Novel inhibitors of histone methyltransferase nuclear localization

Abstract

The present invention relates to novel therapeutic compositions and methods for treating cancer, in particular the use of proteinaceous inhibitors, including novel bicyclic peptide inhibitors, for use in 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 proteinaceous molecules corresponding to the nuclear localization site of SETDB1 and their use for preventing the nuclear localization of SETDB1 polypeptide. The present invention also relates to the use of the compositions and proteinaceous molecules for treating or preventing cancer in a subject. The present invention also generally relates to methods and agents for predicting response to therapy. More specifically, the present disclosure relates to methods, agents, and kits for analyzing the cellular distribution of SETDB1 and stratifying subjects as likely to be responders or non-responders to therapy, particularly immunotherapy.

[0002] The various bibliographic references referred to by numbers in this specification are listed at the end of the description. [Background technology]

[0003] Reference in this specification to any prior publication (or information derived therefrom) or any known matter is not, and should not be construed as, an acknowledgement or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0004] There is now a great deal of evidence showing how tumors co-opt certain immune checkpoint pathways as the primary mechanism of immune resistance, particularly against tumor antigen-specific T cells. Because many immune checkpoints are initiated by ligand-receptor interactions, they can be easily blocked by antibodies or regulated by recombinant forms of the ligand or receptor. Therefore, agonists of costimulatory receptors or antagonists of inhibitory signals, both of which lead to the amplification of antigen-specific T cell responses, are the primary drugs currently in clinical trials.

[0005] In this context, cancer immunotherapy is considered a breakthrough in the field of cancer treatment, shifting from targeting tumors to targeting the immune system (Couzin-Frankel., Science. 2013. 342(6165):1432-3). Blockade of immune checkpoints with antibodies targeting PD1, PD-L1, or CTLA-4 has resulted in promising clinical results and a manageable safety profile. However, only a small proportion of patients respond to these therapies. Therefore, there is a need to improve cancer immunotherapy through new approaches and / or by combining anti-checkpoint antibodies with other treatments (see Jenkins et al., BJC. 2018; 118, 9-16; Sharma et al., Cell. 2017; 168(4):707-723). Furthermore, anti-checkpoint antibodies can induce side effects, mainly autoimmune, such that implementing combination therapies that may help to reduce the administered dose and consequently the adverse events, would be an invaluable medical aid.

[0006] SET domain-divergent histone lysine methyltransferase 1 (SETDB1, also known as ESET, and KMT1A) is an H3K9 methyltransferase involved in gene silencing. Recently, SETDB1 has been reported to be an oncogene in many 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 and arginine residues of histone proteins. The attachment of methyl groups occurs primarily at specific lysine or arginine residues on histones H3 and H4. The class of lysine-specific histone methyltransferases is further subdivided into SET domain-containing and non-SET domain-containing histone methyltransferases. Methylation of the N-terminal lysine residues of histone H3, particularly at positions 4, 9, 27, 36, and 79, to form mono-, di-, or trimethylated lysines, has been well documented. Currently, over 30 histone methyltransferases have been described.

[0007] The use of inhibitors of DNMTs or HDACs 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). Indeed, it has been suggested that DNA demethylating agents can prime solid tumors for T cell-mediated immune responses and thus act synergistically with antitumor immunotherapies, such as checkpoint inhibitors (Roulois et al., Oncoimmunology. 2016; 5(3):e1090077). Additionally, incidental clinical findings suggest that patients with non-small cell lung cancer who are pretreated with 5-azacytidine have a better clinical response to subsequent anti-PD1 therapy (Juergens et al., Cancer Discov. 2011;1:598-607), and mouse models of melanoma have been shown to respond better to the combination of 5-azacytidine and anti-CTLA-4 antibody therapy than to either 5-azacytidine alone or anti-CTLA-4 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 such epigenetic regulators in cancer immunology and immunotherapy remains poorly understood. The range of demethylating agents is diverse, and identifying genes whose reactivation predicts or mediates response remains challenging. Typically, the immunomodulatory effects of treatment with 5-azacytidine, a DNMT, are complex and depend on the clinical setting and type of patient (see Frosig and Hadrup, Mediators Inflamm. 2015; 871641).

[0009] Therefore, elucidating the precise molecular mechanisms by which epigenetic regulators exert their effects on the immune response to cancer or tumors is highly desirable. Summary of the Invention

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

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

[0012] In yet another aspect, there is provided a method of inhibiting or reducing nuclear localization of a SETDB1 polypeptide in a cell, the method comprising contacting the cell with a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to the nuclear localization sequence (NLS) of a SETDB1 polypeptide. In this regard, the present invention also includes mimetics comprising an amino acid sequence according to Formula I, which are particularly effective in inhibiting or reducing nuclear localization of SETDB1. Accordingly, the inventors believe that SETDB1 peptidomimetics comprising an amino acid sequence according to Formula I can be used to inhibit nuclear localization of SETDB1.

[0013] In a further aspect, the present invention provides a method for treating or preventing cancer in a subject, the method comprising administering to the subject an agent that inhibits or prevents binding between a SETDB1 polypeptide and an importin-α polypeptide.

[0014] In some embodiments, the cancer is associated with at least a portion of the SETDB1 polypeptide being present in the cell nucleus. As illustrative examples, the cancer may be selected from the group including 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, there is provided a method for producing an agent that inhibits or reduces nuclear localization of a SETDB1 polypeptide, the method comprising: a) contacting a cell with an agent; b) detecting a reduction or inhibition of nuclear localization of the SETDB1 polypeptide in the cell compared to a normal or reference level of nuclear localization in the absence of the agent.

[0016] In some embodiments, the agent inhibits binding of a SETDB1 polypeptide to an IMPα polypeptide, but does not inhibit binding of any other polypeptide to the IMPα polypeptide.

[0017] In some embodiments, the agent binds directly to the IMPα polypeptide.

[0018] In some embodiments, the agent reduces the amount of SETDB1 that is transported to the cell nucleus, thus reducing the amount of SETDB1 that is present in the cell nucleus.

[0019] In some embodiments, the agent is a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS from a SETDB1 polypeptide.

[0020] In some embodiments, the proteinaceous molecule comprises, consists of, or consists essentially of the amino acid sequence of formula (I). Preferably, the proteinaceous molecule comprises, consists of, or consists essentially of the amino acid sequence set forth in any one of SEQ ID NOs: 1-6, or has at least 85% sequence identity to the sequence set forth in any one of SEQ ID NOs: 1-6. Even more preferably, the proteinaceous molecule comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 6.

[0021] In some embodiments, the cells are cancer stem cells or non-cancer stem cell tumor cells.

[0022] In some embodiments, the cancer is selected from the group including 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.

[0023] In some embodiments, the proteinaceous molecule comprises, consists of, or consists essentially of an amino acid sequence corresponding to residues 206-232 of the human SETDB1 polypeptide (i.e., the human SETDB1 sequence of UniProtKB Accession No. Q15047). In some alternative embodiments, the proteinaceous molecule is a fragment of a polypeptide capable of nuclear localization.

[0024] In some preferred embodiments, the proteinaceous molecule comprises 50 or fewer amino acid residues.

[0025] In some embodiments, the proteinaceous molecule is distinguished from SETDB1 by the addition, deletion, and / or substitution of at least one (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, etc.) amino acid at residues 206-232 of SETDB1.

[0026] In another aspect, the present invention provides a method for producing a proteinaceous molecule that inhibits or reduces nuclear localization of a SETDB1 polypeptide, the method comprising: a) contacting a cell with a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to residues 206-232 of SETDB1; b) detecting a reduction or inhibition of nuclear localization of the SETDB1 polypeptide in the cell compared to a normal or reference level of nuclear localization in the absence of the proteinaceous molecule.

[0027] In yet another aspect, the present invention provides an isolated or purified proteinaceous molecule represented by formula (I): Z1GKKRX1KX2WHX3GTLIX4IQTVGX5GKKX6KVKZ2 Formula (I) Z1 and Z2 are independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integers therebetween) and a protecting moiety; 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; X5 is selected from Pro, Lys, and modified forms thereof; X6 is selected from Tyr, Lys, and modified forms thereof.

[0028] In some embodiments, Z1 is absent.

[0029] In some of the same and some alternative embodiments, Z2 is absent.

[0030] In some of the same and some alternative embodiments, X1 is Thr.

[0031] In some of the same and some alternative embodiments, X2 is Thr.

[0032] In some of the same and some alternative embodiments, X3 is Lys.

[0033] In some of the same and some alternative embodiments, X4 is Ala.

[0034] In some of the same and some alternative embodiments, the X5 is a Pro.

[0035] In some of the same and some alternative embodiments, X6 is Tyr.

[0036] In some of the same and some alternative embodiments, X1 is Arg.

[0037] In some of the same and some alternative embodiments, X2 is Leu.

[0038] In some of the same and some alternative embodiments, X3 is Gly.

[0039] In some of the same and some alternative embodiments, the X4 is a Pro.

[0040] In some preferred embodiments, the proteinaceous molecule comprises, consists of, or consists essentially of the amino acid sequence set forth in SEQ ID NO:1 or SEQ ID NO:2.

[0041] In some preferred embodiments, the proteinaceous molecule of formula I further comprises at least one membrane-permeable moiety. By way of example, the membrane-permeable moiety may be a lipid moiety. In some embodiments of this type, the membrane-permeable moiety is a myristoyl group.

[0042] 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.

[0043] Additionally, the inventors have discovered that nuclear-localized SETDB1 (also referred to herein as "nuclear SETDB1" or "nuclear SETDB1") co-localizes with at least one nuclear polypeptide (e.g., ATF7IP), and that this co-localization is a surrogate marker for increased disease severity and resistance to therapy (e.g., immunotherapy). These findings have been embodied in methods and kits for predicting the likelihood of response to therapy in a subject, as described below.

[0044] Thus, in one aspect, the present disclosure provides methods for predicting the likelihood of a subject's response to a therapy (e.g., immunotherapy). These methods generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1 in cells expressing SETDB1 in the subject, thereby predicting the subject's likelihood of response to the therapy. The cells expressing SETDB1 are preferably tumor cells. The therapy can be immunotherapy.

[0045] In some embodiments, the methods include detecting the presence of SETDB1 in the nucleus of the cell, or a level of SETDB1 in the nucleus of the cell, which indicates an aberrant or abnormal nuclear level of SETDB1 and / or correlates with an increased likelihood of resistance to the therapy, thereby determining that the subject has an increased likelihood of resistance to the therapy. In some embodiments, the methods include detecting a higher level of SETDB1 in the nucleus of the cell compared to a control, thereby determining that the subject has an increased likelihood of resistance to the therapy. In some embodiments, the methods include comparing levels of SETDB1 between different cellular components (e.g., the nucleus and cytoplasm), thereby determining that the subject has an increased likelihood of resistance to the therapy.

[0046] Preferably, the method includes detecting a higher level of SETDB1 in the nucleus of the cell relative to a control (e.g., compared to the nucleus of a corresponding normal control cell, or compared to the level of SETDB1 in the cytoplasm of the cell), indicating that the subject has a likelihood of increased resistance to the therapy. In non-limiting examples of these embodiments, a higher level of SETDB1 in the nucleus of the cell represents a level that is at least about 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% (and all integers 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, a higher level of SETDB1 in the nucleus of the cell represents a higher level of SETDB1 in the nucleus of the cell than outside the nucleus of the cell (e.g., in the cytoplasm, referred to herein as "cytoplasmic"). Representative examples of this type exhibit a ratio of nuclear 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 includes detecting higher 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).

[0047] In other embodiments, the method includes detecting the presence of SETDB1 in the nucleus of the cell, or the level of SETDB1 in the nucleus of the cell, which indicates a normal nuclear level of SETDB1 and correlates with a likelihood of increased susceptibility to the therapy, thereby determining that the subject has a likelihood of increased susceptibility to the therapy. In some of the same and other embodiments, the method includes 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 cytoplasmic SETDB1 in the cell), which indicates a normal nuclear level of SETDB1 and indicates that the subject has a likelihood of increased susceptibility to the therapy. In some of the same and other embodiments, the method includes detecting the presence of cytoplasmic SETDB1 in the cell, thereby determining that the subject has a likelihood of increased susceptibility to the therapy. Preferably, the method includes detecting the level of cytoplasmic SETDB1 in the cell relative to a control (e.g., relative to the cytoplasmic SETDB1 of a corresponding normal cell, or relative to the level of SETDB1 in the nucleus of the subject's cell), which indicates a normal extranuclear level of SETDB1 and indicates that the subject has a likelihood of increased susceptibility to the therapy. In non-limiting examples of these embodiments, the level of cytoplasmic SETDB1 in the cell represents a level that is about the same as the level of cytoplasmic SETDB1 in a corresponding normal control cell (e.g., about 85% to about 115%, and all integers therebetween). In some of these or other non-limiting examples of these embodiments, the level of cytoplasmic SETDB1 in the cell represents a higher level of cytoplasmic SETDB1 than in the nucleus. In representative examples of this type, a higher level represents a ratio of cytoplasmic SETDB1 to nuclear 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 these and other non-limiting examples, the method comprises detecting normal levels of cytoplasmic SETDB1 in greater than 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the subject's cells.

[0048] Suitably, in any of the above embodiments, the method comprises detecting co-localization of SETDB1 with a nuclear binding partner of SETDB1 (e.g., ATF7IP). In some examples of these embodiments, the method comprises contacting a sample comprising cells or a lysate of cells from a subject with a first antigen binding molecule that specifically binds to SETDB1 and a second antigen binding molecule (e.g., ATF7IP) that specifically binds to the nuclear binding partner, and detecting the presence in the sample of a complex comprising the first antigen binding molecule and the second antigen binding molecule, thereby determining 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 compared to a control (e.g., a matched normal control cell), indicating that the subject has an increased likelihood of resistance to the therapy. In other embodiments, the method comprises detecting a level of the complex in the nucleus compared to a control (e.g., a matched normal or immunocompetent control cell), which level indicates a normal level of the complex and indicates that the subject has an increased likelihood of susceptibility to the therapy.

[0049] Another aspect of the present disclosure provides methods for determining the likelihood of resistance to a therapy (e.g., immunotherapy) in a subject. These methods, as generally understood, consist of, or consist essentially of, detecting co-localization of SETDB1 with a nuclear binding partner of SETDB1 (e.g., ATF7IP) in a sample from the subject (e.g., a sample comprising SETDB1-expressing cells, such as tumor cells, or a lysate thereof), or a level of co-localization that indicates an aberrant or abnormal level of co-localization and correlates with the likelihood of increased resistance to the therapy, thereby determining that the subject has an increased likelihood of resistance to the therapy. In some embodiments, the method comprises detecting a higher level of co-localization in the sample compared to a control (e.g., a reference sample comprising corresponding normal control SETDB1-expressing cells, or a lysate thereof), thereby determining that the subject has an increased likelihood of resistance to the therapy. In other embodiments, the method includes detecting approximately the same level of colocalization in the sample compared to a control (e.g., a reference sample containing corresponding SETDB1-expressing cells with abnormal levels of nuclear SETDB1), thereby determining that the subject has a likelihood of increased resistance to the therapy.

[0050] In a related aspect, the present disclosure provides methods for determining the likelihood of resistance to a therapy (e.g., immunotherapy) in a subject. These methods, as generally understood, consist of, or consist essentially of, detecting in a sample from the subject (e.g., a sample containing SETDB1-expressing cells, such as tumor cells, or a lysate thereof), the presence of a complex comprising SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP), or a level of the complex that indicates aberrant or abnormal levels of co-localization of the complex and correlates with the likelihood of increased resistance to the therapy, thereby determining that the subject has an increased likelihood of resistance to the therapy.

[0051] In some embodiments, the method includes detecting a higher level of the complex in the sample compared to a control (e.g., a reference sample comprising corresponding normal control SETDB1-expressing cells, or a lysate thereof), thereby determining that the subject has an increased likelihood of resistance to the therapy.

[0052] In other embodiments, the method includes detecting approximately the same level of the complex compared to a control (e.g., a reference sample containing corresponding SETDB1-expressing cells having abnormal levels of nuclear SETDB1), thereby determining that the subject has an increased likelihood of resistance to the therapy.

[0053] Yet another aspect of the present disclosure provides methods for determining a subject's likelihood of susceptibility to a therapy (e.g., immunotherapy). These methods generally consist of, or consist essentially of, detecting in a subject's sample (e.g., a sample containing SETDB1-expressing cells, such as tumor cells, or a lysate thereof), an absence of colocalization between SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP), or a level of colocalization that indicates a normal level of colocalization and correlates with a likelihood of increased susceptibility to the therapy, thereby determining that the subject has a likelihood of increased susceptibility to the therapy. In some embodiments, the methods involve detecting approximately the same level of colocalization in the subject's sample (e.g., a sample containing tumor cells, or a lysate thereof) compared to a control (e.g., a reference sample containing corresponding normal control SETDB1-expressing cells, or a lysate thereof), thereby determining that the subject has a likelihood of increased susceptibility to the therapy. In other embodiments, the methods involve detecting a lower level of colocalization compared to a control (e.g., a reference sample containing corresponding control SETDB1-expressing cells with abnormal levels of nuclear SETDB1 or a lysate thereof).

[0054] In a related aspect, the disclosure provides methods for determining the likelihood of susceptibility to a therapy (e.g., immunotherapy) in a subject. These methods generally consist of, or consist essentially of, detecting in a subject's sample (e.g., a sample containing SETDB1-expressing cells, such as tumor cells, or a lysate thereof), the absence of, or a normal level of, colocalization between SETDB1 and a nuclear binding partner of SETDB1 (e.g., ATF7IP) and a level of colocalization that indicates a likelihood of increased susceptibility to the therapy and that correlates with a likelihood of increased susceptibility to the therapy, thereby determining that the subject has a likelihood of increased susceptibility to the therapy.

[0055] In some embodiments, the method includes detecting approximately the same level of the complex compared to a control (e.g., a reference sample comprising corresponding normal or immunocompetent control SETDB1-expressing cells or a lysate thereof), thereby determining that the subject has a likelihood of increased susceptibility to the therapy.

[0056] In other embodiments, the method includes detecting a lower level of the complex compared to a control (e.g., a reference sample comprising corresponding control SETDB1-expressing cells having abnormal levels of nuclear SETDB1, or a lysate thereof), thereby determining that the subject has a likelihood of increased susceptibility to the therapy.

[0057] In another aspect, the disclosure provides methods for analyzing 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 co-localization of SETDB1 with a nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα) in a cell, thereby determining the localization of SETDB1 in the cell. In some embodiments, the presence of co-localization indicates nuclear localization of SETDB1. In other embodiments, the absence of co-localization indicates cytoplasmic localization of SETDB1. In yet other embodiments, the method comprises detecting a normal level of co-localization compared to a control (e.g., the level of co-localization in matched normal or immunocompetent cells, or a lysate thereof), indicating that there is higher cytoplasmic localization of SETDB1 than nuclear localization of SETDB1. In yet other embodiments, the method comprises detecting a higher level of colocalization compared to a control (e.g., the level of colocalization in a matched normal cell or a lysate thereof), indicating that there is greater nuclear localization of SETDB1 than cytoplasmic localization of SETDB1. In representative examples of these embodiments, the colocalization is represented by a complex comprising SETDB1 and a nuclear binding partner of SETDB1.

[0058] Yet another aspect of the present disclosure provides methods for stratifying a subject as likely to be a responder or non-responder to a therapy (e.g., immunotherapy). These methods generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1 in a sample from the subject to determine whether the subject has an increased likelihood of sensitivity or resistance to the therapy, thereby stratifying the subject as likely to be a responder or non-responder to the therapy, as broadly described above and elsewhere herein.

[0059] Further aspects of the present disclosure provide methods for managing treatment of a subject with a therapy (e.g., immunotherapy), which methods include, consist of, or consist essentially of selecting a subject for treatment with the therapy based on the subject being likely to be a responder to the therapy, or selecting a subject not to treat with the therapy based on the subject being likely to be a non-responder to the therapy, and treating or not treating the subject with the therapy based on that selection, wherein the selection is based on the stratification methods described broadly above and elsewhere herein.

[0060] In another aspect of the present disclosure, methods are provided for predicting a subject's therapeutic outcome from a therapy (e.g., immunotherapy). These methods generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1, as broadly described above and elsewhere herein, in a sample from the subject to determine whether the subject has a likelihood of increased sensitivity or resistance to the therapy, thereby predicting the subject's therapeutic outcome. In some embodiments, the method comprises detecting the presence or level of nuclear-localized SETDB1 relative to a control, which correlates with a likelihood of increased resistance to the therapy, as broadly described above and elsewhere herein, and predicting a negative therapeutic outcome. Preferably, a negative therapeutic outcome is greater disease severity or progressive disease. In other embodiments, the method comprises detecting the absence or level of nuclear-localized SETDB1 relative to a control, which correlates with a likelihood of increased sensitivity to the therapy, as broadly described above and elsewhere herein, and predicting a positive therapeutic outcome. A positive therapeutic outcome may be selected from a partial or complete response to the therapy and stable disease. In any of these embodiments, the method suitably further comprises predicting a clinical outcome for 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.

[0061] Further aspects of the present disclosure provide methods of 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 of the subject with a therapy (e.g., immunotherapy), where the sample comprises SETDB1-expressing cells (e.g., tumor cells), and analyzing the cellular localization of SETDB1 in the sample as broadly described above and elsewhere herein, where a lower level of nuclear-localized SETDB1 compared to a control sample from the subject taken before treatment indicates an increased clinical benefit of the therapy to the subject (e.g., less disease severity, delayed disease progression, reduced rate of disease progression, or absence or amelioration of disease), and where a similar or higher level of nuclear-localized SETDB1 compared to the control sample indicates no or minimal clinical benefit of the therapy to the subject.

[0062] Yet further aspects of the present disclosure provide methods for determining the status of a disease in a subject, which generally comprise, consist of, or consist essentially of analyzing the cellular localization of SETDB1, as broadly described above and elsewhere herein, in a sample from the subject, thereby determining the status of the disease in the subject, wherein the presence or level of nuclear-localized SETDB1, which correlates with the likelihood of increased resistance to therapy, as broadly described above and elsewhere herein, indicates a higher severity or progression of the disease in the subject, and wherein absence or a level of nuclear-localized SETDB1, which correlates with the likelihood of increased sensitivity to therapy, as broadly described above and elsewhere herein, indicates the absence of disease or a less severe or progression of the disease in the subject.

[0063] Yet another aspect of the present disclosure provides kits for detecting the location of SETDB1 in the cellular location (e.g., cytoplasm or nucleus) of a cell, predicting the likelihood of a cell's response to a therapy (e.g., immunotherapy), determining a subject's likelihood of resistance to a therapy (e.g., immunotherapy), determining a subject's likelihood of susceptibility to a therapy (e.g., immunotherapy), stratifying a subject as likely to be a responder or non-responder to a therapy (e.g., immunotherapy), administering a subject's treatment with a therapy (e.g., immunotherapy), monitoring disease in a subject after treatment with a therapy, determining disease status in a subject, and / or determining a subject's immune status. 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, preferably comprising a detectable label.

[0064] Suitably, the kit further comprises instructional materials for carrying out any one or more of the methods described above and / or broadly elsewhere herein.

[0065] Yet another aspect of the present disclosure provides a complex comprising SETDB1, a core-binding partner of SETDB1 (e.g., ATF7IP, IMPα), a first antigen-binding molecule that specifically binds to SETDB1 in the complex, and a second antigen-binding molecule that binds to the core-binding partner in the complex. In some embodiments, the complex is located in a cell or a lysate thereof. Preferably, the complex further comprises a third antigen-binding molecule that binds to each of the first and second antigen-binding molecules in the complex, and is suitably detectably labeled.

[0066] In a further aspect, the present disclosure provides a cell or lysate thereof comprising a complex as broadly described above and elsewhere herein.

[0067] 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 explanation of the drawings]

[0068] [Figure 1] Interaction SETDB1 / IMPA1 and SETDB1 / ATF7IP:MSETC dose response. (A) Exemplary fields of imaging are shown with an orange 10 mM scale bar. Cells were permeabilized by incubation with 0.5% Triton X-100 for 20 min and probed with mouse anti-ATF7IP, rabbit anti-SETDB1, and goat anti-IMPα1, and visualized with donkey anti-rabbit AF568, anti-mouse 488, and anti-goat 647. Coverslips were mounted on glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. (B-E) Bar graphs show the nuclear or cytoplasmic fluorescence intensity of SETDB1 using SEM, and the ratio of nuclear to cytoplasmic staining (Fn / c) of SETDB1. Graphs show the cytoplasmic PCC of SETDB1 and ATF7IP (B) and SETDB1 and IMPα1 (C). The graphs show the nuclear fluorescence intensity of SETDB1 (D) and ATF7IP (E). [Figure 2]Effect of SETDB1 prototype peptide on proliferation in MDA-MB-231 cells. MDA-MB-231 cells were treated with 047 and 047-1 for up to 72 hours. At 48 hours (A) and 72 hours (B), the medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. Absorbance was recorded at 450 nm for 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 both 48 hours and 72 hours, the medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. Absorbance was recorded at 450 nm for 1 hour using a microplate spectrophotometer. [Figure 3] Effect of SETDB1 prototype peptide 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). Exemplary images are represented by a 10 μM scale bar. (047=PEP1, 047-1=PEP2). [Figure 4] Effect of SETDB1 NLS linear peptide versus bicyclic peptide on proliferation in cancer cell lines. (A) Structural characterization of the IMPα1 (gray), SETDB1 (green), and ATF7IP (blue) trimeric complex. (B) Electrophoretic mobility shift assay showing the interaction between IMPα1 and MSETC. (C) Table showing epigenetic induction transcription pathways in mesenchymal breast cancer cell lines. (D-E) MDA-MB-231 and MDA-MB-231 brain cells were treated with 047-1 or MSETC bicyclic peptide for 72 hours. After incubation, the medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. Absorbance was recorded at 450 nm for 1 hour using a microplate spectrophotometer. [Figure 5](A, B) Proliferation assay of the TNBC cell lines MDA-MB-231 (A) and CT26 (B), which are responsive to MSETC and TBAB-MSETC inhibitor immunotherapy. Cells were treated with MSETC or MSETC-TBAB bicyclic peptide for 72 hours. After 72 hours, the medium was removed and replaced with 100 μL / cell of WST-1 cell proliferation reagent. Absorbance was recorded at 450 nm for 1 hour using a microplate spectrophotometer. (C)–(F) Proliferation assay of the lung cancer cell line LLC using the TBAB-MSETC (C), MSETC (D), MSETC-D (E), and MSETD-DR (F) inhibitors. After 72 hours, the medium was removed and replaced with 100 μL / well of WST-1 cell proliferation reagent. Absorbance was recorded at 450 nm for 1 hour using a microplate spectrophotometer. [Figure 6] Bicyclic peptide treatment does not affect cell viability in healthy PBMC samples. Healthy PBMCs were treated overnight with various concentrations of MSETC (2.5-40 μM) before staining with the LIVE / DEAD™ 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. [Figure 7]MDA-MB-231 cells were treated with 10 mM MSETC. (A) Graph of nuclear fluorescence intensity (NFI) for the markers C-REL, G9A, PKCb1, and the custom antibody PDL1-PTM1. MDA-MB-231 cells were treated with MSETC or control and permeabilized before being probed with antibodies specific for C-REL, G9A, PKCb1, and the custom antibody PDL1-PTM1. Coverslips were mounted on glass microscope slides using ProLong nucblue glass antifade reagent (Life Technologies). Digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA), and graphs represent the mean nuclear fluorescence intensity in the nuclear or cytoplasmic compartment with significant differences calculated according to one-way ANOVA pairwise comparisons. (B) H1299 cells treated with MSETC, the general importin-α1 inhibitor BIMAX, or the SETDB1 catalytic inhibitor MTH, or vehicle were 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. [Figure 8] SETDB1 is cytoplasmic and has low expression in healthy PBMCs. Healthy donor PBMCs were isolated from liquid biopsies and stained for SETDB1. An exemplary field of imaging of PBMCs is shown with an orange 10 μM scale bar. Cells were permeabilized with 0.5% Triton X-100 for 20 minutes, probed with rabbit anti-SETDB1, and visualized with anti-rabbit 568. Coverslips were mounted onto glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. [Figure 9]SETDB1 is cytoplasmic with low expression in healthy donor tonsil tissue. Healthy tonsil tissue was stained using the BondRX automated staining platform. An exemplary field of tissue imaging is shown with an orange 10 μM scale bar. Tissue sections were probed with rabbit anti-SETDB1 and visualized with anti-rabbit 568. Coverslips were mounted onto glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. [Figure 10] SETDB1, a novel epigenetic oncogene, is enriched in NSCLC and other advanced cancers. (A) Percentage of TCGA cases with SETDB1 high- or low-level amplification (amp); BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CESC, cervical squamous cell carcinoma and 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, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; OV, ovarian serum cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PRAD, prostate adenocarcinoma; SARC, sarcoma; STAD, gastric adenocarcinoma; SKCM, skin cutaneous melanoma; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; UCEC, endometrial carcinoma; UCS, uterine carcinosarcoma. (B) SETB1 levels in 492 lung adenocarcinoma and 485 lung squamous cell carcinoma tissues compared with normal tissues (59 and 50, respectively) (http: / / gepia.cancer-pku.cn / ). (C) Kaplan-Meiers survival analysis of SETDB1 expression in NSCLC patients with low and high tumor expression of SETDB1, calculated from the significant difference shown (http: / / kmplot.com / analysis / ). [Figure 11]SETDB1 signature in CTCs from a metastatic patient cohort undergoing immunotherapy. Liquid biopsies were stratified into "resistant" (A) and "responder" (B) cohorts based on response to immunotherapy. CTCs were 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 AF568 and anti-mouse 488 secondary antibodies. Coverslips were mounted on glass microscope slides using ProLong nucblue transparent antifade reagent (Life Technologies). Protein targets were localized by confocal laser scanning microscopy. Single 0.5 μm sections were acquired using an ASI Digital Pathology System with a 100x oil immersion lens running ASI software. The final image was obtained by averaging four consecutive images of the same section. (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 number, and fluorescence intensity (protein expression). [Figure 12] Nuclear SETDB1 is enriched in immunotherapy-resistant patients and not expressed in healthy nuclei. (A) PIE: Total CTCs or % CTC population per 10 mL in metastatic cancer liquid biopsies. CSV+SETDB1+CD45-. (B) Representative digital pathology high-resolution immunofluorescence images of SETDB1 expression in immunotherapy-resistant and -responsive stage IV metastatic FFPE tumors. (C) Graph showing nuclear staining (NFI) of SETDB1 and PCC (colocalization) of SETDB1, ATF7IP, and IMPα1. (D) Representative digital pathology high-resolution immunofluorescence images of SETDB1 and 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 shown. (E) Chart analysis of CD8+PD1+ T cells positive for the end-stage exhaustion marker EOMESAC in responder or resistant patients with SETDB1 intensity. Rsp: responsive, Rsi: resistant. [Figure 13] Increased SETDB1 nuclear expression in "resistant" patient cohort samples. (A) FFPE samples were processed on BONDRX using the Opal staining kit targeting melanoma cancer markers [HMB45+M2-7C10+M2-9E3] (ML) and SETDB1. Green represents melanoma cancer markers [HMB45+M2-7C10+M2-9E3], and magenta represents SETDB1. (B-D) Bar graphs show the ratio of nuclear to cytoplasmic staining for SETDB1 by SEM, integrated nuclear intensity, or integrated cytoplasmic intensity. Significant differences are indicated as calculated by the Kruskal-Wallis nonparametric test. [Figure 14] SETDB1 / IMPα1:MSETC interaction dose response. An exemplary field of imaging of MDA-MB-231 cells is shown with an orange 10 μM scale bar. Cells were treated with the SETDB1 bicyclic inhibitor MSETC at concentrations ranging from 1.25 mM to 20 mM or vehicle control, 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 and anti-mouse AF 488. Coverslips were mounted on glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. (B) Bar graphs show the nuclear or cytoplasmic fluorescence intensity of SETDB1 using SEM, and the ratio of nuclear to cytoplasmic staining of SETDB1 (Fn / c). (C) Graphs show the nuclear or cytoplasmic PCC of SETDB1 / IMPα1. Significant differences are indicated as calculated by the Kruskal-Wallis non-parametric test. The red dotted line represents 50% inhibition. [Figure 15]MDA-MB-231 cells were treated with MSETC at concentrations ranging from 5 μM to 0.078 μM. This figure shows high-resolution imaging of DUOLINK cells stained with SETDB1 and IMPα1. MDA-MB-231 cells were treated with MSETC or control, permeabilized, and probed with the DUOLINK ligation assay. Coverslips were mounted on glass microscope slides using ProLong nucblue glass antifade reagent (Life Technologies). MSETC and IMPα1 DUOLINK digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA). Graphs represent mean DOT fluorescence intensity, with significant differences calculated according to Kruskal-Wallis one-way ANOVA. The IC50 is marked with a red dotted line. [Figure 16] The dual-targeting inhibitor of MSETC is selective for SETDB1 and superior to catalytic inhibitors. (A) MDA-MB-231 cells were treated with 5 mM MSETC, the catalytic SETDB1 inhibitor Mithramycin A (MTH), or vehicle control, permeabilized by incubation with 0.5% Triton X-100 for 20 minutes, probed with rabbit anti-H3k27ac and mouse anti-H3k9me3, and visualized with donkey anti-rabbit AF568 and anti-mouse 488. Coverslips were mounted on glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized using ASI Digital Pathology. Bar graphs show the nuclear fluorescence intensity of H3k9me3 and H3k27ac, indicating significant differences as calculated by the Kruskal-Wallis nonparametric test. (B) Cells were treated with MSETC or vehicle. Cells were permeabilized and stained for H3.3Ser1p and H3k4me2, and digital pathology was used to analyze expression. Significance was determined using Mann-Whitney. [Figure 17](A) H1299 (lung cancer cells) or MDA-MB-231 cells were treated with MSETC (either MSETC or MSETC-D-isomer version (MSETC-D) or MSETC-D-isomer-letero-inverso (MSETC-DR)), the general IMPα1 inhibitor BIMAX, or the SETDB1 catalytic inhibitor MTH. Data from high-resolution imaging of DUOLINK cells stained with SETDB1 IMPα1. MDA-MB-231 or H1299 cells were probed with the DUOLINK ligation assay. Coverslips were mounted on glass microscope slides using ProLong nucblue glass antifade reagent (Life Technologies). MSETC and IMPα1 DUOLINK digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA). Graphs represent mean DOT fluorescence intensity, with significant differences calculated according to Kruskal-Wallis one-way ANOVA. [Figure 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, 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 and anti-mouse 488. Coverslips were mounted on glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. Bar graphs show the nuclear intensities of H3k9me3 and H3k27ac with significant differences, as calculated by the Kruskal-Wallis nonparametric test. (B) H1299 cells were treated with 5 mM MSETC or vehicle control, 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 AF568, anti-mouse 488, and anti-goat 647. Coverslips were mounted on glass microscope slides using ProLong Clear Antifade reagent (Life Technologies). Protein targets were localized by ASI Digital Pathology. (C) Bar graphs show the nuclear intensity or PCC of antibody pairs, with significance calculated by the Kruskal-Wallis nonparametric test. [Figure 19] Increased expression of interferon signaling genes in MCF-7 cells after treatment with bicyclic peptides. Real-time qPCR analysis of MCF-7 cells treated with bicyclic peptides (5 μM) for 24 h before stimulation with phorbol 12-myristate 13-acetate (PMA; 20 ng / mL; Sigma) or poly(I:C) (500 ng / mL; Sigma) for 48 h. Ct was 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. [Figure 20-1]Increased expression of viral mimicry and immunogenic / immunovisible genes in epithelial and mesenchymal MCF-7 cells after treatment with bicyclic peptides. Real-time qPCR analysis of MCF-7 cells treated with bicyclic peptides (5 μM) for 24 hours before stimulation with phorbol 12-myristate 13-acetate (PMA; 20 ng / mL; Sigma) for 48 hours to induce a mesenchymal signature. Ct values ​​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<0.005, unpaired t-test. [Figure 20-2] This is a continuation of Figure 20-1. [Figure 20-3] This is a continuation of Figure 20-2. [Figure 21] Bicyclic peptide (MSETC) + / - αPD1 therapy in the 4T1 model of metastatic breast cancer. (A) Treatment regimen using a Balb / c 4T1 breast cancer model. (B) Mouse weight (g) over the 20-day treatment period. (C) Tumor volume (n=4-5 / group) of mice treated with vehicle (saline) or MSETC (20 mg / kg) in conjunction with αPD1 or isotype control (10 mg / kg). (D) Tumor volume of individual mice at 20 days post-inoculation (data presented in C; *p<0.05, **p<0.01, Tukey's post-hoc test). (E) Representative images of tumors harvested at 20 days post-inoculation. [Figure 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 20 days post-inoculation. (B) Final lung, liver, and spleen weights 20 days post-inoculation. *p<0.05, Tukey's post-hoc test. [Figure 23] Combination MSETC and αPD1 therapy reduces lung metastases in the 4T1 model of metastatic breast cancer. (A) Representative images of lungs fixed in Bouin's solution. (B) Number of lung nodules (n=4-5 / group) in mice treated with vehicle (saline) or MSETC (20 mg / kg) in combination with αPD1 or isotype control (10 mg / kg) at 20 days post-inoculation. *p<0.05, one-way ANOVA. [Figure 24] Combination therapy with MSETC and αPD1 reduces primary tumor CSV expression in metastatic breast cancer. (A) Representative images of FFPE primary tumor samples processed on BONDRX using Opal staining kits targeting cytokeratin, CSV, or SETDB1. (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 as calculated by the Kruskal-Wallis nonparametric test. [Figure 25] Combination therapy with MSETC and αPD1 reduces lung CSV expression in metastatic breast cancer. (A) Representative images of FFPE lung samples processed on BONDRX using Opal staining kits targeting cytokeratin, CSV, or SETDB1. (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 as calculated by the Kruskal-Wallis nonparametric test. [Figure 26] Combination therapy of MSETC and αPD1 increases primary tumor TRM, TEM, TCM, and T effector cell populations in metastatic breast cancer. (A) Representative images of FFPE primary tumor samples processed on BONDRX using the Opal staining kit targeting either CD44, CD103, CD69, CD62L, or CD8. Stained sections were analyzed for tissue-resident memory (B), effector memory (C), central memory (D), or effector (E) T cell populations. Significant differences are indicated as calculated by the Kruskal-Wallis nonparametric test. Four to five tissue sections were analyzed for each group, and more than 500 cells were counted per section. [Figure 27]MSETC reprograms the histone code in a 4T1 tumor model. (A) Primary tumor samples from a 4T1 tumor model treated as indicated were analyzed on BONDRX using the Opal staining kit targeting CSV (tumor marker), H3k27ac, and H3k9me3. (B) The graph shows the nuclear fluorescence intensity (NFI) of H3k28ac and H3k9me3. Significant differences are indicated as calculated by the Kruskal-Wallis nonparametric test. [Figure 28] Administration of C-DR (5 mg / kg) versus MSETC (15 mg / kg) in combination with anti-PD1 reduces tumor burden in a 4T1 syngeneic tumor model. (A) Treatment regimen using a Balb / c 4T1 breast cancer model. (B) Mouse weight (g) before culling on day 9. Data are presented as mean ± SEM. (C) Tumor volume in mice treated with MSETC-DR (5 mg / kg) versus mice treated with MSETC (15 mg / kg) in conjunction with αPD1 (10 mg / kg) (n = 5 / group). Data are presented as fold change relative to day 0. a: **p<0.01 MSETC and MSETC-DR vs. αPD1, b: **p<0.01 MSETC and MSETC-DR vs. αPD1, c: **p<0.01 and *p<0.05 MSETC and MSETC-DR vs. αPD1, respectively, d: **p<0.01 MSETC and MSETC-DR vs. αPD1, two-way ANOVA, Dunnett's post-hoc test. (D) Representative images of tumors harvested on day 9. (E) Liver, spleen, and lung weights (mg) on ​​day 9. [Figure 29] Effect of MSETC and MSETC-DR on cell migration in the MCF-7 PMA / TGFβ-induced model. (A) Wound healing (scratch assay) analysis of the effect of MSETC and MSETC-DR on cell migration over a 24-hour period. Black line: water control, blue line: MSETC (20 μM), orange line: MSETC-DR (20 μM). (B) Graphical representation of relative wound density (%) at 24 hours as depicted in (A). **p<0.01, ****p<0.0001, One-way ANOVA, Dunnett's post-hoc test (n=6 / group). DETAILED DESCRIPTION OF THE INVENTION

[0069] 1.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can 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.

[0070] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., 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.

[0071] The term "about" as used herein refers to a normal range of error for the respective value, readily known to one of ordinary skill in the art. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself.

[0072] The terms "antagonist" or "inhibitor" refer 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 that target. In some embodiments, the terms can refer to an antibody that prevents the target to which it binds, e.g., PD-1, from carrying out its biological function.

[0073] The term "antibody" as used herein is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired biological activity.

[0074] The "amount" or "level" of a biomarker is the detectable level in a sample. These can be measured by methods known to those of skill in the art and also disclosed herein. The expression level or amount of the biomarker being assessed can be used to determine response to treatment.

[0075] Terms such as "concurrent administration" or "administering simultaneously" or "co-administration" refer to the administration of a single composition containing two or more active agents, or the administration of each active agent as a separate composition, and / or delivery by separate routes, contemporaneously, simultaneously, or sequentially, within a sufficiently short period of time that effective results are comparable to those obtained when all such active agents are administered as a single composition. "Concurrently" means that the active agents are administered together at substantially the same time, preferably in the same formulation. "Concurrently" means that the active agents are administered close in time, either before or after another agent (e.g., one agent is administered within about 1 minute to about 1 day). Any contemporaneous time is useful. However, in many cases, when not administered simultaneously, the agents are administered within about 1 minute to about 8 hours, preferably less than about 1 hour to about 4 hours. When administered simultaneously, the agents are preferably administered to the same site on a subject. The term "same site" includes the exact location, but may be within about 0.5 cm to about 15 cm, preferably within about 0.5 cm to about 5 cm. As used herein, the term "separately" means that the agents are administered at intervals, for example, from about one day to several weeks or months. The active agents may be administered in any order. As used herein, the term "sequentially" means that the agents are administered sequentially, for example, at intervals of minutes, hours, days, or weeks, or multiple intervals. If desired, the active agents may be administered in a regular, repeated cycle.

[0076] The term "agent" includes compounds that induce a desired pharmacological and / or physiological effect. This term also encompasses pharmacologically acceptable active ingredients of these compounds specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, and the like. It should be understood that when the above term is used, it includes not only the active agent itself, but also pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, and the like. The term "agent" should not be construed narrowly, but extends to small molecules, SETDB1 bicyclic peptidomimetics (such as peptides, polypeptides, and proteins), and compositions containing them, as well as genetic molecules (RNA, DNA, and their mimetics and chemical analogs), and cellular agents.

[0077] As used herein, "and / or" refers to and includes all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted in the alternative (or).

[0078] As used herein, the terms "bind," "specifically bind to," or "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody, which determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target with higher affinity, avidity, more readily, and / or longer duration than it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of less than 1 mM, less than 100 nM, less than 10 nM, less than 1 nM, or less than 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 can include, but does not require, exclusive binding.

[0079] As used herein, the term "biomarker" refers to an indicator that can be detected in a sample, for example, a predictive, diagnostic, and / or prognostic indicator.

[0080] The terms "cancer" and "cancerous" refer to or describe the physiological condition in a subject that is typically characterized by uncontrolled 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 squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal 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 (low-grade). These include, but are not limited to, high-grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade / follicular NHL; intermediate-grade diffuse NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's macroglobulinemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL), hairy cell leukemia; chronic myeloblastic leukemia; and post-transplant lymphoproliferative disorder (PTLD), as well as abnormal blood vessel growth associated with phakomatosis, edema (such as that associated with brain tumors), Meigs' syndrome, brain, and head and neck cancers and associated metastases. In certain embodiments, cancers amenable to treatment with 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 carcinoma, 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 particular embodiments, the cancer is melanoma or lung cancer, preferably metastatic melanoma or metastatic lung cancer.

[0081] "Chemotherapeutic agents" include compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate, and fluticasone. nib (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (SCH 66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents (such as thiotepa and CYTOXAN® cyclophosphamide); alkyl sulfonates (such as busulfan, improsulfan, and piposulfan); aziridines (such as benzodopa, carboquone, meturedopa, and uredopa); ethyleneimines and methylameramines (including altrelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine); acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cryptoterone acetate;5a-reductases (including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat; dolastatins; aldesleukin, talc; duocarmycins (including synthetic analogs, KW-2189 and CB1-TM1); eloterobin; pancratistatin; sarcodictiin; spongistatins; nitrogen mustards (chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethrin) mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trophosfamide, uracil mustard, etc.; nitrosoureas (carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine, etc.); antibiotics (enediyne antibiotics, etc.) (e.g., calicheamicin, particularly calicheamicin g1I and calicheamicin w1I (Angew. Chem. Inti. Ed. Engl. 1994) 33:183-186); dynemicins (including dynemicin A); bisphosphonates (such as clodronate); esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxo rubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins (mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, etc.); antimetabolites (methotrexate and 5-fluorouracil (5-FU), etc.);Folic acid analogues (denopterin, methotrexate, pteropterin, trimetrexate, etc.); purine analogues (fludarabine, 6-mercaptopurine, thiamiprine, thioguanine, etc.); pyrimidine analogues (ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.); androgens (calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.); antiadrenergics (aminoglutethimide, mitotane, trilostane, etc.); folic acid supplements (furoic acid, etc.); aceglatone; aldofosin Sulfamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziconazole; elfomitine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids (such as maytansine and ansamitocin); mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicon; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veraculin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (Cremophor-free), an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, III.), and TAXOTERE® (docetaxel; Sanofi-Aventis);Chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs (such as cisplatin and carboplatin); vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; etatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids (such as retinoic acid); and pharmaceutically acceptable salts, acids, and derivatives of any of the above;

[0082] Chemotherapeutic agents include (i) antihormonal agents that act to regulate or inhibit hormone action on tumors, such as antiestrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®, tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) anti-tumor agents, such as, for example, 4(5 aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 2-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestany, fadrozole, RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (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 (1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase inhibitors; (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, especially those inhibiting signal transduction pathways involved in abnormal cell proliferation. those that inhibit the expression of genes in the tract (e.g., PKC-α, Ralf, and H-Ras, etc.); (vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME® and HER2 expression inhibitors); (viii) vaccines such as gene therapy vaccines (e.g., ALLOVECTIN®, LEUVECTIN®, and VAXID®); PROLEUKIN®, rIL-2; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); ABARELIX® rmRH;and (ix) pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0083] Chemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech / Biogen Idee), pertuzumab (OMNITARG®, 2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody-drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Additional humanized monoclonal antibodies with therapeutic potential include apolizumab, acelizumab, atlizumab, bapineuzumab, bivatuzumab, mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidofusituzumab, situzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motavizumab, natalizumab, nimotuzumab, norobizumab, Numaravizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pefusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resivizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tuxituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and interleukin-12 Also included is anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), a recombinant, exclusively human sequence, full-length IgG11 antibody genetically modified to recognize the p40 protein.

[0084] 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, alternatively 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 HB 8506), MAb 455 (ATCC CRL HB 8507), 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; ERBUTIX®) and reconstituted human 225 (H225) (see WO 96 / 40210, Imclone Systems, Inc.). 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); antibodies that bind to EGFR as described in U.S. Pat. No. 5,891,996, humanized and chimeric antibodies; 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 (EMD / Merck) directed against EGFR that competes with both EGF and TGF-α for EGFR binding; the human EGFR antibody, HuMax-EGFR (GenMab); the fully human antibodies known 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 with cytotoxic agents to generate immunoconjugates (see, e.g., EP 659439 A2, Merck Patent GmbH). EGFR antagonists include those described in U.S. Patent Nos. 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, 6,140,332, and 5,800,000. 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO98 / 14451, WO98 / 50038, WO99 / 09016, and WO99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals); PD 183805 (Cl 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 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; 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 and N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl]ethyl]amino]methyl]-2-furanyl)-4-quinazolinamine) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); and dual EGFR / HER2 tyrosine kinase inhibitors (such as lapatinib (TYKERB®, GSK572016, or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl]ethyl]amino]methyl]2-furanyl)-4-quinazolinamine)).

[0085] Chemotherapeutic agents include the EGFR-targeted drugs described in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitors such as TAK165 available from Takeda; CP-724,714 (Pfizer and OSI), an oral selective inhibitor of ErbB2 receptor tyrosine kinase; dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; 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 ISIS-5132, an antisense agent available from ISIS Pharmaceuticals that inhibits Raf-1 signaling; imatinib mesylate (GLEEVEC®, GlaxoSmithKline). non-HER-targeted TK inhibitors such as sunitinib (SUTENT®, available from Pfizer); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT®, 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; CGP 59326, CGP 60261, and CGP pyrrolopyrimidines such as 62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., that bind to a nucleic acid encoding a HER); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787 (Novartis / Schering AG); pan-HER inhibitors such as CI-1033 (Pfizer);Affinitac (ISIS 3521; Isis / Lilly); imatinib mesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), including rapamycin (sirolimus, RAPAMUNE®); or the following patent publications: U.S. Pat. No. 5,804,396, WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid); WO 1997 / 38983 (Warner Lambert); WO 1999 / 06378 (Warner Lambert); WO1999 / 06396 (Warner Lambert); WO1996 / 30347 (Pfizer, Inc); WO1996 / 33978 (Zeneca); WO1996 / 3397 (Zeneca) and WO1996 / 33980 (Zeneca);

[0086] Chemotherapeutic agents include dexamethasone, interferon, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG (raw), bevacizumab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, erotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, and lenacolone. Also included are lidomide, levamisole, mesna, methoxysalen, nandrolone, nelarabine, nofetumomab, oprelvekin, palifermin, pamidronate, pegademase, 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.

[0087] Chemotherapeutic agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, and hydrocortisone-17-butyrate. , hydrocortisone-17-valerate, aclomethasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN) Immunoselective anti-inflammatory peptides (ImSAIDs) such as BioTherapeutics, LLC; antirheumatic drugs such as azathioprine; tumor necrosis factor α (TNF-α) blockers such as cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide, minocycline, sulfasalazine, and etanercept (Enbrel); interferon-like peptides (IL-16) such as infliximab (Remicade), adalimumab (Humira), certolizumab pulegol (Cimzia), golimumab (Simponi), and anakinra (Kineret). interleukin-1 (IL-1) blockers, T-cell costimulation blockers such as abatacept (Orencia), interleukin-6 (IL-6) blockers such as tocilizumab (ACTEMERA®); interleukin-13 (IL-13) blockers such as lebrikizumab; interferon-α (IFN) blockers such as rontalizumab; beta-7 integrin blockers such as rhuMAb beta-7; IgE pathway blockers such as anti-Mi prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa / 82 blockers such as anti-lymphotoxin-α (LTa); radioisotopes (e.g., At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi212 、P 32 、Pb 212and radioactive isotopes of Lu; miscellaneous investigational agents such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavins, flavanols, procyanidins, buteric acid, and their derivatives; autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapacone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®); clodronate (e.g., BONEFOS®) bisphosphonates such as benzodiazepine (BMP) or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines such as the THERATOPE® vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors such as oblimersen sodium (GENASENSE®); pixantrone; lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids, or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is the abbreviation for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, which is the abbreviation for the treatment regimen of oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0088] Chemotherapeutic agents also include nonsteroidal anti-inflammatory drugs (NSAIDs), which have analgesic, antipyretic, and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives (ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, naproxen, etc.), acetic acid derivatives (indomethacin, sulindac, etodolac, diclofenac, etc.), enolic acid derivatives (piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, etc.), fenamic acid derivatives (mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, etc.), and COX-2 inhibitors (celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, valdecoxib, etc.). NSAIDs may be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue injury, fever, ileus, and renal colic.

[0089] As used herein, the term "colocalization" or "colocalization" refers to two or more molecules that have identical or overlapping localization within a cell. Colocalization of molecules and proteins can be detected using any suitable method known in the art, including, for example, fluorescence microscopy in fixed or live cells. For example, SETDB1 and a SETDB1 nuclear binding partner (e.g., ATF7IP, IMPα) can be colocalized within a cell using fluorescently labeled anti-SETDB1 and anti-nuclear binding partner primary antibodies, and optionally one or more secondary antibodies. Methods for colocalization of cellular molecules are well known.

[0090] The terms "cell proliferative disorder," "proliferative disorder," and "hyperproliferative disorder" are used interchangeably herein to refer to disorders 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.

[0091] 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 to identify effective dosages for subjects or subgroups or other groups of subjects. For purposes herein, a companion diagnostic refers to a reagent, such as a reagent for detecting or measuring SETDB1 cellular localization (e.g., as described herein) in a sample. A companion diagnostic refers to a reagent, and also to an assay performed using the reagent.

[0092] As used herein, the term "complex" refers to an assembly or aggregation of molecules (e.g., peptides, polypeptides, etc.) that directly and / or indirectly contact each other. In certain embodiments, "contact," or more specifically, "direct contact," means that two or more molecules are sufficiently close that attractive non-covalent interactions, such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions, dominate the molecular interaction. In such embodiments, a molecular complex (e.g., a peptide and a polypeptide) forms under conditions such that the complex is thermodynamically favorable (e.g., compared to the unaggregated or uncomplexed states of its constituent molecules). The term "polypeptide complex" or "protein complex," as used herein, refers to a trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, decamer, decadecamer, dodecamer, or higher-order oligomer. In certain embodiments, the polypeptide complex is formed by self-assembly of SETDB1 and its core binding partner (e.g., ATF7IP, IMPα).

[0093] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" mean the inclusion of a recited step or element, or group of steps or elements, but not the exclusion of any other step or element, or group of steps or elements. Thus, use of terms such as "comprising" indicates that the recited elements are required or mandatory, but that other elements are optional and may or may not be present. "Consisting of" means including and limited to what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or mandatory, but that other elements may or may not be present. "Consisting essentially of" means including any elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or mandatory, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0094] The terms "correlate" or "correlating" refer to determining the relationship between one type of data and another type of data or condition (e.g., response to a therapy). In some embodiments, "correlate" or "correlating" refers to comparing, in any manner, the performance and / or results of a first analysis or protocol with the performance and / or results of a second analysis or protocol. For example, the results of the first analysis or protocol may be used in performing a second protocol and / or to determine whether to perform a second analysis or protocol. With respect to embodiments of polypeptide analyses or protocols, the results of a polypeptide expression or cellular localization analysis or protocol may be used to determine whether to implement a particular treatment regimen.

[0095] "Corresponds to" or "corresponding to" refers to an amino acid sequence that exhibits substantial sequence similarity or identity with a reference amino acid sequence. Generally, the amino acid sequence exhibits at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 97, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or even up to 100% sequence similarity or identity to at least a portion of the reference amino acid sequence.

[0096] "Derivative" means a molecule, such as a polypeptide, that is derived from the basic molecule by modification, for example, by conjugation or complexation with other chemical moieties, or by art-recognized post-translational modification techniques. The term "derivative" also includes within its scope changes made to the parent sequence, including additions or deletions, that provide for functionally equivalent molecules.

[0097] As used herein, the term "unit dosage form" refers to physically discrete units suited as unitary dosages for the subjects to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle.

[0098] An "effective amount" is at least the minimum amount necessary to achieve measurable improvement or prevention of a particular disorder. The effective amount herein may vary depending on factors such as the patient's condition, age, sex, and weight, as well as the ability of the antibody to elicit a desired response in an individual. An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as elimination or reduction of the risk, reduction in severity, or delay in onset of disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes presented during the progression of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing one or more symptoms resulting from the disease, improving the quality of life of people suffering from the disease, reducing the dose of other drugs required to treat the disease, enhancing the effectiveness of other drugs, such as through targeting, slowing the progression of the disease, and / or prolonging survival. In the case of cancer or tumors, an effective amount of a drug may have the effect of reducing the number of cancer cells, reducing the size of a tumor, inhibiting (i.e., slowing or preferably stopping) the invasion of cancer cells into peripheral organs, inhibiting (i.e., slowing or preferably stopping) tumor metastasis, inhibiting (i.e., slowing or preferably stopping) tumor growth, and / or alleviating to some extent one or more symptoms associated with cancer or tumors. In the case of infectious diseases, an effective amount of a drug may have the effect of reducing the titer of a pathogen (bacteria, virus, etc.) in the circulation or tissue, reducing the number of cells infected with a pathogen, inhibiting (i.e., slowing or preferably stopping) pathogen infection of an organ, inhibiting (i.e., slowing or preferably stopping) pathogen growth, and / or alleviating to some extent one or more symptoms associated with an infectious disease. An effective amount can be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve prophylactic or therapeutic treatment either directly or indirectly. As understood in a clinical context, an 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 administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in combination with one or more other agents, a desired result can be or is achieved.

[0099] A patient "effective response" or patient "responsiveness" to treatment with a drug and similar terms refers to a clinical or therapeutic benefit conferred on a patient at risk for or suffering from a disease or disorder, such as cancer. In one embodiment, such benefit includes any one or more of extending survival (including overall survival and progression-free survival), producing an objective response (including a complete or partial response), or ameliorating 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: extending survival (including overall survival and progression-free survival), producing an objective response (including a complete or partial response), or ameliorating signs or symptoms of cancer.

[0100] The term "expression" refers to the biosynthesis of a gene product. For example, in the case of a coding sequence, expression involves transcription of the coding sequence into mRNA and translation of the mRNA into one or more polypeptides. Conversely, expression of a non-coding sequence involves transcription of the non-coding sequence only into transcripts. The term "expression" is also used herein to refer to the presence of a protein or molecule at a particular location, and thus may be used interchangeably with "localization."

[0101] The term "exhaustion" and its grammatical equivalents refer to T cell exhaustion as a state of T cell dysfunction resulting from persistent TCR signaling, which occurs during many chronic infections and cancer development. It is distinguished from anergy in that it results from persistent signaling rather than defective or insufficient signaling. It is defined by poor effector function, persistent expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors. Exhaustion can result from both extrinsic negative regulatory pathways (e.g., immunomodulatory cytokines) and cell-intrinsic negative regulatory (co-stimulatory) pathways (PD-1, B7-H3, B7-H4, etc.).

[0102] 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, if necessary, the translation of the resulting mRNA transcript into a protein. Thus, as will be clear from the context, expression of a coding sequence results from the transcription and translation of the coding sequence. Conversely, expression of a non-coding sequence results from the transcription of the non-coding sequence.

[0103] As used herein, the term "higher," with respect to a measured value of a biomarker or biomarker complex, refers to a statistically significant and measurable difference in the level of a measured value of a biomarker or biomarker complex compared to the level of a measured value of another biomarker or biomarker complex, or compared to a control level in which the measured value of the biomarker or biomarker complex is greater than the level of the other biomarker or 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%.

[0104] The term "host cell" includes an individual cell or cell culture that can be or has been a recipient of any recombinant vector or isolated polynucleotide of the invention. A host cell includes progeny of a single host cell, and progeny may not necessarily be completely identical (in morphology or total DNA complement) due to natural, accidental, or deliberate mutation and / or variation. A host cell includes cells transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell containing a recombinant vector of the invention is a recombinant host cell.

[0105] "Hybridization" is used herein to refer to the pairing of complementary nucleotide sequences to produce DNA-DNA or DNA-RNA hybrids. Complementary base sequences are sequences related by the 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 regard, the terms "match" and "mismatch" as used herein refer to the potential for hybridization of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridize efficiently, such as the classical AT and GC base pairs described above. Mismatches are other combinations of nucleotides that do not hybridize efficiently. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which can be Watson-Crick, Hoogsteen, or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) in the strands of an oligomeric compound. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. Hybridization can occur under a variety of circumstances known to those of skill in the art.

[0106] The term "immunotherapy" refers to any therapy in which one or more components of a human or animal immune system are intentionally modulated to directly or indirectly achieve some therapeutic benefit, including systemic and / or local effects, and prophylactic and / or therapeutic effects. Immunotherapy can include administering one or more immunotherapeutic agents, alone or in any combination, to a human or animal subject, whether systemic, local, or both, by any route (e.g., orally, intravenously, cutaneously, by injection, by inhalation, etc.). Immunotherapy can include inducing, increasing, decreasing, halting, preventing, blocking, or otherwise modulating cytokine production, and / or activating or inactivating cytokines or immune cells, and / or modulating immune cell levels, and / or delivering one or more therapeutic or diagnostic substances to specific locations or types of cells or tissues in the body, and / or destroying specific cells or tissues. Immunotherapy can be used to achieve a local effect, a systemic effect, or a combination of both.

[0107] The term "immunotherapeutic agent" as used herein refers to any drug, 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 anti-cancer therapies. Thus, immunotherapy is a therapy that directly or indirectly stimulates or enhances the immune system's response to cancer cells and / or reduces side effects that may be caused by other anti-cancer drugs. Immunotherapy is also referred to in the art as immunotherapy, biological therapy, biological response modifier therapy, and biologic therapy. 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, immunotherapeutic treatment may consist of administering a certain amount of immune cells (T cells, NK cells, DCs, B cells, etc.) to a subject. Immunotherapeutic agents may be nonspecific, i.e., they may generally enhance the immune system so that the body is more effective in fighting the growth and / or spread of cancer cells, or they may be specific, i.e., they may target the cancer cells themselves. Immunotherapy regimens may combine the use of nonspecific immunotherapeutic agents 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 main therapy for cancer treatment, as well as in addition to main therapy, in which case they function as adjuvants to enhance the effectiveness of other therapies (e.g., cancer vaccines). In this latter context, nonspecific immunotherapeutic agents can also function to reduce the side effects of bone marrow suppression induced by other therapies, such as certain chemotherapeutic agents. Nonspecific immunotherapeutic agents can act on key immune system cells and induce secondary responses, such as increased production of cytokines and immunoglobulins. Alternatively, the agent itself may contain cytokines. Nonspecific immunotherapeutic agents are generally classified as cytokines or noncytokine adjuvants. Some cytokines have been found to be applied in cancer treatment as general nonspecific immunotherapies designed to strengthen the immune system or as adjuvants administered 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 the common types of IFNs, IFN-alpha (IFN-α), IFN-beta (IFN-β), and IFN-gamma (IFN-γ). IFNs can act directly on cancer cells, for example, by slowing their proliferation, promoting their development into cells with more normal behavior, and / or increasing their production of antigens, thus making them easier for the immune system to recognize and destroy. IFNs can also act indirectly on cancer cells, for example, by slowing angiogenesis, strengthening the immune system, and / or stimulating natural killer (NK) cells, T cells, and macrophages. Recombinant IFN-alpha is commercially available as Roferon (Roche Pharmaceuticals) and Intron A (Schering Corporation). Interleukins contemplated by the present disclosure include IL-2, IL-4, IL-11, and IL-12. Examples of commercially available recombinant interleukins include Proleukin® (IL-2; Chiron Corporation) and Neumega® (IL-12; Wyeth Pharmaceuticals). Zymogenetics, Inc. (Seattle, Wash.) 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 alfa, darbopoietin). Treatment with one or more growth factors can help stimulate the production of new blood cells in subjects undergoing conventional chemotherapy. Thus, treatment with CSFs can help reduce chemotherapy-associated side effects and allow higher doses of chemotherapy agents to be used.Various recombinant colony-stimulating factors are commercially available, e.g., Neupogen® (G-CSF; Amgen), Neulasta (perfilgrastim; Amgen), Leukine (GM-CSF; Berlex), Procrit (erythropoietin; Ortho Biotech), Epogen (erythropoietin; Amgen), and Aranesp (erythropoietin). In addition to having a specific or non-specific target, immunotherapeutics can be active, i.e., they can stimulate the body's own immune response, including humoral and cellular immune responses, or they can be passive, i.e., they can involve immune system components such as antibodies, effector immune cells, antigen-presenting cells, etc., generated outside the body. In certain embodiments, passive immunotherapy involves the use of one or more monoclonal antibodies specific for particular antigens found on the surface of cancer cells or immune cells or specific for particular cell growth factors. Monoclonal antibodies can be used in the treatment of cancer in several ways, for example, by targeting specific cell growth factors such as those involved in angiogenesis to enhance a subject's immune response to a particular type of cancer, or by enhancing the delivery of other anti-cancer drugs to cancer cells when bound or conjugated to agents such as chemotherapeutic agents, radioactive particles, or toxins.

[0108] Monoclonal antibodies currently used as cancer immunotherapeutics include alemtuzumab (LEMTRADA®), bevacizumab (AVASTIN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), pertuzumab (OMNITARG®, 2C4), trastuzumab (HERCEPTIN®), tositumomab (Bexxar®), abciximab (REOPRO®), adalimumab (HUMIRA®), apolizumab, and acetaminophen. Mab, atlizumab, bapineuzumab, basiliximab (SIMULECT®), bavituximab, belimumab (BENLYSTA®), briakinumab, canakinumab (ILARIS®), cedelizumab, certolizumab pegol (CIMZIA®), sifutuzumab, situzumab, cixutumumab, clazakizumab, crenezumab, daclizumab (ZENAPAX®), dalotuzumab, denosumab (PROLIA®, XGEVA®), eculizumab (SOLIRIS®) Registered trademark), efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, golimumab (SIMPONI®), ipilimumab, imgatuzumab, infliximab (REMICADE®), labetuzumab, lebrikizumab, lexatumumab, lintuzumab, lucatumumab, lurizumab pegol, lumletuzumab, mapatumumab, matuzumab, mepolizumab, mogamulizumab, motavizumab, motavizumab, muronomab, natalizumab (TYSABRI®), necitumumab (PORTRAZZA®) (Registered Trademark)), nimotuzumab (THERACIM®), norobizumab, numavizumab, olokizumab, omalizumab (XOLAIR®), onartuzumab (also known as MetMAb), palivizumab (SYNAGIS®), pascolizumab, pecfusituzumab, pectuzumab, pembrolizumab (KEYTRUDA®), pexelizumab, priliximab, ralvizumab, ranibizumab (LUCENTIS®), reslivizumab, reslizumab, resivizumab, lobatumumab, rontalizumab,Immunotherapy options include, but are not limited to, lovelizumab, ruplizumab, sarilumab, secukinumab, seribantumab, sifalimumab, sibrotuzumab, siltuximab (SYLVANT®), siplizumab, sontuzumab, tadocizumab, talizumab, tefibazumab, tocilizumab (ACTEMRA®), toralizumab, tuxitusumab, umavizumab, urtoxazumab, ustekinumab (STELARA®), vedolizumab (ENTYVIO®), visilizumab, zanolimumab, and zalutumumab. In certain embodiments, immunotherapy comprises T cell therapy, representative examples of which include adoptive T cell therapy, tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy, and allogeneic T cell transplantation. Non-limiting examples of T cell therapy are described in U.S. Patent Publication Nos. 2014 / 0154228 and 2002 / 0006409, U.S. Patent No. 5,728,388, and WO 2008 / 081035. T cells for immunotherapy can be derived from any source known in the art. For example, T cells can be differentiated in vitro from a hematopoietic stem cell population, or T cells can be obtained from a subject. T cells can be obtained, for example, from peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors, or combinations thereof. Alternatively, or in addition, T cells can be derived from one or more T cell lines available in the art. T cells can also be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as FICOLL™ separation and / or apheresis. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748.

[0109] As used herein, the terms "increase" or "increased," with respect to a biomarker or biomarker complex level, 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 compared to a control level. The increase is preferably at least about a 10% increase, or at least about a 20% increase, or at least about a 30% increase, or at least about a 40% increase, or at least about a 50% increase.

[0110] 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.

[0111] As used herein, "educational materials" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the disclosed compositions and methods. The educational materials of the disclosed kits may, for example, be affixed to a container containing a therapeutic or diagnostic agent of the disclosure, or may be shipped with a container containing a therapeutic or diagnostic and / or prognostic agent of the disclosure.

[0112] As used herein, the term "isolated" refers to material that is substantially or essentially free from components that normally accompany it in its natural state. For example, an "isolated peptide" refers to the in vitro isolation and / or purification of a SETDB1 peptide mimetic from its natural cellular environment and association with other cellular components. "Substantially free" means that a preparation of the peptide is 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% pure. In preferred embodiments, a preparation of the peptide has less than about 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% (by dry weight) of molecules not of interest to the present invention (also referred to herein as "contaminating molecules"). When the peptide is recombinantly produced, it is desirably substantially free of culture medium, i.e., culture medium representing less than about 20, 15, 10, 5, 4, 3, 2, or 1% of the volume of the preparation. The present invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 mg dry weight.

[0113] As used herein, the term "label" 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 antibody, to facilitate detection of the reagent to which it is conjugated or fused. The label may itself be detectable (e.g., a radioisotope label or a fluorescent label) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition that results in a detectable product.

[0114] As used herein, the term "localized" and its grammatical equivalents means accumulating in or being restricted to a specific or restricted space or region, e.g., a subcellular region such as a particular cell, tissue, organelle, or cellular component (e.g., nucleus, cytoplasm, nuclear membrane, plasma membrane, etc.).

[0115] As used herein, the term "lower," with respect to the measurement of a biomarker or biomarker complex, refers to a statistically significant and measurable difference in the level of the measurement of the biomarker or biomarker complex compared to the level of the measurement of another biomarker or biomarker complex, or compared to a control level in which the measurement of the biomarker or biomarker complex is lower than the level of the other biomarker or 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%.

[0116] "Derived" means in possession. Samples so obtained include, for example, polypeptide extracts isolated or derived from a particular source, including cell lysates. For example, an extract can be isolated directly from a biological fluid or tissue of a subject.

[0117] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably to refer to a polymer 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 a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. The terms do not exclude modifications, such as glycosylation, acetylation, phosphorylation, and the like. Soluble forms of the subject peptides are particularly useful. The definition includes peptides containing one or more analogs of an amino acid, including, for example, non-naturally occurring amino acids or polypeptides with substituted linkages.

[0118] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation that is in a form that effectively effects the biological activity of the active ingredient and that contains no additional ingredients that are unacceptably toxic to the subject to which the composition or formulation is administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a mammalian subject to provide an effective dose of the active ingredient employed.

[0119] By "pharmaceutically acceptable carrier" is meant a material that is not biologically or otherwise undesirable, i.e., a pharmaceutical vehicle that may be administered to a subject, along with a selected active agent, without causing any or substantial adverse reactions. Carriers may include excipients and other additives, such as diluents, detergents, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, transfection agents, and the like.

[0120] Similarly, a "pharmacologically acceptable" salt, ester, amide, prodrug, or derivative of a compound provided herein is a salt, ester, amide, prodrug, or derivative that is not biologically or otherwise unsuitable.

[0121] As used herein, the terms "prevent," "prevented," or "preventing" refer to prophylactic treatment that increases a subject's resistance to developing a disease or condition, or in other words, reduces or eliminates the disease or condition entirely, or prevents its worsening, thereby reducing the likelihood that the subject will develop the disease or condition, and initiating post-treatment of the disease or condition. These terms also include within their scope the prevention of a disease or condition from occurring in subjects who may be susceptible to the disease or condition but have not yet been diagnosed with the disease or condition.

[0122] "Radiation therapy" refers to the use of directed gamma or beta radiation to induce sufficient damage to cells so as to limit their ability to function normally or to destroy them completely. It will be understood that many methods for determining dosage and duration of treatment are known in the art. A typical treatment is given as a single dose, with typical dosages ranging from 10 to 200 units (Gray) per day.

[0123] The terms "reduce," "inhibit," "suppress," "reduce," and equivalent terms used with respect to the level of a substance and / or phenomenon in a first sample compared to a second sample mean that the amount of the substance and / or phenomenon in the first sample is lower than in the second sample by any amount that is statistically significant using any art-recognized statistical analysis method. In one embodiment, reduction may be determined subjectively, for example, when a patient refers to their subjective perception of disease symptoms such as pain, fatigue, etc. In another embodiment, reduction may be determined objectively, for example, when the number of CSC and / or non-CSC tumor cells in a sample from the patient is lower than the number of CSC and / or non-CSC tumor cells in a previous sample from the patient. In another embodiment, the amount of a substance and / or phenomenon in a first sample is at least 10% lower than the amount of the same substance and / or phenomenon in a second sample. In another embodiment, the amount of a substance and / or phenomenon in a first sample is at least 25% lower than the amount of the same substance and / or phenomenon in a second sample. In yet another embodiment, the amount of a substance and / or phenomenon in a first sample is at least 50% lower than the amount of the same substance and / or phenomenon in a second sample. In a further embodiment, the amount of a substance and / or phenomenon in a first sample is at least 75% lower than the amount of the same substance and / or phenomenon in a second sample. In yet another embodiment, the amount of a substance and / or phenomenon in a first sample is at least 90% lower than the amount of the same substance and / or phenomenon in a second sample. Alternatively, the difference may be expressed as an "n-fold" difference.

[0124] As used herein, a cancer subject treated with a therapy is considered to "respond," "have a response," "have a positive response," or "be responsive" to the therapy if the subject shows evidence of an anti-cancer effect according to a set of objective criteria accepted in the art, or reasonable modifications thereof, including a clinically significant benefit, such as prevention or reduction in the severity of symptoms, or delay in the progression of cancer. It will be understood that the foregoing terms can also be used in reference to cancer. A variety of different objective criteria for assessing the efficacy of anti-cancer treatments on cancer are known in the art. The World Health Organization (WHO) criteria (Miller, AB, et al., Cancer 1981;47(1):207-14) and their modified version, Response Evaluation Criteria in Solid Tumors (RECIST) (Therasse P, et al., J Natl Cancer Inst 2000;92:205-16) and its revised version (Eisenhauer EA, New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer 2009;45(2):228-47) are a set of objective criteria based on imaging measurements of tumor lesion size and number and the detection of new lesions, for example, from computed tomography (CT), magnetic resonance imaging (MRI), or conventional radiographs. The dimensions of selected lesions (referred to as target lesions) are used to calculate the change in tumor burden between images from different time points. The calculated response is then classified as a complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD). CR is the complete disappearance (-100%) of the tumor, while PD is an increase of approximately 20%-25% or more (depending on the specific criteria) and / or the appearance of new lesions. PR is a significant reduction (at least approximately 30%) in the size of tumor lesions (without the appearance of new lesions), but is less than a complete response. SD lies between PR and PD.(See Tables 1 and 2 for details.) These criteria are widely used as primary endpoints in phase II trials evaluating the efficacy of anticancer drugs, e.g., as surrogates for overall survival. However, anatomical imaging alone using WHO, RECIST, and RECIST 1.1 criteria is designed to detect the early effects of cytotoxic agents and has certain limitations, especially when evaluating the activity of new cancer therapies that stabilize disease. Clinical response patterns in subjects treated with immunotherapeutic or molecularly targeted anticancer agents can extend beyond those of cytotoxic agents and may emerge after an initial increase in tumor burden or the appearance of new lesions. For example, meaningful tumor responses to immune checkpoint inhibitors can occur after a delay, sometimes even after PD as defined by WHO or RECIST. The criteria designating immune-related response criteria (irRC) were defined in an attempt to capture additional favorable response patterns observed with immunotherapy (Wolchok, JD, et al.).

[0125] Guidelines for the evaluation of immune therapy activity in solid tumors: immune-related response criteria. Clin. Care Res. 15, 7412-7420). Four patterns associated with favorable survival have been identified: a decrease in baseline lesions without new lesions; persistent stable disease; an initial increase in total tumor burden but eventual response; and a decrease in total tumor burden during or after the appearance of new lesions. The latter two are distinct from 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). Notably, 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 is the complete disappearance of all lesions, whether measurable or not, and no new lesions. irPR is a 50% reduction in tumor burden compared to baseline. irSD is disease that does not meet the criteria for irCR or irPR in the absence of progressive disease (irPD). irPD is a 25% increase in tumor burden compared to the nadir (lowest recorded tumor burden) (Wolchok, supra). irCR, irPR, and irPD require confirmation by repeated, serial assessments for at least 4 weeks from the date of initial documentation. irCR, irPR, and irSD include all subjects with CR, PR, or SD according to WHO criteria, as well as subjects who transition from WHO PD to these irRC categories. However, some subjects classified as having PD according to WHO or RECIST criteria are instead classified as having PR or SD according to irRC, identifying them as likely to have good survival. irRC is applicable to immune checkpoint inhibitors and other immunotherapeutic agents.Those skilled in the art will understand that additional response criteria are known in the art and take into account various factors, such as the degree of tumor arterial enhancement and / or changes in tumor density, as indicators of tumor viability, with a decrease in arterial enhancement and a decrease in tumor density being indicators of a reduction in 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 J M. Semin Liver Dis 30:52-60, 2010). The Choi criteria and modified Choi criteria take into account a decrease in tumor density on CT scan. 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. Such criteria may be particularly useful for certain cancer types and / or certain classes of therapeutic agents. For example, changes in tumor size may be minimal in tumors such as lymphoma, sarcoma, hepatoma, mesothelioma, and gastrointestinal stromal tumors despite effective treatment. CT tumor density, contrast enhancement, or MRI characteristics may be more informative than size. In certain embodiments, functional imaging, such as using positron emission tomography (PET), may be used. For example, the PET Response Criteria in Solid Tumors (PERCIST) may be used, in which treatment response is assessed by metabolic changes evaluated by (18)F-FDG PET imaging, and a decrease in tracer uptake is considered significant (Wahl RL, et al., J Nucl Med 2009;50,Suppl 1:122S-50S). It should also be understood 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 treated with a therapy is considered to be "unresponsive," "not responsive," "has a negative response," or "is non-responsive" to the therapy if the therapy does not provide a clinically significant benefit, such as preventing or reducing the severity of symptoms, or increases the rate of cancer progression.

[0126] For purposes of this disclosure, a cancer subject treated with an immunotherapy (e.g., an immune checkpoint inhibitor) as monotherapy or in combination with one or more other active agents (e.g., a complement inhibitor, an additional anti-cancer agent, or both) is considered to "respond," "have a response," or "be responsive" to the treatment if the subject has a complete response, a partial response, or stable disease according to at least the immune-related response criteria. (A cancer subject may also respond according to RECIST, RECIST 1.1, WHO, and / or other criteria such as those described above.) Similarly, the cancer in such cases is said to "respond," "be responsive," or "sensitive" to the treatment. A cancer subject is considered to "not respond," "not have a response," or "non-responsive" to the treatment if the subject has progressive disease according to the immune-related response criteria. A cancer subject may also not respond according to RECIST, RECIST 1.1, WHO, and / or other criteria such as those described above. Similarly, the cancer in such cases is said to be "unresponsive," or "non-responsive," "insensitive," or "resistant" to the treatment. A cancer is also considered to have become resistant to a treatment if it initially responds but subsequently exhibits progressive disease in the presence of the treatment. Thus, for example, for methods and products described herein relating to cancer response to treatment (e.g., methods of predicting likelihood of response, methods of classifying subjects according to predicted response, methods of increasing likelihood of response), response is 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. Response criteria may be shown to correlate with benefit, such as increased overall survival or other clinically significant benefit. It will be understood that improvements or revisions of existing response criteria, including additional favorable patterns of clinical activity (e.g., correlated with increased overall survival), applicable to or otherwise useful with immune checkpoint inhibitors, may be developed in the future. In certain embodiments, any such response criteria may be specified for use in the methods described herein.

[0127] As used herein, the terms "salt" and "prodrug" include any pharmaceutically acceptable salt, ester, hydrate, or any other compound that, upon administration to a recipient, is capable of yielding (directly or indirectly) a SETDB1 peptidomimetic of the invention, or an active metabolite or residue thereof. Suitable pharmaceutically acceptable salts include salts of pharmaceutically acceptable inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, carbonic acid, boric acid, sulfamic acid, and hydrobromic acid, or salts of pharmaceutically acceptable inorganic acids such as 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. Basic salts include, but are not limited to, those formed with pharmaceutically acceptable cations such as sodium, potassium, lithium, calcium, magnesium, ammonium, and alkylammonium. Basic nitrogen-containing groups may also be quaternized with such agents 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 other agents. However, it will be understood that non-pharmaceutically acceptable salts, since these may be useful in the preparation of pharmaceutically acceptable salts, are also within the scope of the present invention. The preparation of salts and prodrugs can be carried out by methods known in the art. For example, metal salts can be prepared by reacting the compounds of the present invention with a metal hydroxide. Acid salts can be prepared by reacting the appropriate acid with a SETDB1 peptidomimetic of the present invention.

[0128] The term "sample" as used herein includes any biological specimen that may be extracted, raw, processed, diluted, or concentrated from a subject. Samples may include, but are not limited to, biological fluids such as whole blood, serum, red blood cells, white blood cells, plasma, saliva, urine, stool (i.e., feces), tears, sweat, sebum, nipple aspirate, ductal lavage, tumor effusion, synovial fluid, ascites, peritoneal fluid, amniotic fluid, cerebrospinal fluid, lymphatic fluid, fine needle aspirate, amniotic fluid, any other bodily fluid, cell lysate, cell secretion, inflammatory fluid, semen, and vaginal secretion. Samples may include tissue samples and biopsies, tissue homogenates, and the like. Advantageous samples may include samples containing detectable amounts of any one or more biomarkers as taught herein. Preferably, samples are readily obtainable by minimally invasive methods, allowing for removal or isolation of the sample from a subject. In certain embodiments, the sample contains blood, particularly peripheral blood, or a fraction or extract thereof. Typically, the sample comprises blood cells such as mature, immature, or developing leukocytes, including lymphocytes, polymorphonuclear leukocytes, neutrophils, monocytes, reticulocytes, basophils, coelomycetes, hemocytes, eosinophils, megakaryocytes, macrophages, dendritic cells, natural killer cells, or fractions (e.g., nucleic acid or protein fractions) of such cells. In certain embodiments, the sample comprises leukocytes, including peripheral blood mononuclear cells (PBMCs).

[0129] As used herein, a "reference sample," "reference cell," "reference tissue," "control sample," "control cell," or "control tissue" refers to a sample, cell, tissue, standard, or level used 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 disease-free part (e.g., tissue or cells) of the body of the same subject or individual. For example, a healthy and / or disease-free cell or tissue adjacent to a diseased cell or tissue (e.g., a cell or tissue adjacent to a tumor). In another embodiment, the reference sample is obtained from untreated tissue 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 disease-free part (e.g., tissue or cells) of the body of an individual other than the subject or individual. In yet another embodiment, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from untreated tissue and / or cells of the body of an individual other than the subject or individual.

[0130] A "tissue sample" or "cell sample" refers to a collection of similar cells obtained from the tissue of a subject or individual. The source of a 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; bodily fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells from any time during a subject's pregnancy or development. A tissue sample can also be primary or cultured cells or cell lines. Optionally, a tissue or cell sample is obtained from a diseased tissue / organ. A tissue sample may contain compounds not naturally mixed with natural tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, etc.

[0131] As used herein, the term "scaffold" or "molecular scaffold" refers to a chemical moiety that is attached to a peptide in a composition of the present invention through an alkylamino bond and a thioether bond (if cysteine ​​is present) . As used herein, the term "scaffold molecule" or "molecular scaffold molecule" refers to a molecule that can react with a peptide or peptide ligand to form a derivative of the present invention that has an alkylamino and, in certain embodiments, a thioether bond. Thus, a scaffold molecule has the same structure as a scaffold moiety, except that each reactive group (e.g., residue) of the molecule is replaced by an alkylamino and a thioether bond to the peptide in the scaffold moiety.

[0132] "SETDB1" refers to the protein also known as "SET domain-divergent histone lysine methyltransferase 1," "ESET," and "KMT1A." As used herein, the term encompasses full-length and / or unprocessed SETDB1, as well as any intermediates resulting from processing within a cell. SETDB1 can exist as a soluble protein. Thus, as used herein, the term may refer to the full-length protein. The term also encompasses naturally occurring variants of SETDB1 (e.g., splice variants or allelic variants). 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, in UniProtKB Accession No. Q15047.

[0133] The terms "subject," "subject," "host," or "individual," as used interchangeably herein, refer to any subject for whom therapy or prevention is desired, particularly a vertebrate subject, and even more particularly a mammalian subject. Suitable vertebrates within the scope of the present disclosure include, but are not limited to, any member of the primate family (e.g., humans, monkeys, and apes), including, but not limited to, species of monkeys such as macaques (e.g., cynomolgus monkeys (Macaca fascicularis) and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus), as well as marmosets (Callithrix species), squirrel monkeys (Saimiri species), and tamarins (Saguinus species), and chimpanzees (Pan pantheon). troglodytes), rodents (e.g., mice, rats, guinea pigs), lagomorphs (e.g., rabbits, hares), bovines (e.g., cattle), ovines (e.g., sheep), caprines (e.g., goats), porcines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), felines (cats), birds (e.g., chickens, turkeys, ducks, geese, canaries, companion birds such as budgerigars), marine mammals (e.g., dolphins, whales), reptiles (snakes, frogs, lizards, etc.), and fish. A preferred subject is a human in need of therapy and / or the analytical / determining methods of the present disclosure. However, it will be understood that the foregoing terms do not imply the presence of symptoms.

[0134] As used herein, the terms "stratification" and "classification" are used interchangeably herein to refer to the separation of subjects into different strata or classes based on the characteristics of a particular physiological or pathophysiological state or condition. For example, stratifying a subject population according to whether they are likely to respond to a therapy (e.g., immunotherapy) involves assigning subjects based on the level of response to therapy biomarkers, including SETDB1, in cancer cells.

[0135] As used herein, the terms "treatment," "treating," and the like refer to a clinical intervention designed to alter the natural history of the individual or cell being treated during its clinical pathological course. Desired effects of treatment include reducing the rate of disease progression, improving or mitigating the disease state, and achieving remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with a T cell dysfunction disorder are alleviated or eliminated, including, but not limited to, reducing (or destroying) the proliferation of cancerous cells, reducing pathogen infection, reducing symptoms caused by the disease, improving the quality of life of those suffering from the disease, reducing the dose of other medications required to treat the disease, and / or prolonging the survival of the individual.

[0136] As used herein, the term "tumor" refers to any neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized in part by uncontrolled cell growth. As used herein, the term "cancer" refers to non-metastatic and metastatic cancers, including early-stage and late-stage cancers. The term "pre-cancerous" refers to a condition or growth that typically precedes or develops into cancer. The term "non-metastatic" refers to a cancer that is benign or remains at the primary site and has not infiltrated the lymphatic or vascular system or tissues outside the primary site. Generally, a non-metastatic cancer is any cancer that is a stage 0, I, or II cancer. "Early-stage cancer" refers to a cancer that is not invasive or metastatic, or is classified as a stage 0, I, or II cancer. The term "late stage cancer" generally refers to stage III or IV cancer, but can also refer to stage II cancer or substages of stage II cancer. Those skilled in the art will understand that the classification of stage II cancer as either early stage or late stage 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, gastric cancer, liver cancer, bladder cancer, urinary tract cancer, thyroid cancer, renal cancer (kidney 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 exemplary embodiments, the cancer is breast cancer or melanoma.

[0137] Unless otherwise specified, each embodiment described herein applies to each and every embodiment.

[0138] 2. Proteinaceous molecules The present invention is based, in part, on the determination that proteinaceous molecules corresponding to the nuclear localization sequence (NLS) site of the SETDB1 polypeptide are useful for reducing the nuclear localization of the SETDB1 polypeptide. The inventors have determined that the mechanism by which nuclear shuttling of SETDB1 occurs is via a complex with importin-α (IMPα). Thus, the inventors believe that inhibition of the binding between the SETDB1 polypeptide and the IMPα polypeptide may be used to treat or prevent cancer.

[0139] 2.1 SETDB1 proteinaceous molecule In one aspect, the present invention provides a proteinaceous molecule corresponding to the NLS of a SETDB1 polypeptide, such as a polypeptide comprising an amino acid sequence corresponding to residues 206-232 of wild-type human SETDB1 polypeptide.

[0140] The amino acid sequence of human SETDB1 (Uniprot accession number Q15047) is presented in SEQ ID NO: 1, shown below with the NLS identified in bold and underlined type. [ka]

[0141] Thus, in one aspect of the present invention there is provided an isolated or purified proteinaceous molecule represented by formula (I): Z1GKKRTKTWHKGTLIAIQTVGX1GKKYKVKZ2 Formula (I) During the ceremony, Z1 and Z2 are independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integers therebetween) and a protecting moiety; X1 is selected from non-polar / neutral amino acid residues including A, G, I, L, M, F, P, W, V, and Nle.

[0142] In some embodiments, Z1 is absent. In other embodiments, Z1 consists 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.

[0143] In some embodiments, Z2 is absent. In other embodiments, Z2 consists of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues. In some embodiments, the amino acid residues in Z2 are independently selected from any amino acid residue.

[0144] 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 particular embodiments, X1 is selected from Pro and Leu, particularly Pro.

[0145] In some embodiments, the isolated or purified proteinaceous molecule of Formula I comprises, consists of, or consists essentially of the amino acid sequence represented by any one of SEQ ID NOs: 2 or 3. GKKRTKTWHKGTLIAIQTVGPGKKYKVK SEQ ID NO: 2 GKKRTKTWHKGTLIAIQTVGLGKKYKVK SEQ ID NO: 3

[0146] In some embodiments, the proteinaceous 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 to the amino acid sequence of SEQ ID NO: 2 or 3. In some embodiments, the proteinaceous 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 to the amino acid sequence of SEQ ID NO: 2 or 3.

[0147] In some embodiments where the proteinaceous molecules of the invention comprise an N-terminus and / or a C-terminus, the proteinaceous molecules of the invention have a primary, secondary or tertiary amide, hydrazide, hydroxamide or free carboxyl group at the C-terminus, and / or a primary amine or acetamide at the N-terminus. In some embodiments, the proteinaceous molecules of the invention are cyclic peptides and therefore may not comprise N- and / or C-terminal amino acid residues.

[0148] The present invention also contemplates proteinaceous molecules that are variants of SEQ ID NOs: 1 and 3. Such "variant" proteinaceous molecules include proteinaceous molecules derived from SEQ ID NO: 2 or SEQ ID NO: 3 by the deletion or addition of one or more amino acids to the N-terminus and / or C-terminus of the proteinaceous molecule, the deletion or addition of one or more amino acids at one or more sites in the proteinaceous molecule, or the substitution of one or more amino acids at one or more sites in the proteinaceous molecule.

[0149] Variant proteinaceous molecules encompassed by the present invention are biologically active, i.e., retain the desired biological activity of the proteinaceous molecule. Such variants may result, for example, from genetic polymorphism or human manipulation.

[0150] The proteinaceous molecule of SEQ ID NO: 1 or 2 can be altered in a variety of ways, including amino acid substitution, deletion, truncation, and insertion. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of SEQ ID NO: 2 or 3 may be prepared by mutagenesis of a nucleic acid encoding the amino acid sequence of any one 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. Pat. No. 4,873,192; and Watson et al., 1987). Guidance regarding appropriate amino acid substitutions that do not affect the biological activity of the proteinaceous molecule of interest can be found in the model of Dayhoff et al., 1978. Methods for screening gene products of combinatorial libraries generated by point mutation or truncation, and for screening cDNA libraries for gene products with selected properties, are known in the art. Such methods are adaptable for rapid screening of gene libraries generated by combinatorial mutagenesis of proteinaceous molecules of SEQ ID NO: 2 or 3. Recursive ensemble mutagenesis (REM), a technique for increasing the frequency of functional variants in libraries, can be used in conjunction 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.

[0151] The variant proteinaceous molecules of the present invention may contain conservative amino acid substitutions at various positions along their sequence compared to a parent (e.g., reference) amino acid sequence, such as SEQ ID NO: 2 or 3. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art, as explained in more detail below.

[0152] Acidic: The residue has a negative charge at physiological pH due to loss of a proton, and when the peptide is in aqueous medium at physiological pH, the residue is attracted to aqueous solution so as to seek a surface position in the conformation of the peptide in which it is contained. Amino acids with acidic side chains include glutamic acid and aspartic acid.

[0153] Basic: The residue has a positive charge due to association with a proton at physiological pH or within 1 or 2 pH units thereof (e.g., histidine), and when the peptide is in aqueous medium at physiological pH, the residue is attracted to aqueous solution so as to seek a surface position in the conformation of the peptide in which it is contained. Amino acids with basic side chains include arginine, lysine, and histidine.

[0154] Charged: The residues are charged at physiological pH and therefore include amino acids with acidic or basic side chains (such as glutamic acid, aspartic acid, arginine, lysine, and histidine).

[0155] Hydrophobic: The residue is uncharged at physiological pH and is repelled from aqueous solution when the peptide is in aqueous medium at physiological pH, such that the residue seeks an interior position in the conformation of the peptide in which it is contained. Amino acids with hydrophobic side chains include tyrosine, valine, isoleucine, leucine, methionine, norleucine, phenylalanine, and tryptophan.

[0156] Neutral / polar: The residue is not charged at physiological pH, but is not significantly repelled from aqueous solution so that when the peptide is in aqueous medium at physiological pH, the residue seeks an interior position in the conformation of the peptide in which it is contained. Amino acids with neutral / polar side chains include asparagine, glutamine, cysteine, histidine, serine, and threonine.

[0157] This specification also characterizes certain amino acids as "small" because their side chains are not large enough to confer hydrophobicity, even if they lack a polar group. With the exception of proline, "small" amino acids are those with four or fewer carbons if at least one polar group is present on the side chain, and three or fewer carbons otherwise. Amino acids with small side chains include glycine, serine, alanine, and threonine. The genetically encoded secondary amino acid proline is a special example due to its known effect on the secondary conformation of peptide chains. The structure of proline differs from all other natural amino acids in that its side chain is attached not only to the nitrogen of the α-amino group but also to the α-carbon. However, some amino acid similarity matrices (e.g., the PAM120 and PAM250 matrices disclosed by Dayhoff et al., 1979 and Gonnet et al., 1992) include proline in the same group as glycine, serine, alanine, and threonine. Therefore, for purposes of the present invention, proline is classified as a "small" amino acid.

[0158] The degree of attraction or repulsion required for classification as polar or nonpolar is arbitrary, and therefore, amino acids specifically contemplated by the present invention have been classified as one or the other. Most amino acids not specifically named can be classified based on known behavior.

[0159] Amino acid residues can be further subclassified as cyclic or acyclic, and aromatic or non-aromatic, which are self-explanatory classifications based on the residue's side chain substituents, as well as small or large. A residue is considered small if it contains a total of four or fewer carbon atoms, including the carboxyl carbon, if an additional polar substituent is present, or three or fewer carbon atoms if no additional polar substituent is present. Small amino acid residues are, of course, always non-aromatic. Depending on their structural characteristics, amino acid residues may belong to more than one class. Table 1 presents subclassifications according to this scheme for naturally occurring protein amino acids. [Table 1]

[0160] Conservative amino acid substitutions also include groupings based on side chains. For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, isoleucine, and norleucine; the group of amino acids with aliphatic hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. For example, replacing leucine with isoleucine or valine, replacing aspartic acid with glutamic acid, replacing threonine with serine, or similar replacement of amino acids with structurally related amino acids will not significantly affect the properties of the resulting variant peptides of the present invention. Whether an amino acid change results in a proteinaceous molecule that inhibits or reduces the nuclear localization of SETDB1 can be readily determined by assaying its activity. Conservative substitutions are shown in Table 2 under the headings of exemplary and preferred substitutions. Amino acid substitutions that fall within the scope of the present invention are generally achieved by selecting substitutions that do not significantly alter their effect on maintaining (a) the structure of the peptide backbone in the area of ​​the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity. [Table 2]

[0161] Alternatively, similar amino acids for conservative substitution can be divided into three categories based on the identity of their side chains. As described in Zubay, Biochemistry, third edition, Wm. C. Brown Publishers (1993), 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.

[0162] Thus, essential amino acid residues in the proteinaceous molecules of the present invention are typically substituted with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the coding sequence of a proteinaceous molecule of the present invention, such as by saturation mutagenesis, and the resulting mutants can be screened for the activity of the parent polypeptide, e.g., as described herein, to identify mutants that retain that activity. After mutagenesis of the coding sequence, the encoded proteinaceous molecule can be recombinantly expressed and its activity determined. A "non-essential" amino acid residue is a residue that can be altered from the reference sequence of a proteinaceous molecule of the present embodiments without eliminating or substantially altering one or more of its activities. Preferably, the alteration does not substantially alter one of these activities, e.g., the activity is at least 20%, 40%, 60%, 70%, or 80% of the wild-type. In contrast, an "essential" amino acid residue is a residue that, when altered from the wild-type sequence of the x proteinaceous molecule of the present embodiments, results in a loss of activity of the parent molecule such that less than 20% of the wild-type activity is present.

[0163] Thus, the present invention also contemplates variants of the proteinaceous molecules of SEQ ID NO: 2 or 3, which variants are distinguished from the parent sequence by the addition, deletion, or substitution of one or more amino acid residues. Generally, variants will exhibit 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 to the reference proteinaceous molecule sequence set forth in SEQ ID NO: 2 or 3, as determined, for example, by sequence alignment programs described elsewhere herein using default parameters. Desirably, variants have at least about 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 proteinaceous molecule sequence set forth in SEQ ID NO: 2 or 3, as determined, for example, by a sequence alignment program described herein using default parameters. Variants of SEQ ID NO: 2 or 3 within the scope of the variant proteinaceous molecules of the invention may generally differ from the parent molecule by at least one, but less than 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the proteinaceous molecules of the invention differ from the corresponding sequence of SEQ ID NO: 2 or 3 by at least one, but less than 5, 4, 3, 2, or 1 amino acid residue. In some embodiments, the amino acid sequence of a variant proteinaceous molecule of the invention comprises a proteinaceous molecule of Formula I. In a particular embodiment, the mutant proteinaceous molecule of the present invention inhibits or reduces the nuclear localization of SETDB1.

[0164] When sequence comparison requires alignment, the sequences are typically aligned for maximum similarity or identity. Deletions or insertions, or "looped-out" sequences from mismatches, are generally considered to be differences. Differences are preferably differences or changes in non-essential residues or conservative substitutions.

[0165] In some embodiments, calculations of sequence similarity or sequence identity between sequences are performed as follows: To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (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 comparison purposes). In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 40%, more usually at least 50% or 60%, and even more usually 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. If a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. For amino acid sequence comparison, if a position in the first sequence is occupied by the same or a similar amino acid residue (i.e., a conservative substitution) at the corresponding position in the second sequence, the molecules are similar at that position.

[0166] The percent identity between two sequences is a function of the number of identical amino acid residues shared by the sequences at each position, taking into account the number of gaps and the length of each gap that need 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 each position, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences.

[0167] Comparison of sequences and determination of percent identity or similarity between sequences can be accomplished using mathematical algorithms. In certain embodiments, percent identity or similarity between amino acid sequences is determined using the Needleman and Wunsch (1970) algorithm, which has been incorporated into the GAP program in the GCG software package (Devereaux, et al., 1984), using either 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. In some embodiments, percent identity or similarity between amino acid sequences can be determined using the Meyers and Miller (1989, Cablos, 4:11-17) algorithm, which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

[0168] The present invention also contemplates isolated or purified proteinaceous molecules encoded by polynucleotide sequences that hybridize under stringency conditions as defined herein, in particular under medium, high or very high stringency conditions, preferably under high or very high stringency conditions, to a polynucleotide sequence encoding a proteinaceous molecule of SEQ ID NO: 2 or 3, or its non-coding strand. The present invention also contemplates isolated nucleic acid molecules comprising a polynucleotide sequence that hybridizes under stringency conditions as defined herein, in particular under medium, high or very high stringency conditions, preferably under high or very high stringency conditions, to a polynucleotide sequence encoding a proteinaceous molecule of SEQ ID NO: 2 or 3, or its non-coding strand.

[0169] As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing and can include low stringency, medium stringency, high stringency, and very high stringency conditions.

[0170] Guidance for carrying out hybridization reactions can be found in Ausubel, et al. (1998) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.), particularly sections 6.3.1-6.3.6. Both aqueous and non-aqueous methods can be used. References herein to low stringency conditions include and encompass at least about 1% v / v to at least about 15% v / v formamide and at least about 1 M to at least about 2 M salt for hybridization at 42°C, and at least about 1 M to at least about 2 M salt for washing at 42°C. Low stringency conditions can also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO (pH 7.2), 7% sodium dodecyl sulfate (SDS) for hybridization at 65° C., and (i) 2× sodium chloride / sodium citrate (SSC), 0.1% SDS, or (II) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 5% SDS for washing at room temperature. One embodiment of low stringency conditions includes hybridization in 6×SSC at about 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at at least 50° C. (for low stringency conditions, the temperature of the washes 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 can also include 1% bovine serum albumin (BSA), 1 mM EDTA, 0.5 M NaHPO (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1% SDS, or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 5% SDS for washing at 60-65° C.One embodiment 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 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 washes at 55° C. High stringency conditions can also include 1% BSA, 1 mM EDTA, 0.5 M NaHPO (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC, 0.1% SDS, or (II) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO (pH 7.2), 1% SDS for washes at temperatures above 65° C. One embodiment 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.

[0171] In some aspects of the present invention, there is provided an isolated or purified proteinaceous molecule of the present invention, which is encoded by a polynucleotide sequence that hybridizes under high stringency conditions to a proteinaceous molecule of SEQ ID NO: 2 or 3, or to a non-coding strand thereof. In certain embodiments, the isolated or purified proteinaceous molecule of the present invention is encoded by a polynucleotide sequence that hybridizes under very high stringency conditions to a proteinaceous molecule of SEQ ID NO: 2 or 3, or to a non-coding strand thereof. One embodiment of very high stringency conditions comprises hybridizing in 0.5 M sodium phosphate, 7% SDS at 65°C, followed by one or more washes in 0.2xSSC, 1% SDS at 65°C. In some embodiments, the amino acid sequence of the variant proteinaceous molecule of the present invention comprises the amino acid sequence of Formula I. In certain embodiments, the proteinaceous molecule of the present invention inhibits or reduces nuclear-localized SETDB1.

[0172] Other stringency conditions are well known in the art, and those skilled in the art will recognize that various factors can be manipulated to optimize hybridization specificity. Optimizing the stringency of the final wash can help ensure high hybridization. For detailed examples, see Ausubel, et al. (1998) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.), especially pages 2.10.1 to 2.10.16, and Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Press), especially sections 1.101 to 1.104.

[0173] Stringent washing is typically performed at temperatures between about 42°C and 68°C, although one of skill in the art will appreciate that other temperatures may be suitable for stringent conditions. The maximum hybridization rate is typically determined by the T m It occurs at temperatures about 20 to 25 degrees Celsius lower than T m It is well known in the art that T is the melting temperature, or the temperature at which two complementary polynucleotide sequences dissociate. m Methods for estimating the T of a perfectly matched DNA duplex are well known in the art (see Ausubel, et al. (1998) Current Protocols in Molecular Biology (John Wiley and Sons, Inc.) page 2.10.8). m can be estimated approximately by the following formula: T m =81.5+16.6(log 10 M) + 0.41 (% G + C) - 0.63 (% formamide) - (600 / length) In the formula, M is Na +The concentration of guanosine and cytosine bases is preferably in the range of 0.01M to 0.4M, %G+C is the sum of guanosine and cytosine bases as a percentage of the total number of bases in the range of 30% to 75% G+C, %formamide is the percent of formamide concentration by volume, and length is the number of base pairs in the DNA duplex. m decreases by approximately 1°C for every 1% increase in the number of randomly mismatched base pairs. Washing is generally performed at T m -15°C, T for medium stringency m It is carried out at -30°C.

[0174] In one example of a hybridization procedure, a membrane (e.g., a nitrocellulose or nylon membrane) containing immobilized DNA is hybridized overnight at 42°C in hybridization buffer (50% deionized formamide, 5x SSC, 5x 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 labeled probe. The membrane is then subjected to two successive medium stringency washes (i.e., 2x SSC, 0.1% SDS at 45°C for 15 minutes, followed by 2x SSC, 0.1% SDS at 50°C for 15 minutes), followed by two successive higher stringency washes (i.e., 0.2x SSC, 0.1% SDS at 55°C for 12 minutes, followed by 0.2x SSC and 0.1% SDS solution at 65-68°C for 12 minutes).

[0175] The proteinaceous molecules of the present invention also encompass proteinaceous molecules comprising amino acids with modified side chains, the incorporation of unnatural amino acid residues and / or their derivatives during peptide synthesis, and the use of cross-linking agents and other methods to impose conformational constraints on the proteinaceous molecules of the present invention. Examples of side chain modifications include modifications of amino groups such as acylation with acetic anhydride, acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride, amidation with methyl acetimidate, carbamoylation of amino groups with cyanate, pyridoxylation 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).

[0176] The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide.

[0177] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0178] Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include 4-aminobutyric acid, 6-aminohexanoic acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 4-amino-3-hydroxy-6-methylheptanoic acid, t-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-isomers of the amino acids. A list of unnatural amino acids contemplated by the present invention is shown in Table 3. [Table 3-1] [Table 3-2]

[0179] In some embodiments, a proteinaceous molecule of the invention comprises at least one unnatural amino acid.

[0180] 2.2 Bicyclic proteinaceous molecules In some embodiments, the proteinaceous molecule is a bicyclic molecule comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide has the following amino acid sequence: Z1C1GKKRX1KX2WHC2X3GTLC3IX4IQTVGX5GKKX6KVKZ2 Formula (II) During the ceremony, 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 proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integers therebetween) and a protecting moiety; 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; X5 is selected from Pro, Lys, and modified forms thereof; X6 is selected from Tyr, Lys, and modified forms thereof.

[0181] In some preferred embodiments, both Z1 and Z2 are absent.

[0182] In some of the same and alternative embodiments, X1 is selected from Thr.

[0183] In some of the same and alternative embodiments, X2 is selected from Thr.

[0184] In some of the same and some alternative embodiments, X3 is selected from Lys.

[0185] In some of the same and some alternative embodiments, X4 is selected from Ala.

[0186] In some of the same and alternative embodiments, X5 is selected from Pro.

[0187] In some of the same and some alternative embodiments, X6 is selected from Tyr.

[0188] In some more specific embodiments, the proteinaceous molecule is a bicyclic molecule comprising a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide, such that at least two polypeptide loops are formed on the molecular scaffold, wherein the polypeptide has the following amino acid sequence: Z1X1C1GKKRTKTWHC2KGTLIAIQTVGX2GC3KKYKVKZ2 Formula (III) or a modified derivative or a pharmaceutically acceptable salt thereof, During the ceremony, 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 proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integers therebetween) and a protecting moiety; X1 is absent or is alanine; X2 is selected from non-polar / neutral amino acid residues including A, G, I, L, M, F, P, W, V, and Nle.

[0189] In some embodiments, Z1 is absent. In other embodiments, Z1 consists 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.

[0190] In some embodiments, Z2 is absent. In other embodiments, Z2 consists of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid residues. In some embodiments, the amino acid residues in Z2 are independently selected from any amino acid residue.

[0191] In some embodiments, Z2 is absent.

[0192] 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 certain embodiments, X1 is selected from Pro and Leu, particularly Pro.

[0193] In some other embodiments of this type, the polypeptide comprises an amino acid sequence selected from formula (III) or (IV), as shown below. Z1X1C1GKKRTKTWHC2KGTLC3IAIQTVGX1GKKYKVKZ2 Formula (IV) Z1X1C1GKKRTKTWHC2KGTLIAIQC3TVGX1GKKYKVKZ2 Formula (V)

[0194] In some embodiments, the proteinaceous molecule of Formula II, III, or IV comprises, consists of, or consists essentially of an amino acid sequence selected from Table 4. [Table 4]

[0195] In some embodiments, the proteinaceous molecules of Formulas (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 to the amino acid sequence of any one of SEQ ID NOs: 3-5. In some embodiments, the proteinaceous molecules of Formulas 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 to the amino acid sequence of SEQ ID NOs: 4-6.

[0196] In some embodiments, the proteinaceous molecules of the present invention are alternative types of cyclic molecules. Without wishing to be bound by theory, cyclization of peptides is believed to reduce the peptide's susceptibility to degradation. In certain embodiments, SETDB1 bicyclic peptidomimetics are cyclized using N-C cyclization (head-to-tail cyclization), preferably via an amide bond. Such SETDB1 bicyclic peptidomimetics do not have N- or C-terminal amino acid residues. In certain embodiments, SETDB1 bicyclic peptidomimetics have an amide-cyclized peptide backbone. In other embodiments, the peptides are cyclized using side chain-to-side chain cyclization, preferably via a disulfide bond, a diselenide bond, a selenosulfur bond, a thioether bond (e.g., a lanthionine bond, a selenoether bond, a triazole bond, a lactam bond, or a dimethylene bond), particularly a disulfide bond.

[0197] Molecular scaffolds The SETDB1 bicyclic peptidomimetics of the present invention comprise, consist essentially of, or consist of a polypeptide covalently attached to a molecular scaffold. Molecular scaffolds are described, for example, in International PCT Patent Publication No. 2009 / 098450 and the references cited therein, particularly WO2004 / 077062 and WO2006 / 078161. As described in the aforementioned documents, the molecular scaffold can be a small molecule, such as a small organic molecule.

[0198] In some embodiments, molecular scaffolds may be or be based on naturally occurring monomers such as nucleosides, sugars, or steroids. For example, molecular scaffolds may include short polymers of such entities, such as dimers or trimers.

[0199] In one embodiment, the molecular scaffold is a compound of known toxicity, e.g., low toxicity. Examples of suitable compounds include cholesterol, nucleotides, steroids, or existing drugs such as temazepam.

[0200] In some embodiments, the molecular scaffold may be a macromolecule, hi some embodiments, the molecular scaffold is a macromolecule composed of amino acids, nucleotides, or carbohydrates.

[0201] In some embodiments, the molecular scaffold comprises a reactive group that can react with a functional group of a polypeptide to form a covalent bond.

[0202] The molecular scaffold may include chemical groups such as amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimides, maleimides, alkyl halides, and acyl halides.

[0203] In some embodiments, the scaffold is an aromatic molecular scaffold (i.e., a scaffold comprising (hetero)aryl groups). These aromatic rings can optionally contain one or more heteroatoms (e.g., one or more of N, O, S, and P), such as thienyl, pyridyl, and furanyl rings. The aromatic rings can be optionally substituted. The aryl rings can also be optionally substituted. Suitable substituents include alkyl groups (which can be optionally substituted), other aryl groups (which themselves can be substituted), heterocycles (saturated or unsaturated), alkoxy groups (which are meant to include aryloxy groups (e.g., phenoxy groups)), hydroxy groups, aldehyde groups, nitro groups, amine groups (e.g., unsubstituted or mono- or di-substituted with aryl or alkyl groups), carboxylic acid groups, carboxylic acid derivatives (e.g., carboxylic acid esters, amides, etc.), halogen atoms (e.g., Cl, Br, and I), and the like.

[0204] Suitably, the scaffold comprises a tris-substituted (hetero)aromatic or (hetero)alicyclic moiety, such as a tris-methylene substituted (hetero)aromatic or (hetero)alicyclic moiety. The (hetero)aromatic or (hetero)alicyclic moiety is preferably a six-membered ring structure, and is preferably tris-substituted such that the scaffold has a three-fold axis of symmetry.

[0205] In some embodiments, the scaffold is a tris-methylene(hetero)aryl moiety, such as a 1,3,5-trismethylenebenzene moiety. In these embodiments, the corresponding scaffold molecule preferably has a leaving group on the methylene carbon. The methylene group then 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. Thus, for example, benzyl halides are 100 to 1000 times more reactive toward nucleophilic substitution than alkyl halides not connected to a (hetero)aromatic group.

[0206] In this type of embodiment, the scaffold and scaffold molecule have the general formula: [ka] wherein LG represents a leaving group as further described below for the scaffold molecule, or LG (including the adjacent methylene group that forms the R1 portion of the alkylamino group) represents the alkylamino bond to the peptide in the conjugate of the invention.

[0207] In some embodiments, the LG group may be a halogen, such as, but not limited to, a bromine atom, in which case the scaffold molecule is 1,3,5-tris(bromomethyl)benzene (TBMB). Another suitable molecular scaffold molecule is 2,4,6-tris(bromomethyl)mesitylene. Similar to 1,3,5-tris(bromomethyl)benzene, it contains three additional methyl groups attached to the benzene ring. In this scaffold, the additional methyl groups can form additional contacts with the peptide, thus adding additional structural constraints. Thus, a different diversity range than 1,3,5-tris(bromomethyl)benzene can be achieved.

[0208] Another preferred molecule for forming a scaffold for reacting with peptides by nucleophilic substitution is 1,3,5-tris(bromoacetamido)benzene (TBAB). [ka]

[0209] 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 can be unsubstituted or substituted with one or more substituents. The substituents can be saturated or unsaturated, aromatic or non-aromatic, and examples of suitable substituents include those described above in the discussion of substituents on alkyl and aryl groups. Furthermore, since two or more ring substituents can be linked to form another ring, 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).

[0210] In some alternative embodiments, the molecular scaffold may have a tetrahedral geometry such that reaction of the four functional groups of the encoded peptide with the molecular scaffold produces no more than two product isomers. Other geometries are also possible, and indeed a nearly infinite number of scaffold geometries are possible, providing greater possibilities for peptide ligand diversification.

[0211] The peptides used to form the bicyclic peptides of the invention contain a cysteine ​​that is used to form a thioether bond to the scaffold, replacing the terminal -SH group of the cysteine ​​with -NH2.

[0212] The bicyclic peptides of the present invention have several advantageous properties that allow them to be considered as valuable drug-like molecules for injection, inhalation, nasal, ocular, oral or topical administration. Such advantageous properties include: - species cross-reactivity, a typical requirement for preclinical pharmacodynamic and pharmacokinetic evaluation; - Protease stability, where the bicyclic peptide ligand ideally exhibits stability against plasma proteases, epithelial ("membrane-anchored") proteases, gastric and intestinal proteases, lung surface proteases, intracellular proteases, etc. Protease stability should be maintained across different species so that bicycle peptide candidates can be developed in animal models and confidently administered to humans. - A desirable solubility profile, which is the ratio of charged and hydrophilic to hydrophobic residues and intra- / inter-molecular H-bonds, which is important for formulation and absorption purposes. -Optimal elimination half-life. Depending on the clinical indication and treatment regimen, it may be necessary to develop bicyclic peptides for short exposure in acute disease management settings or to develop bicyclic peptides with enhanced retention, thus making them ideal for the management of more chronic disease states and cancer.

[0213] Another factor driving a desirable elimination half-life is the requirement of sustained exposure to the drug for maximum therapeutic efficacy compared with the associated toxicity of sustained exposure.

[0214] In some embodiments, the molecular scaffold may comprise or consist of tris(bromomethyl)benzene, particularly 1,3,5-tris(bromomethyl)benzene ("TBMB"), or a derivative thereof. In some particularly preferred embodiments, the molecular scaffold is 1,3,5-(tribromomethyl)benzene).

[0215] In some other embodiments, the molecular scaffold is 2,4,6-tris(bromomethyl)mesitylene. This molecule is similar to 1,3,5-tris(bromomethyl)benzene, but contains three additional methyl groups attached to the benzene ring. This has the advantage that the additional methyl groups can form additional contacts with the polypeptide and thus impose additional structural constraints.

[0216] Scaffold reactive groups that can be used on molecular scaffolds to react with the thiol groups of cysteine ​​are alkyl halides (also called halogen alkanes or haloalkanes).

[0217] Examples include bromomethylbenzene (a scaffold reactive group exemplified by TBMB) or iodoacetamide. Another scaffold reactive group used to selectively couple compounds to cysteines in proteins is maleimide. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, and tris-(maleimido)benzene. Selenocysteine ​​is also a natural amino acid that has similar reactivity to cysteine ​​and can be used in the same reactions. Therefore, whenever cysteine ​​is mentioned, it is typically acceptable to substitute selenocysteine ​​unless the context suggests otherwise.

[0218] 2.3 Further N- or C-terminal modifications. In some of the above embodiments, the N-terminus and / or C-terminus of the proteinaceous molecule may be further modified. For example, additional amino acids or other substituents, if present, may be added to the N-terminus or C-terminus of the proteinaceous molecule of the invention. In some embodiments, the proteinaceous molecule of the invention may form part of a longer sequence with additional amino acids added to either or both the N-terminus and C-terminus.

[0219] For certain uses and methods of the present invention, a proteinaceous molecule with a high level of stability may be desired, for example, to increase the half-life of the proteinaceous molecule in a subject. Thus, in some embodiments, the proteinaceous molecule of the present invention comprises a stabilizing or protecting moiety. The stabilizing or protecting moiety can be coupled at any point on the peptide. Suitable stabilizing or protecting moieties include, but are not limited to, polyethylene glycol (PEG), glycans, or capping moieties comprising an acetyl group, pyroglutamate, or amino group. In preferred embodiments, the acetyl group and / or pyroglutamate is coupled to the N-terminal amino acid residue of the proteinaceous molecule. In certain embodiments, the N-terminus of the proteinaceous molecule is acetamide. In preferred embodiments, the amino group is coupled to the C-terminal amino acid residue of the proteinaceous molecule. In certain embodiments, the proteinaceous molecule has a primary, secondary, or tertiary amide, hydrazide, or hydroxamide at the C-terminus, particularly 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 proteinaceous molecule or via the amino group of a lysine side chain or other suitably modified side chain, in particular via the N-terminal amino acid residue, such as the amino group of a residue, or via the amino group of a lysine side chain.

[0220] In a preferred embodiment, the proteinaceous molecules of the present invention have a primary amide or free carboxyl group (acid) at the C-terminus and a primary amine or acetamide at the N-terminus.

[0221] Although the proteinaceous molecules of the present invention can inherently permeate membranes, membrane permeation can be further increased by conjugation of a membrane-permeating moiety to the proteinaceous molecule. Thus, in some embodiments, the proteinaceous molecules of the present invention comprise a membrane-permeating moiety. The membrane-permeating moiety can be attached at any point on the proteinaceous molecule.

[0222] Suitable membrane-permeable moieties include lipid moieties, cholesterol, and proteins, such as cell-penetrating peptides and polycationic peptides, especially lipid moieties.

[0223] Suitable cell membrane-permeable peptides may include, for example, peptides described in US2009 / 0047272, US2015 / 0266935, and US2013 / 0136742. Accordingly, suitable cell-permeable peptides include 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 WCCWK 18 (Di-CWK 18 ; SEQ ID NO: 10), WTLNSAGYLLGKINLKALAALAKKIL (Transportan; 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), K 16 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), PEVKKKKRKPEYP (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 tegument protein VP22; HSV-1 tegument protein VP22r fused to a nuclear export signal (NES); mutant B-subunit of Escherichia coli endotoxin EtxB (H57S); detoxified exotoxin A (ETA); protein transduction domain of HIV-1 Tat protein, GRKKRRQRRRPPQ (SEQ ID NO: 39); 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 (SEQ ID NO: 43);Ku70-derived peptides including peptides selected from the group including 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), MANLGYWLIALFVTMWTDVGLCKKRPKP (SEQ ID NO: 48); pVEC, LLIILRRRIRKQAHAHSK (SEQ ID NO: 49); Pep-I, KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 66); SynBI, RGGRLSYSRRRFSTSTGR (SEQ ID NO: 50); Transportan, GWTLNSAGYLLGKINLKAIAAIAKK IL (SEQ ID NO: 51); Transportan-10, AGYLLGKINLKALAALAKKIL (SEQ ID NO: 52); CADY, Ac-GLWRALWRLLRSLWRLLWRA-cysteamide (SEQ ID NO: 53); Pep-7, SDLWEMMMVSIACQY (SEQ ID NO: 54); HN-1, TSPLNIHNGQKL (SEQ ID NO: 55); VT5, DPKGDPKGVTVTVTVTVTGKGDPKPD (SEQ ID NO: 56); pISL, RVIRVWFQNKRCKDKK (SEQ ID NO: 57), or GALFLGFLGGAAGSTMGAWSQPKSKRKV (MGP; SEQ ID NO: 58);

[0224] In a preferred embodiment, the membrane-permeable moiety is C 10 ~C 20 Fatty acyl groups, especially stearoyl (octadecanoyl; C 18 ), palmitoyl (hexadecanoyl; C 16 ) or myristoyl (tetradecanoyl; C 14 ), particularly a lipid moiety such as myristoyl. In preferred embodiments, the membrane-permeable moiety is coupled to the N- or C-terminal amino acid residue, or via the amino group of a lysine side chain of the SETDB1 bicyclic peptidomimetic or other suitably modified side chain, particularly the N-terminal amino acid residue of the SETDB1 bicyclic peptidomimetic or via the amino group of a lysine side chain. In certain embodiments, the membrane-permeable moiety is coupled through the amino group of the N-terminal amino acid residue.

[0225] Thus, in another aspect of the present invention there is provided an isolated or purified proteinaceous molecule represented by formula VI: MP (Formula VI) During the ceremony, M is the membrane-permeable moiety, P is an isolated or purified proteinaceous molecule represented by any one of formulas (I)-(V).

[0226] In some embodiments, M is coupled at any point on the proteinaceous molecule, in particular to the N- or C-terminal amino acid residue, or to an amino group of a lysine side chain or other suitably modified side chain of the proteinaceous molecule, more particularly via the N-terminal amino acid residue of the proteinaceous molecule, or via an amino group of a lysine side chain, in particular via the amino group of the N-terminal amino acid residue.

[0227] Suitable membrane-permeable moieties and embodiments of proteinaceous molecules represented by any one of formulae (I)-(V) are as described herein.

[0228] 2.4 Salts and Prodrugs The proteinaceous molecules of the present invention may be in the form of salts or prodrugs. The salts of the proteinaceous molecules of the present invention are preferably pharmaceutically acceptable, although it will be understood that non-pharmaceutically acceptable salts are also within the scope of the present invention.

[0229] The proteinaceous molecules of the present invention may be in crystalline form and / or in the form of solvates, such as hydrates. Solvation may be carried out using methods known in the art.

[0230] 2.5 Linear peptide synthesis The peptides of the invention may be prepared using recombinant DNA techniques or by chemical synthesis.

[0231] In some embodiments, the proteinaceous molecules of the present invention are prepared using recombinant DNA techniques. For example, the proteinaceous molecules of the present invention can be prepared by a procedure comprising: (a) preparing a construct comprising a polynucleotide sequence encoding the proteinaceous molecule of the present invention and operably linked to a regulatory element; (b) introducing the construct into a host cell; (c) culturing the host cell to express the polynucleotide sequence, thereby producing the encoded proteinaceous molecule of the present invention; and (d) isolating the proteinaceous molecule of the present invention from the host cell. The proteinaceous molecules of the present invention can be prepared recombinantly 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. Patent No. 5,976,567 (the entire contents of which are incorporated herein by reference).

[0232] Accordingly, the present invention also contemplates nucleic acid molecules encoding the proteinaceous molecules of the present invention. Accordingly, in a further aspect of the present invention there is provided an isolated nucleic acid molecule comprising a polynucleotide sequence encoding a proteinaceous molecule of the present invention or which is complementary to a polynucleotide sequence encoding a proteinaceous molecule of the present invention, such as a proteinaceous molecule of any one of formulae I-IV of SEQ ID NOs: 1-5, or a variant proteinaceous molecule described herein.

[0233] The isolated nucleic acid molecule of the present invention can be DNA or RNA. When the nucleic acid molecule is in a DNA form, it can be genomic DNA or cDNA. The RNA form of the nucleic acid molecule of the present invention is generally mRNA.

[0234] While nucleic acid molecules are typically isolated, in some embodiments, nucleic acid molecules may be incorporated into, ligated to, or otherwise fused or associated with other genetic molecules, such as expression vectors. Generally, expression vectors comprise transcriptional and translational regulatory nucleic acid operably linked to a polynucleotide sequence. Thus, in another aspect of the invention, there is provided an expression vector comprising a polynucleotide sequence encoding a SETDB1 bicyclic peptidomimetic of the invention, such as a proteinaceous molecule of any one of Formulas I-IV of SEQ ID NOS: 1-5, or a variant proteinaceous molecule described herein.

[0235] Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulating the expression of the nucleic acid. Vectors optionally contain a generic expression cassette containing at least one independent terminator sequence, sequences that allow replication of the cassette in eukaryotes, prokaryotes, or both (e.g., shuttle vectors), and selectable markers in both prokaryotic and eukaryotic systems. Vectors may be suitable for replication and integration in 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 which are incorporated by reference.

[0236] Expression vectors containing regulatory elements derived from eukaryotic viruses, such as retroviruses, are typically used to express nucleic acid sequences in eukaryotic cells. SV40 vectors include pSVT7 and pMT2. Bovine papillomavirus-derived vectors include pBV-IMTHA, and Epstein-Barr virus-derived vectors include pHEBO and p205. Other exemplary vectors include pMSG, pAV009 / A+, pMTO10 / A+, pMAMneo-5, baculovirus pDSVE, and any other vector that allows protein expression under the direction of the SV-40 early promoter, SV-40 late promoter, metallothionein promoter, mouse mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown to be effective for expression in eukaryotic cells.

[0237] Although various vectors can be used, it should be noted that viral expression vectors are useful for modifying eukaryotic cells due to the high efficiency with which viral vectors transfect target cells and integrate into the target cell genome.Exemplary expression vectors of this type can be derived from viral DNA sequences, including but not limited to adenovirus, adeno-associated virus, herpes simplex virus, and B, C, and D retroviruses, as well as spumaviruses and modified lentiviruses.Suitable expression vectors for transfecting animal cells are described, for example, by Wu and Ataai (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.

[0238] The polypeptide or peptide-encoding portion of an expression vector may comprise a naturally occurring sequence or a variant thereof engineered using recombinant techniques. In one example of a variant, the codon composition of a polynucleotide encoding a proteinaceous molecule of the present invention is modified to enable enhanced expression of the proteinaceous molecule of the present invention in a mammalian host using methods that exploit codon usage bias or codon translation efficiency in specific mammalian cell or tissue types, as described, for example, in WO 99 / 02694 and WO 00 / 42215. Briefly, these latter methods are based on the observation that the translation efficiency of different codons varies between different cells or tissues, and these differences, along with the codon composition of a gene, can be exploited to modulate protein expression in specific cell or tissue types. Thus, for the construction of a codon-optimized polynucleotide, at least one existing codon in a parent polynucleotide is replaced with a synonymous codon that has a higher translation efficiency in the target cell or tissue than the existing codon it replaces. While it is preferable to replace all existing codons in a parent nucleic acid molecule with synonymous codons with higher translation efficiency, this is not necessary, as even partial replacement can achieve increased 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.

[0239] The expression vector is compatible with the cell into which it is introduced so that the proteinaceous molecule of the present invention can be expressed by the cell. The expression vector is introduced into the cell by any suitable means, depending on the specific selection of the expression vector and the cell used. Such introduction means are well known to those skilled in the art. For example, introduction can be achieved by contact (e.g., in the case of viral vectors), electroporation, transformation, transduction, conjugation or triparental mating, transfection, infectious membrane fusion with cationic lipids, high-velocity bombardment with DNA-coated microprojectiles, incubation with calcium phosphate-DNA precipitates, direct microinjection into single cells, etc. Other methods are also available and known to those skilled in the art. Alternatively, the vector can be introduced via cationic lipids, such as liposomes. Such liposomes are commercially available (e.g., LIPOFECTIN®, LIPOFECTAMINE™, etc., supplied by Life Technologies, Gibco BRL, Gaithersburg, Md.).

[0240] In some embodiments, the proteinaceous molecules of the invention may be produced intracellularly by introduction of one or more expression constructs, such as expression vectors, comprising a polynucleotide sequence encoding the proteinaceous molecule of the invention.

[0241] The present invention contemplates the recombinant production of the proteinaceous molecules of the invention in host cells such as mammalian cells (e.g., Chinese hamster ovary (CHO) cells, mouse myeloma (NS0) 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).

[0242] For therapeutic applications, the present invention also contemplates producing the proteinaceous molecules of the present invention in vivo in cells of a subject, e.g., in SETDB1-overexpressing cells such as vertebrate cells, particularly mammalian cells or avian cells, particularly mammalian cells.

[0243] In some embodiments, the proteinaceous molecules of the present invention are prepared using standard peptide synthesis methods, such as solution synthesis or solid-phase synthesis. Chemical synthesis of the proteinaceous molecules of the present invention can be performed manually or using an automated synthesizer. For example, linear peptides can be synthesized using solid-phase peptide synthesis using either Boc or Fmoc chemistry, as described in Merrifield (1963), Schnolzer et al. (1992), and Cardoso et al. (2015), 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.

[0244] In other embodiments, the proteinaceous molecules of the present invention can be cyclized. Cyclization can be performed using several techniques, for example, as described in Davies (2003), the entire contents of which are incorporated by reference. In certain embodiments, linear peptides are synthesized using solid-phase peptide synthesis involving Boc chemistry, starting with an N-terminal cysteine ​​residue and terminating with a C-terminal thioester. After deprotection and cleavage from the resin, the peptides are cyclized via a thiolactone intermediate, followed by rearrangement to the amine-cyclized peptide.

[0245] 2.6 Synthesis of Bicyclic Peptides The bicyclic peptides of the present invention can be synthetically produced by standard techniques and subsequently reacted with molecular scaffolds in vitro. When this is done, standard chemistry can be used. This allows for rapid large-scale preparation of soluble material for further downstream experimentation or validation. Such methods can be achieved using conventional chemistry, such as that disclosed in Timmerman et al. (supra).

[0246] Thus, the present invention also relates to the production of a polypeptide or conjugate selected as described herein, which production comprises optional further steps as explained below, which in one embodiment are carried out on the final product polypeptide / conjugate made by chemical synthesis.

[0247] Optionally, amino acid residues in the polypeptide of interest may be substituted when preparing the conjugate or complex.

[0248] Peptides can also be extended, for example, to incorporate additional loops, thus introducing multiple specificities. To extend a peptide, it can simply be chemically extended at its N- or C-terminus using orthogonally protected lysines (and analogs) or within a loop using standard solid- or solution-phase chemistry. Activatable N- or C-termini can be introduced using standard protein chemistry. Alternatively, addition can be performed by fragment condensation or native chemical ligation, as described, for example, in (Dawson et al., Science 1994, 266:776-779), or enzymatically using subtiligases, as described, for example, in (Chang et al., Proc Natl Acad Sci USA 1994, 91(26):12544-8 or Hikari et al., Bioorganic & Medicinal Chemistry Letters, 2008, 18(22), 6000-6003).

[0249] Alternatively, the peptide may be extended or modified by further conjugation via a disulfide bond. This has the added advantage of allowing the peptides to dissociate from one another in the reducing environment of a cell. In this case, a molecular scaffold (e.g., TBMB) can be added during the chemical synthesis of a first peptide to react with three cysteine ​​groups, and then an additional cysteine ​​can be added to the N-terminus of the first peptide so that this cysteine ​​reacts only with the free cysteine ​​of the second peptide.

[0250] Similar techniques apply equally to the synthesis / coupling of two bicyclic and bispecific macrocycles, potentially creating tetraspecific molecules. Furthermore, the addition of other functional or effector groups can be achieved in the same manner using appropriate chemistry, coupling at the N-terminus or C-terminus, or via side chains. In one embodiment, coupling is performed in a way that does not block the activity of either entity.

[0251] In some embodiments, the SETDB1 bicyclic peptidomimetics of the present invention can be produced intracellularly by introducing one or more expression constructs, such as expression vectors, that contain a polynucleotide sequence encoding a SEDB1 bicyclic peptidomimetic of the present invention.

[0252] The present invention contemplates recombinant production of the SETDB1 bicyclic peptidomimetics of the invention in host cells such as mammalian cells (e.g., Chinese hamster ovary (CHO) cells, mouse myeloma (NS0) 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., Escherichia coli cells, Corynebacterium glutamicum, or Pseudomonas fluorescens cells).

[0253] For therapeutic applications, the present invention also contemplates producing the SETDB1 bicyclic peptidomimetics of the present invention in vivo in cells of a subject, e.g., SETDB1-overexpressing cells such as vertebrate cells, particularly mammalian cells or avian cells, particularly mammalian cells.

[0254] In some embodiments, the SETDB1 bicyclic peptidomimetics of the present invention are prepared using standard peptide synthesis methods, such as solution synthesis or solid-phase synthesis. Chemical synthesis of the SETDB1 bicyclic peptidomimetics of the present invention can be performed manually or using an automated synthesizer. For example, linear peptides can be synthesized using solid-phase peptide synthesis using either Boc or Fmoc chemistry, as described in Merrifield (1963) J Am Chem Soc, 85(14):2149-2154, Schnolzer, et al. (1992) Int J Pept Protein Res, 40180-193, 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.

[0255] 3. Pharmaceutical Compositions In accordance with the present invention, proteinaceous molecules are useful in compositions and methods for the treatment or prevention of conditions associated with nuclear localization of SETDB1, such as cancer.

[0256] Thus, in some embodiments, the proteinaceous molecule of the present invention may be in the form of a pharmaceutical composition, which comprises the proteinaceous molecule of the present invention and a pharmaceutically acceptable carrier or diluent.

[0257] The proteinaceous molecules of the present disclosure may be formulated into pharmaceutical compositions as neutral or salt forms.

[0258] As will be understood by those skilled in the art, the selection of a pharmaceutically acceptable carrier or diluent depends on the route of administration and the nature of the condition and subject being treated. The specific carrier or delivery system and administration route can be easily determined by those skilled in the art. Careful selection of the carrier or delivery system and administration route is required to ensure that the activity of the proteinaceous molecule is not depleted during the preparation of the formulation and that the proteinaceous molecule can reach the site of action intact. The pharmaceutical compositions of the present invention can be administered via various routes, including, but not limited to, intravenous, oral, rectal, topical, intranasal, intraocular, transmucosal, intestinal, enteral, intramuscular, subcutaneous, intramedullary, intrathecal, intracerebroventricular, intracerebral, intravaginal, intravesical, or intraperitoneal administration. In some preferred embodiments, the pharmaceutical compositions of the present invention can be administered intravenously. In some other preferred embodiments, the pharmaceutical compositions of the present invention can be administered orally.

[0259] Pharmaceutical forms suitable for injectable use include sterile injectable solutions or dispersions and sterile powders for preparing sterile injectable solutions. Such forms must be stable under the conditions of manufacture and storage and can be preserved against reduction, oxidation, and microbial contamination.

[0260] Those skilled in the art will be able to easily determine suitable formulations for the proteinaceous molecules of the present invention using conventional approaches. Techniques for formulation and administration can 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, 2nd edition, the contents of which are incorporated by reference in their entirety.

[0261] Identification of preferred pH ranges and suitable excipients, such as antioxidants, is routine in the art, as described, for example, in Katdare and Chaubel (2006) 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; glutamate; and carbonate buffers. Suitable antioxidants 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.

[0262] For injection, the proteinaceous molecules of the invention can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's 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.

[0263] The compositions of the present invention can be formulated for administration in liquid form containing acceptable diluents (such as saline and sterile water), or in the form of lotions, creams, or gels containing acceptable diluents or carriers to impart the desired texture, consistency, viscosity, and appearance. Acceptable diluents and carriers are well known 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 oils, pH balancers, cellulose derivatives, emulsifiers such as nonionic organic bases and inorganic bases, preservatives, wax esters, steroid alcohols, triglyceride esters, phospholipids such as lecithin and cephalin, polyhydric alcohol esters, fatty alcohol esters, hydrophilic lanolin derivatives, and hydrophilic beeswax derivatives.

[0264] Alternatively, the proteinaceous molecules of the present invention can be readily formulated using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration, which are also contemplated for the practice of the present invention. Such carriers allow the bioactive agents of the present invention to be formulated into dosage forms such as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by the patient to be treated. These carriers may be selected from sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline, and pyrogen-free water.

[0265] Pharmaceutical preparations for parenteral administration include aqueous solutions of the proteinaceous molecules of the present invention in water-soluble form. Additionally, suspensions of the proteinaceous molecules of the present invention can be prepared as appropriate 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 can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound, allowing for the preparation of highly concentrated solutions.

[0266] Sterile solutions can be prepared by combining the required amount of active compound in an appropriate solvent with other excipients as described above, as needed, followed by sterilization, such as filtration. Generally, dispersions are prepared by incorporating the various sterilized active compounds into a sterile vehicle containing a basic dispersion medium and the necessary excipients, as described above. Sterile dry powders can be prepared by vacuum or freeze-drying a sterile solution containing the active compound and other necessary excipients, as described above.

[0267] Oral pharmaceutical preparations can be prepared by combining the proteinaceous molecules of the present invention with solid excipients, optionally adding suitable auxiliary agents, and then processing the resulting granular mixture to obtain tablets or dragee cores. Suitable excipients include, inter alia, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone (PVP). If desired, disintegrants such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof (e.g., sodium alginate) may be added. Such compositions can be prepared by any method of pharmacy; however, all methods include the step of bringing one or more therapeutic agents, as described above, into association with a carrier, which constitutes one or more necessary ingredients. In general, the pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0268] The dragee core is provided with a suitable coating.For this purpose, a concentrated sugar solution can be used, which can optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture.Dyes or pigments can be added to the tablet or dragee coating for identification or to characterize different combinations of particle dosages.

[0269] Orally usable pharmaceuticals include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and plasticizers such as glycerol or sorbitol. Push-fit capsules can contain active ingredients in a mixture with fillers such as lactose, binders such as starch, and / or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as fatty oils, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers can be added.

[0270] The proteinaceous molecules of the present invention may be incorporated into modified release preparations and formulations, such as polymeric microparticle formulations, as well as oil or gel-based formulations.

[0271] In certain embodiments, the proteinaceous molecules of the invention may be administered locally rather than systemically by injecting the proteinaceous molecules directly into tissue, preferably the subcutaneous tissue or the omental tissue, often in a depot or sustained release formulation.

[0272] Furthermore, the proteinaceous molecules of the present invention can be administered in targeted drug delivery systems, such as particles that are suitably targeted to and selectively taken up by cells or tissues. In some embodiments, the proteinaceous molecules of the present invention are contained in or otherwise associated with a vehicle selected from liposomes, micelles, dendrimers, biodegradable particles, artificial DNA nanostructures, lipid-based nanoparticles, and carbon or gold nanoparticles. In illustrative examples of this type, the vehicle is selected from poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol) (PEG), PLA-PEG copolymers, and combinations thereof.

[0273] In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration.

[0274] It is advantageous to formulate compositions in dosage unit form for ease of administration and uniformity of dosage. Determining the novel dosage unit forms of the present invention is dictated by and directly dependent upon the unique characteristics of the active agent, the particular therapeutic effect to be achieved, and the limitations inherent in the technology of compounding active agents for the treatment of diseases in living subjects having pathological conditions that compromise physical health, as disclosed in detail herein.

[0275] Although the proteinaceous molecules of the present invention may be the only active ingredient administered to a subject, it is within the scope of the present invention to administer other cancer therapies simultaneously with the proteinaceous molecules. For example, a proteinaceous molecule of any one of Formulas I-IV of any one of SEQ ID NOS: 2-6, or a variant described herein, may be administered simultaneously with one or more cancer therapies, non-limiting examples of which include radiation therapy, surgery, chemotherapy, hormone ablation therapy, pro-apoptotic therapy, and immunotherapy. The proteinaceous molecules of the present invention may be used therapeutically before treatment with a cancer therapy, after treatment with a cancer therapy, or together with a cancer therapy.

[0276] Suitable radiotherapy includes radiation and waves that induce DNA damage, such as gamma irradiation, X-rays, ultraviolet irradiation, microwaves, electron radiation and radioisotopes.Typically, therapy can be achieved by irradiating local tumor site with the above-mentioned radiation forms.All of these factors are most likely to cause widespread damage to DNA, DNA precursors, DNA replication and repair, chromosome assembly and maintenance.

[0277] X-ray dose ranges range from daily doses of 50-200 roentgens over prolonged periods, such as 3-4 weeks, to single doses of 2000-6000 roentgens. Dose ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells. Suitable radiation therapies may include conformal external beam radiation therapy (50-100 Gray given in fractions over 4-8 weeks), either single injection or fractionated high-dose brachytherapy, permanent interstitial brachytherapy, and systemic radioisotopes such as strontium-89. In some embodiments, radiation therapy may be administered in conjunction with a radiosensitizer. Suitable radiosensitizers may include, but are not limited to, efaproxiral, etanidazole, fluosol, misonidazole, nimorazole, temoporfin, and tirapazamine.

[0278] Suitable chemotherapeutic agents include alkylating agents (e.g., cisplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulfan, and nitrosoureas), antimetabolites (e.g., fluoropyridines such as 5-fluorouracil and tegafur, antifolates such as raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea), antitumor antibiotics (e.g., anthracidines such as adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, and mithramycin), antimitotic agents (e.g., vinca alkaloids such as vincristine, vinblastine, vindesine, and vinorelbine, and taxoids such as pazitaxel and docetaxel), and topoisomerase inhibitors (e.g., etoposide and epipodophyllotoxins such as teniposide; 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, leuprorelin, and buserelin), progestogens (e.g., megestrol acetate), aromatase inhibitors (e.g., anastrozole, letrozole, vorozole, and exemestane), and cytostatic agents such as inhibitors of 5a-reductase, such as finasteride; agents that inhibit cancer cell invasion (e.g., metalloproteinase inhibitors such as marimastat and inhibitors of urokinase plasminogen activator receptor function);Inhibitors of growth factor function, for example, such inhibitors include growth factor antibodies, growth factor receptor antibodies (e.g., the anti-erbb2 antibody trastuzumab (HERCEPTIN™) and the anti-EGFR antibody cetuximab (C225)), farnesyltransferase inhibitors, MEK inhibitors, tyrosine kinase inhibitors and serine / threonine kinase inhibitors, such as other inhibitors of the epidermal growth factor family (e.g., N-(3-chloro-4-fluorouracil)-2-one). N-(3-ethynylphenyl)-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-(3-morpholinopropoxy)quinazolin-4-amine (Cl1033)), e.g., in platelet-derived components. inhibitors of the growth factor family, and for example, inhibitors of the hepatocyte growth factor family; antiangiogenic agents such as those that inhibit the effects of vascular endothelial growth factor (e.g., the anti-vascular endothelial growth factor antibody bevacizumab (AVASTIN™), compounds such as those disclosed in International Patent Publications WO 97 / 22596, WO 97 / 30035, WO 97 / 32856, and WO 98 / 13354), and compounds that inhibit integrin anb3 function by other mechanisms (e.g., linomide, compounds acting through the cyclin-dependent kinase inhibitors such as palvocidib, abemacidib, ribodiib and albodiib; vascular damaging agents such as combretastatin A4 and the compounds disclosed in WO 99 / 02166, WO 00 / 40529, WO 00 / 41669, WO 01 / 92224, WO 02 / 04434 and WO 02 / 08213; antisense therapy, e.g., directed against the above targets such as ISIS 2503, antiras antisense;and gene therapy approaches, including approaches to replace abnormal genes such as abnormal p53 or abnormal GDEPT (gene-directed enzyme prodrug therapy), approaches using, for example, cytosine deaminase, thymidine kinase, or bacterial nitroreductase enzymes, as well as approaches to increase a patient's resistance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy.

[0279] Suitable immunotherapeutic approaches may include, but are not limited to, ex vivo and in vivo approaches to increase the immunogenicity of a patient's tumor cells, such as transfection with cytokines including interleukin-2, interleukin-4, or granulocyte colony-stimulating factor; approaches to reduce T cell anergy; approaches using transfected immune cells such as cytokine-transfected dendritic cells; approaches using cytokine-transfected tumor cell lines; and approaches using anti-idiotypic antibodies. These approaches generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of the malignant cell. The antibody alone may function as the effector of therapy or may recruit other cells to actually promote cell killing. Antibodies may also be conjugated to drugs or toxins (chemotherapeutic agents, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and simply function as targeting agents. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a malignant cell target. Various effector cells include cytotoxic T cells and NK cells.

[0280] 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. 2013 / 173223, 2013 / 079174, 2010 / 077634, 2011 / 066389, 2010 / 036959, 2007 / 005874, 2004 / 004771, 2006 / 133396, and 2011 / 066389. Examples of antibodies include, but are not limited to, antibodies described in Chinese Patent Publications Nos. 013 / 181634, 2012 / 145493, and Chinese Patent Publication No. 101104640, clone EH12, and clone 29E.2A3; CA-170; CA-327; BMS-202 (N-[2-[[[2-methoxy-6-[(2-methyl[1,1'-biphenyl]-3yl)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), particularly (D)PPA-1; AUNP-12; and peptides described in WO2014 / 151634 (the entire contents of which are incorporated by reference).

[0281] In some embodiments, the immune effector is a molecule that targets PD-1, including, but not limited to, an anti-PD-1 antibody, non-limiting examples of which include nivolumab, pembrolizumab, BGB-A317, WO2016 / 106159, WO2009 / 114335, WO2004 / 004771, WO2013 / 173223, WO 2015 / 112900, WO2008 / 156712, WO2011 / 159877, WO2010 / 036959, WO2010 / 089411, WO2006 / 133396, WO2012 / 145493, WO2002 / 078731, anti-mouse PD-1 antibody clone J43, anti-mouse antibody clone RMP1-14, ANB 011 (TSR-042), AMP-514 (MEDI0680), WO2006 / 121168, WO2001 / 014557, WO2011 / 110604, WO2011 / 110621, WO2004 / 072286Al, WO2004 / 056875, WO2010 / 036959, WO2010 / 029434, and WO2013 / 0 2209; AMP-224; compounds described in WO2011 / 082400; molecules and antibodies described in U.S. Pat. No. 6,808,710; molecules and antibodies described in WO2013 / 019906; molecules described in WO2003 / 011911; and compounds described in WO2013 / 132317 (the entire contents of which are incorporated by reference).

[0282] 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 the antibodies described in International Patent Publication No. WO 2010 / 036959, the entire contents of which are incorporated by reference, and rHigM12B7.

[0283] In some embodiments, the immune effector is an agent that targets CTLA-4, including, but not limited to, anti-CTLA-4 antibodies such as ipilimumab, tremelimumab, antibodies described in WO00 / 37504A2, WO01 / 14424A2, US2003 / 0086930A1; and compounds described in WO2006 / 056464A2, the contents of which are incorporated by reference in their entireties.

[0284] Examples of other cancer therapies include phytotherapy, cryotherapy, toxin therapy, or pro-apoptotic therapy. Those skilled in the art will appreciate that this list is not exhaustive of the types of treatment modalities available for cancer and other hyperplastic lesions.

[0285] It is well known that chemotherapy and radiation therapy target rapidly dividing cells and / or disrupt the cell cycle or cell division. These treatments are provided as part of the treatment of some forms of cancer, aiming to slow their progression or ameliorate the symptoms of the disease through curative treatment. However, these cancer treatments can result in an immunocompromised state and subsequent pathogenic infections. Therefore, the present invention also extends to combination therapy using a proteinaceous molecule of any one of Formulas I-IV or any one of SEQ ID NOS: 1-5, or a variant thereof, as described herein, a cancer therapy, and an anti-infective agent that is effective against infections that develop or are at high risk of developing from the immunocompromised state resulting from the cancer therapy. Anti-infective agents may include compounds that kill or inhibit the growth of microorganisms such as viruses, bacteria, yeasts, fungi, and protozoa, and are therefore preferably selected from antibacterial agents, which may include, but are not limited to, antibiotics, amebicides, antifungals, antiprotozoal agents, antimalarials, antituberculosis agents, and antivirals. Anti-infectives also include within their scope anthelmintics and nematicides. Exemplary antibiotics include quinolones (e.g., amifloxacin, cinoxacin, ciprofloxacin, enoxacin, fleroxacin, flumequine, lomefloxacin, nalidixic acid, norfloxacin, ofloxacin, levofloxacin, lomefloxacin, oxolinic acid, pefloxacin, losoxacin, temafloxacin, tosufloxacin, sparfloxacin, dinafloxacin, gatifloxacin, moxifloxacin; gemifloxacin; and garenoxacin), tetracyclines, glycylcyclines, and oxazolidinones (e.g., chlortetracycline, demedocidine, doxycycline, lymecycline, methacycline, minocycline, oxytetracycline, tetracycline, tigecidin;linezolid, eperezolid), glycopeptides, aminoglycosides (e.g., amikacin, arbekacin, butirosin, dibekacin, fortimicin, gentamicin, kanamycin, menomycin, netilcin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin), β-lactams (e.g., imipenem, meropenem, biapenem, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefixime, cefmenoxime, Cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefpimizole, cefpiramide, cefpodoxime, cefsulodin, ceftazidime, ceftaram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile, cephalexin, cephaloglycin, cephaloridine, cephalothin, cephapirin, cephradine, cefinetazole, cefoxitin, cefotetan, aztreonam, carumonam, flomoxef, Moxalactam, amdinocillin, amoxicillin, ampicillin, azlocillin, carbenicillin, benzylpenicillin, carfecillin, doxacillin, didoxacillin, methidrine, mezlocillin, nacillin, oxacillin, penicillin G, piperacillin, sulbenicillin, temocillin, ticalderin, cefditoren, SC004, KY-020, cefdinir, ceftibuten, FK-312, S-1090, CP-0467, BK-218, FK-037, DQ-2556, FK-518, cefozopran lanin, ME1228, KP-736, CP-6232, Ro09-1227, OPC-20000, LY206763), rifamycins, macrolides (e.g., azithromycin, clarithromycin, erythromycin, oleandomycin, rokitamycin, rosaramycin, roxithromycin, troleondomycin), ketolides (e.g., telithromycin, cethromycin), coumermycin, lincosamides (e.g., clindamycin, lincomycin), and chloramphenicol;

[0286] Exemplary antiviral agents include abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir sulfate, lamivudine / zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valacidovir hydrochloride, zalcitabine, zanamivir, and zidovudine.

[0287] Suitable antiprotozoal or antiprotozoal agents include, but are not limited to, atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. The anthelmintic agent may be at least one selected from mebendazole, pyrantel pamoate, albendazole, ivermectin, and thiabendazole. Exemplary antifungal agents may be selected from amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposome, fluconazole, flucytosine, griseofulvin microsize, griseofulvin ultramicrosize, itraconazole, ketoconazole, nystatin, and terbinafine hydrochloride. Suitable antimalarial drugs include, but are not limited to, chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. Antituberculosis drugs include, but are not limited to, clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine, and streptomycin sulfate.

[0288] As mentioned above, the proteinaceous molecule may be mixed with a suitable pharmaceutically acceptable carrier in a dosage unit form for convenient and effective administration in an effective amount. In some embodiments, the unit dosage form may contain the active peptide of the present invention in an amount ranging from about 0.25 pg to about 2000 mg. The active peptide of the present invention may be present in an amount of about 0.25 pg to about 2000 mg / mL of the carrier. In embodiments where the pharmaceutical composition contains one or more additional active ingredients, the dosage will be determined with reference to the usual dosage and method of administration of the ingredient.

[0289] 4.Treatment method The inventors have determined that blocking the nuclear localization of SETDB1 is an effective treatment for diseases such as cancer (e.g., metastatic cancer). Accordingly, in some embodiments, the present invention provides methods of treating cancer in a subject, the method comprising administering to the subject an agent that prevents or reduces the nuclear localization of SETDB1.

[0290] In some embodiments, the agent comprises a proteinaceous molecule described above or elsewhere herein, for example, the proteinaceous molecule may comprise an amino acid sequence corresponding to any one of formulas (I)-(IV).

[0291] Without wishing to be bound by theory, the present inventors have determined that SETDB1 is enriched in the nuclei of metastasis-initiating tumor cells and dysfunctional terminally exhausted CD8+ T cells in subjects who are resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy). Conversely, SETDB1 has been found to be enriched in the cytoplasm and / or cell surface of subjects who have responded to immunotherapy (e.g., immune checkpoint inhibitor therapy). Therefore, it is proposed that preventing the nuclear localization of SETDB1 polypeptide in a subject's cells will prevent or reduce the likelihood that the subject will be resistant to immunotherapy (e.g., immune checkpoint inhibitor therapy).

[0292] Thus, in another aspect of the present invention, there is provided a method for inhibiting or reducing nuclear localization of SETDB1, the method comprising contacting a cell with a proteinaceous molecule as described above or elsewhere herein.

[0293] In some embodiments, the proteinaceous molecule is an isolated or purified proteinaceous molecule represented by any one of formulas (I)-(IV), in particular a proteinaceous molecule of any one of SEQ ID NOs: 2-6, or a variant proteinaceous molecule described herein.

[0294] In another aspect of the present invention there is provided the use of an isolated or purified proteinaceous molecule of the present invention, in particular a proteinaceous molecule of any one of formulae I to IV, SEQ ID NOs: 2 to 6, or a variant proteinaceous molecule as described herein, for therapy or in the manufacture of a medicament for therapy. The present invention also provides an isolated or purified proteinaceous molecule of the present invention, in particular a proteinaceous molecule of formulae (I) to (IV), SEQ ID NOs: 2 to 6, or a variant proteinaceous molecule as described herein, for use in therapy.

[0295] The present invention also provides methods for inhibiting or reducing the nuclear localization of SETDB1 in SETDB1-overexpressing cells, comprising contacting the cells with an inhibitor of binding between a SETDB1 polypeptide and an importin-α polypeptide. In some embodiments, the inhibitor comprises a proteinaceous 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 proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS for inhibiting or reducing the nuclear localization of SETDB1 in SETDB1-overexpressing cells; the use of a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an NLS for use in inhibiting or reducing the nuclear localization of SETDB1 in SETDB1-overexpressing cells; and the manufacture of a medicament for such use.

[0296] In some embodiments of any one of the above aspects, the SETDB1-overexpressing cells are cancer stem cells or non-cancer stem cell tumor cells, particularly cancer stem cell tumor cells.

[0297] Suitable embodiments of proteinaceous molecules are as described herein.

[0298] Proteinaceous molecules comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site as described herein, particularly any one of Formulae (I)-(IV), SEQ ID NOs: 2-6, or variant proteinaceous molecules, are useful for inhibiting the nuclear localization of SETDB1. Accordingly, the inventors believe that proteinaceous molecules are useful for treating or preventing cancer in a subject. Accordingly, in another aspect, there is provided a method for treating or preventing cancer in a subject, wherein the cancer comprises at least one SETDB1-overexpressing cell, comprising administering to the subject a proteinaceous 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 proteinaceous 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 to the manufacture of a medicament for this purpose. Also contemplated is a SETDB1 bicyclic peptidomimetic comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site for use in treating or preventing cancer in a subject, wherein the cancer comprises at least one SETDB1-overexpressing cell.

[0299] The cancer can be any cancer associated with overexpression of SETDB1. Suitable cancers 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; particularly breast cancer, lung cancer or melanoma; particularly breast cancer or melanoma, especially breast cancer.

[0300] In some embodiments, proteinaceous molecules comprising, consisting of, or consisting essentially of amino acid sequences corresponding to the acetylation sites described herein are useful for treating, preventing, and / or alleviating symptoms of malignant tumors, particularly metastatic cancer. In preferred embodiments, SETDB1 bicyclic peptidomimetics are used to treat, prevent, and / or alleviate symptoms of metastatic cancer. Suitable types of metastatic cancer 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, the brain cancer is glioma. In preferred embodiments, the metastatic cancer is metastatic breast cancer, lung cancer, or melanoma, particularly metastatic breast cancer or melanoma, particularly metastatic breast cancer.

[0301] Proteinaceous molecules are useful in methods involving SETDB1-overexpressing cells. In certain embodiments, the SETDB1-overexpressing cells are selected from breast cells, prostate cells, testicular cells, lung cells, bladder cells, pancreatic cells, colon cells, melanoma cells, leukemia cells, retinoblastoma cells, liver cells, ovarian cells, kidney cells, or brain cells; particularly breast cells, lung cells, or melanoma cells; particularly breast cells or melanoma cells, especially breast cells. In a preferred embodiment, the SETDB1-overexpressing cells are breast epithelial cells, particularly breast ductal epithelial cells.

[0302] In some embodiments, the SETDB1 overexpressing cells are cancer stem cells or non-cancer stem cell tumor cells, particularly cancer stem cell tumor cells, particularly breast cancer stem cell tumor cells. In some embodiments, the cancer stem cell tumor cells express CD24 and CD44, particularly CD44 高 , CD24 低 is expressed.

[0303] 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 proteinaceous molecule to the subject, wherein the tumor sample comprises cancer stem cell tumor cells and optionally non-cancer stem cell tumor cells.

[0304] Proteinaceous molecules comprising, consisting of, or consisting essentially of amino acid sequences corresponding to the lysine methylation sites described herein are suitable for treating individuals diagnosed with cancer, individuals suspected of having cancer, individuals known to be susceptible and believed to be at high risk of developing cancer, or individuals believed to develop a recurrence of a previously treated cancer. The cancer may be hormone receptor-negative. In some embodiments, the cancer is hormone receptor-negative and therefore resistant to hormonal or endocrine therapy. In some embodiments, 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.

[0305] There are numerous conditions associated with SETDB1 overexpression for which a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to a lysine methylation site as described herein may be useful. Accordingly, in another aspect of the present invention, there is provided a method for treating or preventing a condition in a subject in which inhibition or reduction of nuclear localization of SETDB1 is an effective treatment, comprising administering to the subject a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to an acetylation site. The present invention also provides a proteinaceous molecule comprising, consisting of, or essentially consisting of an amino acid sequence corresponding to an acetylation site for the treatment or prevention of a condition in a subject in which inhibition or reduction of nuclear localization of SETDB1 is an effective treatment; the use of a proteinaceous molecule comprising, consisting of, or essentially consisting of an amino acid sequence corresponding to an acetylation site for use in the treatment or prevention of a condition in a subject in which inhibition or reduction of nuclear localization of SETDB1 is an effective treatment; and the use of a proteinaceous molecule comprising, consisting of, or essentially consisting of an amino acid sequence corresponding to an acetylation site in the manufacture of a medicament for this purpose.

[0306] Non-limiting examples of conditions associated with SETDB1 overexpression include cancer, infectious diseases, autoimmune diseases, and respiratory diseases.

[0307] In some embodiments, the infectious disease is a pathogenic infection. The infectious disease may be selected from, but is not limited to, viral, bacterial, yeast, fungal, helminthic, or protozoan infections. Viral infections contemplated by the present invention include, but are not limited to, infections 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; particularly, but not limited to, infections caused by HIV, hepatitis, influenza virus, Japanese encephalitis virus, Epstein-Barr virus, and respiratory syncytial virus.Bacterial infections include 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, Bordete. lla species, Borrella species, Campylobacter species, Caulobacter species, Chlorobium species, Chromatium species, Clostridium species, Corynebacterium species, Cytophaga species, Deinococcus species, Escherichia species, Francisella species, Helicobacter species, Haemophilus species, Hyphomicrobium species, Lepto Infections encompassed by the present invention include, but are not limited to, infections caused by spira, Usteria, Micrococcus, Myxococcus, Nitrobacter, Oscillatoria, Prochlorous, Proteus, Pseudomonas, Rhodospirillum, Rickettsia, Shigella, Spirillum, Spirochaeta, Streptomyces, Thiobacillus, Treponema, Vibrio, Yersinia, Nocardia, and Mycobacterium species, particularly infections caused by Neisseria, Meningococcal, Haemophilus, Salmonella, Streptococcal, Legionella, and Mycobacterium species. Protozoal infections encompassed by the present invention include, but are not limited to, those caused by Plasmodium, Lishmania, Trypanosoma, Toxoplasma, Entamoeba, and Glardia species. Helminth infections can 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.

[0308] Suitable autoimmune disorders include autoimmune rheumatic diseases (e.g., rheumatoid arthritis, Sjögren's syndrome, scleroderma, lupus, such as systemic lupus erythematosus (SLE) and lupus nephritis, polymyositis-dermatomyositis, cryoglobulinemia, antiphospholipid syndrome, and psoriatic arthritis), autoimmune gastrointestinal and liver diseases (e.g., inflammatory bowel diseases, 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., anti-neotrophil cytoplasmic antibody (ANCA)-negative vasculitis and ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and microscopic polyangiitis), autoimmune neuropathies (e.g., multiple sclerosis, opsoclonus-myoclonus syndrome, myasthenia gravis, autoimmune diseases (such as neuromyelitis optica, Parkinson's disease, Alzheimer's disease, and autoimmune polyneuropathy), kidney disorders (such as glomerulonephritis, Goodpasture's syndrome, and Buerger's disease), autoimmune skin disorders (such as psoriasis, urticaria, hives, pemphigus vulgaris, pemphigus major, and cutaneous lupus erythematosus), hematological disorders (such as thrombocytopenic purpura, thrombotic thrombocytopenic purpura, retroperitoneal purpura, and autoimmune lytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases (such as inner ear disease and hearing loss), Behcet's disease, Raynaud's syndrome, organ transplant, and autoimmune endocrine disorders (such as diabetes-related autoimmune diseases such as type 1 diabetes, Addison's disease, and autoimmune thyroid diseases (e.g., Graves' disease and thyroiditis)).

[0309] Suitable respiratory disorders include, but are not limited to, chronic obstructive pulmonary disease (CORD) or asthma, particularly allergic asthma.

[0310] In some embodiments, the method further comprises detecting overexpression of the SETDB1 gene in a tumor sample obtained from the subject prior to administering to the subject a proteinaceous molecule of the present invention, wherein the tumor sample comprises cancer stem cell tumor cells and optionally non-cancer stem cell tumor cells.

[0311] In certain embodiments, any one of the above methods involves the administration of one or more additional active agents described in Section 3 above, such as an additional cancer therapy and / or an anti-infective agent, particularly an additional cancer therapy.

[0312] The proteinaceous molecules of the present invention, particularly a proteinaceous molecule of any one of Formulae (I)-(IV), or any one of SEQ ID NOs: 2-6, or a variant proteinaceous molecule described herein, are useful for inhibiting or reducing acetylation of SETDB1. In some embodiments, the acetylation is catalyzed by an acetyltransferase, particularly 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; particularly p300.

[0313] Thus, in a further aspect of the present invention, there is provided a method for inhibiting nuclear localization of SETDB1 in a subject, the method comprising administering a proteinaceous molecule as described herein. The present invention also extends to the use of a proteinaceous molecule as described herein for inhibiting or reducing binding of a SETDB1 polypeptide by binding of an importin polypeptide. In a preferred embodiment, the proteinaceous molecule of the present invention precedes the importin polypeptide's transport of SETDB1 into the nuclear compartment.

[0314] The present invention also contemplates a method for producing a proteinaceous molecule that inhibits or reduces nuclear localization of a SETDB1 polypeptide, the method comprising: a) contacting a cell with a proteinaceous molecule comprising, consisting of, or consisting essentially of an amino acid sequence corresponding to the NLS of SETDB1; b) detecting a reduction or inhibition of nuclear localization of the SETDB1 polypeptide in the cell compared to a normal or reference level of nuclear localization in the absence of the proteinaceous molecule.

[0315] In another aspect, the present invention provides a method for producing a SETDB1 bicyclic peptidomimetic that inhibits or reduces the nuclear localization of SETDB1, wherein methylation of a methylation site in SETDB1 increases its nuclear localization in a cell, the method comprising: a) contacting a cell with a SETDB1 bicyclic peptidomimetic comprising, consisting of, or consisting essentially of an amino acid sequence set forth in any one of Formulas (I), or a bicyclic peptide of any one of Formulas (II)-(V); b) detecting a reduction or inhibition of nuclear localization of the nuclear-localizable polypeptide in the cell compared to a normal or reference level of nuclear localization in the absence of the SETDB1 bicyclic peptidomimetic.

[0316] In some embodiments, the SETDB1 bicyclic peptidomimetic is distinguished from the native wild-type SETDB1 sequence by the addition of at least three cysteine ​​residues.

[0317] Reduction or inhibition of SETDB1 nuclear localization 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), e.g., those 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 contents of which are incorporated by reference in their entireties.

[0318] Those skilled in the art will be well aware of suitable assays that can be used to assess the nuclear localization of a polypeptide, such as SETDB1, and to identify SETDB1 bicyclic peptidomimetics that inhibit or reduce the nuclear localization of the polypeptide. Screening for active agents in accordance with the present invention can be accomplished by any suitable method. For example, the method may include contacting cells 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 cells.

[0319] Alternatively, one may screen for inhibition of the functional activity of a polypeptide of interest, or a reduction in the level of a transcript encoded by the polynucleotide, or inhibition of the activity or expression of a downstream cellular target of the polypeptide or transcript, wherein the activity is associated with nuclear localization of SETDB1. Detecting such inhibition can be achieved using techniques including, but not limited to, ELISA, immunofluorescence, Western blot, immunoprecipitation, immunostaining, slot 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 transcriptase 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. Polypeptide methylation can be determined using an antibody directed against the methylated polypeptide, e.g., an antibody directed against a methylated lysine residue.

[0320] Active molecules can be further tested in animal models to identify those molecules with the most potent in vivo effects. These molecules can serve as lead molecules for further development of pharmaceuticals, for example, by subjecting the compounds to sequential modification, molecular modeling, and other routine procedures used in rational drug design.

[0321] 5. Methods of detection and prognosis / prediction According to the present disclosure, nuclear localization of SETDB1 can be used as a biomarker of response to therapy (e.g., immunotherapy). In some embodiments, nuclear localization of SETDB1 is determined by detecting colocalization of SETDB1 with a SETDB1 binding partner (e.g., ATF7IP or IMPα). Nuclear localization of SETDB1 is suitably assessed in SETDB1-expressing cells, such as, but not limited to, tumor cells. Representative subject samples containing SETDB1-expressing cells include tissue samples such as solid tumors. In some embodiments, the sample is obtained prior to treatment with a therapy. In some embodiments, the tissue sample is formalin-fixed and paraffin-embedded, archived, fresh, or frozen.

[0322] The presence and / or level / or 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) (also collectively referred to herein as "response to therapy biomarkers" or "RTT biomarkers") 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 RTT biomarker at a cellular location in a first sample is increased or elevated compared to its presence / absence and / or level / amount at the cellular location in a second sample. In certain embodiments, the presence / absence and / or level / amount of the RTT biomarker at a cellular location in a first sample is decreased or reduced compared to its presence and / or expression level / amount at the cellular location in a second sample.

[0323] In some embodiments of any of these methods, elevated or higher level / amount refers to an overall increase of any of about 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 compared to the level / amount of the RTT biomarker at a cellular location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain embodiments, elevated or higher level / amount refers to an increase in the level / amount of an RTT biomarker at a sample cellular location, which increase is at least about any of 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.75-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold the level / amount of an RTT biomarker at a cellular location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue. In some embodiments, elevated or higher level / amount refers to an overall increase at a cellular location of more than about 1.5-fold, about 1.75-fold, about 2-fold, about 2.25-fold, about 2.5-fold, about 2.75-fold, about 3.0-fold, or about 3.25-fold compared to a cellular location in a reference sample, reference cell, reference tissue, control sample, control cell, control tissue, control tissue, or internal control.

[0324] In some embodiments of any of these methods, elevated or higher level / amount refers to the ratio between the level / amount of the RTT biomarker at a first cellular location (e.g., nuclear SETDB1) and the level / amount of the RTT biomarker at a second cellular location (e.g., cytoplasmic SETDB1), which ratio is greater than any of approximately 0.55, 0.60, 0.65, 0.70, 0.75, 0.85, 0.90, or 0.95.

[0325] In some embodiments of any of these methods, elevated or higher levels / amounts refer to higher levels of the RTT biomarker in more than about any of 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the subject's cells.

[0326] In some embodiments of any of these methods, a reduced or lower level / amount refers to an overall reduction of any of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more in the level of the RTT biomarker at a cellular location compared to the level / amount at a cellular location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue, as detected by standard art-known methods, such as those described herein. In certain embodiments, a reduced or lower level / amount refers to a decrease in the level / amount of the RTT biomarker at a cellular location in a sample, wherein the decrease is at least about 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold, 0.1-fold, 0.05-fold, or 0.01-fold of the level / amount of the RTT biomarker at a cellular location in a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.

[0327] The presence and / or level / amount of various RTT biomarkers in a sample can be analyzed by a number of methodologies, including but not limited to immunohistochemistry ("IHC"), Western blot analysis, immunoprecipitation, molecular binding assays, ELISA, ELIFA, fluorescence activated cell sorting ("FACS"), proteomics, many of which are known in the art and understood by the skilled artisan.

[0328] According to some embodiments, the presence and / or level / amount is measured by observing protein levels / amounts. In certain embodiments, the method comprises contacting the sample with an RTT biomarker (e.g., an anti-SETDB1 antibody), optionally in combination with an antibody against at least one SETDB1 binding partner (e.g., ATF7IP or IMPα), under conditions that allow binding of the biomarker, and detecting whether a complex is formed between the antibody / antibodies and the biomarker.

[0329] 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 (e.g., immunotherapy).

[0330] In certain embodiments, the presence and / or level / amount of biomarker protein in a sample is examined 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 a protein in a sample, including the cellular localization of the protein. In some embodiments, the level / amount of an RTT biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (such as a tumor sample) using an antibody; and b) determining the level / amount of the biomarker in 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 stained sample or a tissue sample from a non-cancerous subject).

[0331] In some embodiments, the presence and / or level / amount of RTT biomarkers is assessed on a tumor or tumor sample. As used herein, a tumor or tumor sample may include some or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample may further include tumor areas occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (e.g., continuous peritumoral anaplastic stroma). Tumor-associated intratumoral cells and / or tumor-associated stroma may include immune-infiltrated areas (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent to and / or adjacent to the main tumor mass. In some embodiments, RTT biomarker expression is assessed on tumor cells.

[0332] In an alternative method, a sample can be contacted with an antibody specific for the RTT biomarker under conditions sufficient to form an antibody-biomarker complex, and the complex can then be detected. The presence or level / amount of the biomarker can be detected in several ways, for example, by Western blotting and ELISA procedures for assaying a wide variety of tissues and samples, including plasma or serum. A wide range of immunoassay techniques using such assay formats are available (see, e.g., U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653). This includes both non-competitive single-site and two-site or "sandwich" assays, as well as traditional competitive binding assays. These assays also include direct binding of labeled antibodies to target biomarkers.

[0333] In certain embodiments, samples are normalized for both differences in the amount of assayed biomarkers and variability in the quality of the samples used, as well as variability between assay runs. Such normalization can be achieved by detecting and incorporating the expression of certain normalizing biomarkers, including the expression products of well-known housekeeping genes. Alternatively, normalization can be based on the average or median signal of all assayed proteins or a large subset thereof (global normalization approach). For each protein, the measured normalized amount of the tumor protein of interest is compared to the amount found in a reference set. The normalized level of each protein per tumor tested per subject can be expressed as a percentage of the expression level measured in the reference set. The presence and / or level / amount measured in a particular subject sample analyzed falls within some percentile within this range, which can be determined by methods well known in the art.

[0334] In some embodiments, the sample is a clinical sample. In other embodiments, the sample is used in a diagnostic assay. In some embodiments, the sample is obtained from a primary or metastatic tumor. Tissue biopsy is often used to obtain a representative portion of tumor tissue. Alternatively, tumor cells can be obtained indirectly in the form of tissue or fluid known or believed to contain the tumor cells of interest. For example, samples of lung cancer lesions can be obtained by resection, bronchoscopy, fine needle aspiration, bronchial brushing, or from sputum, pleural fluid, or blood. Proteins can be detected from cancer or tumor tissue or from other body samples such as urine, sputum, serum, or plasma. Cancer cells can be shed from cancer lesions and appear in such body samples. By screening such body samples, simple early diagnosis of these cancers can be achieved. In addition, response to therapy can be more easily monitored by testing such body samples for RTT biomarkers.

[0335] 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 obtained at one or more time points different from when the test sample was 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 an earlier time point than when the test sample was obtained. Such a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful when the reference sample is obtained during the initial diagnosis of cancer and the test sample is obtained later when the cancer becomes metastatic or resistant to treatment with a therapy.

[0336] In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more healthy individuals who are not the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combination of multiple samples from one or more individuals with a disease (e.g., cancer) who are not the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled sample of normal tissue or biological fluid, such as blood, from one or more individuals who are not the subject or individual. In certain embodiments, the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a pooled sample of tumor tissue or pooled blood samples from one or more individuals with a disease (e.g., cancer) who are not the subject or individual.

[0337] 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., biopsy tissue). 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.).

[0338] In some embodiments, a tumor tissue sample (the term "tumor sample" is used interchangeably herein) may include some or all of the tumor area occupied by tumor cells. In some embodiments, a tumor or tumor sample may further include tumor areas occupied by tumor-associated intratumoral cells and / or tumor-associated stroma (e.g., continuous peritumoral anaplastic stroma). Tumor-associated intratumoral cells and / or tumor-associated stroma may include immune-infiltrated areas (e.g., tumor-infiltrating immune cells as described herein) immediately adjacent to and / or adjacent to the main tumor mass.

[0339] In some embodiments, tumor cell staining is expressed as a percentage of all tumor cells that exhibit nuclear staining of any intensity. Infiltrating immune cell staining can be expressed as a percentage of the total tumor area occupied by immune cells that exhibit staining of any intensity. Total tumor area encompasses malignant cells as well as tumor-associated stroma, including immune-infiltrated areas immediately adjacent to and adjacent to the main tumor mass. Furthermore, infiltrating immune cell staining can be expressed as a percentage of all tumor-infiltrating immune cells.

[0340] In some embodiments of any of the methods, the disease is a tumor. In some embodiments, the tumor is a malignant cancerous tumor (i.e., cancer). In some embodiments, the tumor and / or cancer is a solid tumor or a non-solid or soft tissue tumor. Examples of soft tissue tumors include leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, adult acute lymphoblastic leukemia, acute myeloid leukemia, mature B-cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, prolymphocytic leukemia, or hairy cell leukemia) or lymphoma (e.g., non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, or Hodgkin's disease). Solid tumors include any cancer of body tissues other than blood, bone marrow, or the lymphatic system. Solid tumors can be further divided into those of epithelial cell origin and those of non-epithelial cell origin. Examples of epithelial cell solid tumors include tumors of the gastrointestinal tract, colon, colorectum (e.g., basal 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, lip, nasopharynx, skin, uterus, male reproductive organs, urinary tract (e.g., urothelial carcinoma, dysplastic urothelial carcinoma, transitional cell carcinoma), bladder, and skin. Solid tumors of non-epithelial origin include sarcoma, brain tumor, and bone tumor. In some embodiments, the cancer is non-small cell lung cancer (NSCLC). In some embodiments, the cancer is secondary or tertiary locally advanced or metastatic non-small cell lung cancer. In some embodiments, the cancer is adenocarcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the 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, the cancer is a primary tumor. In some embodiments, the cancer is a metastatic tumor at a second site derived from any of the above cancer types.

[0341] In some embodiments, the RTT biomarker is detected in a sample using a method selected from the group consisting of FACS, Western blot, ELISA, immunoprecipitation, immunohistochemistry, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, HPLC, 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 for isolating / enriching such cell populations may be used, including but not limited to cell sorting. In some embodiments, the level / amount of nuclear SETDB1 is reduced in a sample from an individual who responds to treatment with a therapy, preferably an immunotherapy (e.g., one 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 elevated in a sample from an individual who does not respond or responds poorly to therapy, preferably an immunotherapy (e.g., one 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 from individuals who do not respond or respond poorly to therapy, preferably immunotherapy (e.g., 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 elevated in samples from individuals who respond to treatment with therapy, preferably immunotherapy (e.g., therapy comprising an anti-immune checkpoint molecule antibody, such as an anti-PD-1 antagonist antibody).

[0342] Also provided herein are predictive / prognostic methods and kits based on the determination that SETDB1 co-localizes in the nucleus with a nuclear binding partner of SETDB1 (e.g., ATF7IP) and that this co-localization contributes, at least in part, to resistance or non-responsiveness to therapy (e.g., immunotherapy) and / or disease state (e.g., disease severity or progression). These methods preferably include: (i) obtaining a sample from a subject, where the sample contains SETDB1-expressing cells (e.g., tumor cells); (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 SETDB1-binding partner in the sample; and (iii) detecting localization of the first and second antigen-binding molecules in the nuclei of the SETDB1-expressing cells, where localization of the first and second antigen-binding molecules in the nuclei of the SETDB1-expressing cells indicates that the SETDB1-expressing cells have a likelihood of increased resistance to therapy, that the subject is likely to be a non-responder to therapy, that the subject has been selected for not being treated with therapy, and / or that the treatment outcome for the subject is predictive of a likely negative treatment outcome.

[0343] Localization of SETDB1 and its nuclear binding partner within the nuclei of SETDB1-expressing cells can be performed by any suitable localization technique, for example, IHC, typically using an anti-SETDB1 antibody bearing a detectable moiety or label that is distinct from the anti-SETDB1 binding partner antibody. In some embodiments, a spatial proximity assay (also referred to as a "proximity assay") is used, which can be used to assess the formation of a complex between SETDB1 and its nuclear binding partner. Proximity assays rely on the principle of "proximity probing," where an analyte, typically an antigen, is detected by the simultaneous binding of multiple (i.e., two or more, generally two, three, or four) binding agents or probes, which generate a signal upon proximity due to binding to the analyte (hence, "proximity probes").

[0344] In some embodiments, at least one of the proximity probes comprises a nucleic acid domain (or moiety) linked to the analyte-binding domain (or moiety) of the probe, and signal generation involves interactions between the nucleic acid moiety and / or further functional moieties carried by the other probes. Signal generation therefore depends on interactions between the probes (more specifically, via the nucleic acids or other functional moieties / domains carried by them) and therefore only occurs when both of the required two (or more) probes are bound to the analyte, thereby providing improved specificity to the detection system.

[0345] The concept of proximity probing has been developed in recent years and many assays based on this principle are now well known in the art.

[0346] Proximity assays are typically used to assess the proximity of two specific proteins or portions thereof, e.g., proteins that bind to each other, fusion proteins, and / or proteins that are located in close proximity. One such assay, known as proximity ligation assay (PLA) and used in some embodiments of the present disclosure, features two antibodies (raised in different species) bound to targets of interest (see Nature Methods 3, 995-1000 (2006)). A PLA probe, a species-specific secondary antibody with a unique oligonucleotide strand attached, is then allowed to bind to the appropriate primary antibody. If the targets are in close proximity, the oligonucleotide strand of the PLA probe can interact with additional ssDNA and DNA ligase, resulting in cycling and amplification via rolling circle amplification (RCA). When using highly engineered DNA polymerases such as Phi29 DNA polymerase, circular DNA templates can be replicated hundreds to thousands of times longer, resulting in the production of ssDNA molecules hundreds of nanometers to microns in length (see Angewandte Chemie International Edition, 2008, 47, 6330-6337). After amplification, the replicated DNA can be detected via a detection system. A visible signal thus indicates the proximity of the intended target. These assays feature the use of several DNA-antibody conjugates and enzymes such as DNA ligase and DNA polymerase.

[0347] In another embodiment, a dual binder (DB) assay is used, which utilizes a bispecific detection agent consisting of two Fab fragments with fast off-rate kinetics joined by a flexible linker (Van Dieck et al., 2014 Chemistry & Biology Vol. 21(3):357-368). In principle, because a dual binder contains Fab fragments with fast off-rate kinetics, if only one of the Fab fragments is bound to its epitope, the dual binder will be washed away (the simultaneous cooperative binding of both Fab fragments of the dual binder prevents dissociation of the dual binder and results in positive staining / visibility).

[0348] According to another approach, disclosed in International PCT Publication No. 2014 / 139980, which is incorporated herein by reference, proximity assays and tools are described that use a biotin ligase substrate and enzyme to perform proximity assays. The method provides for detection of target molecules and proximity while maintaining the cellular context of the sample. The use of a biotin ligase, such as an enzyme derived from E. coli, and a peptide substrate, such as an amino acid substrate for the enzyme, provides sensitive and specific detection of protein-protein interactions in FFPE samples. Because biotin ligase can efficiently biotinylate an appropriate peptide substrate in the presence of biotin, and the reaction can occur only when the enzyme is in physical contact with the peptide substrate, the biotin ligase and substrate can be separately conjugated to two antibodies that each recognize a target of interest.

[0349] In some embodiments, the level / amount of one or more biomarker proteins and / or their cellular location / distribution may be compared to a reference, which may include a sample from a subject not receiving therapy (e.g., immunotherapy). In some embodiments, the reference may include a sample from the same subject before receiving therapy (e.g., immunotherapy). In some embodiments, the reference may include reference values ​​from one or more samples of other subjects receiving therapy (e.g., immunotherapy). For example, a population of subjects may be treated, and an average, mean, or median value of the level / amount of at least one RTT biomarker and / or their cellular location / distribution may be generated from the population as a whole. A set of samples obtained from diseases with common characteristics (e.g., the same cancer type and / or stage, or exposure to a common therapy) may be studied from a population, such as in a clinical outcome study. This set may be used to derive a reference (e.g., reference number) to which a subject's sample can be compared.

[0350] Certain aspects of the present disclosure relate to measuring the level / amount of one or more RTT biomarkers in a sample. In some embodiments, the sample may contain 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. A tumor sample may contain cancer cells, lymphocytes, leukocytes, stroma, blood vessels, connective tissue, basal lamina, and any other cell types 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 isolate one or more cell types (e.g., leukocytes). In some embodiments, the sample may be used without separating or isolating cell types.

[0351] Tumor samples may be obtained from a subject by any method known in the art, including, but not limited to, biopsy, endoscopy, or surgical procedures. In some embodiments, tumor samples may be prepared by methods such as freezing, fixing (e.g., by using formalin or a similar fixative), and / or embedding in paraffin wax. In some embodiments, tumor samples may be sectioned. In some embodiments, fresh tumor samples (i.e., not prepared by the methods described above) may be used. In some embodiments, tumor samples may be prepared by incubation in a solution to preserve the integrity of mRNA and / or protein.

[0352] In some embodiments, responsiveness to a therapy may refer to any one or more of an increase in survival (including overall survival and progression-free survival), an objective response (including a complete or partial response), or an improvement in signs or symptoms of cancer. In some embodiments, responsiveness may refer to an improvement in one or more factors according to the published RECIST set of guidelines for determining tumor status in cancer subjects, i.e., response, stabilization, or progression. For a more detailed discussion of these guidelines, see Eisenhauer et al. (2009 Eur. J. Cancer 45:228-47), Topalian et al. (2012 N Engl. J. Med. 366:2443-54), Wolchok et al. (2009 Clin. Can. Res. 15:7412-20), and Therasse et al. (2000 J. Natl. Cancer Inst. 92:205-16). A responsive subject may refer to a subject whose cancer shows improvement, for example, according to one or more factors based on the RECIST criteria. A non-responsive subject may refer to a subject whose cancer does not show improvement, for example, according to one or more factors based on the RECIST criteria.

[0353] In some embodiments, traditional response criteria may not be sufficient to characterize the activity of anticancer therapies, which may produce delayed responses that may precede early, overt radiological progression, including the appearance of new lesions. Therefore, modified response criteria have been developed that account for the possible appearance of new lesions and allow for confirmation of radiological progression at subsequent evaluations. Thus, in some embodiments, responsiveness may refer to improvement in one of multiple factors according to immune-related response criteria (irRC). See, e.g., Wolchok et al. (2009, supra). In some embodiments, new lesions are added to the defined tumor burden and are tracked, for example, for radiological progression at subsequent evaluations. In some embodiments, the presence of non-target lesions is included in the assessment of complete response, but not in the assessment of radiological progression. In some embodiments, radiological progression may be determined solely based on measurable disease and / or confirmed by serial evaluations approximately four weeks after the first documented date.

[0354] 6. Subject classification and treatment management The present disclosure extends to methods of selecting or identifying individuals who are suitable candidates for treatment with a therapy (e.g., immunotherapy) for the treatment of a disease (e.g., cancer). Such individuals include subjects who are predicted to be responsive to the therapy and therefore are more likely to benefit from administration of the therapy than other subjects with different characteristics (e.g., non-responsiveness to the therapy). In certain embodiments, suitable candidates are those who are reasonably likely to benefit, or at least sufficiently likely to benefit, from the therapy to justify administering the therapy in light of its risks and side effects. The present disclosure also encompasses methods of selecting or identifying individuals who are suitable candidates for treatment with a therapy (e.g., immunotherapy) for the treatment of a disease (e.g., cancer). Such individuals include subjects who are predicted to be non-responsive or poorly responsive to the therapy and therefore are less likely to benefit from administration of the therapy than other subjects with different characteristics (e.g., responsiveness to the therapy), or are less likely or substantially not likely to benefit from such treatment, for which reason it may be desirable to use a different or additional therapy. In some embodiments, whether a subject is a suitable candidate for treatment with a therapy is determined based on assaying at least one RTT biomarker in a sample obtained from the subject, as described herein.

[0355] In some aspects, described herein are methods for determining the likelihood that a subject in need of treatment for a disease (e.g., cancer) will respond to treatment with a therapy (e.g., immunotherapy), and / or for identifying and / or selecting subjects to receive such treatment, e.g., based on assaying at least one RTT biomarker. In specific embodiments, the therapy is preferably an anti-immune checkpoint inhibitor immunotherapy. The phrase "immune checkpoint inhibitor treatment," also referred to as "immune checkpoint inhibitor treatment," "immune checkpoint inhibitor therapy," or "immune checkpoint inhibitor therapy," encompasses embodiments relating to treatment with a single immune checkpoint inhibitor and embodiments relating to treatment with a combination of two or more immune checkpoint inhibitors. In some embodiments, immune checkpoint inhibitor treatment involves inhibiting two or more different immune checkpoint pathways using a single agent or using two or more separate agents.

[0356] 7. Kit In other embodiments of the present invention, a therapeutic kit is provided that includes a SETDB1 bicycle peptidomimetic and an anti-cancer spray. In some embodiments, the therapeutic kit further includes a package insert containing instructions for co-administering the SETDB1 bicycle peptidomimetic and an anti-cancer agent to treat T cell dysfunction, or to enhance immune function (e.g., immune effector function, T cell function, etc.) in an individual with cancer, or to treat or delay the progression of cancer, or to treat an infection in an individual. In some embodiments, the anti-cancer agent includes 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).

[0357] In some embodiments, the SETDB1 peptide and, optionally, the chemotherapeutic agent are in the same container or separate containers. Suitable containers include, for example, bottles, vials, bags, and syringes. The containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and a label on or associated with the container can provide instructions for use. The kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructional materials for use. In some embodiments, the kit further includes one or more other agents (e.g., chemotherapeutic agents and anti-neoplastic agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes.

[0358] In other embodiments of the present invention, diagnostic kits for determining expression of biomarkers are provided, comprising reagents that allow for the detection and / or quantification of the biomarkers. Such reagents include, for example, a compound or substance, or a set of compounds or substances, that allow for the quantification of the biomarkers. In certain embodiments, the compound, substance, or set of compounds or materials allows for the determination of the expression level of a gene (e.g., a T cell function biomarker gene) and includes, but is not limited to, extraction of RNA material, determination of the level of corresponding RNA, etc., primers for the synthesis of corresponding cDNA, primers for amplification of DNA, and / or probes that can specifically hybridize to the RNA (or corresponding cDNA) encoded by the gene, TaqMan probes, proximity assay probes, ligases, antibodies, etc.

[0359] The kit may also optionally include appropriate reagents for detection, such as labels, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, etc. For example, a nucleic acid-based detection kit may include (i) a T cell function biomarker polynucleotide (which may be used as a positive control), and (ii) a primer or probe that specifically hybridizes to the biomarker polynucleotide. It may also include enzymes suitable for amplifying nucleic acids, including various polymerases (e.g., reverse transcriptase, Taq, Sequenase™ DNA ligase, etc., depending on the nucleic acid amplification technique used), deoxynucleotides, and buffers to provide the reaction mixture necessary for amplification. Such kits also generally include, in suitable means, separate containers for each individual reagent and enzyme, as well as each primer or probe. Alternatively, a protein-based detection kit may include (i) a biomarker polypeptide (which may be used as a positive control), and (ii) an antibody that specifically binds to the biomarker polypeptide. The kits may also feature various devices (e.g., one or more) and reagents (e.g., one or more) for performing one of the assays described herein, and / or printed instructional materials for using the kit to quantify 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, chip or chamber, microarray, or kit adapted for use with the assays described in the Examples or below, e.g., the RT-PCR or Q PCR techniques described herein.

[0360] In another aspect, the present invention extends to kits for determining the level / amount and / or cellular localization of an RTT biomarker disclosed herein, comprising reagents that allow for detection and / or quantification of the biomarker. Such reagents include, for example, a compound or substance, or a set of compounds or substances, that allow for quantification of the biomarker. In certain embodiments, the compound, material, or set of compounds or materials allows for determination of the level of the biomarker and includes, but is not limited to, extraction of RNA material, corresponding RNA, etc., primers for synthesis of corresponding cDNA, primers for amplification of DNA, and / or probes that can specifically hybridize to RNA encoded by a gene (or corresponding cDNA), TaqMan probes, proximity assay probes, ligases, antibodies, etc.

[0361] The kit may also optionally include appropriate reagents for detection, such as labels, positive and negative controls, wash solutions, blotting membranes, microtiter plates, dilution buffers, etc. 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 the SETDB1 polypeptide, and / or (iii) at least one nuclear binding partner of SETDB1 (e.g., ATF7IP, IMPα). The antigen-binding molecules are suitably detectably labeled. The kit may also feature various devices (e.g., one or more) and reagents (e.g., one or more) for performing one of the assays described herein, and / or printed instructional materials for using the kit to quantify the level / amount of an RTT biomarker. The reagents described herein, which may optionally be associated with a detectable label, may be presented in the form of a microfluidic card, chip or chamber, microarray, or kit adapted for use with the assays described herein.

[0362] Materials suitable for packaging the components of the diagnostic kit may include crystal, plastic (polyethylene, polypropylene, polycarbonate, etc.), bottles, vials, paper, envelopes, etc. Additionally, the kits of the present invention may include instructional materials for the simultaneous, sequential, or separate use of the different components included in the kit. The instructional materials may be in the form of printed material or may be in the form of an electronic support capable of storing instructions so as to be readable by a subject, such as an electronic storage medium (magnetic disk, tape, etc.), optical medium (CD-ROM, DVD), etc. Alternatively, or in addition, the medium may include an internet address providing the instructional materials.

[0363] In order that the present invention may be readily understood and put into practice, certain preferred embodiments will now be described by way of the following non-limiting examples. [Example]

[0364] Example 1 Prototypic linear SETDB1 peptide inhibitors 7.1 SETDB1 localizes to the nucleus of cancer cells. We found that three cancer cell lines (H1299, 4T1, and LLC3) exhibited significant nuclear expression of SETDB1 and ATF7IP. Furthermore, there was significant colocalization of SETDB1 and ATF7IP or SETDB1 and IMPα1 in all three cancer cell lines tested (Figure 1). Nuclear localization of epigenetic enzymes is a hallmark of aggressive metastatic cancers. Therefore, we hypothesized that targeting the nuclear axis of the epigenetic enzyme SETDB1 and its major nuclear interaction partner ATF7IP would directly inhibit the mesenchymal, therapy-resistant signature. To do this, we targeted the nuclear localization sequence of SETDB1.

[0365] 7.2 Generation of SETDB1 NLS mimetic peptide inhibitors. In light of the above data, we developed novel SETDB1 NLS-mimetic peptide inhibitors based on the newly identified nuclear localization sequence (NLS) motif of SETDB1. Notably, the NLS region of SETDB1 is highly conserved across species. [Table 5]

[0366] Two SETDB1 nuclear localization sequence (NLS) peptidomimetics were generated: (i) peptide 047_wt, which corresponds to residues of the wild-type full-length human SETDB1 amino acid sequence (including the naturally occurring alanine residue at position 15), and (ii) peptide 047_A15P (set forth 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 may enhance the stability of the peptide and its interaction with its target. Thus, the novel peptide sequence has the following amino acid sequence: Peptide 047_wt Myristoyl-GKKRTKTWHKGTLIAIQTVGPGKKYKV [SEQ ID NO: 64] [ka]

[0367] 7.3 SETDB1 NLS peptide inhibitors suppress cell proliferation. 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.

[0368] Both linear SETDB1 peptide inhibitors (i.e., peptide 047_A15P and peptide 047_wt) can inhibit the growth of the MDA-MB-231 TNBC metastatic cancer cell line with an IC of approximately 20 mM or less (Figure 2A, B). Similarly, the linear SETDB1 peptide inhibitors can inhibit the growth of the RPMI-7951 metastatic melanoma cancer cell line (Figure 2C, D).

[0369] 7.4 SETDB1 NLS peptide inhibitors reduce metastatic markers. Next, we sought to examine the effects of inhibiting SETDB1 nuclear translocation on protein markers of mesenchymal and metastatic cancer. In particular, CSV and SNAIL are both markers of metastatic and invasive cancer. We also examined the direct target, SETDB1. Immunofluorescence analysis of protein expression in MDA-MB-231 TNBC cancer cells demonstrated that both linear peptide inhibitors at different concentrations induced a significant reduction in the levels of mesenchymal metastatic markers CSV and SNAIL, as well as nuclear SETDB1 expression (Figure 3). These data indicate that targeting and inhibiting the SETDB1 nuclear axis directly affects mesenchymal and metastatic markers that mediate cancer progression and metastatic spread.

[0370] Materials and Methods IFA microscopy To examine the SETDB1, ATF7IP, and IMPα1 signatures 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 1% BSA in PBS, and probed with either ATF7IP, IMPα1, or SETDB1. Antibodies were visualized with donkey anti-rabbit AF 488, anti-mouse AF 568, or donkey anti-goat 647. Coverslips were mounted on glass microscope slides using ProLong Glass Antifade reagent (Life Technologies). Protein targets were localized by digital pathology laser scanning microscopy. Single 0.5 μm sections were obtained using an ASI digital pathology microscope with a 100x oil immersion lens running ASI software. The final image was obtained by averaging four consecutive images of the same section. Digital images were analyzed using automated ASI software (Applied Spectral Imaging, Carlsbad, CA) to automatically determine the distribution and intensity with automatic thresholding and background correction of mean nuclear fluorescence intensity (NFI), allowing for specific targeting of the expression of the protein of interest. Digital images were also analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA) to determine total cellular fluorescence or cell surface-only fluorescence for non-permeabilized cells. Digital images were analyzed using ImageJ software (ImageJ, NIH, Bethesda, MD, USA) to determine either total nuclear fluorescence intensity (TNFI) or total cytoplasmic fluorescence intensity (TCFI). Pearson coefficient correlation (PCC) was calculated for each antibody pair using ImageJ software with automatic thresholding and manual selection of a region of interest (ROI) specific to the cell nucleus. PCC values ​​range as follows: -1 = reciprocal of colocalization, 0 = no colocalization, and +1 = complete colocalization. Significant differences between data sets were determined using the Mann-Whitney non-parametric test (GraphPad Prism, GraphPad Software, San Diego, CA).

[0371] Using this methodology, we also investigated the signatures of SETDB1, CSV, and SNAIL in MDA-MB231 TNBC cells.

[0372] Cell culture method All breast cancer cell lines used were procured from ATCC. MDA-MB-231 or MDB-MB-231-Br cell lines were maintained and cultured in DMEM (Invitrogen) supplemented with 10% FBS, 2 mM L-glutamine, and 1% PSN. MCF-7 cells were stimulated with 1.29 ng / ml phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich) or 5 ng / ml recombinant TGF-β1 (R&D Systems) for 60 hours. For inhibitor studies, 2 × 10 cells were cultured. 5 Cells were seeded in 2 mL of complete medium in 6-well plates and incubated overnight at 37°C / 5% CO2. Cells were treated with SETDB1 linear peptide. For microscopy, 4 × 10 4 Cells were seeded onto coverslips and treated with inhibitors as described above. At each time point, coverslips were washed, fixed with 3.7% formaldehyde (Sigma), and stored at 4°C until processing.

[0373] Cell viability assay - MDA-MB2321 and RPMI-7951 MDA-MB-231 or RPMI-7951 cell lines were cultured at 4 × 10 3Cells / well were seeded into 96-well flat-bottom tissue culture plates in a fi...

Claims

1. A method for preventing or reducing nuclear localization of a SETDB1 polypeptide, comprising contacting the SETDB1 polypeptide and / or an IMPα polypeptide with an agent that inhibits the 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 of treating or preventing cancer in a subject, 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 present in the cell nucleus.

5. 5. The method of 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 IMPα polypeptide.

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

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

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

11. 11. The method of claim 10, wherein said proteinaceous molecule comprises, consists of or consists essentially of an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 6, or a sequence having at least 85% sequence identity to a sequence set forth in any one of SEQ ID NOs: 1 to 6.

12. 12. The method of claim 10 or 11, wherein said proteinaceous molecule comprises, consists of or consists essentially of the amino acid sequence set forth 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. 14. The method of claim 13, wherein the additional cancer therapy is a chemotherapeutic agent.

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

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

17. 17. The method of any one of claims 1 to 16, wherein said proteinaceous 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 said proteinaceous molecule is a fragment of a nuclear localization sequence (NLS).

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

20. 20. The method of claim 19, wherein the proteinaceous molecule is distinguished 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 206-232 of the full-length human SETDB1 protein.

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

22. 22. The method of claim 21, wherein the proteinaceous molecule is distinguished 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 206-232 of SETDB1.

23. 1. An isolated or purified proteinaceous molecule represented by formula I, GKKRX １ KX ２ WHX ３ GTLIX ４ IQTVGPGKKX ５ KVK During the ceremony, Z １ and Z ２ is independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween), and a protecting moiety; X １ is selected from Thr, Arg, and modified forms thereof; X ２ is selected from Thr, Leu, and modified forms thereof; X ３ is selected from Lys, Gly, and modified forms thereof; X ４ is selected from Ala, Pro, and modified forms thereof; X ５ is selected from Tyr, Lys, and modified forms thereof.

24. Z １ 24. The proteinaceous molecule of claim 23, wherein is absent.

25. Z ２ 25. The proteinaceous molecule of claim 23 or 24, wherein is absent.

26. X １ The proteinaceous molecule according to any one of claims 23 to 25, wherein is Thr.

27. X ２ The proteinaceous molecule according to any one of claims 23 to 26, wherein is Thr.

28. X ３ The proteinaceous molecule according to any one of claims 19 to 23, wherein is Lys.

29. X ４ The proteinaceous molecule of any one of claims 19 to 24, wherein is Ala.

30. X ５ The proteinaceous molecule according to any one of claims 19 to 25, wherein is Tyr.

31. X １ The proteinaceous molecule according to any one of claims 19 to 21, wherein is Arg.

32. X ２ is Leu.

33. X ３ is Gly.

34. X ４ The proteinaceous molecule according to any one of claims 27 to 29, wherein is Pro.

35. X ５ The proteinaceous molecule according to any one of claims 27 to 30, wherein is Lys.

36. 32. The proteinaceous molecule of any one of claims 27 to 31, wherein said proteinaceous molecule of formula I comprises, consists of or consists essentially of an amino acid sequence represented by any one of SEQ ID NOs: 1 or 2. 【Chemical 1】

37. 37. The proteinaceous molecule of any one of claims 27 to 36, wherein said proteinaceous molecule of formula I further comprises at least one membrane-permeable moiety.

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

39. 39. The proteinaceous molecule of claim 38, wherein the membrane-permeable moiety is a myristoyl group.

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

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

42. 1. A SETDB1 bicyclic peptidomimetic comprising: a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences; and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold, wherein the SETDB1 bicyclic peptidomimetic has an amino acid sequence of formula (II): Z １ X １ C １ GKKRTKTWHC ２ KGTLIAIQTVGX ２ GC ３ KKYKVKZ ２ Formula (II) or a modified derivative or a pharmaceutically acceptable salt thereof, During the ceremony, C １ , C ２ , and C ３ represent the first, second and third cysteine ​​residues, respectively; Z １ and Z ２ is independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween), and a protecting moiety; X １ is absent or is alanine; X ２ is selected from any non-polar / neutral amino acid residue (e.g., proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

43. X １ 43. The SETDB1 bicyclic peptidomimetic of claim 42, wherein is absent.

44. X ２ is selected from proline or leucine.

45. X ２ The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 44, wherein is proline.

46. Z １ The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 45, wherein is absent.

47. Z ２ The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 46, wherein is absent.

48. 48. The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 47, wherein the peptide binds to importin.

49. The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 48, wherein the peptidomimetic prevents or disrupts the complex between SETDB1 and importin.

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

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

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

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

54. The modified derivatives may include N-terminal and / or C-terminal modifications, 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 spacer groups, replacement of one or more oxidation-resistant 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, etc.

54. The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 53, comprising one or more modifications selected from: substitution of one or more amino acid residues with amino acid residues, N-alkylation of one or more aminide bonds in the bicyclic peptide ligand, replacement of one or more peptide bonds with surrogate bonds, peptide backbone length modification, replacement of the hydrogen on the α-carbon of one or more amino acid residues with another chemical group, and post-synthetic biorthogonal modification of amino acids such as cysteine, lysine, glutamine and tyrosine with suitable amine-, thiol-, carboxylic acid- and phenol-reactive reagents.

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

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

57. 57. The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 56, wherein said modified derivative comprises replacing one or more amino acid residues with one or more non-natural amino acid residues.

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

59. 59. The SETDB1 bicyclic peptidomimetic of any one of claims 42 to 58, wherein substantially all of the amino acids, except for glycine, are D-amino acids.

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

61. 61. The SETDB1 bicyclic peptidomimetic of any one of claims 42-60, wherein all of the amino acids are in retro-inverso form.

62. 62. The SETDB1 bicyclic peptidomimetic of claim 61, having the amino acid sequence set forth in SEQ ID NO: 11 (vkykkcGpgvtqisiltGkchwtktrkkcG).

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

64. 64. A pharmaceutical composition comprising the SETDB1 bicyclic peptidomimetic of any one of claims 42 to 63 in combination with one or more excipients.

65. 1. A method of reducing nuclear localization of SETDB1 in a SETDB1 overexpressing cell, comprising treating the cell with a SETDB1 bicyclic peptidomimetic having the amino acid sequence: Z １ X １ C １ GKKRTKTWHC ２ KGTLIAIQTVGX ２ GC ３ KKYKVKZ ２ Formula (II) or a modified derivative or a pharmaceutically acceptable salt thereof, During the ceremony, C １ , C ２ , and C ３ represent the first, second and third cysteine ​​residues, respectively; Z １ and Z ２ is independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween), and a protecting moiety; X １ is absent or is alanine; X ２ is selected from any non-polar / neutral amino acid residue (e.g., proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

66. X １ 66. The SETDB1 bicyclic peptidomimetic of claim 65, wherein is absent.

67. X ２ is selected from proline or leucine.

68. X ２ The SETDB1 bicyclic peptidomimetic of any one of claims 65 to 67, wherein is proline.

69. Z １ The SETDB1 bicyclic peptidomimetic of any one of claims 65 to 68, wherein is absent.

70. Z ２ The SETDB1 bicyclic peptidomimetic of any one of claims 65 to 69, wherein 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. 72. The method of any one of claims 65 to 71, wherein the molecular scaffold is 1,3,5-(tribromomethyl)benzene, or TBAB.

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

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

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

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

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

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

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

80. 80. The method of claim 78 or 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. 81. The method of any one of claims 65 to 80, further comprising administering at least one additional cancer therapy.

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

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

84. 1. A SETDB1 bicyclic peptidomimetic having the following amino acid sequence for use in therapy: Z １ X １ C １ GKKRTKTWHC ２ KGTLIAIQTVGX ２ GC ３ KKYKVKZ ２ Formula (III) or a modified derivative or a pharmaceutically acceptable salt thereof, During the ceremony, C １ , C ２ , and C ３ represent the first, second and third cysteine ​​residues, respectively; Z １ and Z ２ is independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween), and a protecting moiety; X １ is absent or is alanine; X ２ is selected from any non-polar / neutral amino acid residue (e.g., proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

85. X １ 85. The composition of claim 84, wherein is absent.

86. X ２ 86. The composition of claim 84 or 85, wherein is selected from proline or leucine.

87. X ２ The composition of any one of claims 84 to 86, wherein is proline.

88. Z １ The composition of any one of claims 84 to 87, wherein is absent.

89. Z ２ The composition of any one of claims 84 to 88, wherein is absent.

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

91. 1. A SETDB1 bicyclic peptidomimetic having the following amino acid sequence for use in the treatment of cancer: Z １ X １ C １ GKKRTKTWHC ２ KGTLIAIQTVGX ２ GC ３ KKYKVKZ ２ Formula (III) or a modified derivative or a pharmaceutically acceptable salt thereof, During the ceremony, C １ , C ２ , and C ３ represent the first, second and third cysteine ​​residues, respectively; Z １ and Z ２ is independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween), and a protecting moiety; X １ is absent or is alanine, X ２ is selected from any non-polar / neutral amino acid residue (e.g., proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

92. X １ 92. The composition of claim 91, wherein is absent.

93. X ２ 93. The composition of claim 91 or 92, wherein is selected from proline or leucine.

94. X ２ The composition of any one of claims 91 to 93, wherein is proline.

95. Z １ The composition of any one of claims 91 to 94, wherein is absent.

96. Z ２ The composition of any one of claims 91 to 95, wherein is absent.

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

98. 1. A bicyclic peptide having the amino acid sequence: Z １ X １ C １ GKKRTKTWHC ２ KGTLIAIQTVGX ２ GC ３ KKYKVKZ ２ Formula (III) or a modified derivative or a pharmaceutically acceptable salt thereof, During the ceremony, C １ , C ２ , and C ３ represent the first, second and third cysteine ​​residues, respectively; Z １ and Z ２ is independently absent or independently selected from at least one of a proteinaceous moiety comprising from about 1 to about 50 amino acid residues (and all integer residues therebetween), and a protecting moiety; X １ is absent or is alanine, X ２ is selected from any non-polar / neutral amino acid residue (e.g., proline, leucine, alanine, glycine, isoleucine, methionine, phenylalanine, tryptophan, valine, and norleucine).

99. X １ The use of claim 98, wherein is absent.

100. X ２ 100. The use of claim 98 or 99, wherein is selected from proline or leucine.

101. X ２ The use according to any one of claims 98 to 100, wherein is proline.

102. Z １ The use according to any one of claims 98 to 101, wherein is absent.

103. Z ２ The use according to any one of claims 98 to 102, wherein 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 for predicting the likelihood of response to a therapy (e.g., immunotherapy) in a subject, comprising, consisting of, or consisting essentially of analyzing the cellular localization of SETDB1 in cells expressing SETDB1 in the subject, thereby predicting 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 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 the level of SETDB1 in the nucleus of the cell, which indicates an aberrant or abnormal nuclear level of SETDB1 and correlates with the likelihood of increased resistance to the therapy, thereby determining that the subject has an increased likelihood of resistance to the therapy.

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

110. The method of claim 109, comprising detecting a higher level of SETDB1 in the nucleus of the cell compared to a control, thereby determining that the subject has an increased likelihood of 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 of the cell (e.g., nucleus, cytoplasm), thereby determining that the subject has a likelihood of increased 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 compared to a control (e.g., compared to the nucleus of a corresponding normal cell, or compared to the level of SETDB1 outside the nucleus of the subject's cell, such as in the cytoplasm of the cell), which indicates that the subject has a likelihood of increased resistance to the therapy.

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

114. The method of claim 112 or 113, wherein the higher level of SETDB1 in the nucleus of the cell represents a higher level of SETDB1 in the nucleus of the cell than outside the nucleus of the cell (e.g., in the cytoplasm or extranucleus).

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

95.

116. 116. The method of any one of claims 105-115, comprising detecting higher 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), which indicates the subject has an increased likelihood of 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, which indicates a normal nuclear level of SETDB1 and correlates with the likelihood of increased sensitivity to the therapy, thereby determining that the subject has the likelihood of increased sensitivity to the therapy.

118. The method of any one of claims 105 to 108 and 117, comprising detecting a level of SETDB1 in the nucleus of the cell compared to a control (e.g., compared to the nucleus of a corresponding normal cell, or compared to the level of SETDB1 outside the nucleus of the subject's cell), which level indicates a normal nuclear level of SETDB1 and indicates that the patient has a likelihood of increased sensitivity to the therapy.

119. 119. The method of any one of claims 105-108, 117, and 118, comprising detecting the presence of SETDB1 outside the nucleus (e.g., cytoplasm) of the cell, thereby determining that the subject has a likelihood of increased sensitivity to the therapy.

120. The method of any one of claims 105 to 108, 117, 118 and 119, comprising detecting a level of SETDB1 outside the nucleus of the cell compared to a control (e.g., compared to the outside of the nucleus of a corresponding normal cell, or compared to the level of SETDB1 inside the nucleus of the subject's cell), which level indicates a normal extracellular level of SETDB1 and indicates that the patient has a likelihood of increased sensitivity to the therapy.

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

122. A method according to any one of claims 119 to 121, wherein the level of SETDB1 outside the nucleus of the cell represents a higher level of SETDB1 outside (e.g., on the surface and / or in the cytoplasm) of the nucleus of the cell than inside the nucleus of the cell.

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

95.

124. 124. The method of any one of claims 105-108 and 117-123, comprising detecting higher levels of extranuclear SETDB1 in more 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), which indicates that the subject has a likelihood of increased sensitivity to the therapy.

125. 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 (e.g., ATF7IP).

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

127. The method of claim 126, comprising detecting a higher level of the complex compared to a control (e.g., a corresponding normal control cell), indicating that the subject has the potential for increased resistance to the therapy.

128. The method of claim 125, comprising detecting a level of the complex in the nucleus compared to a control (e.g., a corresponding normal control cell), which level indicates a normal level of the complex and indicates that the subject has the potential for increased sensitivity to the therapy.

129. 129. A method for stratifying a subject as likely to be a responder or non-responder to a therapy (e.g., immunotherapy), comprising, consisting of, or consisting essentially of, analyzing the cellular localization of SETDB1 in a sample from the subject as defined in any one of claims 105 to 128 to determine whether the subject has a likelihood of increased sensitivity or resistance to the therapy, thereby stratifying the subject as likely to be a responder or non-responder to the therapy.

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

131. A method for predicting a subject's therapeutic outcome from a therapy (e.g., immunotherapy), 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 a likelihood of increased sensitivity or resistance to said therapy, thereby predicting said therapeutic outcome in the patient.

132. A kit for detecting the location of SETDB1 in a cellular location (e.g., cytoplasm or nucleus) of a cell, predicting the likelihood of a cell's response to a therapy (e.g., immunotherapy), determining the likelihood of a subject's resistance to a therapy (e.g., immunotherapy), determining the likelihood of a subject's sensitivity to a therapy (e.g., immunotherapy), stratifying a subject as likely to be a responder or non-responder to a therapy (e.g., immunotherapy), managing treatment of a subject with a therapy (e.g., immunotherapy), monitoring disease in a subject after treatment with a therapy, determining the disease status of a subject, and / or determining the immune status of a subject, wherein the kit comprises, consists of, or consists 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 core binding partner of SETDB1 (e.g., ATF7IP).

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

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

136. 10. A composition, method or kit according to any one of the preceding claims, wherein all of the amino acids are of the D isoform (ie vkykkcGpgvtqisiltGkchwtktrkkcG) and in retro-inverso form.

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