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WO2015117164A1 - Macrophages associés à une tumeur et procédés et compositions pour cibler une thérapie anticancéreuse et identifier des répondeurs potentiels - Google Patents

Macrophages associés à une tumeur et procédés et compositions pour cibler une thérapie anticancéreuse et identifier des répondeurs potentiels Download PDF

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WO2015117164A1
WO2015117164A1 PCT/US2015/014340 US2015014340W WO2015117164A1 WO 2015117164 A1 WO2015117164 A1 WO 2015117164A1 US 2015014340 W US2015014340 W US 2015014340W WO 2015117164 A1 WO2015117164 A1 WO 2015117164A1
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cells
tumor
tams
cell
patient
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Ming Li
Ruth A. FRANKLIN
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Memorial Sloan Kettering Cancer Center
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Memorial Sloan Kettering Cancer Center
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70517—CD8
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present disclosure relates generally to tumor-associated macrophages and to interactions between these macrophages and lymphocytes present in the tumor and more specifically CD8+ T cells.
  • Cancer is characterized by unregulated proliferation of aberrant cells.
  • Surgery, radiation therapy, and chemotherapy have been the standard accepted approaches for treatment of cancer.
  • these approaches are often associated with particularly high toxicity, adverse effects, and poor treatment outcomes.
  • Limitations associated with conventional cancer treatments highlight the need for the development of more effective and less harmful therapies.
  • Cancer immunotherapy has gained substantial attention in the last decade, with many research and clinical studies showing promising therapeutic outcomes.
  • Compared to conventional surgery, radiation, and chemotherapy, immunotherapy is based on a radically different concept by targeting the immune system rather than the tumor itself. Indeed, as a consequence of genetic and epigenetic alterations, tumors express tumor specific or tumor-associated antigens that are recognized by the adaptive immune system including T lymphocytes (Coulie PG at al. Nature reviews Cancer 2014, 14(2): 135-146).
  • Some of the advantages of cancer immunotherapy include: the ability of immune cells to induce (a) specific killing of tumor cells with minimal harm to healthy cells; (b) a systemic antitumor immune response that can control metastases; and (c) immunological memory which could provide protection against cancer recurrence.
  • Tumor immune infiltrates include macrophages, dendritic cells (DC), mast cells, natural killer (NK) cells, naive and memory lymphocytes, B cells and effector T cells (T lymphocytes), primarily responsible for the recognition of antigens expressed by tumor cells and subsequen destruction of the tumor cells by T cells.
  • Tumor progression is characterized by an accumulation of macrophages, both in experimental and clinical settings, yet the precise origin and function of these cells are largely unknown.
  • T LymphocytesT lymphocytes are critical in the development of all cell- mediated immune reactions. Helper T- cells control and modulate the development of immune responses. Cytotoxic T-cells (killer T-cells) are effector cells which play an important role in immune reactions against intracellular parasites and viruses by means of lysing infected target T-cells. Additionally, cytotoxic T-cells have been implicated in protecting cancer development and progression through an immune surveillance mechanism. T suppressor cells block the induction and/or activity of T helper cells. Antigen-presenting cells (APC) are capable of stimulating T cell division or may be virally-infected cells within the body that become a target for cytotoxic T-cells.
  • APC Antigen-presenting cells
  • Cytotoxic or suppressor T-cells usually recognize antigen in association with class I Major Histocompatibiliry Complex (MHC) products which are expressed on all nucleated cells.
  • MHC Major Histocompatibiliry Complex
  • Class II products are expressed mostly on antigen-presenting cells and on some lymphocytes. T- cells can also be divided into two major subpopulations on the basis of their cell membrane glycoproteins as defined with monoclonal antibodies.
  • the CD4 + subset which expresses a 62 kD glycoprotein usually recognizes antigen in the context of class II antigens
  • the CD8 + subset expresses a 76 Kd glycoprotein and is restricted to recognizing antigen in the context of Class I MHC.
  • Tumor Associated Macrophages (TAMs)Macrophages are tissue-resident innate immune cells present in most metazoan species. Characterized by their ability to engulf dying cells, noxious substances and microbes, macrophages control tissue development and homeostasis as well as host defense responses against pathogens (Martinez et aL, 2009; Medzhitov, 2010; Murray and Wynn, 2011).
  • Tumor-associated macrophages in mice are typically defined by expression of the cell surface markers CD 11 b, F4JSO, and the CSF1 receptor, CDl 15 (Qian and Pollard, 2010).
  • Gene expression studies in a breast tumor model suggest that they closely resemble embryonic macrophages, and the trophic functions of macrophages found during development might be subverted by tumors to promote their growth (Ojalvo et al., 2009).
  • TAMs have been proposed to take on an AAM phenotype, and may act downstream of type 2 T cell responses as effector cell types to foster tumor progression (DeNardo et al., 2009; Wynn et al., 2013).
  • PD-1 programmed death 1 polypeptide
  • PD-1 is an inhibitory member of the CD28 family of receptors that in addition to PD-1 includes CD28, CTLA-4, ICOS and BTLA.
  • PD-1 is expressed on several types of immune cells, activated B cells, T cells, and myeloid cells (Okazaki et al, Curr. Opin.
  • This type of immune suppression can be reversed by targeted inhibition of immunologic checkpoints, such as the PD-1 and PD-L1 interaction.
  • immunologic checkpoints such as the PD-1 and PD-L1 interaction.
  • PD-1 expressing T cells contact cells expressing its ligands, functional activities in response to antigenic stimuli, including proliferation, cytokine secretion, and cytotoxicity are reduced.
  • PD-1/PD-L1 interactions downregulate immune responses during tumor progression, chronic antigen stimulation, such as that which occurs during tumorigenesis, results in T cells that express elevated levels of PD-1 and are dysfunctional with respect to activity towards the chronic antigen (Kim et al., Curr. Opin. 1 mm. (2010)). This is termed "T cell exhaustion”.
  • PD-1 inhibitors include BMS936558 (nivolumab), MK-3475 (pembrolizumab), pidilizumab;
  • PD-L1 inhibitors include MPDL3280A, BMS-936559, MEDI4736, MSB 00107180 PDL-1.
  • clinical investigators have observed that PD-1 inhibitors are effective in only a fraction of the patients. Given the potential for high-grade adverse events and the cost of therapy, it would be highly desirable to develop surrogate markers for stratifying patients according to the likelihood that they would respond to anti-PD-1 anti-PDLl therapy. Accordingly, there is a need for biomarkers that could help stratify patients afflicted with solid tumors according to the likelihood that they could be successfully treated with immunologic checkpoint .inhibitors.
  • a method for stratifying a subject suffering from a solid tumor as a suitable candidate for therapy with a checkpoint inhibitor comprising; assessing the percentage of cytotoxic phenotype (CD8+) tumor-associated T cells based on the total number of tumor-associated T cells or on signal intensity in a biological sample derived from said patient that express the marker Programmed cell Death- 1 (PD-1) and that do not express appreciable amounts of Granzyme B (GzmB); identifying the subject as a suitable candidate for treatment with said inhibitor by taking into account if a first substantial percentage of the number or signal intensity of said CD8+ T cells express PD-1 (PD-1+) and a second substantial percentage of the number or signal intensity of the CD8+PD-1+ T cells do not express appreciable amounts of GzmB; or conversely not identifying the patient as a suitable candidate for treatment with said inhibitor if a first substantial percentage of the number or signal intensity of said CD8+ T cells do not express appreciable amounts of PD-1 or a
  • the first substantial percentage of the number or signal intensity is at least about 30% of said CD8+ T cells expressing PD-1 (PD-1+) and the second substantial percentage of the number or signal intensity is at least about 60% of the CD8+PD-1+T cells not expressing appreciable amounts of GzmB.
  • an appreciable amount of PD-1 is at least about 1000-fold higher in expression compared to baseline if measured in flow cytometry experiments; a not appreciable amount of PD-1 or GzmB is an amount at least about 1000-fold lower than that of cells that express PD-1 or GzmB if measured in flow cytometry experiments.
  • the checkpoint inhibitor is at least one member selected from the group consisting of PD-1 inhibitors, Programmed cell Death- 1 Ligand (PD-Ll) inhibitors and decoys of either PD-1 or PD-Ll and combinations of two or more of the foregoing.
  • PD-1 inhibitors Programmed cell Death- 1 Ligand (PD-Ll) inhibitors
  • decoys of either PD-1 or PD-Ll and combinations of two or more of the foregoing.
  • the solid tumor is selected from the group consisting of melanoma, renal cell carcinoma, nonsmall-cell lung carcinoma, breast cancer, bladder cancer, head and neck cancer, ovarian cancer, urothelial cancer, gallbladder cancer, and esophageal cancer.
  • the disclosed methods further comprise assessing whether a third substantial percentage of the number or signal intensity of the macrophages resident in said tumor are tumor associated macrophages (TAMs) rather than noncancer associated tissue-resident macrophages and taking into account the assessment of said TAMs when identifying the subject as a suitable candidate for treatment with a checkpoint inhibitor.
  • TAMs tumor associated macrophages
  • the TAMs are characterized by high expression of major histocompatibility complex Class II (MHCII) and a monocytic marker.
  • MHCII major histocompatibility complex Class II
  • a high expression of a monocytic marker and MHC II respectively is at least about 1000-fold higher fold compared to baseline (negative control) if measured in flow cytometry experiments.
  • the third substantial percentage is at least 20% of the tumor- infiltrating leukocytes being TAMs characterized by high expression of major histocompatibility complex Class II (MHCII) and a monocytic marker.
  • MHCII major histocompatibility complex Class II
  • a percentage of said TAMs higher than 20% expressing PD-1 is used to nonetheless identify the subject as a suitable candidate for therapy with PD-1 or PD-L1 when the patient does not clearly satisfy that at least a substantial percentage of the CD8+ T cells express PD-1 and/or at least a substantial percentage of the CD8+ PD-1+ T-cells express no appreciable amounts of GzmB.
  • the patient is a human and the monocytic marker is CD 14.
  • a method of treating a patient afflicted by a solid tumor comprising administering to said patient an immunologic checkpoint inhibitor, the patient having been identified as a suitable candidate for said treatment by a prior determination that a first substantial percentage of the number or signal intensity of CD8+ T cells in the tumor of said patient express PD-1 (PD-1+) and a second substantial percentage of the number or signal intensity of the CD8+PD-1+ T cells do not express appreciable amounts of GzmB.
  • aspects of the disclosure are directed to a method for treating breast cancer in a patient afflicted with a solid tumor, the tumor comprising tumor-associated macrophages (TAMs) expressing high levels of MHCII and also expressing one or more cell adhesion molecules selected from the group consisting of Vcaml and Itgb8, the method comprising:
  • Yet other aspects are directed to a method for determining whether a cancer patient is a candidate for treatment with a Notch signaling pathway inhibitor or a CSF-1 inhibitor comprising:
  • TAMs determining the cell count of, or signal intensity generated by, TAMs in a tumor sample of said patient, said TAMs expressing high levels of a monocytic marker, MHCII and one or more cell adhesion molecules selected from the group consisting of Vcaml and Itgb8; and
  • Figure 1 includes a series of graphical representations of data showing that macrophages constitute the dominant myeloid cell population in mammary tumors.
  • Figure 1A includes two scatterplots of macrophage distribution according to expression of cell surface markers (CD1 lb, MHCII, Ly6C, and Ly6G). Three groups are identified. On the right side (images labeled I, II, and III) are representative images of cells sorted from populations I-III, after cytospin and Giemsa staining at an original magnification of 40x.
  • Figure IB shows graphs of flow cytometric analysis of populations I-III from Figure 1A.
  • Figure 1C is a plot showing principal component analysis (PC3) of population I: TAM (mammary) and CDl lb+ splenic DC (sp) gene expression compared to populations collected by the Immunological Genome Project (GSE 15907).
  • Figure ID is a table summary of results from support vector machine (SVM) classifier.
  • Figure IE is a graph depicting Zbtb46 (a dendritic cell marker) and the transcription factor and oncogene homolog V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (Majb) mR A expression in TAMs and CDl lb + splenic DCs (spDCs).
  • Figure IF are graphs showing flow cytometric analysis of DC and macrophage signature genes expressed on TAMs and CDl lb + spDCs.
  • Figure 2 is a series of graphical representations of data showing that expansion of TAMs is associated with increased tumor burden.
  • Figure 2A is a group of scatterplots of flow cytometric analysis of myeloid cell populations found in wild- type (WT) and PyMT mice.
  • Figure 2B is a group of graphs showing cell surface expression of DC and macrophage signature genes expressed on MTMs and CD1 lb splenic DCs.
  • Figure 2C is a dot plot showing flow cytometry analysis of TAM and MTM populations.
  • Figure 3 is a series of graphical representations of data showing that TAMs differentiate from the blood-borne precursors.
  • Figure 3 A is a schematic representation of experimental design.
  • Figure 3B are scatterplots of myeloid cell populations present in a CD45.1 + parabiont (host).
  • Figure 4 is a series of graphical representations of data showing that fewer inflammatory monocytes are needed to repopulate TAMs than MTMs.
  • Figure 4A are scatterplots of flow cytometry surface marker analysis of cells from Ccr2 +/+ PyMT and Ccr2 _/" PyMT mice.
  • Figure 4C are scatterplots of flow cytometric analysis of tumor- associated myeloid cells from WT PyMT and CCR2 DTR PyMT mice.
  • Figure 5 is a series of graphical representations of data showing that TAMs proliferate upon their differentiation from inflammatory monocytes.
  • Figure 5A includes a scatterplot (left) (CD l ib and MHCII expression) and a graph (right) analysis of TAM and MTM proliferation by Ki67.
  • Figure 5B are scatterplots and a dot plot of TAM and MTM proliferation by EdU incorporation.
  • Figure 5C are scatterplots of flow cytometry analysis of congenically marked CCR2 + bone marrow (BM) cells following the transfer into CCR2 DTR PyMT mice gated on total live cells (top panel) or total transferred cells (bottom panel).
  • BM bone marrow
  • Figure 5D are graphs of percentages of cell surface marker positive cells in TAMs and CCCR2 + BM monocytes.
  • Figure 5E is a graph of percentage of F4/80 positive cells.
  • Figure 5F is a graph of transferred cells (% of CD45 + cells) versus days post monocyte transfer.
  • Figure 5G is a graph of cell proliferation (via Ki67) after transfer.
  • Figure 5H is a schematic representation of the findings from the CCR2 GFP monocyte transfer experiment.
  • Figure 6 is a series of graphical representation of data showing that TAMs are phenotypically different from AAMs and can be identified by Vcaml expression.
  • Figure 6A is an image showing gene expression data of sorted TAMs and MTMs from PyMT mice. Data are from 3 replicate experiments. Differentially expressed genes were determined with a p value threshold of 0.05. Fold change is depicted using a log scale.
  • Figure 6B is a histogram of Vcaml and Mrcl expression on TAMs and MTMs.
  • Figure 6C is a histogram of Vcaml expression on CCR2 GFP bone marrow monocytes and their progenies after transfer into diphtheria toxin-treated CCR2 DTR PyMT recipients.
  • Figure 6D is a schematic representation of three intermediate stages of TAM differentiation from monocytes.
  • Figure 7 is a series of graphical representations of data showing that TAM differentiation requires RBPJ.
  • Figure 7A is an image showing differential expression of Notch-dependent genetic programming between MTMs and TAMs in PyMT mice. Data are from 3 replicate experiments. Differentially expressed genes were determined with a p-value threshold of 0.05. Fold change is depicted using a log scale.
  • Figure 7B is a scatterplot of representative flow cytometric data of myeloid populations in 20-week-old CDl lc cre Rbpf fl PyMT and wild type (WT) PyMT mice. Plots are gated on CD45 B220 " cells.
  • Figure 7C is a bar graph showing relative CD1 lb mRNA expression on MHCII + population shown in Figure 7B (MFI, mean fluorescence intensity).
  • Figure 7D is an image of a volcano plot of differentially expressed genes between MHCII + populations in Figure 7B.
  • Figure 7E are histograms of Vcaml and Mrcl expression on TAMs and MTMs from WT PyMT mice and MHCII + cells from CDl lc cre Rbpf fl PyMT. Dot plot shows back- gating of Mrcl - population from CD1 lc cre PyMT mice.
  • Figure 8 is a series of graphical representations of data showing that TAMs promote tumor tolerance by modulating the CD8 + T cell response.
  • Figure 8C are scatterplots of flow cytometric analysis of GzmB and PD-1 expression in CD8 + T cells infiltrating PyMT tumors at 8, 16, and 20 weeks. GzmB expression was determined by intracellular staining.
  • Figure 8E are scatterplots of flow cytometric analysis of PD-1 and GzmB expression in CD8 + T cells from WT PyMT or CD1 lc cre Rbpf /fl PyMT mice.
  • Figure 9 is a series of graphical representations of data showing that TAMs and PD- 1 + CD8 + T cells are enriched in the tumor tissue of Human Renal Cell Carcinoma (RCC) patients.
  • Figure 9A are scatterplots of TAMs (isolated from control tissue and renal cell carcinoma (RCC) tissue) gated on CD45 + , CD68 + and HLADR .
  • Figure 9B is a scatter plot of CD45 + CD3 + CD8 + T cells in RCC patients showing increased expression of PD- 1.
  • Figure 10 is a series of graphical representations of data showing that TAMs from human RCC patients are derived from inflammatory monocytes, and suppress GzmB expression in PD1 + CD8 + T cells.
  • Figure 10A are scatterplots of CD14 + HLADR + cells isolated from tumor tissues from two representative human RCC patients.
  • Figure 10B are scatterplots of flow cytometric analysis of GzmB and PD-1 expression in CD45 CD3 CD8 + T cells isolated from 2 different RCC tumors.
  • Figure 11 is a series of graphical representations of data showing that monocyte- derived TAMs and PD-1 GzmB " T cells are present in the tumor tissue of human breast cancer patients.
  • Figure 11 A is an image of immunofluorescence staining of CD 14 and HLADR in breast cancer tissue microarrays (TMAs). Arrows indicate the representative monocyte-derived TAMs.
  • Figure 11B is an image of immunofluorescence staining of PD-1 and GzmB in breast cancer TMAs.
  • Figure 12 are histograms of PD-Ll expression in T cells, B cells, and TAMs isolated from RCC patient.
  • the top curve in the upper two images (B-cells and TAMs) corresponds to PD-Ll expression curve; the bottom curve in the upper two images (B-cells and TAMs) corresponds FMO ctl (fluorescence minus one control). In the third image the two curves are substantially superimposed.
  • antibody refers to an immunoglobulin molecule which specifically binds with an antigen or to an antigen-binding fragment thereof.
  • antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive (antigen-binding) fragments or portions of intact immunoglobulins.
  • the antibodies may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab)2, as well as single chain antibodies (scFv) humanizedantibodies, chimeric antibodies, human recombinant antibodies and bi- and tri-specific antibodies.
  • T cell exhaustion is a state of T cell dysfunction that arises during many chronic infections and cancer. It is characterized by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion impedes control of infection and tumors.
  • CD8+ cytotoxic T cells that are exhausted are characterizes by a lack of ability to kill, as measured for example by substantially reduced or no expression of Granzyme B (GzmB) in a substantial fraction or percentage of s T cell population resident in a tumor.
  • GzmB Granzyme B
  • Immunosurveillance refers to a process by which cells of the immune system seek and recognize pathogens, such as bacteria and viruses, or pre-cancerous or cancer cells.
  • Subject means any animal (mammalian, human or other) patient that can be afflicted with cancer.
  • Tumor immunity refers to the process in which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is “treated” when such evasion is attenuated, and the tumors are recognized and attacked by the immune system.
  • An example of tumor recognition is tumor binding
  • examples of tumor attack are tumor shrinkage and tumor clearance.
  • Immunogenicity refers to the ability of a particular substance to provoke an immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the clearance of the tumor cells by the immune response.
  • T cell refers to a thymus derived lymphocyte that participates in a variety of cell- mediated adaptive immune reactions.
  • Helper T cell refers to a CD4 + T cell; helper T cells recognize antigen bound to MHC Class II molecules. There are at least two types of helper T cells, Thl and Th2, which produce different cytokines.
  • Cytotoxic T cell refers to a T cell that usually bears CD8 molecular markers on its surface (CD8+) and that functions in cell-mediated immunity by destroying a target cell having a specific antigenic molecule on its surface. Cytotoxic T cells also release granzyme, a serine protease that can enter target cells via the perforin- formed pore and induce apoptosis (cell death). Other names for cytotoxic T cell include CTL, cytolytic T cell, cytolytic T lymphocyte, killer T cell, or killer T lymphocyte. Targets of cytotoxic T cells may include virus-infected cells, cells infected with bacterial or protozoal parasites, or cancer cells. Most cytotoxic T cells have the protein CD8 present on their cell surfaces. CD8 is attracted to portions of the Class I MHC molecule. Typically, a cytotoxic T cell is a CD8 + cell.
  • Enhance T-cell function means to induce, cause or stimulate a T-cell to have a sustained or amplified biological function, or renew or reactivate exhausted or inactive T-cells.
  • Examples of enhanced T-cell function include: increased secretion of ⁇ -interferon from CD8 + T-cells, increased proliferation, increased antigen responsiveness (e.g. viral, pathogen, or tumor clearance) and increased expression of relative to such levels before the intervention.
  • the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 1 20%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.
  • T cell dysfunctional disorder refers to a disorder or condition of T-cells characterized by decreased responsiveness to antigenic stimulation.
  • a T-cell dysfunctional disorder is a disorder that is specifically associated with inappropriately increased signaling through PD- 1.
  • a T-cell dysfunctional disorder is one in which T-cells are anergic or have decreased ability to secrete cytokines, proliferate, or execute cytotoxic or cytolytic activity.
  • the decreased responsiveness results in ineffective control of a pathogen or tumor expressing an immunogen.
  • T cell dysfunctional disorders characterized by T-cell dysfunction include unresolved acute infection, chronic infection and tumor immunity.
  • Percentage when used to refer to a particular group of T cells means that fraction, whether expressed in percent or not, of an overall T cell population (such as the T cells resident in a tumor), which the particular T cell comprises.
  • Overall T cell population may be quantified by expression of a T cell antigen or marker characteristic of a group of T cells or of a certain property or by intensity of its signal (which is indicative of the number of T cells expressing the antigen or marker).
  • An overall T cell population may also be quantified by quantifying different T cell subtypes based on the expression, for instance, of particular cell surface proteins, or transcription factors.
  • the methods of the present disclosure may further comprise the steps of i) determining the density of cytotoxic T cells within tumor tissue or cell ii) comparing the cytotoxic T cell densities with predetermined reference values or with the density of all T cells resident in the tumor.
  • signal intensity can be used to describe a percentage or fraction. "Percentage" in reference to a macrophage population has an analogous meaning except the common antigen will be one (or more characteristic of macrophages rather than T cells.
  • “Stratify” when used to refer to patients means to sort patients according to some assessed risk or likely benefit, e.g., into those who are more (or less) likely to benefit from an anti-cancer therapy. The methods of present disclosure may thus be employed for stratifying cancer patients with regard to their susceptibility to treatment with a PD-1 inhibitor or other checkpoint.
  • tumors that contain high levels of TAM's also have cytotoxic T cells that do not perform their cytotoxic effector function. Patients with such tumors are likely to benefit from anti-PD-1 or anti-PDL-1 therapy.
  • “Therapeutically effective amount” refers to a sufficient amount of an agent to provide a desired biological result in alleviating, curing or palliating a disease or disorder. That result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease or disorder, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.
  • Biological sample can be a tumor sample such as a biopsy, or needle aspiration or an extract or otherwise manipulated sample of an excised tumor sample.
  • the present disclosure provides a method for stratifying cancer patients, notably those afflicted with solid tumors as likely to benefit from (and therefore good candidates for) therapy with checkpoint inhibitors, such as PD-1 or PD-1L inhibitors or decoy molecules based on whether at least a substantial percentage of CD 8+ tumor-infiltrating lymphocytes that express PD-1 and simultaneously fail to express GzmB; conversely, the patients are classified sa unlikely to benefit from such therapy if their CD8+ lymphocytes express GzmB, especially if a substantial percentage of their CD8+ lymphocytes also express low amounts of PD-1.
  • checkpoint inhibitors such as PD-1 or PD-1L inhibitors or decoy molecules based on whether at least a substantial percentage of CD 8+ tumor-infiltrating lymphocytes that express PD-1 and simultaneously fail to express GzmB; conversely, the patients are classified sa unlikely to benefit from such therapy if their CD8+ lymphocytes express GzmB, especially if a substantial percentage
  • solid tumors include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryon
  • Some of the most common solid tumors for which the methods of the present disclosure would be useful include of head-and-neck cancer, rectal adenocarcinoma, glioma, medulloblastoma, urothelial carcinoma, pancreatic adenocarcinoma, endometrial cancer, ovarian cancer, prostate adenocarcinoma, non-small cell lung cancer (squamous and adenocarcinoma), small cell lung cancer, melanoma, breast carcinoma, renal cell carcinoma, hepatocellular carcinoma. Investigations with PD-1 inhibitors, PD-1L inhibitors and decoys have concentrated on melanoma, renal cell carcinoma, nonsmall-cell lung carcinoma, breast cancer, and head and neck cancer.
  • the present disclosure provides a method for blocking cytotoxic T-cell exhaustion by a PD-1 or mediated by PD-1L expressing cell and adjusting the tumor microenvironment to inhibit tumor growth in a subject which has been previously determined to be a suitable candidate for checkpoint therapy as outlined above, the method comprising administering to the subject an amount of acheckpoint molecule effective to restore cytolytic activity in said T cells.
  • the present disclosure provides a method of inhibiting, suppressing or preventing immunosuppression of an anti-tumor or anti-cancer immune response in a subject mediated by TAMS that e, the method comprising administering to the subject an effective amount of an inhibitor of a cell adhesion molecule such as an inhibitor of Vcaml or Itgb8.
  • the present disclosure provides a method of treating a subject having a disease, disorder or condition associated with high percentage of TAMs by neutralizing same.
  • the present disclosure relates to methods for the selection of susceptibility and/or for the identification of (medical) responders versus non- responders.
  • the present disclosure provides a method of predicting whether a subject diagnosed with cancer will likely respond to an immune checkpoint inhibitor such as a PD-1 inhibitor, a PD-L1 inhibitor or a decoy molecule of either one.
  • the method comprises assessing the percentage of CD8+ tumor infiltrating lymphocytes in said subject that express PD1 and that are deficient in expression of GzmB; and considering the subject a good candidate for such treatment if at least a substantial percentage of CD8+ tumor infiltrating T cells from the subject express PD-1 and are deficient in expression of GzmB; conversely, identifying the subject as not a good candidate if at least a substantial percentage of CD8+ tumor infiltrating lymphocytes from said subject do not express PD-1 and/or express GzmB.
  • the method may comprise assessing the percentage of TAMs among the macrophages (or cells of myeloid origin) in said tumor is elevated (e.g., at least 20 or 30% or more).
  • the TAMs can be assessed for expression of PD-L1.
  • a high percentage of TAMs e.g., those expressing CDl lc and MHCII and one or more cell adhesion molecules) weighs in favor of considering a patient a candidate for treatment with a checkpoint inhibition if the patient's CD8+ lymphocytes also express high amounts of PD-1 and low amounts of GzmB.
  • the present disclosure relates to a method for stratifying cancer patients that are susceptible to treatment with PD-1 inhibitors.
  • the cancer patient is or has been identified (characterized or stratified) with a method as defined herein, for example with the flow cytometry, immunofiouresence, or IHC analysis described herein. Said identification or stratification method may be carried out prior to and/or during said treatment with a PD-1 inhibitor. Indeed, the same criteria used for stratification may be employed to assess treatment efficacy.
  • the methods of this invention may find use in treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer.
  • a variety of cancers may be treated, or their progression may be delayed, including but are not limited to solid tumors, such as those enumerated above.
  • the tumor has elevated levels of T-cell infiltration.
  • Macrophages Constitute the Major Myeloid Cell Population in Mammary Tumors Tumor progression is characterized by an accumulation of macrophages, both in experimental and clinical settings, yet the precise origin and function of these cells are largely unknown.
  • MMTV-pyMT Mammary Tumor Virus - promoter driven Polyoma Middle T antigen
  • MMTV-pyMT is a transgenic spontaneous mouse model that develops metastatic breast cancer (Guy et al, 1992a).
  • CDl lc is a fairly specific marker for DCs in steady state lymphoid organs
  • many tissue macrophages and activated monocytes in inflammatory settings express CDl lc (Miller et al., 2012).
  • Recent studies have revealed that DCs are derived from dedicated precursors, raising the possibility of defining DCs and macrophages by their respective differentiation pathways (Schraml et al., 2013).
  • This lineage-based classification of mononuclear phagocytes is corroborated by the identification of 'signature genes' expressed by multiple subsets of DCs and macrophages (Gautier et al, 2012; Miller et al, 2012).
  • TAMs defined in the present disclosure do not express CD115 on their surface and express only low levels of CD1 lb, while highly expressing MHCII and CD 11 c, they can be classified as macrophages instead of DCs based on their gene-expression profile. These findings highlight the importance of comprehensive transcriptome analyses to uncover tissue myeloid cells identity. Nevertheless the present inventors have identified that the subset of TAMs of interest here express MHC II and a monocyte marker, in humans CD 14.
  • Tumor-associated macrophages are enriched as tumors progress
  • Tissue -resident myeloid cells populate mammary tissues during steady state and are required for various stages of mammary gland development (Gouon-Evans et al, 2000). While macrophage populations are often defined by CDl lb expression, the TAMs described in this disclosure contained low levels of CDl lb expression ( Figures 1A and 1 B), and thus are being defined herein s MHCII hi and CDE116 low .
  • MHCII hl CDl lb hl cells Upon tumor growth, there was a decrease in the proportion of MHCII hl CDl lb hl cells normally found in untransformed wild-type (WT) mammary glands and an increase in TAMs (Figure 2A).
  • MHCII hl CDl lb hl cells were defined as "mammary tissue macrophages" or "MTMs” because they also phenotypically resembled macrophages.
  • TAM expansion was associated with the growth of individual tumors ( Figure 2C and 2D), indicating that CDl lb 10 TAMs, but not CDl lb hi MTMs, are bona fide tumor-associated macrophages that accumulate with increased tumor burden.
  • TAMs arise from a blood-borne precursor
  • TAMs tumor-associated macrophage population
  • a mammary tissue-resident macrophage population could result from either expansion of a mammary tissue-resident macrophage population or by active recruitment of macrophage precursors from the blood.
  • tissue resident macrophage populations are derived from embryonic precursors and are maintained independently from the blood through in situ proliferation (Hashimoto et al., 2013; Jenkins et al., (2011)).
  • a small MHCll hi CDl lb l0 population was observed in wild type mammary glands ( Figure 2A). To distinguish between these mechanisms, congenically-marked PyMT mice were connected using parabiosis.
  • the non-host chimerism of T cells in the blood and tumor was approximately 50% (Figure 3D)) as previously reported (Liu et al, 2009), indicating a shared blood circulation.
  • CCR2 DTR PyMT mice expressing diphtheria toxin receptor (DTR) under control of the Ccr2 locus were generated (Hohl et al., 2009).
  • DT treatment resulted in 96% depletion of tumor-associated monocytes, compared to 80%> depletion in Ccr2 ⁇ _ mice.
  • both MTM and TAM numbers were significantly reduced ( Figure 4C and 4D), suggesting that TAMs are derived from CCR2 + monocytic precursors, but require less input from the blood compared to MTMs.
  • TAMs were derived from a CCR2 + monocytic precursor, but that they required less input from the blood compared to the MTM population. This raised the possibility that a higher proliferative capacity of TAMs compared to MTMs might account for their differing precursor requirement. Indeed, TAMs expressed higher levels of Ki67 staining and EdU incorporation relative to MTMs ( Figure 5A and 5B). CCR2 monocytes differentiate into TAMs in vivo
  • TAMs are distinguished from MTMs by VCAM1 Expression
  • TAMs did not express AAM markers such as Yml, Fizzl, and Mrcl; instead, MTMs more closely resembled AAMs (Figure 6A).
  • Vcaml and Mrcl (CD206) proteins were detected in TAMs and MTMs, respectively ( Figure 6B).
  • TAMs differentiated from MTMs The lack of Mrcl expression on transferred monocytes at all examined time points suggested that TAM differentiation from monocytes was a distinct pathway, rather than MTM conversion.
  • Vcaml up-regulation was detected on TAMs as a late differentiation event (Figure 6C).
  • TAMs exhibit potent proliferative capacity upon their differentiation from inflammatory monocytes, which renders their accumulation in tumors less dependent on monocytic precursors.
  • TAM differentiation and proliferation represent an innate immune response to tumor growth.
  • Notch signaling is a conserved developmental pathway instrumental in cell fate specification in the hematopoietic lineage (Radtke et al., 2013).
  • canonical Notch signaling mediated by the key transcriptional regulator, RBPJ has recently been shown to control the lineage commitment, homeostasis and terminal differentiation of DC subsets (Catonet al, 2007; Lewis et al, 2011; Satpathy et al, 2013).
  • Mrcl + cells found within the CDl lb hi population in CD1 PyMT mice indicated that MTM differentiation was not compromised.
  • non- PyMT mice were analyzed.
  • MHCII hi CDl lb hi MTMs from WT and COl lc cie Rbpf /J1 mammary glands expressed Mrcl .
  • MHCII hi CDl lb 10 myeloid cells were present in mammary glands from WT mice. However, these cells did not express Vcaml, and their differentiation was not affected in CDl mice, suggesting that they are distinct from MHCII hi CDl lb 10 TAMs.
  • Macrophages found within developing tumors are generally defined as alternatively activated, to fit within the M1/M2 paradigm of macrophage polarization initially described in the context of infections models (Martinez et ai., 2009).
  • a macrophage phenotype that does not fall neatly into either the classically-activated/"M 1" or alternatively-activatedJ"M2" category is described, supporting claims that the Ml vs. M2 classification may be too restrictive in many settings (Mosser and Edwards, 2008).
  • the methods of this disclosure highlight the existence of a novel macrophage differentiation pathway active in tumors that is distinct from previously characterized pathways operating in infection models.
  • TAMs do not fit the classical Ml definition either as they do not express Tnf or Nos2 (data not shown), the findings disclosed here may be relevant in the context of studies showing a role for RBPJ in macrophage polarization away from M2 to a more Ml phenotype (Xu et ai., 2012).
  • TAMs Promote T Cell Tolerance in Mammary Tumors
  • CD1 PyMT mice had reduced tumor burden (Figure 8B).
  • Ccr2 ⁇ /_ PyMT mice which exhibited reduced MTMs ( Figure 4B)
  • tumor development was unaffected ( Figure X), implying a nonredundant function for RBPJ dependent TAMs in promoting tumor growth.
  • CDl lc + myeloid cells act as antigen-presenting cells, forming stable, yet unproductive, interactions with tumor-infiltrating T cells (Engelhardt et ah, Cancer Cell 21, 402-417 (2012)). It was hypothesized that one tumor promoting function of TAMs may be their control of the adaptive immune response.
  • Granzyme B (GzmB) is a cytolytic molecule important for tumor immunosurveillance.
  • PD-1 Programmed Death- 1
  • Figure X is an inhibitory co-receptor denoting "exhausted" T cells.
  • PD-1 GzmETCD8 T cells This observation paralleled TAM expansion, with PD-1 + cells making up -50% of late stage tumor-infiltrating CD8 + T cells ( Figure X).
  • the PD-1 population decreased, whereas the GzmB + population increased ( Figure X).
  • the T cell phenotype in Ccvl 1 mice was unchanged.
  • mice described here suggests an important function for TAMs in promoting immune tolerance to tumors through modulation of the CD8 + T cell response.
  • RBPJ was depleted in conventional DCs in these mice, Notch signaling in DCs primarily control CD4 + T cell priming and T helper 17 cell differentiation (Lewis et al., 2011; Satpathy et al., 2013).
  • findings of this disclosure are in line with a recent study in which a subset of myeloid cells that phenotypically resemble TAMs have been shown to interact with CD8 + T cells in the tumor microenvironment (Engelhardt et al., 2012).
  • TGF- ⁇ signaling in T cells inhibits the expression of GzmB and promotes the acquisition of an "exhausted" T cell phenotype characterized by high expression of PD-1 (Donkor et al., 201 1). Therefore TGF- ⁇ activation by TAMs may provide an important and unexplored mechanism by which TAMs directly influence T cell responses within developing tumors.
  • TAMs may also be promising targets for cancer immunotherapy. Indeed, recent studies have shown therapeutic potential for CSFl R-b locking antibodies in both breast cancer and glioblastoma multiforme (DeNardo, 201 1 ; Pyonteck et ai., 2013). Based on studies described herein, this treatment may work by primarily targeting macrophage precursors, as monocytes express CSFl R on their surface and TAMs express negligible surface CSFl R, but express intracellular CSFR1 comparable to their monocyte precursors.
  • Cancer treatment based on neutralization of a subset of TAM in and/or within vicinity of tumor tissue.
  • the present disclosure is directed to methods for neutralizing a subset of TAM in and/or within the vicinity of tumor tissue through localized delivery.
  • the present disclosure is directed to compositions for neutralizing TAM, for example compositions comprising nanoparticles.
  • a biopsy of tumor tissue is first obtained.
  • the composition of macrophages within the biopsy sample can be determined using well-known methods such as immunohistochemistry (IHC), immunofluorescence or flow cytometry, to measure the abundance of a specific subset of TAM within the tumor tissue that express high levels of CD1 lc, MHC II, and one or more adhesion molecules (e.g., Vcaml , Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially one or both of Vcaml and Itgb8).
  • IHC immunohistochemistry
  • MHC II MHC II
  • adhesion molecules e.g., Vcaml , Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially one or both of Vcaml and Itgb8.
  • this patient would be considered a candidate for the TAM neutralization therapy as described below.
  • this patient would also be considered a candidate for the TAM neutralization therapy as described below.
  • TAM tumorigenic effects
  • Activation of cell adhesion molecules such as CAMs and integrins, has been shown to be involved in pathological processes like tumor growth and metastasis formation.
  • the adhesion molecules are functional targets in addition to being markers for this TAM subset.
  • methods for neutralizing TAM include inhibition of the expression of cell adhesion molecules in TAM, including one or more of Vcaml, Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and/or Itgb8 through blocking their extracellular domains.
  • the blocking of these adhesion molecules can be achieved by delivering to the tumor specific binding molecules that bind to one or more of the foregoing cell adhesion molecules, especially Vcaml and/or Itgb8, and inhibit their bindings to their ligands.
  • Nonlimiting examples of such molecules are humanized antibodies or fully human recombinant antibodies.
  • Antibodies against cell adhesion molecules such as human Vcaml, Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and Itgb8, can be developed by standard methods by those skilled in the art or can be purchased commercially.
  • FDA has approved the clinical use of an anti-ltgb4al antibody (Natalizumab) for treating multiple sclerosis.
  • the patent and scientific literature discloses antibodies against Vcaml, Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal and such antibodies are commercially available.
  • Vcaml Catalog #: MAB2144, clone P3C4, Millipore
  • Itgb8 Catalog #: ARP45250_P050, Aviva Systems Biology
  • Itga8 Catalog #: HPA003432, Sigrna-Aldrich
  • Itga9 Catalog #: ABIN394538, Antibodies-online
  • Itgb5 Catalog #: ab 15459, Abeam
  • Itga6 Catalog #: AM32339PUN, Acris Antibodies
  • Itgav Catalog #: MBS670197, MybioSource
  • Itgax Catalog #: GTX50770, GeneTex
  • Itgal Catalog #: ABIN1105768, Antibodies-online.
  • Vcaml antibodies have been described, e.g., in VVO 93/14220 and Integrin alpha v beta-8 antibodies have been described in WO 20131 026004. Since cell adhesion molecules are important factors in a variety of diseases including inflammation and atherosclerosis, there are many other commercially available antibodies against them, and some of them are currently in clinical trials, so they are suitable for human use.
  • the present disclosure describes a method to deliver antibodies against one or more adhesion molecules (e.g., Vcarnl, Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and Itgb8) locally to the tumor tissue, or alternatively systemically, to neutralize TAM.
  • adhesion molecules e.g., Vcarnl, Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and Itgb8
  • NPs antibody- functionalized nanoparticles
  • a humanized monoclonal Antibody against Vcam-1 can be delivered through NPs to tumor tissues in a breast cancer patient.
  • NPs can be liposomes, albumin-bound NPs, polymeric NPs, denrimers metal NPs and molecular targeted NPs.
  • albumin NPs have been used as anticancer agents with the 2005 approval of Abraxane to treat metastatic breast cancer (Miele E et al., Intl. J. of Nanomedicine 2009:499-105).
  • An antibody to Vcam-1 can be conjugated to albumin NP, and then the conjugated NPs can be delivered intravenously in a patient with breast cancer.
  • an anti- VCAM-1 antibody can also be conjugated to docetaxel-loaded NPs of biodegradable copolymers.
  • methods for neutralizing TAM include inhibition of the expression of one or more of the foregoing cell adhesion molecules in TAM (e.g., Vcaml , Itgb8, Itga8, Itga9, !tgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and Itgb8) through silencing of the expression of these genes using gene therapy.
  • anti-sense or RNAi oligos can be designed against one or more of the adhesion molecules listed above by using standard method or through various vendors.
  • Dharmacon provides on a commercial basis a full line of siRNAs against human cell adhesion molecules, including Vcaml (Catalog #: L- 013351-00-0005, Dharmacon), Itgb8 (Catalog #: L-008014-00-0005, Dharmacon), Itga8 (Catalog #: L-010699-00-0005, Dharmacon), Itga9 (Catalog #: L-008005-00-0005, Dharmacon), Itgb5 (Catalog #: L-004125-00-0005, Dharmacon), Itga6 (Catalog #: L- 007214-00-0005, Dharmacon), Itgav (Catalog #: L-004565-00-0005, Dharmacon), Itgax (Catalog #: L-008009-00-0005, Dharmacon), agal (Catalog #: L-008007-00-0005, Dcaml (Catalog #: L-
  • the present method of delivering anti-sense or RNAi oligos against one or more adhesion molecules e.g., Vcaml , Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or II gal
  • one or more adhesion molecules e.g., Vcaml , Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or II gal
  • NPs nanoparticles
  • an anti-VCAM-1 siRNA can be packaged in protamine, which can then be joined to an antibody fragment against a surface marker protein of certain breast cancer cells, such as HER2, or be joined to an antibody fragment against CD1 lc protein, which is highly expressed on the surface of the subset of TAM that are reported here, using published protocols (Yao YD et al, Sci Transl. Med. 2012 4(130): 130ra48).
  • the functionalized NPs carrying anti-sense or RNAi oligos against adhesion molecules can be injected or infused intravenously or directly to the tumor tissue by following standard procedures for administering antibody based therapeutics.
  • methods for neutralizing TAM within the subset disclosed herein include inhibition of the expression of cell adhesion molecules in TAM (e.g., Vcaml , Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and Itgb8), through small molecule inhibitors.
  • cell adhesion molecules in TAM e.g., Vcaml , Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially Vcaml and Itgb8
  • small molecule inhibitors of major cell adhesion molecules have been developed.
  • B 105192 a small molecule inhibitor for VCAM1 , has shown potential in cancer therapy (Ramirez P et al, Blood 2009: 114: 1340-43).
  • the present disclosure includes a method for delivering small molecule inhibitors against one or more of the foregoing adhesion molecules locally to the tumor tissue to neutralize TAMs. In one embodiment, this could be achieved through nanoparticies (NPs). Natural polymers such as albumin, chitosan and heparin have been used for the delivery of small-molecule drugs.
  • NPs include albumin- based formulation of paclitaxel, N-(2-hydroxypropyi)-methacrylamide copolymer (HPMA), polystyrene-maleic anhydride copolymer (SMA), polyethylene glycol (PEG) and poly-L-glutamic acid (PGA), and are summarized and reviewed by Chen ZG. (Chen ZG, Trends Mol Med. 2010 16(12):594-602).
  • an anti-VCAM-1 small molecule inhibitor can be conjugated to an albumin based formulation.
  • the functionalized NPs carrying the small molecule inhibitors can be injected or infused intravenously or directly to the tumor tissue by following standard procedures for administering antibody-based therapeutics.
  • Each of the foregoing TAM-neutralizing agents are administered in effective amounts. Such amounts are either known or can be determined by methods known in the art.
  • nanoparticies will be coated with at least l,000/cm2 of specific binding molecule of molecules and preferably with a higher density such as over 2000, over 3000 or over 6,000 molecules per /cm2.
  • the number of nanoparticies contained in the composition delivered to the tumor can be for exam pie 0.5 x 106 or higher, such as 1 x 10 6 , 10 7 , 10 8 , etc.
  • Notch signaling pathway promotes cell survival, angiogenesis and treatment resistance in numerous cancers, making it a promising target for cancer therapy, It also crosstalks with other important oncogenes, such that a single intervention in the Notch pathway can affect several signaling pathways.
  • Notch pathway plays a major role in TAM differentiation while MTM differentiation is independent of Notch.
  • Notch signaling especially the Notch transcription factor, RBPJ, is specifically required for the differentiation of inflammatory monocytes to macrophages during tumor development, and is not required for macrophage differentiation in mammary glands during homeostasis. Indeed, inflammatory monocytes are unable to terminally differentiate into TAM in the absence of Notch signaling.
  • prevalence of TAM in tumor tissue from a cancer patient compared to prevalence of MTM from the same tissue may be used as a clinical marker to identify breast cancer patients who are likely to benefit from Notch therapy.
  • prevalence of TAM as measured within a population of patients, e.g., the percentile of TAM prevalence of this patient within a population, may also be used as a clinical marker to identify cancer patients who are likely to benefit from Notch therapy.
  • the present disclosure is directed to a method to identify a cancer patient who is likely to be a responder to Notch therapy and therefore a candidate for such therapy.
  • a biopsy of a tumor tissue sample is obtained.
  • the macrophage composition of the biopsy tissue sample can be measured by a variety of standard methods such as immunohisto chemistry, immunofluorescence, or flow cytometry.
  • the count of cell population representing the subset of TAM with high expression of CD 11c, MHC II, and one or more of adhesion molecules e.g., one or more of Vcaml, Itgb8, Itga8, Itga9, Itgb5, Itga6, Itgav, Itgax, and/or Itgal, especially one or both of Vcaml and Itgb8) can be quantified or semi-quantified by these methods.
  • this patient would be considered a candidate who is likely to benefit from Notch therapy.
  • the prevalence of the present TAM subset can be compared to that of MTM's in the same tumor.
  • TAM cells within the total number of TAM and MTM cells from the same tumor tissue sample is above a certain value (e.g., higher than 75%, to higher than 50%, to higher than 25%, to higher than 10%>), this patient would also be considered a candidate who is likely to benefit from Notch therapy.
  • CSF1 plays an important role in the differentiation of monocytes into macrophages.
  • CSF1 plays an important role in the differentiation of monocytes into macrophages.
  • CSF1 overexpression accelerates tumor growth and metastasis (Un E et al., J Exp Med 193, 727-40)
  • Recent studies have shown therapeutic potential for CSF1 R-blocking antibodies in both breast cancer and glioblastoma multiforme (DeNardo DG et al.
  • CSFlR-b locking therapy may work by primarily targeting macrophage precursors, as monocytes express CSF1R on their surface and TAMs express negligible surface CSF1R, but express intracellular CSFR1 comparable to their monocyte precursors.
  • a high TAM prevalence in a cancer patient represents a heightened differentiation of monocyte precursors to TAM
  • a high prevalence of TAM in tumor tissues from a cancer patient may be used as a clinical marker to identify cancer patients who are likely to benefit from CSFl receptor inhibition therapy.
  • the present disclosure includes a method to identify a cancer patient who is likely to be a responder to CSFl R-b locking therapy.
  • a tumor tissue sample is obtained from the patient.
  • the macrophage composition of the biopsy tissue sample can be measured by a variety of standard methods such as immunohistochemistry, immunofluorescence, flow cytometry by those skilled in the art.
  • the count of cell population representing the subset of TAM with high expression of CD 1 1c, MHC II, and one or more of adhesion molecules can be quantified or semi- quantified by these methods.
  • the count of this subset of TAM in this patient can be compared to a reference value or a reference distribution (e.g., distribution of cell counts of this subset of TAM within a population of cancer patients).
  • this patient would be considered a candidate who is likely to benefit from CSFl receptor blockade therapy.
  • the prevalence of the present TAM subset can be compared to that of MTM's in the same tumor.
  • TAM cells within the total number of TAM and MTM cells from the same tumor tissue sample is above a certain value (e.g., higher than 75%, to higher than 50%, to higher than 25%, to higher than 10%), this patient would also be considered a candidate who is likely to benefit from CSFl receptor blockade therapy.
  • a certain value e.g., higher than 75%, to higher than 50%, to higher than 25%, to higher than 10%
  • This patient would also be considered a candidate who is likely to benefit from CSFl receptor blockade therapy.
  • Stratifying cancer patients according to whether they would benefit from checkpoint inhibitor therapy based on a high percentage of TAMs and/or CD8+ cytotoxic Tt cells that are dysfunctional to determine whether they would benefit from checkpoint targeted therapy
  • the prevalence of TAMs has been associated with a high proportion of cytotoxic T cells expressing CD8 which have dysfunctional effector function and appear to be exhausted.
  • Programmed death 1 is an immune inhibitory receptor expressed on several immune cells, particularly cytotoxic T cells. It interacts with two ligands, programmed death ligand 1 (PD-Ll) (B7-H1, CD274) and PD-L2 (B7-DC). While PD-L2 is expressed primarily on macrophages and dendritic cells, PD-Ll is expressed on tumor cells, as well as other immune cells. PD-1 is expressed in tumor- associated lymphocytes from many types of cancers, such as infiltrating T cells. The interaction of these ligands with PD-1 inhibits T-cell activation and effector T cell function in many cancer types as well.
  • High PD-Ll expression has been observed in a variety of human cancers, and interaction of PD-Ll with PD-1 can induce inhibition or apoptosis of tumor-specific T cells.
  • PD-Ll expression correlated with poor prognosis and reduced overall survival irrespective of subsequent treatment.
  • Blockade of PD-1 /PD-Ll ligation using antibodies to PD-Ll has been shown to restore and augment T cell activation in many systems. Patients with advanced cancer benefit from therapy with a monoclonal antibody to PD-Ll (Brahmer et al., New Engl. J. Med. (2012)).
  • Anti-PD-1 monoclonal antibodies that block binding of PD-Ll to PD-1 have been shown to have anti-tumor activity against a variety of tumor types, with early human clinical data suggesting that patients whose tumors express PD-Ll are more likely to respond to anti-PD-1 therapy (Iwai et al., PNAS 99: 12293-12297 (2002); Ohigashi et al, Clin Cancer Res 11 :2947-2953 (2005); Ghebeh et al, Neoplasia 8: 190-198 (2006); Hamanishi, J et al, PNAS 104:3360-3365 (2007); Yang et al. Invest Ophthalmol Vis Sci. 49(6):2 18-2525 (2008); Gao et al, Clin Cancer Res 15:971-979 (2009); Brahmer J.R. et al, J Clin Oncol. 28:3167-3175 (2010).
  • PD-1- and PD-Ll -directed therapy is currently undergoing investigation in several types of malignancy, inhibitor therapy (for example using anti PD-1 or anti- PD-L1 antibodies) has been most studied in patients with metastatic melanoma.
  • Antibodies targeting PD-1 in clinical development include nivolumab (Opdivo also known as BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (also known as MK-3475, formerly lambrolizumab), pidilizumab (CT-011) and AMP-224 (Amplimmune/Glaxo SmithKline), the latter, a PD-1 decoy, are also under investigation (Table 1).
  • Pembrolizumab has gained FDA approval for patients with advanced or unresectable melanoma (Table 1, below).
  • the first antibody to target PD-Ll in clinical trials was MDX-1105. Additional PD- Ll antibodies in clinical development include MPDL3280A (Genentech), MEDI4736 (Medlmmune), and MSB0010718C (EMD Serono). Table 1 lists the most extensively studied anti PD-1 and PD-Ll inhibitory agents but the listing is by way of example and not limitation. . For an instructive review of this topic and its applicability to many types of cancers see Kim JW and Eder JP, Oncology, 28 (11 Suppl 3), (2014) incorporated by reference in its entirety While these therapies have shown great promise only a fraction of the patients respond.
  • nivolumab and ipilimumab were investigated in a Phase I trial. Although the combination of nivolumab and ipilimumab has been the most extensively reported PD-1 /PD-Ll combination study thus far, ongoing trials are combining PD-1/PD-L1 agents with small-molecule targeted therapy, chemotherapy, and vaccines in a variety of tumor types and disease settings.
  • the present disclosure proposes a method for stratifying cancer patients to partition them into likely responders and unlikely responders to targeted therapy with PD-1 and/or PD-L1 inhibitors, each alone or in combination with other anti-cancer agents.
  • the experimental results described here indicate that the existence of the TAMs described herein (which have been found to express high amounts of PD-L1— see Example 12) together with the exhausted T cell phenotype CD8+PD-l+GzmB " forms a basis for stratifying patients that would benefit from administration of checkpoint inhibitors for example anti-PD-l/anti-PDL-1 antibodies.
  • the results described herein are consistent between an animal model of breast carcinoma and tissue samples from human renal cell carcinoma and from human breast cancer, indicating that the findings made and conclusions drawn here are applicable to diverse types of solid tumors, such as those exemplified elsewhere in this disclosure.
  • Example 10 Based on Example 10, it is clear that patients can be classified as less likely to benefit from PD-1 therapy if they have high GzmB expression in the CD8+ T cells despite high PD-1 expression. These patients might not be good candidates for PD-1 or PD-L1 inhibitor therapy. It is anticipated that there will be another category of patients who will have intermediate values of PD-1 and GzmB expression in their CD8+ T cells from the tumor. Nonlimiting examples of such intermediate values are for example 20%-30% CD8+PD-1+ and 30-60 % GzmB " among CD8+PD-1+ T cells. The treating physician might decide in favor of PD-1/PD-L1 inhibitor therapy if it is anticipated that further deterioration of the effector function of the CD8+ T cells will occur in the future. Other factors such as the patient's co-morbidities and performance status may influence the chosen treatment option but the values of the markers disclosed herein will be taken into account.
  • Measurements of these markers may also be indicated as a method for testing treatment efficacy after treatment has commenced. Tumor infiltrating T cells may be collected and tested in the same manner. If these markers, especially GzmB are increasing, in that the percentage of T cells that express GzmB and PD-1 is decreasing the treatment is likely effective and vice versa.
  • mice MMTV-PyMT mice (Jackson laboratory) were backcrossed to the C57B1J6 background for 10 generations.
  • the CD45.1 + congenic mice were purchased from Taconic Farms.
  • 114 ⁇ ' ⁇ , Rag! '1 " , and R26-stop-YFP reporter mice were purchased from Jackson laboratory.
  • Rbpf ,fl mice were provided by Tasuku Honjo and crossed to CD1 lc cre mice provided by Boris Reizis.
  • Ccrl'- -, CCR2 DTR , and CCR2 GFP reporter mice were provided by Eric Pamer. Littermate controls were used in all experiments when possible.
  • Rbpf ,fl mice were used as wild type controls in
  • mice were maintained in a specific pathogen-free facility and animal experimentation was conducted in accordance with institutional guidelines. Parabiosis
  • mice were generated as reported (Waskow, 2010) using female 6-8 week old age- and weight-matched CD45.1 + or CD45.2 + PyMT mice and maintained for approximately 12 weeks post- surgery. Briefly, matching skin incisions were made from the elbow to the knee of each mouse. Forelimb and hindlimb connections were made with sutures and skin incisions were closed using woundclips.
  • CCR2 + monocytes 16-week-old CCR2 DTR PyMT mice were injected i.p. with 5 ⁇ g/kg diphtheria toxin (DT, Sigma) every three days for approximately three weeks.
  • DT diphtheria toxin
  • bone marrow from CCPv2 GFP mice was collected and depleted of neutrophils, T cells, B cells, and DCs using rat anti-mouse antibodies for Ly6G (Eric Pamer), CD4, CD8, and MHCII (Bio X Cell) in combination with goat anti-rat BioMag beads (Qiagen).
  • the depleted bone marrow was then sorted based on GFP-positivity.
  • the GFP+ cells were injected i.v. into congenically-marked CCR2 DTR recipient mice that received DT 24 hours prior to transfer.
  • Tumor tissues from sacrificed mice were prepared by mechanical disruption followed by 1.25 hr treatment with 1.5 mg/ml Collagenase Type 3 (Worthington Biochemical) and 4 ⁇ g/ml DNase I (Sigma) at 37 C with periodic vortexing. Digested tissues were mashed through 70 mm filters, layered in a 44% and 66% Percoll gradient (Sigma), and centrifuged at 3000 rpm for 30 min without brake. Cells at the interface were collected and analyzed by flow cytometry. Flow cytometry
  • Fluorochrome-conjugated or biotinylated antibodies against CD45.1 (clone 104), CD45.2 (A20), MHCII (M5/114.15.2), CDl lc (N418), TCR-13 (H57-597), CD4 (RM4-5), CD8 (17A2), PD-1 (RMP1-30), CD115 (AFS98), F4/80 (BM8), c-Kit (2B8), nBTLA (8F4), and Flt3 (A2F10) were purchased from eBioscience.
  • Antibodies against mCD45 (clone 30-F11), Ly6C (AL-21), Ly6G (1A8), B220 (RA3-6B2), CD26 (H194-112), CD64 (X54-5/7.1), Ki67 (B56), and VcamlJCD106 (429) were purchased from BD Biosciences.
  • Anti-CD l ib (clone Ml/70) and anti- GzmB (GB11) were purchased from Invitrogen.
  • Anti-MerTK was purchased from R&D Systems (#BAF591).
  • AntiMrcl/ CD206 was purchased from BioLegend (C068C2). All antibodies were tested with their respective isotype controls. Splenocytes were depleted of erythrocytes by hypotonic lysis.
  • FACS sorted cells (populations I-III) were loaded on slides using the Cytospin 4 (Thermo Scientific) and dried slides were stained with the Diff-Quik Wright-Giemsa Stain Set (Dade Behring). Images were captured at an original magnification of 40x using a Zeiss Axioplan 2 widefield microscope. Microarray and computational analysis
  • CDl lb + splenic DCs and TAMs were isolated from 20-week-old PyMT mice by FACS and RNA was prepared with the miRNeasy Mini Kit according to the manufacturer's instructions (Qiagen). MTMs, TAMs, and macrophages from CO ⁇ ⁇ c c Rbpf 11 PyMT mice were sorted directly into Trizol (Sigma). RNA isolation, amplification, labelling, and hybridization to M430 2.0 chips (Affymetrix) were carried out at the Genomics Core Facility of Memorial Sloan-Kettering Cancer Center.
  • Probe expression levels were calculated by quantile normalization and summarized using median polish as implemented in the robust multi-array (RMA) procedure (Irizarry et al., 2003) available via the affy BioConductor package for the R statistical software project. Differentially expressed genes were determined using the limma BioConductor package with a p-value threshold of 0.05. Expression levels were preprocessed prior to principal component analysis (PCA), which entailed mean-centering of gene-level log-transformed expression values.
  • PCA principal component analysis
  • the SVM was trained on the same data set as in the PCA analysis to categorize dendritic cells and macrophages, again employing a Leave-one-out cross validation scheme, and the resulting model was used to predict the probabilities of the test set of belonging to either of the classes.
  • Zbtb46 5'-agagagcacatgaagcgaca-3' and 5'- ctggctgcagacatgaacac-3'; Mafb 5'-ggggccaaattgacatacac-3' and 5' cttaggacgcaaagcctgtc-3'; Fizzl 5' -cccttctcatctgcatctcc-3' and 5'- aggaggcccatctgttcata-3' Mrcl 5'-atgccaagtgggaaatctg-3' and 5'tgtagcagtggcctgcatag- 3' Yml 5' -ccagcatatgggcatacctt-3' and 5' -
  • Paraffin-embedded breast cancer sections were dewaxed by xylene, and rehydrated by gradient ethanol solutions. . The antigen retrieval was conducted, and the sections were blocked by bovine serum albumin plus serum, and stained with programmed cell death protein 1 (PD-1) and Granzyme B (GzmB) or CD 14 and HLADR antibodies. A Leica upright confocal microscope was used to capture images.
  • PD-1 programmed cell death protein 1
  • GzmB Granzyme B
  • CD 14 and HLADR antibodies CD 14 and HLADR antibodies
  • Macrophages Constitute the Major Myeloid Cell Population in Mammary Tumors
  • MMTV-PyMT spontaneous mammary tumor model was used to study the role of myeloid cells in the development and progression of cancer (Guy et al, Mol. Cell. Biol. 12, 954-961, (1992)). Mice were bred onto the C57BL16 background, and it was found that myeloid cells made up more than 50% of the CD45 + leukocyte population in filtrating advanced mammary tumors of 20-week-old PyMT mice ( Figure 1A).
  • This myeloid population was heterogeneous, with three major populations (I, II, & III) distinguishable by morphology and differential cell surface expression of MHCII, CD1 lb, Ly6C, Ly6G, CD1 lc, CD115, and F4/80 ( Figures 1A and IB). Whereas population II shared many features with Ly6C+ inflammatory monocytes and population III phenotypically resembled neutrophils, the largest subset, population I, expressed classical dendritic cell (DC) markers, MHCII and CD 11c, as well as a macrophage marker, F4/80. This finding highlights the confusion currently surrounding identification of myeloid cell subsets during tumor progression.
  • DC dendritic cell
  • Tissue Macrophages are PhenotypicaUy Distinct from TAMs
  • Macrophage populations in mammary glands and other tissues are often defined by CD l ib expression.
  • the TAMs described in this disclosure had low levels of CD l ib expression ( Figures 1A and IB).
  • myeloid cell populations present in PyMT mice were determined by flow cytometry at 8, 16, or 20 weeks of age when tumors evolved from hyperplastic lesions to invasive carcinomas (data not shown).
  • WT untransformed wild type
  • MHCll hi CDl lb hi cells were defined as "mammary tissue macrophages" or "MTMs".
  • MTMs mammary tissue macrophages
  • PyMT mice all 10 mammary glands are transformed, but often with different latencies (Lin et al., 2003).
  • TAMs was associated with the growth of individual tumors isolated from the same mouse ( Figures 2C and 2D). FACS plots were gated on CD45 + leukocytes. Error bars present in the Figure 2 represent standard error measurement (s.e.m.).
  • TAMs originated from a precursor in the blood or expanded from a tissue -resident population
  • the bloodstreams of mice were connected using parabiosis.
  • the animals were sacrificed soon after tumors became palpable, approximately 12 weeks after surgery (Figure 3 A).
  • Circulating monocytes are critical progenitors for macrophages (Geissmann et al., 2010b). To determine whether TAMs and MTMs were maintained by Ly6C + inflammatory monocytes expressing the chemokine receptor CCR2, PyMT mice were crossed to the CCR2-deficient background, and the relative abundance of tumor associated myeloid cells was assessed at 16 and 20 weeks of age.
  • Ccr2 _/_ mice exhibit reduced numbers of circulating inflammatory monocytes due to impaired bone marrow egressAt 16 weeks, the frequencies of monocytes and MTMs in Ccr2 _/ ⁇ PyMT mice were reduced to about 16% and 29%, respectively, of the levels observed in Ccr2 +/+ PyMT mice ( Figures 4A and 4B), implying that MTMs are constitutively repopulated by inflammatory monocytes.
  • the percentage of TAMs in Ccr2 _/" PyMT mice was slightly decreased compared to controls ( Figures 3C and 3D), although this reduction was not statistically significant.
  • CCR2 DTR mice in which the expression of diphtheria toxin receptor (DTR) was under the control of the Ccr2 locus were used (Hohl et al, 2009).
  • CCPv2 DTR mice were crossed to the PyMT background.
  • CCR2 DTR PyMT mice were treated with DT every three days for three weeks (total of 7 treatments), which depleted more than 95% tumor-associated monocytes (Figures 4C and 4D). Under these conditions, both MTMs and TAMs were substantially reduced ( Figures 4C and 4D).
  • data were pooled from at least 3 independent experiments.
  • TAMs Proliferate upon Their Differentiation from Inflammatory Monocytes
  • TAMs from 16-week-old PyMT mice expressed higher levels of the cellular proliferation marker Ki67 than MTMs from the same animal ( Figure 5 A).
  • acute labeling of dividing cells with the modified nucleoside EdU in 16- week-old PyMT mice revealed approximate 3 times more EdU-positive TAMs than MTMs ( Figure 5B), demonstrating high self-renewal capacity of TAMs in developing tumors.
  • TAMs are not AAMs, and can be Distinguished from MTMs by Vcaml Expression
  • MTMs and TAMs were purified by fluorescence-activated cell sorting, and gene-expression profiling experiments were performed with Affymetrix oligonucleotide arrays.
  • several other integrins and the integrin receptor Vcaml were upregulated in TAMs ( Figure 6A).
  • Vcaml and Mrcl also known as CD206, an AAM-associated molecule
  • CD206 an AAM-associated molecule
  • mice were crossed to mice with the IL-4-deficient background. I14 ⁇ '- PyMT mice had normal proportions of CD1 lb lo Vcaml + TAMs. Furthermore, when mice were crossed onto RagT 1' background, it was found that TAM differentiation was not dependent on the adaptive immune system, with an increase in TAM proportion in Ragl '1' mice due to the loss of T and B cells. There was no change in MTM proportion in Ragl '1' animals. These observations suggest that TAMs are not AAMs, and their differentiation is not secondary to tumor-elicited adaptive immune responses.
  • TAMs expressed increased levels of several genes positively regulated by the Notch signaling pathway (Satpathy et al, 2013) and conversely, MTMs expressed higher levels of genes negatively regulated by Notch signaling (Figure 7A), implying a functional Notch dependent program in TAMs.
  • CDl lc cre transgenic mice that efficiently deleted floxed DNA sequences in TAMs but not in monocytes or neutrophils.
  • CDl lc cre transgenic mice were crossed with mice carrying floxed RBPJ alleles (Rbpf fl ), which were further bred to the PyMT background. Strikingly, CDl lc cre PyMT mice exhibited a selective loss of the MHCll hi CDl lb l0 TAM population ( Figure 7B). However, a MHCll hl CDl lb hl population still remained in these animals ( Figures 7B and 7C).
  • TAMs Promote T Cell Tolerance to Mammary Tumors
  • CD1 PyMT mice showed a significant decrease in total MHCII + cells (Figure 8A), which was accompanied by a delay in tumor growth ( Figure 8B).
  • Figure 8A tumor development was unaffected in Ccr2 _/ ⁇ PyMT mice ( Figures 4A and 4B), which showed a reduction in MTMs but not TAMs.
  • CDl lc + myeloid cells act as antigen- presenting cells, stably interacting with T cells in the tumor microenvironment, but that this interaction results in functionally inactive T cells (Engelhardt et al., 2012). Therefore, it was hypothesized that one critical function of TAMs in promoting tumor growth may be their control of the adaptive immune response. To address this question, phenotype of cytotoxic CD8 + T cells was analyzed during tumor development. Granzyme B (GzmB) is a cytolytic molecule involved in the induction of tumor immunosurveiUance and marks CD8 + T cells with a cytotoxic effector phenotype.
  • Granzyme B GzmB
  • PD-1 is an inhibitory co-receptor that denotes T cells that have acquired an "exhausted” phenotype.
  • PD-l + GzmB " CD8 T cells As PyMT tumors progressed, an increase in PD-l + GzmB " CD8 T cells was observed (Figure 8C). The observed increase in T cell exhaustion paralleled the expansion of TAMs, with PD-l + GzmB " CD8 + making up -50% of tumor-infiltrating CD8 + T cells ( Figure 8D).
  • CD1 PyMT mice lacking mature TAMs the PD- l GzmB " exhausted population was markedly reduced, and the PD-l " GzmB + effector population was substantially increased ( Figures 8E and 8F).
  • TAMs may not need take TAMs into account in the stratification process but can simply check for CD8+PD-l+GzmB " T cells. It may be desirable however to check for both TAMs and exhausted cytotoxic T cells displaying the forgoing phenotype.
  • TAMs and PD-1 +CD8+ T Cells are Enriched in the Tumor Tissue
  • cancer cells isolated from patients diagnosed with renal cell carcinoma were tested for the presence of TAMs and PD-1 CD8 + T cells.
  • TAMs were evaluated based on the expression of CD45, CD68, and HLADR expression in samples isolated from normal kidney tissue and cancer kidney tissue from the same patient.
  • samples were assessed for CD68 and HLADR expression.
  • Flow cytometric analysis of CD68 and HLADR expression showed that compared to the normal kidney tissue (control), increased number of CD68 HLADR + TAMs was present in tumor tissue of -20% RCC patients ( Figure 9A).
  • CD3 + T cells were initially separated based on the expression of CD4 and CD8 co-receptors.
  • CD8 + cells were evaluated for the content of PD-1 + cells.
  • increased number of CD8 + T cells that express the co-inhibitory receptor PD-1 were observed in the tumor tissue of RCC patients.
  • TAMs from Human RCC Patients Are Derived from Inflammatory Monocytes, and Suppress GzmB Expression in PD-1+CD8+ T Cells
  • CD14 HLADR + monocytes are considered to constitute a group of inflammatory monocytes.
  • TAMs isolated from human RCC patients are derived from inflammatory monocytes
  • cancer cells isolated from tumor tissues from two RCC patients both had T cells that were PD-1+ but one had T cells that were GzmB+ and the other did not) were evaluated.
  • Patient RCC314 contained a limited percentage of TAMs, while the RCC321 patient was
  • CD3 CD8 PD-1 + cells were evaluated for the GzmB expression.
  • the tumor tissue of RCC314 patient contained two sizeable populations of CD3 CD8 PD-1 + cells with or without the expression of GzmB, whereas
  • CD3 CD8 PD-1 + T cells from RCC321 patient contained mostly GzmB-negative population ( Figure 10B).
  • tumor tissue from breast cancer patients was tested for the presence of TAMs and PDl GzmB cells.
  • Immunofluorescence staining of CD 14 and HLADR in breast cancer tissue microarray showed only few HLADR + cells in the normal breast tissue, while tumor breast tissue contained abundant CD14 + HLADR + monocyte-derived TAMs ( Figure 11A - arrows indicate the representative monocyte-derived TAMs).
  • PD-1 and GzmB exhibited reciprocal relationship within breast cancer cells.
  • Cell expressing high levels of GzmB contained low levels of PD-1, while cells expressing high amounts of PD-1 exhibited low levels of GzmB ( Figure 11B).
  • Example 12 TAMs express PD-1 ligand PD-L1
  • PD-L1 and PD-L2 two ligands for PD-1 have been identified, PD-L1 and PD-L2.
  • PD-L1 is the predominant ligand, while PD-L2 has a much more restricted expression pattern (Rozali et al., Clin Dev Immunol, 2012:656340).
  • an inhibitory signal is transmitted into the T cell, which reduces cytokine production and suppresses T-cell proliferation.
  • TAMs suppress GzmB expression in PD-1+CD8+T cells.
  • TAMs possibly express the ligand for PD- 1, PD-1L. Indeed, TAMs showed significant increase of PD-L1 expression in human RCC patients. PD-L1 expression was detected in TAMs, not in B or T lymphocytes ( Figure 12).
  • Notch-RBP-J signaling controls the homeostasis of CD8- dendritic cells in the spleen. J Exp Med 204, 1653- 1664.
  • LIBSVM A Library for Support Vector Machines. Acm Transactions on Intelligent Systems and Technology 2.
  • CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages. Cancer Cell 16,91-102.
  • Inflammatory monocytes facilitate adaptive CD4 T cell responses during respiratory fungal infection.
  • Colony- stimulating factor 1 promotes progression of mammary tumors to malignancy. J Exp Med 193, 727- 740.
  • CSF-1 R inhibition alters macrophage polarization and blocks glioma progression. Nature medicine 19, 1264- 1272. Qian, B.Z., and Pollard, J.W. (2010). Macrophage diversity enhances tumor progression and metastasis. Cell 141, 39-51.
  • the mononuclear phagocyte system a new classification of macrophages, monocytes, and their precursor cells. Bulletin of the World Health Organization 46, 845- 852. Wang, H.W., and Joyce, J.A. (2010). Alternative activation of tumor-associated macrophages by IL-4: priming for protumoral functions. Cell Cycle 9,4824-4835.

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Abstract

La présente invention concerne des procédés pour stratifier un sujet souffrant d'une tumeur solide en fonction d'un pourcentage de lymphocytes T associés à une tumeur CD8+ et l'utilisation de ces informations dans l'identification de si le patient est un candidat adapté pour le traitement avec des inhibiteurs de point de contrôle immunitaire tels que PD-1 ou PD-Ll. Le procédé comprend (a) l'évaluation du pourcentage de lymphocytes T associés à une tumeur à phénotype cytotoxique (CD8+) dans un échantillon biologique dérivé dudit patient qui expriment le marqueur de mort cellulaire programmée 1 (PD-1) et qui n'expriment pas des quantités appréciables de Granzyme B (GzmB); et (b) l'identification du sujet en tant que candidat adapté pour traitement avec ledit inhibiteur en fonction du pourcentage du nombre desdits lymphocytes T CD8+ qui expriment ou non PD-1 (PD-1+) et du pourcentage du nombre de lymphocytes T CD8+PD-1+ qui n'expriment pas des quantités appréciables de GzmB. Des macrophages associés à une tumeur exprimant MHC II et un marqueur monocytaire peuvent également être utilisés dans la stratification.
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WO2017059280A1 (fr) * 2015-10-02 2017-04-06 The University Of North Carolina At Chapel Hill Nouveaux inhibiteurs de pan-tam et doubles inhibiteurs de mer/axl
US9920123B2 (en) 2008-12-09 2018-03-20 Genentech, Inc. Anti-PD-L1 antibodies, compositions and articles of manufacture
CN109475629A (zh) * 2016-05-20 2019-03-15 伊莱利利公司 用notch和pd-1或pd-l1抑制剂的组合治疗
WO2019053244A1 (fr) * 2017-09-15 2019-03-21 Universität Zürich Biomarqueurs pour la thérapie par inhibiteur de point de contrôle de sensibilité
WO2020018434A1 (fr) * 2018-07-17 2020-01-23 Scripps Health Compositions et procédés pour perturber un réseau de macrophages
CN111094977A (zh) * 2017-07-13 2020-05-01 古斯塔夫·鲁西研究所 监测抗pd-1/pd-l1治疗的肿瘤患者中肿瘤淋巴细胞浸润和预后的基于影像组学的成像工具
WO2020207771A1 (fr) * 2019-04-10 2020-10-15 Universität Zürich Procédé de détermination de la probabilité qu'un patient réponde à une immunothérapie anticancéreuse
CN113293191A (zh) * 2021-05-28 2021-08-24 山东大学齐鲁医院 Gfp转染肿瘤细胞的方法在检测肿瘤相关巨噬细胞吞噬功能中的应用
CN113466453A (zh) * 2021-05-13 2021-10-01 天津市肿瘤医院（天津医科大学肿瘤医院） Hcc组织切片中巨噬细胞包绕肿瘤细胞簇结构及其作为肿瘤标志物的用途和识别方法
WO2022192353A1 (fr) * 2021-03-09 2022-09-15 Mayo Foundation For Medical Education And Research Biomarqueurs pour l'identification et le traitement de patients atteints d'un cancer
CN116381234A (zh) * 2022-11-06 2023-07-04 复旦大学 Mmp14作为软脑膜转移瘤分子标志物和治疗靶点的应用
WO2025107242A1 (fr) * 2023-11-23 2025-05-30 成都福实生物科技有限公司 Utilisation d'un sous-groupe de macrophages sensibles à la force mécanique dans une estimation diagnostique ou pronostique pour le cancer du pancréas
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US10526309B2 (en) 2015-10-02 2020-01-07 The University Of North Carolina At Chapel Hill Pan-TAM inhibitors and Mer/Axl dual inhibitors
WO2017059280A1 (fr) * 2015-10-02 2017-04-06 The University Of North Carolina At Chapel Hill Nouveaux inhibiteurs de pan-tam et doubles inhibiteurs de mer/axl
CN109475629A (zh) * 2016-05-20 2019-03-15 伊莱利利公司 用notch和pd-1或pd-l1抑制剂的组合治疗
CN111094977A (zh) * 2017-07-13 2020-05-01 古斯塔夫·鲁西研究所 监测抗pd-1/pd-l1治疗的肿瘤患者中肿瘤淋巴细胞浸润和预后的基于影像组学的成像工具
CN111094977B (zh) * 2017-07-13 2024-02-13 古斯塔夫·鲁西研究所 监测抗pd-1/pd-l1治疗的肿瘤患者中肿瘤淋巴细胞浸润和预后的基于影像组学的成像工具
WO2019053244A1 (fr) * 2017-09-15 2019-03-21 Universität Zürich Biomarqueurs pour la thérapie par inhibiteur de point de contrôle de sensibilité
WO2020018434A1 (fr) * 2018-07-17 2020-01-23 Scripps Health Compositions et procédés pour perturber un réseau de macrophages
WO2020207771A1 (fr) * 2019-04-10 2020-10-15 Universität Zürich Procédé de détermination de la probabilité qu'un patient réponde à une immunothérapie anticancéreuse
WO2022192353A1 (fr) * 2021-03-09 2022-09-15 Mayo Foundation For Medical Education And Research Biomarqueurs pour l'identification et le traitement de patients atteints d'un cancer
EP4304613A4 (fr) * 2021-03-09 2025-01-15 Mayo Foundation for Medical Education and Research Biomarqueurs pour l'identification et le traitement de patients atteints d'un cancer
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CN113466453B (zh) * 2021-05-13 2023-06-27 天津市肿瘤医院（天津医科大学肿瘤医院） Hcc组织切片中巨噬细胞包绕肿瘤细胞簇结构及其作为肿瘤标志物的用途和识别方法
CN113293191A (zh) * 2021-05-28 2021-08-24 山东大学齐鲁医院 Gfp转染肿瘤细胞的方法在检测肿瘤相关巨噬细胞吞噬功能中的应用
CN116381234A (zh) * 2022-11-06 2023-07-04 复旦大学 Mmp14作为软脑膜转移瘤分子标志物和治疗靶点的应用
WO2025107242A1 (fr) * 2023-11-23 2025-05-30 成都福实生物科技有限公司 Utilisation d'un sous-groupe de macrophages sensibles à la force mécanique dans une estimation diagnostique ou pronostique pour le cancer du pancréas
US12365901B2 (en) 2023-11-23 2025-07-22 Hong Jiang Application of mechanical-force sensitive macrophage subset in pancreatic cancer diagnosis or prognosis evaluation
WO2025234833A1 (fr) * 2024-05-10 2025-11-13 Lunit Inc. Méthode et dispositif de prédiction de réponse de traitement contre le cancer à un inhibiteur de point de contrôle immunitaire

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