Novel inhibitors of histone methyltransferase nuclear localization

Abstract

The present invention relates to novel therapeutic compositions and methods for the treatment of cancer. Specifically, the use of protein inhibitors including novel bicyclic peptide inhibitors for the treatment of cancer.

Description

Technical Field

[0001] The present invention generally relates to compositions for at least partially inhibiting the nuclear localization of SETDB1. The present invention also provides protein molecules corresponding to the SETDB1 nuclear localization site, and their use in preventing the nuclear localization of the SETDB1 polypeptide. The present invention also relates to the use of the compositions and protein molecules for treating or preventing cancer in a subject. The present invention also generally relates to methods and agents for predicting the response to therapy. More specifically, the present disclosure relates to methods, agents and kits for analyzing the cellular distribution of SETDB1 for stratifying subjects into possible responders or non-responders to therapy, particularly immunotherapy.

[0002] The various bibliographic references cited in the specification are listed at the end of the specification by number. Background Art

[0003] Reference in this specification to any prior art disclosure (or information derived therefrom) or to any known material is not and should not be taken as an acknowledgement or acknowledgment or any form of suggestion that the prior art disclosure (or information derived therefrom) or known material forms part of the common general knowledge in the research field to which this specification relates.

[0004] There is now a wealth of evidence to illustrate how tumors co-opt certain immune checkpoint pathways as a major mechanism of immune resistance, particularly against T cells specific for tumor antigens. Because many immune checkpoints are triggered by ligand-receptor interactions, they may be susceptible to blockade by antibodies or modulation by recombinant forms of the ligand or receptor. Therefore, agonists of co-stimulatory receptors or antagonists of inhibitory signals are the main agents currently in clinical testing, both of which lead to the expansion of antigen-specific T cell responses.

[0005] In this context, cancer immunotherapy has been regarded as a breakthrough in the field of cancer treatment, shifting from targeting tumors to targeting the immune system (Couzin-Frankel., "Science (Science)" 2013.342 (6165): 1432-3). Blocking immune checkpoints with antibodies targeting PD1, PD-L1 or CTLA-4 has achieved promising clinical results and manageable safety characteristics. However, only a small number of patients respond to these therapies. Therefore, it is necessary to improve cancer immunotherapy by new methods and / or by combining anti-checkpoint antibodies with other treatments (see Jenkins et al., "British Journal of Cancer (BJC.)" 2018; 118, 9-16; Sharma et al., "Cell (Cell)" 2017; 168 (4): 707-723). In addition, anti-checkpoint antibodies may induce side effects, mainly autoimmunity, so that the implementation of combination therapy may help to reduce the dose administered, and thus reduce adverse events, which is still a valuable medical help.

[0006] SET domain bifurcated histone lysine methyltransferase 1 (SETDB1, also known as ESET and KMT1A) is an H3K9 methyltransferase involved in gene silencing. In recent years, SETDB1 has been reported as an oncogene for a variety of cancers. Histone methyltransferases (HMTs) are histone modifying enzymes (e.g., histone-lysine N-methyltransferases and histone-arginine N-methyltransferases) that catalyze the transfer of one, two or three methyl groups to lysine residues and arginine residues of histones. The attachment of methyl groups occurs primarily at specific lysine residues or arginine residues on histones H3 and H4. Lysine-specific histone methyltransferases are further subdivided into SET domain-containing and SET domain-free. There is a large amount of literature documenting the methylation of the N-terminal lysine residues of histone H3, particularly in positions 4, 9, 27, 36 and 79, to form monomethylated, dimethylated or trimethylated lysines. More than 30 histone methyltransferases have been described.

[0007] The use of DNMT or HDAC inhibitors has also recently been proposed in combination with other cancer therapies, such as immunotherapy (Wrangle et al., Oncotarget. 4(11):2067-2079, Chiapinelli et al., Cell 2015;162(5):974-86; Licht, Cell 2015;162(5):938-9; and Sharma, supra). In fact, it has been shown that DNA demethylating agents may induce T cell-mediated immune responses in solid tumors and may therefore synergize with anti-tumor immunotherapies such as checkpoint inhibitors (Roulois et al., Oncoimmunology. 2016;5(3):e1090077). Moreover, incidental clinical findings suggest that non-small cell lung cancer patients pretreated with 5-azacytidine have better clinical responses to subsequent anti-PD1 therapy (Juergens et al., Cancer Discov. 2011; 1:598-607), and melanoma mouse models have been shown to respond better to the combination of 5-azacytidine and anti-CTLA-4 antibody therapy than to either 5-azacytidine or anti-CTLA4 antibody therapy alone (see Chiappinelli et al., Cell 2015; 162:974-86; and Roulois et al., Cell 2015; 162:961-973).

[0008] However, the role of this epigenetic regulator in cancer immunology and immunotherapy remains poorly understood. Demethylating agents are diverse, and identification of genes that reactivate predictions or mediate responses remains elusive. In general, the immunomodulatory effects of treatment with 5-azacytidine, a DNMT, are complex and depend on the clinical setting and patient type (see Frosig and Hadrup, Mediators Inflamm. 2015; 871641).

[0009] Therefore, there is a great need to elucidate the precise molecular mechanisms underlying the effects of epigenetic regulators on the immune response to cancer or tumors. Summary of the invention

[0010] The present disclosure is based in part on the discovery that translocation of SETDB1 into the nucleus of cells, including tumor cells, is associated with increased severity of cancer and resistance to therapy. Accordingly, the inventors have discovered inhibitors of SETDB1 nuclear localization.

[0011] Therefore, in one aspect of the present invention, a method for inhibiting or reducing nuclear localization of a SETDB1 polypeptide in a cell is provided, the method comprising contacting the cell with an agent that inhibits the binding of the SETDB1 polypeptide to an importin alpha polypeptide.

[0012] In yet another aspect, a method of inhibiting or reducing nuclear localization of a SETDB polypeptide in a cell is provided, the method comprising contacting the cell with a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to a nuclear localization sequence (NLS) of a SETDB1 polypeptide. In this aspect, the invention also includes a mimetic comprising an amino acid sequence according to Formula I, which is particularly effective in inhibiting or reducing nuclear localization of SETDB1. Thus, the inventors have contemplated that a SETDB1 peptide mimetic comprising an amino acid sequence according to Formula I can be used to inhibit nuclear localization of SETDB1.

[0013] In another aspect, the present invention provides a method of treating or preventing cancer in a subject, the method comprising administering to the subject an agent that inhibits or prevents the binding of a SETDB1 polypeptide to an importin alpha polypeptide.

[0014] In some embodiments, the cancer is associated with at least some SETDB1 polypeptide present in the nucleus. By way of illustrative example, the cancer may be selected from the group comprising: breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, colon cancer, liver cancer, metastatic brain cancer, melanoma, retinoblastoma, ovarian cancer and renal cell carcinoma.

[0015] In yet another aspect, a method for producing an agent for inhibiting or reducing nuclear localization of a SETDB1 polypeptide is provided, the method comprising:

[0016] a) contacting the cell with an agent; and

[0017] b) detecting a reduction or inhibition of the nuclear localization of the SETDB1 polypeptide in the cell relative to a normal or reference level of nuclear localization in the absence of the agent.

[0018] In some embodiments, the agent inhibits the binding of the SETDB1 polypeptide to the IMPα polypeptide but does not inhibit the binding of any other polypeptide to the IMPα polypeptide.

[0019] In some embodiments, the agent binds directly to the IMPα polypeptide.

[0020] In some embodiments, the agent reduces the amount of SETDB1 transported into the nucleus of the cell. Accordingly, the agent reduces the amount of SETDB1 present in the nucleus of the cell.

[0021] In some embodiments, the agent is a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to the NLS of a SETDB1 polypeptide.

[0022] In some embodiments, the protein molecule comprises, consists of, or consists essentially of: an amino acid sequence of formula (I). More preferably, the protein molecule comprises, consists of, or consists essentially of: an amino acid sequence as shown in any one of SEQ ID NOs: 1-6 or having at least 85% sequence identity to a sequence as shown in any one of SEQ ID NOs: 1-6. Even more preferably, the protein molecule comprises, consists of, or consists essentially of: an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 6.

[0023] In some embodiments, the cell is a cancer stem cell or a non-cancer stem cell tumor cell.

[0024] In some embodiments, the cancer is selected from the group comprising breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, colon cancer, liver cancer, metastatic brain cancer, melanoma, retinoblastoma, ovarian cancer, and renal cell carcinoma.

[0025] In some embodiments, the protein molecule comprises, consists of, or consists essentially of an amino acid sequence corresponding to residues 206 to 232 of a human SETDB1 polypeptide (i.e., the human SETDB1 sequence of UniProtKB Accession No. Q15047). In some alternative embodiments, the protein molecule is a fragment of a nuclear localization polypeptide.

[0026] In some preferred embodiments, the protein molecule comprises 50 amino acid residues or less.

[0027] In some embodiments, the protein molecule differs from SETDB1 by the addition, deletion and / or substitution of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) amino acid of residues 206 to 232 of SETDB1.

[0028] In another aspect, the present invention provides a method for producing a protein molecule that inhibits or reduces the nuclear localization of a SETDB1 polypeptide, the method comprising:

[0029] a) contacting a cell with a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to residues 206 to 232 of SETDB1; and

[0030] b) detecting a reduction or inhibition of the nuclear localization of the SETDB1 polypeptide in the cell relative to a normal or reference level of nuclear localization in the absence of the protein molecule.

[0031] In still another aspect, the present invention provides an isolated or purified protein molecule represented by formula (I):

[0032] Z1GKKRX1KX2WHX3GTLIX4IQTVGX5GKKX6KVKZ2

[0033] Formula (I)

[0034] Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween);

[0035] X1 is selected from Thr, Arg and modified forms thereof;

[0036] X2 is selected from Thr, Leu and modified forms thereof;

[0037] X3 is selected from Lys, Gly and modified forms thereof;

[0038] X4 is selected from Ala, Pro and modified forms thereof;

[0039] X5 is selected from Pro, Lys and modified forms thereof; and

[0040] X6 is selected from Tyr, Lys and modified forms thereof.

[0041] In some embodiments, Z1 is not present.

[0042] In some identical embodiments and some alternative embodiments, Z2 is absent.

[0043] In some identical embodiments and some alternative embodiments, X1 is Thr.

[0044] In some identical embodiments and some alternative embodiments, X2 is Thr.

[0045] In some identical embodiments and some alternative embodiments, X3 is Lys.

[0046] In some identical embodiments and some alternative embodiments, X4 is Ala.

[0047] In some identical embodiments and some alternative embodiments, the X5 is a Pro.

[0048] In some identical embodiments and some alternative embodiments, X6 is Tyr.

[0049] In some identical embodiments and some alternative embodiments, X1 is Arg.

[0050] In some identical embodiments and some alternative embodiments, X2 is Leu.

[0051] In some identical embodiments and some alternative embodiments, wherein X3 is Gly.

[0052] In some identical embodiments and some alternative embodiments, the X4 is a Pro.

[0053] In some preferred embodiments, the protein molecule comprises, consists of, or consists essentially of the amino acid sequence shown in SEQ ID NO:1 or SEQ ID NO:2.

[0054] In some preferred embodiments, the protein molecule of Formula I further comprises at least one membrane permeable moiety. For example, the membrane permeable moiety can be a lipid moiety. In some embodiments of this type, the membrane permeable moiety is myristoyl.

[0055] In some embodiments, the membrane permeable moiety may be coupled to the N-terminal or C-terminal amino acid residue. Preferably, the membrane permeable moiety is coupled to the N-terminal amino acid residue.

[0056] In addition, the inventors have discovered that nuclear localized SETDB1 (also referred to herein as "nuclear SETDB1" or "nuclear SETDB1") colocalizes with at least one nuclear polypeptide (e.g., ATF7IP), and that this colocalization is a surrogate marker for increased disease severity and resistance to therapy (e.g., immunotherapy). These findings have been put into practice in methods and kits for predicting the likelihood of a subject's response to therapy, as described below.

[0057] Thus, in one aspect, the present disclosure provides methods for predicting the likelihood of a subject's response to a therapy (e.g., an immunotherapy). These methods are generally understood to consist of, or consist essentially of analyzing the cellular localization of SETDB1 in cells expressing SETDB1 in the subject to thereby predict the likelihood of the subject's response to the therapy. The cells expressing SETDB1 are suitably tumor cells. The therapy may be an immunotherapy.

[0058] In some embodiments, the method comprises detecting the presence of SETDB1 in the nucleus of the cell, or a level of SETDB1 in the nucleus of the cell, the level indicating an abnormal or abnormal nuclear level of SETDB1 and / or which is associated with an increased likelihood of developing resistance to the therapy, thereby determining that the subject has an increased likelihood of developing resistance to the therapy. In some embodiments, the method comprises detecting a higher level of SETDB1 in the nucleus of the cell relative to a control, thereby determining that the subject has an increased likelihood of developing resistance to the therapy. In some embodiments, the method comprises comparing the level of SETDB1 between different cellular components (e.g., nucleus and cytoplasm), thereby determining that the subject has an increased likelihood of developing resistance to the therapy.

[0059] Suitably, the method comprises detecting a higher level of SETDB1 in the nucleus of the cell relative to a control (e.g., relative to the level of SETDB1 in the nucleus of a corresponding normal control cell or relative to the level of SETDB1 in the cytoplasm of the cell), which indicates an increased likelihood that the subject will develop resistance to the therapy. In non-limiting examples of these embodiments, the higher level of SETDB1 in the nucleus of the cell means that its level is at least about 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% (and every integer therebetween) of the level of SETDB1 in the nucleus of a corresponding normal control cell. In some of the same or other non-limiting examples of these embodiments, the higher level of SETDB1 in the nucleus of the cell means that the level of SETDB1 in the nucleus of the cell is higher than the level of SETDB1 outside the nucleus of the cell (e.g., in the cytoplasm, referred to herein as "cytoplasmic"). In representative examples of this type, the elevated level indicates a ratio of nuclear SETDB1 to cytoplasmic SETDB1 of greater than about 0.55, 0.60, 0.65, 0.70, 0.75, 0.85, 0.90, or 0.95. In some of the same and other non-limiting examples, the method comprises detecting elevated levels of nuclear SETDB1 in greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the subject's cells (e.g., tumor cells).

[0060] In other embodiments, the method comprises detecting the absence of SETDB1 in the nucleus of the cell or a level of SETDB1 in the nucleus of the cell, the level indicating a normal nuclear level of SETDB1, and this is associated with an increased likelihood of sensitivity to the therapy, thereby determining that the subject has an increased likelihood of sensitivity to the therapy. In some of the same and other embodiments, the method comprises detecting a level of SETDB1 in the nucleus of the cell relative to a control (e.g., relative to the nucleus of a corresponding normal cell or relative to the level of cytoplasmic SETDB1 of the cell), the level indicating a normal nuclear level of SETDB1, and this indicates an increased likelihood of sensitivity to the therapy in the subject. In some of the same and other embodiments, the method comprises detecting the presence of cytoplasmic SETDB1, thereby determining that the subject has an increased likelihood of sensitivity to the therapy. Suitably, the method comprises detecting a level of cytoplasmic SETDB1 in a cell relative to a control (e.g., relative to the cytoplasmic SETBD1 of a corresponding normal cell or relative to the level of SETDB1 in the nucleus of a subject's cell), the level indicating a normal extranuclear level of SETDB1, and indicating an increased likelihood of sensitivity to the therapy in the subject. In non-limiting examples of these embodiments, the level of cytoplasmic SETDB1 of a cell indicates that its level is about the same as the level of cytoplasmic SETDB1 of a corresponding normal control cell (e.g., its level is about 85% to about 115% of the latter, and every integer therebetween). In some of the same or other non-limiting examples of these embodiments, the level of cytoplasmic SETDB1 of a cell is higher than the level of nuclear cytoplasmic SETDB1. In representative examples of this type, the higher level indicates that the ratio of cytoplasmic SETDB1 to nuclear SETDB1 is greater than about 0.55, 0.60, 0.65, 0.70, 0.75, 0.85, 0.90 or 0.95. In some of the same and other non-limiting examples, the method includes detecting normal levels of cytoplasmic SETDB1 in more than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the cells of the subject.

[0061] Suitably, in any of the above embodiments, the method comprises detecting the co-localization of SETDB1 with a nuclear binding partner of SETDB1 (e.g., ATF7IP). In some instances of these embodiments, the method comprises contacting a sample comprising a cell or a lysate of the subject with a first antigen binding molecule that specifically binds to SETDB1 and a second antigen binding molecule that specifically binds to the nuclear binding partner (e.g., ATF7IP), and detecting the presence of a complex comprising the first antigen binding molecule and the second antigen binding molecule in the sample to thereby determine that the subject has an increased likelihood of resistance to the therapy. In some embodiments, the method comprises detecting a higher level of the complex relative to a control (e.g., corresponding to a normal control cell), which indicates an increased likelihood that the subject has resistance to the therapy. In other embodiments, the method comprises detecting the level of the complex in the nucleus relative to a control (e.g., corresponding to a normal or immunocompetent control cell), the level indicating a normal level of the complex, and this indicates an increased likelihood that the subject has sensitivity to the therapy.

[0062] Another aspect of the present disclosure provides methods for determining the likelihood that a subject will develop resistance to a therapy (e.g., an immunotherapy). These methods are generally understood to be, consist of, or consist essentially of: detecting colocalization of SETDB1 with a nuclear binding partner of SETDB1 (e.g., ATF7IP) in a sample of the subject (e.g., a sample containing cells expressing SETDB1, such as tumor cells or lysates thereof), or a level of colocalization, the level indicating an abnormal or abnormal level of colocalization, and being associated with an increased likelihood of developing resistance to the therapy, thereby determining that the subject has an increased likelihood of developing resistance to the therapy. In some embodiments, the method comprises detecting a higher level of colocalization in the sample relative to a control (e.g., a reference sample containing cells expressing SETDB1 corresponding to a normal control or a lysate thereof), thereby determining that the subject has an increased likelihood of developing resistance to the therapy. In other embodiments, the method comprises detecting approximately the same level of colocalization in the sample relative to a control (e.g., a reference sample containing cells expressing SETDB1 corresponding to a normal control or a lysate thereof), thereby determining that the subject has an increased likelihood of developing resistance to the therapy.

[0063] In a related aspect, the present disclosure provides methods for determining the likelihood that a subject will develop resistance to a therapy (e.g., an immunotherapy). These methods are generally understood to be, consist of, or consist essentially of: detecting the presence of a complex comprising SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP) in a sample of the subject (e.g., a sample comprising cells expressing SETDB1, such as tumor cells or a lysate thereof), or a level of the complex that indicates an abnormal or abnormal level of the complex and is associated with an increased likelihood of developing resistance to the therapy, thereby determining that the subject has an increased likelihood of developing resistance to the therapy.

[0064] In some embodiments, the method comprises detecting higher levels of the complex in the sample relative to a control (e.g., a reference sample comprising cells expressing SETDB1 corresponding to a normal control, or a lysate thereof), to thereby determine that the subject has an increased likelihood of developing resistance to the therapy.

[0065] In other embodiments, the method comprises detecting approximately the same level of the complex relative to a control (e.g., a reference sample comprising corresponding SETDB1 expressing cells or lysates thereof having abnormal levels of nuclear SETDB1) to thereby determine that the subject has an increased likelihood of developing resistance to the therapy.

[0066] Yet another aspect of the present disclosure provides methods for determining the likelihood of a subject developing sensitivity to a therapy (e.g., an immunotherapy). These methods generally consist of or consist essentially of detecting the absence of colocalization of SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP) in a sample of the subject (e.g., a sample containing cells expressing SETDB1, such as tumor cells or a lysate thereof), or a level of colocalization that indicates a normal level of colocalization and is associated with an increased likelihood of developing sensitivity to the therapy, thereby determining that the subject has an increased likelihood of developing sensitivity to the therapy. In some embodiments, the method comprises detecting approximately the same level of colocalization in the sample of the subject (e.g., a sample containing tumor cells or a lysate thereof) relative to a control (e.g., a reference sample containing cells expressing SETDB1 corresponding to a normal control or a lysate thereof), thereby determining that the subject has an increased likelihood of developing sensitivity to the therapy. In other embodiments, the method comprises detecting a lower level of colocalization relative to a control (e.g., a reference sample containing cells expressing SETDB1 corresponding to a control or a lysate thereof having an abnormal level of nuclear SETDB1).

[0067] In a related aspect, the present disclosure provides methods for determining the likelihood of a subject developing sensitivity to a therapy (e.g., an immunotherapy). These methods generally consist of or consist essentially of detecting the absence of a complex comprising SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP) in a sample of the subject (e.g., a sample comprising cells expressing SETDB1, such as tumor cells or a lysate thereof), or a level of the complex that indicates a normal level of the complex and is associated with an increased likelihood of developing sensitivity to the therapy, to thereby determine that the subject has an increased likelihood of developing sensitivity to the therapy.

[0068] In some embodiments, the method comprises detecting approximately the same level of the complex relative to a control (e.g., a reference sample comprising cells expressing SETDB1 or a lysate thereof corresponding to a normal or immunocompetent control) to thereby determine the subject has an increased likelihood of sensitivity to the therapy.

[0069] In other embodiments, the method comprises detecting lower levels of the complex relative to a control (e.g., a reference sample comprising corresponding control SETDB1-expressing cells or lysates thereof having abnormal levels of nuclear SETDB1) to thereby determine that the subject has an increased likelihood of developing sensitivity to the therapy.

[0070] In another aspect, the present disclosure provides methods for analyzing the cellular localization of SETDB1 (e.g., in tumor cells). These methods generally comprise, consist of, or consist essentially of detecting the presence, absence, or level of colocalization of SETDB1 with a nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα) in a cell to thereby determine the localization of SETDB1 in the cell. In some embodiments, the presence of colocalization indicates nuclear localization of SETDB1. In other embodiments, the absence of colocalization indicates cytoplasmic localization of SETDB1. In still other embodiments, the method comprises detecting a normal level of colocalization relative to a control (e.g., corresponding to the level of colocalization in a normal or immunocompetent cell or lysate thereof), which indicates that cytoplasmic localization of SETDB1 is greater than nuclear localization of SETDB1. In still other embodiments, the method comprises detecting a higher level of colocalization relative to a control (e.g., corresponding to the level of colocalization in a normal cell or lysate thereof), which indicates that nuclear localization of SETDB1 is greater than cytoplasmic localization of SETDB1. In representative examples of these embodiments, colocalization is represented by a complex comprising SETDB1 and a nuclear binding partner of SETDB1.

[0071] Still another aspect of the present disclosure provides methods for stratifying subjects into likely responders or non-responders to a therapy (e.g., an immunotherapy). These methods generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1 in a sample of the subject (as described broadly above and elsewhere herein) to determine whether the subject has an increased likelihood of developing sensitivity or resistance to the therapy, to thereby stratify the subject into likely responders or non-responders to the therapy.

[0072] Additional aspects of the present disclosure provide methods for managing treatment of a subject with a therapy (e.g., an immunotherapy). These methods generally comprise, consist of, or consist essentially of: selecting to treat the subject with the therapy based on the subject as a possible responder to the therapy, or selecting not to treat the subject with the therapy based on the subject as a possible non-responder to the therapy, and treating the subject with or not treating the subject with the therapy based on the selection, wherein the selection is based on a stratified approach as described above and elsewhere herein.

[0073] In another aspect of the present disclosure, methods for predicting the treatment outcome of a subject with a therapy (e.g., an immunotherapy) are provided. These methods generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1 in a sample of the subject (as described above and elsewhere herein) to determine whether the subject has an increased likelihood of developing sensitivity or resistance to the therapy, thereby predicting the treatment outcome of the subject. In some embodiments, the method comprises detecting the presence or level of nuclear localized SETDB1 relative to a control, which is associated with an increased likelihood of developing resistance to the therapy (as described above and elsewhere herein), and predicting a negative treatment outcome. Suitably, a negative treatment outcome is a higher severity of disease or a higher progressive disease. In other embodiments, the method comprises detecting the absence or level of nuclear localized SETDB1 relative to a control, which is associated with an increased likelihood of developing sensitivity to the therapy (as described above and elsewhere herein), and predicting a positive treatment outcome. A positive treatment outcome may be selected from a partial or complete response to the therapy and a stable disease. In any of these embodiments, the method suitably further comprises predicting the clinical outcome of the subject based on the predicted treatment outcome. In a non-limiting example of this type, the subject is a cancer subject, and the clinical outcome is selected from tumor response (TR), overall survival (OS), progression-free survival (PFS), disease-free survival, time to tumor recurrence (TTR), time to tumor progression (TTP), relative risk (RR), toxicity, or side effects.

[0074] Additional aspects of the disclosure provide methods for monitoring disease in a subject after treatment with a therapy. These methods generally comprise, consist of, or consist essentially of: obtaining a sample from the subject after treatment with a therapy (e.g., an immunotherapy), wherein the sample comprises cells (e.g., tumor cells) expressing SETDB1; analyzing the sample for cellular localization of SETDB1 (as described extensively above and elsewhere herein), wherein a lower level of nuclear localized SETDB1 relative to a control sample of the subject obtained prior to treatment indicates an increased clinical benefit of the therapy to the subject (e.g., reduced disease severity, delayed disease progression, reduced rate of disease progression, or absence of disease or improved disease), and wherein a similar or higher level of nuclear localized SETDB1 relative to the control sample indicates no or negligible clinical benefit of the therapy to the subject.

[0075] Yet another aspect of the present disclosure provides methods for determining a disease state in a subject. These methods generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1 in a sample of the subject (as described broadly above and elsewhere herein) to thereby determine the disease state of the subject, wherein the presence or level of nuclear localized SETDB1 is associated with an increased likelihood of resistance to therapy (as described broadly above and elsewhere herein), which indicates that the subject has a higher degree of severity of the disease or a more rapid disease progression, and wherein the absence or level of nuclear localized SETDB1 is associated with an increased likelihood of sensitivity to therapy (as described broadly above and elsewhere herein), which indicates that the subject does not have the disease or has a lower degree of severity of the disease or a more rapid disease progression.

[0076] Yet another aspect of the present disclosure provides a kit for detecting the localization of SETDB1 in a cell location (e.g., cytoplasm or nucleus) of a cell, for predicting the likelihood of a cell's response to a therapy (e.g., immunotherapy), for determining the likelihood of a subject's resistance to a therapy (e.g., immunotherapy), for determining the likelihood of a subject's sensitivity to a therapy (e.g., immunotherapy), for stratifying a subject into a possible responder or non-responder to a therapy (e.g., immunotherapy), for managing a subject treated with a therapy (e.g., immunotherapy), for monitoring a subject's disease after treatment with a therapy, for determining the state of a subject's disease, and / or for determining the subject's immune state. These kits generally comprise, consist of, or consist essentially of a first antigen binding molecule that specifically binds to SETDB1. In some embodiments, the kit comprises a second antigen binding molecule that specifically binds to a nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα). In some embodiments, the kit further comprises a third antigen binding molecule that binds to the first and second antigen binding molecules, the third antigen binding molecule suitably comprising a detectable label.

[0077] Suitably, the kit further comprises instructional material for carrying out any one or more of the methods broadly described above and / or elsewhere herein.

[0078] Still another aspect of the present disclosure provides a complex comprising SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα), a first antigen binding molecule specifically bound to the SETDB1 of the complex, and a second antigen binding molecule bound to the nuclear binding partner of the complex. In some embodiments, the complex is localized in a cell or a lysate thereof. Suitably, the complex further comprises a third antigen binding molecule bound to each of the first antigen binding molecule and the second antigen binding molecule of the complex, the third antigen binding molecule being suitably detectably labeled.

[0079] In a further aspect, the disclosure provides a cell or a lysate thereof comprising the complex as broadly described above and elsewhere herein.

[0080] In certain embodiments of any of the above aspects, the therapy comprises an immunotherapy (e.g., an immune checkpoint inhibitor, such as an antagonist antigen binding molecule that specifically binds to an immune checkpoint molecule). In an illustrative example of this type, the immunotherapy comprises an antagonist antigen binding molecule that specifically binds to PD-1. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1. Interaction SETDB1 / IMPA1 and SETDB1 / ATF7IP:MSETC dose response. (A) Example areas imaged are depicted with a 10 μM scale bar in orange. Cells were permeabilized by incubation with 0.5% Triton X-100 for 20 minutes, probed with mouse anti-ATF7IP, rabbit anti-SETDB1, and goat anti-IMPα1, and visualized with donkey anti-rabbit AF 568, anti-mouse 488, anti-goat 647. Coverslips were mounted on glass microscope slides with ProLong Clear antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. (BE) Bar graphs show the nuclear or cytoplasmic fluorescence intensity of SETDB1 in the context of SEM, and the ratio of nuclear to cytoplasmic staining of SETDB1 (Fn / c). The figure shows the cytoplasmic PCC of SETDB1 with ATF7IP (B) and SETDB1 with IMPα1 (C). The graph shows the nuclear fluorescence intensity of SETDB1 (D) and ATF7IP (E).

[0082] Figure 2 Effect of SETDB1 prototype peptide on MDA-MB-231 cell proliferation. MDA-MB-231 cells were treated with 047 and 047-1 for up to 72 hours. At the time points of 48 (A) and 72 hours (B), the culture medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. The absorbance was recorded at 450nm at 1 hour using a microplate spectrophotometer. RPMI-7951 melanoma cells were treated with 047 and 047-1 (C, D) for up to 72 hours. At two time points of 48 and 72 hours, the culture medium was removed and replaced with 100 μl / well of WST-1 cell proliferation reagent. The absorbance was recorded at 450nm at 1 hour using a microplate spectrophotometer.

[0083] Figure 3 Effect of SETDB1 prototype peptides on CSV, SNAIL and SETDB1 protein expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with vehicle alone or two different concentrations of peptides. Cells were stained with a panel targeting SETDB1, CSV and SNAIL, imaged (A) and quantified (B). Example images are depicted with a 10 μM scale bar. (047=PEP1, 047-1=PEP2).

[0084] Figure 4.Effects of SETDB1 NLS linear peptides and bicyclic peptides on proliferation of cancer cell lines. (A) Structural characterization of the IMPα1 (grey); SETDB1 (green); ATF7IP (blue) trimeric complex. (B) Electrophoretic mobility shift assay shows the interaction between IMPα1 and MSETC. (C) Table depicting epigenetically induced transcriptional pathways in mesenchymal breast cancer cell lines. (DE) MDA-MB-231 and MDA-MB-231 brain cells were treated with 047-1 or MSETC bicyclic peptides for 72 hours. After incubation, the culture medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. The absorbance was recorded at 450 nm at 1 hour using a microplate spectrophotometer.

[0085] Figure 5 .(A, B) MSETC, TBAB-MSETC inhibition of immunotherapy response system TNBC cell line MDA-MB-231 (A) and CT26 (B) proliferation assay. The cells were treated with MSETC or MSETC-TBAB bicyclic peptide for 72 hours. After 72 hours, the culture medium was removed and replaced with 100 μL / cell of WST-1 cell proliferation reagent. The absorbance was recorded at 450nm at 1 hour using a microplate spectrophotometer. (C)-(F) Lung cancer cell line LLC proliferation assay using TBAB-MSETC (C), MSETC (D), MSETC-D (E) and MSETD-DR (F) inhibitors. After 72 hours, the culture medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. The absorbance was recorded at 450nm at 1 hour using a microplate spectrophotometer.

[0086] Figure 6 . Bicyclic peptide treatment did not affect cell viability in healthy PBMC samples. TM Healthy PBMCs were treated with various concentrations of MSETC (2.5-40 μM) overnight prior to staining with the Fixable Aqua Dead Cell Stain Kit (405 nm excitation). All samples were acquired on an LSR Fortessa cytometer and data were analyzed using FlowJo v10 software.

[0087] Figure 7.MDA-MB-231 cells were treated with 10mM MSETC. (A) Nuclear fluorescence intensity (NFI) of markers C-REL, G9A, PKCb1 and custom antibody PDL1-PTM1. MDA-MB-231 cells were treated with MSETC or controls and permeabilized before detection with antibodies specific for C-REL, G9A, PKCb1 and custom antibody PDL1-PTM1. Coverslips were mounted on glass microscope slides with ProLongnucblue glass anti-fading reagent (Life Technologies). Digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA), and the graphs represent the average nuclear fluorescence intensity in the nuclear or cytoplasmic compartments, where significant differences were calculated based on single factor Anova paired comparisons. (B) H1299 cells were treated with MSETC, the universal importin α1 inhibitor BIMAX, or the SETDB1 catalytic inhibitor MTH, or vehicle and stained for off-target effects by high-resolution imaging of DUOLINK cells stained with PKCθ and IMPα1; LSD1 and IMPα1; G9A and IMPα1; and ACE2 and IMPα1. H1299 cells were probed with the DUOLINK ligation assay.

[0088] Figure 8 SETDB1 is cytoplasmic and lowly expressed in healthy PBMCs. Healthy donor PBMCs were isolated from liquid biopsies and stained for SETDB1. Example regions imaged for PBMCs are depicted with a 10 μM scale bar in orange. Cells were permeabilized with 0.5% Triton X-100 for 20 min and probed with rabbit anti-SETDB1 and visualized with anti-rabbit 568. Coverslips were mounted on glass microscope slides with ProLong Clear antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology.

[0089] Fig. 9 SETDB1 is cytoplasmic and lowly expressed in healthy donor tonsil tissue. Healthy tonsil tissue was stained using the BondRX automated staining platform. Example areas of tissue imaging are depicted with a 10 μM scale bar in orange. Tissue sections were probed with rabbit anti-SETDB1 and visualized with anti-rabbit 568. Coverslips were mounted on glass microscope slides with ProLong Clear antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology.

[0090] Fig.10.SETDB1, as a novel epigenetic oncogene, is enriched in NSCLC and other advanced cancers. (A) Proportion of TCGA cases with high or low-level amplification (amp) of SETDB1, BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and endocervical adenocarcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; GBM, glioblastoma multiforme; HNSC, head and neck squamous cell carcinoma; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LGG, brain low-grade glioma; LIHC, hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; SARC, sarcoma; STAD, gastric adenocarcinoma; SKCM, cutaneous melanoma of the skin; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma. (B) Comparison of SETB1 levels in 492 lung adenocarcinoma and 485 lung squamous cell carcinoma tissues with those in normal tissues (n=59 and 50, respectively) http: / / gepia.cancer-pku.cn / ). (C) Kaplan-Meier survival analysis of SETDB1 expression in NSCLC patients with low and high tumor expression of SETDB1, calculated from ( http: / / kmplot.com / analysis / ), where significant differences are indicated.

[0091] Fig.11 .SETDB1 signature of CTCs in a metastatic patient cohort undergoing immunotherapy. Liquid biopsies were stratified into two groups, "resistant" (A) and "responders" (B), based on the response to immunotherapy. CTCs were permeabilized by incubation with 0.5% Triton X-100 for 20 minutes and then probed with rabbit anti-SETDB1, mouse anti-CSV, and visualized with donkey anti-rabbit AF 568, anti-mouse 488 secondary antibodies. Coverslips were mounted on glass microscope slides with ProLong nucblue clear antifade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5 μm sections were obtained using an ASI digital pathology system, running ASI software, using a 100x oil immersion lens. The final image was obtained by averaging four consecutive images of the same slice. (C) Digital images were analyzed using both ASI software for Population Dynamics and ImageJ software (ImageJ, NIH, Bethesda, MD, USA) to determine the ratio of nuclear to cytoplasmic staining (Fn / C), total cell counts, and fluorescence intensity (protein expression).

[0092] Fig.12.Nuclear SETDB1 is enriched in immunotherapy-resistant patients and is not expressed in healthy cell nuclei. (A) PIE is depicted; total CTC or CTC population CSV+SETDB1+CD45-% per 10mL in metastatic cancer liquid biopsy. (B) Representative digital pathology high-resolution immunofluorescence images of SETDB1 expression in immunotherapy-resistant and responding stage IV metastatic FFPE. (C) The figure depicts nuclear staining (NFI) of SETDB1, PCC (co-localization) of SETDB1, ATF7IP and IMPα1. (D) Representative digital pathology high-resolution immunofluorescence images of SETDB1, CSV in patient liquid biopsies. Fn / c, NFI and CFI of SETDB1 in IO-resistant and responder stage IV and healthy donor (HD) liquid biopsies are depicted. (E) Expression of the terminal failure marker EOMES in responders or resistant patients with SETDB1 intensity AC Graphical analysis of positive CD8+PD1+ T cells. Rsp: responsive, Rsi: resistant.

[0093] Fig.13 . SETDB1 nuclear expression is increased in samples from the "resistant" patient cohort. (A) FFPE samples were processed on BONDRX with the Opal staining kit targeting melanoma cancer markers [HMB45+M2-7C10+M2-9E3] (ML) and SETDB1. Green is melanoma cancer markers [HMB45+M2-7C10+M2-9E3], and magenta is SETDB1. (BD) Bar graphs are depicted showing the ratio of nuclear staining to cytoplasmic staining of SETDB1 in the case of SEM, integrated nuclear staining intensity, or integrated cytoplasmic intensity. Significant differences are indicated by Kruskal-Wallis non-parametric test calculations.

[0094] Fig.14. Interaction SETDB1 / IMPα1:MSETC dose response. Example regions imaged by MDA-MB-231 cells are depicted with an orange 10 μM scale bar. Cells were treated with SETDB1 bicyclic inhibitor MSETC or vehicle control at concentrations of 1.25 mM to 20 mM and permeabilized by incubation with 0.5% Triton X-100 for 20 minutes, probed with rabbit anti-SETDB1 and mouse anti-IMPα1, and visualized with donkey anti-rabbit AF 568, anti-mouse 488. Coverslips were mounted on glass microscope slides with ProLong Clear anti-fading reagent (Life Technologies). Protein targets were located by ASI digital pathology. (B) Bar graphs show nuclear or cytoplasmic fluorescence intensity of SETDB1 in the case of SEM, and the ratio of nuclear staining to cytoplasmic staining of SETDB1 (Fn / c). (C) Figures show nuclear or cytoplasmic PCC of SETDB1 / IMPα1. Significant differences were indicated by Kruskal-Wallis nonparametric test calculations. The red dashed line indicates 50% inhibition.

[0095] Fig.15 .MDA-MB-231 cells were treated with MSETC; wherein the concentration ranged from 5 μM to 0.078 μM. This figure depicts high-resolution imaging of DUOLINK cells stained with SETDB1 and IMPα1. MDA-MB-231 cells were treated with MSETC or controls, permeabilized, and probed with DUOLINK ligation assays. Coverslips were mounted on glass microscope slides with ProLong nucblue glass antifade reagent (Life Technologies). MSETC and IMPα1 DUOLINK digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, Maryland, USA), and the graphs represent mean point fluorescence intensity, with significant differences calculated according to Kruskal-Wallis one-way ANOVA. IC50 is marked with a red dotted line.

[0096] Fig.16.MSETC dual-targeting inhibitors are selective for SETDB1 and are superior to catalytic inhibitors. (A) MDA-MB-231 cells were treated with 5mM MSETC, catalytic SETDB1 inhibitor mithramycin A (MTH) or vehicle control and permeabilized by incubation with 0.5% Triton X-100 for 20 minutes, probed with rabbit anti-H3k27c and mouse anti-H3k9me3, and visualized with donkey anti-rabbit AF 568 and anti-mouse 488. Coverslips were mounted on glass microscope slides with ProLong Clear anti-fading reagent (Life Technologies). Protein targets were located by ASI digital pathology. Bar graphs show nuclear fluorescence intensity of H3k9me3 and H3k27a, with significant differences indicated by Kruskal-Wallis nonparametric test calculations. (B) Cells were treated with MSETC or vehicle. Cells were permeabilized and stained for H3.3Ser1p and H3k4me2, and expression was analyzed using digital pathology. Mann-Whitney was used to determine significance.

[0097] Fig.17 (A) H1299 (lung cancer cells) or MDA-MB-231 cells were treated with MSETC (MSETC or MSETC-D-isomeric version (MSETC-D) or MSETC-D-isomeric-retro-inverso (MSETC-DR), the general IMPα1 inhibitor BIMAX or the SETDB1 catalytic inhibitor MTH. Data are from high-resolution imaging of DUOLINK cells stained with SETDB1 a IMPα1. MDA-MB-231 cells or H1299 cells were probed with the DUOLINK ligation assay. Coverslips were mounted on glass microscope slides with ProLong nucblue glass antifade reagent (Life Technologies). MSETC and IMPα1 DUOLINK digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA) and graphs represent mean spot fluorescence intensities with significant differences calculated according to Kruskal-Wallis one-way ANOVA.

[0098] Fig.18(A) H1299 cells (human non-small cell lung cancer cell line) were treated with 5 mM MSETC, the catalytic SETDB1 inhibitor mithramycin A (MTH) or vehicle control and permeabilized by incubation with 0.5% Triton X-100 for 20 minutes, probed with rabbit anti-SETDB1 and mouse anti-CSV, and visualized with donkey anti-rabbit AF 568, anti-mouse 488. Coverslips were mounted on glass microscope slides with ProLongClear antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. Bar graphs show the nuclear intensity of H3k9me3 and H3k27a, with significant differences indicated calculated by Kruskal-Wallis nonparametric test. (B) H1299 cells were treated with 5 mM MSETC or vehicle control and permeabilized by incubation with 0.5% Triton X-100 for 20 minutes, probed with rabbit anti-SETDB1 and mouse anti-ATF7IP or goat anti-IMPα1, and visualized with donkey anti-rabbit AF 568, anti-mouse 488, and anti-goat 647. Coverslips were mounted on glass microscope slides with ProLong Clear antifading reagent (Life Technologies). Protein targets were located by ASI digital pathology. (C) Bar graphs show nuclear intensity or PCC for antibody pairs, with significant differences calculated by Kruskal-Wallis nonparametric test.

[0099] Fig.19 Expression of interferon signaling genes is increased in MCF-7 cells following treatment with bicyclic peptide. Real-time qPCR analysis of MCF-7 cells treated with bicyclic peptide (5 μM) for 24 h prior to stimulation with phorbol 12-myristate 13-acetate (PMA; 20 ng / mL; Sigma) or poly(I:C) (500 ng / mL; Sigma) for 48 h. Cts were converted to arbitrary copy numbers and normalized to the geometric mean of the housekeeping genes ACTB and PPIA. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, unpaired t-test.

[0100] Fig. 20Increased expression of virus mimics and immunogenicity / immunovisibility genes in epithelial and mesenchymal MCF-7 cells following treatment with bicyclotide. Real-time qPCR analysis of MCF-7 cells treated with bicyclotide (5 μM) for 24 h prior to stimulation with phorbol 12-myristate 13-acetate (PMA; 20 ng / mL; Sigma) for 48 h to induce mesenchymal features. Cts were converted to arbitrary copy numbers and normalized to the geometric mean of the housekeeping genes ACTB and PPIA. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, unpaired t-test.

[0101] Fig.21 . Bicyclic peptide (MSETC) + / - αPD1 therapy in the 4T1 model of metastatic breast cancer. (A) Treatment regimen using the Balb / c 4T1 breast cancer model. (B) Mouse body weight (g) during the 20-day treatment period. (C) Tumor volume of mice treated with vehicle (saline) or MSETC (20 mg / kg) in combination with αPD1 or isotype control (10 mg / kg) (n=4-5 / group). (D) Tumor volume of individual mice on day 20 post-inoculation (data also presented in C). * p<0.05, ** p<0.01, Tukey's post test). (E) Representative images of tumors harvested on day 20 after inoculation.

[0102] Fig. 22 MSETC + / - αPD1 therapy does not alter lung, liver, or spleen weights in the 4T1 model of metastatic breast cancer. (A) Representative images of organs harvested on day 20 post-inoculation. (B) Final lung, liver, and spleen weights on day 20 post-inoculation. * p<0.05, Tukey post hoc test.

[0103] Fig.23 MSETC and αPD1 combination therapy reduces lung metastasis in the 4T1 model of metastatic breast cancer. (A) Representative images of lungs fixed in Bouin's solution. (B) Lung nodule counts in mice treated with vehicle (saline) or MSETC (20 mg / kg) in combination with αPD1 or isotype control (10 mg / kg) on ​​day 20 post-inoculation (n=4-5 / group). * p<0.05, one-way ANOVA.

[0104] Fig.24MSETC and αPD1 combination therapy reduces primary tumor CSV expression in metastatic breast cancer. (A) Representative images of FFPE primary tumor samples treated with Opal staining kits targeting cytokeratin, CSV, or SETB1 on BONDRX. (B) Quantification of CSV, cytokeratin, or SETDB1 fluorescence intensity (FI). (CD) Quantification of cytokeratin (CYT), CSV, and nuclear SETDB1 (nSETDB1) positive cells. Significant differences are indicated by Kruskal-Wallis nonparametric test calculations.

[0105] Fig.25 MSETC and αPD1 combination therapy reduces lung CSV expression in metastatic breast cancer. (A) Representative images of FFPE lung samples treated with the Opal staining kit targeting cytokeratin, CSV, or SETB1 on BONDRX. (B) Quantification of CSV, cytokeratin, or SETDB1 fluorescence intensity (FI). (C-D) Quantification of cytokeratin (CYT), CSV, and nuclear SETDB1 (nSETDB1) positive cells. Significant differences are indicated by Kruskal-Wallis nonparametric test calculation.

[0106] Fig.26 MSETC and αPD1 combination therapy increases primary tumor T in metastatic breast cancer RM , T EM , T CM And T effector cell populations. (A) Representative images of FFPE primary tumor samples treated with Opal staining kits targeting CD44, CD103, CD69, CD62L or CD8 on BONDRX. Stained sections were analyzed for tissue-resident memory (B), effector memory (C), central memory (D) or effector (E) T cell populations. Significant differences were indicated by Kruskal-Wallis nonparametric test calculations. 4-5 tissue sections were analyzed per group, with >500 cells counted per section.

[0107] Fig. 27 .MSETC reprograms the histone code in the 4T1 tumor model. (A) Primary tumor samples of the 4T1 tumor model were processed as depicted using the Opal staining kit targeting CSV (tumor marker), H3k27c, and H3k9me3 on BONDRX. (B) The graph depicts the nuclear fluorescence intensity (NFI) of H3k27c and H3k9me3. Significant differences are indicated by Kruskal-Wallis nonparametric test calculations.

[0108] Fig.28.C-DR (5 mg / kg) combined with anti-PD1 can reduce tumor burden in the 4T1 syngeneic tumor model relative to MSETC (15 mg / kg). (A) Treatment regimen using the Balb / c 4T1 breast cancer model. (B) Mouse body weight (g) before picking on day 9. Data are expressed as mean ± SEM. (C) Tumor volume of mice treated with MSETC-DR (5 mg / kg) combined with αPD1 (10 mg / kg) relative to MSETC (15 mg / kg) combined with it (n=5 / group). Data are expressed as fold change relative to day 0. a; ** p<0.01MSETC and MSETC-DR relative to αPD1, b; ** p<0.01MSETC and MSETC-DR relative to αPD1, c: ** p<0.01 and * p<0.05MSETC and MSETC-DR relative to αPD1, d; ** p<0.01 MSETC and MSETC-DR vs. αPD1, two-way ANOVA, Dunnett's post test. (D) Representative images of tumors collected on day 9. (E) Weights of liver, spleen, and lung on day 9 (mg).

[0109] Fig.29 Effects of MSETC and MSETC-DR on cell migration in the MCF-7PMA / TGFβ-inducible model. (A) Wound healing (scratch assay) analysis of the effects of MSETC and MSETC-DR on cell migration over 24 hours. Black line: water control, blue line: MSETC (20 μM), orange line: MSETC-DR (20 μM). (B) Graphical representation of relative wound density (%) at the 24 hour time point as indicated in (A). ** p<0.01, **** p<0.0001, one-way ANOVA, Dunnett's post hoc test (n=6 / group). DETAILED DESCRIPTION

[0110] 1. Definition

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar to or equivalent to the methods and materials described herein may be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0112] The articles "a" and "an" are used herein to refer to one or to more than one (ie, to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0113] As used herein, the term "about" refers to the common error range of the corresponding value that is easily known to those skilled in the art. "About" values ​​or parameters mentioned herein include (and describe) embodiments involving the value or parameter itself.

[0114] The term "antagonist" or "inhibitor" refers to a substance that prevents, blocks, inhibits, neutralizes or reduces the biological activity or effect of another molecule (such as an enzyme or receptor). The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of the target. In some embodiments, the term can refer to an antibody that prevents the target (e.g., PD-1) to which it binds from exerting a biological function.

[0115] The term "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 the antibodies exhibit the desired biological activity.

[0116] The "amount" or "level" of a biomarker is the detectable level in a sample. These can be measured by methods known to those skilled in the art and may also be disclosed herein. The expression level or amount of the assessed biomarker can be used to determine the response to treatment.

[0117] The terms "administration concurrently" or "administering concurrently" or "co-administering" and the like refer to the administration of a single composition containing two or more active substances, or the administration of each active substance as a separate composition and / or delivery by separate routes concurrently or simultaneously or sequentially within a sufficiently short period of time so that the effective result is equivalent to the result obtained when all of these active substances are administered as a single composition. "Concurrently" means that the active agents are administered at substantially the same time, and are desirably administered together in the same formulation. "Contemporaneous" means that the active agents are administered closely in time, for example, one agent is administered within about one minute to about one day before or after another agent. Any contemporaneous time is useful. However, typically, when administered at different times, the agents will be administered within about one minute to about eight hours, and appropriately within less than about one to about four hours. When administered concurrently, they are appropriately administered at the same site of the subject. The term "same site" includes the exact location, but may be within about 0.5 to about 15 centimeters, preferably within about 0.5 to about 5 centimeters. As used herein, the term "separately" means administration at certain intervals, for example, at intervals of about one day to several weeks or months. The active agents can be administered in any order. As used herein, the term "sequentially" means administration in sequence, for example, at intervals of minutes, hours, days or weeks. If appropriate, the active agents can be administered in regular repeating cycles.

[0118] The term "agent" includes compounds that induce a desired pharmacological and / or physiological effect. The term also encompasses pharmaceutically acceptable and pharmacologically active components of these compounds specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, etc. When the above terms are used, it should be understood that this includes the active agent itself as well as pharmaceutically acceptable pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "agent" should not be interpreted narrowly, but extends to small molecules, SETDB1 bicyclic peptide mimetics, such as peptides, polypeptides and proteins and compositions comprising the same, and genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof and cellular agents.

[0119] As used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative (or).

[0120] As used herein, the term "binds", "binds specifically to" or "has specificity for..." refers to a measurable and reproducible interaction of binding between a target and an antibody, such as determining the presence or absence of a target in the presence of a heterogeneous population of molecules including biomolecules. For example, an antibody that binds or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with greater affinity, activity, greater ease and / or longer duration than it binds to other targets. In one embodiment, the degree to which an antibody binds to an unrelated target is less than about 10% of the degree to which the antibody binds to the target, such as measured by radioimmunoassay (RIA). In certain embodiments, the dissociation constant (Kd) of an antibody that specifically binds to a target is <1 mM, <100 nM, <10 nM, <1 nM or <0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In another embodiment, specific binding may include but does not require exclusive binding.

[0121] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, such as a predictive, diagnostic and / or prognostic indicator.

[0122] The terms "cancer" and "carcinoma" refer to or describe the physiological condition in a subject characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include, but are not limited to, squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma; peritoneal cancer; hepatocellular carcinoma; stomach cancer or gastric cancer, including gastrointestinal cancer and gastrointestinal stromal cancer; pancreatic cancer; glioblastoma; cervical cancer; ovarian cancer; liver cancer; bladder cancer; urinary tract cancer; liver cancer; breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer or uterine cancer; salivary gland cancer; renal cancer or kidney cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma; superficial spreading melanoma; lentigo maligna melanoma; acral lentigo melanoma; nodular melanoma; multiple myeloma; and B-cell lymphoma (including low-grade / follicular non-Hodgkin's lymphoma (non-Hodgkin's The following are NHLs: small lymphocytic (SL) NHL, intermediate / follicular NHL; intermediate diffuse NHL; high-grade immunoblastic NHL; high-grade lymphocytic NHL; high-grade small non-cleaved cell NHL; bulky lesion NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia; chronic lymphoblastic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD); and abnormal vascular proliferation associated with nevus hamartomatosis; edema (such as that associated with brain tumors); Meigs' syndrome; brain cancer; and head and neck cancer and related metastases. In certain embodiments, cancers that can be treated 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 carcinoma, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid, head and neck cancer, ovarian cancer, mesothelioma and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastoma, melanoma, breast cancer, gastric cancer, colorectal cancer (CRC) and hepatocellular carcinoma. However, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma and breast cancer, including metastatic forms of those cancers. In specific embodiments, the cancer is melanoma or lung cancer, suitable metastatic melanoma or metastatic lung cancer.

[0123] "Chemotherapeutic agents" include compounds that can be used to treat cancer. Examples of chemotherapeutic agents include erlotinib, Genentech / OSI Pharmaceuticals), bortezomib, Millennium Pharm., disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant ( AstraZeneca), sunitinib, Pfizer / Sugen), letrozole, Novartis), imatinib mesylate, Novartis), finasunate, Novartis), oxaliplatin ( Sanofi), 5-FU (5-fluorouracil), folinic acid, rapamycin (Rapamycin, Sirolimus, Wyeth), Lapatinib, GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), sorafenib (Sorafenib, Bayer Labs), gefitinib, AstraZeneca), AG1478, alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethyleneamines and methylamelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; callisthenes; ystatin); CC-1065 (including its synthetic analogs adozelesin, carzelesin and bizelesin); candida albicans (specifically candida albicans 1 and candida albicans 8); adrenocortical hormones (including prednisone and prednisolone); cyproterone acetate; 5a-reductase (including finasteride and dutasteride); vorinostat, romidepsin romidepsin, panobinostat, valproic acid, mocetinostat, dolastatin; aldesleukin, talc, duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards (eg, chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin gll and calicheamicin wll (Agnew, Chem Inti. Ed. Engl. 1994 33:183-186); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicins; and neocarzinostatin chromophores. chromophore) and related pigment proteins enediyne antibiotic chromophores), aclacinomysin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs, such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calusterone, dromostanolone propionate, propionate, epitiostanol, mepitiostane, testolactone; antiadreners such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone;aldophosphamideglycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A, A) and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), (Cryoethylene-free castor oil), albumin-engineered paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, III.), and (docetaxel, docetaxel; Sanofi-Aventis); chloranbucil; (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine, ); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids or derivatives of any of the above.

[0124] Chemotherapeutic agents also include (i) antihormonal agents used to modulate or inhibit the effects of hormones on cancer, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (comprising tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and (toremifene citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates the production of estrogen in the adrenal glands, for example, 4(5)-imidazole, aminoglutethimide, (megestrol acetate), (exemestane; Pfizer), formestanie, fadrozole, (vorozole), (letrozole; Novartis) and (anastrozole; AstraZeneca); (iii) antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans retinoic acid, fenretinide, and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, particularly those that inhibit the expression of genes in signal transduction pathways involved in abnormal cell proliferation, for example, PKC-a, Ralf, and H-Ras; (vii) ribozymes, such as inhibitors of VEGF expression (e.g., ) and HER2 expression inhibitors; (viii) vaccines, such as gene therapy vaccines, for example, and rIL-2; Topoisomerase 1 inhibitors such as rmRH; and (ix) a pharmaceutically acceptable salt, acid or derivative of any of the above.

[0125] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab ( Genentech); cetuximab ( Imclone); panitumumab, Amgen), rituximab, Genentech / Biogen Idec), pertuzumab, 2C4, Genentech), trastuzumab ( Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate gemtuzumab ozogamicin ( Wyeth). Additional humanized monoclonal antibodies with therapeutic potential include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumabmertansine, 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, and paxillin. ascolizumab, peefusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab

[00136] The invention relates to anti-IL-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a recombinant, fully human, full-length IgG11 antibody genetically modified to recognize the interleukin-12p40 protein.

[0126] Chemotherapeutic agents also include "EGFR inhibitors," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, and may alternatively be referred to as "EGFR antagonists." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB8506), 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 al.), and variants thereof, such as chimeric 225 (C225 or cetuximab; ) and remodeled human 225 (H225) (see WO 96 / 40210, Imclone Systems Inc.); IMC-11F8, a fully human EGFR targeting antibody (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized antibodies and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD 55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with EGF and TGF-a for EGFR binding (EMD / Merck); human EGFR antibody, HuMax-EGFR (GenMab); fully human antibodies, referred to as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3 and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated to cytotoxic agents, thereby generating immunoconjugates (see, e.g., EP659439A2, Merck Patent GmbH). EGFR antagonists include small molecules, such as compounds described in the following U.S. Patents: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, No. 6,140,332, No. 5,866,572, No. 6,399,602, No. 6,344,459, No. 6,602,863, No. 6,391,874, No. 6,344,455, No. 5,760,041, No. 6,002,008 and No. 5,747,498 and the following PCT applications: WO98 / 14451, WO98 / 50038, WO99 / 09016 and WO99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib,. Genentech / OSI Pharmaceuticals); PD 183805 (Cl 1033, 2-acrylamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer); ZD1839, gefitinib 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim Ingelheim); PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); EGFR / HER2 dual tyrosine kinase inhibitors, such as lapatinib ( GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furyl]-4-quinazolinamine).

[0127] Chemotherapeutic agents also include "tyrosine kinase inhibitors", including the EGFR targeted drugs described in the preceding paragraphs; small molecule HER2 tyrosine kinase inhibitors, such as TAK165 available from Takeda; CP-724,714, oral selective inhibitors of ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual HER inhibitors, such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits cells that overexpress both HER2 and EGFR; lapatinib (GSK572016; available from GlaxoSmithKline), 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 phenobarbital (available from ISIS Pharmaceuticals); Pharmaceuticals), which inhibits Raf-1 signaling; non-HER-targeted TK inhibitors, such as imatinib mesylate ( available from GlaxoSmithKline); multitargeted tyrosine kinase inhibitors, such as sunitinib ( available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, available from Novartis / Schering AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines such as PD 153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines such as CGP59326, CGP 60261 and CGP 62706; pyrazolopyrimidine, 4-(anilino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (dibenzoylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrosine containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., molecules that bind to a nucleic acid encoding a HER); quinoxaline (U.S. Pat. No. 5,804,396); tyrosine phosphorylation inhibitors (U.S. Pat. No. 5,804,396); ZD6474 (AstraZeneca); PTK-787 (Novartis AG / Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); affinitak (ISIS 3521; Isis / Lilly); imatinib mesylate PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone); rapamycin (sirolimus, ); or as described in any of the following patent publications: U.S. Patent No. 5,804,396; WO1999 / 09016 (American Cyanamid); WO1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner-Lambert); WO1999 / 06378 (Warner-Lambert); WO1999 / 06396 (Warner-Lambert); WO1996 / 30347 (Pfizer); WO1996 / 33978 (Zenica); WO1996 / 3397 (Zenica) and WO1996 / 33980 (Zenica).

[0128] Chemotherapeutic agents also include dexamethasone, interferon, colchicine, chlorpheniramine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifotin, arsenic trioxide, asparaginase, live BCG, bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erlotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, Lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nilarabine, nofizumab, oprelekin, palifermin, pamidronate disodium, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, valrubicin, zoledronate and zoledronic acid, and pharmaceutically acceptable salts thereof.

[0129] Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone, mometasone, amcinonide, budesonide, desonide, fluocinolone, fluocinolone acetate, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, alclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicasone, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone hexanoate, fluocortolone pivalate and fluprednisolone acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN Biotherapeutics Ltd. (IMULAN BioTherapeutics, LLC); antirheumatic drugs, such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, sulfasalazine, tumor necrosis factor alpha (TNF-a) blockers, such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) blockers, such as anakinra (Kineret), T cell costimulation blockers, such as abatacept (Orencia), interleukin 6 (IL-6) blockers, such as tocilizumab Interleukin-13 (IL-13) blockers, such as lebrikizumab; interferon-alpha (IFN) blockers, such as rontalizumab; β7 integrin blockers, such as rhuMAbβ7; IgE pathway blockers, such as anti-Mi prime; secreted homotrimer LTa3 and membrane-bound heterotrimer LTal / 82 blockers, such as anti-lymphotoxin-alpha (LTa); radioisotopes (e.g., At 211 ,I 131 ,I 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212and radioisotopes of Lu); other investigational agents such as orthoplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, proanthocyanidins, betulinic acid, and their derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, ); β-lapachone; lapachone; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin); podophyllotoxin; tegafur Bexarotene Bisphosphonates, such as clodronate (e.g. or ), etidronate sodium NE-58095, Zoledronic Acid / Zoledronate Alendronate Pamidronate Tirucuronic acid Risedronate and epidermal growth factor receptor (EGF-R); vaccines, Vaccines; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteasome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as olimusin pyranthrone; farnesyl transferase inhibitors, such as lonafarnib (SCH 6636, SARASAR TM ); and a pharmaceutically acceptable salt, acid or derivative of any of the above; and a combination of two or more of the above, such as CHOP, an abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine and prednisolone; and FOLFOX, oxaliplatin (ELOXATIN TM ) in combination with 5-FU and folinic acid.

[0130] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs with analgesic, antipyretic and anti-inflammatory effects. NSAID includes non-selective inhibitors of cyclooxygenase. The specific examples of NSAID 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, lumiracoxib, parecoxib, rofecoxib, rofecoxib and valdecoxib. NSAIDs are used to relieve symptoms of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory joint disease, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain caused by inflammation and tissue damage, fever, intestinal obstruction, and renal colic.

[0131] As used herein, the term "colocalization" or "colocalized" refers to two or more molecules with the same or overlapping localization in a cell. The colocalization of molecules and proteins can be detected using any suitable method known in the art, including, for example, fluorescence microscopy in fixed or living cells. For example, SETDB1 and SETDB1 nuclear binding partners (e.g., ATF7IP, IMPα) can be colocalized in cells using fluorescently labeled anti-SETDB1, anti-nuclear binding partner primary antibodies and optionally one or more secondary antibodies. Methods for cellular molecule colocalization are well known.

[0132] The terms "cell proliferative disorder," "proliferative disorder," and "hyperproliferative disorder" are used interchangeably herein to refer to a disorder associated with some degree of abnormal cell proliferation. In some embodiments, the cell proliferative disorder is cancer. In some embodiments, the cell proliferative disorder is a tumor, including a solid tumor.

[0133] As used herein, "companion diagnostic" refers to a diagnostic method and / or reagent used to identify subjects susceptible to treatment with a particular treatment or to monitor treatment and / or identify an effective dose of a subject or a subgroup or other group of subjects. For purposes herein, a companion diagnostic refers to an agent, such as an agent used to detect or measure SETDB1 cellular localization in a sample (e.g., as described herein). A companion diagnostic refers to an agent and also refers to a test performed with an agent.

[0134] As used herein, the term "complex" refers to an assembly or aggregate of molecules (e.g., peptides, polypeptides, etc.) that are in direct and / or indirect contact with each other. In a specific embodiment, "contact", or more specifically, "direct contact" means that two or more molecules are close enough so that attractive non-covalent interactions (such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, etc.) dominate the interaction of molecules. In such embodiments, molecular complexes (e.g., peptides and polypeptides) are formed under conditions that make the complex thermodynamically favorable (e.g., compared to the non-aggregated or non-complexed state of its component molecules). As used herein, the term "polypeptide complex" or "protein complex" refers to trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers, decamers, elevenmers, twelvemers or higher order oligomers. In a specific embodiment, the polypeptide complex is formed by self-assembly of SETDB1 and the nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα).

[0135] Throughout this specification, unless the context otherwise requires, the term "comprise, comprises and comprising" should be understood to imply the inclusion of the step or element or a group of steps or elements, but not to exclude any other step or element or any other group of steps or elements. Therefore, the use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but other elements are optional and may or may not exist. "Consisting of" means including and limited to any content after the phrase "consisting of". Therefore, the phrase "consisting of" means that the listed elements are required or mandatory, and other elements may not be present. "Substantially consisting of" means including any element listed after the phrase, and is limited to other elements that do not interfere with or promote the activities or behaviors specified for the listed elements in the present disclosure. Therefore, the phrase "substantially consisting of" means that the listed elements are required or mandatory, but other elements are optional and may or may not exist, depending on whether they affect the activities or effects of the listed elements.

[0136] The term "correlate or correlating" refers to determining a relationship between one type of data and another type of data or to a state (e.g., response to a therapy). In some embodiments, "correlate or correlating" means comparing the performance and / or results of a first analysis or protocol to a second analysis or protocol in any manner. For example, the results of a first analysis or protocol can be used when performing a second protocol, and / or the results of a first analysis or protocol can be used to determine whether a second analysis or protocol should be performed. With respect to embodiments of polypeptide analyses or protocols, the results of a polypeptide expression or cellular localization analysis or protocol can be used to determine whether a specific treatment protocol should be performed.

[0137] By "corresponds to" or "corresponding to" is meant an amino acid sequence that exhibits substantial sequence similarity or identity to a reference amino acid sequence. Typically, an amino acid sequence will exhibit 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 a reference amino acid sequence.

[0138] "Derivative" means a molecule, such as a polypeptide, that has been derived from a base molecule by modification, for example by conjugation or complexation with other chemical moieties, or by post-translational modification techniques as will be understood in the art. The term "derivative" also includes within its scope changes made to the parent sequence, including additions or deletions that provide functionally equivalent molecules.

[0139] The term "dosage unit form" as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.

[0140] "Effective amount" is at least the minimum amount required for measurable improvement or prevention of a particular condition. In this article, effective amount can vary according to factors such as disease morbidity, age, sex and body weight of the patient and the ability of the antibody to cause a desired response in an individual. Effective amount is also the amount of any toxic or harmful effect of the treatment that the therapeutic beneficial effect exceeds. For preventive use, the beneficial or desired result includes such as eliminating or reducing risk, alleviating severity or delaying the onset of the disease, including the results such as biochemical, histological and / or behavioral symptoms of the disease, its complications and the intermediate pathological phenotypes that occur during the disease development. For therapeutic use, the beneficial or desired result includes such as reducing one or more symptoms caused by the disease, improving the quality of life of the experimenter with the disease, reducing the dosage of other drugs required for the treatment of the disease, such as enhancing the effect of another drug by targeting, delaying the progress of the disease and / or prolonging the survival period. In the case of cancer or tumor, the effect of an effective amount of the drug can reduce the number of cancer cells; reduce tumor size; inhibit (i.e., slow down and ideally stop to some extent) cancer cell infiltration into peripheral organs; inhibit (i.e., slow down and ideally stop to some extent) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with cancer or tumors to some extent. In the case of infection, the effect of an effective amount of the drug can reduce the titer of pathogens (bacteria, viruses, etc.) in circulation or tissues; reduce the number of cells infected by pathogens; inhibit (i.e., slow down or ideally stop to some extent) pathogen infection of organs; inhibit (i.e., slow down and ideally stop to some extent) pathogen growth; and / or alleviate one or more symptoms associated with infection to some extent. The effective amount can be administered in one or more administrations. For the purposes of the present invention, the effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to directly or indirectly complete a preventive or therapeutic treatment. As understood in the clinical context, the effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administration of one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result can be achieved or accomplished in combination with one or more other agents.

[0141] An "effective response" of a patient or "responsiveness" of a patient to treatment with a drug and similar terms refers to a clinical or therapeutic benefit conferred on a patient at risk of or suffering from a disease or condition such as cancer. In one embodiment, such benefit includes any one or more of the following: prolonging survival (including overall survival and progression-free survival); resulting in an objective response (including complete response or 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 of the following: prolonging survival (including overall survival and progression-free survival); resulting in an objective response (including complete response or partial response); or improving signs or symptoms of cancer.

[0142] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a coding sequence, expression involves transcribing the coding sequence into mRNA and translating the mRNA into one or more polypeptides. In contrast, expression of a non-coding sequence involves only transcribing the non-coding sequence into a transcript. The term "expression" is also used herein to refer to the presence of a protein or molecule at a specific location, and therefore can be used interchangeably with "localization".

[0143] The term "exhaustion" and its grammatical equivalents refer to T cell exhaustion, which is a state of T cell dysfunction caused by continuous TCR signaling that occurs during many chronic infections and cancers. The difference between exhaustion and anergy is that exhaustion is not produced by incomplete or defective signaling, but by continuous signaling. Exhaustion is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state that is different from the state of functional effector or memory T-cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can be caused by both exogenous negative regulatory pathways (e.g., immune regulatory cytokines) and endogenous negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.) of cells.

[0144] The term "expression" with respect to a gene sequence refers to the transcription of the gene to produce an RNA transcript (e.g., mRNA, antisense RNA, siRNA, shRNA, miRNA, etc.), and, where appropriate, the translation of the produced mRNA transcript into a protein, etc. Thus, it is clear from the context that the expression of a coding sequence is produced by the transcription and translation of the coding sequence. In contrast, the expression of a non-coding sequence is produced by the transcription of the non-coding sequence.

[0145] As used herein, the term "higher" with respect to a biomarker or biomarker complex measurement result refers to a statistically significant and measurable difference in the level of a biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or a control level, wherein the measurement result of the biomarker or the biomarker complex is higher than the other biomarker, biomarker complex or control level. The difference is preferably at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% or at least about 50%.

[0146] The term "host cell" includes individual cells or cell cultures that can be or have been recipients of any recombinant vector or isolated polynucleotide of the present invention. Host cells include progeny of a single host cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be identical (morphologically or in terms of total DNA complement) to the original parental cell. Host cells include cells transfected or infected in vivo or in vitro with a recombinant vector or a polynucleotide of the present invention. A host cell comprising a recombinant vector of the present invention is a recombinant host cell.

[0147] "Hybridization" is used herein to refer to the pairing of complementary nucleotide sequences to produce DNA-DNA hybrids or DNA-RNA hybrids. Complementary base sequences refer to those sequences related by base pairing rules. In DNA, A pairs with T, and C pairs with G. In RNA, U pairs with A, and C pairs with G. In this respect, as used herein, the terms "matching" and "mismatching" refer to the hybridization potential of paired nucleotides in complementary nucleic acid chains. Matched nucleotides hybridize efficiently, such as the above-mentioned classical AT and GC base pairs. Mismatching is a combination of other nucleotides that cannot hybridize efficiently. In the present invention, preferred pairing mechanisms relate to hydrogen bonds between complementary nucleosides or nucleotide bases (nucleobases) of oligomeric compound chains, and the hydrogen bonds can be Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bonds. For example, adenine and thymine are complementary nucleobases that are paired by the formation of hydrogen bonds. As known to those skilled in the art, hybridization can occur in different situations.

[0148] The term "immunotherapy" refers to any therapy that intentionally modulates one or more components of the human or animal immune system in order to directly or indirectly achieve certain therapeutic benefits (including systemic and / or local effects, and preventive and / or therapeutic effects). Immunotherapy can involve administering one or more immunotherapeutic agents, either systemically, locally, or both, to a human or animal subject, either alone or in any combination, by any route (e.g., oral, intravenous, cutaneous, injection, inhalation, etc.). Immunotherapy can involve stimulating, increasing, decreasing, stopping, preventing, blocking, or otherwise regulating the production of cytokines, and / or activating or deactivating cytokines or immune cells, and / or regulating the level of immune cells, and / or delivering one or more therapeutic or diagnostic substances to a specific location or specific type of cell or tissue in the body, and / or destroying specific cells or tissues. Immunotherapy can be used to achieve local effects, systemic effects, or a combination of both.

[0149] As used herein, the term "immunotherapeutic agent" refers to any agent, compound or biological agent that indirectly or directly restores, enhances, stimulates or increases the body's immune response to cancer cells and / or reduces the side effects of other anticancer therapies. Therefore, immunotherapy is a therapy that directly or indirectly stimulates or enhances the immune system's response to cancer cells and / or alleviates the side effects that may be caused by other anticancer agents. Immunotherapy is also referred to as immunotherapy, biotherapy, biological response modifier therapy and biotherapy in the art. Examples of common immunotherapeutic agents known in the art include, but are not limited to, cytokines, cancer vaccines, monoclonal antibodies and non-cytokine adjuvants. Alternatively, immunotherapy can be composed of: administering a certain amount of immune cells (T cells, NK cells, cells, DCs, B cells, etc.) to a subject. Immunotherapeutic agents can be nonspecific, i.e., generally enhance the immune system so that the human body becomes more effective in terms of growth and / or diffusion against cancer cells, or the immunotherapeutic agent can be specific, i.e., targeting cancer cells themselves. Immunotherapy regimens can be used in combination with nonspecific and specific immunotherapeutic agents. Nonspecific immunotherapeutic agents are substances that stimulate or indirectly improve the immune system. Nonspecific immunotherapeutic agents have been used alone as the primary therapy for treating cancer, and as a supplement to the primary therapy, in which case, nonspecific immunotherapeutic agents act as adjuvants to enhance the effectiveness of other therapies (e.g., cancer vaccines). Nonspecific immunotherapeutic agents may also play a role in the latter context to reduce the side effects of other therapies, such as bone marrow suppression induced by certain chemotherapeutic agents. Nonspecific immunotherapeutic agents may act on key immune system cells and cause secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the medicament itself may include cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or non-cytokine adjuvants. Many cytokines have been applied to the treatment of cancer, or as a general nonspecific immunotherapy intended to enhance the immune system, or as an adjuvant provided together with other therapies. Suitable cytokines include, but are not limited to, interferons, interleukins, and colony stimulating factors. Interferons (IFNs) contemplated by the present disclosure include common types of IFNs, IFN-α (IFN-alpha), IFN-β (IFN-beta), and IFN-γ (IFN-gamma). IFN can act directly on cancer cells, for example, slowing down its growth, promoting its development into cells with more normal behavior and / or increasing the production of its antigens, thus making cancer cells more easily recognized and destroyed by the immune system. IFN can also act indirectly on cancer cells, for example, by slowing down angiogenesis, enhancing the immune system and / or stimulating natural killer (NK) cells, T cells and macrophages. Recombinant IFN-α can be commercially available as Roferon (Roche Pharmaceuticals) and Intron A (Schering). Interleukins contemplated by the present disclosure include IL-2, IL-4, IL-11 and IL-12. Examples of commercially available recombinant interleukins include. (IL-2; Chiron Corporation) and (IL-12; Wyeth Pharmaceuticals). Zymogenetics (Seattle, Washington) is currently testing a recombinant form of IL-21, which is also contemplated for use in the combinations of the present disclosure. Colony stimulating factors (CSFs) contemplated by the present disclosure include granulocyte colony stimulating factor (G-CSF or filgrastim), granulocyte-macrophage colony stimulating factor (GM-CSF or sargramostim), and erythropoietin (epoetin alpha, darbopoetin). Treatment with one or more growth factors can help stimulate the production of new blood cells in subjects undergoing traditional chemotherapy. Therefore, treatment with CSF can help reduce the side effects associated with chemotherapy and allow the use of higher doses of chemotherapeutic agents. Various recombinant colony stimulating factors are commercially available, for example, (G-CSF; Amgen), Neulasta (pegfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen alfa (erythropoietin, Amgen), Aranesp (erythropoietin). In addition to having specific or non-specific targets, immunotherapeutics can also be active, i.e., stimulate the body's own immune response, including humoral and cellular immune responses, or the immunotherapeutics can be passive, i.e., contain immune system components produced outside the body, such as antibodies, effector immune cells, antigen presenting cells, etc. In specific embodiments, passive immunotherapy involves the use of one or more monoclonal antibodies that are specific for a specific antigen present on the surface of cancer cells or immune cells or for a specific cell growth factor. Monoclonal antibodies can be used to treat cancer in a variety of ways, for example, to enhance a subject's immune response to a particular type of cancer, to interfere with the growth of cancer cells by targeting specific cell growth factors (such as those involved in angiogenesis), or by enhancing the delivery of other anti-cancer agents to cancer cells when linked or conjugated to chemotherapeutic agents, radioactive particles, or toxins.

[0150] Monoclonal antibodies currently used as cancer immunotherapeutics include, but are not limited to, alemtuzumab Bevacizumab Cetuximab Panitumumab Pertuzumab ( 2C4), trastuzumab Tositumomab Abciximab Adalimumab Apolizumab, asekizumab, atelizumab, bassinizumab, basiliximab Baveximab, belimumab Briankinumab, canakinumab Siligizumab, Becelizumab Ceftriaxone, Cetuximab, Cetuximab, Clazakizumab, Crenezumab, Daclizumab Dalotuzumab, denosumab Eculizumab Efalizumab, epratuzumab, erlizumab, panvituzumab, fontuzumab, golimumab Ipilimumab, imgatuzumab, infliximab labetuzumab, lebrikizumab, lexatumumab, rituzumab, lucatumumab, lulizumab pegol, lumretuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, movizumab, motuzumab, muronomab, natalizumab Necitumumab Nimotuzumab Norovimab, Nunavimab, Olokizumab, Omalizumab Onartuzumab (also called MetMAb), palivizumab Paximab, pivoxezumab, pertuzumab, pembrolizumab pexelizumab, priliximab, larivizumab, ranibizumab Resvetuzumab, reslizumab, rafazodone, robatumumab, rontalizumab, rovelizumab, ruplizmnab, salimycin, secukinumab, seribantumab, sifalimumab, sibrotuzumab, sifalimumab Siligizumab, sotumumab, taduzumab, taduzumab, tefizumab, tocilizumab Tolazumab, tucumab, umab, urobutin, ustekinumab Vedolizumab Visilizumab, zanolimumab, zalutumumab. In a specific embodiment, immunotherapy includes T cell therapy, and its representative examples include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy and allogeneic gene T cell transplantation. Non-limiting examples of T cell therapy are described in U.S. Patent Publication No. 2014 / 0154228 and No. 2002 / 0006409, U.S. Patent No. 5,728,388 and International Publication No. WO2008 / 081035. The T cells of immunotherapy can be from any source known in the art. For example, T cells can be differentiated from hematopoietic stem cell colonies in vitro, or T cells can be obtained from subjects. T cells can be obtained from, for example, peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor, and combinations thereof. Alternatively or additionally, T cells may be derived from one or more T cell lines available in the art. Any number of techniques known to the skilled person (such as FICOLL TM T cells are obtained from a unit of blood collected from a subject by separation and / or apheresis. Additional methods for isolating T cells for use in T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748.

[0151] As used herein, the term "increase" or "increased" with respect to the level of a biomarker or biomarker complex refers to a statistically significant and measurable increase in the level of a biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or a control level. The increase is preferably an increase of at least about 10%, or an increase of at least about 20%, or an increase of at least about 30%, or an increase of at least about 40%, or an increase of at least about 50%.

[0152] As used herein, the term "inhibitor" refers to an agent that reduces or inhibits at least one function or biological activity of a target molecule.

[0153] As used herein, "instructional materials" include disclosures, recordings, diagrams, or any other medium of expression that can be used to convey the usefulness of the compositions and methods of the present disclosure. For example, the instructional materials of the kits of the present disclosure can be affixed to a container containing the therapeutic or diagnostic agent of the present disclosure, or shipped with a container containing the therapeutic, diagnostic, and / or prognostic agent of the present disclosure.

[0154] As used herein, the term "isolated" refers to a material that is substantially or essentially free of components that are typically associated with its native state. For example, an "isolated peptide" refers to a SETDB1 peptide mimetic that is separated and / or purified in vitro from its natural cellular environment and from its association with other components of the cell. "Substantially free" refers to a preparation of a peptide that has a purity of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In preferred embodiments, preparations of peptides have less than about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (by dry weight) of molecules that are not the subject of the invention (also referred to herein as "contaminating molecules"). When the peptides are recombinantly produced, the peptides are also desirably substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% of the volume of the preparation. The invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams by dry weight.

[0155] The term "label" as used herein refers to a detectable compound or composition. A label is typically conjugated or fused directly or indirectly to a reagent (such as a polynucleotide probe or an antibody) and facilitates detection of the reagent to which it is conjugated or fused. A label can be detectable per se (e.g., a radioisotope label or a fluorescent label), or, in the case of an enzyme label, can catalyze a chemical alteration of a substrate compound or composition that produces a detectable product.

[0156] As used herein, the term "localized" and its grammatical equivalents mean accumulated or confined to a specific or limited space or region, such as a specific cell, tissue, organelle, or intracellular region, such as a cellular component (e.g., nucleus, cytoplasm, nuclear membrane, plasma membrane, etc.).

[0157] As used herein, the term "lower" with respect to a biomarker or biomarker complex measurement result refers to a statistically significant and measurable difference in the level of a biomarker or biomarker complex compared to the level of another biomarker or biomarker complex or a control level, wherein the measurement result of the biomarker or the biomarker complex is lower than the other biomarker, biomarker complex or control level. The difference is preferably at least about 10%, or at least about 20%, or at least about 30%, or at least about 40% or at least about 50%.

[0158] "Obtain" means to possess. Samples so obtained include, for example, polypeptide extracts isolated from or derived from a particular source, including cell lysates. For example, the extract may be isolated directly from a biological fluid or tissue of a subject.

[0159] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to polymers of amino acid residues and variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. These terms do not exclude modifications, such as glycosylation, acetylation, phosphorylation, and the like. Soluble forms of the subject peptides are particularly useful. Also included within the definition are, for example, peptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) or polypeptides with substituted linkages.

[0160] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in such form as to allow the biological activity of the active ingredient to be effective and does not contain other components that are unacceptably toxic to the subject to which the composition or formulation is to be administered. Such formulations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) refers to an excipient that can be reasonably administered to a subject mammal to provide an effective dose of the active ingredient employed.

[0161] "Pharmaceutically acceptable carrier" means a pharmaceutical vehicle containing non-biologically or otherwise undesirable materials, i.e., materials that can be administered to a subject together with the selected active agent without causing any or substantial adverse reactions. The carrier may include excipients and other additives, such as diluents, detergents, colorants, wetting agents or emulsifiers, pH buffers, preservatives, transfection agents, etc.

[0162] Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug or derivative of a compound as provided herein refers to a salt, ester, amide, prodrug or derivative that is not biologically or otherwise undesirable.

[0163] As used herein, the terms "prevent, prevented or preventing" refer to a preventive treatment that increases a subject's resistance to the development of a disease or condition, or in other words, reduces the likelihood that a subject will develop a disease or condition, as well as treatment after the onset of a disease or condition to completely reduce or eliminate the disease or condition or prevent it from worsening. These terms also include within their scope the prevention of the disease or condition in a subject who may be susceptible to the disease or condition but has not yet been diagnosed with the disease or condition.

[0164] "Radiotherapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit their ability to function normally or to destroy the cells completely. It will be appreciated that there are many methods in the art to determine the dosage and duration of treatment. Typical treatment is a single administration and a typical dosage is 10 to 200 units per day (grey).

[0165] When the terms "reduce", "inhibit", "suppress", "lower" and grammatical equivalents are used to refer to the level of a substance and / or phenomenon of a first sample relative to the level of a substance and / or phenomenon of a second sample, it means that the amount of the substance and / or phenomenon in the first sample is any amount lower than the amount of the substance and / or phenomenon in the second sample, and any recognized statistical analysis method is statistically significant. In one embodiment, the reduction can be determined subjectively, such as when a patient refers to his or her subjective perception of disease symptoms, such as pain, fatigue, etc. In another embodiment, the reduction can be determined objectively, such as when the number of CSCs and / or non-CSC tumor cells in a patient's sample is lower than the number in an earlier sample of the patient. In another embodiment, the amount of the substance and / or phenomenon in the first sample is at least 10% lower than the amount of the same substance and / or phenomenon in the second sample. In another embodiment, the amount of the substance and / or phenomenon in the first sample is at least 25% lower than the amount of the same substance and / or phenomenon in the second sample. In yet another embodiment, the amount of the substance and / or phenomenon in the first sample is at least 50% lower than the amount of the same substance and / or phenomenon in the second sample. In a further embodiment, the amount of a substance and / or phenomenon in the first sample is at least 75% lower than the amount of the same substance and / or phenomenon in the second sample. In yet another embodiment, the amount of a substance and / or phenomenon in the first sample is at least 90% lower than the amount of the same substance and / or phenomenon in the second sample. Alternatively, the difference may be expressed as an "n-fold" difference.

[0166] As used herein, a cancer subject that has been treated with a therapy is considered to be "responsive," "responding," "having a positive response," or "responsive" to a therapy if the subject shows evidence of an anti-cancer effect according to a set of objective criteria recognized in the art or reasonable modifications thereof, including a clinically significant benefit, such as preventing or alleviating the severity of symptoms or slowing the progression of the cancer. It should be understood that the above terms may also be applied to cancer. Various different objective criteria are known in the art for evaluating the effect of anti-cancer therapy on cancer. The World Health Organization (WHO) criteria (Miller, AB et al., Cancer 1981; 47(1): 207-14) and its modified versions, the Response Evaluation Criteria in Solid Tumours (RECIST) (Therasse P et al., J Natl Cancer Inst 2000; 92: 205-16) and its modified versions (Eisenhauer EA, New Response Evaluation Criteria in Solid Tumors: Modification of RECIST Guidelines (Version 1.1). Eur J Cancer 2009; 45(2): 228-47) are a set of objective criteria based on imaging measurements of the size and number of tumor lesions and the detection of new lesions, such as by computed tomography (CT), magnetic resonance imaging (MRI) or conventional radiography. The size of the selected lesion (referred to as the target lesion) is used to calculate the change in tumor burden between images at different time points. The calculated responses are then classified as complete response (CR), partial response (PR), stable disease (SD) or progressive disease (PD). CR is the complete disappearance of the tumor (-100%), and PD is an increase of about 20%-25% or more (depending on the specific criteria) and / or the appearance of new lesions. PR is a significant reduction (at least about 30%) in the size of the tumor lesions (no new lesions appear), but not a complete response. SD is between PR and PD. (See Tables 1 and 2 for details.) These criteria are widely used as the primary endpoint of Phase II trials for evaluating the efficacy of anticancer agents, for example, as an alternative endpoint for overall survival. However, anatomical imaging using WHO, RECIST and RECIST 1.1 standards alone is designed to detect the early effects of cytotoxic agents and has certain limitations, particularly in evaluating the activity of newer cancer therapies for stable disease. The clinical response pattern of subjects treated with immunotherapeutic anticancer agents or molecular targeted anticancer agents may exceed the range of cytotoxic agents and may be manifested after the initial increase in tumor burden or the appearance of new lesions. For example, in some cases, meaningful tumor responses to immune checkpoint inhibitors may occur after a delay after WHO- or RECIST-defined PD.Criteria for designating immune-related response criteria (irRC) were defined in an attempt to capture the otherwise favorable response patterns observed with immunotherapy (Wolchok, JD et al.).

[0167] Guidelines for the evaluation of immunotherapy activity in solid tumors: immune-related response criteria. Clinical Care Res. 15, 7412-7420. Four patterns associated with favorable survival were identified, namely, a reduction in baseline lesions with no new lesions; durable stable disease; an initial increase in total tumor burden, but ultimately a response; and a reduction in total tumor burden during or after the appearance of new lesions, the latter two of which are different from the response patterns considered favorable according to WHO or RECIST criteria. irRC includes criteria for complete response (irCR), partial response (irPR), stable disease (irSD), and progressive disease (irPD). In addition, irRC incorporates measurable new lesions into the "total tumor burden" and compares this variable to baseline measurements, rather than assuming that new lesions necessarily represent progressive disease. In summary, according to the immune-related response criteria, irCR refers to the complete disappearance of all lesions, whether measurable or not, and the absence of new lesions; irPR is a 50% reduction in tumor burden relative to baseline; irSD is disease that does not meet the irCR or irPR criteria in the absence of progressive disease (irPD); irPD is a 25% increase in tumor burden relative to the nadir (the minimum tumor burden recorded) (Wolchok, supra). irCR, irPR, and irPD need to be confirmed by repeated, continuous assessments within at least 4 weeks from the date of the first record. irCR, irPR, and irSD include all subjects with CR, PR, or SD according to WHO criteria, as well as subjects who have moved from WHO PD to these irRC categories. However, some subjects who are classified as having PD according to WHO or RECIST criteria are classified as having PR or SD according to irRC, thereby identifying them as likely to have good survival. irRC is applicable to immune checkpoint inhibitors and other immunotherapeutics. Those of ordinary skill in the art will appreciate that additional response criteria are known in the art that take into account various factors, such as changes in the degree of tumor arterial enhancement and / or tumor density, as indicators of tumor viability, wherein reduced arterial enhancement and reduced tumor density are indicators of reduced viable tumor tissue (e.g., due to tumor necrosis). For example, the modified RECIST criteria (mRECIST) take into account changes in the degree of tumor arterial enhancement (Lencioni R and Llovet JM. Semin Liver Dis 30:52-60, 2010). The Choi criteria and modified Choi criteria take into account a decrease in tumor density on CT.Choi H et al., J Clin Oncol 25:1753-1759, 2007; Nathan PD et al., Cancer Biol Ther 9:15-19, 2010; Smith AD et al., Am J Roentgenol 194:157-165, 2010. This criterion may be particularly useful in certain cancer types and / or certain types of therapeutic agents. For example, tumor size changes may be minimal in tumors such as lymphomas, sarcomas, hepatocarcinomas, mesotheliomas, and gastrointestinal stromal tumors despite effective treatment. CT tumor density, contrast enhancement, or MRI characteristics appear to provide more information than size. In some embodiments, functional imaging may be used, such as using positron emission tomography (PET). For example, the PET response criteria for solid tumors (PERCIST) can be used, in which treatment response is assessed by metabolic changes assessed with (18)F-FDG PET imaging, where decreased tracer uptake indicates (Wahl RL et al., J Nucl Med 2009;50, Suppl 1:122S-50S). It will also be appreciated that response criteria developed for various specific cancer types, such as melanoma, breast cancer, and lung cancer, are known in the art. In contrast, a cancer subject who has been treated with a therapy is considered to be "unresponsive," "lacking a response," having a "negative response," or "non-responsive" to the therapy if the therapy does not provide a clinically significant benefit, such as preventing symptoms or reducing the severity of the symptoms or increasing the rate of progression of the cancer.

[0168] For purposes of this disclosure, for cancer subjects treated with immunotherapy (e.g., immune checkpoint inhibitors) as a monotherapy or in combination with one or more other active agents (e.g., complement inhibitors, additional anticancer agents, or both), if the subject has a complete response, partial response, or stable disease according to at least an immune-related response criterion, the subject is considered to have "responded," "responded," or "responsive" to treatment. (Cancer subjects may also respond according to RECIST, RECIST 1.1, WHO, and / or other criteria (such as those mentioned above). Again, in this case, the cancer is considered to have "responded," "responsive," or "sensitive" to treatment. According to immune-related response criteria, if a cancer subject has progressive disease, the subject is considered to have "no response," "no response," or "no responsiveness" to treatment. Cancer subjects may also respond according to RECIST, RECIST 1.1, WHO, and / or other criteria (such as those mentioned above). 1.1, WHO and / or other standards (such as those mentioned above) do not respond. Similarly, in this case, cancer is considered to be "unresponsive" to treatment, or "non-responsiveness", "insensitivity" or "resistance". If the cancer initially responds, but the subject subsequently exhibits progressive disease during treatment, the cancer is also considered to be resistant to treatment. Therefore, for example, for methods and products related to responses to treatment for cancer described herein (e.g., methods for predicting the possibility of responses, methods for classifying subjects according to predicted responses, methods for increasing the possibility of responses), responses are defined as irCR, irPR or irSD, and unless otherwise specified, lack of response is defined as irPD. In certain embodiments, any useful response criteria may be specified. The response criteria may have been shown to be associated with increased overall survival or other clinically significant benefits. It will be understood that existing response criteria may be refined or revised in the future, for example, to cover additional favorable clinical activity patterns (e.g., associated with increased overall survival) or other useful patterns applicable to immune checkpoint inhibitors. In certain embodiments, any such response criteria may be specified for use in the methods described herein.

[0169] As used herein, the terms "salt" and "prodrug" include any pharmaceutically acceptable salt, ester, hydrate or any other compound that is capable of (directly or indirectly) providing a SETDB1 peptide mimetic of the present invention, or an active metabolite thereof, or a residue thereof, after administration to a recipient. Suitable pharmaceutically acceptable salts include pharmaceutically acceptable inorganic acids such as salts of hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid and hydrobromic acid; or pharmaceutically acceptable organic acids such as salts of acetic acid, propionic acid, butyric acid, tartaric acid, maleic acid, hydroxymaleic acid, fumaric acid, citric acid, lactic acid, mucic acid, gluconic acid, benzoic acid, succinic acid, oxalic acid, phenylacetic acid, methanesulfonic acid, toluenesulfonic acid, benzenesulfonic acid, salicylic acid, sulfanilic acid, aspartic acid, glutamic acid, edetic acid, stearic acid, palmitic acid, oleic acid, lauric acid, pantothenic acid, tannic acid, ascorbic acid and valeric acid. Alkali salts include, but are not limited to, alkali salts formed by pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium. Moreover, basic nitrogen-containing groups can be quaternized with agents such as lower alkyl halides, such as methyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkyl sulfates, such as dimethyl sulfate and diethyl sulfate; and the like. However, it should be understood that non-pharmaceutically acceptable salts also fall within the scope of the present invention, as those can be used to prepare pharmaceutically acceptable salts. The preparation of salts and prodrugs can be performed by methods known in the art. For example, metal salts can be prepared by reacting a compound of the present invention with a metal hydroxide. Acid salts can be prepared by reacting an appropriate acid with the SETDB1 peptide mimetic of the present invention.

[0170] As used herein, the term "sample" includes any biological specimen that can be extracted, untreated, processed, diluted or concentrated from a subject. Samples can include, but are not limited to, biological fluids, such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, feces (i.e., excreta), tears, sweat, sebum, nipple aspirates, ductal lavages, tumor exudates, synovial fluid, ascites, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymphatic fluid, fine needle aspirates, amniotic fluid, any other body fluids, cell lysates, cell secretion products, inflammatory reaction fluid, semen and vaginal secretions. Samples can include tissue samples and biopsies, tissue homogenates, etc. Advantageous samples can include samples containing any one or more biomarkers taught herein in a detectable amount. Suitably, samples are easily obtained by minimally invasive methods, allowing removal or separation of samples from subjects. In certain embodiments, the sample contains blood, particularly peripheral blood, or a portion or extract thereof. Typically, 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 a portion of such cells (e.g., a nucleic acid or protein portion). In a specific embodiment, the sample comprises leukocytes, including peripheral blood mononuclear cells (PBMCs).

[0171] As used herein, "reference sample", "reference cell", "reference tissue", "control sample", "control cell" or "control tissue" refers to a sample, cell, tissue, standard or level for comparison purposes. In one embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of the same subject or individual. For example, healthy and / or non-diseased cells or tissues are adjacent to diseased cells or tissues (e.g., cells or tissues adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissues and / or cells of the body of the same subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell or control tissue is obtained from a healthy and / or non-diseased part (e.g., tissue or cell) of the body of an individual who is not a subject or individual. In even another embodiment, the 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 a subject or individual.

[0172] "Tissue sample" or "cell sample" refers to a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample can be solid tissue from fresh, frozen and / or preserved organs, tissue samples, biopsies and / or aspirates; blood or any blood component, such as plasma; body fluids, such as cerebrospinal fluid, amniotic fluid, peritoneal fluid or interstitial fluid; cells from the subject at any time during pregnancy or development. The tissue sample can also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue / organ. The tissue sample may contain compounds that are not naturally mixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0173] As used herein, the term "scaffold" or "molecular scaffold" refers to a chemical moiety in the composition of the invention that is bonded to a peptide at an alkylamino bond and a thioether bond (when cysteine ​​is present). As used herein, the term "scaffold molecule" or "molecular scaffold molecule" refers to a molecule that is capable of reacting with a peptide or peptide ligand to form a derivative of the invention having an alkylamino bond, and in certain embodiments, also having a thioether bond. Thus, the scaffold molecule has the same structure as the scaffold portion, except that the corresponding reactive groups (such as leaving groups) of the molecule are replaced by the alkylamino bonds and thioether bonds bonded to the peptide in the scaffold portion.

[0174] "SETDB1" refers to a protein also known as "SET domain bifurcated histone lysine methyltransferase 1", "ESET" and "KMT1A". As used herein, the term encompasses full-length and / or unprocessed SETDB1 as well as any intermediates produced by processing in cells. SETDB1 can exist as a soluble protein; therefore, the term as used herein can refer to the full-length protein. The term also encompasses naturally occurring variants (e.g., splice variants or allelic variants) of SETDB1. The protein may additionally contain a tag, such as a His tag or an Fc tag. The amino acid sequence of an exemplary human full-length SETDB1 protein can be found, for example, under UniProtKB Accession No. Q15047.

[0175] The terms "subject", "subject", "host" or "individual" used interchangeably herein refer to any subject in need of therapy or prevention, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates falling within the scope of the present disclosure include, but are not limited to, any member of the subphylum Chordata, including primates (e.g., humans, monkeys and apes, and including monkeys of the genus Macaca (e.g., cynomolgus monkeys such as long-tailed macaques and / or rhesus macaques (Macaca fascicularis)) and baboons (Papio papio) as well as marmosets (species of the genus Marmoset), squirrel monkeys (species of the genus Sciurus) and tamarins (species of the genus Myna), as well as species of apes such as chimpanzees (Panthera troglodytes)), rodents (e.g., The present invention relates to a subject that is a mammal (e.g., a hamster, a mouse, a rat, a guinea pig), a lagomorph (e.g., a rabbit, a hare), a bovine (e.g., a cow), an ovine (e.g., a sheep), a caprine (e.g., a goat), a porcine (e.g., a pig), an equine (e.g., a horse), a canine (e.g., a dog), a feline (e.g., a cat), a bird (e.g., a chicken, a turkey, a duck, a goose, a companion bird such as a canary, a budgie, etc.), a marine mammal (e.g., a dolphin, a whale), a reptile (e.g., a snake, a frog, a lizard, etc.), and a fish. The preferred subject is a human being in need of the therapy and / or analysis / determination methods of the present disclosure. However, it should be understood that the above terms do not imply the presence of symptoms.

[0176] As used herein, the terms "stratification" and "classification" are used interchangeably herein and refer to sorting subjects into different levels or categories based on the characteristics of a particular physiological or pathophysiological state or condition. For example, stratifying a population of subjects according to whether they are likely to respond to a therapy (e.g., immunotherapy) involves assigning subjects based on the response level of a therapy biomarker including SETDB1 in cancer cells.

[0177] As used herein, the terms "treatment" and "treating" and the like refer to clinical interventions designed to alter the natural processes of the treated individual or cell during the course of clinical pathology. Desirable therapeutic effects include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, if one or more symptoms associated with a T cell dysfunction disorder are alleviated or eliminated, including but not limited to reducing the proliferation of cancer cells (or destroying cancer cells), reducing pathogen infection, reducing symptoms caused by the disease, improving the quality of life of patients with the disease, reducing the dosage of other drugs required to treat the disease, and / or prolonging the survival of the individual, the individual is successfully "treated".

[0178] As used herein, the term "tumor" refers to any tumor cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer" and "cancer" refer to or describe a physiological condition in a mammal, which is usually characterized by uncontrolled cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early cancers and late cancers. The term "precancerous" refers to a condition or growth that usually precedes or develops into cancer. The term "non-metastatic" refers to a benign cancer, or remains at the primary site, and does not penetrate into the lymphatic or vascular system or tissues outside the primary site. Generally, non-metastatic cancer is any cancer, i.e., stage 0, stage I or stage II cancer. "Early cancer" means a cancer that is not invasive or metastatic, or is classified as stage 0, stage I or stage II cancer. The term "late cancer" generally refers to stage III or stage IV cancer, but may also refer to a substage of stage II cancer or stage II cancer. It will be understood by those skilled in the art that the classification of stage II cancer as early cancer or late cancer depends on the specific type of cancer. Illustrative examples of cancer include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, pancreatic cancer, colorectal cancer, lung cancer, hepatocellular carcinoma, stomach cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, kidney cancer (renal cancer), carcinoma, retinoblastoma, melanoma, brain cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, endometrial cancer, multiple myeloma, mesothelioma, rectal cancer, and esophageal cancer. In an exemplary embodiment, the cancer is breast cancer or melanoma.

[0179] Each of the embodiments described herein applies mutatis mutandis to each and every instance unless specifically stated otherwise.

[0180] 2. Protein molecules

[0181] The present invention is based in part on the determination that protein molecules corresponding to the nuclear localization sequence (NLS) site of the SETDB1 polypeptide can be used to reduce the nuclear localization of the SETDB1 polypeptide. The inventors identified that the mechanism of nuclear shuttling of SETDB1 occurs through complexing with importin alpha (IMPα). Therefore, the inventors have conceived that inhibiting the binding between SETDB1 polypeptide and IMPα polypeptide can be used to treat or prevent cancer.

[0182] 2.1 SETDB1 protein molecule

[0183] In one aspect, the present invention provides protein molecules corresponding to the NLS of a SETDB1 polypeptide. Examples of this class include polypeptides comprising an amino acid sequence corresponding to residues 206 to 232 of a wild-type human SETDB1 polypeptide.

[0184] The amino acid sequence of human SETDB1 (Uniprot Accession No. Q15047) is presented as SEQ ID NO: 1, with the NLS indicated below in bold and underlined font.

[0185]

[0186]

[0187] Therefore, in one aspect of the present invention, there is provided an isolated or purified protein molecule represented by formula (I).

[0188] Z1GKKRTKTWHKGTLIAIQTVGX1GKKYKVKZ2

[0189] Formula (I)

[0190] in:

[0191] Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween); and

[0192] X1 is selected from non-polar / neutral amino acid residues including A, G, I, L, M, F, P, W, V and Nle.

[0193] In some embodiments, Z1 is absent. In other embodiments, Z1 is composed of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. In some embodiments, the amino acid residues in Z1 are independently selected from any amino acid residue.

[0194] In certain embodiments, Z2 does not exist. In other embodiments, Z2 is composed of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. In certain embodiments, the amino acid residue in Z2 is independently selected from any amino acid residue.

[0195] In some embodiments, X1 is selected from non-polar / neutral amino acid residues, including Ala, Gly, Ile, Leu, Met, Phe, Pre, Trp, Val and Nle. In a specific embodiment, X1 is selected from Pro and Leu, especially Pro.

[0196] In some embodiments, the isolated or purified protein molecule of Formula I comprises, consists of, or consists essentially of an amino acid sequence represented by any one of SEQ ID NO: 2 or 3:

[0197] GKKRTKTWHKGTLIAIQTVGPGKKYKVK SEQ ID NO:2

[0198] GKKRTKTWHKGTLIAIQTVGLGKKYKVK SEQ ID NO:3

[0199] In some embodiments, the protein molecule of Formula I has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity with the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the protein molecule of Formula I has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2 or 3.

[0200] In some embodiments where the protein molecule of the invention comprises an N-terminus and / or a C-terminus, the protein molecule of the invention has a primary amide, secondary amide or tertiary amide, hydrazide, hydroxyamide or free carboxyl group at the C-terminus and / or a primary amine or acetamide at the N-terminus. In some embodiments, the protein molecule of the invention is a cyclic peptide and therefore may not comprise an N-terminal and / or C-terminal amino acid residue.

[0201] The present invention also contemplates protein molecules that are variants of SEQ ID NOs: 1 and 3. Such "variant" protein molecules include protein molecules derived from SEQ ID NO: 2 or SEQ ID NO: 3 by deletion or addition of one or more amino acids at the N-terminus and / or C-terminus of the protein molecule, deletion or addition of one or more amino acids at one or more sites of the protein molecule, or substitution of one or more amino acids at one or more sites of the protein molecule.

[0202] The variant protein molecules encompassed by the present invention have biological activity, that is, they continue to have the desired biological activity of the protein molecule. Such variants can be generated by, for example, genetic polymorphism or human manipulation.

[0203] The protein molecules of SEQ ID NO: 1 or 2 can be changed in various ways, including amino acid substitutions, deletions, truncations and insertions. Methods for such manipulations are well known in the art. For example, amino acid sequence variants of SEQ ID NO: 2 or 3 can be prepared by mutagenesis of nucleic acids encoding the amino acid sequences of any of SEQ ID NO: 2 or 3. Methods for mutagenesis and nucleotide sequence changes are well known in the art (e.g., Kunkel, 1985; Kunkel et al., 1987; U.S. Patent No. 4,873,192; and Morrison et al., 1987). Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein molecule of interest can be found in the model of Dayhoff et al., 1978. Methods for screening gene products of combinatorial libraries prepared by point mutations or truncations, and methods for screening cDNA libraries of gene products having selected properties are known in the art. Such methods are suitable for rapid screening of gene libraries produced by combinatorial mutagenesis of protein molecules of SEQ ID NO: 2 or 3. Recursive ensemble mutagenesis (REM), a technique to increase the frequency of functional mutants in a library, can be used in combination with screening assays to identify active variants (Arkin and Yourvan, 1992; Delgrave et al., 1993). Conservative substitutions, such as exchanging one amino acid for another with similar properties, may be desirable, as discussed in more detail below.

[0204] Compared to a parent (e.g., reference) amino acid sequence, such as SEQ ID NO: 2 or 3, a variant protein molecule polypeptide of the present invention may contain conservative amino acid substitutions at different positions along its sequence."Conservative amino acid substitutions" are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, as discussed in detail below.

[0205] Acidic: The residue has a negative charge due to the loss of a proton at physiological pH, and the residue is attracted to aqueous solution so as to seek a surface position in the conformation of a peptide containing the residue when the peptide is in aqueous medium at physiological pH. Amino acids with acidic side chains include glutamic acid and aspartic acid.

[0206] Basic: A residue has a positive charge due to association with a proton at physiological pH or within one or two pH units thereof (e.g., histidine), and the residue is attracted to aqueous solution so as to seek a surface position in the conformation of a peptide containing the residue when the peptide is in aqueous medium at physiological pH. Amino acids with basic side chains include arginine, lysine, and histidine.

[0207] Charged: Residues are charged at physiological pH and thus include amino acids with acidic or basic side chains such as glutamic acid, aspartic acid, arginine, lysine, and histidine.

[0208] Hydrophobicity: The residue is uncharged at physiological pH, and the residue is repelled by aqueous solution so as to seek an interior position in the conformation of a peptide containing the residue when the peptide is in aqueous medium at physiological pH. Amino acids with hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, norleucine, phenylalanine, and tryptophan.

[0209] Neutral / Polar: The residue is uncharged at physiological pH, but the residue is not sufficiently repelled by aqueous solution so that when a peptide containing the residue is in aqueous medium at physiological pH, the residue will seek an internal position in the conformation of the peptide. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0210] This description also characterizes certain amino acids as "small" because their side chains are not large enough to confer hydrophobicity even in the absence of polar groups. In addition to proline, "small" amino acids are amino acids with four or fewer carbons when there is at least one polar group on the side chain, and amino acids with three or fewer carbons when there is no at least one polar group on the side chain. Amino acids with small side chains include glycine, serine, alanine, and threonine. The genetically encoded secondary amino acid proline is a special case due to its known effect on the secondary conformation of the peptide chain. The structure of proline is different from all other naturally occurring amino acids because its side chain is bound to the nitrogen of the α-amino group as well as the α-carbon. However, several amino acid similarity matrices (e.g., as disclosed in, for example, Dayhoff et al., 1979; and Gonnet et al., PAM120 matrix and PAM250 matrix in 1992) include proline in the same group as glycine, serine, alanine, and threonine. Therefore, for the purposes of the present invention, proline is classified as a "small" amino acid.

[0211] The degree of attraction or repulsion required to be classified as polar or nonpolar is arbitrary, and therefore, the amino acids specifically contemplated by the present invention are classified as one or the other. Most amino acids that are not specifically named can be classified based on known behavior.

[0212] Amino acid residues can be further subdivided into cyclic or noncyclic, and aromatic or nonaromatic, categories that are self-explanatory relative to the side chain substituents of the residue, and small or large. If the residue contains a total of four or fewer carbon atoms (including the carboxyl carbon), the residue is considered small, provided that there are additional polar substituents; if there are no additional polar substituents, then if the residue contains a total of three or fewer carbon atoms (including the carboxyl carbon), the residue is considered small. Of course, small amino acid residues are always nonaromatic. Depending on their structural properties, amino acid residues can be divided into two or more categories. For naturally occurring protein amino acids, the subclassification according to this scheme is presented in Table 1.

[0213] Table 1 Amino acid subclassification

[0214]

[0215]

[0216] Conservative amino acid substitutions also include groupings based on side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, isoleucine, and norleucine; a group of amino acids with aliphatic-hydroxy side chains is serine and threonine; a group of amino acids with amide-containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, it is reasonable to expect that replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, replacing threonine with serine, or similarly replacing amino acids with structurally related amino acids will not have a major effect on the properties of the resulting variant peptides of the present invention. By measuring the activity of SETDB1, it can be easily determined whether an amino acid change will produce a protein molecule that inhibits or reduces the nuclear localization of the SETDB1. Conservative substitutions are shown under the headings of exemplary and preferred substitutions in Table 2. Amino acid substitutions falling within the scope of the present invention are generally achieved by selecting substitutions that do not significantly differ in maintaining the following effects: (a) the structure of the peptide backbone in the substituted region; (b) the charge or hydrophobicity of the molecule at the target site; or (c) the volume of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

[0217] Table 2 Exemplary and preferred amino acid substitutions

[0218]

[0219]

[0220] Alternatively, similar amino acids for conservative substitution can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine and histidine, all of which have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine and asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine and norleucine, as described in Zubay, Biochemistry, 3rd edition, Wm.C.Brown Publishers (1993).

[0221] Therefore, the predicted non-essential amino acid residue in the protein molecule of the present invention is usually replaced by another amino acid residue from the same side chain family. Alternatively, mutations can be randomly introduced along all or part of the coding sequence of the protein molecule of the present invention, such as by saturation mutagenesis, and the resulting mutant can be screened for the activity of the parent polypeptide, as described herein, to identify the mutant retaining the activity. After the mutagenesis of the coding sequence, the encoded protein molecule can be recombinantly expressed and its activity can be determined. "Non-essential" amino acid residues are residues that can be changed from the reference sequence of the embodiment protein molecule of the present invention without eliminating or substantially changing one or more of its activities. Suitably, the change does not substantially change one of these activities, such as these activities are at least 20%, 40%, 60%, 70% or 80% of the activity of the wild type. In contrast, "essential" amino acid residues refer to when changing from the reference sequence of the embodiment x protein molecule of the present invention, causing the activity of the parent molecule to be eliminated so that there is less than 20% of the wild-type activity residue.

[0222] Thus, the present invention also contemplates variants of the protein molecule of SEQ ID NO: 2 or 3, wherein the variant is distinguished from the parent sequence by the addition, deletion or substitution of one or more amino acid residues. Typically, the variant will show at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence similarity with the reference protein molecule sequence (such as, for example, as shown in SEQ ID NO: 2 or 3), as determined by the sequence alignment programs described elsewhere herein using default parameters. Ideally, the variant will have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the parent or reference protein molecule sequence (such as shown in SEQ ID NO: 2 or 3), as determined by the sequence alignment program described herein using default parameters. Variants of SEQ ID NO: 2 or 3 fall within the scope of variant protein molecules of the present invention and can generally differ from the parent molecule by at least 1 amino acid residue, but differ by less than 5, 4, 3, 2 or 1 amino acid residues. In some embodiments, the protein molecules of the present invention differ from the corresponding sequences in SEQ ID NO: 2 or 3 by at least 1 amino acid residue, but differ by less than 5, 4, 3, 2 or 1 amino acid residues. In some embodiments, the amino acid sequence of the variant protein molecules of the present invention comprises a protein molecule of Formula I. In specific embodiments, the variant protein molecules of the present invention inhibit or reduce the nuclear localization of SETDB1.

[0223] If alignment is required for sequence comparison, the sequences are usually aligned for maximum similarity or identity. Deletions or insertions or mismatched "loops" out of the sequence are usually considered differences. Appropriately, differences are differences or changes in non-essential residues or conservative substitutions.

[0224] In some embodiments, calculations of sequence similarity or sequence identity between sequences can be performed as follows:

[0225] In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for the purpose of comparison). In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 40%, more typically at least 50% or 60%, and even typically at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as at the corresponding position in the second sequence, the molecules are identical at the position. For amino acid sequence comparisons, when a position in the first sequence is occupied by the same or similar amino acid residue (i.e., conservative substitution) as at the corresponding position in the second sequence, the molecules are similar at the position.

[0226] The percent identity between two sequences is a function of the number of identical amino acid residues shared by the sequences at individual positions, taking into account the number of gaps and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. In contrast, the percent similarity between two sequences is a function of the number of identical and similar amino acid residues shared by the sequences at individual positions, taking into account the number of gaps and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0227] The comparison of sequences and the determination of the identity percentage or similarity percentage between sequences can be realized using a mathematical algorithm. In certain embodiments, the Needleman and Wünsch, (1970) algorithm in the GAP program incorporated into the GCG software package (Devereaux et al., 1984) is used, using a Blosum 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6 to determine the identity or similarity percentage between amino acid sequences. In certain embodiments, the Meyers and Miller algorithm (1989, Cablos, 4: 11-17) incorporated into the ALIGN program (version 2.0) can be used, using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 to determine the identity or similarity percentage between two amino acid sequences.

[0228] The present invention also contemplates an isolated or purified protein molecule, the protein molecule being encoded by a polynucleotide sequence, the polynucleotide sequence hybridizing with a polynucleotide sequence encoding a protein molecule of SEQ ID NO: 2 or 3 or a non-coding strand thereof under stringent conditions as defined herein, in particular hybridizing under medium, high or very high stringency conditions, preferably hybridizing under high or very high stringency conditions. The present invention also contemplates an isolated nucleic acid molecule, the isolated nucleic acid molecule comprising a polynucleotide sequence, the polynucleotide sequence hybridizing with a polynucleotide sequence encoding a protein molecule of SEQ ID NO: 2 or 3 or a non-coding strand thereof under stringent conditions as defined herein, in particular hybridizing under medium, high or very high stringency conditions, preferably hybridizing under high or very high stringency conditions.

[0229] As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing, and can encompass low stringency, medium stringency, high stringency, and very high stringency conditions.

[0230] Guidelines for conducting hybridization reactions can be found in Ausubel et al., (1998) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.), specifically sections 6.3.1-6.3.6. Both aqueous and non-aqueous methods can be used. Low stringency conditions referred to herein include and encompass at least about 1% v / v to at least about 15% v / v formamide and at least about 1M to at least about 2M salt for hybridization at 42°C, and at least about 1M to at least about 2M salt for washing at 42°C. Low stringency conditions may also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% sodium dodecyl sulfate (SDS) for hybridization at 65°C, and (i) 2× sodium chloride / sodium citrate (SSC), 0.1% SDS for washing at room temperature; or (II) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. An example of low stringency conditions includes hybridization in 6×SSC at about 45°C, followed by washing twice in 0.2×SSC, 0.1% SDS at at least 50°C (the washing temperature under low stringency conditions can be increased to 55°C). Moderate stringency conditions include and encompass at least about 16% v / v to at least about 30% v / v formamide and at least about 0.5 M to at least about 0.9 M salt for hybridization at 42°C, and at least about 0.1 M to at least about 0.2 M salt for washing at 55°C. Moderate stringency conditions may also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65°C, and (i) 2×SSC, 0.1% SDS for washing at 60-65°C; or (II) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 5% SDS. An example of moderate stringency conditions includes hybridization in 6×SSC at about 45°C, followed by one or more washes in 0.2×SSC, 0.1% SDS at 60°C. High stringency conditions include and encompass at least about 31% v / v to at least about 50% v / v formamide and about 0.01 M to about 0.15 M salt for hybridization at 42°C, and about 0.01 M to about 0.02 M salt for washing at 55°C.High stringency conditions may also include 1% BSA, 1 mM EDTA, 0.5 M NaHPO4 (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS for washing at temperatures above 65° C.; or (II) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO4 (pH 7.2), 1% SDS. An example of high stringency conditions includes hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C.

[0231] In some aspects of the present invention, an isolated or purified protein molecule of the present invention is provided, the protein molecule being encoded by a polynucleotide sequence that hybridizes under high stringency conditions with a polynucleotide sequence encoding a protein molecule of SEQ ID NO: 2 or 3 or its non-coding strand. In certain embodiments, the isolated or purified protein molecule of the present invention is encoded by a polynucleotide sequence that hybridizes under very high stringency conditions with a polynucleotide sequence encoding a protein molecule of SEQ ID NO: 2 or 3 or its non-coding strand. An embodiment of very high stringency conditions comprises hybridization at 65°C with 0.5M sodium phosphate, 7% SDS, followed by washing one or more times at 65°C with 0.2×SSC, 1% SDS. In some embodiments, the amino acid sequence of the variant protein molecule of the present invention comprises the amino acid sequence of Formula I. In specific embodiments, the protein molecule of the present invention inhibits or reduces nuclear localization of SETDB1.

[0232] Other stringent conditions are well known in the art, and those skilled in the art will recognize that various factors can be manipulated to optimize the specificity of hybridization. The strict optimization of the final wash can be used to ensure high hybridization. About specific examples, see Ausubel et al., (1998) "Contemporary Molecular Biology Experiment Guide" (John Wiley & Sons Publishing Company), specifically 2.10.1 to 2.10.16 pages, and Sambrook et al., (1989) "Molecular Cloning: Laboratory Manual (Molecular Cloning: A Laboratory Manual)" (Cold Spring Harbour Press (Cold Spring Harbour Press)), specifically 1.101 to 1.104 chapters.

[0233] Although stringent washes are typically performed at temperatures of about 42°C to 68°C, those skilled in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization rates typically occur below the T of formation of DNA-DNA hybrids. m It is well known in the art that Tm is the melting temperature, or the temperature at which two complementary polynucleotide sequences dissociate. It is used to estimate T m The method of is well known in the art (see Ausubel et al., (1998) Current Molecular Biology Laboratory Manual (John Wiley & Sons), at page 2.10.8). Generally, the T of a completely matched duplex of DNA m It can be estimated as an approximate value by the following formula:

[0234] T m =81.5+16.6(log 10 M) + 0.41 (G + C%) – 0.63 (formamide%) – (600 / length)

[0235] Where: M is Na + The concentration of is preferably in the range of 0.01M to 0.4M; G+C% is the percentage of the sum of guanosine and cytosine bases to the total number of bases, ranging between 30% and 75% G+C; Formamide% is the volume percentage of formamide concentration; Length is the number of base pairs in the DNA duplex. For every 1% increase in the number of randomly mismatched base pairs, the T of the double-stranded DNA increases by 1%. m The temperature will decrease by about 1°C. Washing is usually done at T m Perform at -15°C for high stringency, or at T m Perform at -30°C to obtain moderate stringency.

[0236] In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose membrane or a nylon membrane) containing immobilized DNA is hybridized overnight at 42° C. in a hybridization buffer (50% deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% ficoll, 0.1% polyvinylpyrrolidone and 0.1% BSA), 0.1% SDS and 200 mg / mL denatured salmon sperm DNA) containing a labeled probe. The membrane is then subjected to two consecutive medium stringency washes (i.e., 2×SSC, 0.1% SDS at 45° C. for 15 minutes, followed by 2×SSC, 0.1% SDS at 50° C. for 15 minutes), followed by two consecutive higher stringency washes (i.e., 0.2×SSC, 0.1% SDS at 55° C. for 12 minutes, followed by 0.2×SSC and 0.1% SDS solution at 65-68° C. for 12 minutes).

[0237] The protein molecules of the present invention also encompass protein molecules comprising amino acids with modified side chains, incorporation of non-natural amino acid residues and / or derivatives thereof during peptide synthesis, and the use of cross-linking agents and other methods of imposing conformational constraints on the protein molecules of the present invention. Examples of side chain modifications include modification of amino groups, such as by acylation with acetic anhydride; acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; amidation with methylacetimidate; formylation of amino groups with cyanate; pyridoxalation of lysine with pyridoxal-5-phosphate followed by reduction with sodium borohydride; reductive alkylation by reaction with an aldehyde followed by reduction with sodium borohydride; and trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS).

[0238] Carboxyl groups can be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatization to, for example, the corresponding amide.

[0239] The guanidine group of arginine residues can be modified by forming heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal, and glyoxal.

[0240] Examples of incorporation of unnatural amino acids and their derivatives during peptide synthesis include, but are not limited to, the use of 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, tert-butylglycine, norleucine, norvaline, phenylglycine, ornithine, N δ -Acetyl-L-ornithine, sarcosine, 2-thienylalanine, N ε -Acetyl-L-lysine, N ε -Methyl-L-lysine, N ε -Dimethyl-L-lysine, N ε -Formyl-L-lysine and / or the D-isomer of an amino acid. A list of unnatural amino acids contemplated by the present invention is shown in Table 3.

[0241] Table 3 Unconventional amino acids

[0242]

[0243]

[0244] In some embodiments, protein molecules of the invention comprise at least one unnatural amino acid.

[0245] 2.2 Bicyclic protein molecule

[0246] In some embodiments, the protein molecule is a bicyclic molecule comprising a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two loop sequences, and the molecular scaffold forms covalent bonds with the cysteine ​​residues of the polypeptide, so that at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide comprises the following amino acid sequence:

[0247] Z1C1GKKRX1KX2WHC2X3GTLC3IX4IQTVGX5GKKX6KVKZ2

[0248] Formula (II)

[0249] in:

[0250] C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively;

[0251] Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween);

[0252] X1 is selected from Thr, Arg and modified forms thereof;

[0253] X2 is selected from Thr, Leu and modified forms thereof;

[0254] X3 is selected from Lys, Gly and modified forms thereof;

[0255] X4 is selected from Ala, Pro and modified forms thereof;

[0256] X5 is selected from Pro, Lys and modified forms thereof; and

[0257] X6 is selected from Tyr, Lys and modified forms thereof.

[0258] In some preferred embodiments, both Z1 and Z2 are absent.

[0259] In some identical embodiments and some alternative embodiments, X1 is selected from Thr.

[0260] In some identical embodiments and some alternative embodiments, X2 is selected from Thr.

[0261] In some identical embodiments and some alternative embodiments, X3 is selected from Lys.

[0262] In some identical embodiments and some alternative embodiments, X4 is selected from Ala.

[0263] In some identical embodiments and some alternative embodiments, X5 is selected from Pro.

[0264] In some of the same embodiments and some alternative embodiments, X6 is selected from Tyr.

[0265] In some specific embodiments, the protein molecule is a bicyclic molecule comprising a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two loop sequences, and the molecular scaffold forms covalent bonds with the cysteine ​​residues of the polypeptide, so that at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide comprises the following amino acid sequence:

[0266] Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2

[0267] Formula (III)

[0268] or a modified derivative thereof, or a pharmaceutically acceptable salt thereof;

[0269] in:

[0270] C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively;

[0271] Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integers therebetween);

[0272] X1 is absent or is alanine; and

[0273] X2 is selected from non-polar / neutral amino acid residues including A, G, I, L, M, F, P, W, V and Nle.

[0274] In some embodiments, Z1 is absent. In other embodiments, Z1 is composed of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. In some embodiments, the amino acid residues in Z1 are independently selected from any amino acid residue.

[0275] In certain embodiments, Z2 does not exist. In other embodiments, Z2 is composed of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid residues. In certain embodiments, the amino acid residue in Z2 is independently selected from any amino acid residue.

[0276] In some embodiments, Z2 is not present.

[0277] In some embodiments, X2 is selected from non-polar / neutral amino acid residues, including Ala, Gly, Ile, Leu, Met, Phe, Pro, Trp, Val and Nle. In a specific embodiment, X1 is selected from Pro and Leu, especially Pro.

[0278] In some other embodiments of this type, the polypeptide comprises an amino acid sequence selected from formula (III) or (IV), as shown below:

[0279] Z1X1C1GKKRTKTWHC2KGTLC3IAIQTVGX1GKKYKVKZ2

[0280] Formula (IV)

[0281] Z1X1C1GKKRTKTWHC2KGTLIAIQC3TVGX1GKKYKVKZ2

[0282] Formula (V)

[0283] In some embodiments, the protein molecule of Formula II, III or IV comprises, consists of, or consists essentially of an amino acid sequence selected from Table 4.

[0284] Table 4 Bicyclic protein molecules

[0285] Amino acid sequence SEQ ID NO: <![CDATA[C1GKKRTKTWHC2KGTLIAIQTVGX1GC3KKYKVK]]> 4 <![CDATA[C1GKKRTKTWHC2KGTLC3IAIQTVGX1GKKYKVK]]> 5 <![CDATA[C1GKKRTKTWHC2KGTLIAIQC3TVGX1GKKYKVK]]> 6

[0286] In some embodiments, the protein molecules of Formula (II)-(V) have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence similarity with the amino acid sequence of any one of SEQ ID NOs: 3-5. In some embodiments, the protein molecules of Formula II-IV have at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NOs: 4-6.

[0287] In some embodiments, the protein molecules of the present invention are alternative types of cyclic molecules. Without wishing to be bound by theory, the cyclization of peptides is believed to reduce the susceptibility of peptide degradation. In a specific embodiment, the SETDB1 bicyclic peptide mimics use N to C cyclization (head to tail cyclization), preferably through an amide bond to cyclize. Such SETDB1 bicyclic peptide mimics do not have an N-terminal or C-terminal amino acid residue. In a specific embodiment, the SETDB1 bicyclic peptide mimics have an amide cyclized peptide backbone. In other embodiments, the peptide is cyclized using a side chain to side chain cyclization, preferably through a disulfide bond, a diselenide bond, a selenosulfur bond, a thioether bond, such as a lanthionine bond, a selenoether bond, a triazole bond, a lactam bond or a dimethylene bond cyclization; especially through a disulfide bond to cyclize.

[0288] Molecular scaffold

[0289] The SETDB1 bicyclic peptide mimetics of the present invention comprise, consist essentially of, or consist of a polypeptide covalently bound to a molecular scaffold. The molecular scaffold is described in, for example, International PCT Patent Application No. WO 2009 / 098450 and references cited therein, particularly WO 2004 / 077062 and WO 2006 / 078161. As noted in the aforementioned documents, the molecular scaffold may be a small molecule, such as a small organic molecule.

[0290] In some embodiments, the molecular scaffold may be or may be based on a natural monomer, such as a nucleoside, a sugar or a steroid. For example, the molecular scaffold may comprise a short polymer of such an entity, such as a dimer or trimer.

[0291] In one embodiment, the molecular scaffold is a compound of known toxicity, such as low toxicity. Examples of suitable compounds include cholesterol, nucleotides, steroids, or existing drugs such as temazepam.

[0292] In some embodiments, the molecular scaffold can be a macromolecule. In some embodiments, the molecular scaffold is a macromolecule composed of amino acids, nucleotides or carbohydrates.

[0293] In some embodiments, the molecular scaffold comprises a reactive group capable of reacting with one or more functional groups of a polypeptide to form a covalent bond.

[0294] The molecular scaffolds may comprise chemical groups such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, olefins, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides and acyl halides.

[0295] In some embodiments, the scaffold is an aromatic molecular scaffold (i.e., a scaffold containing (hetero) aromatic groups). These aromatic rings may optionally contain one or more heteroatoms (e.g., one or more of N, O, S, and P), such as thienyl rings, pyridyl rings, and furanyl rings. Aromatic rings may be optionally substituted. The aryl rings may also be optionally substituted. Suitable substituents include alkyl groups (which may be optionally substituted), other aryl groups (which themselves may be optionally substituted), heterocycles (saturated or unsaturated), alkoxy groups (which are meant to include aryloxy groups (e.g., phenoxy groups)), hydroxyl groups, aldehyde groups, nitro groups, amine groups (e.g., unsubstituted, or monosubstituted or disubstituted with aryl or alkyl groups), carboxylic acid groups, carboxylic acid derivatives (e.g., carboxylates, amides, etc.), halogen atoms (e.g., Cl, Br, and I), etc.

[0296] Suitably, the scaffold comprises a trisubstituted (hetero)aromatic or (hetero)alicyclic moiety, such as a trimethylene substituted (hetero)aromatic or (hetero)alicyclic moiety. The (hetero)aromatic or (hetero)alicyclic moiety is suitably a six-membered ring structure, preferably trisubstituted, such that the scaffold has a 3-fold symmetry axis.

[0297] In some embodiments, the scaffold is a trimethylene (hetero) aryl moiety, for example, a 1,3,5-trimethylene benzene moiety. In these embodiments, the corresponding scaffold molecule appropriately has a leaving group on the methylene carbon. Then, the methylene forms the R1 portion of the alkylamino bond as defined herein. In these methylene-substituted (hetero) aromatic compounds, the electrons of the aromatic ring can stabilize the transition state during nucleophilic substitution. Therefore, for example, benzyl halides are 100-1000 times more reactive with nucleophilic substitution than alkyl halides not connected to (hetero) aromatic groups.

[0298] In embodiments of this type, the scaffolds and scaffold molecules have the general formula:

[0299]

[0300] wherein LG represents a leaving group of a scaffold molecule as further described below, or LG (including the adjacent methylene group forming the R1 portion of the alkylamino group) represents an alkylamino bond to a peptide in a conjugate of the invention.

[0301] In some embodiments, the above-mentioned group LG can be a halogen, such as but not limited to a bromine atom, in which case the scaffold molecule is 1,3,5-tri(bromomethyl)benzene (TBMB). Another suitable molecular scaffold molecule is 2,4,6-tri(bromomethyl)-mesitylene. It is similar to 1,3,5-tri(bromomethyl)benzene, but contains three additional methyl groups connected to the benzene ring. In the case of this scaffold, the additional methyl groups can form additional contacts with the peptide and thus add additional structural constraints. Therefore, compared to 1,3,5-tri(bromomethyl)benzene, it achieves a different range of diversity.

[0302] Another preferred molecule is 1,3,5-tris(bromoacetamido)benzene (TBAB) for reaction with peptides to form scaffolds via nucleophilic substitution:

[0303]

[0304] In some alternative embodiments, the scaffold is a non-aromatic molecular scaffold (e.g., a scaffold comprising a (hetero)alicyclic group). As used herein, "(hetero)alicyclic" refers to a homocyclic or heterocyclic saturated ring. The ring may be unsubstituted, or it may be substituted with one or more substituents. The substituents may be saturated or unsaturated, aromatic or non-aromatic, and examples of suitable substituents include the substituents mentioned above in the discussion of alkyl and aryl substituents. In addition, two or more ring substituents may be combined to form another ring, so that as used herein, "ring" is meant to include fused ring systems. In these embodiments, the alicyclic scaffold is preferably 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (TATA).

[0305] In some alternative embodiments, the molecular scaffold may have a tetrahedral geometry, such that the four functional groups of the encoded peptide react with the molecular scaffold to produce no more than two product isomers. Other geometries are also possible; in fact, the number of scaffold geometries may be almost unlimited, which provides greater possibilities for the diversification of peptide ligands.

[0306] The peptides used to form the bicyclic peptides of the present invention contain a cysteine ​​that is used to form a thioether bond with the scaffold, wherein the terminal -SH group of the cysteine ​​is replaced by -NH2.

[0307] The bicyclic peptides of the present invention have many favorable properties that make them considered as beneficial drug-like molecules for injection, inhalation, nasal administration, ocular administration, oral administration or topical administration. Such favorable properties include:

[0308] -species cross-reactivity, which is a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluations;

[0309] - Protease stability, e.g., ideally, the bicyclic peptide ligand should exhibit stability to plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across species so that bicyclic peptide candidates can be developed in animal models and administered to humans with confidence;

[0310] - Desired solubility properties, which is a function of the ratio of charged and hydrophilic to hydrophobic residues and intra / inter-molecular H-bonding, which is important for formulation and absorption purposes; and

[0311] - Optimal elimination half-life. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides with short exposure in acute disease management settings, or to develop bicyclic peptides with enhanced retention. Therefore, they are the best choice for the management of more chronic disease states and cancer.

[0312] Other factors driving the desired elimination half-life are the need for sustained exposure to achieve maximum therapeutic efficacy versus attendant toxicological effects resulting from sustained agent exposure.

[0313] In some embodiments, the molecular scaffold may comprise or may consist of tri(bromomethyl)benzene, in particular 1,3,5-tri(bromomethyl)benzene ("TBMB") or a derivative thereof. In some particularly preferred embodiments, the molecular scaffold is 1,3,5-(tribromomethyl)benzene.

[0314] In some other embodiments, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)mesitylene but contains three additional methyl groups attached to the benzene ring. The advantage is that the additional methyl groups can form additional contacts with the polypeptide and thus add additional structural constraints.

[0315] Scaffold reactive groups that can be used on the molecular scaffold to react with the thiol group of cysteine ​​are alkyl halides (or also known as halogenoalkane or haloalkane).

[0316] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or iodoacetamide. Other scaffold reactive groups for selectively coupling compounds to cysteine ​​in proteins are maleimides. Examples of maleimides that can be used as molecular scaffolds in the present invention include: tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, tris-(maleimido)benzene. Selenocysteine ​​is also a natural amino acid that has similar reactivity to cysteine ​​and can be used for the same reaction. Therefore, unless the context suggests otherwise, it is generally acceptable to replace selenocysteine ​​whenever cysteine ​​is mentioned.

[0317] 2.3 Additional N-terminal or C-terminal modifications.

[0318] In some of the above embodiments, additional modifications may be made to the N-terminus and / or C-terminus of the protein molecule. For example, additional amino acids or other substituents may be added to the N-terminus or C-terminus (if present) of the protein molecule of the invention. In some embodiments, the protein molecule of the invention may form part of a longer sequence in which additional amino acids are added to one or both of the N-terminus and the C-terminus.

[0319] For specific uses and methods of the present invention, it is desirable to have a protein molecule with a high level of stability, for example, to increase the half-life of the protein molecule in a subject. Therefore, in some embodiments, the protein molecule of the present invention comprises a stabilizing portion or a protecting portion. The stabilizing portion or the protecting portion can be coupled at any point of the peptide. Suitable stabilizing or protecting portions include, but are not limited to, polyethylene glycol (PEG), polysaccharides or end-capping molecules, including acetyl, pyroglutamic acid or amino groups. In a preferred embodiment, the acetyl and / or pyroglutamic acid are coupled to the N-terminal amino acid residue of the protein molecule. In a specific embodiment, the N-terminus of the protein molecule is acetamide. In a preferred embodiment, the amino group is coupled to the C-terminal amino acid residue of the protein molecule. In a specific embodiment, the protein molecule has a primary amide, a secondary amide or a tertiary amide, a hydrazide or a hydroxyamide at the C-terminus, specifically a primary amide at the C-terminus. In a preferred embodiment, PEG is coupled to the N-terminal or C-terminal amino acid residue of the protein molecule, or is coupled through the amino group of a lysine side chain or other appropriately modified side chain, particularly through the N-terminal amino acid residue, such as through the amino group of the residue, or through the amino group of a lysine side chain.

[0320] In a preferred embodiment, the protein molecule of the invention has a primary amine or a free carboxyl (acid) group at the C-terminus and a primary amine or acetamide group at the N-terminus.

[0321] Although the protein molecules of the present invention can inherently permeate membranes, membrane permeability can be further increased by conjugating membrane permeation moieties to the protein molecules. Therefore, in some embodiments, the protein molecules of the present invention comprise membrane permeation moieties. The membrane permeation moieties can be coupled at any point on the protein molecule.

[0322] Suitable membrane permeating moieties include lipid moieties, cholesterol, and proteins, such as cell permeating peptides and polycationic peptides; particularly lipid moieties.

[0323] Suitable cell penetrating peptides may include, for example, peptides described in US2009 / 0047272, US2015 / 0266935, and US 2013 / 0136742. Thus, 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 analogs of Arg and Lys residues, such as YGRKKRPQRRR (HIV TAT 47-57 ; SEQ ID NO:7), RRWRRWWRRWWRRWRR (W / R; SEQ ID NO:9), CWK 18 (AlkCWK 18 ; SEQ ID NO:9), K 18 WKK 18 (Di-CWK 18 ; SEQ ID NO: 10), WTLNSAGYLLGKINLKALAALAKKIL (transporter; SEQ ID NO: 11), GLFEALEELWEAK (DipaLytic; SEQ ID NO: 12), K 16 GGCRGDMFGCAK 16 RGD (K16RGD; SEQ ID NO:13), K 16 GGCMFGCGG (PI; SEQ ID NO:14), K 16 ICRRARGDNPDDRCT (P2; SEQ ID NO:15), KKWKMRRNQFWVKVQRbAK(B)bA (P3; SEQ ID NO:16), VAYISRGGVSTYYSDTVKGRFTRQKYNKRA (P3a; SEQ ID NO:17), IGRIDPANGKTKYAPKFQDKATRSNYYGNSPS (P9.3; SEQ ID NO:18), KETWWETWWTEWSQPKKKRKV (Pep-1; SEQ ID NO:19), PLAEIDGIELTY (Plae; SEQ ID NO:20), K16 GGPLAEIDGIELGA(Kplae;SEQ ID NO:21)、K 16GGPLAEIDGIELCA(cKplae; SEQ ID NO:22), WEAK(LAKA)2-LAKH(LAKA)2LKAC(HA2; SEQ ID NO:23), (LARL)6NHCH3(LARL46; SEQ ID NO:24), KLLKLLLKLWLLKLLL (Hel-11-7; SEQ ID NO:25), (KKKK)2GGC (KK; SEQ ID NO:26), (KWKK)2GCC (KWK; SEQ ID NO:27), (RWRR)2GGC (RWR; SEQ ID NO:28), PKKKRKV (SV40 NLS7; SEQ ID NO:29), PEVKKKRKPEYP (NLS12; SEQ ID NO:30), TPPKKKRKVEDP (NLS12a; SEQ ID NO:31), GGGGPKKKRKVGG (SV40 NLS13; SEQ ID NO:32), GGGFSTSLRARKA (AV NLS13; SEQ ID NO:33), CKKKKKKSEDEYPYVPN (AV RME NLS17; SEQ ID NO:34), CKKKKKKKSEDEYPYVPNFSTSLRARKA (AV FP NLS28; SEQ ID NO:35), LVRKKRKTEEESPLKDKDAKKSKQE (SV40 N1 NLS24; SEQ ID NO:36) and K9K2K4K8GGK5 (Loligomer; SEQ ID NO:37); HSV-1 envelope protein VP22; HSV-1 envelope protein VP22r fused to a nuclear export signal (NES); mutant B subunit of Escherichia coli enterotoxin EtxB (H57S); detoxifying exotoxin A (ETA); protein transduction domain of HIV-1 Tat protein, GRKKRRQRRRPPQ (SEQ ID NO:38); Drosophila melanogaster antennapedia domain Antp (amino acids 43-58), RQIKIWFQNRRMKWKK (SEQ ID NO:39); Buforin II, TRSSRAGLQFPVGRVHRLLRK (SEQ ID NO:40); hClock (amino acids 35-47) (human clock protein DNA binding peptide), KRVSRNKSEKKRR (SEQ ID NO:41); MAP (model amphipathic peptide), KIALKIALKALKAALKIA (SEQ ID NO:42); K-FGF,AAVALLPAVLIALIAP (SEQID NO:43);Ku70 derived peptides comprising a peptide selected from the group comprising: VPMLKE (SEQ ID NO:44), VPMLK (SEQ ID NO:45), PMLKE (SEQ ID NO:46) or PMLK (SEQ ID NO:47); Prion, mouse Prpe (amino acids 1-28), MANLGYWLIALFVTM WTDVGLCKKRPKP (SEQ ID NO:48); pVEC, LLIILRRRIRKQAHAHSK (SEQ ID NO:49); Pep-I, KETWWETWWTEWSQPKKKRKV (SEQ ID NO:66); SynBI, RGGRLSYSR RRFSTSTGR (SEQ ID NO:50); Transportin, GWTLN SAGYLLGKINLKAIAAIAKKIL (SEQ ID NO:51); Transportin-10, AGYLLGKINLKALAALAKKIL (SEQ ID NO:52). NO:52); CADY, Ac-GLWRALWRLLRSLWRLLWRA-mercaptoethylamine (SEQ ID NO:53); Pep-7, SDLWEMMM VSIACQY (SEQ ID NO:54); HN-1, TSPLNIHNGQKL (SEQ ID NO:55); VT5, DPKGDPK GVTVTVTVTGKGDPKPD (SEQ ID NO:56); pISL, RVIRVWFQNKRCKDKK (SEQ ID NO:57) or GALFLGFLGGAAGSTMGAWSQPKSKRKV (MGP; SEQ ID NO:58). ;

[0324] In a preferred embodiment, the membrane permeable moiety is a lipid moiety, such as C 10 -C 20 Fatty acyl, specifically stearoyl (octadecanoyl; C 18 ), palmitoyl (hexadecanoyl; C 16 ) or myristoyl (tetradecanoyl; C 14 ); more specifically myristoyl. In a preferred embodiment, the membrane permeable moiety is coupled to the N-terminal or C-terminal amino acid residue, or is coupled via the amino group of the lysine side chain or other appropriately modified side chains of the SETDB1 bicyclic peptide mimetic, in particular the N-terminal amino acid residue of the SETDB1 bicyclic peptide mimetic, or is coupled via the amino group of the lysine side chain. In a specific embodiment, the membrane permeable moiety is coupled via the amino group of the N-terminal amino acid residue.

[0325] Therefore, in another aspect of the present invention, there is provided an isolated or purified protein molecule represented by Formula VI:

[0326] MP

[0327] (Formula VI)

[0328] in:

[0329] M is the membrane permeate fraction; and

[0330] P is an isolated or purified protein molecule represented by any one of formulas (I)-(V).

[0331] In some embodiments, M is coupled at any point on the protein molecule; in particular, it is coupled to the N-terminal or C-terminal amino acid residue or through the amino group of the lysine side chain or other appropriately modified side chain of the protein molecule, more particularly, the N-terminal amino acid residue of the protein molecule or through the amino group of the lysine side chain; most particularly, it is coupled through the amino group of the N-terminal amino acid residue.

[0332] Suitable membrane permeating moieties and embodiments of protein molecules represented by any of Formulae (I)-(V) are described herein.

[0333] 2.4 Salts and Prodrugs

[0334] The protein molecules of the present invention may be in the form of salts or prodrugs. The salts of the protein molecules of the present invention are preferably pharmaceutically acceptable, but it will be understood that non-pharmaceutically acceptable salts also fall within the scope of the present invention.

[0335] The protein molecules of the present invention may be in crystalline form and / or in the form of a solvate, such as a hydrate. Solvation may be performed using methods known in the art.

[0336] 2.5 Synthesis of linear peptides

[0337] The peptides of the present invention can be prepared using recombinant DNA technology or by chemical synthesis.

[0338] In some embodiments, the protein molecules of the present invention are prepared using recombinant DNA technology. For example, the protein molecules of the present invention can be prepared by a procedure comprising the following steps: (a) preparing a construct comprising a polynucleotide sequence encoding the protein molecule of the present invention and operably connected to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the polynucleotide sequence to thereby produce the encoded protein molecule of the present invention; and (d) isolating the protein molecule of the present invention from the host cell. The protein molecules of the present invention can be recombinantly prepared using standard protocols, for example, as described in Klint et al., (2013); Sambrook et al., (1989); Ausubel et al., (1998); Coligan et al., (1997); and U.S. Pat. No. 5,976,567, the entire contents of which are hereby incorporated by reference.

[0339] Therefore, the present invention also contemplates nucleic acid molecules encoding protein molecules of the present invention. Therefore, in another aspect of the present invention, an isolated nucleic acid molecule is provided, the nucleic acid molecule comprising a polynucleotide sequence encoding a protein molecule of the present invention, or a polynucleotide sequence complementary to a polynucleotide sequence encoding a protein molecule of the present invention (e.g., a protein molecule of any one of Formulas I-IV of SEQ ID NOs: 1-5 or a variant protein molecule as described herein).

[0340] The isolated nucleic acid molecule of the present invention can be DNA or RNA. When the nucleic acid molecule is in the form of DNA, it can be genomic DNA or cDNA. The RNA form of the nucleic acid molecule of the present invention is usually mRNA.

[0341] Although nucleic acid molecules are usually isolated, in some embodiments, nucleic acid molecules can be integrated into other genetic molecules (such as expression vectors) or connected to other genetic molecules, or fused or associated with other genetic molecules in other ways. Generally, expression vectors include transcription and translation regulatory nucleic acids operably connected to polynucleotide sequences. Therefore, in another aspect of the present invention, an expression vector is provided, which comprises a polynucleotide sequence encoding a SETDB1 bicyclic peptide mimetic of the present invention (such as a protein molecule of any one of Formulas I-IV of SEQ ID NO: 1-5 or a variant protein molecule as described herein).

[0342] Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences and promoters that can be used to regulate the expression of nucleic acids. The vector optionally includes a universal expression cassette containing at least one independent terminator sequence, a sequence (e.g., a shuttle vector) that allows replication of the cassette in eukaryotes, prokaryotes or both, and a selection marker for both prokaryotic and eukaryotic systems. The vector can be suitable for replication and integration of prokaryotes, eukaryotes or both. See Giliman and Smith (1979); Roberts et al., (1987); Berger and Kimmel; Sambrook et al., supra; and Ausubel et al., supra, the entire contents of the document are incorporated by reference.

[0343] Expression vectors containing regulatory elements of eukaryotic viruses (such as retroviruses) are generally used to express nucleic acid sequences in eukaryotic cells. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papillomavirus include pBV-1MTHA, and vectors derived from Epstein Barr virus include pHEBO and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE and any other vectors that allow protein expression under the guidance of SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter or other promoters that are effective in expressing in eukaryotic cells.

[0344] Although a variety of vectors can be used, it should be noted that viral expression vectors can be used to modify eukaryotic cells because viral vectors can efficiently transfect target cells and integrate them into the target cell genome. Illustrative expression vectors of this type can be derived from viral DNA sequences, including but not limited to adenoviruses, adeno-associated viruses, herpes simplex viruses, and retroviruses, such as B, C, and D retroviruses, as well as foamy viruses and modified slow viruses. Suitable expression vectors for transfecting animal cells are described in, for example, Wu and Atai (2000); Vigna and Naldini (2000); Kay et al., (2001); Athanasopoulos et al., (2000); and Walther and Stein (2000), the entire contents of which are incorporated by reference.

[0345] The part of the coding polypeptide or peptide of the expression vector can include a naturally occurring sequence or a variant thereof, which has been engineered using recombinant technology. In one example of a variant, the codon composition of the polynucleotide encoding the protein molecule of the present invention is modified using a method utilizing codon usage preference or codon translation efficiency in a specific mammalian cell or tissue type, to allow the protein molecule of the present invention to enhance expression in a mammalian host, as described in, for example, International Publication Nos. WO 99 / 02694 and WO 00 / 42215. In short, the latter method is based on the following observations: there are differences in the translation efficiency of different codons between different cells or tissues, and these differences and the codon composition of genes can be used to regulate the expression of proteins in specific cells or tissue types. Therefore, for constructing a codon-optimized polynucleotide, at least one existing codon of the parental polynucleotide can be replaced by a synonymous codon, and the translation efficiency of the synonymous codon in the target cell or tissue is higher than that of the replaced existing codon. Although it is preferred to replace all existing codons in the parental nucleic acid molecule with a synonymous codon with a higher translation efficiency, this is not necessary, because even partial replacement can increase expression. Suitably, the substitution step affects 5%, 10%, 15%, 20%, 25%, 30%, more preferably 35%, 40%, 50%, 60%, 70% or more of the existing codons of the parent polynucleotide.

[0346] The expression vector is compatible with the cell into which it is introduced so that the protein molecule of the present invention can be expressed by the cell. The expression vector is introduced into the cell by any suitable means, which will depend on the specific selection of the expression vector and the cell used. This mode of introduction is well known to those skilled in the art. For example, it can be introduced by contact (for example, in the case of a viral vector), electroporation, transformation, transduction, conjugation or triparental mating, transfection, membrane fusion infected with cationic lipids, high-speed bombardment of microparticles coated with DNA, incubation with calcium phosphate-DNA precipitates, direct microinjection into single cells and the like. Other methods can also be used and known to those skilled in the art. Alternatively, the vector can also be introduced by cationic lipids (for example, liposomes). Such liposomes are commercially available (for example, LIPOFECTAMINE TM etc., supplied by Life Technologies, Inc., Gibco BRL, Gaithersburg, Md.).

[0347] In some embodiments, the protein molecules of the present invention can be produced in cells by introducing one or more expression constructs (eg, expression vectors) comprising a polynucleotide sequence encoding the protein molecules of the present invention.

[0348] The present invention contemplates recombinant production of the protein molecules of the present invention in host cells, such as mammalian cells (e.g., Chinese hamster ovary (CHO) cells, mouse myeloma (NSO) cells, baby hamster kidney (BHK) cells, or human embryonic kidney (HEK293) cells), yeast cells (e.g., Pichia pastorls cells, Saccharomyces cerevisiae cells, Schizosaccharomyces pombe cells, Hansenula polymorpha cells, Kluyveromyces lactis cells, Yarrowia lipolytica cells, or Arxula adeninivorans cells), or bacterial cells (e.g., E. coli cells, Corynebacterium glutamicum, or Pseudomonas fluorescens cells).

[0349] For therapeutic applications, the present invention also envisages producing the protein molecules of the present invention in vivo in cells of a subject, for example SETDB1 overexpressing cells, such as vertebrate cells, in particular mammalian or avian cells, especially mammalian cells.

[0350] In some embodiments, the protein molecules of the present invention are prepared using standard peptide synthesis methods, such as solution synthesis or solid phase synthesis. The chemical synthesis of the protein molecules of the present invention can be performed manually or using an automatic synthesizer. For example, linear peptides can be synthesized using solid phase peptide synthesis using Boc or Fmoc chemistry, as described by Merrifield (1963); Schnolzer et al., (1992); and Cardoso et al., (2015); the entire contents of the document are incorporated by reference. After deprotection and cleavage from the solid support, the linear peptide is purified using a suitable method (such as preparative chromatography).

[0351] In other embodiments, protein molecules of the present invention can be cyclized. Cyclization can be performed using a variety of techniques, such as described in, for example, Davies (2003), the entire contents of which are incorporated by reference. In a specific embodiment, linear peptides are synthesized starting from a cysteine ​​residue at the N-terminus and ending with a thioester at the C-terminus using solid phase peptide synthesis involving Boc chemistry. After deprotection and cleavage from the resin, the peptide is cyclized through a thiolactone intermediate, which is then rearranged into an amine cyclized peptide.

[0352] 2.6 Synthesis of bicyclic peptides

[0353] The bicyclic peptides of the present invention can be synthetically produced by standard techniques followed by in vitro reaction with a molecular scaffold. When this is done, standard chemistry can be used. This enables rapid large-scale preparation of soluble materials for additional downstream experiments or validation. Such methods can be accomplished using conventional chemistry, such as that disclosed in Timmerman et al. (supra).

[0354] Therefore, the present invention also relates to the preparation of a polypeptide or conjugate selected as described herein, wherein said preparation comprises optional additional steps as explained below. In one embodiment, these steps are performed on the final product polypeptide / conjugate prepared by chemical synthesis.

[0355] Optionally, when making conjugates or complexes, amino acid residues in the polypeptide of interest may be substituted.

[0356] The peptide can also be extended to incorporate, for example, another loop and thus introduce multiple specificities. To extend the peptide, the peptide can be chemically extended using standard solid phase or solution phase chemistry with orthogonally protected lysine (and analogs) only at the N-terminus or C-terminus of the peptide or within a loop. Standard protein chemistry can be used to introduce an activatable N-terminus or C-terminus. Alternatively, addition can be performed by fragment condensation or native chemical ligation, for example as described in (Dawson et al., Science 1994, 266:776-779), or enzymatically, for example using subtilisinase, as described in (Chang et al., Proc Natl Acad Sci USA, 1994, 91(26):12544-8; or see Hikari et al., Bioorganic & Medicinal Chemistry Letters, 2008, 18(22), 6000-6003).

[0357] Alternatively, the peptide can be extended or modified by additional conjugation via disulfide bonds. This has the advantage of allowing the first and second

[0358] The peptides dissociate from each other once in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TBMB) can be added during the chemical synthesis of the first peptide to react with the three cysteine ​​groups; then an additional cysteine ​​can be attached to the N-terminus of the first peptide so that this cysteine ​​reacts only with the free cysteine ​​of the second peptide.

[0359] Similar techniques are equally applicable to the synthesis / coupling of two bicyclic macrocycles and bispecific macrocycles, potentially generating tetraspecific molecules. In addition, the addition of other functional groups or effector groups can be accomplished in the same manner using appropriate chemistry, either at the N-terminus or C-terminus, or through side chain coupling. In one embodiment, the coupling is performed in such a way that the activity of either entity is not blocked.

[0360] In some embodiments, the SETDB1 bicyclic peptide mimetics of the present invention can be produced in cells by introducing one or more expression constructs (eg, expression vectors) comprising a polynucleotide sequence encoding the SEDB1 bicyclic peptide mimetics of the present invention.

[0361] The present invention contemplates recombinant production of the SETDB1 bicyclic peptide mimetics of the present invention in host cells, such as mammalian cells (e.g., Chinese hamster ovary (CHO) cells, mouse myeloma (NSO) cells, baby hamster kidney (BHK) cells, or human embryonic kidney (HEK293) cells), yeast cells (e.g., Pichia pastoris cells, Saccharomyces cerevisiae cells, Schizosaccharomyces pombe cells, Hansenula polymorpha cells, Kluyveromyces lactis cells, Yarrowia lipolytica cells, or Akkermansia adenovirus cells), or bacterial cells (e.g., Escherichia coli cells, Corynebacterium glutamicum cells, or Pseudomonas fluorescens cells).

[0362] For therapeutic applications, the present invention also contemplates producing the SETDB1 bicyclic peptide mimetics of the present invention in vivo in cells of a subject, for example SETDB1 overexpressing cells, such as vertebrate cells, particularly mammalian or avian cells, especially mammalian cells.

[0363] In some embodiments, the SETDB1 bicyclic peptide mimetics of the present invention are prepared using standard peptide synthesis methods, such as solution synthesis or solid phase synthesis. The chemical synthesis of the SETDB1 bicyclic peptide mimetics of the present invention can be performed manually or using an automatic synthesizer. For example, linear peptides can be synthesized using solid phase peptide synthesis using Boc or Fmoc chemistry, as described in: Merrifield (1963) Journal of the American Chemical Society (J Am Chem Soc), 85 (14): 2149-2154; Schnolzer et al., (1992) International Journal of Peptide and Protein Research (Int J Pept Protein Res), 40: 180-193 and Cardoso et al., (2015) Mol Pharmacol, 88 (2): 291-303, the entire contents of which are incorporated by reference. After deprotection and cleavage from the solid support, the linear peptide is purified using a suitable method (such as preparative chromatography).

[0364] 3. Pharmaceutical Composition

[0365] According to the present invention, protein molecules can be used in compositions and methods for treating or preventing conditions involving nuclear localization of SETDB1 (eg, cancer).

[0366] Therefore, in some embodiments, the protein molecule of the present invention may be in the form of a pharmaceutical composition, wherein the pharmaceutical composition comprises the protein molecule of the present invention and a pharmaceutically acceptable carrier or diluent.

[0367] The protein molecules of the present invention can be formulated into pharmaceutical compositions in neutral or salt form.

[0368] As will be appreciated by those skilled in the art, the selection of pharmaceutically acceptable carriers or diluents will depend on the nature of the route of administration, the condition and the subject to be treated. Those skilled in the art can easily determine specific carriers or delivery systems and routes of administration. Careful selection of carriers or delivery systems and routes of administration should be made to ensure that the activity of the protein molecule will not be exhausted during the preparation of the formulation, and that the protein molecule can reach the site of action intactly. The pharmaceutical composition of the present invention can be administered by a variety of routes, including but not limited to intravenous, oral, rectal, topical, intranasal, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intracerebral, intravaginal, intravesical or intraperitoneal administration. In some preferred embodiments, the pharmaceutical composition of the present invention can be administered intravenously. In some other preferred embodiments, the pharmaceutical composition of the present invention can be administered orally.

[0369] Pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for the preparation of sterile injectable solutions. Such forms should be stable under the conditions of manufacture and storage and should be preserved against reduction, oxidation, and microbial contamination.

[0370] Those skilled in the art will be able to readily determine the appropriate formulations of the protein molecules of the invention using conventional methods. Formulation and administration techniques may be found, for example, in Remington (1980) Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., latest edition; and Niazi (2009) Handbook of Pharmaceutical Manufacturing Formulations, Informa Healthcare, New York, second edition, the entire contents of which are incorporated by reference.

[0371] The identification of preferred pH ranges and suitable excipients (such as antioxidants) is routine in the art, for example as described in Katdare and Chaubel (2006) in Excipient Development for Pharmaceutical, Biotechnology and Drug Delivery Systems (CRC Press). Buffer systems are routinely used to provide a desired range of pH values ​​and may include, but are not limited to, carboxylic acid buffers such as acetate, citrate, lactate, tartrate, and succinate; glycine; histidine; phosphate; tris (hydroxymethyl) aminomethane (Tris); arginine; sodium hydroxide; glutamic acid; and carbonate buffers. Suitable antioxidants may include, but are not limited to, phenolic compounds such as butylated hydroxytoluene (BHT) and butylated hydroxyanisole; vitamin E; ascorbic acid; reducing agents such as methionine or sulfites; metal chelators such as ethylenediaminetetraacetic acid (EDTA); cysteine ​​hydrochloride; sodium bisulfite; sodium metabisulfite; sodium sulfite; ascorbyl palmitate; lecithin; propyl gallate; and alpha-tocopherol.

[0372] For injection, the protein molecules of the invention can be formulated in aqueous solution, suitably in a physiologically compatible buffer such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0373] The compositions of the present invention can be formulated for administration in the form of a liquid containing an acceptable diluent (such as saline and sterile water), or in the form of an emulsion, cream or gel containing an acceptable diluent or carrier to impart the desired texture, consistency, viscosity and appearance. Acceptable diluents and carriers are familiar to those skilled in the art and include, but are not limited to, ethoxylated and non-ethoxylated surfactants, fatty alcohols, fatty acids, hydrocarbon oils (such as palm oil, coconut oil and mineral oil), cocoa butter wax, silicone oil, pH balancing agents, cellulose derivatives, emulsifiers (such as nonionic organic and inorganic bases), preservatives, wax esters, steroids, triglycerides, phospholipids (such as lecithin and cephalin), polyol esters, fatty alcohol esters, hydrophilic lanolin derivatives and hydrophilic beeswax derivatives.

[0374] Alternatively, the protein molecules of the present invention can be formulated into dosages suitable for oral administration using pharmaceutically acceptable carriers well known in the art, which is also the practice contemplated by the present invention. Such carriers enable the bioactive agents of the present invention to be formulated into dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. These carriers can be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline and pyrogen-free water.

[0375] Pharmaceutical formulations for parenteral administration include aqueous solutions of the protein molecules of the present invention in water-soluble form. In addition, suspensions of the protein molecules of the present invention can be prepared into suitable oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil or synthetic fatty acid esters such as ethyl oleate or triglycerides. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound to allow the preparation of high concentration solutions.

[0376] Sterile solutions can be prepared by merging the required amount of active compound with other excipients as described above in an appropriate solvent, and then sterilizing (such as filtering) processing. Typically, dispersions are prepared by incorporating various sterilized active compounds into sterile vehicles, which contain basic dispersion media and required excipients as described above. Sterile dry powders can be prepared by vacuum or freeze drying sterile solutions, which contain active compounds as described above and other required excipients.

[0377] Pharmaceutical preparations for oral use can be obtained by combining the protein molecules of the present invention with solid excipients, and processing the granular mixture after adding suitable adjuvants (if necessary) to obtain tablets or dragee cores. Suitable excipients are specifically fillers, such as sugars, including lactose, sucrose, mannitol or sorbitol; cellulose preparations, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone (PVP). If necessary, disintegrants such as cross-linked polyvinylpyrrolidone, agar or alginic acid or its salts, such as sodium alginate, can be added. Such compositions can be prepared by any pharmaceutical method, but all methods include the step of associating one or more therapeutic agents as described above with a carrier constituting one or more necessary ingredients. Generally, the pharmaceutical composition of the present invention can be prepared in a manner known per se, for example, by means of conventional mixing, dissolving, granulating, making dragees, grinding, emulsifying, encapsulating, embedding or lyophilizing processes.

[0378] Suitable coatings are provided for dragee cores. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talcum powder, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyes or pigments may be added to tablets or dragee coatings to facilitate identification or characterization of different combinations of granule dosages.

[0379] Drugs that can be used orally include press-fit capsules made of gelatin, and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Such push-fit capsules can contain active ingredients, which are mixed with fillers such as lactose, adhesives such as starch, and / or lubricants such as talcum or magnesium stearate and optionally stabilizers. In soft capsules, the active compound can be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycol. In addition, stabilizers can also be added.

[0380] The protein molecules of the invention can be incorporated into modified release preparations and formulations, such as polymer microsphere formulations, and oil-based formulations or gel-based formulations.

[0381] In specific embodiments, the protein molecules of the invention may be administered locally rather than systemically, such as by direct injection of the protein molecule into a tissue, preferably the subcutaneous tissue or omental tissue, typically in a long-acting or sustained release formulation.

[0382] In addition, the protein molecules of the present invention can be used in targeted drug delivery systems, such as in particles, which can be appropriately targeted to cells or tissues and selectively absorbed by the cells or tissues. In some embodiments, the protein molecules of the present invention are contained in or otherwise associated with a medium selected from the following: liposomes, micelles, dendritic molecules, biodegradable particles, artificial DNA nanostructures, lipid-based nanoparticles, and carbon or gold nanoparticles. In illustrative examples of this type, the medium is selected from poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly (lactic acid-co-glycolic acid) (PLGA), polyethylene glycol (PEG), PLA-PEG copolymers, and combinations thereof.

[0383] In cases of local administration or selective uptake, the effective local concentration of the agent may not be related to plasma concentration.

[0384] It is advantageous to formulate the compositions in dosage unit form for ease of administration and uniformity of dosage. The determination of the novel dosage unit form of the present invention is dictated by and directly dependent upon the unique characteristics of the active materials, the specific therapeutic effect to be achieved, and the limitations inherent in the art of compounded active materials for treating diseases in living subjects having diseased conditions with compromised physical health as disclosed in detail herein.

[0385] Although the protein molecules of the present invention may be the only active ingredient administered to a subject, other cancer therapies administered simultaneously with the protein molecules are also within the scope of the present invention. For example, the protein molecules of any one of Formulas I-IV, any one of SEQ ID NOs: 2-6, or the variants described herein can be administered simultaneously with one or more cancer therapies, non-limiting examples of which include radiotherapy, surgery, chemotherapy, hormone ablation therapy, pro-apoptotic therapy, and immunotherapy. The protein molecules of the present invention can be used therapeutically before treatment with a cancer therapy, can be used therapeutically after a cancer therapy, or can be used therapeutically together with a cancer therapy.

[0386] Suitable radiotherapy includes radiation and waves that induce DNA damage, for example, gamma-irradiation, X-rays, UV irradiation, microwaves, electron emission and radioisotopes. Typically, therapy can be achieved by irradiating the local tumor site with the above forms of radiation. Most likely, all of these factors cause extensive damage to DNA, its precursors, the replication and repair of DNA, and the assembly and maintenance of chromosomes.

[0387] The dosage range of X-rays is from a dosage of 50-200 roentgens per day for an extended period of time (such as 3-4 weeks) to a single dose of 2000-6000 roentgens. The dosage range of radioisotopes varies greatly and depends on the half-life of the isotope, the intensity and type of the radiation emitted, and the uptake of tumor cells. Suitable radiotherapy may include, but is not limited to, conformal external beam radiotherapy (50-100 grays, given in fractions over 4-8 weeks), single or fractionated high-dose brachytherapy, permanent interstitial brachytherapy, and systemic radioisotopes, such as strontium 89. In certain embodiments, radiotherapy may be administered with a radiosensitizer. Suitable radiosensitizers may include, but are not limited to, efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin, and tirapazamine.

[0388] Suitable chemotherapeutic agents may include, but are not limited to, antiproliferative / antineoplastic drugs and combinations thereof, including alkylating agents (e.g., cisplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, and nitrosoureas), antimetabolites (e.g., antifolates, such as 5-fluorouracil and tegafur, fluoropyridines such as raltitrexed, methotrexate, cytarabine, and hydroxyurea), antitumor antibiotics (e.g., anthracene drugs, such as doxorubicin, bleomycin, doxorubicin, daunorubicin, epirubicin, idarubicin, mitomycin-C, dactinomycin, and mithramycin), antimitotic agents (e.g., vinca alkaloids, such as vincristine, vinblastine, vindesine, and vinorelbine, and taxanes, such as paclitaxel and docetaxel), and topoisomerase inhibitors (e.g., epipodophyllotoxins, such as etoposide and teniposide, amsacrine, topotecan, and camptothecin); cytostatic agents, Such as antiestrogens (e.g., tamoxifen, toremifene, raloxifene, droloxifene and idoxifene), estrogen receptor downregulators (e.g., fulvestrant), antiandrogens (e.g., bicalutamide, flutamide, nilutamide and cyproterone acetate), UH antagonists or LHRH agonists (e.g., goserelin, leuprolide and buserelin), progestins (e.g., megestrol acetate), aromatase inhibitors (e.g., anastrozole, letrozole, vorozole and exemestane) and 5a reductase inhibitors, such as finasteride; agents that inhibit cancer cell invasion (e.g., metalloproteinase inhibitors, such as marimastat and urokinase plasminogen activator receptor function inhibitors); growth factor function inhibitors, such as such inhibitors include growth factor antibodies, growth factor receptor antibodies (e.g., anti-erbB2 antibody trastuzumab (HERCEPTIN TM ) and the anti-EGFR antibody cetuximab (C225)), farnesyl transferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine / threonine kinase inhibitors, such as other inhibitors of the epidermal growth factor family (e.g., other EGFR family tyrosine kinase inhibitors, such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, AZD1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (Cl 1033), such as inhibitors of the platelet-derived growth factor family and inhibitors of the hepatocyte growth factor family; anti-angiogenic agents, such as agents that inhibit the action of vascular endothelial growth factor (e.g., the anti-vascular endothelial growth factor antibody bevacizumab (AVASTIN TM), compounds disclosed in International Patent Publication Nos. WO 97 / 22596, WO 97 / 30035, WO 97 / 32856 and WO 98 / 13354), and compounds that act by other mechanisms (e.g., linoamide, integrin anb3 function inhibitors and angiostatin); cyclin-dependent kinase inhibitors such as palbocidib, abemacidib, riboddib and alvadoddib; vascular damaging agents such as Compretin A4 and International Patent Publication Nos. WO 99 / 02166, WO 00 / 40529, WO 00 / 41669, WO 01 / 92224, WO Compounds disclosed in 02 / 04434 and WO02 / 08213; antisense therapy, such as therapy directed against the above targets, such as ISIS2503, an anti-ras antisense therapy; and gene therapy methods, including, for example, methods of replacing abnormal genes, such as abnormal p53 or abnormal GDEPT (gene-directed enzyme prodrug therapy) methods, such as methods using cytosine deaminase, thymidine kinase or bacterial nitroreductase, and methods of improving a patient's tolerance to chemotherapy or radiation therapy, such as multidrug resistance gene therapy.

[0389] Suitable immunotherapy methods may include, but are not limited to, in vitro and in vivo methods to increase the immunogenicity of patient tumor cells, such as transfection with cytokines, including interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor; methods to reduce T cell anergy; methods using transfected immune cells, such as dendritic cells transfected with cytokines; methods using tumor cell lines transfected with cytokines; and methods using anti-idiotypic antibodies. These methods generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Immune effectors can be antibodies that have specificity for some markers on the surface of malignant cells, for example. A single antibody can serve as an effector of therapy or it can recruit other cells to actually promote cell killing. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chains, cholera toxin, pertussis toxin, etc.) and serve only as targeting agents. Alternatively, effectors can be lymphocytes carrying surface molecules that interact directly or indirectly with malignant cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0390] In some embodiments, the immune effector is a molecule that targets PD-L1, including but not limited to anti-PD-L1 antibodies, non-limiting examples of which include atezolizumab, avelumab, durvalumab, BMS-936559, BMS-935559, International Patent Publication Nos. WO 2013 / 173223, WO 2013 / 079174, WO 2010 / 077634, WO 2011 / 066389, WO 2010 / 036959, WO 2007 / 005874, WO 2004 / 004771, WO 2006 / 133396, WO 2013 / 181634, WO Antibodies described in No. 2012 / 145493 and Chinese Patent Publication No. CN101104640, clone EH12 and clone 29E.2A3; CA-170; CA-327; BMS-202 (N-[2-[[[2-methoxy-6-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]-3-pyridinyl]methyl]amino]ethyl]-acetamide); BMS-8 (1-[[3-bromo-4-[(2-methyl[1,1'-biphenyl]-3-yl)methoxy]phenyl]methyl]-2-piperidinecarboxylic acid); peptides described in Chang et al., (2015), in particular (D)PPA-1; AUNP-12; and peptides described in WO 2014 / 151634, the entire contents of which are incorporated by reference.

[0391] In some embodiments, the immune effector is a molecule targeting PD-1, including but not limited to anti-PD-1 antibodies, non-limiting examples of which include nivolumab, pembrolizumab, BGB-A317, WO 2016 / 106159, WO 2009 / 114335, WO 2004 / 004771, WO 2013 / 173223, WO 2015 / 112900, WO 2008 / 156712, WO 2011 / 159877, WO 2010 / 036959, WO 2010 / 089411, WO 2006 / 133396, WO 2012 / 145493, WO Antibodies described in WO 2002 / 078731, anti-mouse PD-1 antibody clone J43, anti-mouse antibody clone RMP1-14, ANB011 (TSR-042), AMP-514 (MEDI0680), WO 2006 / 121168, WO 2001 / 014557, WO 2011 / 110604, WO 2011 / 110621, WO2004 / 072286 Al, WO 2004 / 056875, WO 2010 / 036959, WO 2010 / 029434 and WO 2013 / 02209; AMP-224; compounds described in WO 2011 / 082400; molecules and antibodies described in U.S. Pat. No. 6,808,710; WO The molecules and antibodies described in WO 2013 / 019906; the molecules described in WO 2003 / 011911; and the compounds described in WO 2013 / 132317, the entire contents of which are incorporated by reference.

[0392] In some embodiments, the immune effector is a molecule that targets PD-L2, including but not limited to anti-PD-L2 antibodies, non-limiting examples of which include antibodies described in International Patent Publication No. WO 2010 / 036959, the entire contents of which are incorporated by reference; and rHigM12B7.

[0393] In some embodiments, the immune effector is a molecule that targets CTLA-4, including but not limited to anti-CTLA-4 antibodies, such as ipilimumab, tremelimumab, antibodies described in WO 00 / 37504A2, WO 01 / 14424A2, US2003 / 0086930 A1; and compounds described in WO 2006 / 056464 A2, the entire contents of which are incorporated by reference.

[0394] Examples of other cancer therapies include phytotherapy, cryotherapy, toxin therapy, or pro-apoptotic therapy.One skilled in the art will appreciate that this list is not an exhaustive list of the types of treatment modalities that may be used for cancer and other proliferative disorders.

[0395] As is well known, chemotherapy and radiotherapy target rapidly dividing cells and / or destroy the cell cycle or cell division. These treatments are part of the treatment of various forms of cancer, and are intended to delay disease progression or reverse disease symptoms by curative treatment. However, these cancer therapies may lead to immunocompromised states and subsequent pathogenic infections, and therefore, the present invention is also extended to combination therapy, which uses any one of Formulas I-IV or SEQ ID NO: 1-5 protein molecules or variants described herein, cancer therapy and anti-infective agents, which are effective for infections caused by or at risk of immunocompromised symptoms caused by cancer therapy. Anti-infective drugs are suitably selected from antibacterial agents, which may include but are not limited to compounds that kill or inhibit the growth of microorganisms (such as viruses, bacteria, yeasts, fungi, protozoa, etc.), and therefore include antibiotics, anti-amoebic drugs, antifungal drugs, antiprotozoal drugs, antimalarials, anti-tuberculosis drugs and antivirals. Anti-infective drugs are also included in the scope of anthelmintics and nematicides.Exemplary antibiotics include quinolones (e.g., amloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, rosofloxacin, temafloxacin, toloxacin, sparfloxacin, dinaflopax, gatifloxacin, moxifloxacin, gemifloxacin, and garenoxacin), tetracyclines, glycylcyclines, and oxazolidinones (e.g., chlortetracycline, demeclocycline, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tadalafil, tadalafil, oxadiazine, chlortetracycl ... , linezolid, eprazolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirocin, dibekacin, fortimicin, gentamicin, kanamycin, manmomycin, netilmicin, ribostamicin, sisomicin, spectinomycin, streptomycin, tobramycin), beta-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefdroxil, cefamandole, ceftriaxone, cefuroxime, ceftriaxone, cefazolin, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, cefoperazone, cefoperazone, cefotaxime, cefotiam, Ceftriaxone, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, cefditoren, ceftizole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefazolin, cefacetonitrile, cephalexin, cefuroxime, cephalothin, cephapirin, cefadroxil, cefuroxime, cefotaxime, cephalothin, cefpirin, cefadroxil, cefotaxime, cefotaxime, cefoxitin, cefotetan, aztreonam, carumonam, fluoxef, latamoxef, amdinocilin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecilin ), doxacillin, didoxacillin, methicillin, mezlocillin, nacillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticarcillin, cefditoren, SC004, KY-020, cefdinir, cefbutan, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefazolin, ME1228, KP-736, CP-6232, Ro 09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rotamycin, roxamicin, roxithromycin, troleandomycin), ketolides (e.g., telithromycin, sirithromycin), kumamycins, lincosamides (e.g., clindamycin, lincomycin), and chloramphenicol.

[0396] Illustrative antiviral drugs include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, formosan sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine, lamivudine / zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacidovir hydrochloride, zalcitabine, zanamivir, and zidovudine.

[0397] Suitable anti-amoebic or anti-protozoal drugs include, but are not limited to, atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isosulfate. Anthelmintics may be at least one selected from the following: mebendazole, pyrantel pamoate, albendazole, ivermectin, and thiabendazole. Illustrative antifungal agents may be selected from amphotericin B, amphotericin B-cholesterol sulfate complex, amphotericin B lipid complex, amphotericin B liposomes, fluconazole, flucytosine, griseofulvin microparticles, griseofulvin ultramicroparticles, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. Suitable antimalarials include, but are not limited to, chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. Anti-tuberculosis drugs include, but are not limited to, clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampicin, rifapentine, and streptomycin sulfate.

[0398] As previously described, protein molecules can be compounded with suitable pharmaceutically acceptable carriers in dosage unit form to conveniently and effectively administer in effective amounts. In certain embodiments, unit dosage forms can include active peptides of the present invention in the range of about 0.25 pg to about 2000 mg. Active peptides of the present invention can be present in the amount of carriers of about 0.25 pg to about 2000 mg / mL. In embodiments where the pharmaceutical composition comprises one or more additional active ingredients, dosage is determined with reference to the usual dose and mode of administration of the composition.

[0399] 4. Treatment Methods

[0400] The inventors have determined that blocking the nuclear localization of SETDB1 would be an effective treatment for diseases such as cancer (e.g., metastatic cancer). Therefore, in some embodiments, the present invention provides a method for treating cancer in a subject, the method comprising administering to the subject an agent that prevents or reduces the nuclear localization of SETDB1.

[0401] In some embodiments, the agent comprises a protein molecule described above or elsewhere herein. For example, the protein molecule may comprise an amino acid sequence corresponding to any one of formula (I)-(IV).

[0402] Without wishing to be bound by theory, the inventors have determined that SETDB1 is enriched in the nuclei of metastatic initiating tumor cells and dysfunctional terminally exhausted CD8+T cells in subjects resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). In contrast, SETDB1 was found to be enriched in the cytoplasm and / or cell surface of subjects responsive to immunotherapy (e.g., immune checkpoint inhibitor therapy). Therefore, it has been proposed that by preventing nuclear localization of SETDB1 polypeptides in cells of a subject, the likelihood of resistance to immunotherapy (e.g., immune checkpoint inhibitor therapy) can be prevented or reduced.

[0403] Thus, in another aspect of the invention, there is provided a method of inhibiting or reducing nuclear localization of SETDB1, the method comprising contacting a cell with a protein molecule as described above and elsewhere herein.

[0404] In some embodiments, the protein molecule is an isolated or purified protein molecule represented by any one of formula (I)-(IV); in particular, a protein molecule of any one of SEQ ID NOs: 2-6 or a variant protein molecule described herein.

[0405] In another aspect of the present invention, there is provided the use of the isolated or purified protein molecules of the present invention, in particular the protein molecules of any one of formula I-IV, SEQ ID NO: 2-6 or the variant protein molecules described herein, for use in therapy or for the preparation of a medicament for therapy. The present invention also provides the isolated or purified protein molecules of the present invention, in particular the protein molecules of formula (I)-(IV), SEQ ID NO: 2-6 or the variant protein molecules described herein, for use in therapy.

[0406] The present invention also provides a method for inhibiting or reducing nuclear localization of SETDB1 in a SETDB1 overexpressing cell, the method comprising contacting the cell with an inhibitor of binding between a SETDB1 polypeptide and an importin-α polypeptide. In some embodiments, the inhibitor comprises a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS. The present invention also contemplates the use of a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS for inhibiting or reducing nuclear localization of SETDB1 in a SETDB1 overexpressing cell; the use of a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS for inhibiting or reducing nuclear localization of SETDB1 in a SETDB1 overexpressing cell; and for preparing a medicament for such use.

[0407] In some embodiments of any of the aforementioned aspects, the SETDB1 overexpressing cell is a cancer stem cell or a non-cancer stem cell tumor cell, in particular a cancer stem cell tumor cell.

[0408] Suitable examples of protein molecules are described herein.

[0409] A protein molecule comprising, consisting of or consisting essentially of an amino acid sequence corresponding to an acetylation site as described herein, in particular a protein molecule of any one of formula (I)-(IV), SEQ ID NO: 2-6 or a variant protein molecule can be used to inhibit nuclear localization of SETDB1. Therefore, the inventors believe that the protein molecule can be used to treat or prevent cancer in a subject. Therefore, in another aspect, a method for treating or preventing cancer in a subject, wherein the cancer comprises at least one SETDB1 overexpressing cell, is provided, the method comprising administering to the subject a protein molecule comprising, consisting of or consisting essentially of an amino acid sequence corresponding to an acetylation site. The present invention also extends to the use of a protein molecule comprising, consisting of or consisting essentially of an amino acid sequence corresponding to an acetylation site for treating or preventing cancer in a subject, wherein the cancer comprises at least one SETDB1 overexpressing cell; and the preparation of a medicament for this purpose. Also contemplated is the use of a SETDB1 bicyclic peptide mimetic comprising, consisting of or consisting essentially of an amino acid sequence corresponding to an acetylation site for treating or preventing cancer in a subject, wherein the cancer comprises at least one SETDB1 overexpressing cell.

[0410] The cancer may be any cancer involving overexpression of SETDB1. Suitable cancers may include, but are not limited to, breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, colon cancer, liver cancer, ovarian cancer, kidney cancer or brain cancer, or melanoma or retinoblastoma; in particular breast cancer, lung cancer or melanoma; most in particular breast cancer or melanoma; more in particular breast cancer.

[0411] In some embodiments, protein molecules comprising, consisting of or consisting essentially of an amino acid sequence corresponding to an acetylation site as described herein can be used to treat, prevent and / or alleviate symptoms of malignant tumors, particularly metastatic cancers. In a preferred embodiment, SETDB1 bicyclic peptide mimetics are used to treat, prevent and / or alleviate symptoms of metastatic cancers. Suitable types of metastatic cancers include, but are not limited to, metastatic breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, colon cancer, liver cancer, ovarian cancer, kidney cancer or brain cancer, or melanoma or retinoblastoma. In some embodiments, brain cancer is a glioma. In a preferred embodiment, metastatic cancer is metastatic breast cancer, lung cancer or melanoma; in particular, metastatic breast cancer or melanoma; most particularly, metastatic breast cancer.

[0412] The protein molecule can be used in a method involving a SETDB1 overexpressing cell. In a specific embodiment, the SETDB1 overexpressing cell is selected from a breast, prostate, testis, lung, bladder, pancreas, colon, melanoma, leukemia, retinoblastoma, liver, ovary, kidney or brain cell; in particular a breast, lung or melanoma cell; most particularly a breast or melanoma cell; more particularly a breast cell. In a preferred embodiment, the SETDB1 overexpressing cell is a mammary epithelial cell, in particular a mammary ductal epithelial cell.

[0413] In some embodiments, the SETDB1 overexpressing cells are cancer stem cells or non-cancer stem cell tumor cells; in particular, cancer stem cell tumor cells; most particularly, breast cancer stem cell tumor cells. In some embodiments, the cancer stem cell tumor cells express CD24 and CD44, specifically low CD44. 高 、CD24 低 .

[0414] In some embodiments, the method further comprises detecting overexpression of the SETDB1 gene in a tumor sample obtained from the subject prior to administering the protein molecule to the subject, wherein the tumor sample comprises cancer stem cell tumor cells and optionally non-cancer stem cell tumor cells.

[0415] Protein molecules comprising, consisting of, or consisting essentially of amino acid sequences corresponding to lysine methylation sites as described herein are suitable for treating individuals diagnosed with cancer, individuals suspected of having cancer, individuals known to be susceptible and considered likely to develop cancer, or individuals considered to have recurred cancer from a previously treated cancer. Cancer can be hormone receptor negative. In some embodiments, cancer is hormone receptor negative and is therefore resistant to hormone or endocrine therapy. In some embodiments where the cancer is breast cancer, the breast cancer is hormone receptor negative. In some embodiments, the breast cancer is estrogen receptor negative and / or progesterone receptor negative.

[0416] There are many situations involving overexpression of SETDB1 in which a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to a lysine methylation site as described herein may be useful. Thus, in another aspect of the present invention, a method of treating or preventing a condition associated with inhibition of SETDB1 nuclear localization or reduction of effective treatment in a subject is provided, the method comprising administering to the subject a protein molecule consisting of, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site. The present invention also provides the use of a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site for treating or preventing a condition associated with inhibition of SETDB1 nuclear localization in a subject or reduction of effective treatment; a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site for treating or preventing a condition associated with inhibition of SETDB1 nuclear localization in a subject or reduction of effective treatment; and a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site for preparing a medicament for this purpose.

[0417] Non-limiting examples of conditions involving overexpression of SETDB1 include cancer, infection, autoimmune disorders, and respiratory disorders.

[0418] In some embodiments, the infection is a pathogenic infection. The infection may be selected from, but is not limited to, a virus, a bacterium, a yeast, a fungus, a worm or a protozoan infection. The viral infection contemplated by the present invention includes, but is not limited to, an infection caused by HIV, hepatitis, influenza virus, Japanese encephalitis virus, Epstein-Barr virus, herpes simplex virus, filovirus, human papillomavirus, human T-cell lymphotropic virus, human retrovirus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, rubella virus, mumps virus, adenovirus, enterovirus, rhinovirus, Ebola virus, West Nile virus and respiratory syncytial virus; especially an infection caused by HIV, hepatitis, influenza virus, Japanese encephalitis virus, Epstein-Barr virus and respiratory syncytial virus. Bacterial infections include, but are not limited to, infections caused by Neisseria species, Meningococcal species, Haemophilus species, Salmonella species, Streptococcal species, Legionella species, Mycoplasma species, Bacillus species, Staphylococcus species, Chlamydia species, Actinomyces species, Anabaena species, Bacteriodes species, Bdellovibrio species, Bordetella species, Borrella species, Campylobacter species, Caulobacter species, Chlorobium species, Chromatium species, Clostridium species, species), Corynebacterium species, Cytophaga species, Deinococcus species, Escherichia species, Francisella species, Helicobacterspecies, Haemophilus species, Hyphomicrobium species, Leptospira species, Usteria species, Micrococcus species, Myxococcus species, Nitrobacter species, Oscillatoria species, Prochloron species, Proteus species, Pseudomonas species, Rhodospirillum species, Rickettsia species, Shigella species, Spirillum species, Spirochaeta species, Streptomyces species, Thiobacillus species, Treponema species, Vibriospecies, Yersinia species, Nocardia species, species) and mycobacterium species; in particular infections caused by Neisseria, meningococci, Haemophilus, Salmonella, Streptococcus, Legionella and mycobacteria. Protozoan infections encompassed by the present invention include, but are not limited to, infections caused by Plasmodium species, Lishmania species, Trypanosoma species, Toxoplasma species, Entamoeba species and Giardia species. Helminthal infections may include, but are not limited to, infections caused by Schistosoma species. Fungal infections contemplated by the present invention include, but are not limited to, infections caused by Histoplasma species and Candida species.

[0419] Suitable autoimmune disorders include, but are not limited to, autoimmune rheumatic disorders (e.g., rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus, such as systemic lupus erythematosus (SLE) and lupus nephritis, polymyositis dermatomyositis, cryoglobulinemia, antiphospholipid antibody syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver disorders (e.g., inflammatory bowel disease, such as ulcerative colitis and Crohn's disease, autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (e.g., antineutrophil cytoplasmic antibody (ANCA)-negative vasculitis and ANCA-associated vasculitis), including Churg-Strauss vasculitis, Wegener's granulomatosis, and inflammatory bowel disease. granulomatosis, and microscopic polyangiitis), autoimmune nervous system disorders (e.g., multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, neuromyelitis, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), renal disorders (e.g., glomerulonephritis, Goodpasture's syndrome, and Berger's disease), disease), autoimmune skin disorders (e.g., psoriasis, urticaria, hives, pemphigus vulgaris, bullous pemphigoid, and cutaneous lupus erythematosus), blood disorders (e.g., thrombocytopenic purpura, thrombotic thrombocytopenic purpura, post-transfusion purpura, and autoimmune hemolytic anemia), atherosclerosis, uveitis, autoimmune hearing disorders (e.g., inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplantation, and autoimmune endocrine disorders (e.g., diabetes-related autoimmune diseases such as type I diabetes, Addison's disease, and autoimmune thyroid diseases (e.g., Graves' disease and thyroiditis)).

[0420] Suitable respiratory disorders include, but are not limited to, chronic obstructive pulmonary disease (CORD) or asthma, especially allergic asthma.

[0421] In some embodiments, the method further comprises detecting overexpression of the SETDB1 gene in a tumor sample obtained from the subject before administering the protein molecule of the present invention to the subject, wherein the tumor sample comprises cancer stem cell tumor cells and optionally non-cancer stem cell tumor cells.

[0422] In specific embodiments, any of the above methods involve administration of one or more additional active agents as described in Section 3 above, such as additional cancer therapies and / or anti-infective agents, particularly additional cancer therapies.

[0423] Protein molecules of the invention as described herein, in particular protein molecules or variant protein molecules of any one of formula (I)-(IV) or SEQ ID NO: 2-6, can be used to inhibit or reduce the acetylation of SETDB1. In some embodiments, the acetylation is catalyzed by an acetyltransferase; in particular, by a histone acetyltransferase. In some embodiments, the histone acetyltransferase is GCN5, Hat1, ATF-2, Tip60, MOZ, MORF, HBO1, p300, CBP, SRC-1, ACTR, TIF-2, SRC-3, TAF1, TFIIIC and / or CLOCK; in particular, p300.

[0424] Thus, in a further aspect of the invention, a method of inhibiting nuclear localization of SETDB1 in a subject is provided, the method comprising administering a protein molecule as described herein. The invention also extends to the use of a protein molecule as described herein for inhibiting or reducing the binding of a SETDB1 polypeptide to an importin polypeptide. In a preferred embodiment, the protein molecule of the invention prevents the importin polypeptide from transporting SETDB1 to the nuclear compartment.

[0425] The present invention also contemplates a method for producing a protein molecule that inhibits or reduces nuclear localization of a SETDB1 polypeptide, the method comprising:

[0426] a) contacting a cell with a protein molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS of SETDB1; and

[0427] b) detecting a reduction or inhibition of the nuclear localization of the SETDB1 polypeptide in the cell relative to a normal or reference level of nuclear localization in the absence of the protein molecule.

[0428] In another aspect, the present invention provides a method for producing a SETDB1 bicyclic peptide mimetic that inhibits or reduces the nuclear localization of SETDB1, wherein the methylation of the methylation site of SETDB1 increases its nuclear localization in a cell, the method comprising:

[0429] a) contacting a cell with a SETDB1 bicyclic peptide mimetic comprising, consisting of, or consisting essentially of an amino acid sequence as shown in any one of formula (I) or a bicyclic peptide of any one of formulas (II)-(V); and

[0430] b) detecting a reduction or inhibition of nuclear localization of the nuclear localization polypeptide in a cell relative to a normal or reference level of nuclear localization in the absence of the SETDB1 bicyclic peptide mimetic.

[0431] In some embodiments, the SETDB1 bicyclic peptide mimetic differs from the native wild-type SETDB1 sequence by the addition of at least three cysteine ​​residues.

[0432] Reduction or inhibition of nuclear localization of SETDB1 can be determined using standard techniques in the art, non-limiting examples of which include immunofluorescence, immunohistochemical staining, chromatin immunoprecipitation (ChIP), ChIP-seq, chromatin accessibility assays such as DNase-seq, FAIRE-seq, and ATAC-seq assays, as described in Satelli et al., (2016) Sci Rep, 6:28910; Bajetto et al., (2000) Brain Research Protocols, 5(3):273-281; ​​and Sung et al., (2014) BMC Cancer, 14:951, the entire contents of which are incorporated by reference.

[0433] Those skilled in the art will be well aware of suitable assays for assessing the nuclear localization of polypeptides such as SETDB1 and identifying SETDB1 bicyclic peptide mimetics that inhibit or reduce the nuclear localization of polypeptides. Screening for active agents according to the present invention may be achieved by any suitable method. For example, the method may comprise contacting a cell expressing a polynucleotide corresponding to a gene encoding a polypeptide of interest such as SETDB1 with an agent suspected of having inhibitory activity, and screening for inhibition or reduction of the level of the polypeptide of interest in the nucleus of the cell.

[0434] Alternatively, one may screen for inhibition of the functional activity of a polypeptide of interest or reduction of the level of a transcript encoded by a polynucleotide, or inhibition of the activity or expression of a downstream cellular target of a polypeptide or transcript, wherein the activity is associated with nuclear localization of SETDB1. Detection of such inhibition may be accomplished using techniques including, but not limited to, ELISA, immunofluorescence, Western blot, immunoprecipitation, immunostaining, slit or dot blot assays, scintillation proximity assays, fluorescent immunoassays using antigen binding molecule conjugates or antigen conjugates of fluorescent substances such as fluorescein or rhodamine, RIA, Ouchterlony double diffusion analysis, immunoassays using avidin-biotin or streptavidin-biotin detection systems, nucleic acid detection assays including reverse transcription polymerase chain reaction (RT-PCR), cell proliferation assays such as WST-1 proliferation assays, and immunoblot analysis of cells treated with SETDB1 Half-Way ChIP. Methylation of a polypeptide may be determined using antibodies against methylated polypeptides, such as antibodies against methylated lysine residues.

[0435] Active molecules can be further tested in animal models to identify those with the most potent in vivo effects. These molecules can serve as lead molecules for additional development of drugs, for example, by sequential modification of compounds, molecular modeling, and other routine procedures employed in rational drug design.

[0436] 5. Detection and prognostic / predictive methods

[0437] According to the present disclosure, nuclear localization of SETDB1 can be used as a biomarker responsive to therapy (e.g., immunotherapy). In some embodiments, nuclear localization of SETDB1 is determined by detecting co-localization of SETDB1 with a SETDB1 binding partner (e.g., ATF7IP or IMPα). Nuclear localization of SETDB1 is appropriately assessed in cells expressing SETDB1, such as, but not limited to, tumor cells. Representative cell-containing subject samples expressing SETDB1 include tissue samples, such as solid tumors. In some embodiments, the sample is obtained prior to treatment with therapy. In some embodiments, the tissue sample is formalin-fixed and paraffin-embedded, archived, fresh or frozen.

[0438] The presence and / or level / amount of a biomarker (e.g., SETDB1 (e.g., nuclear SETDB1, cytoplasmic SETDB1, etc.) or a complex comprising SETDB1 and a SETDB1 binding partner (e.g., ATF7IP, IMPα), collectively referred to herein as a "biomarker responsive to therapy" or "RTT biomarker") at a cellular location (e.g., cytoplasm or nucleus) can be determined qualitatively and / or quantitatively based on any suitable criteria known in the art. In certain embodiments, the presence and / or level / amount of the biomarker at a cellular location in a first sample is increased or elevated compared to the presence / absence and / or level / amount of the RTT biomarker at a cellular location in a second sample. In certain embodiments, the presence / absence and / or level / amount of the biomarker at a cellular location in a first sample is decreased or reduced compared to the presence and / or expression level / amount of the RTT biomarker at a cellular location in a second sample.

[0439] In some embodiments of any of the methods, an elevated or higher level / amount refers to an overall increase of about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level / amount of the RTT biomarker at a cellular location, e.g., as detected by standard art-known methods as described herein. In certain embodiments, an increase or higher level / amount refers to an increase in the level / amount of an RTT biomarker at a sample cell location, wherein the increase is at least about 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.75 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 25 times, 50 times, 75 times, or 100 times the level / amount of an RTT biomarker at a cell location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, an increase or higher level / amount refers to an overall increase at a cell location that is greater than about 1.5 times, about 1.75 times, about 2 times, about 2.25 times, about 2.5 times, about 2.75 times, about 3.0 times, or about 3.25 times that of a cell location in a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, or internal control.

[0440] In some embodiments of any of the methods, an elevated or higher level / amount refers to a ratio of the level / amount of an RTT biomarker at a first cellular location (e.g., nuclear SETDB1) to the level / amount of an RTT biomarker at a second cellular location (e.g., cytoplasmic SETDB1), wherein the ratio is greater than any of about 0.55, 0.60, 0.65, 0.70, 0.75, 0.85, 0.90, or 0.95.

[0441] In some embodiments of any of the methods, elevated or higher levels / amounts refer to greater than about any of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the cells in the subject having a higher level of the RTT biomarker.

[0442] In some embodiments of any method, reduction or lower level / amount refers to an overall reduction of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the level / amount of RTT in a cell location as detected by standard art-known methods as described herein, compared to the level / amount at a cell location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In certain embodiments, reduction or lower level / amount refers to a reduction in the level / amount of RTT biomarker in a cell location in a sample, wherein the reduction is at least about 0.9x, 0.8x, 0.7x, 0.6x, 0.5x, 0.4x, 0.3x, 0.2x, 0.1x, 0.05x or 0.01x of the level / amount of RTT biomarker in a cell location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0443] The presence and / or level / amount of various RTT biomarkers in a sample can be analyzed by a variety of methods, many of which are known in the art and will be understood by those skilled in the art, including but not limited to immunohistochemistry ("IHC"), western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), proteomics.

[0444] According to some embodiments, the presence and / or level / amount is measured by observing the protein level / amount. In certain embodiments, the method comprises contacting the sample with an antibody to a RTT biomarker (e.g., an anti-SETDB1 antibody), optionally in combination with an antibody to at least one SETDB1 binding partner (e.g., ATF7IP or IMPα), under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibody / antibody and the biomarker.

[0445] Such methods can be in vitro or in vivo methods.In some embodiments, one or more anti-RTT biomarker antibodies are used to select subjects eligible for treatment with a therapy (eg, immunotherapy).

[0446] In certain embodiments, the presence and / or level / amount of biomarker proteins in a sample are checked using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method for determining or detecting the presence or level / amount of proteins in a sample, including the cellular localization of proteins. In some embodiments, the level / amount of RTT biomarkers is determined using a method comprising: (a) performing IHC analysis on a sample (such as a tumor sample) with an antibody; and b) determining the level / amount of biomarkers at a cellular location (e.g., nucleus or cytoplasm) in the sample. In some embodiments, the IHC staining intensity is determined relative to a reference. In some embodiments, the reference is a reference value. In some embodiments, the reference is a reference sample (e.g., a control cell line staining sample or a tissue sample from a non-cancerous subject).

[0447] In some embodiments, the presence and / or level / amount of RTT biomarkers are assessed on a tumor or tumor sample. As used herein, a tumor or tumor sample may encompass part or all of a tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample may further encompass a tumor area occupied by tumor-related intratumor cells and / or tumor-related matrix (e.g., continuous peritumoral desmoplastic matrix). Tumor-related intratumor cells and / or tumor-related matrix may include an immune infiltration area (e.g., tumor-infiltrating immune cells as described herein) adjacent to and / or adjacent to the main tumor mass. In some embodiments, the expression of RTT biomarkers is assessed on tumor cells.

[0448] In alternative methods, the sample can be contacted with an antibody specific for the RTT biomarker under conditions sufficient to form an antibody-biomarker complex, and then the complex is detected. The presence or level / amount of the biomarker can be detected in a variety of ways, such as by Western blotting and ELISA procedures to determine various tissues and samples, including plasma or serum. A variety of immunoassay techniques using this assay format are available (see, for example, U.S. Patents Nos. 4,016,043, 4,424,279, and 4,018,653). These include single-site and dual-site or "sandwich" detections of non-competitive types, as well as traditional competitive binding assays. These assays also include the direct binding of labeled antibodies to target biomarkers.

[0449] In certain embodiments, the samples are normalized for the differences in the amount of the biomarkers measured and the variability of the quality of the samples used and the variability between the assay runs. This normalization can be achieved by detecting and incorporating the expression of certain normalized biomarkers, including the expression products of well-known housekeeping genes. Alternatively, normalization can be based on the mean or median signal of all assayed proteins or most of their subsets (global normalization method). On a protein-by-protein basis, the measured normalized amount of the subject's tumor protein is compared with the amount in the reference set. The normalized level of each protein of each tested tumor of each subject can be expressed as a percentage of the expression level measured in the reference set. The presence and / or level / amount measured in the specific subject sample to be analyzed will fall into a certain percentile within this range, which can be determined by methods well known in the art.

[0450] In certain embodiments, the sample is a clinical sample. In other embodiments, the sample is used in a diagnostic assay. In certain embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is generally used to obtain a representative tumor tissue piece. Alternatively, tumor cells can be obtained indirectly in the form of a tissue or body fluid known to be or believed to contain tumor cells of interest. For example, lung cancer lesion samples can be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushing or from sputum, pleural effusion or blood. Protein can be detected from cancer or tumor tissue or other body samples such as urine, sputum, serum or plasma. Cancer cells can fall off from cancer lesions and appear in such body samples. By screening such body samples, simple early diagnosis of these cancers can be performed. In addition, by detecting the RTT biomarkers of such body samples, it is also easier to monitor the response to therapy.

[0451] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or a combined plurality of samples from the same subject or individual, the sample being obtained at one or more time points different from the time when the test sample is obtained. For example, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from the same subject or individual at a time point earlier than the time 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 when the cancer is initially diagnosed and the test sample is obtained later when the cancer metastasizes or becomes resistant to treatment with the therapy.

[0452] In certain embodiments, 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 of non-subject or individual. In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a combination of multiple samples from one or more individuals of non-subject or individual suffering from disease (for example, cancer). In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a sample of the merging of normal tissue or biological fluid (such as blood) from one or more individuals of non-subject or individual. In certain embodiments, reference sample, reference cell, reference tissue, control sample, control cell or control tissue is a sample of the merging of tumor tissue from one or more individuals of non-subject or individual suffering from disease (for example, cancer) or the blood sample of merging.

[0453] In some embodiments, the sample is a tissue sample from an individual. In some embodiments, the tissue sample is a tumor tissue sample (e.g., a biopsy). In some embodiments, the tissue sample is lung tissue. In some embodiments, the tissue sample is kidney tissue. In some embodiments, the tissue sample is skin tissue. In some embodiments, the tissue sample is pancreatic tissue. In some embodiments, the tissue sample is stomach tissue. In some embodiments, the tissue sample is bladder tissue. In some embodiments, the tissue sample is esophageal tissue. In some embodiments, the tissue sample is mesothelial tissue. In some embodiments, the tissue sample is breast tissue. In some embodiments, the tissue sample is thyroid tissue. In some embodiments, the tissue sample is colorectal tissue. In some embodiments, the tissue sample is head and neck tissue. In some embodiments, the tissue sample is osteosarcoma tissue. In some embodiments, 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, etc.).

[0454] In some embodiments, a tumor tissue sample (the term "tumor sample" is used interchangeably herein) may encompass a portion or all of a tumor region occupied by tumor cells. In some embodiments, a tumor or tumor sample may further encompass a tumor region occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (e.g., continuous peritumoral desmoplastic stroma). Tumor-associated intratumoral cells and / or tumor-associated stroma may include immune-infiltrated regions (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent to and / or adjacent to the main tumor mass.

[0455] In some embodiments, tumor cell staining is expressed as a percentage of all tumor cells that show nuclear staining of any intensity. Infiltrating immune cell staining can be expressed as a percentage of the total tumor area that shows immune cells that stain at any intensity. The total tumor area encompasses malignant cells and tumor-associated stroma, including areas of immune infiltration immediately adjacent to and adjacent to the main tumor mass. Additionally, infiltrating immune cell staining can be expressed as a percentage of all tumor-infiltrating immune cells.

[0456] In some embodiments of the above methods, the disease is a tumor. In certain embodiments, the tumor is a malignant tumor (i.e., cancer). In certain embodiments, the tumor and / or cancer is a solid tumor or a non-solid tumor or a soft tissue tumor. The example of a soft tissue tumor includes leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphocytic leukemia, acute myeloid leukemia, mature B cell acute lymphocytic leukemia, chronic lymphocytic leukemia, protolymphocytic leukemia or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T cell lymphoma or Hodgkin's disease). Solid tumors include any body tissue cancer outside the blood, bone marrow or lymphatic system. Solid tumors can be further divided into epithelial cell derived tumors and non-epithelial cell derived tumors. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectum (e.g., basaloid colorectal cancer), breast, prostate, lung, kidney, liver, pancreas, ovary (e.g., endometrioid ovarian cancer), head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male genitalia, urinary organs (e.g., urothelial carcinoma, dysplastic urothelial carcinoma, transitional cell carcinoma), bladder and skin. Solid tumors of non-epithelial origin include sarcomas, brain tumors and osteomas. In some embodiments, cancer is non-small cell lung cancer (NSCLC). In some embodiments, cancer is second-line or third-line locally advanced or metastatic non-small cell lung cancer. In some embodiments, cancer is adenocarcinoma. In some embodiments, cancer is squamous cell carcinoma. In some embodiments, cancer is non-small cell lung cancer (NSCLC), glioblastoma, neuroblastoma, melanoma, breast cancer (e.g., triple-negative breast cancer), gastric cancer, colorectal cancer (CRC) or hepatocellular carcinoma. In some embodiments, cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site originating from any of the above types of cancer.

[0457] In some embodiments, the RTT biomarker in the sample is detected using a method selected from the group consisting of: FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blot, immunodetection method, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC and combinations thereof. In some embodiments, the RTT biomarker is detected in a blood sample. In some embodiments, the RTT biomarker is detected in circulating tumor cells in a blood sample. Any suitable method can be used to separate / enrich such cell populations, including but not limited to cell sorting. In some embodiments, the level / amount of nuclear SETDB1 is reduced in a sample of an individual who responds to a treatment performed on a certain therapy, and the therapy is appropriately immunotherapy (e.g., a therapy comprising an anti-immune checkpoint molecule antibody, such as an anti-PD-1 antagonist antibody). In some embodiments, the level / amount of nuclear SETDB1 is increased in a sample of an individual who does not respond to or responds weakly to a treatment performed on a certain therapy, and the therapy is appropriately immunotherapy (e.g., a therapy comprising an anti-immune checkpoint molecule antibody, such as an anti-PD-1 antagonist antibody). In some embodiments, the level / amount of extranuclear SETDB1 is reduced in samples of individuals who have no response or weak response to a therapy, and the therapy is an appropriate immunotherapy (e.g., a therapy comprising an anti-immune checkpoint molecule antibody, such as an anti-PD-1 antagonist antibody). In some embodiments, the level / amount of extranuclear SETDB1 is increased in samples of individuals who have responded to a therapy, and the therapy is an appropriate immunotherapy (e.g., a therapy comprising an anti-immune checkpoint molecule antibody, such as an anti-PD-1 antagonist antibody).

[0458] Also provided herein are predictive / diagnostic methods and kits based on the following determination: SETDB1 colocalizes with a nuclear binding partner of SETDB1 (e.g., ATF7IP) in the nucleus, and such colocalization results in, at least in part, resistance or non-responsiveness to a therapy (e.g., immunotherapy) and / or a disease state (e.g., severity or progression of a disease). These methods suitably comprise: (i) obtaining a sample from a subject, wherein the sample comprises cells (e.g., tumor cells) expressing SETDB1; (ii) contacting the sample with a first antigen binding molecule that binds to SETDB1 in the sample and a second antigen binding molecule that binds to a binding partner of SETDB1 in the sample; and (iii) detecting localization of the first antigen binding molecule and the second antigen binding molecule in the nucleus of a cell expressing SETDB1, wherein localization of the first antigen binding molecule and the second antigen binding molecule in the nucleus of a cell expressing SETDB1 indicates an increased likelihood that the cell expressing SETDB1 is resistant to the therapy, that the subject may be a non-responder to the therapy, that the subject is selected not to be treated with the therapy, and / or predicting that the treatment outcome of the subject may be a negative treatment outcome.

[0459] The localization of SETDB1 and the nuclear binding partner of SETDB1 in the nucleus of a cell expressing SETDB1 can be performed using any suitable localization technique, such as by IHC, typically using an anti-SETDB1 antibody having a detectable portion or label different from the anti-SETDB1 binding partner antibody. In some embodiments, a spatial proximity assay (also referred to as a "proximity assay") is employed, which can be used to assess the formation of a complex between SETDB1 and the nuclear binding partner of SETDB1. The proximity assay relies on the principle of "proximity probing", in which an analyte (usually an antigen) is detected by the simultaneous binding of multiple (i.e., two or more, typically two, three or four) binding agents or probes, which can generate a signal when the binding agents or probes are brought into proximity by binding to the analyte (hence the term "proximity probe").

[0460] In some embodiments, at least one of the proximity probes comprises a nucleic acid domain (or portion) connected to an analyte binding domain (or portion) of the probe, and the generation of the signal involves an interaction between the nucleic acid portion and / or another functional portion carried by the other probes. Thus, signal generation depends on the interaction between the probes (more specifically, the nucleic acids or other functional portions / domains carried by them), and therefore only occurs when both necessary two (or more) probes are bound to the analyte, thereby improving the specificity of the detection system.

[0461] The concept of proximity detection has been developed in recent years and many assays based on this principle are well known in the art.

[0462] Proximity assays are often used to assess whether two specific proteins or portions thereof are in close proximity, such as proteins that bind to each other, fusion proteins, and / or proteins that are located in close proximity. One such assay, referred to as a proximity ligation assay (PLA), and used in some embodiments of the present disclosure, is characterized by two antibodies (produced in different species) that bind to the target of interest (see Nature Methods 3, 995-1000 (2006)). PLA probes are species-specific secondary antibodies attached with unique oligonucleotide chains, which are then combined with appropriate primary antibodies. In the case where the targets are in close proximity, the oligonucleotide chains of the PLA probes can interact with additional ssDNA and DNA ligases, allowing for circulation and amplification by rolling circle amplification (RCA). When a highly processed DNA polymerase, such as Phi29 DNA polymerase, is used, the replication time of the circular DNA template can be extended by hundreds to thousands of times, and thus ssDNA molecules with lengths of hundreds of nanometers to micrometers are produced (see Angewandte Chemie International Edition, 2008, 47, 6330-6337). After amplification, the replicated DNA can be detected by a detection system. Thus, a visible signal indicates that the target of interest is very close. These assays are characterized by the use of several DNA antibody conjugates and enzymes such as DNA ligases and DNA polymerases.

[0463] In other embodiments, a dual binder (DB) assay is used, which utilizes a bispecific detector consisting of two Fab fragments with fast dissociation rate kinetics connected by a flexible linker (Van dieck et al., 2014 "Chemistry & Biology" Vol. 21 (3): 357-368). In principle, since the dual binder contains Fab fragments with fast dissociation rate kinetics, if only one Fab fragment binds to its epitope, the dual binder will be washed away (the simultaneous cooperative binding of the two Fab fragments of the dual binder can prevent the dissociation of the dual binder and lead to positive staining / visibility).

[0464] According to another method disclosed in International PCT Publication No. WO2014 / 139980, which is encompassed in the practice of the present disclosure, proximity assays and tools are described that employ biotin ligase substrates and enzymes for proximity assays. The method provides detection of target molecules and proximity while maintaining the cellular environment of the sample. Using a biotin ligase, such as an enzyme from Escherichia coli, and a peptide substrate, such as an amino acid substrate for the enzyme, protein-protein interactions in FFPE samples can be sensitively and specifically detected. Because the biotin ligase can efficiently biotinylate an appropriate peptide substrate in the presence of biotin, and the reaction can only occur when the enzyme is in physical contact with the peptide substrate, the biotin ligase and the substrate can be separately bound to two antibodies that recognize the target of interest.

[0465] In some embodiments, the level / amount of one or more biomarker proteins and / or its cellular location / distribution can be compared with a reference, which may include samples from subjects who have not received therapy (e.g., immunotherapy). In some embodiments, the reference may include samples from the same subject before receiving therapy (e.g., immunotherapy). In some embodiments, the reference may include reference values ​​of one or more samples from other subjects receiving therapy (e.g., immunotherapy). For example, a subject population can be treated, and the mean, average or median of the level / amount and / or its cellular location / distribution of at least one RTT biomarker can be generated according to the entire population. A sample collection obtained from a disease with shared characteristics (e.g., the same cancer type and / or stage, or exposed to a common therapy) can be studied according to a population, such as a clinical outcome study. This collection can be used to derive a reference (e.g., a reference value) to be compared with a subject's sample.

[0466] Certain aspects of the present disclosure relate to the measurement of the level / amount of one or more RTT biomarkers in a sample. In some embodiments, the sample may include cancer cells. In some embodiments, the sample may be a peripheral blood sample (e.g., from a subject with a tumor). In some embodiments, the sample is a tumor sample. Tumor samples may include cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal layer, and any other cell type associated with a tumor. In some embodiments, the sample is a tumor tissue sample containing tumor infiltrating leukocytes. In some embodiments, the sample may be processed to separate or separate one or more cell types (e.g., leukocytes). In some embodiments, the sample may be used without separating or separating cell types.

[0467] Tumor samples can be obtained from subjects by any method known in the art, including but not limited to biopsy, endoscopy or surgical procedures. In some embodiments, tumor samples can be prepared by methods such as freezing, fixing (e.g., by using formalin or similar fixatives) and / or embedding in paraffin. In some embodiments, tumor samples can be sliced. In some embodiments, fresh tumor samples (i.e., samples not yet prepared by the above methods) can be used. In some embodiments, tumor samples can be prepared by incubation in a solution to retain mRNA and / or protein integrity.

[0468] In some embodiments, responsiveness to therapy can refer to any one or more of the following: prolonging survival (including overall survival and progression-free survival); causing an objective response (including complete response or partial response); or improving signs or symptoms of cancer. In some embodiments, responsiveness can refer to an improvement in one or more factors according to a published RECIST guideline set for determining the state of a tumor in a cancer subject, i.e., response, stability, or progression. For a more detailed discussion of these guidelines, see Eisenhauer et al., (2009 "European Journal of Nutrition (Eur.J.Cancer)" 45:228-47), Topalian et al., (2012 "New England Journal of Medicine (N.Engl.J.Med.)" 366:2443-54), Wolchok et al., (2009 "Clin.Can.Res.)" 15:7412-20) and Therasse et al., (2000 "National Cancer Institute Journal (J.Natl.Cancer Inst.)" 92:205-16). Responsive subjects can refer to subjects whose cancer shows improvement, for example, according to one or more factors based on RECIST criteria. Non-responsive subjects can refer to subjects whose cancer does not show improvement, for example, according to one or more factors based on RECIST criteria.

[0469] In some embodiments, conventional response criteria may not be sufficient to characterize the activity of anticancer therapy, which may produce a delayed response, which may be before the initial obvious radiological progression (including the appearance of new lesions). Therefore, a modified response standard has been formulated, which takes into account the possible appearance of new lesions and allows confirmation of radiological progression in subsequent assessments. Therefore, in some embodiments, responsiveness can refer to the improvement of one or more factors according to the immune-related response criteria (irRC). See, for example, Wolchok et al., (2009, supra). In some embodiments, new lesions are added to the defined tumor burden and tracked in subsequent assessments, such as for radiological progression. In some embodiments, the presence of non-target lesions is included in the assessment of complete response and is not included in the assessment of radiological progression. In some embodiments, radiological progression can be determined based only on measurable disease, and / or can be confirmed by continuous assessment of about 4 weeks from the date of first recording.

[0470] 6. Subject classification and treatment management

[0471] The present disclosure extends to methods for selecting or identifying individuals, and the individual is a suitable candidate for treatment with a therapy (e.g., immunotherapy) for treating a disease (e.g., cancer). Such individuals include subjects predicted to respond to therapy, and therefore the possibility of the subject benefiting from the administration of therapy is increased compared to other subjects with different characteristics (e.g., no response to therapy). In certain embodiments, a suitable candidate is a person who is reasonably likely to benefit from treatment, or there is at least enough possible benefit to prove that the administration of treatment is reasonable according to its risks and side effects. The present disclosure also encompasses methods for selecting or identifying individuals, and the individual is not a suitable candidate for treatment with a therapy (e.g., immunotherapy) for treating a disease (e.g., cancer). Such individuals include subjects predicted to be unresponsive or weakly responsive to therapy, and therefore relative to other subjects with different characteristics (e.g., responsiveness to therapy), the possibility of benefiting from the administration of therapy is reduced, or the possibility of benefiting from such treatment is low or substantially absent, so that different or additional treatments may need to be used. In some embodiments, as described herein, a determination is made as to whether a subject is an appropriate candidate for treatment with a therapy based on the determination of at least one RTT biomarker in a sample obtained from the subject.

[0472] In some aspects, described herein is a method for determining the likelihood that a subject in need of treatment for a disease (e.g., cancer) will respond to treatment with therapy (e.g., immunotherapy), for example, based on the determination of at least one RTT biomarker, and / or identifying and / or selecting a subject to receive such treatment. In a specific embodiment, the therapy is immunotherapy, appropriately with an anti-immune checkpoint inhibitor. The phrase "treatment with an immune checkpoint inhibitor", also referred to as "immune checkpoint inhibitor treatment", "therapy with an immune checkpoint inhibitor", or "immune checkpoint inhibitor therapy", covers embodiments related to treatment with a single immune checkpoint inhibitor and embodiments related to treatment with a combination of two or more immune checkpoint inhibitors. In some embodiments, immune checkpoint inhibitor treatment includes the use of a single agent or the use of two or more separate agents to inhibit two or more different immune checkpoint pathways.

[0473] 7. Test kit

[0474] In other embodiments of the present invention, a therapeutic kit comprising a SETDB1 bicyclic peptide mimetic and an anticancer spray is provided. In some embodiments, the therapeutic kit further comprises a package insert comprising instructional materials for: administering the SETDB1 bicyclic peptide mimetic and an anticancer agent simultaneously to treat a T cell dysfunction disorder in an individual with cancer, or to enhance their immune function (e.g., immune effector function, T cell function, etc.), or to treat cancer or delay cancer progression, or to treat an infection in an individual. In some embodiments, the anticancer agent comprises a chemotherapeutic agent (e.g., an agent that targets rapidly dividing cells and / or disrupts the cell cycle or cell division, representative examples of which include cytotoxic compounds such as taxanes).

[0475] In some embodiments, the above-mentioned SETDB1 peptide and optionally the chemotherapeutic agent are in the same container or in separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The container can be formed of a variety of materials, such as glass, plastic (such as polyvinyl chloride or polyolefin) or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation, and a label on the container or a label associated with the container may indicate instructions for use. The kit may further include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and packaging inserts with instructional materials for use. In some embodiments, the kit further includes one or more other agents (e.g., chemotherapeutic agents and anti-tumor agents). Suitable containers for the one or more agents include, for example, bottles, vials, bags, and syringes.

[0476] In other embodiments of the present invention, a diagnostic kit for determining the expression of a biomarker is provided, which includes reagents that can detect and / or quantify biomarkers. Such reagents include, for example, compounds or materials that can quantify biomarkers, or a group of compounds or materials. In a specific embodiment, a compound, a material, or a group of compounds or materials can be used to determine the expression level of a gene (e.g., a T cell function biomarker gene), including but not limited to extracting RNA materials, determining the level of corresponding RNA, etc., primers for synthesizing corresponding cDNA, primers for amplifying DNA, and / or probes that can specifically hybridize with RNA (or corresponding cDNA) encoded by a gene, TaqMan probes, proximity assay probes, ligases, antibodies, etc.

[0477] The kit may also optionally include appropriate reagents for detection labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates, dilution buffers, etc. For example, a nucleic acid-based detection kit may include (i) T cell function biomarker polynucleotides (which may be used as positive controls), (ii) primers or probes that specifically hybridize to the biomarker nucleotides. Enzymes suitable for amplifying nucleic acids may also be included, including various polymerases (reverse transcriptase, Taq, sequencing enzymes, etc.). TM DNA ligase, etc., depending on the nucleic acid amplification technology used), deoxynucleotides and buffers to provide the reaction mixture required for amplification. Such kits will also typically contain different containers for each individual reagent and enzyme and each primer or probe in an appropriate manner. Alternatively, a protein-based detection kit may include (i) a biomarker polypeptide (which can be used as a positive control), (ii) an antibody that specifically binds to the biomarker polypeptide. The kit may also have various devices (e.g., one or more devices) and reagents (e.g., one or more reagents) for performing one of the assays described herein; and / or printed instruction materials for using the kit to quantify the expression of T cell function biomarker genes. The reagents described herein, which may optionally be associated with a detectable label, may be presented in the form of a microfluidic card, a chip or chamber, a microarray or a kit, suitable for use in the examples or assays described below, such as the RT-PCR or Q PCR techniques described herein.

[0478] On the other hand, the present invention extends to a kit for determining the level / amount and / or cellular localization of the RTT biomarkers disclosed herein, which includes reagents that allow detection and / or quantification of biomarkers. Such reagents include, for example, compounds or materials that can be quantified for biomarkers, or a group of compounds or materials. In a specific embodiment, the compound, material, or a group of compounds or materials can be used to determine the level of the biomarker, including but not limited to extracting RNA material, determining the level of the corresponding RNA, etc., primers for synthesizing the corresponding cDNA, primers for amplifying DNA and / or probes that can specifically hybridize with RNA (or corresponding cDNA) encoded by the gene, TaqMan probes, proximity assay probes, ligases, antibodies, etc.

[0479] The kit may also optionally include appropriate reagents for detection labels, positive and negative controls, washing solutions, blotting membranes, microtiter plates, dilution buffers, and the like. For example, a protein-based detection kit may include (i) at least one SETDB1 polypeptide (which may be used as a positive control); (ii) one or more antigen binding molecules that specifically bind to a SETDB1 polypeptide; and / or (iii) at least one nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα). The antigen binding molecule is appropriately detectably labeled. The kit may also have various devices (e.g., one or more devices) and reagents (e.g., one or more reagents) for performing one of the assays described herein; and / or printed instructional materials for using the kit to quantify the level / amount of RTT biomarkers. The reagents described herein, which may optionally be associated with a detectable label, may be presented in the form of a microfluidic card, a chip or chamber, a microarray, or a kit suitable for the assays described herein.

[0480] Materials suitable for packaging the components of the diagnostic kit may include crystals, plastics (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, envelopes, etc. In addition, the kit of the present invention may contain instructional materials for the simultaneous, sequential or separate use of the different components contained in the kit. The instructional materials may be in the form of printed materials, or may be in the form of electronic support capable of storing instructions so that the subject can read, such as electronic storage media (disks, tapes, etc.), optical media (CD-ROMs, DVDs), etc. Alternatively or additionally, the medium may contain an Internet address that provides instructional materials.

[0481] In order that the present invention may be readily understood and put into practical effect, particularly preferred embodiments will now be described by way of the following non-limiting examples.

[0482] Examples

[0483] Example 1

[0484] Prototype linear SETDB1 peptide inhibitor

[0485] 7.1 SETDB1 is localized to the nucleus of cancer cells.

[0486] The present inventors found that three cancer cell lines (H1299, 4T1 and LLC3) showed significant nuclear expression of SETDB1 and ATF7IP. In addition, in all three cancer cell lines tested, there was significant co-localization of SETDB1 and ATF7IP or SETDB1 and IMPα1 ( Figure 1 ). Nuclear localization of epigenetic enzymes is a characteristic of invasive metastatic cancers. Therefore, the inventors hypothesized that targeting the nuclear axis of the epigenetic enzyme SETDB1 and its major nuclear interaction partner ATF7IP would directly inhibit mesenchymal therapy resistance characteristics. To this end, the nuclear localization sequence of SETDB1 was targeted.

[0487] 7.2 Generation of SETDB1 NLS-mimetic peptide inhibitors.

[0488] Based on the above data, novel SETDB1 NLS mimetic peptide inhibitors were developed based on the newly identified nuclear localization sequence (NLS) motif of SETDB1. It is worth noting that the NLS region of SETDB1 is highly conserved among species.

[0489]

[0490] Two SETDB1 nuclear localization sequence (NLS) peptide mimetics were generated, (i) peptide 047_wt, corresponding to the residues of the wild-type full-length human SETDB1 amino acid sequence (including the native alanine residue at position 15), and (ii) peptide 047_A15P (as shown in SEQ ID NO: 1), which contains a single amino acid substitution (alanine to proline) at position 15. This amino acid substitution was made because it was hypothesized that proline could enhance the stability of the peptide and its interaction with the target. Thus, the novel peptide sequence has the following amino acid sequence:

[0491] Peptide 047_wt myristoyl-GKKRTKTWHKGTLIAIQTVGPGKKYKV [SEQ ID NO: 64] Peptide 047_A15P myristoyl-GKKRTKTWHKGTLI P IQTVGPGKKYKV [SEQ ID NO: 65]

[0492] 7.3 SETDB1 NLS peptide inhibitors abolish cell proliferation.

[0493] The effects of candidate peptide inhibitors (peptide 047_A15P and peptide 047_wt) on cell proliferation of TNBC cancer cell line MDA-MB-231 and melanoma cancer cell line RPMI-7951 were investigated.

[0494] Both linear SETDB1 peptide inhibitors (i.e., peptide 047_A15P and peptide 047_wt) were able to abolish the proliferation of the MDA-MB-231TNBC metastatic cancer cell line with IC values ​​of about 20 mM or less ( Figure 2 A, B). Similarly, a linear SETDB1 peptide inhibitor was able to abolish the proliferation of the RPMI-7951 metastatic melanoma cancer cell line ( Figure 2 C, D).

[0495] 7.4 SETDB1 NLS peptide inhibitor reduces metastatic markers.

[0496] Next, the inventors attempted to explore the effect of inhibiting SETDB1 nuclear translocation on protein markers of mesenchymal metastatic cancer. Specifically, CSV and SNAIL are two markers of metastatic and invasive cancer. The direct target SETDB1 was also examined. Immunofluorescence analysis of protein expression in MDA-MB-231TNBC cancer cells showed that linear peptide inhibitors at different concentrations induced a significant decrease in the expression levels of mesenchymal metastatic markers CSV, SNAIL and nuclear SETDB1 ( Figure 3 These data suggest that targeting and inhibiting the nuclear axis of SETDB1 directly affects mesenchymal-metastatic markers that mediate cancer progression and metastatic spread.

[0497] Materials and Methods

[0498] IFA microscopy

[0499] To examine the characteristics of SETDB1, ATF7IP and IMPα1 in LLC3 (lung cancer), H1299 (lung cancer) and 4T1 (TNBC) cell lines, cells were permeabilized by incubation with 0.5% Triton X-100 for 15 minutes, blocked with PBS containing 1% BSA, and probed with ATF7IP, IMPα1, S...

Claims

1. A method for preventing or reducing nuclear localization of a SETDB1 polypeptide, the method comprising contacting the SETDB1 polypeptide and / or IMPα polypeptide with an agent that inhibits binding of the SETDB1 polypeptide to the IMPα polypeptide.

2. A method for preventing or reducing nuclear localization of a SETDB1 polypeptide in a cancer cell, the method comprising contacting the cell with an agent that inhibits binding of the SETDB1 polypeptide to an IMPα polypeptide.

3. A method for treating or preventing cancer in a subject, the method comprising administering to the subject an agent that inhibits binding of a SETDB1 polypeptide to an IMPα polypeptide.

4. The method of claim 3, wherein the cancer is associated with at least some SETDB1 polypeptide being present in the cell nucleus.

5. The method according to any one of claims 2 to 4, wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, colon cancer, liver cancer or brain cancer or melanoma or retinoblastoma.

6. The method of any one of claims 1 to 5, wherein the agent inhibits the binding of the SETDB1 polypeptide to the IMPα polypeptide but does not inhibit the binding of any other polypeptide to the IMPα polypeptide.

7. The method of any one of claims 1 to 6, wherein the agent binds directly to the IMPalpha polypeptide.

8. The method of any one of claims 1 to 7, wherein the agent reduces the amount of SETDB1 polypeptide present in the nucleus of the cell.

9. The method of any one of claims 1 to 8, wherein the agent reduces the ratio of nuclear SETDB1 polypeptide to cytoplasmic SETDB1 polypeptide.

10. The method of any one of claims 1 to 9, wherein the agent is a protein molecule comprising, consisting of, or consisting essentially of: Amino acid sequence corresponding to the nuclear localization sequence (NLS) of the SETDB1 polypeptide.

11. The method of claim 10, wherein the protein molecule comprises, consists of, or consists essentially of: An amino acid sequence as set forth in any one of SEQ ID NOs: 1-6, or having at least 85% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-6.

12. The method of claim 10 or claim 11, wherein the protein molecule comprises, consists of, or consists essentially of: The amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:

6.

13. The method of any one of claims 2 to 12, further comprising administering one or more additional cancer therapies.

14. The method of claim 13, wherein the additional cancer therapy is a chemotherapeutic agent.

15. The method of claim 13, wherein the additional cancer therapy is immunotherapy.

16. The method of claim 15, wherein the immunotherapy is an immune checkpoint inhibitor.

17. The method according to any one of claims 1 to 16, wherein the protein molecule comprises, consists of or consists essentially of an amino acid sequence corresponding to residues 206 to 232 of the full-length human SETDB1 protein.

18. The method according to any one of claims 1 to 17, wherein the protein molecule is a fragment of a nuclear localization sequence (NLS).

19. The method of claim 17 or claim 18, wherein the protein molecule comprises 50 amino acid residues or less.

20. The method of claim 19, wherein the protein molecule differs from SETDB1 by the addition, deletion and / or substitution of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) amino acid corresponding to residues 206 to 232 of the full-length human SETDB1 protein.

21. A method for producing a protein molecule that inhibits or reduces nuclear localization of a SETDB1 polypeptide, the method comprising: a) contacting the cell with a protein molecule comprising, consisting of, or consisting essentially of: an amino acid sequence corresponding to residues 206 to 232 of the full-length human SETDB1 protein; as well as b) detecting a reduction or inhibition of the nuclear localization of the SETDB1 polypeptide in the cell relative to a normal or reference level of nuclear localization in the absence of the protein molecule.

22. The method of claim 21, wherein the protein molecule differs from SETDB1 by the addition, deletion and / or substitution of at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) amino acid corresponding to residues 206 to 232 of SETDB1.

23. An isolated or purified protein molecule represented by Formula I: GKKRX1KX2WHX3GTLIX4IQTVGPGKKX5KVK in: Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween); X1 is selected from Thr, Arg and modified forms thereof; X2 is selected from Thr, Leu and modified forms thereof; X3 is selected from Lys, Gly and modified forms thereof; X4 is selected from Ala, Pro and modified forms thereof; and X5 is selected from Tyr, Lys and modified forms thereof.

24. The protein molecule according to claim 23, wherein Z1 is absent.

25. The protein molecule according to claim 23 or 24, wherein Z2 is absent.

26. The protein molecule according to any one of claims 23 to 25, wherein X1 is Thr.

27. The protein molecule according to any one of claims 23 to 26, wherein X2 is Thr.

28. The protein molecule according to any one of claims 19 to 23, wherein X3 is Lys.

29. The protein molecule according to any one of claims 19 to 24, wherein X4 is Ala.

30. The protein molecule according to any one of claims 19 to 25, wherein X5 is Tyr.

31. The protein molecule according to any one of claims 19 to 21, wherein X1 is Arg.

32. The protein molecule of claim 27, wherein X2 is Leu.

33. A protein molecule according to claim 27 or claim 28, wherein X3 is Gly.

34. The protein molecule according to any one of claims 27 to 29, wherein X4 is Pro.

35. The protein molecule according to any one of claims 27 to 30, wherein X5 is Lys.

36. The protein molecule of claims 27 to 31, wherein the protein molecule of Formula I comprises, consists of, or consists essentially of: The amino acid sequence represented by any one of SEQ ID NO: 1 or 2:

37. The protein molecule according to any one of claims 27 to 36, wherein the protein molecule of Formula I further comprises at least one membrane permeable moiety.

38. The protein molecule of claim 37, wherein the membrane permeable moiety is a lipid moiety.

39. The protein molecule of claim 38, wherein the membrane permeating moiety is myristoyl.

40. The protein molecule according to any one of claims 33 to 39, wherein the membrane permeable moiety is coupled to the N-terminal or C-terminal amino acid residue.

41. The protein molecule of claim 40, wherein the membrane permeable moiety is coupled to the N-terminal amino acid residue.

42. A SETDB1 bicyclic peptide mimetic, comprising a polypeptide and a molecular scaffold, wherein the polypeptide comprises at least three cysteine ​​residues separated by at least two ring sequences, and the molecular scaffold forms covalent bonds with the cysteine ​​residues of the polypeptide, so that at least two polypeptide rings are formed on the molecular scaffold, wherein the SETDB1 bicyclic peptide mimetic comprises an amino acid sequence of formula (II): Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2 Formula (II) or a modified derivative thereof, or a pharmaceutically acceptable salt thereof, in: C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively; Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween); X1 is absent or is alanine; and X2 is selected from any non-polar / neutral amino acid residue (eg, proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

43. The SETDB1 bicyclic peptide mimetic of claim 42, wherein X1 is absent.

44. The SETDB1 bicyclic peptide mimetic according to claim 42 or claim 43, wherein X2 is selected from proline or leucine.

45. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 44, wherein X2 is proline.

46. ​​The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 45, wherein Z1 is absent.

47. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 46, wherein Z2 is absent.

48. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 47, wherein the peptide binds to an importin protein.

49. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 48, wherein the peptide mimetic prevents or disrupts complexation between SETDB1 and importin.

50. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 49, comprising the following amino acid sequence: CGKKRTKTWHCKGTLIAIQTVGPGCKKYKV [SEQ ID NO: 8].

51. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 50, wherein the molecular scaffold is 1,3,5-(tribromomethyl)benzene) or TBAB.

52. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 51, further comprising an N-terminal cell penetrating peptide.

53. The SETDB1 bicyclic peptide mimetic of claim 52, wherein the cell penetrating peptide is Myr.

54. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 53, wherein the modified derivative comprises one or more modifications selected from the group consisting of: N-terminal and / or C-terminal modification; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acids with one or more isosteric or isoelectronic amino acids; replacement of one or more hydrophobic amino acid residues with other non-natural isosteric or isoelectronic amino acids); addition of a spacer group; replacement of one or more antioxidant amino acid residues; replacement of one or more amino acid residues with alanine; replacement of one or more L amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within the bicyclic peptide ligand; replacement of one or more peptide bonds with a surrogate bond; modification of the peptide backbone length; replacement of a hydrogen on the alpha carbon of one or more amino acid residues with another chemical group; and post-synthetic biorthogonal modification of amino acids such as cysteine, lysine, glutamate and tyrosine with suitable amine, thiol, carboxylic acid and phenol reactive reagents.

55. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 54, wherein the modified derivative comprises an N-terminal modification, such as an N-terminal acetyl group.

56. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 55, wherein the modified derivative comprises a C-terminal modification, such as a C-terminal amide group.

57. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 56, wherein the modified derivative comprises replacement of one or more amino acid residues with one or more non-natural amino acid residues.

58. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 57, wherein the modified derivative comprises one or more D-amino acids.

59. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 58, wherein the amino acids other than glycine are substantially D-amino acids.

60. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 59, wherein two or more of the amino acids are in retro-inverso form.

61. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 60, wherein all amino acids are in retro-inverso form.

62. The SETDB1 bicyclic peptide mimetic according to claim 61, wherein the amino acid sequence is shown in SEQ ID NO: 11 (vkykkcGpgvtqisiltGkchwtktrkkcG).

63. The SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 62, wherein the pharmaceutically acceptable salt is selected from hydrochloride or acetate.

64. A pharmaceutical composition comprising a SETDB1 bicyclic peptide mimetic according to any one of claims 42 to 63 in combination with one or more excipients.

65. A method for reducing nuclear localization of SETDB1 in a SETDB1 overexpressing cell, the method comprising contacting the cell with a SETDB1 bicyclic peptide mimetic having the following amino acid sequence: Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2 Formula (II) or a modified derivative thereof, or a pharmaceutically acceptable salt thereof, in: C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively; Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween); X1 is absent or is alanine; and X2 is selected from any non-polar / neutral amino acid residue (eg, proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

66. The SETDB1 bicyclic peptide mimetic of claim 65, wherein X1 is absent.

67. The SETDB1 bicyclic peptide mimetic according to claim 65 or claim 66, wherein X2 is selected from proline or leucine.

68. The SETDB1 bicyclic peptide mimetic according to any one of claims 65 to 67, wherein X2 is proline.

69. The SETDB1 bicyclic peptide mimetic according to any one of claims 65 to 68, wherein Z1 is absent.

70. The SETDB1 bicyclic peptide mimetic according to any one of claims 65 to 69, wherein Z2 is absent.

71. The method of any one of claims 65 to 70, comprising the following amino acid sequence: CGKKRTKTWHCKGTLIAIQTVGPGCKKYKV [SEQ ID NO: 8].

72. The method of any one of claims 65 to 71, wherein the molecular scaffold is 1,3,5-(tribromomethyl)benzene) or TBAB.

73. The method of any one of claims 65 to 72, wherein the modified derivative comprises one or more D-amino acids.

74. The method of any one of claims 65 to 73, wherein the amino acids are substantially D-amino acids.

75. The method of any one of claims 65 to 74, wherein the peptide is formulated in a lipid nanoparticle.

76. The method of any one of claims 65 to 75, further comprising an N-terminal cell penetrating peptide.

77. The method of claim 76, wherein the cell penetrating peptide is myristic acid.

78. A method of treating or preventing cancer in a subject, wherein the cancer comprises at least one SETDB1 overexpressing cell, the method comprising administering to the subject a SETDB1 bicyclic peptide mimetic according to any one of claims 43 to 62.

79. The method of any one of claims 65 to 78, wherein the SETDB1 overexpressing cell is a cancer cell, a cancer stem cell, or a non-cancer stem cell tumor cell.

80. The method of claim 78 or claim 79, wherein the cancer is selected from breast cancer, prostate cancer, lung cancer, bladder cancer, pancreatic cancer, colon cancer, liver cancer or brain cancer or melanoma or retinoblastoma.

81. The method of any one of claims 65 to 80, further comprising administering at least one additional cancer therapy.

82. The method of claim 81, wherein the additional cancer therapy is a chemotherapeutic agent and / or an immunotherapy.

83. The method of claim 81, wherein the immunotherapy is an immune checkpoint inhibitor.

84. A composition comprising a SETDB1 bicyclic peptide mimetic having the following amino acid sequence: Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2 Formula (III) or a modified derivative thereof, or a pharmaceutically acceptable salt thereof, for use in therapy; in: C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively; Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween); X1 is absent or is alanine; and X2 is selected from any non-polar / neutral amino acid residue (eg, proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

85. The composition of claim 84, wherein X1 is absent.

86. A composition according to claim 84 or claim 85, wherein X2 is selected from proline or leucine.

87. A composition according to any one of claims 84 to 86, wherein X2 is proline.

88. A composition according to any one of claims 84 to 87, wherein Z1 is absent.

89. according to the composition described in any one of claims 84 to 88, wherein Z2 does not exist.

90. The composition of any one of claims 84 to 89, comprising the following amino acid sequence: CGKKRTKTWHCKGTLIAIQTVGPGCKKYKV [SEQ ID NO: 8].

91. A composition comprising a SETDB1 bicyclic peptide mimetic having the following amino acid sequence: Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2 Formula (III) or a modified derivative thereof, or a pharmaceutically acceptable salt thereof; for use in treating cancer; in: C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively; Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween); X1 is absent or is alanine; and X2 is selected from any non-polar / neutral amino acid residue (eg, proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

92. The composition of claim 91, wherein X1 is absent.

93. A composition according to claim 91 or claim 92, wherein X2 is selected from proline or leucine.

94. A composition according to any one of claims 91 to 93, wherein X2 is proline.

95. according to the composition described in any one of claims 91 to 94, wherein Z1 does not exist.

96. A composition according to any one of claims 91 to 95, wherein Z2 is absent.

97. A composition according to any one of claims 91 to 96, comprising the following amino acid sequence: CGKKRTKTWHCKGTLIAIQTVGPGCKKYKV [SEQ ID NO: 8].

98. Use of a composition comprising a bicyclic peptide having the following amino acid sequence: Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2 Formula (III) or a modified derivative thereof, or a pharmaceutically acceptable salt thereof; for use in preparing a drug for treating cancer; in: C1, C2, and C3 represent the first, second, and third cysteine ​​residues, respectively; Z1 and Z2 are independently absent or independently selected from at least one of a protein moiety and a protecting moiety comprising about 1 to about 50 amino acid residues (and all integer residues therebetween); X1 is absent or is alanine; and X2 is selected from any non-polar / neutral amino acid residue (eg, proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

99. The use according to claim 98, wherein X1 is absent.

100. The use of claim 98 or claim 99, wherein X2 is selected from proline or leucine.

101. The use according to any one of claims 98 to 100, wherein X2 is proline.

102. The use according to any one of claims 98 to 101, wherein Z1 is absent.

103. The use according to any one of claims 98 to 102, wherein Z2 is absent.

104. The composition of any one of claims 98 to 103, comprising the following amino acid sequence: CGKKRTKTWHCKGTLIAIQTVGPGCKKYKV [SEQ ID NO: 8].

105. A method of predicting the likelihood of a subject's response to a therapy (e.g., an immunotherapy), the method comprising, consisting of, or consisting essentially of: The cellular localization of SETDB1 in cells expressing SETDB1 in the subject is analyzed to thereby predict the likelihood of the patient's response to the therapy.

106. The method of claim 105, wherein the cell expressing SETDB1 is a tumor cell.

107. The method of claim 1 or claim 2, wherein the therapy is immunotherapy.

108. The method of any one of claims 105 to 107, comprising detecting the presence of SETDB1 in the nucleus of the cell, or a level of SETDB1 in the nucleus of the cell, which level is indicative of an abnormal or abnormal nuclear level of SETDB1, and which is associated with an increased likelihood of developing resistance to the therapy, thereby determining that the subject has an increased likelihood of developing resistance to the therapy.

109. The method of claim 108, comprising detecting and comparing the levels of SETDB1 between different compartments of the cell (eg, nucleus, cytoplasm) to thereby determine that the subject has an increased likelihood of developing resistance to therapy.

110. The method of claim 109, comprising detecting higher levels of SETDB1 in the nucleus of the cell relative to a control, thereby determining that the subject has an increased likelihood of developing resistance to the therapy.

111. The method of any one of claims 105 to 110, comprising comparing the level of SETDB1 between different cellular compartments (e.g., nucleus, cytoplasm) of the cells to thereby determine that the subject has an increased likelihood of developing resistance to the therapy.

112. The method of any one of claims 105 to 111, comprising detecting a higher level of SETDB1 in the nucleus of the cell relative to a control (e.g., relative to the nucleus of a corresponding normal cell or relative to the level of SETDB1 outside the nucleus of the subject's cells, such as the cytoplasm of the cells), which indicates an increased likelihood that the subject has developed resistance to the therapy.

113. The method of claim 112, wherein the elevated level of SETDB1 in the nucleus of the cell means that the level is at least about 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900% or 1000% (and every integer therebetween) of the level of SETDB1 in the nucleus of a corresponding response control cell.

114. The method of claim 112 or claim 113, wherein the higher level of SETDB1 in the nucleus of the cell means that the level of SETDB1 in the nucleus of the cell is higher than the level of SETDB1 outside the nucleus (eg, cytoplasm or extranuclear) of the cell.

115. The method of claim 114, wherein the higher level indicates that the ratio of nuclear SETDB1 to extranuclear SETDB1 is greater than about 0.55, 0.65, 0.70, 0.75, 0.85, 0.90, or 0.

95.

116. The method of any one of claims 105 to 115, comprising detecting elevated levels of nuclear SETDB1 in greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the subject's cells (e.g., tumor cells), indicating an increased likelihood that the subject has developed resistance to the therapy.

117. The method of any one of claims 105 to 108, comprising detecting the absence of SETDB1 in the nucleus of the cell or a level of SETDB1 in the nucleus of the cell that is indicative of a normal nuclear level of SETDB1 and that is associated with an increased likelihood of sensitivity to the therapy, to thereby determine that the subject has an increased likelihood of sensitivity to the therapy.

118. The method of any one of claims 105 to 108 and 117, comprising detecting the level of SETDB1 in the nucleus of the cell relative to a control (e.g., relative to the nucleus of a corresponding normal cell or relative to the level of SETDB1 outside the nucleus of a cell of the subject), the level being indicative of normal nuclear levels of SETDB1 and indicating an increased likelihood that the patient will develop sensitivity to the therapy.

119. The method of any one of claims 105 to 108, 117 and 118, comprising detecting the presence of SETDB1 outside the nucleus (eg, cytoplasm) of the cell to thereby determine that the subject has an increased likelihood of developing sensitivity to the therapy.

120. The method of any one of claims 105 to 108, 117, 118 and 119, comprising detecting the level of SETDB1 outside the nucleus of the cell relative to a control (e.g., relative to the level of SETDB1 outside the nucleus of a corresponding normal cell or relative to the level of SETDB1 inside the nucleus of a cell of the subject), the level being indicative of normal extranuclear levels of SETDB1 and indicating an increased likelihood that the patient will develop sensitivity to the therapy.

121. The method of claim 119 or claim 120, wherein the level of SETDB1 outside the nucleus of the cell indicates a level that is approximately the same as the level of SETDB1 outside the nucleus of a corresponding normal control cell (e.g., a level that is about 85% to about 115% of the latter, and every integer therebetween).

122. The method of any one of claims 119 to 121, wherein the level of SETDB1 outside the nucleus of the cell means that the level of SETDB1 outside the nucleus (e.g., surface and / or cytoplasm) of the cell is higher than the level of SETDB1 within the nucleus of the cell.

123. The method of claim 122, wherein the higher level indicates a ratio of extranuclear SETDB1 to nuclear SETDB1 greater than about 0.55, 0.65, 0.70, 0.75, 0.85, 0.90, or 0.

95.

124. The method of any one of claims 105 to 108 and 117 to 123, comprising detecting elevated levels of extranuclear SETDB1 in greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the subject's cells (e.g., tumor cells), indicating an increased likelihood that the subject will be sensitive to the therapy.

125. The method of any one of claims 105 to 124, comprising detecting the presence, absence or level of co-localization of SETDB2 with a nuclear binding partner of SETDB1 (eg, ATF7IP).

126. The method of claim 125, comprising contacting a sample comprising cells of the subject or a lysate of the cells with a first antigen binding molecule that specifically binds to SETDB1 and with a second antigen binding molecule that specifically binds to the nuclear binding partner, and detecting the presence of a complex comprising SETDB1, the nuclear binding partner, the first antigen binding molecule and the second antigen binding molecule in the sample to thereby determine that the subject has an increased likelihood of developing resistance to the therapy.

127. The method of claim 126, comprising detecting higher levels of the complex relative to a control (e.g., corresponding to normal control cells), which indicates an increased likelihood that the subject has developed resistance to the therapy.

128. The method of claim 125, comprising detecting a level of the complex in the nucleus relative to a control (e.g., corresponding to a normal control cell), the level being indicative of a normal level of the complex and indicating an increased likelihood that the subject will develop sensitivity to the therapy.

129. A method for stratifying subjects into likely responders or non-responders to a therapy (e.g., an immunotherapy), the method comprising, consisting of, or consisting essentially of: Analyzing the cellular localization of SETDB1 in a sample from said subject, as defined according to any one of claims 105 to 128, to determine whether said subject has an increased likelihood of developing sensitivity or resistance to said therapy, to thereby stratify said subject into a likely responder or non-responder to said therapy.

130. A method for managing a subject treated with a therapy (e.g., an immunotherapy), the method comprising, consisting of, or consisting essentially of: Selecting to treat the subject with the therapy based on the subject being a possible responder to the therapy, or selecting not to treat the subject with the therapy based on the subject being a possible non-responder to the therapy, and treating the subject with the therapy or not treating the subject with the therapy based on the selection, wherein the selection is based on the stratification method according to claim 129.

131. A method for predicting a treatment outcome of a subject treated with therapy (e.g., immunotherapy), the method comprising, consisting of, or consisting essentially of: Analyzing the cellular localization of SETDB1 in a sample from said subject, as defined in any one of claims 105 to 128, to determine whether said subject has an increased likelihood of developing sensitivity or resistance to said therapy, to thereby predict the patient's treatment outcome.

132. A kit for detecting the localization of SETDB1 in a cellular location (e.g., cytoplasm or nucleus) of a cell, for predicting the likelihood of a cell's response to a therapy (e.g., immunotherapy), for determining the likelihood of a subject developing resistance to a therapy (e.g., immunotherapy), for determining the likelihood of a subject developing sensitivity to a therapy (e.g., immunotherapy), for stratifying a subject into a likely responder or non-responder to a therapy (e.g., immunotherapy), for managing treatment of a subject with a therapy (e.g., immunotherapy), for monitoring a subject's disease after treatment with a therapy, for determining the status of a subject's disease and / or for determining the immune status of a subject, the kit comprising, consisting of, or consisting essentially of: A first antigen-binding molecule that specifically binds to SETDB1.

133. The kit of claim 132, further comprising a second antigen binding molecule that specifically binds to a nuclear binding partner of SETDB1 (eg, ATF7IP).

134. The kit of claim 133, further comprising a third antigen binding molecule that binds to the first antigen binding molecule and the second antigen binding molecule, the third antigen binding molecule suitably comprising a detectable label.

135. The kit of any one of claims 132 to 134, further comprising instructional material for performing the method of any one of claims 105 to 131.

136. The composition, method or kit of any of the preceding claims, wherein all amino acids are of D subtype (ie, vkykkcGpgvtqisiltGkchwtktrkkcG) and are in retro-inverso form.

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