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Definitions

• This invention relates generally to immunopotentiating compositions. More particularly, the present invention relates to the use of lysine demethylase (LSD) inhibitors for enhancing the immune effector function of functionally repressed T-cells that have undergone epithelial to mesenchymal transition (EMT).
• LSD inhibitors are used to enhance susceptibility of exhausted T-cells to reinvigoration by PD-1 binding antagonists.
• the compositions of the present invention find utility in treating a range of disorders including T-cell dysfunctional disorders such as pathogenic infections and hyperproliferative disorders.
• PD-1 is an immune checkpoint regulator that is expressed in various immune cells including T-cells, B-cells, natural killer (NK) cells, NK T (NKT) cells, monocytes, macrophages, and dendritic cells (DCs) following their activation.
• PD-1 binds to its two ligands: programmed cell death 1 ligand-1 (PD-L1; B7-H1; CD274) and PD-L2 (B7-DC; CD273), both of which are B7 family members.
• PD-L1 is constitutively expressed in a wide range of cells including hematopoietic and non-hematopoietic cells.
• PD-L2 expression is restricted to professional antigen presenting cells (APCs; monocytes, macrophages, and DCs) and a certain subset of B cells.
• APCs professional antigen presenting cells
• IFNs interferons
• a, b, and y are potent regulators of both PD-L1 and PD-L2 expression.
• PD-1 is induced by T-cell receptor (TCR) signaling, and when PD-1 binds to PD-L1 or PD-L2, it inhibits TCR/CD28 signaling and T-cell activation.
• TCR T-cell receptor
• These immunoregulatory roles of PD- 1 are responsible for limiting excessive T-cell activation to prevent immune-mediated tissue damage.
• prolonged TCR stimulation and PD-1 expression lead to T-cell exhaustion, which is a state of T-cell dysfunction defined by poor T-cell effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T-cells, and which is commonly associated with inefficient control of tumors and persistent viral infections (Wherry, EJ., 2011. Nature Immunology 12: 492-499).
• the PD-1 pathway is an important determinant of the outcome of the T-cell response, regulating the balance between effective host defense and immunopathology, implicating the potential for manipulating the PD-1 pathway against various human diseases.
• Blockade of the PD-1 pathway has been used to reinvigorate exhausted T-cells and restore anti-tumor or anti-pathogen immune responses. Indeed, antibodies that block the PD-1 pathway have shown promising clinical results in a significant number of advanced-stage cancer patients. However, clinical trial data to date show a high variety of response rates among different types of cancers to PD-1 immune checkpoint inhibition therapy, with a range of 18% to 87%.
• the present invention arises from the unexpected finding that increased translocation of phosphorylated lysine demethylase 1 (also referred to herein as "LSDlp” or “nuclear LSD”) in the nucleus of T-cells (e.g., CD8 + T-cells) induces epithelial to mesenchymal transition (EMT) of the cells with repression of their immune effector function, including decreased expression of biomarkers of T-cell activation and effector capacity (e.g.
• interleukin-2 interleukin-2
• IFN-g interferon-g
• TNF-a tumor necrosis factor-a
• IL-2 interleukin-2
• IFN-g interferon-g
• TNF-a tumor necrosis factor-a
• EOMES Eomesodermin
• LSDlp and EOMES are in close proximity in the nucleus and form a complex that is predicted to be a repressor of T-cell function, including stimulating the T-cell to adopt and/or maintain an exhausted phenotype.
• the present inventors have also found that exposure of these mesenchymal, functionally repressed T-cells to LSD inhibitors, including LSD1 inhibitors, results in epigenetic reprogramming of the T-cells with remarkable de-repression of their immune effector function, including elevated expression of biomarkers of T-cell activation and effector capacity (e.g. , IFN-y, TNF-a, Ki67 and TBET), decreased expression of biomarkers of T-cell exhaustion (e.g., EOMES), as well as increased activation and proliferation of T-cells, including effector and memory T-cells.
• LSD inhibitors including LSD1 inhibitors
• the present invention provides compositions for enhancing T-cell (e.g. , CD8 + T-cell or CD4 + T-cell) function, or for treating a T-cell dysfunctional disorder.
• These compositions generally comprise, consist or consist essentially of a LSD inhibitor and a PD-1 binding antagonist.
• the LSD inhibitor is suitably selected from inhibitors of LSD enzymatic activity and inhibitors of LSD nuclear translocation.
• the LSD inhibitor is suitably a LSD1 inhibitor and in specific examples, the LSD1 inhibitor is a specific or selective LSD1 inhibitor.
• the PD-1 binding antagonist suitably inhibits the binding of PD-1 to PD-L1 and/or PD-L2.
• the PD-1 binding antagonist is an anti-PD-1 antagonist antibody, illustrative examples of which include nivolumab, pembrolizumab, lambrolizumab and pidilizumab.
• the PD-1 binding antagonist is an immunoadhesin (e.g. , AMP-224).
• compositions further comprise an ancillary agent for trePCT/A 112018/051268 n the treatment of, a T-cell dysfunctional disorder.
• the ancillary agent is a chemotherapeutic agent, which suitably targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g. , a cytotoxic compound such as a taxane).
• the compositions are typically pharmaceutical compositions or formulations, which optionally comprise a pharmaceutically acceptable carrier.
• Another aspect of the present invention provides methods of enhancing T-cell function. These methods generally comprise, consist or consist essentially of contacting a T-cell with a LSD inhibitor (e.g. , a LSD1 inhibitor) and a PD-1 binding antagonist, to thereby enhance T- cell function.
• a LSD inhibitor e.g. , a LSD1 inhibitor
• a PD-1 binding antagonist e.g., a PD-1 binding antagonist
• the T-cell has a mesenchymal phenotype.
• the T-cell has aberrant expression of nuclear LSD.
• the T-cell expresses nuclear LSD at a higher level than the level of expression of TBET in the same T-cell, and/or at a higher level than in an activated T-cell.
• the T-cell is one exhibiting T-cell exhaustion or anergy.
• the T-cell expresses a higher level of EOMES than TBET and/or has elevated expression of PD-1.
• the T-cell may be a CD4 + T -cell or a CD8 + T-cell.
• the T-cell is a CD8 + T-cell.
• the present inventors propose that since nuclear LSD-mediated EMT occurs both in tumor cells and in T-cells, which are unrelated cell types, nuclear LSD-mediated epigenetic reprogramming is also likely to occur more broadly, including in other immune effector cells that express PD-1 (e.g., T-cells, B-cells, NK cells, NKT cells, monocytes, macrophages and DCs), to thereby repress their immune effector function.
• PD-1 e.g., T-cells, B-cells, NK cells, NKT cells, monocytes, macrophages and DCs
• the present invention provides methods of enhancing immune effector function of an immune effector cell that expresses PD-1. These methods generally comprise, consist or consist essentially of contacting the immune effector cell with a LSD inhibitor (e.g.
• the enhanced immune effector function includes any one or more of increased recognition of an antigen or an antigen peptide derived from an antigen in the context of MHC class II molecules by T-cell receptors, increased release of cytokines and/or the activation of CD4 + lymphocytes, increased release of cytokines and/or the activation of CD8 + lymphocytes (CTLs) and/or B-cells, increased recognition of an antigen or an antigen peptide derived from an antigen in the context of MHC class I molecules by T-cell receptors, increased elimination of cells presented in the context of MHC class I molecules, /.e., cells characterized by presentation of an antigen with class I MHC, for example, via apoptosis or perforin-mediated cell lysis, increased production of cytokines such as IFN-g and TNF-a, and increased specific cytolytic
• the immune effector cell has aberrant expression of nuclear LSD.
• the immune effector expresses nuclear LSD at a higher level than the level than in a control iWQ 2019/104381 cell (e.g., an immune effector cells with normal or non- rePCT/A II 2018/0 1268 ec to r function).
• the present invention provides methods of treating a T-cell dysfunctional disorder in a subject. These methods generally comprise, consist or consist essentially of administering concurrently to the subject a LSD inhibitor (e.g., a LSD1 inhibitor) and a PD-1 binding antagonist in effective amounts to treat the T-cell dysfunctional disorder.
• a LSD inhibitor e.g., a LSD1 inhibitor
• a PD-1 binding antagonist in effective amounts to treat the T-cell dysfunctional disorder.
• the LSD inhibitor and PD-1 binding antagonist are administered in synergistically effective amounts.
• the T-cell dysfunctional disorder is a disorder or condition of T- cells characterized by decreased responsiveness to antigenic stimulation and/or increased inhibitory signal transduction through PD-1.
• the T- cell dysfunctional disorder is one in which the T-cells have decreased ability to secrete cytokines, proliferate, or execute cytolytic activity.
• the decreased responsiveness to antigenic stimulation results in ineffective control of a pathogen or tumor.
• the T-cell dysfunctional disorder is one in which T-cells are anergic.
• T-cell dysfunctional disorders include unresolved acute infection, chronic infection and tumor immunity.
• the T-cell dysfunctional disorder is a cancer or infection that comprises a T-cell (e.g., a CD8 + or CD4 + T-cell) with a mesenchymal phenotype.
• the T-cell expresses nuclear LSD at a higher level than the level of expression of TBET in the same T-cell, and/or at a higher level than in an activated T-cell.
• the T-cell is one exhibiting T-cell exhaustion or anergy.
• the T-cell expresses a higher level of EOMES than TBET and/or has elevated expression of PD-1.
• the T-cell is a tumor-infiltrating lymphocyte.
• the T-cell is a circulating lymphocyte.
• the cancer is skin cancer (e.g. , melanoma), lung cancer, breast cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, colorectal cancer, glioblastoma, neuroblastoma, or hepatocellular carcinoma.
• the cancer is a metastatic cancer.
• the metastatic cancer is metastatic melanoma or metastatic lung cancer.
• the methods further comprise further administering concurrently to the subject, with the LSD inhibitor (e.g. , a LSD1 inhibitor) and the PD-1 binding antagonist, an ancillary agent (e.g. , a chemotherapeutic agent) or ancillary therapy (e.g. , ablation or cytotoxic therapy) for treating, or for aiding in the treatment of, a T-cell dysfunctional disorder.
• the methods comprise further administering concurrently to the subject, with the LSD inhibitor (e.g.
• a chemotherapeutic agent which suitably targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g., a cytotoxic compound such as a taxane).
• the present invention provides methods of treating or delaying the progression of cancer in a subject. These methods generally comprise, consist or consist essentially of administering concurrently to the subject a LSD inhibitor (e.g., a LSD1 inhibitor) and a PD-1 binding antagonist in effective amounts to treat or delay the progression of the cancer.
• a LSD inhibitor e.g., a LSD1 inhibitor
• a PD-1 binding antagonist in effective amounts to treat or delay the progression of the cancer.
• the subject has been diagnosed with cancer, wherein a T-cell in a tumor sample of the cancer from the subject expresses nuclear LSD at a higher level than the level of expression of TBET in the same T-cell, and/or at a higher level than in an activated T-cell.
• the present invention provides methods of enhancing immune function (e.g., immune effector function) in an individual having cancer.
• immune function e.g., immune effector function
• a LSD inhibitor e.g., a LSD1 inhibitor
• a PD-1 binding antagonist in effective amounts to enhance the immune function.
• the individual has been diagnosed with cancer, wherein a T-cell in a tumor sample of the cancer taken from the individual expresses nuclear LSD at a higher level than the level of expression of TBET in the same T-cell, and/or at a higher level than in an activated T-cell.
• methods of treating infection e.g. , with a bacteria or virus or other pathogen. These methods generally comprise, consist or consist essentially of administering concurrently to the individual a LSD inhibitor (e.g. , a LSD1 inhibitor) and a PD-1 binding antagonist in effective amounts to treat the infection.
• the infection is with virus and/or bacteria.
• the infection is with a pathogen.
• the infection is an acute infection.
• the infection is a chronic infection.
• the present invention provides methods of enhancing immune function (e.g., immune effector function, T-cell function etc. ) in an individual having an infection.
• These methods generally comprise, consist or consist essentially of administering concurrently to the individual a LSD inhibitor (e.g. , a LSD1 inhibitor) and a PD-1 binding antagonist in effective amounts to enhance the immune function.
• the individual has been diagnosed with the infection, wherein a T-cell in a sample taken from the individual expresses nuclear LSD at a higher level than the level of expression of TBET in the same T-cell, and/or at a higher level than in an activated T-cell.
• Another aspect of the present invention provides use of a LSD inhibitor (e.g. , a LSD1 inhibitor) and a PD-1 binding antagonist for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g. , immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection.
• the LSD inhibitor and PD-1 binding antagonist are generally used in the manufacture of medicaments for this purpose.
• the LSD inhibitor and PD-1 binding antagonist are formulated for concurrent administration.
• the present invention provides use of a LSD inhibitor (e.g., a LSD1 inhibitor), a PD-1 binding antagonist and an ancillary agent (e.g. , a chemotherapeutic agent) for treating, or for aiding in the treatment of, a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection.
• the LSD inhibitor, PD-1 binding antagonist and ancillary agent e.g. , a chemotherapeutic agent
• the LSD inhibitor, PD- 1 binding antagonist and ancillary agent are formulated for concurrent administration.
• the ancillary agent is a chemotherapeutic agent, which suitably targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g., a cytotoxic compound such as a taxane).
• the methods for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g. , immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection comprise detecting an elevated level of nuclear LSD (i.e. , a LSD, suitably LSDlp, localized in the nucleus) in iWO_?Q19/104381ative to the level of TBET in the same T-cell or the level oPCX/AU2018/051268 activated T-cell) in a sample obtained from the subject, prior to the concurrent administration.
• a LSD suitably LSDlp, localized in the nucleus
• the methods for treating a T-cell dysfunctional disorder comprise detecting an elevated level of nuclear LSD (/.e., a LSD, suitably LSDlp, localized in the nucleus) in a T cell (e.g., relative to the level of TBET in the same T-cell or the level of nuclear LSD in an activated T-cell) and an elevated level of EOMES in the nucleus of the T cell (e.g. , relative to the level of TBET in the same T-cell or the level of EOMES in the nucleus of an activated T-cell) in a sample obtained from the subject, prior to the concurrent administration.
• these methods comprise detecting an elevated level of a complex comprising a LSD (e.g., a LSD1, suitably LSDlp) and EOMES, suitably in the nucleus of the T-cell.
• kits comprising a medicament comprising a LSD inhibitor (e.g. , a LSD1 inhibitor) and an optional pharmaceutically acceptable carrier, and a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• a LSD inhibitor e.g., a LSD1 inhibitor
• a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• immune function e.g., immune
• the package insert comprises instructional material for concurrent administration of the medicament with another medicament comprising an ancillary agent and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g. , immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• the ancillary agent is a chemotherapeutic agent, which suitably targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g. , a cytotoxic compound such as a taxane).
• kits comprising a medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier, and a package insert comprising instructional material for concurrent administration of the medicament with another medicament comprising a LSD inhibitor (e.g. , a LSD1 inhibitor) and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g. , immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• a LSD inhibitor e.g. , a LSD1 inhibitor
• an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g. , immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• the package insert comprises instructional material for concurrent administration of the medicament with another medicament comprising an ancillary agent and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• the ancillary agent is a chemotherapeutic agent, which suitably targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g., a cytotoxic compound such as a taxane).
• kits comprising a first medicament comprising a LSD inhibitor (e.g. , a LSD1 inhibitor) and an optional pharmaceutically acceptable carrier, and a second medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing iWQ 2019/104381 (e.g., immune effector function, T-cell function etc. ) in aiPCT/AlJ2018/051268 cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• a LSD inhibitor e.g., a LSD1 inhibitor
• a second medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing iWQ 2019/104381 (e.g., immune effector function, T-cell function etc. ) in aiPCT/AlJ2018/051268 cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• kits further comprise a package insert comprising instructional material for administering concurrently the first medicament and the second medicament for treating a T- cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T- cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• the package insert comprises instructional material for concurrent administration of the medicament with another medicament comprising an ancillary agent and an optional pharmaceutically acceptable carrier for treating a T- cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T- cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• the ancillary agent is a chemotherapeutic agent, which suitably targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g., a cytotoxic compound such as a taxane).
• kits comprising a first medicament comprising a LSD inhibitor (e.g., a LSD1 inhibitor) and an optional pharmaceutically acceptable carrier, a second medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier and a third medicament comprising a chemotherapeutic agent and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• a LSD inhibitor e.g., a LSD1 inhibitor
• a second medicament comprising a PD-1 binding antagonist and an optional pharmaceutically acceptable carrier
• a third medicament comprising a chemotherapeutic agent and an optional pharmaceutically acceptable carrier for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc. ) in an individual having cancer, for treating or delaying the progression of cancer, or
• kits further comprise a package insert comprising instructional material for administering concurrently the first medicament, second and third medicaments for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g., immune effector function, T-cell function etc.) in an individual having cancer, for treating or delaying the progression of cancer, or for treating infection in an individual.
• the chemotherapeutic agent targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g., a cytotoxic compound such as a taxane).
• CD8 + T cells in the individual have enhanced priming, activation, proliferation and/or cytolytic activity as compared to before the administration of the combination.
• the number of CD8 + T cells is elevated as compared to before administration of the combination.
• the CD8 + T cell is an antigen-specific CD8 + T cell.
• Treg function is suppressed as compared to before
• T cell exhaustion is decreased as compared to before administration of the combination of LSD inhibitor (e.g., a LSD1 inhibitor) and PD-1 binding antagonist.
• number of Treg cells is decreased as compared to before administration of the combination of the LSD inhibitor (e.g., a LSD1 inhibitor) and PD-1 binding antagonist.
• plasma IFN-g is increased as compared to before administration of the combination of the LSD inhibitor (e.g., a LSD1 inhibitor) and PD-1 binding antagonist.
• plasma TNF-a is increased as compared to before administration of the combination of the LSD inhibitor (e.g., a LSD1 inhibitor) and PD-1 binding antagonist.
• the number of memory T effector cells is increased as compared to before administration of the combination of LSD inhibitor (e.g., a LSD1 inhibitor) and PD-1 binding iWQ 2019/104381 me embodiments, memory T effector cell activation and/cPC.T/AU2018/051268 increased as compared to before administration of the combination of the LSD inhibitor (e.g ., a LSD1 inhibitor) and PD-1 binding antagonist.
• memory T effector cells are detected in peripheral blood. In some embodiments, detection of memory T effector cells is by detection of CXCR3.
• the LSD inhibitor e.g., a LSD1 inhibitor
• PD-1 binding antagonist are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
• the treatment further comprises administering an ancillary agent (e.g., a
• chemotherapeutic agent for treating or delaying progression of cancer in an individual.
• the individual has been treated with a chemotherapeutic agent (e.g., a compound that targets rapidly dividing cells and/or disrupt the cell cycle or cell division, suitably a cytotoxic compound such as a taxane) before the combination treatment with the LSD inhibitor and PD-1 binding antagonist.
• a chemotherapeutic agent e.g., a compound that targets rapidly dividing cells and/or disrupt the cell cycle or cell division, suitably a cytotoxic compound such as a taxane
• the individual treated is refractory to a chemotherapeutic agent
• chemotherapeutic agent treatment Some embodiments of the methods, uses, compositions, and kits described throughout the application, further comprise administering a chemotherapeutic agent for treating or delaying progression of cancer.
• a further aspect of the present invention provides methods of diagnosing the presence of a T-cell dysfunctional disorder in a subject. These methods generally comprise, consist or consist essentially of:
• CD8 + T-cell or CD4 + T-cell CD8 + T-cell or CD4 + T-cell
• a first binding agent that binds to a LSD e.g., a nuclear LSD such as LSDlp
• a second binding agent that binds to EOMES in the sample e.g., a nuclear LSD such as LSDlp
• localization of the first and second binding agents in the nucleus of the T-cell is indicative of the presence of the T-cell dysfunctional disorder in the subject.
• the present invention provides methods of diagnosing the presence of a T-cell dysfunctional disorder in a subject. These methods generally comprise, consist or consist essentially of:
• CD8 + T-cell or CD4 + T-cell CD8 + T-cell or CD4 + T-cell
• a first binding agent that binds to a LSD e.g., a nuclear LSD such as LSDlp
• a second binding agent that binds to EOMES in the sample e.g., a nuclear LSD such as LSDlp
• WO 2019/104381 further aspect of the present invention provides method PCT/A IZOlS/OSlliiS treatment of a subject with a T-cell dysfunctional disorder. These methods generally comprise, consist or consist essentially of:
• obtaining a sample from the subject following treatment of the subject with a therapy for the T-cell dysfunctional disorder wherein the sample comprises a T-cell (e.g. , CD8 + T-cell or CD4 + T-cell);
• a T-cell e.g. , CD8 + T-cell or CD4 + T-cell
• a lower level of LSD— EOMES complex detected in the sample relative to a level of LSD— EOMES complex detected in a control sample taken from the subject prior to the treatment is indicative of an increased clinical benefit (e.g., enhanced immune effector function such as enhanced T-cell function) to the subject, and
• a higher level of LSD— EOMES complex detected in the sample relative to a level of LSD— EOMES complex detected in a control sample taken from the subject prior to the treatment is indicative of no or negligible clinical benefit (e.g., enhanced immune effector function such as enhanced T-cell function) to the subject.
• kits for diagnosing the presence of a T-cell dysfunctional disorder in a subject.
• kits generally comprise, consist or consist essentially of: (i) a first binding agent that binds to a LSD (e.g., a nuclear LSD such as LSDlp), (ii) a second binding agent that binds to EOMES; and (iii) a third agent comprising a label, which is detectable when each of the first and second binding agents is bound to a LSD— EOMES complex.
• the third agent is a binding agent that binds to the first and second binding agent.
• the present invention provides a complex comprising a LSD (e.g. , a nuclear LSD such as LSDlp) and EOMES, a first binding agent that is bound to the LSD of the complex, a second binding agent bound to EOMES of the complex; and (iii) a third agent comprising a label, which is detectable when each of the first and second binding agents is bound to the LSD— EOMES complex.
• a LSD e.g. , a nuclear LSD such as LSDlp
• EOMES e.g. a nuclear LSD such as LSDlp
• a first binding agent that is bound to the LSD of the complex
• a second binding agent bound to EOMES of the complex e.g. a nuclear LSD such as LSDlp
• a third agent comprising a label, which is detectable when each of the first and second binding agents is bound to the LSD— EOMES complex.
• the LSD— EOMES complex is located in a T
• the present invention provides a T-cell that comprises a complex comprising a LSD (e.g., a nuclear LSD such as LSDlp) and EOMES, a first binding agent that is bound to the LSD of the complex, a second binding agent bound to EOMES of the complex; and (iii) a third agent comprising a label, which is detectable when each of the first and second binding agents is bound to the LSD— EOMES complex.
• the third agent is a binding agent that binds to the first and second binding agent.
• respective binding agents are preferably antibodies.
• FIG. 1 is a graphical representation showing efficacy of the LSD1 inhibitor, phenelzine sulfate in inhibiting demethylation and cell proliferation of a breast cancer cell line.
• Figure 2 is a graphical and photographic representation showing the efficacy of dual phenelzine sulfate (phenelzine) and anti-PDl antibody (PD1) therapy on circulating tumor cells (CTCs) and cancer stem cells (CSC) as well as tumor burden.
• phenelzine phenelzine
• PD1 circulating tumor cells
• CSC cancer stem cells
• A Statistics of tumor volumes on day 15 post-treatment. A Mann-Whitney non-parametric t-test was used to compare control vs other groups (*p ⁇ 0.02, **p ⁇ 0.008).
• Group A Control, Group C: PD1 (10 mg/kg), Group D: Phenelzine (40 mg/kg), Group F: PDl+Phenelzine. Illustration of the treatment protocol is also shown.
• B phenelzine sulfate
• PD1 circulating tumor cells
• CSC cancer stem cells
• Figure 3 is a photographic and graphical representation showing that dual epigenetic-immunotherapy inhibits metastatic progression in 4T1 Mouse model.
• A. 4T1 Treatment FFPE from each treatment group (Group A Control, Group C: PD1 (lOmg/kg), Group D:
• FFPE tissues were fixed and Immunofluorescence microscopy was performed probing with rabbit LSDl(Slllp); mouse anti CSV and goat anti SNAIL and visualized with a donkey anti-rabbit AF 488, anti-mouse 568 and anti-goat 633.
• Cover slips were mounted on glass microscope slides with ProLong Diamond Antifade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy.
• TNFI Total Nuclear Fluorescent Intensity
• TCFI Total Cytoplasmic Fluorescent Intensity
• TFI Total Fluorescent Intensity
• Graphs for both organs are presented here. Graphs represent the TNFI values for LSD1, ALDH1A and TCFI for CSV measured using ImageJ to select the nucleus minus background. Representative images for the Lung dataset are shown.
• Figure 4 is a graphical representation showing that dual epigenetic-immunotherapy re-educates and re-programs innate macrophage repertoire.
• Group A Control
• Group C PD1 (10 mg/kg)
• Group D Phenelzine (40 mg/kg)
• Group F PDl+Phenelzine).
• Figure 5 is a graphical representation showing that dual epigenetic-immunotherapy re-educates and re-programs the T-cell repertoire.
• A. Cells stimulated with PMA/ionomycin for 4 hours in the presence of Brefeldin A. Cells were surfaced stained with CD45, CD3, CD4, CD8,
• a Mann-Whitney non-parametric t-test was used to compare control vs other groups (*p ⁇ 0.05, n 2-5).
• FIG. 6 is a graphical and photographic representation depicting nuclear LSD1 complexes with EOMES in exhausted T-cell signatures.
• A. Nanostring transcript analysis was performed on isolated CD8 + T-cells isolated from a 4T1 metastatic cancer mouse model treated with either Group A Control, Group C: PD1 (10 mg/kg), Group D: Phenelzine (40 mg/kg), or Group F: PDl+Phenelzine. Displayed are the effects on expression of mRNA of genes associated with exhaustion or activation T-cell markers.
• B Immunofluorescence microscopy was performed on CD8 + T-cells fixed and probed with primary anti-LSDl, anti-EOMES and anti-CD8 antibodies and DAPI.
• C. Displays plot-profiles for EOMES/LSD1. Plot-profiles were plotted with the use of ImageJ software measuring a series of fluorescent intensities along a line spanning the nucleus. The pattern of the two plots can give an insight into the nature of the relationship between the two fluorochromes.
• Figure 8 is a graphical representation showing that dual epigenetic-immunotherapy re-programs gene expression programs in CD8 + T-cells.
• % genes affected is shown for each of the listed gene pathways and for each treatment group or combination. This is in purified CD8 + T-cells the 4T1 metastatic mouse model.
• C This figure depicts the effect of expression or inhibition on the adaptive, innate, inflammation, cancer progression and T-cell function message by treatment with phenelzine, PD1 or combination in purified CD8 + T-cells in the 4T1 metastatic mouse model.
• D ATAQ Sequencing showing chromatin accessibility changes directly mediated by LSD1 in purified CD8 + T-cells from the 4T1 metastatic mouse model.
• Figure 9 is a photographic and graphical representation showing that EOMES and LSD1 form a complex in exhausted CD8+ T-Cells.
• Figure 10 presents the amino acid sequence of human EOMES.
• the red bold text is a predicted monopartite nuclear localization sequence (NLS), the text highlighted in yellow (with the lysine target in bold red) represents potential methylation sites near the sequence of the NLS with a support vector machine (SVM) probability of ⁇ 0.7 or higher.
• SVM support vector machine
• Figure 11 is a photographic and graphical representation showing triple therapy efficacy on CTC/CSC and tumor burden.
• A Statistics of tumor volumes on day 15 post-treatment. A Mann-Whitney non-parametric t-test was used to compare control vs other groups (*p ⁇ 0.02, **p ⁇ 0.008).
• Group A Control, Group B: Abraxane (30mg/kg), Group C: PD1 (10 mg/kg), Group D: Phenelzine (40 mg/kg), Group E: Abraxane+PDl, Group F: PDl+Phenelzine, Group G:
• Graph represents the TNFI values for LSD1, SNAIL and TCFI for CSV measured using ImageJ to select the nucleus minus background (n > 20 individual cells
• Group A Control
• Group B Abraxane (30 mg/kg)
• Group C PD1 (10 mg/kg)
• Group D Phenelzine (40 mg/kg)
• Group E Abraxane + PD1
• Group F PDl+Phenelzine
• Group G Abraxane+Phenelzine
• Group H Triple Therapy.
• E. Immunofluorescence microscopy was performed on cells fixed and probed with primary anti-CD133, anti-ALDHIA and anti ABCB5 antibodies and DAPI.
• Activation refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change.
• T cells such activation refers to the state of a T cell that has been sufficiently stimulated tWO : 2019/104381 proliferation.
• Activation of a T cell may also induce cytokPCT/AlI2018_/ () 5_1268 detectable effector functions, including performance of regulatory or cytolytic effector functions. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process. Activation can also be associated with induced cytokine production, and detectable effector functions.
• activated T-cell means a T-cell that is currently undergoing cell division, detectable effector functions, including cytokine production, performance of regulatory or cytolytic effector functions, and/or has recently undergone the process of "activation".
• administering concurrently or “co administering” and the like refer to the administration of a single composition containing two or more actives, or the administration of each active as separate compositions and/or delivered by separate routes either contemporaneously or simultaneously or sequentially within a short enough period of time that the effective result is equivalent to that obtained when all such actives are administered as a single composition.
• simultaneous is meant that the active agents are administered at substantially the same time, and desirably together in the same formulation.
• temporary it is meant that the active agents are administered closely in time, e.g. , one agent is administered within from about one minute to within about one day before or after another. Any contemporaneous time is useful.
• the agents when not administered simultaneously, the agents will be administered within about one minute to within about eight hours and suitably within less than about one to about four hours.
• the agents are suitably administered at the same site on the subject.
• the term "same site” includes the exact location, but can be within about 0.5 to about 15 centimeters, preferably from within about 0.5 to about 5 centimeters.
• the term "separately” as used herein means that the agents are administered at an interval, for example at an interval of about a day to several weeks or months.
• the active agents may be administered in either order.
• the term “sequentially” as used herein means that the agents are administered in sequence, for example at an interval or intervals of minutes, hours, days or weeks. If appropriate the active agents may be administered in a regular repeating cycle.
• agent includes a compound that induces a desired pharmacological and/or physiological effect.
• the term also encompass pharmaceutically acceptable and
• pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
• active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc.
• agent is not to be construed narrowly but extends to small molecules, proteinaceous molecules such as peptides, polypeptides and proteins as well as compositions comprising them and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular agents.
• agent includes a cell that is capable of producing and secreting a polypeptide referred to herein as well as a polynucleotide comprising a nucleotide sequence that encodes that polypeptide.
• agent extends to nucleic acid constructs including vectors such as viral or non-viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells.
• Amplification generally refers to the process of producing multiple copies of a desired sequence.
• Multiple copies mean at least two copies.
• a “copy” does not
• the "amount” or “level” of a biomarker is a detectable level in a sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to treatment.
• the term "anergy” refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T-cell receptor (e.g. increase in intracellular Ca 2+ in the absence of ras-activation). T-cell anergy can also result upon stimulation with antigen in the absence of co-stimulation, resulting in the cell becoming refractory to subsequent activation by the antigen even in the context of co-stimulation. The unresponsive state can often be overridden by the presence of IL-2. Anergic T-cells do not undergo clonal expansion and/or acquire effector functions.
• antagonist refers to a substance that prevents, blocks, inhibits, neutralizes, or reduces a biological activity or effect of another molecule, such as an enzyme or receptor.
• specific antagonist refers to a compound with high specificity for its target (e.g. for a LSD such as LSD1 including nuclear LSD, or for PD-1). Specificity of a particular antagonist or inhibitor is defined as a ratio of the IC50 values of the particular antagonist or inhibitor for the target of interest versus another target.
• an antagonist that is specific for PD-1 will have an IC50 value for target A (e.g., PD-1) lower than that for target B (e.g., PD-L1 or PD-L2).
• an inhibitor that is specific for LSD1 will have an IC50 value for target A (e.g., LSD1) lower than that for target B (e.g. , LSD2).
• the IC50 value for target A is at least 10 times lower than the IC50 value of the same inhibitor for target B.
• the IC50 value for target A is 100 times lower, or in other example is 1000 times lower.
• the IC50 value for target A is 10,000 times lower than the IC50 value of the same inhibitor for target B.
• the term “selective” is used herein to refer to compounds that inhibit or display antagonism towards a LSD without displaying substantial inhibition or antagonism towards another LSD or another enzyme such as a monoamine oxidase (MAO) (e.g. , MAO A or MAO B).
• MAO monoamine oxidase
• a compound that is selective for LSD1 exhibits a LSD1 selectivity of greater than about 2-fold, 5-fold, 10-fold, 20-fold, 50-fold or greater than about 100-fold with respect to inhibition or antagonism of another LSD (/.e., a LSD other than LSD1 such as LSD2) or of another enzyme (e.g., a MAO).
• selective compounds display at least 50- fold greater inhibition or antagonism towards a specified LSD than towards another LSD or another enzyme (e.g. , a MAO).
• selective compounds inhibit or display at least 100-fold greater inhibition or antagonism towards a specified LSD than towards another LSD or another enzyme (e.g., a MAO).
• selective compounds display at least 500-fold greater inhibition or antagonism towards L a specified LSD than towards another LSD or another enzyme (e.g., a MAO). In still other embodiments, selective compounds display at least 5 ⁇ Q_20_19/ 104381 r inhibition or antagonism towards a specified LSD than tcPCT/AU2018/051268 or another enzyme (e.g., a MAO).
• antigen antibody refers to an antibody that binds to a target and prevents or reduces the biological effect of that target.
• the term can denote an antibody that prevents the target, e.g. , PD-1, to which it is bound from performing a biological function.
• an "anti-PD-1 antagonist antibody” refers to an antibody that is able to inhibit PD-1 biological activity and/or downstream events(s) mediated by PD-1.
• Anti-PD-1 antagonist antibodies encompass antibodies that block, antagonize, suppress or reduce (to any degree including significantly) PD-1 biological activity, including inhibitory signal transduction through PD-1 and downstream events mediated by PD-1, such as PD-L1 binding and downstream signaling, PD-L2 binding and downstream signaling, inhibition of T cell proliferation, inhibition of T cell activation, inhibition of IFN secretion, inhibition of IL-2 secretion, inhibition of TNF secretion, induction of IL-10, and inhibition of anti-tumor immune responses.
• the term "anti-PD-1 antagonist antibody” it will be explicitly understood that the term "anti-PD-1 antagonist antibody"
• antagonist PD-1 antibody encompasses all the previously identified terms, titles, and functional states and characteristics whereby PD-1 itself, a PD-1 biological activity, or the consequences of the biological activity, are substantially nullified, decreased, or neutralized in any meaningful degree.
• an anti-PD-1 antagonist antibody binds PD-1 and upregulates an anti-tumor or anti-pathogen immune response. Examples of anti-PD-1 antagonist antibodies are provided herein.
• antibody herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.
• an "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with research, diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
• an antibody is purified (1) to greater than 95% by weight of antibody as determined by, for example, the Lowry method, and in some embodiments, to greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of, for example, a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or nonreducing conditions using, for example,
• Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present.
• isolated antibody will be prepared by at least one purification step.
• “Native antibodies” are usually heterotetra meric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• VH variable domain
• Each light chain has a variable ⁇ WO 2019/104381 id (v L ) and a constant domain at its other end; the constePCT/AU2018/051268 ht chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.
• constant domain refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site.
• the constant domain contains the CHI, C H and C H domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
• variable region or “variable domain” of an antibody refers to the amino- terminal domains of the heavy or light chain of the antibody.
• variable domain of the heavy chain may be referred to as "V H .”
• variable domain of the light chain may be referred to as "Vi_.” These domains are generally the most variable parts of an antibody and contain the antigen binding sites.
• variable refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR).
• HVRs hypervariable regions
• FR framework regions
• the variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
• the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et a/., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
• the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
• the "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("K”) and lambda ("l”), based on the amino acid sequences of their constant domains.
• IgG immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
• antibodies can be assigned to different classes.
• immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGi, IgG , IgG , IgG 4 , IgAi, and IgA .
• the heavy chain constant domains that correspond to the different classes of immunoglobulins are called a, g, e, y, and p, respectively.
• An antibody may be part of a larger fusion molecule, formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.
• WO 2019/104381 he terms "full length antibody,” “intact antibody” and “whPCT/A _2018/051268 e d herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
• naive T-cells refers to immune cells that comprise antigen- inexperienced cells, e.g. , immune cells that are precursors of memory T effector cells.
• naive T cells may be differentiated, but have not yet encountered their cognate antigen, and therefore are activated T cells or memory effector T cells.
• naive T cells may be characterized by expression of CD62L, CD27, CCR7, CD45RA, CD28, and CD127, and the absence of CD95, or CD45RO isoform.
• a "naked antibody” for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
• Antibody fragments comprise a portion of an intact antibody, preferably comprising the antigen binding region thereof.
• the antibody fragment described herein is an antigen-binding fragment.
• Examples of antibody fragments include Fab, Fab, and fragments thereof.
• Fab', F(ab'2, and Fv fragments diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
• Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual "Fc” fragment, whose name reflects its ability to crystallize readily.
• Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.
• Fv is the minimum antibody fragment which contains a complete antigen-binding site.
• a two-chain Fv species consists of a dimer of one heavy- and one light- chain variable domain in tight, non-covalent association.
• one heavy- and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three FIVRs of each variable domain interact to define an antigen-binding site on the surface of the VFI-VL dimer.
• FIVRs confer antigen-binding specificity to the antibody.
• a single variable domain or half of an Fv comprising only three FIVRs specific for an antigen
• the Fab fragment contains the heavy- and light-chain variable domains and also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
• Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CFI1 domain including one or more cysteines from the antibody hinge region.
• Fab'-SFI is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group.
• F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.
• Single-chain Fv or “scFv” antibody fragments comprise the VFI and VL domains of antibody, wherein these domains are present in a single polypeptide chain.
• the scFv polypeptide further comprises a polypeptide linker between the VFI and VL domains which enables the scFv to form the desired structure for antigen binding.
• scFv see, e.g. ,
• diabodies refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
• VH heavy-chain variable domain
• VL light-chain variable domain
• Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO
• Triabodies and tetrabodies are also described in Hudson et a/.,
• the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
• such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
• the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
• a selected target binding sequence can be further altered, for example, to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc. , and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention.
• each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
• monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
• the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
• the monoclonal antibodies to be used in accordance with the invention may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et at. , Hybridoma, 14 (3): 253-260 (1995), Harlow et at. , Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
• the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to
• Chimeric antibodies include PRIMATTZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g. , immunizing macaque monkeys with the antigen of interest.
• Humanized forms of non-human (e.g. , murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
• a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from a HVR of the recipient are replaced by residues from a HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
• donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
• FR residues of the human immunoglobulin are replaced by corresponding non human residues.
• humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
• the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human
• a "human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.
• Human antibodies can be produced using various techniques known in the art, including phage- display libraries. Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al. , J. Mol. Biol., 222: 581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et ai, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et ai , J. Immunol., 147(l):86-95 (1991). See also van Dijk and van de Winkel, Curr.
• Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to ⁇ WQ ?01?/104381ge, but whose endogenous loci have been disabled, e.g., PCT/AU2018/051268 3 (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding XENOMOUSETM technology). See also, for example, Li et a/., Proc. Natl. Acad. Sci. USA, 103 : 3557-3562 (2006) regarding human antibodies generated via a human B-cell hybridoma technology.
• a "species-dependent antibody” is one which has a stronger binding affinity for an antigen from a first mammalian species than it has for a homologue of that antigen from a second mammalian species.
• the species-dependent antibody "binds specifically" to a human antigen (e.g., has a binding affinity (Kd) value of no more than about lxlO -7 M, preferably no more than about lxlO -8 M and preferably no more than about lxlO -9 M) but has a binding affinity for a homologue of the antigen from a second nonhuman mammalian species which is at least about 50- fold, or at least about 500-fold, or at least about 1000-fold, weaker than its binding affinity for the human antigen.
• the species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a humanized or human antibody.
• hypervariable region refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops.
• antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (LI, L2, L3).
• H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
• HVR delineations are in use and are encompassed herein.
• the Kabat Complementarity Determining Regions are based on sequence variability and are the most commonly used (Kabat et a/., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).
• the AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software.
• the "contact" HVRs are based on an analysis of the available complex crystal structures. The residues from each of these HVRs are noted below.
• HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (LI), 46-56 or 50- 56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (HI), 50-65 or 49-65 (H2) and 93-102, 94- W0 201?/1043_81 H3) in the VH.
• the variable domain residues are numbere?CT/AU2018/051268t et al. , supra, for each of these definitions.
• FR residues are those variable domain residues other than the FIVR residues as herein defined.
• the FR of a variable domain generally consists of four FR domains: FR1, FR2, FR3, and FR4. Accordingly, the FIVR and FR sequences generally appear in the following sequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3 (L3)-FR4.
• variable domain residue numbering as in Kabat or "amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al. , supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
• a heavy chain variable domain may include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82.
• the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a "standard" Kabat numbered sequence.
• the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest. 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
• the "EU numbering system” or "EU index” is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et a/., supra).
• the "EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody.
• linear antibodies refers to the antibodies described in Zapata et al. (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.
• antigen and its grammatically equivalents expressions (e.g., "antigenic”) refer to a compound, composition, or substance that may be specifically bound by the products of specific humoral or cellular immunity, such as an antibody molecule or T-cell receptor.
• Antigens can be any type of molecule including, for example, haptens, simple antibodies, and many others.
• antigens include, but are not limited to, viral antigens, bacterial antigens, fungal antigens, protozoa and other parasitic antigens, tumor antigens, antigens involved in autoimmune disease, allergy and graft rejection, toxins, and other miscellaneous antigens.
• the term “binds”, “specifically binds to” or is “specific for” refers to measurable and reproducible interactions such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules.
• an antibody that binds to or specifically binds to a target is an antibody that binds this target with greater affinity, avidity, more readily, and/or with greater duration than it binds to other targets.
• the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target as measured, e.g., by a radioimmunoassay (RIA).
• an antibody that specifically binds to a target has a ?CJ/Ay2018/051268t (Kd) of ⁇ 1 mM, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, or ⁇ 0.1 nM.
• Kd ?CJ/Ay2018/051268t
• an antibody specifically binds to an epitope on a protein that is conserved among the protein from different species.
• specific binding can include, but does not require exclusive binding.
• binding agent refers to an agent that binds to a target antigen and does not significantly bind to unrelated compounds.
• binding agents that can be effectively employed in the disclosed methods include, but are not limited to, lectins, proteins, and antibodies, such as monoclonal antibodies, chimeric antibodies, or polyclonal antibodies, or antigen-binding fragments thereof, as well as aptamers, Fc domain fusion proteins, and aptamers having or fused to hydrophobic protein domain, e.g., Fc domain, etc.
• the binding agent is an exogenous antibody.
• An exogenous antibody is an antibody not naturally produced in a mammal, e.g. in a human, by the mammalian immune system.
• biomarker refers to an indicator, e.g. , predictive, diagnostic, and/or prognostic, which can be detected in a sample.
• the biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., T-cell dysfunctional disorder) characterized by certain, molecular, pathological, histological, and/or clinical features.
• a biomarker is a gene.
• Biomarkers include, but are not limited to, polynucleotides (e.g.
• DNA, and/or RNA DNA, and/or RNA
• polynucleotide copy number alterations e.g., DNA copy numbers
• polypeptides e.g., polypeptide and polynucleotide modifications
• carbohydrates e.g., glycolipid-based molecular markers.
• biomarker signature refers to one or a combination of biomarkers whose expression is an indicator, e.g. , predictive, diagnostic, and/or prognostic.
• the biomarker signature may serve as an indicator of a particular subtype of a disease or disorder (e.g. , T-cell dysfunctional disorder) characterized by certain molecular, pathological, histological, and/or clinical features.
• the biomarker signature is a "gene signature.”
• the term “gene signature” is used interchangeably with “gene expression signature” and refers to one or a combination of polynucleotides whose expression is an indicator, e.g., predictive, diagnostic, and/or prognostic.
• the biomarker signature is a "protein signature.”
• protein signature is used interchangeably with “protein expression signature” and refers to one or a combination of polypeptides whose expression is an indicator, e.g. , predictive, diagnostic, and/or prognostic.
• cancer and “cancerous” refer to or describe the physiological condition in subjects that is typically characterized by unregulated cell growth.
• examples of cancer include but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies.
• cancers include, but not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic (WQ .2019/104381 ; a rci no m a , penile carcinoma, melanoma, superficial spreaPCT/AU2018/051268tigo maligna melanoma
• Macroglobulinemia may be chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phacomatoses, edema (such as that associated with brain tumors), Meigs' syndrome, brain, as well as head and neck cancer, and associated metastases.
• CLL chronic lymphocytic leukemia
• ALL acute lymphoblastic leukemia
• PTLD post-transplant lymphoproliferative disorder
• abnormal vascular proliferation associated with phacomatoses such as that associated with brain tumors
• Meigs' syndrome such as that associated with brain tumors
• brain as well as head and neck cancer, and associated metastases.
• cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma.
• breast cancer colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkin's lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma.
• NDL non-Hodgkin'
• the cancer is selected from : small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet, in some embodiments, the cancer is selected from : non-small cell lung cancer, colorectal cancer, glioblastoma and breast carcinoma, including metastatic forms of those cancers. In specific embodiments, the cancer is melanoma or lung cancer, suitably metastatic melanoma or metastatic lung cancer.
• the cell proliferative disorder is cancer. In some embodiments, the cell proliferative disorder is a tumor, including a solid tumor.
• chemotherapeutic agent includes compounds useful in the treatment of cancer.
• chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin
• alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine
• adrenocorticosteroids including prednisone and prednisolone
• cyproterone acetate including finasteride and dutasteride
• dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino- doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcello
• demecolcine diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; phenamet; pirarubicin;
• losoxantrone podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
• TAXOL paclitaxel
• ABRAXANE® Cremophor- free
• albumin-engineered nanoparticle formulations of paclitaxel American Pharmaceutical Partners, Schaumberg, III.
• TAXOTERE® docetaxel, doxetaxel; Sanofi-Aventis
• mitoxantrone vincristine
• NAVELBINE® vinorelbine
• novantrone teniposide
• edatrexate edatrexate
• daunomycin aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids and derivatives of any of the above.
• Chemotherapeutic agent also includes (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)- imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), tWO.2019/104381 -ozole, RIVISOR® (vorozole), FEMARA
• ribozymes such as VEGF expression inhibitors (e.g. , ANGIOZYME®) and FIER2 expression inhibitors;
• vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,
• LEUVECTIN® LEUVECTIN®, and VAXID®
• PROLEUKIN® PROLEUKIN®
• rIL-2 a topoisomerase 1 inhibitor
• Chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab
• Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol,
• cidfusituzumab cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, peefusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizum
• Chemotherapeutic agent also includes "EGFR inhibitors,” which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an "EGFR antagonist.”
• EGFR inhibitors refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity
• Examples of such agents include antibodies and small molecules that bind to EGFR.
• antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533, Mendelsohn et at.
• chimerized 225 C225 or Cetuximab; ERBUTIX®
• H225 reshaped human 225
• IMC-11F8 a fully human, EGFR-targeted antibody
• EMD7200 a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-a for EGFR binding (EMD/Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies
• the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659439A2, Merck Patent GmbH).
• EGFR antagonists include small molecules such as compounds described in U.S. Pat. Nos. 5,616,582,
• EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSI Pharmaceuticals); PD 183805 (Cl 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7- [3-(4-morpholinyl)propoxy]-6-quin-azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli- ne, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-
• Chemotherapeutic agents also include "tyrosine kinase inhibitors" including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKIine), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI- 1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non
• pyrrolopyrimidines such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines, 4- (phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloyl methane, 4,5-bis (4- fluoroanilino)phthalimide); tyrphostines containing nitrothiophene moieties; PD-0183805 (Warner- Lamber); antisense molecules (e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No.
• Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprel
• Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate
• Rontalizumab e.g., Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE pathway blockers such as Anti- Mi prime; Secreted homotrimeric LTa3 and membrane bound heterotrimer LTal ⁇ 2 blockers such as Anti-lymphotoxin a (LTa); radioactive isotopes (e.g.
• miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-I8-OCH 3 , or farnesyl transferase inhibitors (L-739749, L- 744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechine gallate, theaflavins, flavanols, procyanidins, betulinic acid and derivatives thereof; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; acetylcamptothecin, scop
• celecoxib or etoricoxib include proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASARTM); and pharmaceutically acceptable salts, acids or derivatives of iWO 2019/104381 ; as well as combinations of two or more of the above suc?.
• T/AU2018/051268 abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATINTM) combined with 5-FU and leucovorin.
• ELOXATINTM oxaliplatin
• Chemotherapeutic agents also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects.
• NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase.
• Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumirac
• osteoarthritis inflammatory arthropathies, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain, headache and migraine, postoperative pain, mild-to-moderate pain due to inflammation and tissue injury, pyrexia, ileus, and renal colic.
• a "companion diagnostic” refers to a diagnostic method and or reagent that is used to identify subjects susceptible to treatment with a particular treatment or to monitor treatment and/or to identify an effective dosage for a subject or sub-group or other group of subjects.
• a companion diagnostic refers to reagents, such as a reagent for detecting, measuring or localizing a T-cell function biomarker (e.g., as described herein) in a sample.
• the companion diagnostic refers to the reagents and also to the test(s) that is/are performed with the reagent.
• the term “complex” refers to an assemblage or aggregate of molecules (e.g. , peptides, polypeptides, etc. ) in direct and/or indirect contact with one another.
• “contact”, or more particularly, “direct contact” means two or more molecules are close enough so that attractive noncovalent interactions, such as Van der Waal forces, hydrogen bonding, ionic and hydrophobic interactions, and the like, dominate the interaction of the molecules.
• a complex of molecules e.g. , a peptide and polypeptide
• the complex is formed under conditions such that the complex is thermodynamically favored (e.g.
• polypeptide complex or “protein complex,” as used herein, refers to a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, undecamer, dodecamer, or higher order oligomer.
• the polypeptide complexes are formed by self-assembly of a LSD (e.g. , a LSD1 such as LSDlp) and EOMES.
• correlate refers to determining a relationship between one type of data with another or with a state (e.g., T-cell activation status, mesenchymal state, immune status, etc.) .
• state e.g., T-cell activation status, mesenchymal state, immune status, etc.
• correlate or “correlating” is meant comparing, in any way, the performance and/or results of a first analysis or protocol with the performance and/or results of a second analysis or protocol. For example, one may use the results of a first analysis or protocol in carrying out a second protocols and/or one may use the results of a first analysis or protocol to determine whether a second analysis or protocol should be performed.
• polypeptide analysis or protocol one may use the results of the polypeptide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed.
• polynucleotide analysis or protocol one may use the results of the polynucleotide expression analysis or protocol to determine whether a specific therapeutic regimen should be performed. .
• amino acid sequence that displays substantial sequence similarity or identity to a reference amino acid sequence.
• amino acid sequence will display at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to at least a portion of the reference amino acid sequence.
• cytolytic activity refers to ability of a cell, e.g. , a CD8 + cell or an NK cell, to lyse target cells. Such cytolytic activity can be measured using standard techniques, e.g., by radioactively labeling the target cells.
• cytotoxic agent refers to any agent that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise hinders a cellular function).
• Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g. , At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.
• radioactive isotopes e.g. , At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu
• chemotherapeutic agents e.g. , At 211 , I 131 ,
• Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism.
• the cytotoxic agent is a taxane.
• the taxane is paclitaxel or docetaxel.
• the cytotoxic agent is a platinum agent. In some embodiments, the cytotoxic agent is an antagonist of EGFR. In representative examples of this type, the antagonist of EGFR is N-(3- ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In some
• the cytotoxic agent is a RAF inhibitor.
• the RAF iWO 2019/104381, F and/or CRAF inhibitor In other non-limiting examples, PCT/AU2018/051268 vemurafenib.
• the cytotoxic agent is a PI3K inhibitor.
• cytotoxic therapy refers to therapies that induce cellular damage including but not limited to radiation, chemotherapy, photodynamic therapy,
• a cytotoxic therapeutic may induce DNA damage when applied to a cell.
• delay of a disease means to defer, hinder, slow, retard, stabilize, and/or postpone development of the disease (such as a T-cell dysfunctional disorder).
• This delay can be of varying lengths of time, depending on the history of the disease and/or individual being treated.
• a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease.
• a late stage cancer such as development of metastasis, may be delayed.
• detection includes any means of detecting, including direct and indirect detection.
• diagnosis is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition (e.g., T-cell dysfunctional disorder).
• diagnosis may refer to identification of a particular type of T-cell dysfunctional disorder.
• Diagnosis may also refer to the classification of a particular subtype of T-cell dysfunctional disorder, e.g. , by histopathological criteria, or by molecular features (e.g. , a subtype characterized by expression of one or a combination of biomarkers (e.g., particular genes or proteins encoded by said genes)).
• a method of aiding diagnosis of a disease or condition can comprise measuring certain biomarkers in a biological sample from an individual.
• a “disorder” is any condition that would benefit from treatment including, but not limited to, chronic and acute disorders or diseases including those pathological conditions which predispose a subject to the disorder in question.
• the term "dysfunction" in the context of immune dysfunction refers to a state of reduced immune responsiveness to antigenic stimulation.
• the term includes the common elements of both exhaustion and/or anergy in which antigen recognition may occur, but the ensuing immune response is ineffective to control infection or tumor growth.
• disfunctional also includes refractory or unresponsive to antigen recognition, specifically, impaired capacity to translate antigen recognition into down stream T-cell effector functions, such as proliferation, cytokine production (e.g., IL-2, IFN-g, TNF- a, etc. ) and/or target cell killing.
• cytokine production e.g., IL-2, IFN-g, TNF- a, etc.
• an "effective amount” is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder.
• An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual.
• An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically lW.Q-2019/104381.
• beneficial or desired results includeP.QT/Ay2018/05_1268 eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
• beneficial or desired results include clinical results such as decreasing one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival.
• an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (/.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (/.e., slow to some extent and desirably stop) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the cancer or tumor.
• an effective amount of the drug may have the effect in reducing pathogen (bacterium, virus, etc.
• an effective amount can be administered in one or more administrations.
• an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
• an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
• an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
• an "effective response" of a patient or a patient's “responsiveness" to treatment with a medicament and similar wording refers to the clinical or therapeutic benefit imparted to a patient at risk for, or suffering from, a disease or disorder, such as cancer.
• such benefit includes any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.
• a patient who "does not have an effective response" to treatment refers to a patient who does not have any one of extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.
• Enhancing 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.
• enhancing T-cell function include any one or more of: increased secretion of IFN-y, increased secretion of TNF-a, increased secretion of IL-2 from CD8 + T-cells, increased proliferation, increased antigen responsiveness (e.g. , viral, pathogen, or tumor clearance) relative to such levels before the intervention.
• the level of enhancement is as least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of ordinary skill in the art.
• epithelial phenotype is understood in the art, aPCT/AU201_8/Q51268 by morphological, molecular and/or functional characteristics.
• epithelial cells generally have a rounded or cobblestone appearance, express the epithelial marker E-cadherin, are rapidly dividing and/or have relatively low levels of motility, invasiveness and/or anchorage-independent growth as compared with mesenchymal cells.
• EMT epithelial-to-mesenchymal transition
• mesenchymal phenotype which is a normal process of embryonic development.
• EMT is also the process whereby injured epithelial cells that function as ion and fluid transporters become matrix remodeling mesenchymal cells. In carcinomas, this transformation typically results in altered cell morphology, the expression of mesenchymal proteins and increased invasiveness.
• the criteria for defining EMT in vitro involve the loss of epithelial cell polarity, the separation into individual cells and subsequent dispersion after the acquisition of cell motility (see, Vincent-Salomon et ai , Breast Cancer Res.
• Classes of molecules that change in expression, distribution, and/or function during EMT, and that are causally involved, include growth factors (e.g., transforming growth factor-b (TGF-b), wnts), transcription factors (e.g., Snail, SMAD, LEF, and nuclear b-catenin), molecules of the cell-to-cell adhesion axis (cadherins, catenins), cytoskeletal modulators (Rho family), and extracellular proteases (matrix metalloproteinases, plasminogen activators) (see, Thompson et a/., Cancer Research 65, 5991-5995, Jul. 15, 2005).
• EMT refers to a process whereby epithelial cancer cells take on a mesenchymal phenotype, which may be associated with metastasis. These mesenchymal cells may display reduced adhesiveness, increased motility and invasiveness and are relatively resistant to immunotherapeutic agents, chemotherapeutic agents and/or radiation (e.g., treatments that target rapidly dividing cells).
• epitope refers to that portion of a molecule capable of being recognized by and bound by an antibody at one or more of the antibody's antigen-binding regions. Epitopes often consist of a surface grouping of molecules such as amino acids or sugar side chains and have specific three-dimensional structural characteristics as well as specific charge characteristics.
• the epitope can be a protein epitope. Protein epitopes can be linear or conformational. In a linear epitope, all of the points of interaction between the protein and the interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein.
• non-linear epitope or “conformational epitope” comprises non-contiguous polypeptides (or amino acids) within the antigenic protein to which an antibody specific to the epitope binds. Once a desired epitope on an antigen is determined, it is possible to generate antibodies to that epitope, e.g., using the techniques described in the present specification.
• the generation and characterization of antibodies may elucidate information about desirable epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope.
• An approach to achieve this is to conduct competition and cross-competition studies to find antibodies that compete or cross- compete with one another for binding to a target antigen (e.g., PD-1), e.g., the antibodies compete for binding to the antigen.
• a target antigen e.g., PD-1
• exhaustion refers to T-cell exhaustion as a state of T-cell dysfunction that arises from sustained TCR signaling that occurs during many chronic infections and cancer. It is distinguished from anergy in that it arises not through incomplete or deficient signaling, but from sustained signaling. It is defined by poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that ( WQ 2 l?/104381 c tor or memory T-cells. Exhaustion prevents optimal cont?CT/AU2018/051268 tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g.,
• RNA transcript e.g. , mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.
• expression of a coding sequence results from transcription and translation of the coding sequence.
• expression of a non-coding sequence results from the transcription of the non-coding sequence.
• level of expression or “expression level” in general are used interchangeably and generally refer to the amount of a biomarker in a sample. “Expression” generally refers to the process by which information (e.g. , gene-encoded and/or epigenetic) is converted into the structures present and operating in the cell. Therefore, as used herein,
• expression may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide). Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications (e.g., post-translational modification of a polypeptide) shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post-translational processing of the polypeptide, e.g. , by proteolysis.
• Expressed genes include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (e.g. , transfer and ribosomal RNAs).
• Elevated expression refers to an increased expression or increased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., T-cell dysfunctional disorder) or an internal control (e.g., housekeeping biomarker).
• a control such as an individual or individuals who are not suffering from the disease or disorder (e.g., T-cell dysfunctional disorder) or an internal control (e.g., housekeeping biomarker).
• Reduced expression refers to a decreased expression or decreased levels of a biomarker in an individual relative to a control, such as an individual or individuals who are not suffering from the disease or disorder (e.g., T-cell dysfunctional disorder) or an internal control (e.g., housekeeping biomarker).
• a control such as an individual or individuals who are not suffering from the disease or disorder (e.g., T-cell dysfunctional disorder) or an internal control (e.g., housekeeping biomarker).
• reduced expression is little or no expression.
• housekeeping biomarker refers to a biomarker or group of biomarkers (e.g. , polynucleotides and/or polypeptides) which are typically similarly present in all cell types.
• the housekeeping biomarker is a "housekeeping gene.”
• a "housekeeping gene” refers herein to a gene or group of genes which encode proteins whose activities are essential for the maintenance of cell function and which are typically similarly present in all cell types.
• a "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell either in vitro or in vivo.
• growth inhibitory agent is growth inhibitory antibody that prevents or reduces proliferation of a cell expressing an antigen to which the antibody binds.
• the growth inhibitory agent may be one which significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents t i? 2019/104381 rre st and M-phase arrest.
• agents that arrest G1 also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.
• Taxanes are anticancer drugs both derived from the yew tree.
• Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel and docetaxel promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in cells.
• immune effector cells in the context of the present invention relates to cells which exert effector functions during an immune reaction.
• such cells secrete cytokines and/or chemokines, kill microbes, secrete antibodies, recognize infected or cancerous cells, and optionally eliminate such cells.
• immune effector cells comprise T-cells (cytotoxic T-cells, helper T-cells, tumor infiltrating T-cells), B-cells, natural killer (NK) cells, lymphokine-activated killer (LAK) cells, neutrophils, macrophages, and dendritic cells.
• immune effector functions in the context of the present invention includes any functions mediated by components of the immune system that result, for example, in the killing of virally infected cells or tumor cells, or in the inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor dissemination and metastasis.
• the immune effector functions in the context of the present invention are T-cell mediated effector functions.
• Such functions comprise in the case of a helper T-cell (CD4 + T-cell) the recognition of an antigen or an antigen peptide derived from an antigen in the context of MFIC class II molecules by T-cell receptors, the release of cytokines and/or the activation of CD8 + lymphocytes (CTLs) and/or B-cells, and in the case of CTL the recognition of an antigen or an antigen peptide derived from an antigen in the context of MFIC class I molecules by T-cell receptors, the elimination of cells presented in the context of MFIC class I molecules, i.e.
• cells characterized by presentation of an antigen with class I MFIC for example, via apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-g and TNF-a, and specific cytolytic killing of antigen expressing target cells.
• immune response refers to any detectable response to a particular substance (such as an antigen or immunogen) by the immune system of a host mammal, such as innate immune responses (e.g., activation of Toll receptor signaling cascade), cell-mediated immune responses (e.g. , responses mediated by T cells, such as antigen-specific T cells, and non specific cells of the immune system), and humoral immune responses (e.g. , responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids).
• innate immune responses e.g., activation of Toll receptor signaling cascade
• cell-mediated immune responses e.g. , responses mediated by T cells, such as antigen-specific T cells, and non specific cells of the immune system
• humoral immune responses e.g. , responses mediated by B cells, such as generation and secretion of antibodies into the plasma, lymph, and/or tissue fluids.
• immunogenic refers to the ability of a substance to cause, elicit, stimulate, or induce an immune response including an enhanced T-cell (e.g. , CD8 + T-cell) immune response, or to improve, enhance, increase or prolong a pre-existing immune response, against a particular antigen, whether alone or when linked to a carrier, in the presence or absence of an adjuvant.
• WO 2019/10_4381j mmunO genicity refers to the ability of a particular substS T/AU2018/051268 immune response. Tumors are immunogenic and enhancing tumor immunogenicity aids in the clearance of the tumor cells by the immune response. Examples of enhancing tumor
• immunogenicity include treatment with a LSD inhibitor (e.g., a LSD1 inhibitor) and a PD-1 binding antagonist.
• a LSD inhibitor e.g., a LSD1 inhibitor
• a PD-1 binding antagonist e.g., a PD-1 binding antagonist
• infection refers to invasion of body tissues by disease-causing microorganisms, their multiplication and the reaction of body tissues to these microorganisms and the toxins they produce. "Infection” includes but are not limited to infections by viruses, prions, bacteria, viroids, parasites, protozoans and fungi.
• Retroviridae human immunodeficiency viruses such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III); and other isolates, such as HIV-LP); Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis, including Norwalk and related viruses); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Fllovlrldae (e.g.,
• Orthomyxoviridae e.g., influenza viruses
• Bunyaviridae e.g., Hantaan viruses, bunya viruses, phleboviruses and Nairo viruses
• Arenaviridae hemorrhagic fever viruses
• Reoviridae e.g., reoviruses, orbiviruses and rotaviruses
• Bimaviridae Hepadnaviridae (Hepatitis B virus);
• Parvoviridae parvoviruses
• Papovaviridae papilloma viruses, polyoma viruses
• Adenoviridae most adenoviruses
• Herpesviridae herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus
• Poxviridae variola viruses, VACV, pox viruses
• Iridoviridae e.g., African swine fever virus
• Representative bacteria that are known to be pathogenic include pathogenic Pasteurella species (e.g., Pasteurella multocida ), Staphylococcus species (e.g., Staphylococcus aureus ), Streptococcus species (e.g. , Streptococcus pyogenes (Group A Streptococcus ), Streptococcus agalactiae (Group B Streptococcus ),
• Pasteurella species e.g., Pasteurella multocida
• Staphylococcus species e.g., Staphylococcus aureus
• Streptococcus species e.g. , Streptococcus pyogenes (Group A Streptococcus ), Streptococcus agalactiae (Group B Streptococcus )
• Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae ), Neisseria species (e.g. , Neisseria gonorrhoeae, Neisseria meningitidis ), Escherichia species (e.g., enterotoxigenic E. coli (ETEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), and enteroinvasive E. coli (EIEC)), Bordetella species, Campylobacter species, Legionella species (e.g.
• ETEC enterotoxigenic E. coli
• EPEC enteropathogenic E. coli
• EHEC enterohemorrhagic E. coli
• EIEC enteroinvasive E. coli
• Legionella pneumophila Pseudomonas species, Shigella species, Vibrio species, Yersinia species, Salmonella species, Haemophilus species (e.g. , Haemophilus influenzae), Brucella species, Francisella species, Bacteroides species,
• Clostridiium species e.g., Clostridium difficile, Clostridium perfringens, Clostridium tetani
• Mycobacteria species e.g. , M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae
• Helicobacter pyloris Borelia burgdorferi, Listeria monocytogenes, Chlamydia trachomatis,
• Non-limiting pathogenic fungi include Cryptococcus neoformans,
• WQ 2019/104381 Z oa helminths, Plasmodium, such as Plasmodium falcipaiPC T/AU2018/051268 malariae, Plasmodium ovale, and Plasmodium vivax; Toxoplasma gondii; Trypanosoma brucei, Trypanosoma cruzi; Schistosoma haematobium, Schistosoma mansoni, Schistosoma japonicum; Leishmania donovani; Giardia intestinalis; Cryptosporidium parvum; and the like.
• kits of the invention include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention.
• the instructional material of the kit of the invention may, for example, be affixed to a container which contains the therapeutic or diagnostic agents of the invention or be shipped together with a container which contains the therapeutic or diagnostic agents of the invention.
• label when used herein refers to a detectable compound or composition.
• the label is typically conjugated or fused directly or indirectly to a reagent, such as a
• the label may itself be detectable (e.g., radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which results in a detectable product.
• leukocytes or "white blood cell” as used herein refers to any immune cell, including monocytes, neutrophils, eosinophils, basophils, and lymphocytes.
• LSD inhibitor means an agent that decreases or inhibits the function or biological activity of a LSD polypeptide (e.g., LSD1 - also known as lysine-specific histone demethylase 1A; lysine (K)-specific demethylase 1 (KDM1); lysine (K)-specific demethylase 1A (KDM1A); BRAF35-HDAC complex protein BHC110; FAD-binding protein BRAF35-HDAC complex, 110 kDa subunit; amine oxidase (flavin containing) domain 2 (AOF2); lysine-specific histone demethylase 1; RP1-184J9.1 - and LSD2 - also known as lysine- specific histone demethylase IB (KDM1B); amine oxidase flavin-containing 1 (AOF1); amine oxidase (flavin-containing) domain 1; fla
• lymphocytes refers to cells of the immune system which are a type of white blood cell. Lymphocytes include, but are not limited to, T-cells (cytotoxic and helper T-cells), B-cells and natural killer cells (NK cells).
• T-cells cytotoxic and helper T-cells
• B-cells cytotoxic and helper T-cells
• NK cells natural killer cells
• tumor infiltrating lymphocyte refers to lymphocytes that are present in a solid tumor.
• circulating lymphocyte refers to lymphocytes that are present in the circulation (e.g., present in blood).
• memory T effector cells is meant a subset of T-cells including CTL and helper T-cells that have previously encountered and responded to their cognate antigen; thus, the term antigen-experienced T-cell is often applied.
• T-cells can recognize foreign microbes, such as bacteria or viruses, as well as cancer cells.
• Memory T effector cells have become "experienced” by having encountered antigen during a prior infection, encounter with cancer, or previous vaccination.
• memory T effector cells can reproduce to mount a faster and stronger immune response than the first time the immune system responded to the microbe. This behavior is utilized in T lymphocyte proliferation assays, which can reveal exposure to specific antigens.
• mesenchymal phenotype is understood in the aPCT/AU2018/051268jfj e d by morphological, molecular and/or functional characteristics.
• mesenchymal cells generally have an elongated or spindle-shaped appearance, express the mesenchymal markers vimentin, fibronectin and N-cadherin, divide slowly or are non-dividing and/or have relatively high levels of motility, invasiveness and/or anchorage-independent growth as compared with epithelial cells.
• MET meenchymal-to-epithelial transition
• EMT electrospray-to-epithelial transition
• MET refers to the reprogramming of cells that have undergone EMT to regain one or more epithelial characteristics (e.g., as described above). For example, such cells typically exhibit reduced motility and/or invasiveness and/or are rapidly dividing, and may thereby regain sensitivity to immunotherapeutics and/or cytotoxic agents.
• multiplex-PCR refers to a single PCR reaction carried out on nucleic acid obtained from a single source (e.g., an individual) using more than one primer set for the purpose of amplifying two or more DNA sequences in a single reaction.
• patient refers to any subject, particularly a vertebrate subject, and even more particularly a mammalian subject, for whom therapy or prophylaxis is desired.
• Suitable vertebrate animals that fall within the scope of the invention include, but are not restricted to, any member of the subphylum Chordata including primates (e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e.g., cynomologus monkeys such as Macaca fascicularis, and/or rhesus monkeys ( Macaca mulatta )) and baboon (Papio ursinus), as well as marmosets (species from the genus Callithrix ), squirrel monkeys (species from the genus Saimiri) and tamarins (species from the genus Saguinus ), as well as species of apes such as chimpanzees (Pan troglodytes )), rodents (e.g.
• primates e.g., humans, monkeys and apes, and includes species of monkeys such from the genus Macaca (e
• a preferred subject is a human in need of eliciting an immune response, including an immune response with enhanced T-cell activation. Flowever, it will be understood that the aforementioned terms do not imply that symptoms are present.
• composition or “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the composition or formulation would be administered. Such formulations are sterile. "Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed.
• PD-1 refers to any form of PD-1 and variants thereof that retain at least part of the activity of PD-1. Unless indicated differently, such as by specific reference to human PD-1, PD-1 includes all mammalian species of native sequence PD-1, e.g.,
• exemplary human PD-1 is PCT/AU201.8/051268 Accession Number Q15116.
• PD-1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2.
• the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners.
• the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.
• PD-1 binding antagonists include anti-PD-1 antibodies, antigen binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides and other molecules that decrease, block, inhibit, abrogate or interfere with signal transduction resulting from the interaction of PD-1 with PD-L1 and/or PD-L2.
• a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T-cells mediated through PD-1 so as to render a dysfunctional T-cell less dysfunctional (e.g. , enhancing effector responses to antigen recognition).
• the PD-1 binding antagonist is an anti-PD-1 antibody.
• a PD-1 binding antagonist is MDX-1106 (nivolumab). In another specific aspect, a PD-1 binding antagonist is MK-3475 (pembrolizumab). In another specific aspect, a PD-1 binding antagonist is CT-011 (pidilizumab). In still another specific aspect, a PD-1 binding antagonist is AMP-224.
• the term "priming” refers to the induction of a first contact of the T-cell (typically a naive T-cell) with its specific antigen (e.g. , by antigen- presenting cells presenting the antigen to T-cells), which causes the differentiation of the T-cell into an effector-T cell (e.g. , a cytotoxic T cell or a T helper cell).
• T-cell typically a naive T-cell
• its specific antigen e.g. , by antigen- presenting cells presenting the antigen to T-cells
• an effector-T cell e.g. , a cytotoxic T cell or a T helper cell.
• radiation therapy is meant the use of directed gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are given as a one-time administration and typical dosages range from 10 to 200 units (Grays) per day.
• sample includes any biological specimen that may be extracted, untreated, treated, diluted or concentrated from a subject.
• Samples may include, without limitation, biological fluids such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, stool (i.e., feces), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor exudates, synovial fluid, ascitic fluid, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymph, fine needle aspirate, amniotic fluid, any other bodily fluid, cell lysates, cellular secretion products, inflammation fluid, semen and vaginal secretions.
• biological fluids such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, stool (i.e., feces), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor exudates, synovial fluid, ascitic fluid, peri
• Samples may include tissue samples and biopsies, tissue homogenates and the like.
• Advantageous samples may include ones comprising any one or more biomarkers as taught herein in detectable quantities.
• the sample is readily obtainable by minimally invasive methods, allowing the removal or isolation of the sample from the subject.
• the sample contains blood, especially peripheral blood, or a fraction or extract thereof.
• the sample comprises blood cells such as mature, immature or developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomocytes, hemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells natural killer cells, or fraction of such cells (e.g., a nucleic acid or protein fraction).
• the sample comprises leukocytes including peripheral blood mononuclear cells (PBMC).
• PBMC peripheral blood mononuclear cells
• WO 2019/104381 refers to a sample, cell, tissue, standard, or level that is used for comparison purposes.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non- diseased part of the body (e.g ., tissue or cells) of the same subject or individual.
• healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue e.g., cells or tissue adjacent to a tumor.
• a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject or individual.
• tissue sample or “cell sample” is meant a collection of similar cells obtained from a tissue of a subject or individual.
• the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
• the tissue sample may also be primary or cultured cells or cell lines.
• the tissue or cell sample is obtained from a disease tissue/organ.
• the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
• sequence identity refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison.
• a “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, I) or the identical amino acid residue (e.g., Ala,
• sequence identity will be understood to mean the "match percentage" calculated by an appropriate method.
• sequence identity analysis may be carried out using the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software.
• small molecule refers to a compound that has a molecular weight of less than 3 kilodalton (kDa), and typically less than 1.5 kilodalton, and more preferably less than about 1 kilodalton.
• Small molecules may be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon-containing) or inorganic molecules.
• extensive libraries of chemical and/or biological mixtures often fungal, bacterial, or algal extracts, may be screened with any of the assays of the invention to identify compounds that modulate a bioactivity.
• a "small organic molecule” is an organic compound (or organic compound complexed with an inorganic (W.Q.2019/104381 metal)) that has a molecular weight of less than 3 kilodal?CT/AlJ2018/Q51268 kilodalton, or even less than about 1 kDa.
• Stringent conditions or “high stringency conditions”, as defined herein, can be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50. degree. C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
• 5xSSC 0.75 M NaCI, 0.075 M sodium citrate
• 50 mM sodium phosphate pH 6.8
• 0.1% sodium pyrophosphate 0.1% sodium
• sustained response refers to the sustained effect on reducing tumor growth after cessation of a treatment.
• the tumor size may remain to be the same or smaller as compared to the size at the beginning of the administration phase.
• the sustained response has a duration at least the same as the treatment duration, at least 1.5x, 2. Ox, 2.5x, or 3. Ox length of the treatment duration.
• the term “synergistic” means that the therapeutic effect of a LSD inhibitor when administered in combination with a PD-1 binding antagonist (or vice-versa ) or when administered in combination with a PD-1 binding antagonist and a chemotherapeutic agent ("anti- PD-1— chemo combination”), is greater than the predicted additive therapeutic effects of the LSD inhibitor and the PD-1 binding antagonist, or the LSD inhibitor and the anti-PD-1— chemo combination, when administered alone.
• compositions generally a pharmaceutical formulation
• which is effective for enhancing immune effector function including any one or more of increased recognition of an antigen or an antigen peptide derived from an antigen in the context of MHC class II molecules by T-cell receptors, increased release of cytokines and/or the activation of CD8 + lymphocytes (CTLs) and/or B-cells, increased recognition of an antigen or an antigen peptide derived from an antigen in the context of MHC class I molecules by T-cell receptors, increased elimination of cells presented in the context of MHC class I molecules, i.e., cells characterized by presentation of an antigen with WO.2_019/1043_8l3xample, via apoptosis or perforin-mediated cell lysis, inaPCT/AU2018/051268 :
• the dose response curve used to determine synergy in the art is described for example by Sande et a/ (see, p. 1080-1105 in A. Goodman et a/., ed., the Pharmacological Basis of Therapeutics, MacMillan Publishing Co., Inc., New York (1980)).
• the optimum synergistic amounts can be determined, using a 95% confidence limit, by varying factors such as dose level, schedule and response, and using a computer-generated model that generates isobolograms from the dose response curves for various combinations of the LSD inhibitor and the PD-1 binding antagonist or anti-PD-1— chemo combination.
• the highest enhancement of immune effector function on the dose response curve correlates with the optimum dosage levels.
• a "T-cell dysfunctional disorder” is 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 inappropriate 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 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.
• treatment refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating or palliating the disease state, and remission or improved prognosis.
• an individual is successfully "treated” if one or more symptoms associated with a T-cell dysfunctional disorder are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, reducing pathogen infection, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging survival of individuals.
• Treg and "regulatory T-cells”, formerly known as suppressor T-cells, refer to T lymphocytes that maintain immunological tolerance.
• Tregs inhibit T cell-mediated immunity and suppress auto-reactive T cells that have escaped negative selection within the thymus.
• Adaptive Treg cells (called Th3 or Tr 1 cells) are thought to be generated during an immune response.
• Naturally occurring Treg cells are thought to be generated during an immune response.
• CD4 + CD25 + FoxP3 + Treg cells are generated in the thymus and have been linked to interactions between developing T-cells with both myeloid (CDllc + ) and plasmacytoid (CD123 + ) dendritic cells that have been activated with the cytokine thymic stromal lymphopoietin (TSLP).
• TSLP cytokine thymic stromal lymphopoietin
• Tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
• the terms 'WQ 2019/104381 ous”, “cell proliferative disorder”, “proliferative disorder” PCT/AU2018/051268 disorder” and “tumor” are not mutually exclusive as referred to herein.
• 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. Examples of tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
• underscoring or italicizing the name of a gene shall indicate the gene, in contrast to its protein product, which is indicated by the name of the gene in the absence of any underscoring or italicizing.
• "LSD1” shall mean the LSD1 gene
• “LSD1” shall indicate the protein product or products generated from transcription and translation and/or alternative splicing of the "LSD1" gene.
• the present invention is based in part of the determination that exposure of functionally repressed T-cells of a mesenchymal phenotype to LSD inhibitors, including LSD1 inhibitors, results in epigenetic reprogramming of the T-cells with de-repression of their immune effector function, including elevated expression of biomarkers of T-cell activation and effector capacity (e.g. , IFN-y, TNF-a, Ki67 and TBET), decreased expression of biomarkers of T-cell exhaustion (e.g. , EOMES), as well as increased activation and proliferation of T-cells, including effector and memory T-cells.
• LSD inhibitor-mediated epigenetic reprogramming confers enhanced susceptibility of exhausted T-cells to reinvigoration by PD-1 binding antagonists.
• compositions and methods that take advantage of a LSD inhibitor (e.g. , an inhibitor of LSD demethylase activity or an inhibitor of LSD nuclear translocation/localization) and a PD-1 binding antagonist to enhance immune effector function, and/or to enhance T-cell (e.g. , CD8 + T-cell or CD4 + T-cell) function, including increasing T-cell activation and enhancing susceptibility of exhausted T-cells to reinvigoration by PD-1 binding antagonists .
• LSD inhibitor e.g. , an inhibitor of LSD demethylase activity or an inhibitor of LSD nuclear translocation/localization
• T-cell e.g. , CD8 + T-cell or CD4 + T-cell
• the methods and compositions of the present invention are thus particularly useful in the treatment of T-cell dysfunctional disorders including cancers and infections.
• the LSD inhibitor includes and encompasses any active agent that reduces the accumulation, function or stability of a LSD; or decrease expression of a LSD gene, and such inhibitors include without limitation, small molecules and macromolecules such as nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, polysaccharides, lipopolysaccharides, lipids or other organic (carbon containing) or inorganic molecules.
• Preferred LSD inhibitors are ones that bind to LSD and inhibit its enzymatic activity and/or its nuclear localization. In specific embodiments, these LSD inhibitors are specific or selective LSD inhibitors.
• the LSD inhibitor is an antagonistic nucleic acid molecule that functions to inhibit the transcription or translation of LSD (e.g., LSD1 or LSD2 ) transcripts.
• LSD LSD1 or LSD2
• Representative transcripts of this type include nucleotide sequences corresponding to any one the following sequences: (1) human LSD1 nucleotide sequences as set forth for example in GenBank WO 2019/104381 M_015013.3, NP_001009999.1, and NM_001009999.2; hPCT/AU_2018/051268 de sequences as set forth for example in GenBank Accession No.
• nucleotide sequences that share at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity with any one of the sequences referred to in (1); (3) nucleotide sequences that hybridize under at least low, medium or high stringency conditions to the sequences referred to in (1); (4) nucleotide sequences that encode any one of the following amino acid sequences: human LSD1 amino acid sequences as set forth for example in GenPept Accession Nos. NP_055828.2, NP_001009999.1 and 060341.2; human LSD2 amino acid sequences as set forth for example in GenPept Accession Nos.
• NP_694587.3 nucleotide sequences that encode an amino acid sequence that shares at least
• Illustrative antagonist nucleic acid molecules include antisense molecules, aptamers, ribozymes and triplex forming molecules, RNAi and external guide sequences.
• the nucleic acid molecules can act as effectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
• Antagonist nucleic acid molecules can interact with any macromolecule, such as DNA, RNA, polypeptides, or carbohydrate chains.
• antagonist nucleic acid molecules can interact with LSD ⁇ e.g., LSD1 or LSD2 ) mRNA or the genomic DNA of LSD ⁇ e.g., LSD1 or LSD2 ) or they can interact with a LSD polypeptide e.g., LSD1 or LSD2).
• LSD LSD1 or LSD2
• mRNA mRNA
• genomic DNA of LSD ⁇ e.g., LSD1 or LSD2
• LSD polypeptide e.g., LSD1 or LSD2
• antagonist nucleic acid molecules are designed to interact with other nucleic acids based on sequence homology between the target molecule and the antagonist nucleic acid molecule.
• the specific recognition between the antagonist nucleic acid molecule and the target molecule is not based on sequence homology between the antagonist nucleic acid molecule and the target molecule, but rather is based on the formation of tertiary structure that allows specific recognition to take place.
• anti-sense RNA or DNA molecules are used to directly block the translation of LSD ⁇ e.g . , LSD1 or LSD2 ) by binding to targeted mRNA and preventing protein translation.
• Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule may be designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule may be designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule.
• the antisense molecules bind the target molecule with a dissociation constant (K d ) less than or equal to 10 -6 , 10 -8 , 10 _1 °, or 10 12 .
• K d dissociation constant
• oligodeoxyribonucleotides derived from the translation initiation site e.g., between -10 and +10 regions are employed.
• WO 2019/104381 ptamers are molecules that interact with a target molecul?CT/AlJ_2018/051268 jfj C way.
• Aptamers are generally small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
• Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin.
• Aptamers can bind very tightly with Kds from the target molecule of less than 10 12 M.
• the aptamers bind the target molecule with a Kd less than 10 -6 , 10 -8 , 10 _1 °, or 10 12 .
• Aptamers can bind the target molecule with a very high degree of specificity.
• aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule.
• an aptamer have a K d with the target molecule at least 10-, 100-, 1000-, 10,000-, or 100,000-fold lower than the K d with a background-binding molecule.
• a suitable method for generating an aptamer to a target of interest is the
• SELEXTM Systematic Evolution of Ligands by Exponential Enrichment
• the SELEXTM method is described in U.S. Pat. No. 5,475,096 and U.S. Pat. No. 5,270,163 (see also WO 91/19813). Briefly, a mixture of nucleic acids is contacted with the target molecule under conditions favorable for binding. The unbound nucleic acids are partitioned from the bound nucleic acids, and the nucleic acid-target complexes are dissociated.
• the dissociated nucleic acids are amplified to yield a ligand-enriched mixture of nucleic acids, which is subjected to repeated cycles of binding, partitioning, dissociating and amplifying as desired to yield highly specific high affinity nucleic acid ligands to the target molecule.
• anti-LSD e.g., anti -LSD1 or LSD2
• ribozymes are used for catalyzing the specific cleavage of LSD (e.g. , LSD1 or LSD2 ) RNA.
• LSD e.g. , LSD1 or LSD2
• the mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage.
• ribozymes that catalyze nuclease or nucleic acid polymerase type reactions, which are based on ribozymes found in natural systems, such as hammerhead ribozymes, hairpin ribozymes, and tetrahymena ribozymes. There are also a number of ribozymes that are not found in natural systems, but which have been engineered to catalyze specific reactions de novo. Representative ribozymes cleave RNA or DNA substrates. In some embodiments, ribozymes that cleave RNA substrates are employed.
• RNA targets Specific ribozyme cleavage sites within potential RNA targets are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features such as secondary structure that may render the oligonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.
• Triplex forming functional nucleic acid molecules are molecules that can interact with either double-stranded or single-stranded nucleic acid.
• triplex molecules When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is generally desirable that the triplex forming molecules bind the target molecule with a K d less than 10 -6 , 10 -8 , 10 1 , or 10 12 .
• External guide sequences are molecules that bind a target nucleic acid molecule forming a complex, and this complex is recognized by RNAse P, which cleaves the target
• Bacterial RNAse P can be recruited to cleave virtually any RNA sequence by using an EGS that causes the target RNA: EGS complex to mimic the natural tRNA substrate.
• EGS/RNAse P-directed cleavage of RNA can be utilized to cleave desired targets within eukaryotic cells.
• RNA molecules that mediate RNA interference (RNAi) of a LSD (e.g. , LSD1 or LSD2 ) gene or LSD (e.g., LSD1 or LSD2) transcript can be used to reduce or abrogate gene expression.
• RNAi refers to interference with or destruction of the product of a target gene by introducing a single-stranded or usually a double-stranded RNA (dsRNA) that is homologous to the transcript of a target gene.
• dsRNA double-stranded RNA interference
• siRNA small interfering RNA
• RNAi can be triggered by 21- to 23-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et a/., 2002 Mol. Cell. 10: 549-561; Elbashir et a/., 2001. Nature 411:494-498), or by micro-RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which are expressed In vivo using DNA templates with RNA polymerase III promoters (Zeng et a , 2002. Mol.
• dsRNA per se and especially dsRNA-producing constructs corresponding to at least a portion of a LSD (e.g. , LSD1 or LSD2 ) gene are used to reduce or abrogate its expression.
• a LSD e.g. , LSD1 or LSD2
• RNAi-mediated inhibition of gene expression may be accomplished using any of the techniques reported in the art, for instance by transfecting a nucleic acid construct encoding a stem-loop or hairpin RNA structure into the genome of the target cell, or by expressing a transfected nucleic acid construct having homology for a LSD (e.g., LSD1 or LSD2 ) gene from between convergent promoters, or as a head to head or tail to tail duplication from behind a single promoter.
• LSD e.g., LSD1 or LSD2
• Any similar construct may be used so long as it produces a single RNA having the ability to fold back on itself and produce a dsRNA, or so long as it produces two separate RNA transcripts, which then anneal to form a dsRNA having homology to a target gene.
• RNAi-encoding nucleic acids can vary in the level of homology they contain toward the target gene transcript, i.e., with dsRNAs of 100 to 200 base pairs having at least about 85% homology with the target gene, and longer dsRNAs, i.e., 300 to 100 base pairs, having at least about 75% homology to the target gene.
• RNA-encoding constructs that express a single RNA transcript designed to anneal to a separately expressed RNA, or single constructs expressing separate transcripts from convergent promoters are suitably at least about 100 nucleotides in length.
• RNA-encoding constructs that express a single RNA designed to form a dsRNA via internal folding are usually at least about 200 nucleotides in length.
• the promoter used to express the dsRNA-forming construct may be any type of promoter if the resulting dsRNA is specific for a gene product in the cell lineage targeted for (WO.2019/104381 -natively, the promoter may be lineage specific in that it i:PCT/AU2 () l_8/p51268 ells of a particular development lineage. This might be advantageous where some overlap in homology is observed with a gene that is expressed in a non-targeted cell lineage.
• the promoter may also be inducible by externally controlled factors, or by intracellular environmental factors.
• RNA molecules of about 21 to about 23 nucleotides which direct cleavage of specific mRNA to which they correspond, as for example described by Tuschl et al. in U.S. 2002/0086356, can be utilized for mediating RNAi.
• Such 21- to 23-nt RNA molecules can comprise a 3' hydroxyl group, can be single-stranded or double stranded (as two 21- to 23-nt RNAs) wherein the dsRNA molecules can be blunt ended or comprise overhanging ends (e.g., 5',
• the antagonist nucleic acid molecule is a siRNA.
• siRNAs can be prepared by any suitable method. For example, reference may be made to International Publication WO 02/44321, which discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3’ overhanging ends, which is incorporated by reference herein. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer. siRNA can be chemically or in v/tro-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin,
• siRNA can also be synthesized in vitro using kits such as Ambion’s SILENCERTM siRNA Construction Kit.
• siRNAs short hairpin RNAs
• Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex’s GENESUPPRESSORTM Construction Kits and Invitrogen's BLOCK-ITTM inducible RNAi plasmid and lentivirus vectors.
• methods for formulation and delivery of siRNAs to a subject are also well known in the art.
• RNAi molecules e.g., LSD ⁇ e.g., LSD1 or LSD2 ) siRNA and shRNA
• LSD ⁇ e.g., LSD1 or LSD2 siRNA and shRNA are described in the art (e.g., Yang, et al., 2010. Proc. Natl. Acad. Sci. USA 107: 21499-21504 and He et a/., 2012. Transcription 3: 3: 1-16) or available commercially from Santa Cruz Biotechnology,
• the present invention further contemplates peptide or polypeptide based inhibitor compounds.
• BHC80 also known as PHD finger protein 21A
• the present invention further contemplates the use of BHC80 or biologically active fragments thereof for inhibiting LSD1 enzymatic activity.
• Amino acid sequences of BHC80 polypeptides, and nucleotide sequences encoding BHC80 polypeptides, are publicly available. In this regard, reference may be made for example to GenBank Accession No.
• NP057705 for a Homo sapiens BHC80 amino acid sequence
• GenBank NM016621 for a nucleotide sequence encoding the amino acid sequence set forth in GenBank Accession No. NP057705
• GenBank Accession No. NP620094 for a Mus musculus
• GenBank NM138755 for a nucleotide secPCT/A U 2018/051268 amino acid sequence set forth in GenBank Accession No. NP620094
• GenBank NM001199647 for a nucleotide sequence encoding the amino acid sequence set forth in GenBank Accession No.
• Illustrative BHC80 polypeptides are selected from the group consisting of: (1) a polypeptide comprising an amino acid sequence that shares at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence similarity with the amino acid sequence listed in any one of the GenBank BHC80 polypeptide entries noted above; (2) a polypeptide comprising an amino acid sequence that shares at least 70,
• a BHC80 polypeptide can be introduced into a cell by delivering a polypeptide per se, or by introducing into the cell a BHC80 nucleic acid comprising a nucleotide sequence encoding a BHC80 polypeptide.
• a BHC80 nucleic acid comprises a nucleotide sequence selected from: (1) a BHC80 nucleotide sequence listed in any one of the GenBank BHC80 polynucleotide entries noted above; (2) a nucleotide sequence that shares at least 70, 71, 72, 73,
• the BHC80 nucleic acid can be in the form of a recombinant expression vector.
• the BHC80 nucleotide sequence can be operably linked to a transcriptional control element(s), e.g. , a promoter, in the expression vector.
• Suitable vectors include, e.g., recombinant retroviruses, lentiviruses, and adenoviruses; retroviral expression vectors, lentiviral expression vectors, nucleic acid expression vectors, and plasmid expression vectors.
• the expression vector is integrated into the genome of a cell. In other cases, the expression vector persists in an episomal state in a cell.
• WO 2019/104381 Uj table expression vectors include, but are not limited to,PCT/AU2018/051268/j ra
• Murine Leukemia Virus spleen necrosis virus
• vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like.
• the present invention also contemplates small molecule agents that reduce enzymatic activity of LSDs ⁇ e.g. , LSD1 or LSD2).
• Small molecule agents that reduce enzymatic activity of LSD1 that are suitable for use in the present invention include monoamine oxidase (MAO) inhibitors that also inhibit LSD1 enzymatic activity; polyamine compounds that inhibit LSD1 enzymatic activity;
• MAO monoamine oxidase
• MAO inhibitors include MAO-A-selective inhibitors, MAO-B- selective inhibitors, and MAO non-selective inhibitors.
• Illustrative examples of MAO inhibitors include reported inhibitors of the MAO-A isoform, which preferentially deaminates 5- hydroxytryptamine (serotonin) (5-HT) and norepinephrine (NE), and/or the MAO-B isoform, which preferentially deaminates phenylethylamine (PEA) and benzylamine (both MAO-A and MAO-B metabolize Dopamine (DA)).
• MAO inhibitors may be irreversible or reversible ⁇ e.g.
• reversible inhibitors of MAO-A RIMA
• MAO-A and/or MAO-B may have varying potencies against MAO-A and/or MAO-B ⁇ e.g. , non-selective dual inhibitors or isoform-selective inhibitors).
• the MAO inhibitors are selected from: clorgyline; L- deprenyl; isocarboxazid (MarplanTM); ayahuasca; nialamide; iproniazide; iproclozide; moclobemide (AurorixTM; 4-chloro-N-(2-morpholin-4-ylethyl)benzamide); phenelzine (NardilTM; ( ⁇ )-2- phenylethylhydrazine); tranylcypromine (ParnateTM; ( ⁇ )-trans-2-phenylcyclopropan-l-amine) (the congeneric of phenelzine); toloxatone; levo-deprenyl (SelegilineTM); harmala; RIMAs ⁇ e.g. , moclobemide, described in Da Prada et at. (1989. J Pharmacol Exp Ther 248:400-414);
• bifemelane desoxypeganine; harmine (also known as telepathine or banasterine); linezolid (ZYVOX, ZYVOXID); pargyline (EUDATIN, SUPIRDYL); dienolide kavapyrone desmethoxyyangonin; 5-(4-Arylmethoxyphenyl)-2-(2-cyanoethyl)tetrazoles; and the like.
• Small molecule LSD1 inhibitors may also be selected from polyamine compounds as described for example by Woster et a/ in U.S. Publication No. 2007/0208082, which is incorporated
• R 3 is selected from the group consisting of -Cs alkyl, C 4 - 5 cycloalkyl, C 3 -
• each f1 ⁇ 2 is independently selected from hydrogen or a Ci-Cs alkyl.
• a suitable polyamine compound is a compound of formula (I), wherein one or both Ri is a C 6 -C 20 aryl, such as a single ring aryl, including without limitation, a phenyl.
• the compound is of the formula (I) and each Ri is phenyl.
• q is I, m and p are 3, and n is 4.
• q is I, m and p are 3, and n is 7.
• a suitable polyamine compound is a compound of formula (I), where at least one or both Ri is a Cs-Qu or a Q-Cs alkyl, such as a linear alkyl.
• One or both Ri may be a Ci-Ce linear alkyl, such as methyl or ethyl.
• each Ri is methyl.
• One or both Ri may comprise or be a C 4 -C 15 cycloalkyl group, such as a cycloalkyl group containing a linear alkyl group, where the cycloalkyl group is connected to the molecule either via its alkyl or cycloalkyl moiety.
• one or both Ri may be cyclopropylmethyl or cyclohexylmethyl.
• one Ri is cyclopropylmethyl or cyclohexylmethyl and the other Ri is a linear alkyl group, such as a linear -Cs unsubstituted alkyl group, including without limitation an ethyl group.
• Ri is a C 3 - 5 branched alkyl group such as isopropyl.
• the substituted alkyl may be substituted with any substituent, including a primary, secondary, tertiary or quaternary amine.
• Ri is a Ci-Ce alkyl group substituted with an amine such that Ri may be e.g.
• alkyl-NH 2 or an alkyl-amine-alkyl moiety such as -(CH 2 ) y NH(CH 2 )zCH 3 where y and z are independently an integer from 1 to 8.
• Ri is -(CH 2 ) 3 NH 2 .
• the compound is of the formula (I) where one or both Ri is a C 7 -C 24 substituted or unsubstituted aralkyl, which in one embodiment is an aralkyl connected to the molecule via its alkyl moiety (e.g. , benzyl).
• both Ri are aralkyl moieties wherein the alkyl portion of the moiety is substituted with two aryl groups and the moiety is connected to the molecule via its alkyl group.
• one or both Ri is a C 7 -C 24 aralkyl wherein the alkyl portion is substituted with two phenyl groups, such as when Ri is WO_2019/1043811 0 r 2,2-dibenzylethyl.
• both Ri of forP . CT/AU2018/051268 diphenylethyl and n is 1, 2 or 5.
• each Ri of formula (I) is 2,2-diphenylethyI, n is 1, 2 or 5 and m and p are each 1.
• At least one Ri is hydrogen.
• the other i may be any moiety listed above for Ri, including an aryl group such as benzyl.
• Any of the compounds of formula (I) listed above include compounds where at least one or both of R 2 is hydrogen or a Q-Cs substituted or unsubstituted alkyl. In one embodiment, each R 2 is an unsubstituted alkyl such as methyl. In another embodiment, each R 2 is hydrogen. Any of the compounds of formula (I) listed above may be compounds where q is 1 and m and p are the same.
• the polyaminoguanidines of formula (I) may be symmetric with reference to the polyaminoguanidine core (e.g , excluding Ri).
• the compounds of formula (I) may be asymmetric, e.g. , when q is 0.
• m and p are 1.
• q is 0.
• n is an integer from 1 to 5.
• the compound is a polyaminobiguanide or N-alkylated polyaminobiguanide.
• An N-alkylated polyaminobiguanide intends a polyaminobiguanide where at least one imine nitrogen of at least one biguanide is alkylated.
• the compound is a polyaminobiguanide of the formula (I), or a salt, solvate, or hydrate thereof, where q is 1, and at least one or each Ri is of the structure:
• each R 3 is independently selected from the group consisting of Ci-Ce alkyl, C6-C20 aryl, C6-C20 heteroaryl, C7-C24 aral kyl, and C7-C24 heteroaralkyl; and each f1 ⁇ 2 is independently hydrogen or a Q-Cs alkyl.
• each R 3 is a Ci-Cs alkyl.
• the alkyl may be substituted with any substituent, including a primary, secondary, tertiary or quaternary amine.
• R 3 is a -Cs alkyl group substituted with an amine such that R 3 may be e.g., alkyl- NH 2 or an alkyl-amine-alkyl moiety such as -(CH 2 ) y NH(CH 2 ) z CH 3 where y and z are independently an integer from 1 to 8.
• R 3 is -(CH2)3N H2.
• R3 may also be a C4-C15 cycloalkyl or a C3-C15 branched alkyl.
• at least one or each R3 is a C6-C20 aryl.
• q is I, m and p are 3, and n is 4.
• q is I, m and p are 3, and n is 7.
• the compound is a polyaminobiguanide of formula (I) where at least one R3 is a C7-C24 aralkyl, which in one embodiment is an aralkyl connected to the molecule via its alkyl moiety.
• each R 3 is an aralkyl moiety where the alkyl portion of the moiety is substituted with one or two aryl groups and the moiety is connected to the molecule via its alkyl moiety.
• each R 3 is an aralkyl where the alkyl portion is substituted with two phenyl or benzyl groups, such as when R 3 is 2,2-diphenylethyl or 2,2-dibenzylethyl.
• each R 3 is 2,2-diphenylethyl and n is 1, 2 or 5.
• each R 3 is 2,2-diphenylethyl and n is 1, 2 or 5 and m and p are each 1.
• each R. 2 is an unsubstituted alkyl, such as methyl.
• each R 2 is a hydrogen
• any of the polyaminobiguanide compounds of formula (I) listed above include compounds where q is 1 and m and p are the same. Accordingly, the polyaminobiguanides of formula (I) may be symmetric with reference to the polyaminobiguanide core. Alternatively, the compounds of formula (I) may be asymmetric. In one embodiment, m and p are 1. In one embodiment, q is 0. In one embodiment, n is an integer from 1 to 5. In one embodiment, q, m and p are each 1 and n is 1, 2 or 5.
• each Ri, R , R 3 , m, n, p and q disclosed in reference to formula (I) intends and includes all combinations thereof the same as if each and every combination of Ri, R , R 3 , m, n, p and q were specifically and individually listed.
• Representative compounds of the formula (I) include, e.g :
• the polyamine compound is represented by the structure according to formula (II):
• n 1, 2 or 3
• each L is independently a linker of from about 2 to 14 carbons in length, for example of about 2, 3, 4, 5, 6, 8, 10, 12 or 14 carbon atoms in length, where the linker backbone atoms may be saturated or unsaturated, usually not more than one, two, three, or four unsaturated atoms will be present in a tether backbone, where each of the backbone atoms may be substituted or unsubstituted (for example with a Ci-C 8 alkyl), where the linker backbone may include a cyclic group (for example, a cyclohex-1, 3-diyl group where 3 atoms of the cycle are included in the backbone);
• each R12 is independently selected from hydrogen and a -Cs alkyl
• each Rn is independently selected from hydrogen, C2-C8 alkenyl, Ci-C 8 alkyl or C3- C 8 branched alkyl (e.g., methyl, ethyl, tert-butyl, isopropyl, pentyl, cyclobutyl, cyclopropylmethyl, 3-methylbutyl, 2-ethylbutyl, 5-NH 2 -pent-l-yl, propyl-l-ylmethyl(phenyl)phosphinate,
• each L is independently selected from: -CHRi 3 -(CH 2 ) m -, - CHRi 3 -(CH 2 )n-CH Ri 3 -, -(CH 2 )mCHRi 3 -, -CH 2 -A-CH 2 - and -(CH 2 ) P -
• m is an integer from 1 to 5;
• A is (CH2) m , ethane-1, 1-diyl or cyclohex-1, 3-diyl;
• n is an integer from 1 to 12;
• R 13 is a -Ce alkyl.
• the alkyl portion of the aralkyl or heteroaralkyl moiety is connected to the molecule via its alkyl moiety.
• (1 ⁇ 2 may be an aralkyl moiety such as 2-phenylbenzyl, 4-phenylbenzyl, 3,3,- diphenylpropyl, 2-(2-phenylethyl)benzyl, 2-methyl-3-phenylbenzyl, 2-napthylethyl, 4- (pyrenyl)butyl, 2-(3-methylnapthyl)ethyl, 2-(l,2-dihydroacenaphth-4-yl)ethyl and the like.
• aralkyl moiety such as 2-phenylbenzyl, 4-phenylbenzyl, 3,3,- diphenylpropyl, 2-(2-phenylethyl)benzyl, 2-methyl-3-phenylbenzyl, 2-napthylethyl, 4- (pyrenyl)butyl, 2-(3-methylnapthyl)ethyl, 2-(l,2-dihydroacenaphth-4-yl)eth
• Rn may be a heteroaralkyl moiety such as 3- (benzoimidazolyl)propanoyl, l-(benzoimidazolyl)methanoyl, 2-(benzoimidazolyl)ethanoyl, 2- (benzoimidazolyl)ethyl and the like.
• the compound of formula (II) comprises at least one moiety selected from the group consisting of t-butyl, isopropyl, 2-ethylbutyl, 1-methylpropyl, 1- methylbutyl, 3-butenyl, isopent-2-enyl, 2-methylpropan-3-olyl, ethylthiyl, phenylthiyl, propynoyl, l-methyl-lH-pyrrole-2-yl; trifluoromethyl, cyclopropanecarbaldehyde, halo-substituted phenyl, nitro-substituted phenyl, alkyl-substituted phenyl, 2,4,6-trimethylbenzyl, halo-5-substituted phenyl (such as para-(F3S)-phenyl, azido and 2-methylbutyl.
• t-butyl isopropyl, 2-ethylbutyl, 1-
• each Rn is independently selected from hydrogen, n-butyl, ethyl, cyclohexyl methyl, cyclopentylmethyl, cyclopropylmethyl,
• the polyamine compound is of the structure of formula
• Li, L2 and L3 are independently selected from -CHRi 3 -(CH 2 ) m -, -CHR13- (CH 2 )n-CH Ri3-, -(CH 2 )m-CHRi3-, -CH 2 -A-CH 2 - and -(CH 2 ) P -
• the polyamine compound is of the structure of formula
• Li is -CHRi3-(CH 2 ) m -;
• L2 is -CH Ri3-(CH 2 ) n -CHRi3-; and
• L3 is -(CH 2 ) m -CHRi3-; where Rn, R12, R13, m and n are as defined above.
• the polyamine compound is of the structure of formula (III) where: Li, L2 and L3 are independently -CH2-A-CH2-; and R12 is hydrogen; where Rn and A are as defined above.
• Rn and A are as defined above.
• at least one of an A and an Rn comprises an alkenyl moiety.
• the polyamine compound is of the structure of formula (III) where: Li, L2 and L3 are independently -(CH2) P - where p is as defined above; and Rn is hydrogen. In particular embodiments, for U and L3, p is an integer from 3 to 7, and for L3 p is an integer from 3 to 14.
• the polyamine compound is of thePCT/AU2018/051268a (III) where: Li, and L 3 are independently -(CH 2 ) P -; L is -CH 2 -A-CH 2 -; and R is hydrogen; where R 12 , p and A are as defined above.
• p is an integer from 2 to 6
• for L 3 A is (CH 2 ) X where x is an integer from 1 to 5, or cyclohex-1, 3-diyl.
• the polyamine compound is of the structure of formula (II), where n is 2, such that the compound has a structure according to formula (IV):
• Li and L 2 are independently selected from -CHRi 3 -(CH 2 )m-CHRi 3 -(CH 2 )n- CHRis-,— (CH 2 ) n , CHR 13 -, -CH 2 -A-CH 2 - and -(CH 2 ) P -
• the polyamine compound is of the structure of formula (IV) where: Li is -(CH 2 ) P -; and L is -(CH 2 ) m -CHRi 3 -; where R 13 , m and p are as defined above.
• Li p is an integer from 3 to 10
• L 2 n is an integer from 2 to 9
• the polyamine compound is of the structure of formula (IV) where: Li and L 2 are -(CH 2 ) P -; where p is as defined above. In particular embodiments, p is an integer from 3 to 7
• the polyamine compound is of the structure of formula (II), where n is 1, such that the compound has a structure according to formula (V) :
• Li is -(CH 2 ) P - where p is as defined above.
• p is an integer from 2 to 6.
• one Rn is an amino-substituted cycloalkyl (e.g. , a cycloalkyl group substituted with a primary, secondary, tertiary or quaternary amine) or a C 2 -Cs alkanoyl (which alkanoyl may be substituted with one or more substituents such as a methyl or an alkylazide group); and the other Rn is a -Cs alkyl or a C 7 -C 24 aralkyl.
• cycloalkyl e.g. , a cycloalkyl group substituted with a primary, secondary, tertiary or quaternary amine
• a C 2 -Cs alkanoyl which alkanoyl may be substituted with one or more substituents such as a methyl or an alkylazide group
• the other Rn is a -Cs alkyl or a C 7 -C 24 aralkyl
• Representative compounds of the formula (II) include, e.g :
• Phenylcyclopropylamine derivatives that are inhibitors of include compounds represented by formula (VI):
• each of R1-R5 is independently selected from H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy,
• heteroarylalkoxy isocyanato, isothiocyanate, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato, trihalomethanesulfonamido, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, and C-amido;
• R6 is H or alkyl; WO 2019/104381 7 j S H, alkyl, or cycloalkyl; PCT/AU2018/051268
• R8 is an -L-heterocyclyl wherein the ring or ring system of the -L-heterocyclyl has from 0 to 3 substituents selected from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L- aryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate, nitro, sulfin,
• R8 is -L-aryl wherein the ring or ring system of the -L-aryl has from 1 to 3 substituents selected from halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L- heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, alkylamino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanate, nitro, sulfinyl, sulfonyl,
• each L is independently selected from -(CH 2 ) n -(CH 2 ) n -, -(CH 2 ) n NH(CH 2 ) n -, - (CH 2 ) n O(CH 2 ) n -, and -(CH 2 ) n S(CH 2 ) n -, and where each n is independently chosen from 0, 1, 2, and 3;
• L is a covalent bond.
• R6 and R7 are hydro.
• one of R1-R5 is selected from -L-aryl, -L-heterocyclyl, and -L-carbocyclyl.
• a compound of the invention is of formula (VI) where:
• each R1-R5 is optionally substituted and independently chosen from -H, halo, alkyl, alkoxy, cycloalkoxy, haloalkyl, haloalkoxy, -L-aryl, -L-heteroaryl, -L-heterocyclyl, -L-carbocyclyl, acylamino, acyloxy, alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, cyano, cyanato, haloaryl, hydroxyl, heteroaryloxy, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thio
• R6 is chosen from -H and alkyl
• R7 is chosen from -H, alkyl, and cycloalkyl
• Rx when present is chosen from -H, alkyl, alkynyl, alkenyl, -L-carbocyclyl, -L-aryl, and -L-heterocyclyl, all of which are optionally substituted (except -H);
• WO 2019/104381 y when present is chosen from -H, alkyl, alkynyl, alkeny
• optionally substituted refers to zero or 1 to 4 optional substituents independently chosen from acylamino, acyloxy, alkenyl, alkoxy, cycloalkoxy, alkyl, alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, carbocyclyl, cyano, cyanato, halo, haloalkyl, haloaryl, hydroxyl, heteroaryl, heteroaryloxy, heterocyclyl, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl, thiocyanato, tri
• R8 is -CORz, such that the compound is of the following structure:
• R1-R7 are described above; and Rz is -L-heterocyclyl which is optionally substituted with from 1-4 optional substituents independently chosen from acylamino, acyloxy, alkenyl, alkoxy, cycloalkoxy, alkyl, alkylthio, cycloalkylthio, alkynyl, amino, aryl, arylalkyl, arylalkenyl, arylalkynyl, arylalkoxy, aryloxy, arylthio, heteroarylthio, carbocyclyl, cyano, cyanato, halo, haloalkyl, haloaryl, hydroxyl, heteroaryl, heteroaryloxy, heterocyclyl, heteroarylalkoxy, isocyanato, isothiocyanato, nitro, sulfinyl, sulfonyl, sulfonamide, thiocarbonyl
• each L is independently chosen from - (CH2) n -(CH2) n - and -(CH 2 ) n -0-(CH 2 ) n where each n is independently chosen from 0, 1, 2, and 3.
• each L is chosen from a bond, -CH 2 CH 2 -, -
• each L is chosen from a bond, -CH 2 CH 2 -, OCH2-, and -CH 2 CH 2 CH 2 - .
• L is chosen from a bond and -CH 2 - .
• WO 2019/104381 xemplary compounds of formula ( ⁇ 1) include: PCT/AU2018/051268
• Exemplary compounds of formula ( ⁇ 1) include: N-cydopropyl-2- ⁇ [(trans)-2- pheny!cyc!opropy!]amino ⁇ acetamide; 2- ⁇ [(trans)-2-phenylcyclopropyl]amino acetamide; N- cyclopropyl-2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ propanamide; 2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ -N-prop-2-ynylacetamide; N-isopropyl-2- ⁇ [(trans)-2- phenyIcyclopropyI]amino ⁇ acetamide; N-(tert-butyl)-2- ⁇ [(trans)-2- phenyIcyclopropyI]amino ⁇ acetamide; N-(2-morpholin-4-yl-2-oxoethyl)-N-[(trans)-2- phenylcyclopropy!]a
• Alternative small molecule LSD inhibitor compounds may be selected from selective LSD1 and LSD1/MAOB dual inhibitors disclosed for example in W02010/043721
• Representative compounds of this type include phenylcyclopropylamine derivatives or homologs, illustrative examples of which include phenylcyclopropylamine with one or two substitutions on the amine group; phenylcyclopropylamine with zero, one or two substitutions on the amine group and one, two, three, four, or five substitution on the phenyl group;
• phenylcyclopropylamine with zero, one or two substitutions on the amine group wherein the phenyl group of PCPA is substituted with (exchanged for) another ring system chosen from aryl or heterocyclyl to give an aryl- or heteroaryl-cyclopropylamine having zero, one or two substituents on the amine group; phenylcyclopropylamine wherein the phenyl group of PCPA is substituted with (exchanged for) another ring system chosen from aryl or heterocyclyl to give an aryl- or heterocyclyl-cyclopropylamine wherein said aryl- or heterocyclyl-cyclopropylamine on said aryl or heterocyclyl moiety has zero, one or two substitutions on the amine group and one, two, three, four, or five substitution on the phenyl group; phenylcyclopropylamine with one, two, three, four, or five substitution on the phenyl group; or any of the above
• Non-limiting embodiments of phenylcyclopropylamine derivatives or analogs include “cyclopropylamine amide” derivatives and "cyclopropylamine” derivatives.
• Specific examples of "cyclopropylamine acetamide” derivatives include, but are not limited to: N-cyclopropyl-2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ acetamide; 2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ acetamide; N-cyclopropyl-2- ⁇ [(trans)-2- phenylcyclopropyl]amino ⁇ propanamide; 2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ -N-prop-2- ynylacetamide; N-isopropyl-2- ⁇ [(trans)-2-phenylcyclopropyl]amino ⁇ acetamide; N-(tert-butyl)-2- ⁇ [(trans)
• cyclopropylamine derivatives, include, but are not limited to: N-4-fluorobenzyl-N- ⁇ (trans)-2-[4-(benzyloxy)phenyl]cyclopropyl ⁇ amine, N-4-methoxybenzyl-N- ⁇ (trans)-2-[4-(benzyloxy)phenyl]cyclopropyl ⁇ amine, N-benzyl-N- ⁇ (trans)-2-[4- (benzyloxy)phenyl]cyclopropyl ⁇ amine, N-[(trans)-2-phenylcyclopropyl]amino-methyl)pyridin-3-ol, N-[(trans)-2-phenylcyclopropyl]-N-(3-methylpyridin-2-ylmethyl)amine, N-[(trans)-2- phenylcyclopropyl]-N-(4-chloropyridin-3-ylmethyl)amine, N-[(trans)-2-phenyl
• LSD1 inhibitors are, e.g. , phenelzine or pargyline
• phenelzine and pargyline include, but are not limited to, compounds where the phenyl group of the parent compound is replaced with a heteroaryl or optionally substituted cyclic group or the phenyl group of the parent compound is optionally substituted with a cyclic group.
• the phenelzine or pargyline derivative or analog thereof has selective LSD1 or dual LSD1/MAOB inhibitory activity as described herein.
• the phenelzine derivative or analog has one, two, three, four or five substituents on the phenyl group.
• the phenelzine derivative or analog has the phenyl group substituted with (exchanged for) an aryl or heterocyclyl group wherein said aryl or heterocyclyl group has zero, one, two, three, four or five substituents.
• the pargyline derivative or analog has one, two, three, four or five substituents on the phenyl group.
• the pargyline derivative or analog has the phenyl group substituted with (exchanged for) an aryl or heterocyclyl group wherein said aryl or heterocyclyl group has zero, one, two, three, four or five substituents.
• the present invention also contemplates tranylcypromine derivatives as described for example by Binda et al. (2010. J. Am. Chem. Soc. 132: 6827-6833, which is hereby incorporated by reference herein in its entirety) as inhibitors of LSD (e.g. , LSD1 and/or LSD2) enzymatic function.
• LSD e.g. , LSD1 and/or LSD2
• LSD1 inhibitor compounds may be selected from tranylcypromine analogs described by Benelkebir et al. (2011. Bioorg. Med. Chem. doi: 10.1016/j.bmc.2011.02.017, which is hereby incorporated by reference herein in its entirety).
• Representative analogs of this type, including o,- m- and p-bromo analogues include: (lR,2S)-2-(4- bromophenyl)cyclopropanamine hydrochloride (Compound 4c), (lR,2S)-2-(3- bromophenyl)cyclopropanamine hydrochloride (Compound 4d), (lR,2S)-2-(2- bromophenyl)cyclopropanamine hydrochloride (Compound 4e), (lR,2S)-2-(biphenyl-4- yl)cyclopropanamine hydrochloride (Compound 4f).
• cyclopropylamine compounds that are useful for inhibiting LSD1 include those disclosed by Fyfe et al. in U.S. Publication No. 2013/0197013, which is incorporated herein by reference in its entirety.
• Illustrative cyclopropylamine inhibitors of LSD1, which are disclosed as being selective for inhibiting LSD1, include compounds according to formula (VI)I:
• X 1 and X 2 are independently C(R2) or N; WO 2019/1043813 ancj c4, w hen present, are independently C(R2) or N; PCT/AU2018/051268
• (G) is a cyclyl group (as shown in formula (VII), the cyclyl group (G) has n substituents (Rl));
• each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from -H, alkyl, alkenyl, alkynyl, cyclyl, -Ll- cyclyl, -Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 independently chosen optional substituents or two (R2) groups can be taken together to form a heterocyclyl or aryl group having 1, 2, or 3 independently chosen optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy, heterocycly
• R3 is -H or a (Ci-Ce)alkyl group
• each LI is independently alkylene or heteroalkylene
• n 0, 1, 2, 3, 4 or 5
• X 1 is CH or N;
• G is a cyclyl group;
• each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;
• WO 2019/104381 a ch LI is independently alkylene or heteroalkylene; PCT/AU2018/051268
• (G) is a cyclyl group
• each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 0, 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;
• each LI is independently alkylene or heteroalkylene; m is 0, 1, 2 or 3; and
• n 0, 1, 2, 3, 4 or 5
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate;
• R3 is -H or a (Ci-Ce)alkyl group; each LI is alkylene or heteroalkylene; and n is 0, 1, 2, 3, 4 or 5,
• X 1 , X 2 , X 3 and X 4 are independently CH or N, provided that at least one of X 1 , X 2 , X 3 and X 4 is N;
• (G) is a cyclyl group; each (Rl) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, -Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl;
• each (R2) is independently chosen from alkyl, alkenyl, alkynyl, cyclyl, -Ll-cyclyl, - Ll-amino, -Ll-hydroxyl, amino, amido, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, hydroxyl, alkoxy, urea, carbamate, acyl, or carboxyl, wherein each (R2) group has 1, 2, or 3 optional substituents, wherein said optional substituents are independently chosen from alkyl, alkanoyl, heteroalkyl, heterocyclyl, haloalkyl, cycloalkyl, carbocyclyl, arylalkoxy,
• heterocyclylalkoxy aryl, aryloxy, heterocyclyloxy, alkoxy, haloalkoxy, oxo, acyloxy, carbonyl, carboxyl, carboxamido, cyano, halogen, hydroxyl, amino, aminoalkyl, amidoalkyl, amido, nitro, thiol, alkylthio, arylthio, sulfonamide, sulfinyl, sulfonyl, urea, or carbamate; each LI is alkylene or heteroalkylene;
• Representative compounds according to formula (VII) are suitably selected from: (trans)-2-(3'-(trifluoromethyl)biphenyl-4-yl)cyclopropanamine; (trans)-2-(terphenyl-4- yl)cyclopropanamine; 4'-((trans)-2-aminocyclopropyl)biphenyl-4-ol; 4'-((trans)-2- aminocyclopropyl)biphenyl-3-ol; (trans)-2-(6-(3-(trifluoromethyl)phenyl)pyridin-3- yl)cyclopropanamine; (Trans)-2-(6-(3,5-dichlorophenyl)pyridin-3-yl)cyclopropanamine; (trans)-2- (6-(4-chlorophenyl)pyridin-3-yl)cyclopropanamine; (trans)-2-(6-(3-chlorophenyl)pyridin-3-yl)cycloprop
• LSD1 inhibitor compounds are selected from
• Ari is a 5 to 7 membered aryl or heteroaryl ring
• Ar 2 and Ar3 are each independently selected from a 5 to 7 membered aryl or heteroaryl ring, optionally substituted with 1 to 3 substituents;
• R3 is selected from hydrogen, -Ci-6alkyl or -OH;
• m is an integer from 1 to 5;
• n is an integer from 1 to 3;
• Ari is a six membered aryl or heteroaryl ring, especially phenyl, pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and 1,2,3-triazine, more especially phenyl;
• Ar 2 is a six membered aryl or heteroaryl ring, especially phenyl, pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and 1,2,3-triazine, especially phenyl; especially where the six membered aryl or heteroaryl ring is optionally substituted with one optional substituent, especially in the 3 or 4 position;
• Ar 3 is a six membered aryl or heteroaryl ring, especially phenyl, pyridine, pyrimidine, pyrazine 1,3,5-triazine, 1,2,4-trazine and 1,2,3-triazine, especially phenyl; especially where the six membered aryl or heteroaryl ring is optionally substituted with one optional substituent, especially in the 3 or 4 position.
• R3 is H, -Ci-3alkyl or -OH, especially H, -CH3 or -OH.
• m is 2 to 5, especially 3 to 5, more especially 4,
• n is 1 or 2, especially 1.
• the compounds of formula (XII) are compounds of formula (Xlla):
• Non-limiting compounds represented by formula (XII) include the following:
• LSD1 inhibitors include, but are not limited to those, e.g., disclosed in Ueda et a/. (2009. J. Am. Chem. Soc. 131(48): 17536-17537) including; Mimasu i (2010. Biochemistry June 22. [Epub ahead of print] PMID: 20568732 [PubMed-as supplied by publisher].
• LSD1 inhibitor compounds are selected from
• A is a hydrocarbon group optionally having substituent(s), or a heterocyclic group optionally having substituent(s);
• R is a hydrogen atom, a hydrocarbon group optionally having substituent(s), or a heterocyclic group optionally having substituent(s); or
• a and R are optionally bonded to each other to form a ring optionally having substituent(s);
• Q 1 , Q 2 , Q 3 and Q 4 are each independently a hydrogen atom or a substituent; Q 1 and Q 2 , and Q 3 and Q 4 , are each optionally bonded to each other to form a ring optionally having substituent(s);
• WO 2019/104381 j S a hydrogen atom, an acyclic hydrocarbon group optionee, T/AU2018/051268 substituent(s), or a saturated cyclic group optionally having substituent(s);
• Y 1 , Y 2 and Y 3 are each independently a hydrogen atom, a hydrocarbon group optionally having substituent(s), or a heterocyclic group optionally having substituent(s);
• X and Y 1 , and Y 1 and Y 2 are each optionally bonded to each other to form a ring optionally having substituent(s);
• Z 1 , Z 2 and Z 3 are each independently a hydrogen atom or a substituent, or a salt thereof.
• A is a phenyl group optionally having 1 to 3 Ci- 6 alkyl groups substituted by 1 to 3 halogen atoms, a biphenylyl group, or a pyrazolyl group;
• R is a hydrogen atom; or A and R are optionally bonded to each other to form a dihydroisoindole ring having 1 or 2 oxo groups;
• Q 1 is a hydrogen atom or a Ci- 6 alkyl group;
• Q 2 , Q 3 and Q 4 are each a hydrogen atom;
• X is a hydrogen atom;
• Y 1 , Y 2 and Y 3 are each independently a hydrogen atom or a C3-8 cycloalkyl group;
• Y 1 and Y 1 are optionally bonded to each other to form, together with the adjacent carbon atom, a piperidine ring optionally having 1 to 3 Ci- 6 alkyl groups; and
• Z 1 , Z 2 and Z 3 are each
• Representative compounds according to formula (XIII) are suitably selected from : (1) N-(4- ⁇ trans-2-[(cyclopropylmethyl)amino]cyclopropyl ⁇ -2-methylphenyl)benzamide, (2) N-(4- ⁇ trans-2-[(cyclopropylmethyl)amino]cyclopropyl ⁇ phenyl)-3-(trifluoromethoxy)benzamide, (3) N- (4- ⁇ trans-2-[(cyclopropylmethyl)amino]cyclopropyl ⁇ phenyl)benzamide, (4) N-(4- ⁇ trans-2- [(cyclopropylmethyl)amino]cyclopropyl ⁇ phenyl)-cyclohexanecarboxamide, (5) N-(4- ⁇ trans-2-[(l,l- dioxidotetrahydro-2H-thiopyran-4-yl)amino]cyclopropyl- ⁇ phenyl)-3-(trifluoromethyl)benzamide, (6) N-(4-
• LSD1 inhibitor compounds are selected from compounds described for example by Munoz et ai in U.S. Publication No. 2014/0213657, which is hereby incorporated by reference herein in its entirety. Representative compounds of this type are represented by formula (XIV) :
• (A) is heteroaryl or aryl
• X is 0, 1, 2, or 3;
• WO 2019/1043813 is a cyclopropyl ring, wherein (A) and (Z) are covalent
• (Z) is -NH-;
• (L) is chosen from a single bond, -CH 2 -, -CH2CH2-, -CH2CH2CH2-, and -CH2CH2CH2CH2--;
• (D) is an aliphatic carbocyclic group or benzocycloalkyl, wherein said aliphatic carbocyclic group or said benzocycloalkyl has 0, 1, 2, or 3 substituents independently chosen from -NH2, -NH(Ci-Ce alkyl), -N(Ci-Ce alkyl)(Ci-Ce alkyl), alkyl, halo, amido, cyano, alkoxy, haloalkyl, and haloalkoxy; or an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt or solvate thereof.
• Non-limiting examples of compounds according to formula (XIV) include N-((trans)- 2-(4-(benzyloxy)phenyl)cyclopropyl)-6-methoxy-2,3-dihydro-lH-i-nden-l-amine; N-((trans)-2-(4- (benzyloxy)phenyl)cyclopropyl)-5,6-dimethoxy-2, 3-dihydro- lH-inden-l-amine; N-((trans)-2-(4- (benzyloxy)phenyl)cyclopropyl)-4,5-dimethoxy-2, 3-dihydro- lH-inden-l-amine; N-((trans)-2- phenylcyclopropyl)-2,3-dihydro-lH-inden-l-amine; 6-methoxy-N-((trans)-2-phenylcyclopropyl)- 2,3-dihydro-lH-inden-l-amine
• LSD1 inhibitor compounds are selected from substituted (E)-N'-(l-phenylethylidene)benzohydrazide analogs, as described for example by Vankayalapati et al. in U.S. Publication No. 2014/0163017, which is hereby incorporated by reference herein in its entirety.
• Representative compounds of this type are represented by formula (XV):
• m is 0 or 1;
• n is an integer from 0 to 3;
• X is selected from the group consisting of OH, NO2 and F;
• Z is selected from the group consisting of N and CH;
• Ri is selected from the group consisting of halo, C 1 -C 3 haloalkyl, and C 1 -C 3 polyhaloalkyl;
• each of f1 ⁇ 2, R 3 , and R 4 is independently selected from the group consisting of hydrogen, halo, hydroxyl, cyano, amino, C2-C6 alkalkoxy, -Ce alkoxy, Q-Ce alkyl, -Ce polyhaloalkyl, and Ci-Ce haloalkyl;
• R5 is selected from the group consisting of NRe R 7 , Q-Ce alkyl, C 3 -C 6 cycloalkyl,
• Cy and substituted with 0-3 groups independently selected from halo hydroxyl, amino, C2-C6 alkalkoxy, Q-Ce alkylalcohol, Q-Ce alkoxy, Q-Ce alkyl, Q-Ce polyhaloalkyl, Q-Ce haloalkyl, C3-C6 cycloalkyl, and Cy; Cy is a heterocycloalkyl selected from the group consisting of aziridinyl, azetidinyl, pyrrolidinyl, piperidinyl, azepanyl, oxazolidinyl, imidazolidinyl, pyrazolidinyl, piperazinyl, oxazinanyl, morpholinyl, hexahydrophyrimidinyl, and
• each of Re and R7 is independently selected from the group consisting of hydrogen, Ci-Ce alkyl, Cs-Ce cycloalkyl, and C3-C6 heterocycloalkyl; or a pharmaceutically acceptable salt thereof.
• Illustrative compounds according to formulas (XV) and (XVI) include:
• LSD1 inhibitor compounds are selected from
• hydroxytyrosol hydroxytyrosol derived and/or substituted compounds, and/or a hydroxytyrosol metabolites, as described for example by McCord et a/ in U.S. Publication No. 2014/0155339, which is hereby incorporated by reference herein in its entirety.
• Representative compounds of this type include:
• LSD1 inhibitor compounds are selected from small molecule compounds described by Casero et ah in U.S. Publication No. 2014/0011857, which is hereby incorporated by reference herein in its entirety. Representative compounds of this type are represented by formula (XVII):
• R is alkyl, aryl, carbocyclic, heterocyclic, aralkyl, alkoxy, aryloxy, haloalkyl, or halo, each of which is optionally substituted, nitro, hydroxy, thio, C(0)N R*RB, or C(0)0RA;
• R4 is H, alkyl, aryl, carbocyclic, heterocyclic, aralkyl, alkoxy, aryloxy, haloalkyl, or halo, each of which is optionally substituted, nitro, hydroxy, thio, C(0)N RARB, or C(0)0RA;
• RS is H, alkyl, aryl, carbocyclic, heterocyclic, aralkyl, alkoxy, aryloxy, haloalkyl, or halo, each of which is optionally substituted, nitro, hydroxy, thio, C(0)N RARB, or C(0)0RA;
• RS is H, alkyl
• LSD1 inhibitors are selected from arylcyclopropylamine compounds described by Munoz et ai in U.S. Publication No. 2013/0231342, which is hereby incorporated by reference herein in its entirety.
• Representative compounds of this type are represented by formula (XVIII):
• (A) is a cyclyl group having n substituents (R3);
• (B) is a cyclyl group or an -(Ll)-cydyl group, wherein said cyclyl group or the cyclyl moiety comprised in said -(Ll)-cyclyl group has n substituents (R2);
• (LI) is -0-, -NH-, -N(alkyl)-, alkylene or heteroalkylene;
• (D) is a heteroaryl group or an -(L2)-heteroaryl group, wherein said heteroaryl group or the heteroaryl moiety comprised in said -(L2)-heteroaryl group has one substituent (Rl), and further wherein said heteroaryl group is covalently bonded to the remainder of the molecule through a ring carbon atom or the heteroaryl moiety comprised in said -(L2)-heteroaryl group is covalently bonded to the (L2) moiety through a ring carbon atom;
• (L2) is -0-, -NH-, -N(alkyl)-, alkylene or heteroalkylene;
• each (R2) is independently selected from alkyl, alkenyl, alkynyl, cyclyl, amino, amido, C-amido, alkylamino, hydroxyl, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, alkoxy, acyl, carboxyl, carbamate or urea;
• each (R3) is independently selected from alkyl, alkenyl, alkynyl, cyclyl, amino, amido, C-amido, alkylamino, hydroxyl, nitro, halo, haloalkyl, haloalkoxy, cyano, sulfinyl, sulfonyl, sulfonamide, alkoxy, acyl, carboxyl, carbamate, or urea; and n is independently 0, 1, 2, 3 or 4.
• Non-limiting examples of compounds according to formula (XVIII) are selected from: 5-(((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)pyrimidin-2-amine; 5- (((trans)-2-(4-(benzyloxy)phenyl)cyclopropylamino)methyl)thiazol-2-amine; 5-((trans)-2-(6-(3- (trifluoromethyl)phenyl)pyridin-3-yl)cyclopropylamino)methyl)pyrimidin-2-amine; 5-((trans)-2-(6- (3-(trifluoromethyl)phenyl)pyridin-3-yl)cyclopropylamino-)methyl)thiazol-2-amine; 3-(5-((trans)- 2-((2-aminopyrimidin-5-yl)methylamino)cyclopropyl)pyridin-2-yl)phenol; 3-(5-((trans)-2-((2-aminopyr
• the LSD inhibitor is an inhibitor of nuclear
• Zi and Z2 are independently absent or are independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues in between), and a protecting moiety; and
• Xi is selected from small amino acid residues, including S, T, A, G and modified forms thereof.
• "Xi" is selected from S and A.
• Xi is selected from S, A and modified forms thereof. In some embodiments, “Xi" is selected from S, A and S(POs).
• Xi is a modified form of S, especially S(POs).
• Zi is a proteinaceous molecule represented by Formula
• X2 is absent or is a protecting moiety
• X 4 is selected from any amino acid residue.
• X3 is selected from basic amino acid residues including R, K and modified forms thereof. In some embodiments, "X3" is R.
• X4" is selected from aromatic amino acid residues, including F, Y, W and modified forms thereof. In some embodiments, "X4" is W.
• the isolated or purified proteinaceous molecule of Formula XIX comprises, consists or consists essentially of an amino acid sequence represented by SEQ ID NO: 2, 3 or 4:
• RWRRTARRKRAKV [SEQ ID NO: 4]
• the isolated or purified proteinaceous molecule of formula XIX comprises, consists or consists essentially of an amino acid sequence represented by SEQ ID NO: 2 or 3.
• the isolated or purified proteinaceous molecule of formula XIX comprises, consists or consists essentially of the amino acid sequence of SEQ ID NO: 5: WO 2019/104381 GRRTSRRKRAKVE [SEQ ID NO: 5] , PCT/AU2018/051268
• the isolated or purified proteinaceous molecule of formula XIX is other than a proteinaceous molecule consisting of the amino acid sequence of SEQ ID NO: 5.
• the molecules comprise at least one membrane permeating moiety.
• the membrane permeating moiety may be conjugated at any point of the proteinaceous molecule.
• Suitable membrane permeating moieties include lipid moieties, cholesterol and proteins, such as cell-penetrating peptides and polycationic peptides; especially lipid moieties.
• Non-limiting examples of cell-penetrating peptides include the peptides described in, for example, US 20090047272, US 20150266935 and US 20130136742. Accordingly, suitable cell penetrating peptides may include, but are not limited to, basic poly(Arg) and poly(Lys) peptides and basic poly(Arg) and poly(Lys) peptides containing non-natural analogues of Arg and Lys residues such as YGRKKRPQRRR [SEQ ID NO: 6] (HIV TAT47-57), RRWRRWWRRWRRWRR (W/R) [SEQ ID NO: 7], CWKi 8 (AlkCWKi 8 ) [SEQ ID NO: 8], Ki 8 WCCWKi 8 (Di-CWKi 8 ) [SEQ ID NO: 9], WTLNSAGYLLGKINLKALAALAKKIL [SEQ ID NO: 10] (Transportan), GLFEALEEL
• IGRIDPANGKTKYAPKFQDKATRSNYYGNSPS [SEQ ID NO: 17] (P9.3), KETWWETWWTEWSQPKKKRKV [SEQ ID NO: 18] (Pep-1), PLAEIDGIELTY [SEQ ID NO: 19] (Plae), KieGGPLAEIDGIELGA [SEQ ID NO: 20] (Kplae), KieGGPLAEIDGIELCA [SEQ ID NO: 21] (cKplae),
• the membrane permeable moiety is conjugated to the N- or C-terminal amino acid residue or through the amine of a lysine side-chain of the proteinaceous molecule, especially the N- terminal amino acid residue of the proteinaceous moiety.
• PD-1 binding antagonists are suitably molecules that inhibit signaling through PD-1 and include molecules that inhibit the binding of PD-1 to its ligand binding partners.
• the PD-1 ligand binding partners are PD-L1 and/or PD-L2.
• the antagonist may be an antibody, an immunoadhesin, a fusion protein, or oligopeptide.
• the PD-1 binding antagonist is preferably an anti-PD-1 antibody (e.g . , a human antibody, a humanized antibody, or a chimeric antibody).
• the anti-PD-1 antibody is selected from the group consisting of MDX-1106 (nivolumab, OPDIVO), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA), CT-011 (pidilizumab), MEDI-4736 (durvalumab) MEDI- 0680 (AMP-514), PDR001, REGN2810, BGB-108, and BGB-A317.
• the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
• the PD-1 binding antagonist is AMP-224.
• Nivolumab also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described in W02006/121168.
• Pembrolizumab also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335.
• CT-011 also known as hBAT, hBAT-1 or Pidilizumab, is an anti-PD-1 antibody described in W02009/101611.
• AMP-224 also known as B7-DCIg, is a PD-L2-FC fusion soluble receptor described in W02010/027827 and WO2011/066342.
• the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).
• an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 53 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 54.
• an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, wherein :
• the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
• the light chain sequences has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence: WO 2019/104381 iVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPiPC_T/AU2018/051268RFSG SGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLN N FY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGE
• the anti-PD-1 antibody is pembrolizumab (CAS Registry Number: 1374853-91-4).
• an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 55 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 56.
• an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, wherein:
• the heavy chain sequence has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the heavy chain sequence:
• the light chain sequences has at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the light chain sequence:
• EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPA RFSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRG EC [SEQ ID NO: 56]
• the present invention also contemplates antibody fragments comprising heavy and light chain FIVRs of a full-length anti-PD-1 antagonist antibody.
• nucleic acids encoding any of the antibodies described herein.
• the nucleic acid further comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PDLl, anti- PD-1, or anti-PDL2 antibodies.
• the vector further comprises a host cell suitable for expression of the nucleic acid.
• the host cell is a eukaryotic cell or a prokaryotic cell.
• the eukaryotic cell is a mammalian cell, such as Chinese Flamster Ovary (CFIO).
• the antibody or antigen binding fragment thereof may be made using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-PD-1, or antigen-binding fragment in a form suitable for expression, under conditions suitable to produce such antibody or fragment, and recovering the antibody or fragment.
• the isolated anti-PD-1 antibody is c PCT/AU2018/051268
• Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
• the tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
• O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.
• Removal of glycosylation sites form an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed.
• the alteration may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site another amino acid residue (e.g., glycine, alanine or a conservative substitution).
• the LSD inhibitor and PD-1 binding antagonist are administered concurrently with an ancillary agent for treating, or for aiding in the treatment of, a T-cell dysfunctional disorder.
• ancillary agents include cytotoxic agents, gene therapy agents, DNA therapy agents, viral therapy agents, RNA therapy agents,
• the ancillary agent may be in the form of adjuvant or neoadjuvant therapy.
• the ancillary agent is a small molecule enzymatic inhibitor or anti metastatic agent.
• the ancillary agent is a side-effect limiting agent (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti nausea agents, etc.).
• the ancillary agent is a radiotherapy agent.
• the ancillary agent is an agent that targets PI3K/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.
• the ancillary agent is an immunotherapeutic, e.g., a blocking antibody, ipilimumab (also known as MDX-010, MDX-101, or Yervoy®), tremelimumab (also known as ticilimumab or CP-675,206), an antagonist directed against B7-H3 (also known as CD276), e.g.
• a blocking antibody MGA271, an antagonist directed against a TGF-b, e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299, a T cell (e.g. , a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR), a T cell comprising a dominant-negative TGF-b receptor, e.g., a dominant-negative TGF-b type II receptor, an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), e.g.
• a TGF-b e.g., metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299
• a T cell e.g. , a cytotoxic T cell or CTL
• CAR chimeric antigen receptor
• an activating antibody urelumab (also known as BMS-663513), an agonist directed against CD40, e.g. , an activating antibody, CP- 870893, an agonist directed against 0X40 (also known as CD134), e.g., an activating antibody, administered in conjunction with an anti-OX40 antibody (e.g., AgonOX), an agonist directed against CD27, e.g.
• an activating antibody CDX-1127, indoleamine-2, 3-dioxygenase (IDO), 1- methyl-D-tryptophan (also known as 1-D-MT), an antibody-drug conjugate (in some embodiments, comprising mertansine or monomethyl auristatin E (MMAE)), an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599), trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech), DMUC5754A, an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), e.g.
• EDNBR endothelin B receptor
• TLR agonist e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, or CpG ODN, TNF-a , IL-1, HMGB1, an IL-10 antagonist, an IL-4 antagonist, an IL-13 antagonist, an HVEM antagonist, an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS, an agent targeting CX3CL1, an agent targeting CXCL10, an agent targeting CCL5, an LFA-1 or ICAM1 agonist, a Selectin agonist, a targeted therapeutic agent, an inhibitor of B-Raf, vemurafenib (also known as Zelboraf®, dabrafenib (also known as Tafinlar®), erlotinib (also known as Tarceva®), an inhibitor of a MEK
• B-Raf vemurafenib
• vemurafenib also known as Zelboraf
• cobimetinib also known as GDC-0973 or XL-518
• trametinib also known as Mekinist®
• K-Ras an inhibitor of K-Ras
• an inhibitor of c-Met an inhibitor of c-Met, onartuzumab (also known as MetMAb)
• Aik an inhibitor of Aik
• AF802 also known as CH5424802 or alectinib
• an inhibitor of a phosphatidylinositol 3-kinase PI3K
• BKM120 idelalisib
• perifosine also known as KRX-0401
• an Akt Akt
• MK2206 GSK690693
• GDC-0941 an inhibitor of mTOR
• sirolimus also known as rapamycin
• temsirolimus also known as CCI-779 or Torisel®
• everolimus also known as RAD001
• ridaforolimus also known as
• the ancillary agent is an anti-infective drug.
• the anti- infective drugs is suitably selected from antimicrobials, which include without limitation compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeast, fungi, protozoa, etc. and thus include antibiotics, amebicides, antifungals, antiprotozoals, antimalarials, antituberculotics and antivirals.
• Anti-infective drugs also include within their scope anthelmintics and nematocides.
• Illustrative antibiotics include quinolones (e.g.
• tetracyclines e.g. , chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecycline; linezolide, eperozolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicins, gentamicin, kanamycin, meomycin, netilmicin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), b-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, ce
• b-lactams e.g., imipe
• Illustrative antivirals include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine,
• lamivudine/zidovudine lamivudine/zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir, and zidovudine.
• amebicides or antiprotozoals include atovaquone, chloroquine hydrochloride, chloroquine phosphate,
• Anthelmintics can be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin and
• Illustrative antifungals can be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposomal, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride.
• Non-limiting examples of antimalarials include chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine.
• Antituberculotics include but are not restricted to clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.
• compositions and formulations comprising a LSD inhibitor (e.g., a LSD1 inhibitor, a nuclear LSD inhibitor, etc.), a PD-1 binding antagonist and a pharmaceutically acceptable carrier.
• a LSD inhibitor e.g., a LSD1 inhibitor, a nuclear LSD inhibitor, etc.
• the pharmaceutical compositions and formulations further comprise an ancillary agent as described for example herein.
• the ancillary agent is one that targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g., a cytotoxic compound such as a taxane).
• compositions and formulations as described herein can be prepared by mixing the active ingredients (e.g., a small molecule, nucleic acid, or polypeptide) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers
• Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as
• octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or iWQ 2019/104381 ; hydrophilic polymers such as polyvinylpyrrolidone; amirP T/AU2018/051268j ne , glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose
• Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).
• sHASEGP soluble neutral-active hyaluronidase glycoproteins
• rHuPH20 HYLENEX®, Baxter International, Inc.
• Certain exemplary sHASEGPs and methods of use, including rHuPH20 are described in US Patent Publication Nos. 2005/0260186 and 2006/0104968.
• a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
• the active agents and optional pharmaceutically acceptable carriers are in the form of lyophilized formulations or aqueous solutions.
• exemplary lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958.
• Aqueous antibody formulations include those described in U.S. Pat. No. 6,171,586 and
• compositions and formulations herein may also contain further active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
• active ingredients are suitably present in combination in amounts that are effective for the purpose intended.
• Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g. , liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems e.g. , liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
• Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
• the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g. , by filtration through sterile filtration membranes.
• the formulations may be administered systemically or locally.
• Suitable routes may, for example, include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
• parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
• the present invention discloses that a LSD inhibitor (e.g. , a LSD1 inhibitor, a nuclear LSD inhibitor, etc. ) and a PD-1 binding antagonist (also referred to herein as the "dual therapy") are useful for treating a T-cell dysfunctional disorder, or for enhancing immune function (e.g. , immune effector function, T-cell function etc.) in an individual having cancer, for treating or (_ .Q,2 19/10438J j ressi on of cancer, or for treating infection in an individua?C.T/AU2018/051268 embodiments, the therapeutic combination is disclosed for treating or delaying the progression of cancer, including metastatic cancer, and for preventing cancer recurrence. Any of the LSD inhibitors and PD-1 binding antagonists known in the art or described herein may be used in this regard.
• the combination therapy further comprises the use or administration of an ancillary agent (e.g. , a chemotherapeutic agent), as described for example herein.
• an ancillary agent e.g. , a chemotherapeutic agent
• the ancillary agent is one that targets rapidly dividing cells and/or disrupt the cell cycle or cell division (e.g. , a cytotoxic compound such as a taxane).
• the combination therapy is referred to herein as "triple therapy").
• the individual to be treated with the combination therapy comprises a T- cell (e.g. , a CD8 + T-cell or CD4 + T-cell) with a mesenchymal phenotype, for example, a T-cell that expresses nuclear LSD at a higher level than the level of expression of TBET in the same T-cell, and/or at a higher level than in an activated T-cell.
• the T-cell may be a tumor-infiltrating lymphocyte or a circulating lymphocyte.
• the T-cell suitably exhibits T-cell exhaustion or anergy and in representative examples of this type, the T-cell expresses a higher level of EOMES than TBET.
• the T-cell has impaired or repressed immune function and suitably expresses biomarkers of reduced T-cell activation (e.g., reduced production and/or secretion of cytokines such as IL-2, IFN-g and TNF-a).
• the T-cell suitably expresses EOMES in the nucleus of the T cell at a higher level than the level of TBET in the same T-cell or the level of EOMES in the nucleus of an activated T-cell.
• nuclear LSD, EOMES and TBET, together with PD-1 which is known marker of T-cell exhaustion, (also referred to herein as "T-cell function biomarkers”) can be used to determine the immune function of T cells in a patient for assessing a patient's T-cell immune status, including susceptibility to treatment with PD-1 binding antagonists.
• the individual is a human.
• the individual has been treated with a PD-1 binding antagonist before the combination treatment with a PD-1 binding antagonist and a LSD inhibitor (e.g., a nuclear translocation inhibitor of LSD).
• a LSD inhibitor e.g., a nuclear translocation inhibitor of LSD
• the individual has cancer that is resistant (has been demonstrated to be resistant) to one or more PD-1 binding antagonists.
• resistance to a PD-1 antagonist includes recurrence of cancer or refractory cancer. Recurrence may refer to the reappearance of cancer, in the original site or a new site, after treatment.
• resistance to a PD-1 binding antagonist includes progression of the cancer during treatment with the PD-1 binding antagonist.
• resistance to a PD-1 binding antagonist includes cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. In some embodiments, the cancer is at early stage or at late stage.
• any one or more of the T-cell function biomarkers are detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, qPCR, RT-qPCR, multiplex qPCR or RT-qPCR, RNA-seq, microarray analysis, SAGE, MassARRAY technique, and FISH, and combinations thereof.
• any of the methods, assays and/PCT/AU2018/051268 1O re of the T-cell function biomarkers are detected in the sample by protein expression.
• protein expression is determined by immunohistochemistry (IHC).
• any one or more of the T-cell function biomarkers are detected using an antibody that binds specifically to a respective biomarker.
• nuclear LSD and/or EOMES biomarkers are detected in the nucleus of a T-cell, for example using IHC.
• a complex comprising nuclear LSD and EOMES biomarkers is detected in the nucleus of a T-cell.
• the combination therapy of the invention comprises administration of a LSD inhibitor and a PD-1 binding antagonist.
• the LSD inhibitor and PD-1 binding antagonist may be administered in any suitable manner known in the art.
• the LSD inhibitor and PD-1 binding antagonist is typically administered concurrently.
• the LSD inhibitor is in a separate composition as the PD-1 binding antagonist.
• the LSD inhibitor is in the same composition as the PD-1 binding antagonist. Accordingly, the combination therapy may involve administering the LSD inhibitor separately, simultaneously or sequentially with PD-1 binding antagonist.
• this may be achieved by administering a single composition or pharmacological formulation that includes both types of agent, or by administering two separate compositions or formulations at the same time, wherein one composition includes the LSD inhibitor and the other, PD-1 binding antagonist.
• the treatment with the LSD inhibitor may precede or follow the treatment with the PD-1 binding antagonist by intervals ranging from minutes to days.
• the LSD inhibitor is applied separately to the PD-1 binding antagonist, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the LSD inhibitor would still be able to exert an advantageously effect on a functionally repressed T-cell (e.g.
• a mesenchymal T-cell as noted above, and in particular, to render the T-cell with enhanced immune function, including susceptibility of the T-cell to reinvigoration by the PD-1 binding antagonist.
• the combination therapy of the invention comprises administration of a LSD inhibitor, a PD-1 binding antagonist and a chemotherapeutic agent.
• a LSD inhibitor, a PD-1 binding antagonist and a chemotherapeutic agent may be administered in any suitable manner known in the art.
• the LSD inhibitor, PD-1 binding antagonist and chemotherapeutic agent may be administered concurrently.
• the LSD inhibitor, PD-1 binding antagonist and chemotherapeutic agent are in separate compositions.
• the LSD inhibitor, PD-1 binding antagonist and chemotherapeutic agent are in the same composition.
• the LSD inhibitor is in the same composition as the PD-1 binding antagonist and the chemotherapeutic agent is in a jWO .2019/104381 jti on.
• the LSD inhibitor is in the PC.T/AU 2018/051268 3 the chemotherapeutic agent and the PD-1 binding antagonist is in a separate composition.
• the combination therapy may involve administering the LSD inhibitor separately, simultaneously or sequentially with PD-1 binding antagonist and chemotherapeutic agent. In some embodiments, this may be achieved by administering a single composition or pharmacological formulation that includes the three types of agent, or by administering separate compositions or formulations at the same time. In other embodiments, the treatment with one agent may precede or follow the treatment with the other two agents by intervals ranging from minutes to days.
• one agent is applied separately to the other agent, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the LSD inhibitor would still be able to exert an advantageously effect on a functionally repressed T-cell (e.g. , a mesenchymal T-cell) as noted above, and in particular, to render the T-cell with enhanced immune function, including susceptibility of the T-cell to reinvigoration by the PD-1 binding antagonist.
• a functionally repressed T-cell e.g. , a mesenchymal T-cell
• the LSD inhibitor and PD-1 binding antagonist and optionally the chemotherapeutic agent may be administered by the same route of administration or by different routes of administration.
• the PD-1 binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
• the LSD inhibitor is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
• the chemotherapeutic agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.
• An effective amount of the LSD inhibitor, PD-1 binding antagonist and optionally the chemotherapeutic agent may be administered for prevention or treatment of disease.
• chemotherapeutic agent may be determined based on the type of disease to be treated, the type of the LSD inhibitor, PD-1 binding antagonist and optionally the chemotherapeutic agent, the severity and course of the disease, the clinical condition of the individual, the individual's clinical history and response to the treatment, and the discretion of the attending physician.
• combination treatment with LSD inhibitor e.g., an enzymatic or nuclear translocation inhibitor of LSD
• PD-1 binding antagonists e.g., anti-PD-1 antibody
• optionally the chemotherapeutic agent are synergistic, whereby an efficacious dose of a PD-1 binding antagonists (e.g.
• anti-PD-1 antibody and/or chemotherapeutic agent in the combination is reduced relative to efficacious dose of the PD-1 binding antagonists (e.g. , anti-PD-1 antibody) and/or chemotherapeutic agent as a single agent.
• the therapeutically effective amount of a peptide or polypeptide active agent e.g. , an antibody, peptide inhibitor, immunoadhesin, etc.
• a peptide or polypeptide active agent e.g. , an antibody, peptide inhibitor, immunoadhesin, etc.
• administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight whether by one or more administrations.
• the antibody used is about 0.01 to about ‘WO 2019/104381 o.O l to about 40 mg/kg, about 0.01 to about 35 mg/kg, ?CT/AU2_0_18/051268 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example.
• the peptide or polypeptide active agent e.g. , an antibody, peptide inhibitor, immunoadhesin, etc.
• other dosage regimens may be useful.
• an anti-PDLl antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg or about 1400 mg on day 1 of 21-day cycles.
• the dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions.
• the dose of peptide or polypeptide active agent e.g. , an antibody, peptide inhibitor, immunoadhesin, etc.
• administered in a combination treatment may be reduced as compared to a single treatment. The progress of this therapy is easily monitored by
• Small molecule compounds are generally administered at an initial dosage of about 0.0001 mg/kg to about 1000 mg/kg daily.
• the dosages may be varied depending upon the requirements of the patient, the severity of the condition being treated, and the compound being employed.
• dosages can be empirically determined considering the type and stage of disease diagnosed in a particular patient.
• the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time.
• the size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound in a particular patient. Determination of the proper dosage for a particular situation is within the skill of the practitioner. Generally, treatment is initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day, if desired.
• Doses can be given daily, or on alternate days, as determined by the treating physician. Doses can also be given on a regular or continuous basis over longer periods of time (weeks, months or years), such as through the use of a subdermal capsule, sachet or depot, or via a patch or pump.
• the LSD inhibitor, PD-1 binding antagonist and optionally an ancillary agent e.g. , a chemotherapeutic agent
• the combination therapy may be administered as symptoms arise.
• routine schedule refers to a predetermined designated period of time.
• the routine schedule may encompass periods of time which are identical or which differ in length, as long as the schedule is predetermined.
• the routine schedule may involve administration of the LSD inhibitor, PD-1 binding antagonist and optional chemotherapeutic agent on a daily basis, every two days, every three days, every four days, every five days, every six days, a weekly basis, a monthly basis or any set number of days or weeks there-between, every two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, etc.
• the predetermined routine schedule may involve concurrent administration of the LSD inhibitor, PD-1 binding iWQ 2019/104381 ptional chemotherapeutic agent on a daily basis for the fir?CT/AU2018/051268/ a monthly basis for several months, and then every three months after that. Any particular combination would be covered by the routine schedule as long as it is determined ahead of time that the appropriate schedule involves administration on a certain day.
• the treatment methods and uses may further comprise an additional therapy.
• the additional therapy may be radiation therapy, surgery (e.g. , lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing.
• the additional therapy is radiation therapy.
• the additional therapy is surgery.
• the additional therapy is a combination of radiation therapy and surgery.
• the additional therapy is gamma irradiation.
• any of the methods described herein e.g., combination treatments including administering an effective amount of a combination of LSD inhibitor, PD-1 binding antagonist and optional chemotherapeutic agent may be tested in various models known in the art, such as clinical or pre-clinical models.
• Suitable pre-clinical models are exemplified herein and further may include without limitation ID8 ovarian cancer, GEM models, B16 melanoma, RENCA renal cell cancer, CT26 colorectal cancer, MC38 colorectal cancer, and Cloudman melanoma models of cancer.
• any of the methods described herein may be tested in a GEM model that develops tumors, including without limitation GEM models of non-small-cell lung cancer, pancreatic ductal adenocarcinoma, or melanoma.
• a GEM model that develops tumors
• pl6/pl9 nul1 allele may be used as a pre-clinical model for pancreatic ductal adenocarcinoma (PDAC).
• PDAC pancreatic ductal adenocarcinoma
• mice are randomly recruited into treatment groups receiving combination LSD inhibitor, PD-1 binding antagonist and optional chemotherapeutic agent treatment or control treatment.
• Tumor size e.g., tumor volume
• overall survival rate is also monitored.
• the cancer in some embodiments, a sample of the patient's cancer as examined using a diagnostic test, as described for example herein) comprises tumor-infiltrating lymphocytes (TILs), wherein the TILs are within or otherwise associated with the cancer tissue.
• TILs are assessed for expression of any one or more of the T-cell function biomarkers disclosed herein.
• nuclear LSD and EOMES can be used as biomarkers of mesenchymal phenotype and T- ⁇ WO_2Q19/104381
• TBET and/or PD-1 can be used as biomarkers c?CT/AU2018/051268 which is characterized for example by high levels of inhibitory co-receptors and lacking the capacity to produce effector cytokines (Wherry, E. J. 2011 Nature immunology 12 : 492-499; Rabinovich et at., 2007 Annual Review of immunology 25 : 267-296).
• the individual has a T-cell dysfunction that manifests in a T-cell dysfunctional disorder.
• the T-cell dysfunctional disorder may be characterized by T-cell anergy or decreased ability to secrete cytokines, proliferate or execute cytolytic activity.
• the T- cell dysfunctional disorder is characterized by repressed T-cell immune function.
• the T-cell dysfunctional disorder is characterized by T-cell of a mesenchymal phenotype.
• the T-cell dysfunctional disorder is characterized by T-cell exhaustion.
• the T-cells are CD4 + and/or CD8 + T cells.
• LSD inhibitor treatment may increase expression of biomarkers of T-cell activation and effector capacity (e.g. , IFN-y, TNF-a, Ki67 and TBET), decrease expression of biomarkers of T-cell exhaustion (e.g., EOMES), and increase activation and proliferation of T-cells, including effector and memory T-cells.
• LSD inhibitor treatment may confer enhanced susceptibility of exhausted T-cells to reinvigoration by PD-1 binding antagonists.
• the combination treatment LSD inhibitor and a PD-1 binding antagonist may increase T- cell (e.g., CD4 + T-cell, CD8 + T-cell, memory T-cell) priming, activation and/or proliferation relative to prior to the administration of the combination.
• T- cell e.g., CD4 + T-cell, CD8 + T-cell, memory T-cell
• the T cells are CD4 + and/or CD8 + T cells.
• activated CD4 + and/or CD8 + T-cells in the individual are characterized by IFN-g producing CD4 + and/or CD8 + T cells and/or enhanced cytolytic activity as compared to before the administration of the
• IFN-g may be measured by any means known in the art, including, e.g. , intracellular cytokine staining (ICS) involving cell fixation, permeabilization, and staining with an antibody against IFN-g.
• ICS intracellular cytokine staining
• Cytolytic activity may be measured by any means known in the art, e.g., using a cell killing assay with mixed effector and target cells.
• CD8 + T-cells are characterized, e.g. , by presence of CD8b expression (e.g., by RT-PCR using e.g., Fluidigm) (Cd8b is also known as T-cell surface
• glycoprotein CD8 beta chain CD8 antigen, alpha polypeptide p3'7; Accession No. is NM_172213).
• CD8 + T cells are from peripheral blood. In some embodiments, CD8 + T cells are from tumor.
• Treg cells are characterized, e.g. , by presence of Fox3p expression (e.g., by RT-PCR e.g., using Fluidigm) (Foxp3 is also known as Forkhead box protein P3; scurfin; FOXP3delta7; immunodeficiency, polyendocrinopathy, enteropathy, X-linked; the accession no. is NM_014009).
• Fox3 is also known as Forkhead box protein P3; scurfin; FOXP3delta7; immunodeficiency, polyendocrinopathy, enteropathy, X-linked; the accession no. is NM_014009
• Treg are from peripheral blood.
• Treg cells are from tumor.
• inflammatory or activated T-cells are characterized, e.g. , by presence of TBET and/or CXCR3 expression or by a TBET: EOMES ratio that correlates with inflammatory or activated T-cells (e.g., by RT-PCR using, e.g. , Fluidigm).
• inflammatory or activated T cells are from peripheral blood.
• inflammatory or activated T cells are from tumor.
• WO 2019/104381- some embodiments of the methods of the present disclc?C.T/AU2018/051268 D8 + T cells exhibit increased release of cytokines selected from the group consisting of IFN-g, TNF-a. Cytokine release may be measured by any means known in the art, e.g., using Western blot,
• ELISA immunohistochemical assays to detect the presence of released cytokines in a sample containing CD4 + and/or CD8 + T-cells.
• the CD4 + and/or CD8 + T cells are effector memory T cells.
• the CD4 + and/or CD8 + effector memory T cells are characterized by having the expression of CD44 high CD62L low .
• Expression of CD44 high CD62L low may be detected by any means known in the art, e.g. , by preparing single cell suspensions of tissue (e.g. , a cancer tissue) and performing surface staining and flow cytometry using commercial antibodies against CD44 and CD62L.
• the CD4 + and/or CD8 + effector memory T cells are characterized by having expression of CXCR3 (also known as C-X-C chemokine receptor type 3; Mig receptor; IP10 receptor; G protein-coupled receptor 9; interferon- inducible protein 10 receptor; Accession No. NM_001504).
• CXCR3 also known as C-X-C chemokine receptor type 3; Mig receptor; IP10 receptor; G protein-coupled receptor 9; interferon- inducible protein 10 receptor; Accession No. NM_001504
• the CD4 + and/or CD8 + effector memory T cells are from peripheral blood.
• the CD4 + and/or CD8 + effector memory T cells are from tumor.
• the administration of an effective amount of a LSD inhibitor and a PD-1 binding antagonist and optionally an ancillary agent such as a chemotherapeutic agent to an individual is characterized by increased levels of inflammatory markers (e.g. , CXCR3) on CD8 + T cells as compared to before administration of the combination therapy.
• CXCR3/CD8 + T cells may be measured by any means known the art.
• CXCR3/CD8 + T cells are from peripheral blood.
• CXCR3/CD8 + T cells are from tumor.
• Treg function is suppressed as compared to before administration of the combination.
• T-cell exhaustion is decreased as compared to before administration of the combination.
• number of Treg is decreased as compared to before administration of the combination.
• the levels of plasma IFN-g is increased as compared to before administration of the combination.
• Treg number may be assessed, e.g., by determining percentage of CD4 + Fox3p + CD45 + cells (e.g. , by FACS analysis).
• absolute number of Treg e.g. , in a sample, is determined.
• Treg are from peripheral blood.
• Treg are from tumor.
• T-cell priming, activation and/or proliferation is increased as compared to before administration of the combination.
• the T-cells are CD4 + and/or CD8 + T cells.
• T-cell proliferation is detected by determining percentage of Ki67 + CD8 + T cells (e.g., by FACS analysis).
• T-cell proliferation is detected by determining percentage of Ki67 + CD4 + T cells (e.g. , by FACS analysis).
• the T-cells are from peripheral blood. In some embodiments, the T-cells are from tumor.
• nuclear LSD and EOMES can be employed as biomarkers of T-cell mesenchymal phenotype and impaired T-cell function.
• PD-1 and TBET may be used as known in the art to assess T-cell exhaustion.
• T-cells can be obtained tWO . 2019/104381 jnjng patient samples which are suitably selected from tis?CJ/AU2018/051268j tumors and fluid samples such as peripheral blood.
• the sample is obtained prior to treatment with the therapeutic combination.
• the tissue sample is formalin fixed and paraffin embedded, archival, fresh or frozen.
• the sample is whole blood.
• the whole blood comprises immune cells, circulating tumor cells and any combinations thereof.
• Presence and/or expression levels/amount of a biomarker can be determined qualitatively and/or quantitatively based on any suitable criterion known in the art, including but not limited to DNA, mRNA, cDNA, proteins, protein fragments and/or gene copy number.
• presence and/or expression levels/amount of a biomarker in a first sample is increased or elevated as compared to
• presence/absence and/or expression levels/amount in a second sample e.g., before treatment with the therapeutic combination.
• presence/absence and/or expression levels/amount of a biomarker in a first sample is decreased or reduced as compared to presence and/or expression levels/amount in a second sample.
• the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. Additional disclosures for determining presence/absence and/or expression levels/amount of a gene are described herein.
• elevated expression refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)), detected by standard art known methods such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
• biomarker e.g., protein or nucleic acid (e.g., gene or mRNA)
• the elevated expression refers to the increase in expression level/amount of a biomarker in the sample wherein the increase is at least about any of 1.5x, 1.75x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, lOx, 25x, 50x, 75x, or lOOx the expression level/amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
• elevated expression refers to an overall increase of greater than about 1.5-fold, about 1.75-fold, about-2 fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold as compared to a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control (e.g., housekeeping gene).
• reduced expression refers to an overall reduction of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or greater, in the level of biomarker (e.g., protein or nucleic acid (e.g., gene or mRNA)), detected by standard art known methods such as those described herein, as compared to a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
• biomarker e.g., protein or nucleic acid (e.g., gene or mRNA)
• reduced expression refers to the decrease in expression level/amount of a biomarker in the sample wherein the decrease is at least about any of 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, O. lx, 0.05x, or O.Olx the expression level/amount of the respective biomarker in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
• Presence and/or expression level/amount of various biomarkers in a sample can be analyzed by a number of methodologies, many of which are known in the art and understood by the skilled artisan, including, but not limited to, immunohistochemistry ("IHC"), Western blot WQ,2J) 1 /10438 !
• Typical protocols for evaluating the status of genes and gene products are found, for example in Ausubel et al., eds., 1995, Current Protocols In Molecular Biology, Units 2 (Northern Blotting), 4 (Southern Blotting), 15 (Immunoblotting) and 18 (PCR Analysis). Multiplexed immunoassays such as those available from Rules Based Medicine or Meso Scale Discovery (“MSD”) may also be used.
• MSD Meso Scale Discovery
• presence and/or expression level/amount of a biomarker is determined using a method comprising: (a) performing gene expression profiling, PCR (such as rtPCR or qRT-PCR), RNA-seq, microarray analysis, SAGE, MassARRAY technique, or FISH on a sample (such as a subject cancer sample); and b) determining presence and/or expression level/amount of a biomarker in the sample.
• the microarray method comprises the use of a microarray chip having one or more nucleic acid molecules that can hybridize under stringent conditions to a nucleic acid molecule encoding a gene mentioned above or having one or more polypeptides (such as peptides or antibodies) that can bind to one or more of the proteins encoded by the genes mentioned above.
• the PCR method is qRT-PCR.
• the PCR method is multiplex-PCR.
• gene expression is measured by microarray.
• gene expression is measured by qRT-PCR.
• expression is measured by multiplex-PCR.
• Methods for the evaluation of mRNAs in cells include, for example, hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
• complementary DNA probes such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques
• nucleic acid amplification assays such as RT-PCR using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like.
• Samples from mammals can be conveniently assayed for mRNAs using Northern, dot blot or PCR analysis.
• such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a "housekeeping" gene such as an actin family member).
• the sequence of the amplified target cDNA can be determined.
• Optional methods include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies.
• mRNAs such as target mRNAs
• test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes.
• the probes are then hybridized to an array of nucleic acids immobilized on a solid support.
• the array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlates with increased or reduced clinical benefit of anti-angiogenic therapy may be arrayed on a solid support.
• Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
• the method comprises contacting the biological sample with antibodies to a biomarker (e.g., anti-PD-1 antibodies, anti-LSD antibodies, anti-TBET antibodies, anti-EOMES antibodies) described herein under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibodies and the biomarker.
• a biomarker e.g., anti-PD-1 antibodies, anti-LSD antibodies, anti-TBET antibodies, anti-EOMES antibodies
• Such method may be an in vitro or in vivo method.
• one or more anti-biomarker antibodies are used to select subjects eligible for combination therapy with a LSD inhibitor and a PD-1 binding antagonist.
• the presence and/or expression level/amount of biomarker proteins in a sample is examined using IHC and staining protocols.
• IHC staining of tissue sections has been shown to be a reliable method of determining or detecting presence of proteins in a sample.
• expression of a T-cell function biomarker in a sample from an individual is elevated protein expression and, in further embodiments, is determined using IHC.
• expression level of biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (such as a subject cancer sample) with an antibody; and b) determining expression level of a biomarker in the sample.
• IHC staining intensity is determined relative to a reference.
• the reference is a reference value.
• the reference is a reference sample (e.g. , control cell line staining sample or tissue sample from non-cancerous patient).
• T-cell function biomarker expression is evaluated on a tumor or tumor sample.
• a tumor or tumor sample may encompass part or all of the tumor area occupied by tumor cells.
• a tumor or tumor sample may further encompass tumor area occupied by tumor associated intratumoral cells and/or tumor associated stroma (e.g., contiguous peri-tumoral desmoplastic stroma).
• Tumor associated intratumoral cells and/or tumor associated stroma may include areas of immune infiltrates (e.g. , tumor infiltrating immune cells as described herein) immediately adjacent to and/or contiguous with the main tumor mass.
• T-cell function biomarker expression is evaluated on tumor cells.
• T-cell function biomarker expression is evaluated on immune cells within the tumor area as described above, such as tumor infiltrating immune cells.
• the sample may be contacted with an antibody specific for said biomarker under conditions sufficient for an antibody-biomarker complex to form, and then detecting said complex.
• the presence of the biomarker may be detected in a number of ways, such as by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum.
• a wide range of immunoassay techniques using such an assay format are available, see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These include both single-site and two-site or "sandwich" assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labeled antibody to a target biomarker.
• Presence and/or expression level/amount of a selected T-cell function biomarker in a tissue or cell sample may also be examined by way of functional or activity-based assays.
• the biomarker is an enzyme (e.g. , LSD)
• assays e.g., demethylase assays
• the samples are normalized for b(PCT/AU2018/051268 3 amount of the biomarker assayed and variability in the quality of the samples used, and variability between assay runs. Such normalization may be accomplished by detecting and incorporating the expression of certain normalizing biomarkers, including well known housekeeping genes.
• normalization can be based on the mean or median signal of all of the assayed genes or a large subset thereof (global normalization approach).
• measured normalized amount of a subject tumor mRNA or protein is compared to the amount found in a reference set. Normalized expression levels for each mRNA or protein per tested tumor per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and/or expression level/amount measured in a particular subject sample to be analyzed will fall at some percentile within this range, which can be determined by methods well known in the art.
• the sample is a clinical sample. In other embodiments, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain a representative piece of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissues or fluids that are known or thought to contain the tumor cells of interest. For instance, samples of lung cancer lesions may be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushings, or from sputum, pleural fluid or blood. Genes or gene products can be detected from cancer or tumor tissue or from other body samples such as urine, sputum, serum or plasma.
• Cancer cells may be sloughed off from cancer lesions and appear in such body samples. By screening such body samples, a simple early diagnosis can be achieved for these cancers. In addition, the progress of therapy can be monitored more easily by testing such body samples for target genes or gene products.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same subject or individual that are obtained at one or more different time points than when the test sample is obtained.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same subject or individual than when the test sample is obtained.
• Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not the subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals with a disease or disorder (e.g ., cancer) who are not the subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject or individual.
• a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumor tWO.2019/104381 plasma or serum samples from one or more individuals v?CT/AU20J ⁇ /051268j r(j er ( e.g . , cancer) who are not the subject or individual.
• the sample is a tissue sample from the individual.
• the tissue sample is a tumor tissue sample (e.g. , biopsy tissue).
• the tissue sample is lung tissue.
• the tissue sample is renal tissue.
• the tissue sample is skin tissue.
• the tissue sample is pancreatic tissue.
• the tissue sample is gastric tissue.
• the tissue sample is bladder tissue.
• the tissue sample is esophageal tissue.
• the tissue sample is mesothelial tissue.
• the tissue sample is breast tissue.
• the tissue sample is thyroid tissue.
• the tissue sample is colorectal tissue.
• the tissue sample is head and neck tissue.
• the tissue sample is
• the tissue sample is prostate tissue. In some embodiments, the tissue sample is ovarian tissue, HCC (liver), blood cells, lymph nodes, and/or bone/bone marrow tissue. In some embodiments, the tissue sample is colon tissue. In some embodiments, the tissue sample is endometrial tissue. In some embodiments, the tissue sample is brain tissue (e.g. , glioblastoma, neuroblastoma, and so forth).
• a tumor tissue sample may encompass part or all of the tumor area occupied by tumor cells.
• a tumor or tumor sample may further encompass tumor area occupied by tumor associated intratumoral cells and/or tumor associated stroma (e.g., contiguous peri-tumoral desmoplastic stroma).
• Tumor associated intratumoral cells and/or tumor associated stroma may include areas of immune infiltrates (e.g., tumor infiltrating immune cells as described herein) immediately adjacent to and/or contiguous with the main tumor mass.
• tumor cell staining is expressed as the percent of all tumor cells showing membranous staining of any intensity.
• Infiltrating immune cell staining may be expressed as the percent of the total tumor area occupied by immune cells that show staining of any intensity. The total tumor area encompasses the malignant cells as well as tumor-associated stroma, including areas of immune infiltrates immediately adjacent to and contiguous with the main tumor mass.
• infiltrating immune cell staining may be expressed as the percent of all tumor infiltrating immune cells.
• the disease or disorder is a tumor.
• the tumor is a malignant cancerous tumor (/.e., cancer).
• the tumor and/or cancer is a solid tumor or a non-solid or soft tissue tumor.
• soft tissue tumors include leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, adult acute lymphoblastic leukemia, acute myelogenous leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease).
• a solid tumor includes any cancer of body tissues other than blood, bone marrow, or the lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin.
• epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectal (e.g., basaloid colorectal carcinoma), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian carcinoma), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gall bladder, labium,
• colorectal e.g., basaloid colorectal carcinoma
• breast prostate
• lung kidney
• liver pancreas
• ovary e.g., endometrioid ovarian carcinoma
• head and neck oral cavity
• stomach duodenum
• small intestine large intestine
• anus
• Solid tumors of non-epithelial origin include sarcomas, brain tumors, and bone tumors.
• the cancer is non-small cell lung cancer (NSCLC).
• the cancer is second-line or third-line locally advanced or metastatic non-small cell lung cancer.
• the cancer is adenocarcinoma.
• the cancer is squamous cell carcinoma.
• the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g. triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.
• the cancer is a primary tumor.
• the cancer is a metastatic tumor at a second site derived from any of the above types of cancer.
• the cancer displays human effector cells (e.g., is infiltrated by human effector cells).
• Methods for detecting human effector cells are well known in the art, including, e.g. , by IHC.
• the cancer displays high levels of human effector cells.
• human effector cells are one or more of NK cells, macrophages, monocytes.
• the cancer is any cancer described herein.
• the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g. triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.
• NSCLC non-small cell lung cancer
• glioblastoma glioblastoma
• neuroblastoma e.g. triple-negative breast cancer
• CRC colorectal cancer
• the cancer displays cells expressing FcR (e.g., is infiltrated by cells expressing FcR).
• FcR e.g., is infiltrated by cells expressing FcR.
• Methods for detecting FcR are well known in the art, including, e.g., by IHC.
• the cancer displays high levels of cells expressing FcR.
• FcR is FcyR.
• FcR is activating FcyR.
• the cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast carcinoma (e.g. triple-negative breast cancer), gastric cancer, colorectal cancer (CRC), or hepatocellular carcinoma.
• NSCLC non-small cell lung cancer
• glioblastoma glioblastoma
• neuroblastoma melanoma
• breast carcinoma e.g. triple-negative breast cancer
• CRC colorectal cancer
• the T-cell function biomarker is detected in the sample using a method selected from the group consisting of FACS, Western blot, ELISA,
• the T- cell function biomarker is detected using FACS analysis.
• the T-cell function biomarker is PD-1.
• the PD-1 expression is detected in blood samples.
• the PD-1 expression is detected on circulating immune cells in blood samples.
• the circulating immune cell is a CD3 + /CD8 + T cell.
• the immune cells prior to analysis, are isolated from the blood samples. Any suitable method to isolate/enrich such population of cells may be used including, but not limited to, cell sorting.
• the PD-1 expression is reduced in samples from individuals that respond to treatment with a LSD inhibitor and/or PD-1 binding antagonist, such as an anti-PD-1 antibody.
• the PD-1 expression is elevated on circulating immune cells, such as
• CD3 + /CD8 + T cells in blood samples.
• diagnostic methods and kits that are based on the determination that LSD and EOMES co-localize in the nucleus and that this co-localization W . Q . ?01?/104381jst in part to EMT of T-cells and repression of their immurPCT/AlJ2018/051268 diagnostic methods suitably comprise: (i) obtaining a sample from a subject, wherein the sample comprises a T-cell (e.g. , CD8 + T-cell or CD4 + T-cell); (ii) contacting the sample with a first binding agent that binds to LSD (e.g. , LSD1, nuclear LSD, etc.
• a T-cell e.g. , CD8 + T-cell or CD4 + T-cell
• a first binding agent that binds to LSD e.g. , LSD1, nuclear LSD, etc.
• the first and second binding-agents suitably bind to epitopes of LSD (e.g., LSD1, nuclear LSD, etc. ) and EOMES polypeptides, respectively.
• LSD e.g., LSD1, nuclear LSD, etc.
• EOMES e.g., EOMES polypeptides
• Any suitable epitope may be chosen in the amino acid sequence of LSD (as set forth for example in GenPept Accession Nos. NP_055828.2, NP_001009999.1, 060341.2 and NP_694587.3), or in the amino acid sequence of EOMES (as set forth for example in GenPept Accession Nos. NP_001265111, NP_005433 and NP_001265112).
• Localization of LSD and EOMES in the nucleus of the T-cell may be performed using any suitable localization technique, e.g., by IHC, typically using an anti-LSD antibody that has a different detectable moiety or label than an anti-EOMES antibody.
• IHC immunohistochemical oxidation-semiconductor
• spatial proximity assays also referred to as "proximity assays” are employed, which can be used to assess the formation of a complex between LSD and EOMES.
• Proximity assays rely on the principle of "proximity probing", wherein an analyte, typically an antigen, is detected by the coincident binding of multiple (i.e., two or more, generally two, three or four) binding agents or probes, which when brought into proximity by binding to the analyte (hence “proximity probes”) allow a signal to be generated.
• proximity probes multiple binding agents or probes
• At least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and generation of the signal involves an interaction between the nucleic acid moieties and/or a further functional moiety which is carried by the other probe(s).
• signal generation is dependent on an interaction between the probes (more particularly by the nucleic acid or other functional moieties/domains carried by them) and hence only occurs when both the necessary two (or more) probes have bound to the analyte, thereby lending improved specificity to the detection system.
• the concept of proximity probing has been developed in recent years and many assays based on this principle are now well known in the art.
• Proximity assays are typically used to assess whether two particular proteins or portions thereof are in close proximity, e.g. , proteins that are bound to each other, fusion proteins, and/or proteins that are positioned in close proximity.
• One such assay known as proximity ligation assay (PLA), and which is used in some embodiments of the present invention, features two antibodies (raised in different species) bound to the targets of interest (see Nature Methods 3, 995-1000 (2006)).
• PLA probes which are species-specific secondary antibodies with a unique oligonucleotide strand attached, are then bound to the appropriate primary antibodies.
• the oligonucleotide strands of the PLA probes can interact with additional ssDNA and DNA ligase such they can be circulated and amplified via rolling circle amplification (RCA).
• RCA rolling circle amplification
• highly processive DNA polymerases such as Phi29 DNA polymerase
• the circular DNA template can be replicated hundreds to thousands of times longer and as a result producing ssDNA molecules from hundreds of nanometers to microns in length (see, Angewandte Chemie International Edition, 2008, 47, 6330-6337).
• WO 2_019/ip4381a n be detected via detection systems.
• a visible s i g n P . T/ A II 2018/ 0 1268 h e targets of interest are in close proximity.
• These assays feature the use of several DNA-antibody conjugates as well as enzymes such as DNA ligase and DNA polymerase.
• a dual binders (DB) assay which utilizes a bi specific detection agent consisting of two Fab fragments with fast off-rate kinetics joined by a flexible linker (Van dieck et at. , 2014 Chemistry & Biology Vol.21(3): 357-368).
• the dual binders comprise Fab fragments with fast off-rate kinetics
• the dual binders are washed off if only one of the Fab fragments is bound to its epitope (simultaneous cooperative binding of both Fab fragments of the dual binder prevents dissociation of the dual binder and leads to positive staining/visibility).
• WO2014/139980 which is encompassed in the practice of the present invention, proximity assays and tools are described, which employ a biotin ligase substrate and an enzyme to perform a proximity assay.
• the method provides detection of target molecules and proximity while maintaining the cellular context of the sample.
• biotin ligase such as an enzyme from E. coli and peptide substrate such as amino-acid substrate for that enzyme provides for a sensitive and specific detection of protein-protein interactions in FFPE samples.
• biotin ligase can efficiently biotinylate appropriate peptide substrate in the presence of biotin and the reaction can only occur when the enzyme makes physical contact with the peptide substrate, biotin ligase and the substrate can be separately conjugated to two antibodies that recognize targets of interest respectively.
• these methods may further comprise measuring the expression level of one or more additional biomarkers of T cell function and/or cellular composition (e.g. , percentage of Treg and/or absolute number of Treg; e.g. , number of CD8 + or CD4 + effector T cells), wherein the additional biomarkers of T cell function include a cytokine, e.g.
• IFN-g a T cell marker, or a memory T cell marker (e.g., a marker of T effector memory cells); and determining the treatment as demonstrating pharmacodynamic activity based on the expression level of the one or more T-cell function biomarkers, the one or more additional biomarkers of T cell function and/or cellular composition in the sample obtained from the subject, as compared with a reference, where an increased expression level of the one or more T-cell function biomarkers, the one or more additional biomarkers of T cell function and/or cellular composition as compared with the reference indicates pharmacodynamic activity to the PD- 1 antagonist treatment.
• Expression level of the biomarker(s) and/or cellular composition may be measured by one or more methods as described herein.
• PD activity may refePCJ/A.U2Q18/051268 treatment (e.g ., a LSD inhibitor in combination with a PD-1 binding antagonist treatment and optionally a chemotherapeutic agent) to the subject.
• An example of a PD activity may include modulation of the expression level of one or more genes.
• monitoring PD activity such as by measuring expression of one or more T-cell function biomarkers, may be advantageous during a clinical trial examining a LSD inhibitor and PD- 1 binding antagonist and optionally a chemotherapeutic agent.
• Monitoring PD activity may be used, for example, to monitor response to treatment, toxicity, and the like.
• the expression level of one or more marker genes, proteins and/or cellular composition may be compared to a reference which may include a sample from a subject not receiving a treatment (e.g. , a LSD inhibitor treatment in combination with a PD-1 binding antagonist and optionally a chemotherapeutic agent).
• a reference may include a sample from the same subject before receiving a treatment (e.g. , a LSD inhibitor treatment in combination with a PD-1 binding antagonist and optionally a chemotherapeutic agent).
• a reference may include a reference value from one or more samples of other subjects receiving a treatment (e.g., a LSD inhibitor treatment in combination with a PD-1 binding antagonist and optionally a chemotherapeutic agent).
• a treatment e.g., a LSD inhibitor treatment in combination with a PD-1 binding antagonist and optionally a chemotherapeutic agent.
• a population of patients may be treated, and a mean, average, or median value for expression level of one or more genes may be generated from the population as a whole.
• a set of samples obtained from cancers having a shared characteristic e.g., the same cancer type and/or stage, or exposure to a common treatment such as a LSD inhibitor treatment in combination with a PD-1 binding antagonist and optionally a chemotherapeutic agent
• This set may be used to derive a reference, e.g., a reference number, to which a subject's sample may be compared. Any of the references described herein may be used as a reference for monitoring
• a sample may include leukocytes.
• the sample may be a peripheral blood sample (e.g. , from a patient having a tumor).
• the sample is a tumor sample.
• a tumor sample may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell type in association with the tumor.
• the sample is a tumor tissue sample containing tumor-infiltrating leukocytes.
• the sample may be processed to separate or isolate one or more cell types (e.g., leukocytes).
• the sample may be used without separating or isolating cell types.
• a tumor sample may be obtained from a subject by any method known in the art, including without limitation a biopsy, endoscopy, or surgical procedure.
• a tumor sample may be prepared by methods such as freezing, fixation (e.g. , by using formalin or a similar fixative), and/or embedding in paraffin wax.
• a tumor sample may be sectioned.
• a fresh tumor sample (/.e. , one that has not been prepared by the methods described above) may be used.
• a tumor sample may be prepared by incubation in a solution to preserve mRNA and/or protein integrity.
• the sample may be a peripheral blood sample.
• a peripheral blood sample may include white blood cells, PBMCs, and the like. Any technique known in the art tWO_2019/104381 OC ytes from a peripheral blood sample may be used. For cP T/A 11201 /0 1268 n p
• a fresh peripheral blood sample i.e., one that has not been prepared by the methods described above may be used.
• a peripheral blood sample may be prepared by incubation in a solution to preserve mRNA and/or protein integrity.
• responsiveness to treatment may refer to any one or more of: extending survival (including overall survival and progression free survival); resulting in an objective response (including a complete response or a partial response); or improving signs or symptoms of cancer.
• responsiveness may refer to improvement of one or more factors according to the published set of RECIST guidelines for determining the status of a tumor in a cancer patient, i.e. , responding, stabilizing, or progressing. For a more detailed discussion of these guidelines, see, Eisenhauer et ai (2009 Eur J Cancer 45 : 228-47), Topalian et at. (2012 N Engl J Med 366: 2443-54), Wolchok et ai (2009 Clin Can Res 15 : 7412-20) and
• a responsive subject may refer to a subject whose cancer(s) show improvement, e.g., according to one or more factors based on RECIST criteria.
• a non-responsive subject may refer to a subject whose cancer(s) do not show
• response criteria may not be adequate to characterize the anti-tumor activity of therapeutic agents of the invention, which can produce delayed responses that may be preceded by initial apparent radiological progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that account for the possible appearance of new lesions and allow radiological progression to be confirmed at a subsequent assessment. Accordingly, in some embodiments, responsiveness may refer to improvement of one of more factors according to immune-related response criteria (irRC). See, e.g. , Wolchok et aL (2009, supra). In some embodiments, new lesions are added into the defined tumor burden and followed, e.g. , for radiological progression at a subsequent assessment.
• irRC immune-related response criteria
• presence of non-target lesions is included in assessment of complete response and not included in assessment of radiological progression.
• radiological progression may be determined only on the basis of measurable disease and/or may be confirmed by a consecutive assessment 34 weeks from the date first documented.
• responsiveness may include immune activation. In some embodiments, responsiveness may include treatment efficacy. In some embodiments,
• responsiveness may include immune activation and treatment efficacy.
• therapeutic kits comprising a LSD inhibitor (e.g., a LSD1 inhibitor, a nuclear LSD inhibitor, etc. ) and a PD-1 binding antagonist.
• a LSD inhibitor e.g., a LSD1 inhibitor, a nuclear LSD inhibitor, etc.
• PD-1 binding antagonist e.g., a PD-1 binding antagonist
• the therapeutic kits further comprise a package insert comprising instructional material for administering concurrently the LSD inhibitor and the PD-1 binding antagonist to treat a T-cell dysfunctional disorder, or to enhance immune function (e.g. , immune effector function, T-cell function etc.) in an individual having cancer, or to treat or delay cancer progression, or to treat infection in an individual .
• the therapeutic kits may further comprise a chemotherapeutic agent (e.g. , an agent that targets rapidly dividing cells and/or W0_2019/104381 ⁇ c
• the LSD inhibitor, PD-1 binding antagonist and optionally chemotherapeutic agents are in the same container or separate containers.
• Suitable containers include, for example, bottles, vials, bags and syringes.
• the container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy).
• the container holds the formulation and the label on, or associated with, the container may indicate directions for use.
• the kits may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructional material for use.
• kits further include one or more of other agents (e.g., a chemotherapeutic agent, and anti-neoplastic agent).
• Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
• diagnostic kits are provided for determining expression of biomarkers, including the T-cell function biomarkers disclosed herein, which include reagents that allow detection and/or quantification of the biomarkers.
• reagents include, for example, compounds or materials, or sets of compounds or materials, which allow quantification of the biomarkers.
• the compounds, materials or sets of compounds or materials permit determining the expression level of a gene (e.g., T-cell function biomarker gene), including without limitation the extraction of RNA material, the determination of the level of a corresponding RNA, etc., primers for the synthesis of a corresponding cDNA, primers for amplification of DNA, and/or probes capable of specifically hybridizing with the RNAs (or the corresponding cDNAs) encoded by the genes, TaqMan probes, proximity assay probes, ligases, antibodies etc.
• a gene e.g., T-cell function biomarker gene
• kits may also optionally include appropriate reagents for detection of labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates, dilution buffers and the like.
• a nucleic acid-based detection kit may include (i) a T-cell function biomarker polynucleotide (which may be used as a positive control), (ii) a primer or probe that specifically hybridizes to a T-cell function biomarker polynucleotide.
• enzymes suitable for amplifying nucleic acids including various polymerases (reverse transcriptase, Taq, SequenaseTM, DNA ligase etc.
• kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
• a protein-based detection kit may include (i) a T-cell function biomarker polypeptide (which may be used as a positive control), (ii) an antibody that binds specifically to a T-cell function biomarker polypeptide.
• the kit can also feature various devices (e.g. , one or more) and reagents (e.g.
• the reagents described herein which may be optionally associated with detectable labels, can be presented in the format of a microfluidics card, a chip or chamber, a microarray or a kit adapted for use with the assays described in the examples or below, e.g., RT-PCR or Q PCR techniques described herein.
• kits of the invention can contain instructional material for the simultaneous, sequential or separate use of the different components contained in the kit.
• the instructional material can be in the form of printed material or in the form of an electronic support capable of storing instructions such that they can be read by a subject, such as electronic storage media (magnetic disks, tapes and the like), optical media (CD-ROM, DVD) and the like.
• the media can contain Internet addresses that provide the instructional material.
• the present inventors examined the LSD1 inhibitor, phenelzine sulfate, in the triple negative, breast cancer cell line MDA-MB-231 to test its efficacy in inhibiting demethylation and cell proliferation. It was observed that phenelzine sulfate inhibited the nuclear axis of LSD1, as measured by H3k4me2 de-methylation as well as inhibiting the proliferation of MDA-MB-231, as measured by the WST-1 assay ( Figure 1A, B).
• the present inventors also examined the effect of the treatment modalities on the innate and adaptive immune repertoire. They observed some inhibition in the infiltration of CD4/CD8+ naive T-cells across all 3 treatment groups and increased infiltration of CD4 + effector memory T-cells (Figure 5A). Anti-PDl, phenelzine sulfate and the combination treatments were shown to enhance CD8 + central memory and effector memory populations. ( Figure 5A).
• a T-cell exhaustion signature was examined in CD8 + T-cells isolated from TME.
• High expression of EOMES (EOMES high ) and low expression of TBET (TBET low ) represent an exhaustive T- cell signature in CD8 + T-cells.
• EOMES a key marker of exhaustion
• Figure 5B Consistent with EOMES inhibition, a pattern of induction was observed in TBET and Ki67 expression, which are markers of T-cell activity and effector status. Both these markers were induced by anti-PDl treatment but to a lesser extent and more strongly by phenelzine sulfate treatment. Notably, the strongest % increase in expression was seen in the combination therapy (Figure 5B).
• CD4 + and CD8 + T-cells were found to be less capable of producing key pro-inflammatory and Thl cytokines IFN-g, IL-2 and TNF-a, consistent with an exhaustive signature.
• all treatment groups /.e. , anti-PDl, phenelzine sulfate and combination
• CD4 + T-cells were observed to have a better pro-inflammatory/Thl response when compared to the control, as well as having CD4 + T-cells that were much more efficient in producing IL-2 and
• the present inventors also examined the effect of the treatment modalities on the expression of T-cell activation markers in CD8 + T cells using the nanostring platform. Compared to control samples, all treatments resulted in decreased expression of Sell (CD62L) and increased CD44 gene expression with more effector memory T cells (CD62L-CD44 hi ) ( Figure 5E).
• NUCLEAR LSD1 COMPLEXES WITH EOM ES IN EXHAUSTED T-CELL SIGNATURES
• LSD1 inhibition had minimal impact on the mRNA of T-cell exhaustion genes such as EOMES.
• phenelzine sulfate which is expected to inhibit the epigenetic activity of LSD1
• inhibited exhaustive signature genes such as CTLA4 and LAG3.
• LSD1 may be capable of regulating the function of target proteins at both the protein and transcript levels. This regulation would be by post-translational modification of the protein and may well impact nuclear localization and binding partners.
• LSD1 inhibition alone or PD1 signaling alone induces or represses distinct gene transcription programs in key signaling pathways (Figure 8A, B).
• inhibition of LSD1 and PD1 induces and represses gene expression programs involved in adaptive, innate and inflammation signals ( Figure 8C).
• LSD1 re-programs the epigenetic template as confirmed by ATAQ-sequence data, which monitors epigenome changes ( Figure 8D). This re programming in turn enables the PD1 signals to be received by the epigenetic template resulting in appropriate mRNA production or repression.
• the present inventors sought to investigate the putative complex that LSD1 may form with EOMES though use of the DUOLINK (Sigma) ligation-IF assay, which confirms the presence of interacting proteins via a ligation reaction measured by fluorescent microscopy.
• LSD1 in tumor- infiltrating lymphocytes (TILs).
• TILs tumor- infiltrating lymphocytes
• This regulation may be indirect or direct.
• LSD1 may indirectly impact on the epigenome via protein: protein interaction.
• LSD1 is complexed to EOMES and this is critical to maintain this exhaustive, transcription factor in the nucleus to mediate exhaustive gene signature transcription programs.
• the present inventors postulate that the interaction between LSD1 with EOMES maintains this transcription factor in a de- methylated state which is crucial for its nuclear retention.
• LSD1 may directly impact on the epigenome and in this scenario, LSD1 is hypothesized to tether to the epigenetic template and re-program the chromatin structure either in an active or repressor state based on iWO 2019/104381 e (H3k4me2/h3k9me2). This re-programmed state is bel i PCT/ All 2018/051268 e to PD1 mediated docking of transcription factors and subsequent mRNA expression or repression.
• mice were treated with vehicle control (Group A), Abraxane (30 mg/kg) (Group B), anti-PDl antibody (10 mg/kg) (Group C), phenelzine sulfate (40 mg/kg) (Group D), Abraxane (30 mg/kg) + PD1 antibody (10 mg/kg) (Group E), phenelzine sulfate (40 mg/kg) + PD1 antibody (10 mg/kg) (Group F), Abraxane (30 mg/kg) + phenelzine sulfate (40 mg/kg) (Group G), and Abraxane (30 mg/kg) + phenelzine sulfate (40 mg/kg) + PD1 antibody (10 mg/kg) (also referred to herein as "triple therapy”) (Group H). It was found that all treatments significantly reduced the primary tumor volume (Figure 11A) with triple therapy providing the most reduction in tumor burden.
• CTC mesenchymal circulating tumor cell
• LSDlp mesenchymal circulating tumor cell
• SNAI1 mesenchymal circulating tumor cell
• the cancer cells were assessed for expression of a chemo-resistant, stem-like biomarker signature biomarker panel (CD133, ALDH1A and ABCB5).
• the % change in expression in the Abraxane treated group showed a marked increase in expression of CD133, ALDH1A and ABCB5 ( Figure HE, Group C).
• the % change in expression in the anti-PDl treated group showed a moderate increase in expression of ALDH1A and CD133 but no change in expression of ABCB5 ( Figure 11E, Group C).
• phenelzine sulfate alone did not significantly affect expression of CD133 and ALDHA1 but strongly inhibited ABCB5 expression ( Figure 2C, Group D).
• WO 2019/104381jarvested cells from primary tumors where then cytospun ?CT/AU201_8/051268- treated with poly-l-lysine and fixed then stored in PBS for IFA Microscopy Analysis.
• Cells were permeabilized by incubating with 1% Triton X-100 for 20 min and were probed with a variety of primary antibodies as described in the figure legends and the corresponding secondary antibodies.
• Cover slips were mounted on glass microscope slides with ProLong Diamond Antifade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5 pm sections were obtained using a Leica DMI8 microscope using lOOx oil immersion lens running LAX software. The final image was obtained by averaging four sequential images of the same section.
• TNFI Total Nuclear Fluorescent Intensity
• TCFI Total Cytoplasmic Fluorescent Intensity
• TFI total Fluorescent Intensity
• TNFI Total Nuclear Fluorescent Intensity
• TCFI Total Cytoplasmic Fluorescent Intensity
• TFI Total Fluorescent Intensity
• Cells were surfaced stained with CD49b, F4/80 and intracellular stained for IFN-y, TNF-a and IL-10.
• NK cells and the macrophages M1/M2
• CD45, CD3, CD4, CD8, CD44, CD62L for naive, effector and central memory.
• Cells were finally resuspended in PBS 2% FBS and flow cytometry performed on FACS Fortessa (BD) or FACS LSRII (BD). Analysis of data was done using FlowJo® analysis software and % cell populations calculated from the raw data. A Mann-Whitney non-parametric t-test was used to compare control vs other groups.
• CD8 + T-cells were isolated from the 4T1 metastatic mouse model with high purity using the StemCell technologies CD8 isolation Kit.
• Qiagen mRNA prep kit was used to generate mRNA and then processed for nanostring analysis using manufactures guidelines and protocols and profiled with the immuno-oncology gene panel.
• the DUO-Link ligation was employed to measure the co-interaction of two proteins of interest (EOMES and LSDlnp) via a ligation/amplification IFA as per the manufactures protocol and SOP. Analysis was carried out measuring the intensity of the corresponding IF which tWO 2 i9/104381jscopy with positive signals corresponding to a successful PCJ/AII 2018/051268j the target two target proteins interacting.
• QR patient group included immune reactive (R) HSCT recipients who acquired stable anti-CMV T cell immunity as indicated by QuantiFERON-CMV reactivity (30.1 IU/mL) and no evidence of viral recrudescence.
• the QNR group included immune non-reactive (NR), HSCT recipients who failed to acquire stable anti-CMV T cell immunity as indicated by QuantiFERON-CMV reactivity ( ⁇ 0.1 IU/mL) and with symptomatic viral recrudescence (single or multiple viral reactivations) or asymptomatic viral recrudescence.
• the QuantiFERON-CMV assay measures the amount of CMV-specific IFN-g secretion in whole blood

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