For therapeutic use, the immunomodulator is preferably combined with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0133] As used herein, the term "pharmaceutically acceptable carrier" refers to buffers, carriers, and excipients suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any standard pharmaceutical carrier, such as phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th edition, Mack Publ. Co., Easton, PA

[1975] . Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic agents, and absorption delaying agents compatible with drug administration. The use of such media and substances for pharmaceutically active substances is known in the art.

[0134] In certain embodiments, pharmaceutical compositions may contain formulation materials used to alter, maintain, or preserve, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamic acid, aspartic acid, arginine, and lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming reactions ions (e.g., sodium); preservatives (e.g., benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide); solvents (e.g., glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (e.g., mannitol or sorbitol); suspending agents; surfactants or wetting agents (e.g., pluronics, PEG, sorbitan esters, polysorbates, such as polysorbate 20, polysorbate, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancers (e.g., sucrose or sorbitol); tonicity enhancers (e.g., alkali metal halides, preferably sodium chloride or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see, Remington's Pharmaceutical Sciences, 18th ed., (Mack Publishing Company, 1990).

[0135] In certain embodiments, the pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see, Anselmo et al. (2016) BIOENG.TRANSL.MED. 1:10-29).

[0136] In certain embodiments, pharmaceutical composition can contain sustained delivery or controlled delivery preparation.It is also well known to those skilled in the art for preparing the technology of sustained delivery or controlled delivery mode (such as liposome carrier, bioerodible microparticle or porous beads and reservoir injection).Sustained release preparation can comprise for example porous polymer microparticle or semi-permeable polymer matrix, and it is a formed product form, for example film or microcapsule.Sustained release matrix can comprise the copolymer of polyester, hydrogel, polylactic acid, L-glutamic acid and γ-ethyl-L-glutamic acid, poly-(2-hydroxyethyl-ethyl acrylate), ethylene vinyl acetate or poly--D (-)-3-hydroxybutyric acid.Sustained release composition can also comprise the liposome that can be prepared by any one of several methods known in the art.

[0137] Pharmaceutical compositions containing the immunomodulators disclosed herein can be in dosage unit form and can be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, and rectal administration. The preferred route of administration is IV infusion. Useful formulations can be prepared by methods known in the pharmaceutical field. For example, see Remington's Pharmaceutical Sciences, 18th edition (Mack Publishing Company, 1990). Formulation ingredients suitable for parenteral administration include sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers, for example, such as acetates, citrates or phosphates; and substances for adjusting tension such as sodium chloride or glucose.

[0138] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage and should be preserved for microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and a suitable mixture thereof.

[0139] The pharmaceutical preparation is preferably sterile. Sterilization can be accomplished by any suitable method, such as filtration through a sterile filtration membrane. In the case where the composition is lyophilized, filtration sterilization can be performed before or after lyophilization and reconstitution.

[0140] The compositions described herein can be administered topically or systemically. Administration is typically parenteral. In preferred embodiments, the pharmaceutical compositions are administered subcutaneously, and in more preferred embodiments, intravenously. Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.

[0141] Typically, the therapeutically effective amount of an active ingredient (e.g., an immunomodulator) is in the range of 0.1 mg / kg to 100 mg / kg, e.g., 1 mg / kg to 100 mg / kg, 1 mg / kg to 10 mg / kg. The dosage will depend on variables such as the type and extent of the disease or indication to be treated, the patient's overall health, the in vivo efficacy of the antibody, the pharmaceutical formulation, and the route of administration. The initial dose can be increased to exceed the upper limit to quickly reach the desired blood level or tissue level. Alternatively, the initial dose can be less than the optimal dose, and the daily dose can be gradually increased during the course of treatment. The human dose can be optimized, for example, in a conventional Phase I dose escalation study designed to run from 0.5 mg / kg to 20 mg / kg. The frequency of administration may vary, depending on factors such as the route of administration, dosage, serum half-life of the immunomodulator, and the disease being treated. Exemplary dosing frequencies are once a day, once a week, and once every two weeks. The preferred route of administration is parenteral, such as intravenous infusion. In certain embodiments, the immunomodulator is lyophilized and subsequently reconstituted in buffered saline when administered.

[0142] VI. Therapy

[0143] Once a suitable immunomodulator has been selected for a given subject, either alone or in combination with an anticancer drug, the subject can be treated according to conventional healthcare practices. In particular, an effective amount of an immunomodulator can be administered to the subject, either alone or in combination with an effective amount of another anticancer drug. As used herein, the term "effective amount" refers to an amount of an active agent (e.g., an immunomodulator) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a specific formulation or route of administration.

[0144] As used herein, "treat," "treating," and "treatment" refer to treating a disease in a subject (e.g., a human). This includes: (a) inhibiting the disease, i.e., preventing its progression; and (b) alleviating the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably include humans.

[0145] Examples of cancers that can be treated by the methods described herein are described in Section I. In certain embodiments, the cancer is a metastatic cancer. In certain embodiments, the cancer is a refractory cancer.

[0146] As mentioned above, it is expected that immunomodulators can be administered alone or in combination with another cancer drug or therapeutic agent. The term "combination" as used herein is understood to mean that two (or more) different treatments are delivered to a subject during the course of the subject's disease so that the effects of the treatment on the patient overlap at a certain time point. In certain embodiments, when the delivery of the second treatment begins, the delivery of one treatment is still occurring, thereby overlapping in terms of administration. This is sometimes referred to as "simultaneous" or "concurrent delivery" in this article. In other embodiments, the delivery of one treatment ends before the delivery of another treatment begins. In certain embodiments of either case, due to combined administration, the treatment is more effective. For example, compared to the first treatment, the second treatment is more effective when the second treatment is administered in the absence of the first treatment, for example, an equal effect is seen with less second treatment, or the second treatment alleviates symptoms to a greater extent, or a similar situation is seen with the first treatment. In certain embodiments, delivery causes a reduction in symptoms or other parameters associated with the condition to be greater than that observed when one treatment is delivered in the absence of another treatment. The effects of the two treatments can be partially added, fully added, or greater than added. Delivery can make it possible to detect the effect of the first treatment delivered when the second treatment is delivered.

[0147] In certain embodiments, immunomodulators are administered in combination with one or more other therapies (e.g., surgery, radiotherapy) or another therapeutic preparation. In certain embodiments, other therapies may include chemotherapy, such as cytotoxic agents. In certain embodiments, other treatments may include targeted therapy, such as tyrosine kinase inhibitors, proteasome inhibitors or protease inhibitors. In certain embodiments, other therapies may include anti-inflammatory, anti-angiogenic, anti-fibrotic or anti-proliferative compounds, such as steroids, biological immunomodulators, monoclonal antibodies, antibody fragments, aptamers, siRNA, antisense molecules, fusion proteins, cytokines, cytokine receptors, bronchodilators, statins, anti-inflammatory agents (e.g., methotrexate) or NSAIDs. In certain embodiments, other therapies may include a combination of different classes of therapies.

[0148] Exemplary cancer drugs that can be administered in combination with the methods or compositions described herein include, for example, antimicrotubule agents, topoisomerase inhibitors, antimetabolites, protein synthesis and degradation inhibitors, mitotic inhibitors, alkylating agents, platinating agents, nucleic acid synthesis inhibitors, histone deacetylase inhibitors (HDAC inhibitors, such as vorinostat (SAHA, MK0683), entinostat (MS-275), panobinostat (LBH589), trichostatin A (TSA), moxistat (MGCD0103), belinostat (PXD101), romidepsin (FK228, depsipeptide), DNA methyltransferase inhibitors, nitrogen mustards, nitrosoureas, ethyleneimines, alkyl sulfonates, triazenes, folic acid analogs, nucleoside analogs, ribonucleotide reductase inhibitors, vinca alkaloids, taxanes, epothilones, intercalating agents, substances capable of interfering with signal transduction pathways, agents that promote apoptosis and In one embodiment, the cytotoxic agents that can be administered using the methods or compositions described herein are platinum-based agents (such as cisplatin), cyclophosphamide, dacarbazine, methotrexate, fluorouracil, gemcitabine, capecitabine, hydroxyurea, topotecan, irinotecan, azacitidine, vorinostat, ixabepilone, bortezomib, taxanes (such as paclitaxel or docetaxel), cells Cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenotoposide, vincristine, vinblastine, vinorelbine, colchicine, anthracyclines (e.g., doxorubicin or epirubicin), daunorubicin, dihydroxyanthraquinone dione, mitoxantrone, mithramycin, actinomycin D, doxorubicin, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, ricin, or maytansine.

[0149] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism to be treated by the methods and compositions of the present invention. Such an organism is preferably a mammal (e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, or non-human primate, such as a monkey, chimpanzee, baboon, and rhesus monkey), and more preferably a human.

[0150] As used herein, the term "treat" includes any effect that results in an improvement in a condition, disease, or disorder, such as alleviating, reducing, regulating, preventing, slowing the progression of a condition, disease, or disorder, improving or eliminating a condition, disease, or disorder, etc. For example, treating a cancer or tumor can mean reducing the growth of a cancer or tumor, regulating a cancer or tumor to prevent the growth of a cancer or tumor, slowing the progression of a cancer or tumor, improving or eliminating the growth of a cancer or tumor. Treatment can be curing, improving, or at least partially alleviating a condition, such as cancer. In certain embodiments, treatment is curing a disease, such as cancer. Unless otherwise indicated, the term "disorder" refers to the term disease, condition, or illness and can be used interchangeably with these terms.

[0151] In this application, when an element or component is referred to as being included in and / or selected from a list of enumerated elements or components, it should be understood that the element or component can be any one of the enumerated elements or components, or the element or component can be selected from a group consisting of two or more enumerated elements or components.

[0152] Throughout this specification, when compositions are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is contemplated that there are additional compositions of the invention that consist essentially of, or consist of, the components, and that there are processes and methods according to the invention that consist essentially of, or consist of, the processing steps.

[0153] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0154] Throughout this specification, when compositions and kits are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is contemplated that there are additional compositions and kits of the invention that consist essentially of, or consist of, the components, and that there are processes and methods of the invention that consist essentially of, or consist of, the processing steps.

[0155] In addition, it should be understood that the elements and / or features of the compositions or methods described herein can be combined in various ways without departing from the spirit and scope of the present invention, whether it is clear or implicit herein. For example, when referring to a particular compound, unless otherwise understood from the context, the compound can be used for the various embodiments in the compositions of the present invention and / or the inventive method. In other words, in this application, embodiments have been described and depicted in a manner that allows for the preparation and drawing of clear and concise applications, but it is intended and will be appreciated that embodiments can be combined or separated in various ways without departing from current teachings and inventions. For example, it should be understood that all features described and depicted herein can be applied to all aspects of the present invention described and depicted herein.

[0156] The articles "a" and "an" are used in this disclosure to refer to one or more (ie, to at least one) of the grammatical object of the article unless the context does not allow. For example, "an element" means one element or more than one element.

[0157] The term "and / or" is used in this disclosure to mean "and" or "or" unless otherwise indicated.

[0158] It should be understood that the expression "at least one" includes each of the items mentioned below individually and various combinations of two or more items, unless otherwise indicated from the context and usage. The expression "and / or" in conjunction with three or more items should be understood to have the same meaning, unless otherwise indicated from the context.

[0159] The use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including grammatical equivalents thereof, should generally be understood as open-ended and non-limiting, e.g., not excluding other undescribed elements or steps, unless otherwise expressly stated or understood from the context.

[0160] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself unless otherwise specifically stated. As used herein, unless otherwise stated or inferred, the term "about" refers to a ±10% variation from the nominal value.

[0161] Where molecular weights of, for example, polymers are provided rather than absolute values, the molecular weights are to be understood as average molecular weights unless otherwise stated or understood otherwise from the context.

[0162] Generally, the percentages stated for compositions are by weight unless otherwise indicated. Additionally, if a variable is not defined, the preceding definition of the variable will prevail.

[0163] Should be understood that, as long as the present invention remains operable, the order of steps or the order in which certain actions are performed is irrelevant.In addition, two or more steps or actions can be performed simultaneously.

[0164] The use of any and all examples or exemplary language, such as "such as" or "including," is intended merely to better illustrate the invention and does not constitute a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. Example

[0165] The present disclosure is further illustrated by the following examples, which should not be construed as limiting the disclosure in scope or spirit to the specific procedures described herein. It should be understood that these examples are provided to illustrate certain embodiments and are not intended to limit the scope of the present disclosure.

[0166] Example 1 - Determination of the composition of the tumor microenvironment

[0167] This example demonstrates the ability of the claimed method to determine the composition of the tumor microenvironment from solid tumors of the lung, breast, and kidney using surface and intracellular markers. hi Tumors with FoxP+Tregs showed immunosuppressive features with tumor spread and thus had poor prognosis and objective response, which could help to formulate treatment strategies for patients.

[0168] In this example, lung, breast, and kidney solid tumor samples were obtained and shipped to Pierian Biosciences in Miltenyi tissue transport buffer in a Therapak controlled rate shipper maintained at 2-8°C. Upon receipt, the tumors were cut into 2-3 mm fragments using a sterile disposable scalpel and placed in tumor dissociation buffer (RPMI1640 + penicillin / streptomycin). The enzymes from the Miltenyi Human Tumor Dissociation Kit and Miltenyi's gentleMACS with heater were then used. Tumor fragments were mechanically dissociated into a single-cell suspension using an Octo dissociator. The single-cell suspension was then filtered through a 70 μM filter and counted. The cell suspension was then centrifuged and resuspended in RPMI 1640 + 10% FBS or 1X PBS + 0.5% BSA at the desired cell concentration.

[0169] After resuspending the cells in a suitable buffer, 80 μL were inoculated into designated wells of a deep-well 96-well plate (2 mL volume). Subsequently, the cells were initially stained with amine-reactive dye Alexa750 for 15 min to distinguish live cells from dead cells. The cells were then washed twice in staining buffer (1XPBS+0.5% BSA) using a Biotek ELx450 deep-well plate washer. The cells were then fixed and permeabilized using the FOXP3 / transcription staining buffer kit from eBioscience according to the manufacturer's recommendations for processing and staining cells in deep-well plates. The cells were then washed twice in staining buffer (1X PBS+0.5% BSA) using a Biotek ELx450 deep-well plate washer. The cells were then stained with a mixture of fluorescently labeled antibodies to detect CD3+, CD4+, CD8+, and Treg (CD4+CD25 hiFoxP3+). The stained cells were incubated in the dark at ambient temperature for 1 h. The cells were then washed twice in staining buffer (1X PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer before being fixed in a final concentration of 1% PFA.

[0170] Stained cells were acquired directly from 96-well deep-well plates using a Thermo-Fisher Attune NxT 16-color flow cytometer. Prior to acquisition, all required information (e.g., sample ID, antibody staining panel, and pre-defined and fixed flow cytometer instrument settings and acquisition parameters) was incorporated into a 96-well plate layout using Ryvett software from Qognit, Inc. This plate layout file was imported into the Attune NxT software prior to acquisition. Following acquisition, FCS files were automatically analyzed and gated in the Ryvett software using pre-defined gating procedures. After manual review of the automated gating, raw data and calculated metrics were exported to CSV files using pre-defined criteria and data extraction procedures. Results are displayed in Figure 4A -B in. Figure 4A An exemplary flow cytometry plot is shown showing CD3+, CD4+, CD8+ and Treg (CD4+CD25 hi FoxP3+) detection. Figure 4B The dot plots provided show the expression of CD3+ T cells (expressed as a percentage of CD45+ leukocytes), CD4+ and CD8+ T cells (expressed as a percentage of CD3+ T cells), and Tregs (expressed as a percentage of CD4+ T cells).

[0171] The above results demonstrate that the methods described herein can be used to detect the presence and abundance of specific types of immune cells in the tumor microenvironment. Understanding the composition of immune cells in the tumor microenvironment, such as the ratio of CD4+ / CD8+ cells and CD8+ / Treg cells, is important for determining the potential of a patient to respond to administered immunotherapy.

[0172] Example 2 - Assessment of the functional capacity of cells from isolated tumors by surface and intracellular staining of targets in basal and induced states

[0173] This example demonstrates that the claimed method can determine functional capacity by measuring both basal and induced levels of functional readouts in identified immune cells from breast, lung, and kidney solid tumors using surface and intracellular markers.

[0174] In this example, lung, breast, and kidney solid tumor samples were obtained and shipped to Pierian Biosciences in Miltenyi tissue transport buffer in a Therapak controlled rate shipper maintained at 2-8°C. Upon receipt, the tumors were cut into 2-3 mm fragments using a sterile disposable scalpel and placed in tumor dissociation buffer (RPMI1640 + penicillin / streptomycin). The enzymes from the Miltenyi Human Tumor Dissociation Kit and Miltenyi's gentleMACS with heater were then used. Tumor fragments were mechanically dissociated into a single-cell suspension using an Octo dissociator. The single-cell suspension was then filtered through a 70 μM filter and counted. The cell suspension was then centrifuged and resuspended in RPMI 1640 + 10% FBS or 1X PBS + 0.5% BSA at the desired cell concentration.

[0175] After resuspending the cells in the appropriate buffer, 80 μL was inoculated into the designated wells of a deep-well 96-well plate (2 mL volume). Subsequently, the cells were initially stained with the amine-reactive dye Alexa750 for 15 min to distinguish live cells from dead cells. The cells were then washed twice in staining buffer (1XPBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. Leukocyte activation mixture and BD GolgiPlug from Becton Dickinson were then used. TM The cells were conditioned for 3 hours. Leukocyte activation mixture and BD GolgiPlug TM A ready-to-use polyclonal cell activation cocktail containing phorbol esters, PMA (phorbol 12-myristate 13-acetate), a calcium ionophore (ionomycin), and the protein transport inhibitor BD GolgiPlug (Brefeldin A). After 3 h, the cells were then fixed and permeabilized using the FOXP3 / Transcript Staining Buffer Kit from eBioscience according to the manufacturer's recommendations for processing and staining cells in deep-well plates. The cells were then washed twice in staining buffer (1X PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer. The cells were then stained with a cocktail of fluorescently labeled antibodies to detect CD3+, CD4+, CD8+, and Treg (CD4+CD25 hi FoxP3+). The stained cells were incubated in the dark at ambient temperature for 1 h. The cells were then washed twice in staining buffer (1X PBS + 0.5% BSA) using a Biotek ELx450 deep-well plate washer before being fixed in PFA at a final concentration of 1%.

[0176] Stained cells were acquired directly from 96-well deep-well plates using a Thermo-Fisher Attune NxT 16-color flow cytometer. Prior to acquisition, all required information (e.g., sample ID, antibody staining panel, and pre-defined and fixed flow cytometer instrument settings and acquisition parameters) was incorporated into the 96-well plate layout using Ryvett software from Qognit, Inc. After acquisition, FCS files were automatically analyzed and gated in the Ryvett software using pre-defined gating programs. After manual review of the automated gating, raw data and calculated metrics were exported to CSV files using pre-defined criteria and data extraction programs. Results are displayed in Figure 5-6 middle. Figure 5 Shown are exemplary flow cytometry graphs demonstrating detection of basal and induced IFNγ and TNFα in CD4+ and CD8+ T cells, and associated dot plots showing expression of IFNγ and TNFα (expressed as percent positive). Figure 6 Shown are exemplary flow cytometry graphs demonstrating detection of granzyme B expression in CD4+ and CD8+ T cells from breast, lung, and kidney tumors.

[0177] IFNγ and TNFα are cytokines that indicate "inflamed" tumors with greater potential for an immune response, with elevated levels of IFNγ and TNFα associated with initial and sustained responses, respectively, both during initial immunotherapy treatment and subsequent follow-up. Granzyme B is an effector molecule found on CD8+ cells and is part of the granzyme B-perforin complex, which CD8+ cells use to kill target tumor cells. Reduced levels of granzyme B are associated with reduced CD8+ cytotoxic potential and are an indicator of T cell exhaustion. Determining the percentage of CD8+ cells expressing granzyme B in tumors and the quantitative level of granzyme B expression are useful for predicting response to immunotherapy and tailoring treatment strategies for patients.

[0178] Example 3 - Evaluation of immune checkpoint and exhaustion markers and their cognate ligands from solid tumors

[0179] In this example, the expression levels of immune checkpoint / exhaustion markers and their cognate ligands were measured on TILs and tumor epithelial cells. Immunotherapy treatment currently relies on a single IHC measurement of the expression of the IMR ligand PD-L1 on tumor cells as a guide for treatment, but this does not fully identify IMR and IMR-L expression on any other cellular components within the tumor microenvironment. The holistic approach described here could improve patient stratification and treatment strategies.

[0180] In this example, lung, breast, and kidney solid tumor samples were obtained and shipped to Pierian Biosciences in Miltenyi tissue transport buffer in a Therapak controlled rate shipper maintained at 2-8°C. Upon receipt, the tumors were cut into 2-3 mm fragments using a sterile disposable scalpel and placed in tumor dissociation buffer (RPMI1640 + penicillin / streptomycin). The enzymes from the Miltenyi Human Tumor Dissociation Kit and Miltenyi's gentleMACS with heater were then used. Tumor fragments were mechanically dissociated into a single-cell suspension using an Octo dissociator. The single-cell suspension was then filtered through a 70 μM filter and counted. The cell suspension was then centrifuged and resuspended in RPMI 1640 + 10% FBS or 1X PBS + 0.5% BSA at the desired cell concentration.

[0181] After resuspending the cells in a suitable buffer, 80 μL was inoculated into the designated wells of a deep-well 96-well plate (2 mL volume). Subsequently, the cells were initially stained with the amine-reactive dye Alexa750 for 15 minutes to distinguish live cells from dead cells. The cells were then washed twice in staining buffer (1XPBS+0.5% BSA) using a Biotek ELx450 deep-well plate washer. The cells were then stained with a mixture of fluorescently labeled antibodies to detect CD326+, CD45+, CD3+, CD4+, CD8+, CD19+, CD56+, and CD14+ cells. Additional labeled antibodies were used to identify the following IMR or IMR-L: CD73, CD112, CD155, CD172ab, CD274, CD279, CD366, TIGIT, TIM-3. The stained cells were incubated in the dark at ambient temperature for 20 minutes. Cells were then washed twice in staining buffer (1X PBS + 0.5% BSA) using a Biotek ELx450 deep well plate washer before being fixed in PFA at a final concentration of 1%.

[0182] Stained cells were acquired directly from 96-well deep-well plates using a Thermo-Fisher Attune NxT 16-color flow cytometer. Prior to acquisition, all required information (e.g., sample ID, antibody staining panel, and predefined and fixed flow cytometer instrument settings and acquisition parameters) was incorporated into the 96-well plate layout using Ryvett software from Qognit, Inc. Following acquisition, FCS files were automatically analyzed and gated in Ryvett software using a predefined gating program. Following manual review of the automated gating, raw data and calculated metrics were exported to CSV files using predefined criteria and data extraction programs.

[0183] like Figure 7As shown in , CD45+ leukocytes, CD326+ epithelial cells, CD14+ monocytes / macrophages, and CD4+ and CD8+ T cells were detected within the tumor microenvironment. Examples of inhibition of checkpoint receptor expression are shown in Figure 8 In a new study, researchers detected the inhibitory IMR TIGIT on a subset of cells in the tumor microenvironment. Specifically, TIGIT was detected on CD4+, CD8+, and CD14+ leukocytes, but not on CD326+ tumor cells.

[0184] The levels of IMR / exhaustion markers and their ligands (CD279 / CD274 [PD1 / PD-L1], TIGIT / CD112-CD155, CD366 [TIM-3] / Galectin-9, CD172a [SIRPa] / CD47, CD73) were measured to summarize the functional status of TILs. Figure 9-11 As shown in Figure 3, reliable separation of IMR / IMR-L-positive TILs from negative TILs and IMR / IMR-L-positive tumor cells from negative tumor cells was observed, with significant heterogeneity across cell types and tumor types (breast, lung, and kidney). This included elevated expression of several IMR-Ls on TILs and IMRs on epithelial and stromal cells, suggesting tumor- and TIL-intrinsic mechanisms regulating checkpoint interactions.

[0185] These results demonstrate that the methods described herein can be used to identify IMR / exhaustion markers and their ligands on specific cell types in the tumor microenvironment. Understanding the composition of IMR / exhaustion markers and their ligands on specific cell types (e.g., immune cells) in the tumor microenvironment is important for determining the potential of a patient to respond to an administered immunotherapy.

[0186] Example 4 - Treatment of Patients with Immunomodulators

[0187] Tumor samples were received from patients and evaluated according to the methods described in Examples 1-3. A panel of IMR / exhaustion markers and their ligands (CD279 / CD274[PD1 / PD-L1], TIGIT / CD112-CD155, CD366[TIM-3] / Galectin-9, CD172a[SIRPa] / CD47, CD73) were measured to summarize the functional status of TILs. The results showed the presence of PD-1 (CD279) on T cells and PD-L1 (CD274) on one or more of dendritic cells, macrophages, and tumor cells. It is considered that if a subject is treated with anti-PD-L1 or anti-PD-1 antibodies, the tumor may regress.

[0188] Example 5 - Treatment of patients with a combination of immunomodulatory agents

[0189] Tumor samples were received and evaluated from patients according to the method described in Examples 1-3. A group of IMR / exhaustion markers and their ligands (CD279 / CD274[PD1 / PD-L1], TIGIT / CD112-CD155, CD366[TIM-3] / Galectin-9, CD172a[SIRPa] / CD47, CD73) were measured to summarize the functional status of TIL. The results showed the presence of PD-1 (CD279) on T cells, and the presence of PD-L1 (CD274) on one or more of dendritic cells, macrophages, and tumor cells. The results also showed the presence of immune checkpoint protein TIGIT on T cells and NK cells, and the presence of corresponding ligands CD112 and CD255 on one or more of dendritic cells, macrophages, and tumor cells. It is considered that if a subject is treated with both anti-PD-L1 or anti-PD-1 antibodies and anti-TIGIT antibodies, the tumor may regress.

[0190] Incorporated by reference

[0191] The entire disclosure of each patent and scientific document mentioned herein is incorporated by reference for all purposes.

[0192] equivalent body

[0193] The present invention may be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. The foregoing embodiments are therefore to be considered in all respects as illustrative rather than restrictive of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all variations that come within the meaning and range of equivalence of the claims are intended to be embraced therein.

Claims

1. A method for determining the composition of a solid tumor microenvironment, the method comprising the steps of: (a) combining a single-cell suspension of cells derived from a solid tumor with a plurality of labeling agents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs), and IMR ligands (IMR-Ls), and allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single-cell suspension to produce labeled cells; and (b) determining the presence and / or amount of labeled cells by cytometry, thereby (i) determining the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor microenvironment and (ii) determining whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L, thereby determining the solid tumor microenvironment.

2. The method of claim 1, wherein the cytometry is selected from the group consisting of flow cytometry, mass cytometry, image cytometry, and single cell technology (SCT).

3. The method of claim 1 or 2, wherein the plurality of labeling agents comprises at least one labeling agent selected from the group consisting of a fluorophore, an infrared label, and a heavy metal label.

4. The method of any of the preceding claims, wherein the immune cells comprise lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B-cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or a combination thereof.

5. The method of any preceding claim, wherein the cell surface markers further comprise cell activation markers.

6. The method of claim 5, wherein the cell type marker comprises a marker expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B-cells, and natural killer cells) and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells).

7. The method of any preceding claim, wherein the cell type marker is a cancer cell marker.

8. The method of claim 7, wherein the cancer cell markers comprise CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

9. The method of claim 5, wherein the cell activation markers include CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197) and / or functional markers, e.g., IFNγ, TNFα and / or other cytokines and / or granzyme B.

10. The method of any one of claims 1 to 9, wherein the IMR or IMR-L marker comprises PD-1 (CD279), PD-L1(CD274), CTLA-4(CD152), LAG3(CD223), OX40(CD134), TIM3(CD366), G ITR(CD357), 4-1BB(CD137), KIR(CD158B), 2B4(CD244), ICOS(CD278), IDO, TIGIT, CD7 3. CD39, CD172a (SIRPa), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY 55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA) and / or CD115 (CSF-1R).

11. The method of any one of claims 1-10, further comprising the step of combining the cells with an immunomodulatory agent.

12. The method of claim 11, further comprising determining the effect of the immunomodulatory agent on the expression of at least a portion of the cell markers on the cancer cells and / or immune cells.

13. A method of determining whether a subject having a solid tumor is likely to respond to an immunomodulatory agent, the method comprising the steps of: (a) combining a single-cell suspension of cells derived from a solid tumor with a plurality of labeling agents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs), and IMR-ligands (IMR-Ls), and allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single-cell suspension to produce labeled cells; (b) combining at least a portion of the single-cell suspension of cells with an immunomodulatory agent; and (c) determining (i) the presence and / or amount of labeled cells by cytometry to determine the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L, and (ii) the effect of an immunomodulator on cellular markers on or in the cancer cells, immune cells, or both cancer cells and immune cells to determine whether the subject is likely to respond to the immunomodulator.

14. The method of claim 13, wherein an immunomodulatory agent is combined with the single cell suspension before, during, or after step (a).

15. The method of claim 13 or claim 14, wherein the cytometry is selected from the group consisting of flow cytometry, mass cytometry, image cytometry and single cell technology (SCT).

16. The method of claims 13-15, wherein the plurality of labeling agents comprises at least one labeling agent selected from the group consisting of a fluorophore, an infrared label, and a heavy metal label.

17. The method of claims 13-17, wherein the immune cells comprise lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B-cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or a combination thereof.

18. The method of claims 13-17, wherein the cell surface markers further comprise cell activation markers.

19. The method of claims 13-18, wherein the cell type markers comprise markers expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B-cells, and natural killer cells) and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells).

20. The method of claims 13-19, wherein the cell type marker is a cancer cell marker.

21. The method of claim 20, wherein the cancer cell markers comprise CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

22. The method of claim 18, wherein the cell activation markers comprise CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197) and / or functional markers, e.g., IFNγ, TNFα and / or other cytokines and / or granzyme B.

23. The method of any one of claims 13-22, wherein the IMR or IMR-L markers include PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, CD39, CD172a (SIRPα), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA), and CD115 (CSF-1R).

24. A method of treating a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of an immunomodulatory agent, thereby treating the solid tumor, wherein the immunomodulatory agent is selected by using a method comprising the steps of: (a) combining a single-cell suspension of cells derived from a solid tumor with a plurality of labeling agents capable of binding to a corresponding plurality of cell surface markers expressed on cancer cells and / or immune cells, wherein the cell surface markers include cell type markers, immunomodulatory receptors (IMRs), and IMR-ligands (IMR-Ls), and allowing the agents to simultaneously bind to cancer cells, immune cells, or both cancer cells and immune cells present in the single-cell suspension to produce labeled cells; (b) combining at least a portion of the single-cell suspension of cells with an immunomodulatory agent; and (c) determining (i) the presence and / or amount of labeled cells by cytometry, thereby determining the presence and / or amount of cancer cells, immune cells, or both cancer cells and immune cells present in the solid tumor, and whether the cancer cells, immune cells, or both cancer cells and immune cells express at least one IMR and / or at least one IMR-L and (ii) the effect of an immunomodulator on cellular markers on or in the cancer cells, immune cells, or both cancer cells and immune cells, thereby determining whether the subject is likely to respond to the immunomodulator.

25. The method of claim 24, wherein an immunomodulatory agent is combined with the single cell suspension before, during, or after step (a).

26. The method of claim 24 or claim 25, wherein the cytometry technique is selected from the group consisting of flow cytometry, mass cytometry, image cytometry, and single cell technology (SCT).

27. The method of any one of claims 24-26, wherein the plurality of labeling agents comprises at least one labeling agent selected from the group consisting of a fluorophore, an infrared label, and a heavy metal label.

28. The method of any one of claims 24-27, wherein the immune cells comprise lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B-cells, and natural killer cells), myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells), or a combination thereof.

29. The method of any one of claims 24-27, wherein the cell surface markers further comprise cell activation markers.

30. The method of any one of claims 24-29, wherein the cell type markers comprise markers expressed on lymphocytes (e.g., T cells (e.g., CD4+ T cells, CD8+ T cells, Tregs), B-cells, and natural killer cells) and / or myeloid cells (e.g., dendritic cells, macrophages, and myeloid-derived suppressor cells).

31. The method of any one of claims 24-30, wherein the cell type marker is a cancer cell marker.

32. The method of claim 31, wherein the cancer cell markers comprise CD44, CD47, CD49f, CD271, CD326, cytokeratin (intracellular), E-cadherin, and / or vimentin.

33. The method of claim 29, wherein the cell activation markers include CD25, CD26, CD27, CD28, CD38, CD40, CD44, CD62L, CD69, CD80, CD86, CD95, CD95L, CD127, CCR7 (CD197) and / or functional markers, e.g., IFNγ, TNFα and / or other cytokines and / or granzyme B.

34. The method of any one of claims 24-33, wherein the IMR or IMR-L markers include PD-1 (CD279), PD-L1 (CD274), CTLA-4 (CD152), LAG3 (CD223), OX40 (CD134), TIM3 (CD366), GITR (CD357), 4-1BB (CD137), KIR (CD158B), 2B4 (CD244), ICOS (CD278), IDO, TIGIT, CD73, CD39, CD172a (SIRPα), B7H4 (B7S1), VISTA (B7-H5), CD355 (CRTAM), KLRG1, CD160 (BY55, NK1, NK28), CD30 (TNFRSF8), CD224 (GGT1), CD226, CD272 (BTLA) and / or CD115 (CSF-1R).

35. The method of any one of claims 1 to 34, wherein a plurality of differently labeled cells are detected simultaneously during the cytometry procedure.

36. The method of any one of claims 1 to 35, wherein a plurality of different cell surface markers are detected simultaneously during the cytometry procedure.

37. The method of claim 36, wherein at least 14 different cell surface markers are detected simultaneously.

38. The method of any one of claims 1 to 37, wherein receptor-ligand interactions between the labeled cells can be detected and optionally quantified.

39. The method of claim 38, wherein the receptor-ligand interaction comprises an interaction between a checkpoint inhibitor and its cognate ligand.

40. The method of claim 39, wherein the receptor-ligand interaction is selected from the group consisting of the interaction between PD-1 and PD-L1, CTLA-4 and B7-1 and / or B7-2, TIM-3 and Gal9, GITR and GITRL, OX-40 and OX40L, CD-27 and CD70, 4-1BB and 4-1BBL, and / or CD-40L and CD40.

41. The method of any one of claims 1-40, wherein the presence and / or amount of a cell activation marker, an IMR marker, an IMR-L marker, or a combination of an activation marker and an IMR and / or IMR-L marker expressed on cancer cells and / or immune cells is determined.

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