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  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
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  • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
  • C07K16/30—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
  • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
  • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
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  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/10—Type of nucleic acid
  • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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  • C12N2310/00—Structure or type of the nucleic acid
  • C12N2310/50—Physical structure
  • C12N2310/53—Physical structure partially self-complementary or closed
  • C12N2310/531—Stem-loop; Hairpin

Definitions

• Table 1 submitted herewith as “Table_l_CRISPR_Positive.txt”, created June 15, 2020 and 348,531 bytes in size
• Table 2 submitted herewith as “Table_2_CRISPR_Negative.txt”, created June 15, 2020 and 292,318 bytes in size
• Table 3 submitted herewith as “Table_3_ORF_Positive.txt”, created June 12, 2020 and 581,299 bytes in size
• Table 4 submitted herewith as
• HLA I or MHC I HLA class I
• PSMB8/LMP2, PSMB9/LMP7 peptide processing
• TAPI peptide transport from the cytosol to the ER
• B2M-HLA I heavy chain HLA-A, HLA-B, and HLA-C
• viruses block HLA I heavy chain insertion into the ER (CMV), resist proteasomal degradation (EBV), interfere with TAP (herpesviruses), or modulate trafficking and turnover of HLA molecules (HIV), among other mechanisms.
• CMV CMV
• EBV resist proteasomal degradation
• TAP TAP
• HLA molecules HLA molecules
• MCC Merkel cell carcinoma
• LT Merkel cell polyomavirus
• ST Merkel cell polyomavirus
• IHC immunohistochemistry
• 84% of MCC lesions have been reported to exhibit surface HLA I downregulation or loss, and similar findings have been observed in MCC cell lines.
• HLA I surface expression in MCC also appears to be highly plastic, as it can be upregulated in vitro by interferons or histone deacetylase (HD AC) inhibitors.
• HD AC histone deacetylase
• the present invention is based, at least in part, on the discovery that inhibiting or blocking one or more biomarkers listed in Tables 1-5, such as MYCL or one or more PRC1.1 complex members like PCGF1, BCORL1, and USP7, results in increased expression of MHC class I molecules, such as HLA I molecules, in cancer cells.
• the present invention involves the modulation (e.g ., upregulation or downregulation) of one or more biomarkers listed in Tables 1-5, such as MYCL and/or one or more PRC1.1 complex members (e.g., PCGF1, BCORLl, and USP7) to increase surface expression of MHC class I molecules, such as HLA I molecules, on cancer cells.
• the one or more biomarkers listed in Tables 1-5 such as MYCL and/or one or more PRC1.1 complex members (e.g, PCGF1, BCORLl, and USP7) have been identified as targets that, when modulated, sensitize cancers to immunotherapy.
• MHC class I such as HLA I
• PRC1.1 complex members e.g, PCGF1, BCORLl, and USP7
• MHC class I such as HLA I
• surface expression is reduced relative to a control and that upon inhibiting targets like a PRC.1.1 component polypeptide or MYCL
• MHC class I such as HLA I
• expression is increased, thereby increasing the susceptibility of these cells to immunotherapies.
• One aspect of the invention provides a method of treating a subject afflicted with a cancer comprising administering to the subject a therapeutically effective amount of an agent that modifies the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 1, 2, 3, 4, or 5 or a fragment thereof, and an immunotherapy.
• the agent decreases the copy number, the expression level and/or the activity of one or more biomarkers listed in Table 1 or 4 or a fragment thereof.
• the agent decreases the copy number, the expression level, and/or the activity of MYCL polypeptide and/or a poly comb repressor complex 1.1 (PRC1.1) polypeptide, or polynucleotide encoding the polypeptide.
• PRC1.1 poly comb repressor complex 1.1
• the polycomb repressor complex 1.1 (PRC 1.1) polypeptide is USP7, BCORLl, PCGF1, KDM2B, SKP1, RING1A, RING1B, RYBP, YAF2, and/or BCOR.
• the agent is a small molecule inhibitor, CRISPR single-guide RNA (sgRNA), RNA interfering agent, antisense oligonucleotide, peptide or peptidomimetic inhibitor, aptamer, antibody, or intrabody.
• the RNA interfering agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA).
• the sgRNA comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequences listed in Tables 1-4.
• the agent comprises an intrabody, or an antigen binding fragment thereof, that specifically binds to the one or more biomarkers and/or a substrate of the one or more biomarkers listed in Table 1, 2, 3, 4, or 5.
• the intrabody, or antigen binding fragment thereof is a murine, chimeric, humanized, composite, or human intrabody, or antigen binding fragment thereof.
• the intrabody, or antigen binding fragment thereof is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab’)2, Fab’, dsFv, scFv, sc(Fv)2, and diabody fragments.
• the agent increases the copy number, the expression level and/or the activity of one or more biomarkers listed in Table 2 or 3 or a fragment thereof.
• the agent increases the sensitivity of the cancer cells to an immunotherapy.
• the immunotherapy is administered before, after, or concurrently with the agent.
• the immunotherapy comprises an anti-cancer vaccine and/or vims.
• the immunotherapy is a cell-based immunotherapy, optionally wherein the cell-based immunotherapy is chimeric antigen receptor (CAR-T) therapy.
• the immunotherapy inhibits an immune checkpoint.
• the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7- H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, IDO, CD39, CD73 and A2aR.
• the immune checkpoint is selected from the group consisting of PD-1, PD-L1, and PD-L2, optionally wherein the immune checkpoint is PD-1.
• the one or more biomarker comprises a nucleic acid sequence having at least 95% identity to a nucleic acid sequence listed in Table 5 and/or encodes an amino acid sequence having at least 95% identity to an amino acid sequence listed in Table 5.
• the subject is a mammal.
• the subject is a human, non-human primate, mouse, rat, or domesticated mammal.
• the agent increases the sensitivity of the cancer to the immunotherapy, optionally wherein (i) the immunotherapy is T-cell-mediated and/or (ii) the agent increases the amount of CD8+ T cells in a tumor comprising the cancer cells. In another embodiment, the agent increases the level of MHC-I on the surface of the cancer cells. In another embodiment, the method also comprises administering to the subject at least one additional cancer therapy or regimen. In yet another embodiment, the at least one additional cancer therapy or regimen is administered before, after, or concurrently with the agent and/or the immunotherapy. In yet another embodiment, the agent is administered in a pharmaceutically acceptable formulation. In still another embodiment, the cancer is a neuroendocrine cancer. In still another embodiment, the neuroendocrine cancer is a Merkel cell carcinoma, neuroblastoma, small cell lung cancer, or neuroendocrine carcinoma.
• Another aspect provides a method of increasing major histocompatibility complex expression in a cancer cell, the method comprising contacting the cancer cell with an agent that modulates the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 1,
• the agent that decreases the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 1 or 4.
• the agent decreases the copy number, the expression level, and/or the activity of MYCL polypeptide and/or a polycomb repressor complex 1.1 (PRC1.1) polypeptide, or polynucleotide encoding the polypeptide.
• the polycomb repressor complex 1.1 (PRC1.1) polypeptide is USP7, BCORL1, PCGF1, KDM2B, SKP1, RING1A, RING1B, RYBP, YAF2, and/or BCOR.
• the agent is a small molecule inhibitor, CRISPR single-guide RNA (sgRNA), RNA interfering agent, antisense oligonucleotide, peptide or peptidomimetic inhibitor, aptamer, antibody, or intrabody.
• the RNA interfering agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA).
• the sgRNA comprises a nucleic acid sequence selected from the group consisting of nucleic acid sequence listed in Tables 1-4.
• the agent comprises an intrabody, or an antigen binding fragment thereof, that specifically binds to the one or more biomarkers and/or a substrate of the one or more biomarkers listed in Table 1, 2, 3, 4, or 5.
• the intrabody, or antigen binding fragment thereof is a murine, chimeric, humanized, composite, or human intrabody.
• the intrabody, or antigen binding fragment thereof is detectably labeled, comprises an effector domain, comprises an Fc domain, and/or is selected from the group consisting of Fv, Fav, F(ab’)2, Fab’, dsFv, scFv, sc(Fv)2, and diabodies fragments.
• the agent increases the copy number, the expression level, and/or the activity of one or more biomarkers listed in Table 2 or 3.
• the agent increases the sensitivity of the cancer cells to the immunotherapy.
• the cancer cells are contacted with the immunotherapy before, after, or concurrently with the agent.
• the immunotherapy comprises an anti-cancer vaccine and/or virus.
• the immunotherapy is a cell-based immunotherapy, optionally wherein the cell-based immunotherapy is chimeric antigen receptor (CAR-T) therapy.
• the immunotherapy inhibits an immune checkpoint.
• the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4,
• the immune checkpoint is selected from the group consisting of PD-1, PD-L1, and PD-L2.
• the immune checkpoint is PD-1.
• the biomarker comprises a nucleic acid sequence having at least 95% identity to a nucleic acid sequence listed in Table 5 and/or encodes an amino acid sequence having at least 95% identity to an amino acid sequence listed in Table 5.
• the one or more biomarker is a human, mouse, chimeric, or a fusion biomarker.
• the immunotherapy is (i) T-cell- mediated and/or (ii) the agent increases the amount of CD8+ T cells in a tumor comprising the cancer cells.
• the agent increases the level of MHC class I surface expression in the cancer cells.
• the method further comprises administering to the subject at least one additional cancer therapy or regimen.
• the at least one additional cancer therapy or regimen is administered before, after, or concurrently with the agent and/or the immunotherapy.
• the agent is administered in a pharmaceutically acceptable formulation.
• the cancer cell is a neuroendocrine cancer cell.
• the neuroendocrine cancer cell is a Merkel cell carcinoma, neuroblastoma, small cell lung cancer, or neuroendocrine carcinoma cell.
• Another aspect of the present invention is a method of identifying a subject afflicted with, or at risk for developing, a cancer that can be treated by modulating the copy number, amount, and/or activity of at least one biomarker listed in Table 1, 2, 3, 4, or 5, the method comprising detecting an increased or decreased level of major histocompatibility complex (MHC) class I expression in a cell from the subject relative to a control, thereby identifying the subject afflicted with, or at risk of developing, a cancer that can be treated by modulating the copy number, amount, and/or activity of at least one biomarker listed in Table 1, 2, 3, 4, or 5, optionally wherein a biological sample comprising the cell from the subject is obtained from the subject.
• MHC major histocompatibility complex
• the agent decreases the copy number, amount, and/or activity of at least one biomarker listed in Table 1 or 4.
• the method also comprises recommending, prescribing, or administering to the identified subject an agent that inhibits the at least one biomarker listed in Table 1 or 4.
• the agent increases the copy number, amount, and/or activity of at least one biomarker listed in Table 2 or 3.
• the method further comprises recommending, prescribing, or administering to the identified subject an immunotherapy.
• the immunotherapy comprises an anti cancer vaccine, an anti-cancer virus, and/or a checkpoint inhibitor.
• the method further comprises recommending, prescribing, or administering to the subject a cancer therapy selected from the group consisting of targeted therapy, chemotherapy, radiation therapy, and/or hormonal therapy.
• the control comprises a sample derived from a cancerous or non-cancerous sample from either the patient or a member of the same species to which the patient belongs.
• the control is a known reference value.
• the cancer is a neuroendocrine cancer.
• the neuroendocrine cancer is a Merkel cell carcinoma, neuroblastoma, small cell lung cancer, or neuroendocrine carcinoma.
• a method for predicting the clinical outcome of a subject afflicted with a cancer expressing one or more biomarkers listed in Table 1, 2, 3, 4, or 5 or a fragment thereof to treatment with an immunotherapy comprising a) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1, 2, 3, 4, or 5 in a subject sample; b) determining the copy number, amount, and/or activity of the at least one biomarker in a control having a good clinical outcome; and c) comparing the copy number, amount, and/or activity of the at least one biomarker in the subject sample and in the control; wherein the presence of, or an insignificant change in the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1, 2, 3, 4, or 5 in the subject sample as compared to the copy number, amount and/or activity in the control, is an indication that the subject has a poor clinical outcome.
• Another aspect provides a method for monitoring the treatment of a subject having or suspected of having cancer with an agent that decreases the copy number and/or amount and/or inhibits the activity of at least one biomarker listed in Table 1 or 4 and an immunotherapy, the method comprising detecting a change or no change in the level of MHC class I expression in a sample derived from the subject at a first time point and the level of MHC class I expression in a sample derived from the subject at a subsequent time point, thereby monitoring the treatment of the subject.
• Yet another aspect provides a method for monitoring the treatment of a subject having or suspected of having cancer with an agent that increases the copy number and/or amount and/or inhibits the activity of at least one biomarker listed in Table 2 or 3 and an immunotherapy, the method comprising detecting a change or no change in the level of MHC class I expression in a sample derived from the subject at a first time point and the level of MHC class I expression in a sample derived from the subject at a subsequent time point, thereby monitoring the treatment of the subject.
• a method for assessing the efficacy of an agent that decreases the copy number, amount, and/or the activity of at least one biomarker listed in Table 1 or 4 in a subject, the method comprising detecting in a subject sample at a first time point a change or no change in the copy number, amount, and/or or activity of at least one biomarker listed in Table 1 or 4 relative to a subsequent time point, wherein a decrease in the copy number, amount, and or activity of at least one biomarker listed in Table 1 or 4 indicates the agent is effective.
• Another aspect provides a method of assessing the efficacy of an agent that increases the copy number, amount, and/or the activity of at least one biomarker listed in Table 2 or 3 in a subject, the method comprising detecting in a subject sample at a first time point a change or no change in the copy number, amount, and/or or activity of at least one biomarker listed in Table 2 or 3 relative to a subsequent time point, wherein a decrease in the copy number, amount, and or activity of at least one biomarker listed in Table 2 or 3 indicates the agent is effective.
• the subject has undergone treatment, completed treatment, and/or is in remission for the cancer.
• treatment comprises administering the agent to the subject.
• the first and/or the subsequent sample comprises ex vivo or in vivo samples.
• the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject.
• the sample comprises cells, serum, peritumoral tissue, and/or intratumoral tissue obtained from the subject.
• the cancer or cancer cell is a neuroendocrine cancer.
• the neuroendocrine cancer is a Merkel cell carcinoma, neuroblastoma, small cell lung cancer, or neuroendocrine carcinoma.
• the cancer or cancer cell is in an animal model of the cancer.
• the animal model is a mouse model.
• the cancer is in a mammalian subject.
• the mammalian subject is a mouse or a human.
• the mammal is a human.
• biomarkers of the present invention such as those listed in Tables 1, 4, and 5, for which inhibition in combination with an immunotherapy, results in a synergistic therapeutic benefit for treating cancers that is unexpected given the lack of such benefit observed for the immunotherapy alone
• certain biomarkers clearly described herein, especially at Tables 1, 4, and 5, whose promoted expression rather than inhibition in combination with an immunotherapy e.g ., identified as being enriched in the sgRNA screen rather than being depleted
• results in a synergistic therapeutic benefit for treating cancers are readily apparent.
• any aspect and embodiment described herein and above can use such biomarkers and their promoted expression in diagnostic, prognostic, therapeutic, etc. applications regarding immunotherapy and cancers.
• a method of killing cancer cells comprising contacting the cancer cells with an agent that promotes rather than inhibits the copy number, the expression level, and/or the activity of one or more such biomarkers listed in Tables, 1, 4, or 5, or a fragment thereof, in combination with an immunotherapy, is provided.
• a method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from promoting the copy number, amount, and/or activity of such at least one biomarker listed in Table 1, 4, or 5 comprising a) obtaining a biological sample from the subject; b) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1; c) determining the copy number, amount, and/or activity of the at least one biomarker in a control; and d) comparing the copy number, amount, and/or activity of the at least one biomarker detected in steps b) and c); wherein the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 1, 4, or 5 in the subject sample relative to the control copy number, amount, and/or activity of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from
• biomarkers of the present invention such as those listed in Tables 2 and 3, for which promotion in combination with an immunotherapy, results in a synergistic therapeutic benefit for treating cancers that is unexpected given the lack of such benefit observed for the immunotherapy alone
• certain biomarkers clearly described herein, especially at Tables 2 and 3, whose inhibited expression rather than promotion in combination with an immunotherapy e.g ., identified as being enriched in the sgRNA screen rather than being depleted
• results in a synergistic therapeutic benefit for treating cancers are readily apparent.
• any aspect and embodiment described herein and above can use such biomarkers and their promoted expression in diagnostic, prognostic, therapeutic, etc. applications regarding immunotherapy and cancers.
• a method of killing cancer cells comprising contacting the cancer cells with an agent that promotes rather than inhibits the copy number, the expression level, and/or the activity of one or more such biomarkers listed in Tables, 2 or 3, or a fragment thereof, in combination with an immunotherapy, is provided.
• a method of determining whether a subject afflicted with a cancer or at risk for developing a cancer would benefit from promoting the copy number, amount, and/or activity of such at least one biomarker listed in Table 2 or 3 comprising a) obtaining a biological sample from the subject; b) determining the copy number, amount, and/or activity of at least one biomarker listed in Table 1; c) determining the copy number, amount, and/or activity of the at least one biomarker in a control; and d) comparing the copy number, amount, and/or activity of the at least one biomarker detected in steps b) and c); wherein the absence of, or a significant decrease in, the copy number, amount, and/or activity of, the at least one biomarker listed in Table 2 or 3 in the subject sample relative to the control copy number, amount, and/or activity of the at least one biomarker indicates that the subject afflicted with the cancer or at risk for developing the cancer would benefit from promoting
• FIG. 1A - FIG. IS show the generation of patient-derived MCC lines that exhibit classic features of MCC and recapitulate their corresponding original tumors.
• FIG. 1A is a graph showing cell culture media optimization in the MCC-336 cell line. Cells were counted at Day 0, 4, and 7.
• FIG. IB are images showing Immunohistochemistry of MCC cell lines with stains for MCC markers SOX2 and CK20.
• MCC-277 One representative virus-positive (MCC-277) and virus negative (MCC-350) line are shown.
• FIG. 1C comprises images showing immunohistochemistry for 9 of the newly generated MCC cell lines, with staining for classical MCC markers SOX2 and CK20.
• FIG. ID shows virpanel data
• FIG. IE is a CoMut plot displaying the top 50 most frequently mutated genes across 7 MCC tumor and cell line pairs.
• FIG. IF shows clustering of MCC tumors and cell lines by mutational profiles. Similarity scores were calculated based on the concordant presence or absence of mutations between tumor and cell line on a 0 to 1 scale, where a score of 1 indicates identical profiles.
• FIG. 1G shows unsupervised hierarchical clustering of RNA-seq samples, comprised of 9 MCC patient tumors and corresponding cell lines. Heatmaps were constructed using a distance matrix on variance-stabilizing transformed expression values. Top track indicates quantification of transcript reads mapping to the MCPy V genome.
• FIG. 1H is a graph showing pairwise Spearman correlations based on RNA-Seq data for corresponding tumor-cell line pairs, tumor-tumor pairs, cell line-cell line pairs, and all other pairings.
• FIG. II is a diagram showing translated unannotated ORFs that can be translated by Ribo-seq.
• FIG. 1 J shows flow cytometry results (left y-axis) for HLA-I surface expression across 11 MCC lines, both at baseline (pink bars) and in response to IFN-y (red bars), compared to isotype control (white bars).
• the overlaid black line plot indicates the percentage of tumor cells that stained positive for HLA-I by IHC of the corresponding original tumor (right y-axis).
• FIG. IK shows IHC staining of 4 original MCC tumor biopsies for HLA class I, HLA-DR, CD4, and CD8.
• FIG. 1L shows flow cytometry experiments measuring HLA- ABC surface expression (W6/32 antibody) in the MCC-301 line and two established MCPyV+ lines, MKL-1 and WaGa.
• FIG. 1M comprises graphs showing the effect of type I and type II interferons on surface MHC I expression in MCC by flow cytometry.
• 5 xl05 MCC cells were treated with the indicated doses of IFNa2b, PTN ⁇ b, or IFNy for 24 hours.
• Representative histogram plots show cells stained with anti-HLA class I or isotype antibodies. The experiment was performed in the MCPyV- line MCC-290 (left) and the MCPyV+ line MCC-301 (right).
• FIG. IN is a graph showing flow cytometry assessment of HLA-DR expression in all 11 MCC lines, both at baseline (light pink) and after IFN-g treatment (red).
• FIG lO shows IHC of MCC tumor archival samples.
• MCC cell lines were derived from post-treatment samples.
• Representative IHC images of two HLA I-low tumors, MCC-301 and MCC-336 are on the right, stained for HLA class I (brown) with SOX2 co-stain (red) to identify MCC cells.
• FIG. IP shows growth curves of newly generated MCC cell lines. One million cells were seeded in triplicate on Day 0 and counted at Day 2 and Day 4.
• FIG. IQ shows IHC images of parental MCC tumors, stained for HLA class I (brown) with SOX2 co-stain (red) to identify MCC cells.
• FIG. IS shows representative multiplex immunofluorescence images of MCC FFPE tumor tissue sections.
• Probes include DAPi nuclear (blue), CDS (white), FOXP3 (yellow), PD-i (orange), PD-L1 (green), and SOX2 (magenta).
• FIG. 2A - FIG. 20 illustrate that transcriptional suppression of multiple class I pathway genes and NLRC5 alterations underlie the loss of MHC I surface expression in this panel of MCC lines.
• FIG. 2A comprises RNA-seq heatmaps of class I antigen presentation gene expression in MCC lines and controls. Counts were normalized by a set of housekeeping genes (Eisenberg and Levanon 2013), using the RUV method (Risso et al. (2014) Nature 32 (9): 896-902. The middle heatmap shows unsupervised clustering by Euclidean distance of the MCC cell line panel, both at baseline and after IFN-g treatment.
• the left heatmap is a reference heatmap of previously established MCC lines MKL-1 and WaGa.
• the right heatmap is a reference heatmap of normal epidermal keratinocytes and dermal fibroblasts.
• FIG. 2B is a volcano plot of differentially expressed genes (genes below FDR cutoff 0.01 are shown in yellow) between baseline and IFN-y-treated MCC cell lines. Differential expression analysis was performed using DESeq2, and negative LFC indicates increased expression in -iTFN-g samples. IFN genes are highlighted in red.
• FIG. 2C shows unsupervised clustering of proteomic expression values for class I pathway genes in 4 of the MCC lines, at baseline and after IFN-g treatment.
• FIG. 2D is a proteomics heatmap depicting the relative expression of key IFN-g pathway components in 4 of the MCC lines, both at baseline and after IFN-g treatment.
• FIG. 2E comprises graphs summarizing a targeted analysis of normalized STAT1 peptide counts (left) and STAT-Y701y phosphosite counts (right) between untreated and IFN-y-treated cell lines.
• FIG. 2F shows scRNA-seq data from MCC-336 (MCPyV + ) and -350 (MCPyV) fresh tumor samples.
• UMAP uniform manifold approximation and projection
• FIG. 2G comprises charts of scRNA-seq expression of MCC markers SOX2,
• FIG. 2H comprises graphs showing scRNA-seq expression of additional HLA class I genes across all clusters (clusters 0-5: MCC; cluster 6: immune cells).
• FIG. 21 shows NLRC5 copy number loss is common in MCC. Log2 copy number ratios are displayed for class I antigen presentation genes (left) and for chromosome 16 (right), where NLRC5 is located. Red and blue signify copy number gain and loss, respectively.
• FIG. 2J comprises graphs showing Pearson correlation plots between class 1 genes and NLRC5 generated from RNA-seq data from the 11 MCC cell lines. P-values not adjusted for multiple comparisons.
• FIG. 2K shows unsupervised clustering of promoter-averaged methylation values of class I pathway genes in 8 of the MCC lines, generated from whole-genome bisulfite sequencing.
• FIG. 2L is a graph of ATAC-seq normalized read coverage in 8 of the MCC lines, focusing on the TSS +/- 5kb of class I genes and the housekeeping gene TBP. All datasets including those from GEO and ENCODE were normalized by RPKM (see Methods).
• FIG. 2M is a graph comparing the percentage of peaks falling within the union DNase- 1 hypersensitivity sites (DHS) between the MCC lines and datasets on Cistrome DB. Comparison to the median level (left) as well as the full distribution (right) are shown.
• DHS DNase- 1 hypersensitivity sites
• FIG. 2N is a graph comparing total, 5-fold and 10-fold enriched peak numbers across MCC lines with the median of Cistrome DB datasets. Dashed line represents peak number of 500.
• FIG. 20 is graph showing peak conservation across samples.
• FIG. 3A - FIG. 3N illustrate that IFNy increases and alters the HLA peptidome.
• FIG. 3A comprises graphs showing the frequency of peptides predicted to bind to each HLA allele in tumor and cell line samples for MCC-277, -290, and -301.
• FIG. 3B shows the number of detected peptides presented on HLA Class I is low for primary tumor and tumor derived cell lines but increased after IFNY treatment.
• FIG. 3C is heatmap showing the correlation of peptide sequences for tumor, cell line and cell line +IFNy in motif space.
• FIG. 3D comprises pie charts showing the allele distribution of peptides detected in tumor and cell line of MCC 2314.
• FIG. 3E comprises graphs showing motif changes for tumor, cell line and cell line + IFNy samples of MCC290 and 301. This Figure also shows 9mer motif changes between untreated and IFN-y-treated samples for MCC-290 (MCPyV) and -301 (MCPyV + ) cell lines.
• FIG. 3F comprises graphs showing the allele distribution of peptides detected in cell lines +/- IFNy. HLA allele distribution of presented peptides detected in cell lines is shown at baseline and after IFN-g treatment. Each HLA allele is represented by a different color.
• FIG. 3G comprises graphs showing the increase of peptide presentation per HLA type upon IFN treatment.
• the Figure shows a summary of changes in peptides presented per HLA gene upon IFN-g treatment across all MCC lines analyzed for HLA-A (left), -B (middle), and -C (right).
• FIG. 3H comprises graphs showing the allele distribution of peptides detected in cell lines +/- IFNy.
• FIG. 31 comprises graphs showing the increase of peptide presentation per HLA type upon IFN treatment.
• FIG. 3J is a readout of the mass spectrum of peptide representing Large T antigen in MCC367.
• FIG 3K shows the number of detected peptides presented on HLA-I in MCC lines at baseline (gray bar) and after IFN-g treatment (red bar).
• CL cell line (left).
• FIG. 3L shows IFN-g secretion by peripheral blood mononuclear cells (PBMCs) from patient MCC-367 co-cultured in an ELISpot with DMSO, HIV-GAG negative control peptide, autologous MCC-367 tumor cells, or the Large T antigen-derived peptide identified in the MCC-367 HLA peptidome in panel F.
• PBMCs peripheral blood mononuclear cells
• FIG. 3M shows a schematic representation of immunopeptidome workflow.
• HLA molecules are immunoprecipitated from tumor and cell line material, peptides are eluted from HLA complex and analyzed by LC-MS/MS. After database searching, peptides are assigned to their most likely allele by prediction in HLAthena.
• FIG. 3N shows motif changes of 9mers between baseline cell line and IFN-y-treated cell line samples.
• FIG. 4A - FIG. 4Q illustrate paired genome-scale CRISPR and ORF screens to identify known and novel regulators of MHC class I surface expression in MCC.
• FIG. 4A shows a genome-scale screening workflow: 150 million MCC-301 cells were transduced with library lentivirus (Brunello CRISPR-KO or human ORFeome v8.14) at low multiplicity of infection, and then selected for 3 days with puromycin. Subsequently, cells were stained with an anti-HLA-ABC antibody (W6/32 clone), and MHC I-high and -low populations (top and bottom 10%) were flow cytometrically sorted. Each screen was repeated in triplicate.
• FIG. 4B comprises graphs showing flow cytometric assessment of HLA I surface expression (W6/32 antibody) in MCC-301 cells transduced with the human ORFeome v8.1 library lentivirus, 2 days and 20 days after transduction.
• Controls include MCC-301 cells transduced with a GFP ORF virus, a no-virus control (media added instead), and untransduced cells.
• FIG. 4C is a chart showing the distribution of the log2-normalized construct scores [log2 (construct reads / total reads * 106 + 1)] for each sorted population in FIG. 4F.
• FIG. 4D shows the results for the gain-of-function ORF screen. Genes were ranked according to their log-fold-change enrichment in MHC I-high versus -low populations. Inset: GSEA analysis displaying select gene sets enriched in the ORF positive hits.
• FIG. 4E shows the results for the loss-of-function CRISPR-KO screen.
• Guide RNA ranks based on log-fold-change enrichment in MHC-I-hi versus low populations were input into the STARS algorithm to generate a gene-level ranking of negative (right) and positive (left) hits.
• FIG. 4F shows sorted populations of cells from of the ORF (left) and CRISPR (right) screens.
• FIG. 4G is a graph showing average LFC enrichment of the 3 highest-scoring sgRNAs for USP7, BCORLl, and PCGF1, with the distribution of a set of control non targeting or intergenic sgRNAs shown as a reference.
• FIG. 4H shows flow cytometry for surface HLA-I (W6/32 antibody) in MCC-301 (left) and MCC-277 (right) cells transduced with the indicated individual ORFs.
• FIG. 41 is a scatterplot of gene-level LFCs (average LFC of all constructs) between two replicates of the ORF screen (top) and CRISPR screen (bottom). Notable screen hits are highlighted in red or blue.
• FIG. 4J is a graph summarising flow cytometry results for surface MHC I in MCC- 301 PRC1.1 KO lines.
• MCC-301 cells were transduced with lentivirus containing Cas9 and either control sgRNA or sgRNAs targeting PRC1.1 components BCORLl, PCGF1, or USP7. Cells were selected with puromycin for 3 days, and knockout was confirmed via Sanger sequencing and Western blot or qRT-PCR. Cells were stained with anti-HLA-ABC (W6/32) and analyzed on a BD LSRFortessa. Each condition was repeated in technical triplicate.
• FIG. 4K is a schematic of PRC1.1 components and MYCL, with yellow indicating screen hits and green indicating screen hits that have also been reported to interact with MCPyV viral antigens.
• FIG. 4L comprises a table and a readout of a TIDE analysis of PRC 1.1 KO lines.
• the table shows the percentage of cells with indels in each knockout line was determined using the TIDE software (Brinkman et al. 2014).
• the TIDE tracing is an example analysis of the PCGFl-2 KO line in MCC-301.
• FIG. 4M shows flow cytometry for surface HLA-I in MKL-1 cells transduced with a dox-inducible control shRNA, MYCL shRNA MYCL, or MYCL shRNA with rescue expression of MYCL.
• FIG. 4N shows a RNA-seq volcano plot showing LFC expression in MKL-1 cells expressing a shRNA against MYCL compared to a scrambled control shRNA.
• Class I APM genes with p adj ⁇ 0.05 and log2-fold change (LFC) > 1 are highlighted in red; other notable class I genes are in black.
• FIG. 40 shows RNA-seq volcano plot showing LFC expression in WaGa cells expressing an shRNA against both ST and LT antigens, compared to a scrambled control shRNA.
• Class I APM genes with p adj ⁇ 0.05 and LFC > 1 are highlighted in red; other notable class I genes in black.
• FIG. 4P shows flow cytometry for surface HLA-I in a double guide PCGF1 KO line after IFN-g treatment.
• FIG. 4Q shows western blot quantification of TAPI and TAP2 in MKL-1 cells in response to varying concentrations of IFN-g.
• FIG. 5A - FIG. 5J illustrate that MYCL suppresses HLA I in MCPyV+ MCC and is relevant in MCPyV- MCC and other cancers.
• FIG. 5A is a volcano plot of MYCL shRNA knockdown versus scrambled shRNA control in MCPyV+ MKL-1 cells. Class I genes with p adj ⁇ 0.05 and LFC > 1 are highlighted in red; other notable class I genes in black.
• FIG. 5B shows the enrichment of the GO term GO ANTIGEN BINDING in GSEA analysis of gene upregulated in MKL-1 shMYCL cells relative to a scrambled shRNA control (FIG. 3E).
• FIG. 5C is a volcano plot of pan-T antigen shRNA knockdown versus scrambled control shRNA in MCPyV+ WaGa line.
• Class I genes with p adj ⁇ 0.05 and LFC > 1 are highlighted in red; other notable class I genes in black.
• FIG. 5D shows differential expression analysis of MKL-1 cells transduced with one of two shRNAs against EP400 (shEP400-2 or shEP400-3), compared to a scrambled shRNA control.
• Red indicates HLA-I genes with LFC > 1 and p adj ⁇ 0.01.
• Triangles indicate genes whose padj values were reported as zero by DeSeq2, and subsequently plotted at the lowest non-zero p adj value in the dataset.
• FIG. 5E shows copy number variations in MYC family genes in 4 of the virus negative MCC lines for which whole-genome sequencing was performed. CN gains and losses are shown in red and blue, respectively. Gray indicates no CNV data.
• FIG. 5F shows unsupervised clustering of RNA-seq expression values of class I pathway genes and MYC family genes across all available cancer cell lines in the Cancer Cell Line Encyclopedia. For each cancer type, the median expression value from all cell lines of that cancer classification was used. Color scale is row-min to row-max.
• FIG. 5G comprises heatmaps of an RNA-seq analysis of HLA class I genes and notable screen hits across a cohort of 52 MCC tumors and unsupervised hierarchical clustering heatmap using Pearson correlations.
• Top track tumor purity scores for each tumor, generated by ESTIMATE (Yoshihara et ak, (2013) Nature Communications 4: 2612).
• Figure 5H shows flow cytometry for surface HLA-I in MCC-301 PRC1.1 KO lines (PCGF1, USP7, and BCORLl). Knockout lines were made using either the highest or second-highest scoring sgRNA for each gene. Western blot for PCGF1 (top) and USP7 (bottom) in WT MCC-301, a control MCC-301 line transduced with a non-targeting sgRNA and Cas9, or the indicated knockout line.
• Figure 51 shows RNA-seq volcano plot showing LFC in gene expression in an MCC- 301 PCGF1- KO line compared to MCC-301 transduced with a non-targeting sgRNA and Cas9 control.
• GSEA plot demonstrating enrichment of PRC2 targets within genes upregulated in the PCGF1- KO line.
• Figure 5J shows western blot showing TAPI protein levels in non-targeting control and PCGF1- KO lines at varying IFN-g concentrations.
• FIG. 6A - FIG. 6L illustrate pharmacologic inhibition of PRC1.1 component USP7 upregulates HLA I in MCPyV+ and MCPyV- MCC and mediates MYCL-mediated HLA I suppression.
• FIG. 6A is a genome browser view of USP7 and PCGF1 with ChIP-seq tracks for MAX (red), EP-400 (blue), MCPyV ST antigen (pink), and activating histone marks H3K4me3 and H3K27Ac (black).
• FIG. 6B is genome browser view of BCOR and BCORLl with ChIP-seq tracks for MAX (red), EP-400 (blue), MCPyV ST antigen (pink), and activating histone marks H3K4me3 and H3K27Ac (black).
• FIG. 6C comprises graphs showing that ChIP-qPCR targets the USP7 and PCGF1 promoters, using MKL-1 chromatin immunoprecipitated with either a MAX (left) or EP400 (right) antibody.
• FIG. 6D shows flow cytometry experiments measuring HLA-I surface levels in MCC lines treated with the USP7 inhibitor XL177A or control compound XL177B.
• Y-axis displays MFI (HLA-ABC) in inhibitor-treated cells, normalized to the mean MFI (HLA-ABC) of DMSO-treated cells.
• Sample preparation and flow cytometry analysis was performed in technical triplicate for each condition. ** isE ⁇ 0.01; * isE ⁇ 0.05; n.s. isE > 0.05.
• Left Pearson correlation coefficients and FDRs of the top genes that are co-dependent with USP7, with Poly comb genes highlighted.
• Right Graphical comparison of dependency of USP7 versus Polycomb genes PCGF1 and RING1 in TP53-WT (blue) and TP53-mut cell lines (red).
• FIG. 6F is a GSEA analysis of genes based on their degree of co-dependency with USP7 within TP53-mut cancer cell lines, as determined by Pearson correlations (Fig 6D). Genes exhibiting higher codependency had the highest enrichment for the terms GO HI S T ONE UB IQUITIN ATION and GO_HISTONE_H2A_UBIQUITINATION
• FIG. 6G shows ChIP-qPCR targeting the USP7 and PCGF1 promoters, using MKL-1 chromatin immunoprecipitated with either a MAX (left) or EP400 (right) antibody. Each condition was repeated in triplicate, and p-values were calculated by performing a one-way ANOVA followed by a post hoc Dunnett multiple comparisons test.
• FIG. 6H shows ELLA I flow cytometry to assess the effect of USP7 inhibitors in MKL-1 p53-WT control lines (left) or p53-KO lines (right).
• Cells were treated with 100 nM XL177A (red), XL177B (black), or DMSO (light gray).
• FIG. 6J shows that the frequency of peptides presented on each ELLA allele in MCC- 301 cells treated with XL177A or XL177B, compared to untreated cells.
• FIG. 6K shows a western blot for p53 in 3 MKL-1 p53 KO lines compared to control lines (WT, SCR, AAVS1).
• FIG. 6L shows distribution of cell cycle phases, determined by flow cytometry, of MKL-1 p53 KO lines treated with XL177A, XL177B, or DMSO.
• regulators of one or more biomarkers listed in Tables 1-5 can be used to modulate surface MHC-I expression on cells, modulate immune responses, and augment tumor immunity and responsiveness to immunotherapies.
• (a) decreasing the copy number, expression level, and/or activity of one or more biomarkers listed in Table 1 or Table 4 and/or (b) increasing the copy number, expression level, and/or activity of one or more biomarkers listed in Table 2 or Table 3 results in increased MHC-I expression on cells and increased immune responses with increased responsiveness to immunotherapies, which is useful for treating disorders that would benefit from increased immune responses like cancer, infection, and the like.
• the instant disclosure provides methods of increasing immune responses such as to treat cancers, e.g ., those cancer types otherwise not responsive or weakly responsive to immunotherapies, with a combination of a negative regulator of one or more biomarkers listed in Tables 1-5 and an immunotherapy.
• the present invention provides exemplary RNA interfering agents and small molecules that inhibit such regulators and can be used in the combination therapy and other methods described herein, such as agents that inhibit the function and/or the ability of one or more biomarkers listed in Tables 1-5.
• methods of screening for modulators of such regulators and methods of diagnosing, prognosing, and monitoring cancer involving such inhibitors/immunotherapy combination therapies are provided.
• an element means one element or more than one element.
• altered amount refers to increased or decreased copy number (e.g, germline and/or somatic) of a biomarker nucleic acid, e.g, increased or decreased expression level in a cancer sample, as compared to the expression level or copy number of the biomarker nucleic acid in a control sample.
• altered amount of a biomarker also includes an increased or decreased protein level of a biomarker protein in a sample, e.g, a cancer sample, as compared to the corresponding protein level in a normal, control sample.
• an altered amount of a biomarker protein may be determined by detecting posttranslational modification such as methylation status of the marker, which may affect the expression or activity of the biomarker protein.
• the amount of a biomarker in a subject is “significantly” higher or lower than the normal amount of the biomarker, if the amount of the biomarker is greater or less, respectively, than the normal level by an amount greater than the standard error of the assay employed to assess amount, and preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or than that amount.
• the amount of the biomarker in the subject can be considered “significantly” higher or lower than the normal amount if the amount is at least about two, and preferably at least about three, four, or five times, higher or lower, respectively, than the normal amount of the biomarker.
• Such “significance” can also be applied to any other measured parameter described herein, such as for expression, inhibition, cytotoxicity, cell growth, and the like.
• altered level of expression of a biomarker refers to an expression level or copy number of the biomarker in a test sample, e.g., a sample derived from a patient suffering from cancer, that is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least twice, and more preferably three, four, five or ten or more times the expression level or copy number of the biomarker in a control sample (e.g, sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples.
• a test sample e.g., a sample derived from a patient suffering from cancer
• a control sample e.g, sample from a healthy subjects not having the associated disease
• the altered level of expression is greater or less than the standard error of the assay employed to assess expression or copy number, and is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more times the expression level or copy number of the biomarker in a control sample (e.g, sample from a healthy subjects not having the associated disease) and preferably, the average expression level or copy number of the biomarker in several control samples.
• a control sample e.g, sample from a healthy subjects not having the associated disease
• the level of the biomarker refers to the level of the biomarker itself, the level of a modified biomarker (e.g, phosphorylated biomarker), or to the level of a biomarker relative to another measured variable, such as a control (e.g, phosphorylated biomarker relative to an unphosphorylated biomarker).
• a modified biomarker e.g, phosphorylated biomarker
• a control e.g, phosphorylated biomarker relative to an unphosphorylated biomarker
• altered activity of a biomarker refers to an activity of the biomarker which is increased or decreased in a disease state, e.g, in a cancer sample, as compared to the activity of the biomarker in a normal, control sample.
• Altered activity of the biomarker may be the result of, for example, altered expression of the biomarker, altered protein level of the biomarker, altered structure of the biomarker, or, e.g, an altered interaction with other proteins involved in the same or different pathway as the biomarker or altered interaction with transcriptional activators or inhibitors.
• altered structure of a biomarker refers to the presence of mutations or allelic variants within a biomarker nucleic acid or protein, e.g, mutations which affect expression or activity of the biomarker nucleic acid or protein, as compared to the normal or wild-type gene or protein.
• mutations include, but are not limited to substitutions, deletions, or addition mutations. Mutations may be present in the coding or non-coding region of the biomarker nucleic acid.
• antibody and “antibodies” refers to antigen-binding portions adaptable to be expressed within cells as “intracellular antibodies.” (Chen etal. (1994) Human Gene Ther. 5:595-601).
• Intracellular antibodies can also be introduced and expressed in one or more cells, tissues or organs of a multicellular organism, for example for prophylactic and/or therapeutic purposes (e.g., as a gene therapy) (see, at least PCT Pubis. WO 08/020079, WO 94/02610,
• Antibodies may be polyclonal or monoclonal; xenogeneic, allogeneic, or syngeneic; or modified forms thereof (e.g. humanized, chimeric, etc.). Antibodies may also be fully human. Preferably, antibodies of the present invention bind specifically or substantially specifically to a biomarker polypeptide or fragment thereof.
• monoclonal antibodies and “monoclonal antibody composition”, as used herein, refer to a population of antibody polypeptides that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of an antigen
• polyclonal antibodies and “polyclonal antibody composition” refer to a population of antibody polypeptides that contain multiple species of antigen binding sites capable of interacting with a particular antigen.
• a monoclonal antibody composition typically displays a single binding affinity for a particular antigen with which it immunoreacts.
• Antibodies may also be “humanized”, which is intended to include antibodies made by a non-human cell having variable and constant regions which have been altered to more closely resemble antibodies that would be made by a human cell. For example, by altering the nonhuman antibody amino acid sequence to incorporate amino acids found in human germline immunoglobulin sequences.
• the humanized antibodies of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.
• the term “humanized antibody”, as used herein, also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• the term “assigned score” refers to the numerical value designated for each of the biomarkers after being measured in a patient sample.
• the assigned score correlates to the absence, presence or inferred amount of the biomarker in the sample.
• the assigned score can be generated manually ( e.g ., by visual inspection) or with the aid of instrumentation for image acquisition and analysis.
• the assigned score is determined by a qualitative assessment, for example, detection of a fluorescent readout on a graded scale, or quantitative assessment.
• an “aggregate score,” which refers to the combination of assigned scores from a plurality of measured biomarkers, is determined.
• the aggregate score is a summation of assigned scores.
• combination of assigned scores involves performing mathematical operations on the assigned scores before combining them into an aggregate score.
• the aggregate score is also referred to herein as the “predictive score.”
• biomarker refers to a measurable entity of the present invention that has been determined to be predictive of effects of combinatorial therapies comprising one or more inhibitors of one or more biomarkers listed in Tables 1-5, for example, one or more biomarkers listed in Tables 1-5, such as MYCL and/or one or more PRC1.1 complex members (e.g., PCGF1, BCORLl, and USP7).
• Biomarkers can include, without limitation, nucleic acids and proteins, including those shown in the Tables, the Examples, the Figures, and otherwise described herein. As described herein, any relevant characteristic of a biomarker can be used, such as the copy number, amount, activity, location, modification (e.g, phosphorylation), and the like.
• blocking antibody or an antibody “antagonist” is one which inhibits or reduces at least one biological activity of the antigen(s) it binds.
• the blocking antibodies or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen(s).
• body fluid refers to fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle, chyme, stool, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal lubrication, vitreous humor, vomit).
• fluids that are excreted or secreted from the body as well as fluids that are normally not (e.g. amniotic fluid, aqueous humor, bile, blood and blood plasma, cerebrospinal fluid, cerumen and earwax, cowper’s fluid or pre-ejaculatory fluid, chyle,
• cancer or “tumor” or “hyperproliferative” refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain characteristic morphological features. Unless otherwise stated, the terms include metaplasias. In some embodiments, such cells exhibit such characteristics in part or in full due to the expression and activity of signaling pathways regulated by one or more biomarkers listed in Tables 1-5. In some embodiments, the cancer cells described herein are not sensitive to at least one of immunotherapies. In some embodiments, the cancer cells are treatable with an agent capable of antagonizing regulators of the biomarkers described herein, such as inhibiting expression and/or function, as described herein.
• Cancer cells are often in the form of a tumor, but such cells may exist alone within an animal, or may be a non-turn origenic cancer cell, such as a leukemia cell.
• cancer includes premalignant as well as malignant cancers.
• Cancers include, but are not limited to, B cell cancer, e.g., multiple myeloma, Waldenstrom's macroglobulinemia, the heavy chain diseases, such as, for example, alpha chain disease, gamma chain disease, and mu chain disease, benign monoclonal gammopathy, and immunocytic amyloidosis, melanomas, breast cancer, lung cancer, bronchus cancer, colorectal cancer, prostate cancer, pancreatic cancer, stomach cancer, ovarian cancer, urinary bladder cancer, brain or central nervous system cancer, peripheral nervous system cancer, esophageal cancer, cervical cancer, uterine or endometrial cancer, cancer of the oral cavity or pharynx, liver cancer, kidney cancer, testicular cancer, biliary tract cancer, small bowel or appendix cancer, salivary gland cancer, thyroid gland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer of hematologic tissues, and the like.
• the heavy chain diseases such as, for
• cancers include human sarcomas and carcinomas, e.g., Merkel cell carcinoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, colorectal cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bro
• cancers are epithelial in nature and include but are not limited to, bladder cancer, breast cancer, cervical cancer, colon cancer, gynecologic cancers, renal cancer, laryngeal cancer, lung cancer, oral cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, or skin cancer.
• the cancer is breast cancer, prostate cancer, lung cancer, or colon cancer.
• the epithelial cancer is non-small-cell lung cancer, nonpapillary renal cell carcinoma, cervical carcinoma, ovarian carcinoma (e.g, serous ovarian carcinoma), or breast carcinoma.
• the epithelial cancers may be characterized in various other ways including, but not limited to, serous, endometrioid, mucinous, clear cell, Brenner, or undifferentiated.
• a “neuroendocrine cancer” or “neuroendocrine tumor” is either one which arises from the neuroendocrine system or from non-endocrine cells that acquire properties of neuroendocrine cells through an oncogenic process.
• Most adult neuroendocrine tumors arise from a known primary site, including the carcinoid, pheochromocytoma, and Merkel's cell tumors.
• Carcinoid tumors can be benign or malignant.
• Carcinoid cancers include stomach, pancreas, colon, liver, lung (e.g, small cell carcinoma), ovarian, breast, testicular, and cervical cancer. Small cell carcinoma originates in large central with a propensity to metastasize early and often.
• Pheochromocytoma is a cancer of the adrenal medulla, which causes overproduction of catecholamine by the adrenal gland.
• Merkel cell carcinoma a neuroendocrine cancer of the skin, is a cancer that forms on or beneath the skin.
• Merkle cell cancers may arise from soft tissues underlying the skin and are fast-growing and often spread to other parts of the body.
• the cancer encompasses Merkle cell carcinoma.
• MCC was first described in 1972 by Toker as a trabecular carcinoma of the skin with carcinoid features (Toker (1972) Arch. Dermatol. 105: 107-110). Toker later reported the presence of neurosecretory granules, membrane bound granules containing dense cores, within the tumor cells. This feature is indistinguishable from tumor cells of neural crest origin and is also present in normal Merkel cells (Tang et al. (1978) Cancer 42:2311-2321). The tumor name was changed to Merkel cell carcinoma to reflect the similarity in appearance of tumor cells to Merkel cells (Toker (1982) Dermatopathol. 4:497-497-500; Rywlin (1982) Am. J Dermatolpathol. 4:513-515).
• MCC is an aggressive neuroendocrine carcinoma of the skin that frequently metastasizes to draining lymph nodes and distant organs including liver, bone, pancreas, lung, and brain (Lewis etal. (2020) Cancer Med. 9:1374-1382).
• MCC typically presents as a rapidly growing, erythematous lesion, in the dermal layer of the skin. The most common presentation of MCC is in older, fair skin, adults with a lifelong history of intense UV exposure from the sun. MCC occurs less frequently in non-sun-exposed skin as well as in children, young adults, and dark skin persons. Latitude closer to the equator is associated with increased incidence of MCC in North American men, but not women, possibly due to occupational sunlight exposure patterns (Stang etal.
• High-grade neuroendocrine MCC cells have a high nuclear to cytoplasmic ratio with scant cytoplasm, giving it the appearance of a small blue cell tumor when stained by hematoxylin and eosin.
• the tumor nuclei have an open, pepper and salt-appearing chromatin pattern with frequent mitotic figures indicative of a high proliferative rate).
• Immunohistochemistry (IHC) staining of MCC for neuroendocrine markers are typically positive for chromogranin, synaptophysin, CD56, and neurofilament.
• MCC also stain specifically for CK20 that typically shows a paranuclear dot-like pattern. In contrast, CK20 staining in normal Merkel cells is more uniformly distributed throughout the cytoplasm.
• CK20 staining can distinguish MCC from other more common neuroendocrine tumors such as small cell lung carcinoma (SCLC) (Leech et al. (2001) J. Clin. Pathol. 54:727-729).
• SCLC stains positive for TTF-1 (thyroid-specific transcription factor 1, encoded by the NKX2-1 gene), while MCC is negative for this stain.
• INSM1 is a useful IHC marker for MCC and Merkel cells, as well as for other neuroendocrine carcinomas (Lilo etal. (2018 )Am. J. Surg. Pathol. 42:1541-1548).
• coding region refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues
• noncoding region refers to regions of a nucleotide sequence that are not translated into amino acids (e.g. , 5' and 3' untranslated regions).
• complementary refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that an adenine residue of a first nucleic acid region is capable of forming specific hydrogen bonds (“base pairing”) with a residue of a second nucleic acid region which is antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that a cytosine residue of a first nucleic acid strand is capable of base pairing with a residue of a second nucleic acid strand which is antiparallel to the first strand if the residue is guanine.
• a first region of a nucleic acid is complementary to a second region of the same or a different nucleic acid if, when the two regions are arranged in an antiparallel fashion, at least one nucleotide residue of the first region is capable of base pairing with a residue of the second region.
• the first region comprises a first portion and the second region comprises a second portion, whereby, when the first and second portions are arranged in an antiparallel fashion, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues of the first portion are capable of base pairing with nucleotide residues in the second portion.
• control refers to any reference standard suitable to provide a comparison to the expression products in the test sample.
• the control comprises obtaining a “control sample” from which expression product levels are detected and compared to the expression product levels from the test sample.
• a control sample may comprise any suitable sample, including but not limited to a sample from a control cancer patient (can be stored sample or previous sample measurement) with a known outcome; normal tissue or cells isolated from a subject, such as a normal patient or the cancer patient, cultured primary cells/tissues isolated from a subject such as a normal subject or the cancer patient, adjacent normal cells/tissues obtained from the same organ or body location of the cancer patient, a tissue or cell sample isolated from a normal subject, or a primary cells/tissues obtained from a depository.
• control may comprise a reference standard expression product level from any suitable source, including but not limited to housekeeping genes, an expression product level range from normal tissue (or other previously analyzed control sample), a previously determined expression product level range within a test sample from a group of patients, or a set of patients with a certain outcome (for example, survival for one, two, three, four years, etc.) or receiving a certain treatment (for example, standard of care cancer therapy).
• a certain outcome for example, survival for one, two, three, four years, etc.
• a certain treatment for example, standard of care cancer therapy
• control samples and reference standard expression product levels can be used in combination as controls in the methods of the present invention.
• control may comprise normal or noncancerous cell/tissue sample.
• control may comprise an expression level for a set of patients, such as a set of cancer patients, or for a set of cancer patients receiving a certain treatment, or for a set of patients with one outcome versus another outcome.
• the specific expression product level of each patient can be assigned to a percentile level of expression, or expressed as either higher or lower than the mean or average of the reference standard expression level.
• control may comprise normal cells, cells from patients treated with combination chemotherapy, and cells from patients having benign cancer.
• control may also comprise a measured value for example, average level of expression of a particular gene in a population compared to the level of expression of a housekeeping gene in the same population.
• control comprises a ratio transformation of expression product levels, including but not limited to determining a ratio of expression product levels of two genes in the test sample and comparing it to any suitable ratio of the same two genes in a reference standard; determining expression product levels of the two or more genes in the test sample and determining a difference in expression product levels in any suitable control; and determining expression product levels of the two or more genes in the test sample, normalizing their expression to expression of housekeeping genes in the test sample, and comparing to any suitable control.
• control comprises a control sample which is of the same lineage and/or type as the test sample.
• control may comprise expression product levels grouped as percentiles within or based on a set of patient samples, such as all patients with cancer.
• a control expression product level is established wherein higher or lower levels of expression product relative to, for instance, a particular percentile, are used as the basis for predicting outcome.
• a control expression product level is established using expression product levels from cancer control patients with a known outcome, and the expression product levels from the test sample are compared to the control expression product level as the basis for predicting outcome.
• the methods of the present invention are not limited to use of a specific cut-point in comparing the level of expression product in the test sample to the control.
• the “copy number” of a biomarker nucleic acid refers to the number of DNA sequences in a cell (e.g ., germline and/or somatic) encoding a particular gene product. Generally, for a given gene, a mammal has two copies of each gene. The copy number can be increased, however, by gene amplification or duplication, or reduced by deletion.
• germline copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in the normal complement of germline copies in a control (e.g., the normal copy number in germline DNA for the same species as that from which the specific germline DNA and corresponding copy number were determined).
• Somatic copy number changes include changes at one or more genomic loci, wherein said one or more genomic loci are not accounted for by the number of copies in germline DNA of a control (e.g, copy number in germline DNA for the same subject as that from which the somatic DNA and corresponding copy number were determined).
• the “normal” copy number (e.g, germline and/or somatic) of a biomarker nucleic acid or “normal” level of expression of a biomarker nucleic acid or protein is the activity/1 evel of expression or copy number in a biological sample, e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow, from a subject, e.g, a human, not afflicted with cancer, or from a corresponding non-cancerous tissue in the same subject who has cancer.
• a biological sample e.g., a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, and bone marrow
• costimulate with reference to activated immune cells includes the ability of a costimulatory molecule to provide a second, non-activating receptor mediated signal (a “costimulatory signal”) that induces proliferation or effector function.
• a costimulatory signal can result in cytokine secretion, e.g, in a T cell that has received a T cell- receptor-mediated signal.
• Immune cells that have received a cell-receptor mediated signal, e.g, via an activating receptor are referred to herein as “activated immune cells.”
• determining a suitable treatment regimen for the subject is taken to mean the determination of a treatment regimen (i.e ., a single therapy or a combination of different therapies that are used for the prevention and/or treatment of the cancer in the subject) for a subject that is started, modified and/or ended based or essentially based or at least partially based on the results of the analysis according to the present invention.
• a treatment regimen i.e ., a single therapy or a combination of different therapies that are used for the prevention and/or treatment of the cancer in the subject
• a subject that is started, modified and/or ended based or essentially based or at least partially based on the results of the analysis according to the present invention.
• a treatment regimen i.e ., a single therapy or a combination of different therapies that are used for the prevention and/or treatment of the cancer in the subject
• the determination can, in addition to the results of the analysis according to the present invention, be based on personal characteristics of the subject to be treated. In most cases, the actual determination of
• diagnosing cancer includes the use of the methods, systems, and code of the present invention to determine the presence or absence of a cancer or subtype thereof in an individual.
• the term also includes methods, systems, and code for assessing the level of disease activity in an individual.
• a molecule is “fixed” or “affixed” to a substrate if it is covalently or non-covalently associated with the substrate such that the substrate can be rinsed with a fluid (e.g. standard saline citrate, pH 7.4) without a substantial fraction of the molecule dissociating from the substrate.
• a fluid e.g. standard saline citrate, pH 7.4
• expression signature refers to a group of one or more coordinately expressed biomarkers related to a measured phenotype.
• the genes, proteins, metabolites, and the like making up this signature may be expressed in a specific cell lineage, stage of differentiation, or during a particular biological response.
• the biomarkers can reflect biological aspects of the tumors in which they are expressed, such as the cell of origin of the cancer, the nature of the non-malignant cells in the biopsy, and the oncogenic mechanisms responsible for the cancer.
• Expression data and gene expression levels can be stored on computer readable media, e.g ., the computer readable medium used in conjunction with a microarray or chip reading device. Such expression data can be manipulated to generate expression signatures.
• “Homologous” as used herein refers to nucleotide sequence similarity between two regions of the same nucleic acid strand or between regions of two different nucleic acid strands. When a nucleotide residue position in both regions is occupied by the same nucleotide residue, then the regions are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position of each region is occupied by the same residue. Homology between two regions is expressed in terms of the proportion of nucleotide residue positions of the two regions that are occupied by the same nucleotide residue.
• a region having the nucleotide sequence 5'- ATTGCC-3' and a region having the nucleotide sequence 5'-TATGGC-3' share 50% homology.
• the first region comprises a first portion and the second region comprises a second portion, whereby, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residue positions of each of the portions are occupied by the same nucleotide residue. More preferably, all nucleotide residue positions of each of the portions are occupied by the same nucleotide residue.
• Immune cell refers to cells that play a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
• lymphocytes such as B cells and T cells
• natural killer cells such as myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
• immunotherapy refers to any treatment that uses certain parts of a subject’s immune system to fight diseases such as cancer.
• the subject’s own immune system is stimulated (or suppressed), with or without administration of one or more agent for that purpose.
• Immunotherapies that are designed to elicit or amplify an immune response are referred to as “activation immunotherapies.”
• Immunotherapies that are designed to reduce or suppress an immune response are referred to as “suppression immunotherapies.” Any agent believed to have an immune system effect on the genetically modified transplanted cancer cells can be assayed to determine whether the agent is an immunotherapy and the effect that a given genetic modification has on the modulation of immune response.
• the immunotherapy is cancer cell-specific.
• immunotherapy can be “untargeted,” which refers to administration of agents that do not selectively interact with immune system cells, yet modulates immune system function.
• untargeted therapies include, without limitation, chemotherapy, gene therapy, and radiation therapy.
• Immunotherapy is one form of targeted therapy that may comprise, for example, the use of cancer vaccines and/or sensitized antigen presenting cells.
• an oncolytic virus is a virus that is able to infect and lyse cancer cells, while leaving normal cells unharmed, making them potentially useful in cancer therapy. Replication of oncolytic viruses both facilitates tumor cell destruction and also produces dose amplification at the tumor site. They may also act as vectors for anticancer genes, allowing them to be specifically delivered to the tumor site.
• the immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g ., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen).
• a cancer antigen or disease antigen e.g ., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen.
• anti-VEGF and mTOR inhibitors are known to be effective in treating renal cell carcinoma.
• Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines.
• antisense polynucleotides can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.
• Immunotherapy can involve passive immunity for short-term protection of a host, achieved by the administration of pre-formed antibody directed against a cancer antigen or disease antigen (e.g., administration of a monoclonal antibody, optionally linked to a chemotherapeutic agent or toxin, to a tumor antigen). Immunotherapy can also focus on using the cytotoxic lymphocyte-recognized epitopes of cancer cell lines. Alternatively, antisense polynucleotides, ribozymes, RNA interference molecules, triple helix polynucleotides and the like, can be used to selectively modulate biomolecules that are linked to the initiation, progression, and/or pathology of a tumor or cancer.
• immunotherapy refers to any chemotherapy that has been demonstrated to induce immunogenic cell death, a state that is detectable by the release of one or more damage-associated molecular pattern (DAMP) molecules, including, but not limited to, calreticulin, ATP and HMGB1 (Kroemer etal. (2013), Annu. Rev. Immunol., 31:51-72).
• DAMP damage-associated molecular pattern
• Specific representative examples of consensus immunogenic chemotherapies include 5’-fluorouracil, anthracy clines, such as doxorubicin, and the platinum drug, oxaliplatin, among others.
• immunotherapy comprises inhibitors of one or more immune checkpoints.
• Immune checkpoint refers to a group of molecules on the cell surface of CD4+ and/or CD8+ T cells that fine-tune immune responses by down-modulating or inhibiting an anti-tumor immune response.
• Immune checkpoint proteins are well-known in the art and include, without limitation, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD 160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP, CD47, CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilins, IDO, CD39, CD73 and A2aR (see, for example, WO 2012/177624).
• the term further encompasses biologically active protein fragment, as well as nucleic acids encoding full-length immune checkpoint proteins and biologically active protein fragments thereof. In some embodiment, the term further encompasses any fragment according to homology descriptions provided herein.
• the immune checkpoint is PD-1.
• PD-1 refers to a member of the immunoglobulin gene superfamily that functions as a coinhibitory receptor having PD-L1 and PD-L2 as known ligands.
• PD-1 was previously identified using a subtraction cloning based approach to select for genes upregulated during TCR-induced activated T cell death.
• PD-1 is a member of the CD28/CTLA-4 family of molecules based on its ability to bind to PD-L1. Like CTLA-4, PD-1 is rapidly induced on the surface of T-cells in response to anti- CD3 (Agata etal. 25 (1996) Int. Immunol.
• PD-1 is also induced on the surface of B-cells (in response to anti-IgM). PD-1 is also expressed on a subset of thymocytes and myeloid cells (Agata etal. (1996) supra; Nishimura etal. (1996) Int. Immunol. 8:773).
• PD-1 has an extracellular region containing immunoglobulin superfamily domain, a transmembrane domain, and an intracellular region including an immunoreceptor tyrosine-based inhibitory motif (ITIM) (Ishida et al. (1992) EMBO J. 11:3887; Shinohara et al.
• ITIM immunoreceptor tyrosine-based inhibitory motif
• immunoinhibitory receptors which also includes gp49B, PIR-B, and the killer inhibitory receptors (KIRs) (Vivier and Daeron (1997) Immunol. Today 18:286). It is often assumed that the tyrosyl phosphorylated ITIM and ITSM motif of these receptors interacts with SH2-domain containing phosphatases, which leads to inhibitory signals.
• MHC polypeptides for example the KIRs
• CTLA4 binds to B7-1 and B7-2. It has been proposed that there is a phylogenetic relationship between the MHC and B7 genes (Henry et al. (1999) Immunol. Today 20(6):285-8).
• Nucleic acid and polypeptide sequences of PD-1 orthologs in organisms other than humans are well-known and include, for example, mouse PD-1 (NM_008798.2 and NP_032824.1), rat PD-1 (NM_001106927.1 and NP_001100397.1), dog PD-1 (XM_543338.3 and XP_543338.3), cow PD-1 (NM_001083506.1 and NP_001076975.1), and chicken PD-1 (XM_422723.3 and XP_422723.2).
• PD-1 polypeptides are inhibitory receptors capable of transmitting an inhibitory signal to an immune cell to thereby inhibit immune cell effector function, or are capable of promoting costimulation ( e.g ., by competitive inhibition) of immune cells, e.g, when present in soluble, monomeric form.
• Preferred PD-1 family members share sequence identity with PD-1 and bind to one or more B7 family members, e.g., B7-1, B7-2, PD-1 ligand, and/or other polypeptides on antigen presenting cells.
• PD-1 activity includes the ability of a PD-1 polypeptide to modulate an inhibitory signal in an activated immune cell, e.g, by engaging a natural PD-1 ligand on an antigen presenting cell. Modulation of an inhibitory signal in an immune cell results in modulation of proliferation of, and/or cytokine secretion by, an immune cell.
• PD-1 activity includes the ability of a PD-1 polypeptide to bind its natural ligand(s), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.
• PD-1 ligand refers to binding partners of the PD-1 receptor and includes both PD-L1 (Freeman et al. (2000) ./. Exp. Med. 192:1027-1034) and PD-L2 (Latchman et al. (2001) Nat. Immunol. 2:261). At least two types of human PD-1 ligand polypeptides exist. PD-1 ligand proteins comprise a signal sequence, and an IgV domain, an IgC domain, a transmembrane domain, and a short cytoplasmic tail. Both PD-L1 (See Freeman et al. (2000) for sequence data) and PD-L2 (See Latchman et al. (2001) Nat. Immunol.
• Both PD-L1 and PD-L2 are expressed in placenta, spleen, lymph nodes, thymus, and heart. Only PD-L2 is expressed in pancreas, lung and liver, while only PD-L1 is expressed in fetal liver. Both PD-1 ligands are upregulated on activated monocytes and dendritic cells, although PD-L1 expression is broader.
• PD-L1 is known to be constitutively expressed and upregulated to higher levels on murine hematopoietic cells (e.g ., T cells, B cells, macrophages, dendritic cells (DCs), and bone marrow-derived mast cells) and non-hematopoietic cells (e.g., endothelial, epithelial, and muscle cells), whereas PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast cells (see Butte et al. (2007) Immunity 27: 111).
• murine hematopoietic cells e.g ., T cells, B cells, macrophages, dendritic cells (DCs), and bone marrow-derived mast cells
• non-hematopoietic cells e.g., endothelial, epithelial, and muscle cells
• PD-L2 is inducibly expressed on DCs, macrophages, and bone marrow-derived mast
• PD-1 ligands comprise a family of polypeptides having certain conserved structural and functional features.
• family when used to refer to proteins or nucleic acid molecules, is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology, as defined herein.
• family members can be naturally or non-naturally occurring and can be from either the same or different species.
• a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
• Members of a family may also have common functional characteristics.
• PD-1 ligands are members of the B7 family of polypeptides.
• B7 family or “B7 polypeptides” as used herein includes costimulatory polypeptides that share sequence homology with B7 polypeptides, e.g, with B7- 1, B7-2, B7h (Swallow et al. (1999) Immunity 11:423), and/or PD-1 ligands (e.g, PD-L1 or PD-L2).
• B7-1 and B7-2 share approximately 26% amino acid sequence identity when compared using the BLAST program at NCBI with the default parameters (Blosum62 matrix with gap penalties set at existence 11 and extension 1 (See the NCBI website).
• B7 family also includes variants of these polypeptides which are capable of modulating immune cell function.
• IgV domains and the IgC domains are art-recognized Ig superfamily member domains. These domains correspond to structural units that have distinct folding patterns called Ig folds. Ig folds are comprised of a sandwich of two £ sheets, each consisting of anti -parallel f3 strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, IgC domains of Ig, TCR, and MHC molecules share the same types of sequence patterns and are called the Cl- set within the Ig superfamily. Other IgC domains fall within other sets. IgV domains also share sequence patterns and are called V set domains. IgV domains are longer than IgC domains and contain an additional pair of b strands.
• Preferred B7 polypeptides are capable of providing costimulatory or inhibitory signals to immune cells to thereby promote or inhibit immune cell responses.
• B7 family members that bind to costimulatory receptors increase T cell activation and proliferation, while B7 family members that bind to inhibitory receptors reduce costimulation.
• the same B7 family member may increase or decrease T cell costimulation.
• PD-1 ligand when bound to a costimulatory receptor, can induce costimulation of immune cells or can inhibit immune cell costimulation, e.g ., when present in soluble form.
• PD-1 ligand polypeptides can transmit an inhibitory signal to an immune cell.
• B7 family members include B7-1, B7-2, B7h, PD-L1 or PD-L2 and soluble fragments or derivatives thereof.
• B7 family members bind to one or more receptors on an immune cell, e.g. , CTLA4, CD28, ICOS, PD-1 and/or other receptors, and, depending on the receptor, have the ability to transmit an inhibitory signal or a costimulatory signal to an immune cell, preferably a T cell.
• PD-1 ligand activity includes the ability of a PD-1 ligand polypeptide to bind its natural receptor(s) (e.g. PD-1 or B7-1), the ability to modulate immune cell costimulatory or inhibitory signals, and the ability to modulate the immune response.
• PD-L1 refers to a specific PD-1 ligand.
• Two forms of human PD-L1 molecules have been identified.
• One form is a naturally occurring PD-L1 soluble polypeptide, i.e., having a short hydrophilic domain and no transmembrane domain, and is referred to herein as PD-L1S.
• the second form is a cell-associated polypeptide, i.e., having a transmembrane and cytoplasmic domain, referred to herein as PD-L1M.
• PD-L1 proteins comprise a signal sequence, and an IgV domain and an IgC domain.
• the signal sequence of PD-L1S is shown from about amino acid 1 to about amino acid 18.
• the signal sequence of PD-L1M is shown from about amino acid 1 to about amino acid 18.
• the IgV domain of PD-L1S is shown from about amino acid 19 to about amino acid 134 and the IgV domain of PD-L1M is shown from about amino acid 19 to about amino acid 134.
• the IgC domain of PD-L1S is shown from about amino acid 135 to about amino acid 227 and the IgC domain of PD-L1M is shown from about amino acid 135 to about amino acid 227.
• the hydrophilic tail of the PD-L1 exemplified in PD-L1S comprises a hydrophilic tail shown from about amino acid 228 to about amino acid 245.
• the PD-L1 polypeptide exemplified in PD-L1M comprises a transmembrane domain shown from about amino acids 239 to about amino acid 259 and a cytoplasmic domain shown from about 30 amino acid 260 to about amino acid 290.
• nucleic acid and polypeptide sequences of PD-L1 orthologs in organisms other than humans are well-known and include, for example, mouse PD-L1 (NM_021893.3 and NP_068693.1), rat PD-Ll (NM_001191954.1 and NP_001178883.1), dog PD-L1 (XM_541302.3 and XP_541302.3), cow PD-Ll (NM_001163412.1 and NP_001156884.1), and chicken PD-L1 (XM_424811.3 and XP_424811.3).
• PD-L2 refers to another specific PD-1 ligand.
• PD-L2 is a B7 family member expressed on various APCs, including dendritic cells, macrophages and bone-marrow derived mast cells (Zhong et al. (2007) Eur. J. Immunol. 37:2405).
• APC-expressed PD-L2 is able to both inhibit T cell activation through ligation of PD-1 and costimulate T cell activation, through a PD-1 independent mechanism (Shin etal. (2005) J. Exp. Med. 201:1531).
• PD-L2 dendritic cell-expressed PD-L2 results in enhanced dendritic cell cytokine expression and survival (Radhakrishnan et al. (2003) J. Immunol. 37: 1827; Nguyen et al. (2002) J. Exp. Med. 196:1393).
• the nucleic acid and amino acid sequences of representative human PD-L2 biomarkers are well-known in the art and are also available to the public at the GenBank database under NM 025239.3 and NP 079515.2.
• PD-L2 proteins are characterized by common structural elements.
• PD-L2 proteins include at least one or more of the following domains: a signal peptide domain, a transmembrane domain, an IgV domain, an IgC domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.
• amino acids 1-19 of PD-L2 comprises a signal sequence.
• a “signal sequence” or “signal peptide” serves to direct a polypeptide containing such a sequence to a lipid bilayer, and is cleaved in secreted and membrane bound polypeptides and includes a peptide containing about 15 or more amino acids which occurs at the N-terminus of secretory and membrane bound polypeptides and which contains a large number of hydrophobic amino acid residues.
• a signal sequence contains at least about 10-30 amino acid residues, preferably about 15- 25 amino acid residues, more preferably about 18- 20 amino acid residues, and even more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g ., valine, leucine, isoleucine or phenylalanine).
• amino acid residues 220-243 of the native human PD-L2 polypeptide and amino acid residues 201-243 of the mature polypeptide comprise a transmembrane domain.
• transmembrane domain includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g, leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zaklakla, W. N.
• amino acid residues 20-120 of the native human PD-L2 polypeptide and amino acid residues 1-101 of the mature polypeptide comprise an IgV domain.
• Amino acid residues 121- 219 of the native human PD-L2 polypeptide and amino acid residues 102-200 of the mature polypeptide comprise an IgC domain.
• IgV and IgC domains are recognized in the art as Ig superfamily member domains. These domains correspond to structural units that have distinct folding patterns called Ig folds.
• Ig folds are comprised of a sandwich of two B sheets, each consisting of antiparallel (3 strands of 5-10 amino acids with a conserved disulfide bond between the two sheets in most, but not all, domains.
• IgC domains of Ig, TCR, and MHC molecules share the same types of sequence patterns and are called the Cl set within the Ig superfamily. Other IgC domains fall within other sets.
• IgV domains also share sequence patterns and are called V set domains. IgV domains are longer than C-domains and form an additional pair of strands.
• amino acid residues 1-219 of the native human PD-L2 polypeptide and amino acid residues 1-200 of the mature polypeptide comprise an extracellular domain.
• extracellular domain represents the N-terminal amino acids which extend as a tail from the surface of a cell.
• An extracellular domain of the present invention includes an IgV domain and an IgC domain, and may include a signal peptide domain.
• amino acid residues 244- 273 of the native human PD-L2 polypeptide and amino acid residues 225-273 of the mature polypeptide comprise a cytoplasmic domain.
• cytoplasmic domain represents the C-terminal amino acids which extend as a tail into the cytoplasm of a cell.
• nucleic acid and polypeptide sequences of PD-L2 orthologs in organisms other than humans are well-known and include, for example, mouse PD-L2 (NM 021396.2 and NP_067371.1), rat PD-L2 (NM_001107582.2 and NP_001101052.2), dog PD-L2 (XM_847012.2 and XP_852105.2), cow PD-L2 (XM_586846.5 and XP_586846.3), and chimpanzee PD-L2 (XM_001140776.2 and XP_001140776.1).
• PD-L2 activity refers to an activity exerted by a PD-L2 protein, polypeptide or nucleic acid molecule on a PD-L2-responsive cell or tissue, or on a PD- L2 polypeptide binding partner, as determined in vivo , or in vitro , according to standard techniques.
• a PD-L2 activity is a direct activity, such as an association with a PD-L2 binding partner.
• a “target molecule” or “binding partner” is a molecule with which a PD-L2 polypeptide binds or interacts in nature, such that PD-L2-mediated function is achieved.
• a PD-L2 target molecule is the receptor RGMb.
• a PD- L2 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the PD- L2 polypeptide with its natural binding partner (i.e., physiologically relevant interacting macromolecule involved in an immune function or other biologically relevant function), e.g ., RGMb.
• RGMb biologically relevant interacting macromolecule involved in an immune function or other biologically relevant function
• the PD-L2 polypeptides of the present invention can have one or more of the following activities: 1) bind to and/or modulate the activity of the receptor RGMb, PD-1, or other PD- L2 natural binding partners, 2) modulate intra-or intercellular signaling, 3) modulate activation of immune cells, e.g. , T lymphocytes, and 4) modulate the immune response of an organism, e.g. , a mouse or human organism.
• Anti-immune checkpoint therapy refers to the use of agents that inhibit immune checkpoint nucleic acids and/or proteins. Inhibition of one or more immune checkpoints can block or otherwise neutralize inhibitory signaling to thereby upregulate an immune response in order to more efficaciously treat cancer.
• agents useful for inhibiting immune checkpoints include antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof.
• Exemplary agents for upregulating an immune response include antibodies against one or more immune checkpoint proteins block the interaction between the proteins and its natural receptor(s); a non-activating form of one or more immune checkpoint proteins (e.g, a dominant negative polypeptide); small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s); fusion proteins (e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin) that bind to its natural receptor(s); nucleic acid molecules that block immune checkpoint nucleic acid transcription or translation; and the like.
• a non-activating form of one or more immune checkpoint proteins e.g, a dominant negative polypeptide
• small molecules or peptides that block the interaction between one or more immune checkpoint proteins and its natural receptor(s)
• fusion proteins e.g. the extracellular portion of an immune checkpoint inhibition protein fused to the Fc portion of an antibody or immunoglobulin
• agents can directly block the interaction between the one or more immune checkpoints and its natural receptor(s) (e.g, antibodies) to prevent inhibitory signaling and upregulate an immune response.
• agents can indirectly block the interaction between one or more immune checkpoint proteins and its natural receptor(s) to prevent inhibitory signaling and upregulate an immune response.
• a soluble version of an immune checkpoint protein ligand such as a stabilized extracellular domain can bind to its receptor to indirectly reduce the effective concentration of the receptor to bind to an appropriate ligand.
• anti-PD-1 antibodies, anti-PD-Ll antibodies, and/or anti-PD-L2 antibodies are used to inhibit immune checkpoints. These embodiments are also applicable to specific therapy against particular immune checkpoints, such as the PD- 1 pathway (e.g ., anti-PD-1 pathway therapy, otherwise known as PD-1 pathway inhibitor therapy).
• USP7 also known as “Ubiquitin Specific Peptidase 7,” refers to a member of the C19 peptidase family that includes ubiquitinyl hydrolases. USP7 deubiquitinates target proteins (e.g., F0X04, p53/TP53, MDM2, ERCC6, DNMT1, UHRF1, PTEN, KMT2E/MLL5 and DAXX), which prevents degradation of the deubiquitinated target protein. Thus, USP7 counteracts the activity of ubiquitin ligases.
• target proteins e.g., F0X04, p53/TP53, MDM2, ERCC6, DNMT1, UHRF1, PTEN, KMT2E/MLL5 and DAXX
• the nucleic acid and amino acid sequences of a representative human USP7 is available to the public at the GenBank database (Gene ID 7874) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for USP7.
• Human USP7 variants include the transcript variant 1 encoding isoform 1 (NM 003470.3 and NP_003461.2), the transcript variant 2 encoding isoform 2 (NM_001286457.2 and NP_001273386.2), the transcript variant 3 encoding isoform 3 (NM_001286458.2 and NP_001273387.1), and the transcript variant 4 encoding isoform 4 (NM_001321858.1 and NP_001308787.1).
• Nucleic acid and polypeptide sequences of USP7 orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM 024349753.1 and XP_024205521.1; XM_016929384.2 and XP_016784873.1; XM_016929385.2 and XP_016784874.1; and XM_016929388.2 and XP_016784877.1), macaque (XM_015125591.2 and XP_014981077.1; XM_015125592.2 and XP_014981078.1; XM_002802389.3 and XP_002802435.1; and XM_002802388.3 and XP_002802434.1), wolf (XM_005621558.3 and XP_005621615.1; and XM_005621559.3 and XP_005621616.1), cow (XM
• USP7 activity includes the ability of a USP7 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its substrate and/or catalyze the ubiquitinase activity.
• USP7 inhibitor(s) includes any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of reducing, inhibiting, blocking, preventing, and/or that inhibits the ability of a USP7 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• such inhibitors may reduce or inhibit the binding/interaction between USP7 and its substrates or other binding partners.
• such inhibitors may increase or promote the turnover rate, reduce or inhibit the expression and/or the stability ( e.g ., the half-life), and/or change the cellular localization of USP7, resulting in at least a decrease in USP7 levels and/or activity.
• such inhibitors may impair the catalytic activity of USP7.
• the inhibitors inhibit the deubiquitinase activity of USP7.
• Such inhibitors may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents).
• RNAi RNA interfering
• Such inhibitors may be specific to USP7 or also inhibit at least one of the binding partners.
• Such inhibitors may include XL177A and/or XL188 (Shauer et ah, Sci Rep 10, 5324 (2020)).
• a USP7 inhibitor is XL177A, which has the following structure:
• the USP7 inhibitor is XL188, which has the following structure: Such inhibitors may also include P-22077 (Cas No. 1247819-59-5). Additional USP7 inhibitors are known in the art, such as in PCT Publ. No. WO 2019/067503, USSN 16/650,727, and PCT Publ. No. WO 2020/086595.
• RNA interference for USP7 polypeptides are well known and commercially available (e.g ., human, rat, or mouse shRNA/siRNA products (Cat. # TL308454V, TR308454, SR305301, TL308454, SR422076, TL308454V, TF308454, TL513496, SR513215,
• USP7 Methods for detection, purification, and/or inhibition of USP7 (e.g., by anti-USP7 antibodies) are also well known and commercially available (e.g, multiple USP7 antibodies from Signalway Antibody (College Park, MD, Cat. # 38401, 27041, and 43178), Sino Biological (Wayne, PA; Cat. # 11681-MM01), Invitrogen (Carlsbad, CA, Cat. # Cat #PA5-17179, Cat #MA5-15585, etc.). USP7 knockout human cell lines are also well known and available at Horizon (Cambridge, UK, Cat. # HD 115-028, HDR02-029, and HDR02-028).
• MYCL also known as “MYCL proto-oncogene, bHLH transcription factor” refers to a bHLH protein and member of the poly comb repression complex (PRC) 1.1 that has DNA binding and transcription factor activity. Efficient DNA binding requires dimerization with another bHLH protein (e.g, MAX).
• PRC poly comb repression complex
• MYCL is intended to include fragments, variants (e.g, allelic variants) and derivatives thereof.
• the nucleic acid and amino acid sequences of a representative human MYCL is available to the public at the GenBank database (Gene ID 4610) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for MYCL.
• Human MYCL variants include the transcript variant 1 encoding isoform 1 (NM_00103308L3 and NP_001028253.1), the transcript variant 2 encoding isoform 2 (NM_005376.5 and NP_005367.2), and the transcript variant 3 encoding isoform 3 (NM_001033082.3 and NP_001028254.2).
• Nucleic acid and polypeptide sequences of MYCL orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM 016959814.2 and XP_016815303.2), Rhesus macaque (XM_028835497.1 and XP_028691330.1; and XM_015136019.2 and XP_014991505.2), dog (XM_022427768.1 and XP_022283476.1; XM_022427769.1 and XP_022283477.1; XM_022427775.1 and XP_022283483.1; XM_022427774.1 and XP_022283482.1; XM 022427778.1 and XP_022283486.1; XM_022427767.1 and XP_022283475.1; XM 022427772.1 and XP_022283480.1; XM_005628887.3
• MYCL activity includes the ability of a MYCL polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind to DNA and/or activate transcription.
• MYCL-regulated pathway includes pathways in which MYCL (and its fragments, domains, and/or motifs thereof, discussed herein) binds to template DNA and activates transcription of at least one gene in the pathway.
• MYCL-regulated pathways include at least those described herein, such as regulation of expression of genes that suppress MHC class I, such as HLA I, surface expression in cancer cells.
• MYCL inhibitor(s) includes any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of reducing, inhibiting, blocking, preventing, and/or that inhibits the ability of a MYCL polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein). In one embodiment, such inhibitors may reduce or inhibit the binding/interaction between MYCL and DNA or MYCL and its binding partners. In another embodiment, such inhibitors may reduce or inhibit MYCL as a transcription factor.
• such inhibitors may increase or promote the turnover rate, reduce or inhibit the expression and/or the stability (e.g ., the half- life), and/or change the cellular localization of MYCL, resulting in at least a decrease in MYCL levels and/or activity.
• such inhibitors may impair the catalytic activity of MCYL.
• the inhibitors inhibit the transcription activation activity of MYCL.
• Such inhibitors may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents).
• RNA interference molecules for MYCL polypeptides are well known and commercially available (e.g ., human, rat, or mouse shRNA/siRNA products (Cat. #
• TL311321, TL513612V, TL311321V, and TL316626V siRNA/shRNA products from Origene
• siRNA/shRNA products Cat. # sc-38071
• Methods for detection, purification, and/or inhibition of MYCL are also well known and commercially available (e.g, multiple MYCL antibodies from Origene (Cat. #
• KDM2B also known as “Lysine Demethylase 2B” refers to histone demethylase that demethylates 'Lys-4' and 'Lys-36' of histone H3.
• KDM2B is a member of the F-box protein family, which is characterized by the “F-box,” an approximately 40 amino acid motif F-box proteins are a component of the ubiquitin protein ligase complex called SCF (SKPl-cullin-F-box). There are three classes of F-box proteins.
• Fbws F-box proteins comprise WD-40 domains
• Fbls F-box proteins comprise containing leucine-rich repeats
• Fbxs F-box proteins comprise either different protein-protein interaction modules or no recognizable motifs.
• KDM2B belongs to the Fbls class. Alternative splicing results in multiple transcript variants.
• KDM2B is intended to include fragments, variants (e.g, allelic variants) and derivatives thereof.
• the nucleic acid and amino acid sequences of a representative human KDM2B is available to the public at the GenBank database (Gene ID 84678) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for KDM2B, including at least 5 different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/Q8NHM5).
• Human KDM2B variants include the transcript variant 1 encoding isoform b (NM_001005366.2 andNP_001005366.1), transcript variant 2 encoding isoform a (NM_032590.5 and NP_115979.3), transcript variant 3 encoding isoform XI (XM_011538867.3 and XP_011537169.1), transcript variant 4 encoding isoform X2 (XM_011538868.3 and XP_011537170.1), transcript variant 5 encoding isoform X4 (XM_005253955.4 and XP_005254012.1), transcript variant 6 encoding isoform X5 (XM_005253956.4 and XP_005254013.1), transcript variant 7 encoding isoform X7 (XM_005253961.5 and XP_005254018.1), transcript variant 8 encoding isoform X6 (XM_011538875.3 and XP_01
• KDM2B activity includes the ability of a KDM2B polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its substrates, and/or mediate its demethylase activity.
• KDM2B substrate(s) refers to binding partners of a KDM2B polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein), e.g ., the proteins listed herein, including SKP1 and a cullin protein.
• KDM2B regulated pathway(s) includes pathways in which KDM2B (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed. KDM2B -regulated pathways include at least those described herein, such as positive or negative regulation of histone modification. .
• agents that decrease the copy number, the expression level, and/or the activity of KDM2B encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a KDM2B polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between KDM2B and its substrates or other binding partners. For example, the agent may increase the recognition and/or binding of KDM2B to histones thereby decreasing demethylation of the histones.
• the agent may decrease the expression of a KDM2B polypeptide.
• such agent may decrease KDM2B’s activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability ( e.g ., the half-life), and/or change the cellular localization of KDM2B, resulting in at least a decrease in KDM2B levels and/or activity.
• Such agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well- known agents), and gene constructs that inhibit endogenous production of KDM2B or its fragments inside cancer cells.
• RNA interfering agents including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well- known agents
• Such agents may be specific to KDM2B or also to at least one of the binding partners, including but not limited to SCF or a cullin polypeptide.
• Antibodies for detection of KDM2B are commercially available (Cat. # AP08592PU-N AP51620PU-N (OriGene); ab234082, ab5199 (Abeam); ab234082 (Santa Cruz).
• RNA interference for KDM2B polypeptides are well known and commercially available (e.g., human, rat, or mouse shRNA/siRNA products (Cat. # TL313046V, SR325364, SR420035, SR325364, TL313046, TG313046, TF514017, TL313046V, TR313046, TR514017, TL514017V, TL514017 and human or mouse gene knockout kit via CRISPR (Cat. # KN413999, KN508731) from Origene (Rockville, MD), and siRNA/shRNA products (Cat. # sc-75005and sc-75006) from Santa Cruz Biotechonology (Dallas, Texas).
• CRISPR Cat. # KN413999, KN508731
• siRNA/shRNA products Cat. # sc-75005and sc-75006 from Santa Cruz Biotechonology (Dallas, Texas).
• KDM2B knockout cell line is commercially available from Horizon (Cambridge, UK, Cat. # HZGHC014730c012).
• BCORL1 also known as “BCL6 corepressor like 1” refers to a transcriptional corepressor that is found tethered to promoter regions by DNA-binding proteins.
• BCORL1 can interact with several class II histone deacetylases to repress transcription. Alternative splicing results in multiple transcript variants.
• the term “BCORL1” is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof.
• nucleic acid and amino acid sequences of a representative human BCORL1 is available to the public at the GenBank database (Gene ID 63035) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for BCORL1, including at least 3 different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/Q5H9F3).
• Human BCORL1 variants include the transcript variant 1 encoding isoform la (NM_001184772.3 and NP_001171701; NM_001379450.1 and NP_001366379; and NM_001379451.1 and NP_001366380.), transcript variant 2 encoding isoform 1 (NM_021946.5 and NP_068765.3), transcript variant 3 encoding isoform XI (XM_005262453.4 and XP 005262510.1; XM_006724777.3 and XP 006724840.1; XM_017029721.1 and XP 016885210.1; XM_006724776.3 and XP 006724839.1; XM_005262455.4 and XP_005262512.2; and XM_017029722.1 and XP_016885211.1), transcript variant 4 encoding isoform X3 (XM_005262456.4 and
• Nucleic acid and polypeptide sequences of BCORL1 orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM 016943863.2 and XP_016799352.1; XM_016943867.1 and XP_016799356.1; XM_016943861.1 and XP_016799350.1; XM_016943870.1 and XP_016799359.; XM_016943862.1 and XP_016799351.1; XM_024353327.1 and XP_024209095.1; XM_016943864.2 and XP_016799353.1; XM_016943868.2 and XP_016799357.1; XM_016943866.2 and XP_016799355.1; and XM_016943865.1 and XP_016799354.1), rhesus macaque (XM_0288
• BCORL1 activity includes the ability of a BCORL1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its substrates, and/or mediate its transcription repression activity.
• BCORL1 substrate(s) refers to binding partners of a BCORL1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein.
• BCORL1 regulated pathway(s) includes pathways in which BCORL1 (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• BCORL1 -regulated pathways include at least those described herein, such as transcription regulation.
• agents that decrease the copy number, the expression level, and/or the activity of BCORL1 encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a BCORL1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between BCORL1 and its substrates or other binding partners.
• the agent may decrease the expression of a BCORL1 polypeptide.
• such agent may decrease BCORLL s activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g ., the half-life), and/or change the cellular localization of BCORL1, resulting in at least a decrease in BCORL1 levels and/or activity.
• Such agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents), and gene constructs that inhibit endogenous production of BCORLl or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to BCORLl or also to at least one of its binding partners.
• RNA interference for BCORL1 polypeptides are well known and commercially available (e.g, human, rat, or mouse shRNA/siRNA products (Cat. # TL306414V, TF306414, TR519839, TR306414,
• BCORLl knockout human cell lines are also well known and available at Horizon (Cambridge, UK, Cat. # HZGHC630358).
• RING1 A also known as “ring finger protein 1” refers to a gene or protein belonging to the RING family. Ring family members are characterized by having a RING domain, a zinc-binding motif related to the zinc finger domain. RING1 A interacts with poly comb group complex proteins BMI, EDR1, and CBX4. Alternative splicing results in multiple transcript variants.
• the term “RING1 A” is intended to include fragments, variants (e.g, allelic variants) and derivatives thereof.
• the nucleic acid and amino acid sequences of a representative human RING1 A is available to the public at the GenBank database (Gene ID 6015) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for RING1 A, including at least 2 different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/Q06587). Human RING1 A variants include the transcript variant 1 encoding isoform 1 (NM_002931.4 and NP_002922.2).
• Nucleic acid and polypeptide sequences of RING1A orthologs in organisms other than humans are well known and include, for example, chimpanzee (NM 001081482.1 and NP_001074951.1; XM_009450849.3 and XP_009449124.1; and XM_016954658.2 and XP_016810147.1), rhesus macaque (NM_001114959.1 and NP_001108431.1; XM_028846856.1 and XP_028702689.1; and XM_015136067.2 and XP_014991553.1), dog (NM_001048128.1 and NP_001041593.1), cow (NM_001105051.1 andNP_001098521.1), mouse (NM_009066.3 and NP_033092.3), rat (NM_212549.2 and NP_997714.2;
• RING1 A activity includes the ability of a RING1 A polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its substrates, and/or mediate its transcription repression activity.
• RING1 A substrate(s) refers to binding partners of a RING1 A polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein), e.g ., the proteins listed herein, including BMI1, EDR1, and CBX4.
• RING1 A regulated pathway(s) includes pathways in which RING1 A (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• RING1 A -regulated pathways include at least those described herein, such as transcription repression.
• agents that decrease the copy number, the expression level, and/or the activity of RING1 A encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a RING1 A polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between RING1 A and its substrates or other binding partners.
• the agent may decrease the expression of a RING1 A polypeptide.
• such agent may decrease RINGlA’s activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g, the half-life), and/or change the cellular localization of RING1 A, resulting in at least a decrease in RING1 A levels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents), and gene constructs that inhibit endogenous production of RING1 A or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to RING1 A or also to at least one of the binding partners, including but not limited to BMI1, EDR1, and CBX4.
• RNA interference for RING1 A polypeptides are well known and commercially available (e.g, human, rat, or mouse shRNA/siRNA products (Cat. TL309810V, SR304071, SR304071, SR304082, TG309787, TG512489, TL309787, among others, and human or mouse gene knockout kit via CRISPR (Cat. KN514834, KN402650) from Origene (Rockville, MD), and siRNA/shRNA products (Cat.
• RING1A knockout cell line is commercially available from Horizon (Cambridge, UK, Cat. # HZGHC00 1111 c003, HZGHC001111 cO 12, and HZGHC001111 cOO 1).
• RING1B also known as “ring finger protein 2” refers to a member of polycomb group complexes (e.g ., PRC 1.1) encoded by the RNF2 gene. RING1B has been shown to interact with and inhibit CP2, a transcription factor. RING1B also interacts with huntingtin interacting protein 2 (HIP2), a ubiquitin-conjugating enzyme and possesses ubiquitin ligase activity. The protein has chromatin binding and ubiquitin-protein transferase.
• the term “RING1B” is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof.
• the nucleic acid and amino acid sequences of a representative human RING1B is available to the public at the GenBank database (Gene ID 6045) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for RING1B, including at least 2 different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/Q99496). Human RING1B encodes the canonical sequence (NM_007212.4 and NP_009143.1).
• Human RING1B variants also include the transcript variant encoding isoform XI (XM_011509852.2 and XP_011508154.1; and XM 011509851.3 and XP Ol 1508153.1) and the transcript variant encoding isoform X2 (XM_005245413.3 and XP_005245470.1).
• Nucleic acid and polypeptide sequences of RING1B orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM_514057.6 and XP_514057.3; XM_003308638.4 and XP_003308686.1; XM_009439605.3 and XP_009437880.1; and XM_009439610.3 and XP_009437885.1), dog (XM_022420969.1 and XP_022276677.1), cow (NM_001101203.1 and NP_001094673.1; XM_024976397.1 and XP_024832165.1; and XM_024976398.1 and XP_024832166.1), mouse (NM_001360844.1 and NP_001347773.1; NM_001360845.1 and NP_001347774.1; NM_001360847.1 and NP_001347776.1; and NM_01
• RING1B activity includes the ability of a RING1B polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its substrates, and/or mediate its ubiquitin ligase activity.
• DFS-29725 The term “RING1B substrate(s)” refers to binding partners of a RING1B polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein), e.g., the proteins listed herein, including C2 and HIP2.
• RING1B regulated pathway(s) includes pathways in which RING1B (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• RING1B-regulated pathways include at least those described herein, such as development and cell proliferation.
• agents that decrease the copy number, the expression level, and/or the activity of RING1B encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a RING1B polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between RING1B and its substrates or other binding partners.
• the agent may decrease the expression of a RING1B polypeptide.
• such agent may decrease RING1B’s activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g., the half-life), and/or change the cellular localization of RING1B, resulting in at least a decrease in RING1B levels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents), and gene constructs that inhibit endogenous production of RING1B or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to RING1B or also to at least one of the binding partners, including but not limited to C2 and HIP2.
• RNA interference for RING1B polypeptides are well known and commercially available (e.g., human, rat, or mouse shRNA/siRNA products (Cat. # TL309787V, SR304082, SR304082, TG309787, TG512489, among others, and human or mouse gene knockout kit via CRISPR (Cat.# KN514934, KN403089) from Origene (Rockville, MD), and siRNA/shRNA products (Cat.
• RING1B knockout human cell lines are also well known and available at Horizon (Cambridge, UK, Cat. # HZGHCOOl 181c002, HZGHCOOl 181c007, HZGHCOOl 181c005, HZGHCOOl 18 IcOOl, HZGHCOOl 18 lc003, among others).
• RYBP also known as “RING1 And YY1 Binding Protein” refers to a member of the polycomb repressive complex 1 (and 1.1). RYBP is a transcription corepressor. Alternative splicing results in multiple transcript variants.
• RYBP is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof.
• nucleic acid and amino acid sequences of a representative human RYBP is available to the public at the GenBank database (Gene ID 23429) and is shown in Table 1.
• GenBank database Gene ID 23429
• Table 1 A single transcript variant encoding RYBP has been identified (see World Wide Web at uniprot.org/uniprot/Q8N488; NM_001005366.2 and NP_001005366.1).
• Nucleic acid and polypeptide sequences of RYBP orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM 016941488.2 and XP_016796977.1), dog (XM_022407339.1 and XP_022263047.1), mouse (NM_019743.3 and NP_062717.2), and chicken (XM_015293232.2 and XP_015148718.1).
• RYBP substrate(s) refers to binding partners of a RYBP polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein.
• RYBP-regulated pathway(s) includes pathways in which RYBP (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• RYBP regulated pathways include at least those described herein, such as the E2F transcription factor network and chromatin regulation and acetylation.
• agents that decrease the copy number, the expression level, and/or the activity of RYBP encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a RYBP polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between RYBP and its substrates or other binding partners.
• the agent may decrease the expression of a RYBP polypeptide.
• such agent may decrease RYBP activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g, the half-life), and/or change the cellular localization of RYBP, resulting in at least a decrease in RYBP levels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well- known agents), and gene constructs that inhibit endogenous production of RYBP or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to RYBP or also to at least one of the binding partners.
• RNA interference for RYBP polypeptides are well known and commercially available (e.g ., human, rat, or mouse shRNA/siRNA products (Cat. # TL309675V, SR308270, TL503156, SR308270, TR309675, R404933, among others and human or mouse gene knockout kit via CRISPR (Cat. # KN406186, KN515228) from Origene (Rockville, MD), and siRNA/shRNA products (Cat. # sc-77379, sc-106751) from Santa Cruz Biotechonology (Dallas, Texas).
• CRISPR Cat. # KN406186, KN515228
• siRNA/shRNA products Cat. # sc-77379, sc-106751 from Santa Cruz Biotechonology (Dallas, Texas).
• RYBP e.g., by anti- RYBP antibodies
• Methods for detection, purification, and/or inhibition of RYBP are also well known and commercially available (e.g, (Cat. # 28645 (Signalway Antibodies); AB INI 156059, AB INI 156058 (antibodies-online.com); RYBP (A-l), RYBP (A-l) X (Santa Cruz).
• Antibodies that specifically bind RYBP are commercially available (Cat.
• RYBP knockout human cell lines are also well known and available at Horizon (Cambridge, UK, Cat. # HZGHC23429).
• PCGF1 also known as “Polycomb Group Ring Finger 1” refers to a member of the PRC 1.1 complex. An important paralog of this gene is COMMD3-BMI1. Alternative splicing results in multiple transcript variants.
• PCGF1 is intended to include fragments, variants (e.g, allelic variants) and derivatives thereof.
• PCGF1 The nucleic acid and amino acid sequences of a representative human PCGFlis available to the public at the GenBank database (Gene ID 84759) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for PCGF1, including at least 2 different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/Q9BSMl). Human PCGF1 variants include transcript variant 1 encoding isoform 1 (NM_032673.3 and NP_116062.2) and transcript variant 2 encoding isoform XI (XM_024453181.1 and XP_024308949.1).
• Nucleic acid and polypeptide sequences of PCGF1 orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM_515562.6 and XP_515562.2), rhesus macaque (XM_015112696.2 and XP_014968182.1), dog (XM_022404797.1 and XP_022260505.1; XM_005630527.3 and XP_005630584.1; XM_005630524.3 and XP_005630581.1; XM_005630526.3 and XP_005630583.1; XM_005630529.3 and XP_005630586.1; XM_532995.6 and XP_532995.2; and XM_022404796.1 and XP_022260504.1), cow (NM_001046447.2 and NP_001039912.2), mouse (XM_030255588.1 and
• PCGF1 substrate(s) refers to binding partners of a PCGF1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein.
• PCGF1 regulated pathway(s) includes pathways in which PCGF1 (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• agents that decrease the copy number, the expression level, and/or the activity of PCGF1 encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a PCGF1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between PCGF1 and its substrates or other binding partners.
• the agent may decrease the expression of a PCGF1 polypeptide.
• such agent may decrease PCGFl’s activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g ., the half-life), and/or change the cellular localization of PCGF1, resulting in at least a decrease in PCGFllevels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents), and gene constructs that inhibit endogenous production of PCGFlor its fragments inside cancer cells. Such agents may be specific to PCGFlor also to at least one of the binding partners.
• Antibodies for detection of PCGFl are commercially available (Cat.
• RNA interference for PCGFlpolypepitdes are well known and commercially available (e.g., human, rat, or mouse shRNA/siRNA products (Cat. # TL302590V, SR313658, TR302590, SR406929, SR313658, TL302590 among others) and human or mouse gene knockout kit via CRISPR (Cat. # KN512948, KN416322) from Origene (Rockville, MD), and siRNA/shRNA products (Cat.
• PCGF1 knockout human cell lines are also well known and available at Horizon (Cambridge, UK, Cat. # HZGHC84759).
• SKP1 also known as “S-phase kinase-associated protein 1” refers to a protein that is a component of SCF complexes, which are involved in the ubiquitination of protein substrates. These complexes are described supra. Alternative splicing results in multiple transcript variants.
• the term “SKP1” is intended to include fragments, variants (e.g, allelic variants) and derivatives thereof.
• SKPlis The nucleic acid and amino acid sequences of a representative human SKPlis available to the public at the GenBank database (Gene ID 6500) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for SKP1, including at least 2 different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/P63208).
• Human SKPlvariants include transcript variant 1 encoding isoform a (NM_006930.3 and NP_008861.2) and transcript variant 2 encoding isoform b (NM_170679.3 andNP_733779.1).
• Nucleic acid and polypeptide sequences of SKP1 orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM 001166401.6 and XP_001166401.1), dog (NM_001252408.1 and NP_001239337.1), cow (NM_001034781.2 and NP_001029953.1), mouse (NM_011543.4 and NP_035673.3; and XM_006532786.2 and XP_006532849.1), rat (NM_001007608.2 and NP_001007609.1), chicken (NM_001006153.1 and NP_001006153.1; XM_025154856.1 and XP_025010624.1;
• NM_025154857.1 and XP_025010625.1) frog (NM_001016519.3 and NP_001016519.1; XM_0 12959026.3 and XP_012814480.1), fruit fly (NM_166858.3 and NP_726692.1; NM_058042.5 and NP_477390.1; NM_001038729.3 and NP_001033818.1; NM_166857.3 and NP_726691.1; NM_166856.3 and NP_726690.1; NM_166861.3 andNP_726695.1; NM_166860.3 andNP_726694.1; NM_166859.3 and NP_726693.1; NM_001297826.1 and NP 001284755.1).
• SKP1 activity includes the ability of a SKP1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its substrates, which as a SCF complex is involved in cell cycle progression, signal transduction and transcription.
• SKP1 substrate(s) refers to binding partners of a SKP1 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein), e.g ., the proteins listed herein, including Cull and F-box proteins.
• SKP1 -regulated pathway(s) includes pathways in which SKP1 (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• SKP1 -regulated pathways include at least those described herein.
• agents that decrease the copy number, the expression level, and/or the activity of SKPl encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a SKPl polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between SKPl and its substrates or other binding partners.
• the agent may decrease the expression of a SKPl polypeptide.
• such agent may decrease SKPl activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g, the half-life), and/or change the cellular localization of SKPl, resulting in at least a decrease in SKPl levels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents), and gene constructs that inhibit endogenous production of SKPl or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to SKPl or also to at least one of the binding partners, including but not limited to F-box proteins and cullin (e.g., CUL1).
• Antibodies for detection of SKPl are commercially available (Cat. # AM06704SU-N, AM06720SU-N (OriGene); ab76502, ab233484, ab228637 (Abeam); sc-136301, sc-5281 (Santa Cruz).
• RNA interference for SKPl polypeptides are well known and commercially available (e.g, human, rat, or mouse shRNA/siRNA products (Cat.
• BCOR also known as “BCL6 Corepressor” refers to a corepressor that interacts with POZ domain of BCL6. BCOR is also known to interact with classes of histone deacetylases. Alternative splicing results in multiple transcript variants.
• BCOR is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof.
• nucleic acid and amino acid sequences of a representative human BCOR is available to the public at the GenBank database (Gene ID 54880) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for BCOR, including at least four different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/ Q6W2J9).
• Human BCOR variants include the transcript variant 3 encoding isoform a (NM_001123383.1 and NP_001116855.1), transcript variant 4 encoding isoform b (NM_001123384.2 and NP_001116856.1), transcript variant 5 encoding isoform c (NM_001123385.2 and NP_001116857), transcript variant 1 encoding isoform a (NM_017745.6 and NP_060215.4), transcript variant XI encoding isoform XI (XM_005272616.1 and XP_005272673.1), transcript variant X6 encoding isoform XI (XM_011543931.2 and XP_011542233.1), transcript variant X5 encoding isoform XI (XM_011543930.1 and XP_011542232.1), transcript variant X2 encoding isoform XI (XM_011543929.2 and XP_011542231.1), transcript
• BCOR substrate(s) refers to binding partners of a BCOR polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein), e.g., the proteins listed herein, including BCL6.
• BCOR regulated pathway(s) includes pathways in which BCOR (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• BCOR-regulated pathways include at least those described herein, such as positive or negative regulation of histone modification.
• agents that decrease the copy number, the expression level, and/or the activity of BCOR encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a BCOR polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between BCOR and its substrates or other binding partners.
• the agent may decrease the expression of a BCOR polypeptide.
• such agent may decrease BCOR’s activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g., the half-life), and/or change the cellular localization of BCOR, resulting in at least a decrease in BCOR levels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well-known agents), and gene constructs that inhibit endogenous production of BCOR or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to BCOR or also to at least one of the binding partners, including but not limited to BL6.
• Antibodies for detection of BCOR are commercially available (Cat. # AP33297PU-N, CF807724 (OriGene); at>135801, ab88112, abl29777, ab245423, among other, (Abeam); sc-514576 (Santa Cruz).
• RNA interference for BCOR polypeptides are well known and commercially available (e.g., human, rat, or mouse shRNA/siRNA products (Cat. # TL306415V, SR310311, TL504552, TL306415, TL306415V, TF306414, TL519839V, among others, and human or mouse gene knockout kit via CRISPR (Cat.
• BCOR knockout human cell lines are also well known and available at Horizon (Cambridge, UK, Cat. # HZGHC004895c005, HZGHC004895c010).
• YAF2 also known as “YY1 -associated factor 2” refers to a zinc finger polypeptide or a YAF2-encoding polynucleotide that is involved in regulating transcription. YAF2 interacts with Yyl and can promote its proteolysis. YAF2 also binds to MYC and inhibits MYC-mediated transactivation. Multiple alternatively spliced transcript variants are known.
• YAF2 is intended to include fragments, variants (e.g., allelic variants) and derivatives thereof.
• nucleic acid and amino acid sequences of a representative human YAF2 is available to the public at the GenBank database (Gene ID 10138) and is shown in Table 1. Multiple transcript variants encoding several different isoforms have been found for YAF2, including at least four different human transcript variants generated by alternative splicing (see World Wide Web at uniprot.org/uniprot/ Q8IY57).
• Human YAF2 variants include the transcript variant 3 encoding isoform 3 (NM_001190977.2 and NP_001177906.1), transcript variant 1 encoding isoform 1 (NM_001190979.2 and NP_001177908.1), transcript variant 4 encoding isoform 4 (NM_001190980.2 and NP_001177909.1), transcript variant 5 encoding isoform 5 (NM_005748.6 and NP_005739.2), transcript variant XI encoding isoform (XI XM_011537728.3 and XP_011536030.1), transcript variant X2 encoding isoform X2 (XM_024448792.1 and XP_024304560.1), transcript variant X3 encoding isoform X3 (XM_006719185.3 and XP_006719248.1), transcript variant X4 encoding isoform X4(XM_011537729.2 and XP_0115360
• Nucleic acid and polypeptide sequences of YAF2 orthologs in organisms other than humans are well known and include, for example, chimpanzee (XM OOl 167723.5 and XP_001167723.1; XM_016923636.1 and XP_016779125.1; XM_016923633.1 and XP_016779122.1; XM_016923635.1 and XP_016779124.1; and XM_016923634.2 and XP_016779123.1), rhesus macaque (XM_015151457.2 and XP_015006943.1), and dog (XM_022410901.1 and XP_022266609.1; XM_014108587.2 and XP_013964062.1).
• YAF2 activity includes the ability of a YAF2 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein) to bind its
• YAF2 substrate(s) refers to binding partners of a YAF2 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein), e.g., the proteins listed herein, including MYC and Yyl.
• YAF2 regulated pathway(s) includes pathways in which YAF2 (and its fragments, domains, and/or motifs thereof, discussed herein) binds to at least one of its substrate, through which at least one cellular function and/or activity and/or cellular protein profiles is changed.
• YAF2-regulated pathways include at least those described herein, such as positive or negative regulation of histone modification.
• agents that decrease the copy number, the expression level, and/or the activity of YAF2 encompasses any natural or non-natural agent prepared, synthesized, manufactured, and/or purified by human that is capable of decreasing the expression level and/or activity of a YAF2 polypeptide (and its fragments, domains, and/or motifs thereof, discussed herein).
• the agent may decrease the binding/interaction between YAF2 and its substrates or other binding partners.
• the agent may decrease the expression of a YAF2 polypeptide.
• such agent may decrease YAF2 activity in enhancing the immune response against tumors.
• such inhibitors may increase the turnover rate, decrease the expression and/or the stability (e.g., the half-life), and/or change the cellular localization of YAF2, resulting in at least a decrease in YAF2 levels and/or activity.
• agents may be any molecule, including but not limited to small molecule compounds, antibodies or intrabodies, RNA interfering (RNAi) agents (including at least siRNAs, shRNAs, microRNAs (miRNAs), piwi, and other well- known agents), and gene constructs that inhibit endogenous production of YAF2 or its fragments inside cancer cells.
• RNAi RNA interfering
• Such agents may be specific to YAF2 or also to at least one of the binding partners, including but not limited to MYC and Yyl.
• Antibodies for detection of YAF2 are commercially available (Cat. # TA329295 TA329928 (OriGene); ab239150, abl77945, and ab250017 (Abeam); ABIN203352, ABIN1501785, ABIN5621096, ABIN6742302, ABIN6736123, ABIN2568985, ABIN2895187 (antibodies-online.com).
• RNA interference for YAF2 polypeptides are well known and commercially available (e.g., human, rat, or mouse shRNA/siRNA products (Cat. # TL316898V, SR306838, TR316898, TL503808V, TL708870V, TR708870, SR404295, TL708870, SR306838, TR503808, TL316898, TL503808, TL316898V) and human or mouse gene knockout kit via CRISPR (Cat.
• immune response includes T cell mediated and/or B cell mediated immune responses.
• exemplary immune responses include T cell responses, e.g., cytokine production and cellular cytotoxicity.
• immune response includes immune responses that are indirectly effected by T cell activation, e.g, antibody production (humoral responses) and activation of cytokine responsive cells, e.g, macrophages.
• immunotherapeutic agent can include any molecule, peptide, antibody or other agent which can stimulate a host immune system to generate an immune response to a tumor or cancer in the subject.
• Various immunotherapeutic agents are useful in the compositions and methods described herein.
• cancer includes the reduce, decrease, limitation, or blockage, of, for example a particular action, function, or interaction.
• cancer is “inhibited” if at least one symptom of the cancer is alleviated, terminated, slowed, or prevented.
• cancer is also “inhibited” if recurrence or metastasis of the cancer is reduced, slowed, delayed, or prevented.
• interaction when referring to an interaction between two molecules, refers to the physical contact (e.g, binding) of the molecules with one another. Generally, such an interaction results in an activity (which produces a biological effect) of one or both of said molecules.
• isolated protein refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
• isolated or purified protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the antibody, polypeptide, peptide or fusion protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
• the language “substantially free of cellular material” includes preparations of a biomarker polypeptide or fragment thereof, in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced.
• the language “substantially free of cellular material” includes preparations of a biomarker protein or fragment thereof, having less than about 30% (by dry weight) of non biomarker protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-biomarker protein, still more preferably less than about 10% of non biomarker protein, and most preferably less than about 5% non-biomarker protein.
• polypeptide, peptide or fusion protein or fragment thereof e.g ., a biologically active fragment thereof
• it is also preferably substantially free of culture medium, i.e ., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
• isotype refers to the antibody class (e.g, IgM, IgGl, IgG2C, and the like) that is encoded by heavy chain constant region genes.
• KD is intended to refer to the dissociation equilibrium constant of a particular antibody-antigen interaction.
• the binding affinity of antibodies of the disclosed invention may be measured or determined by standard antibody-antigen assays, for example, competitive assays, saturation assays, or standard immunoassays such as ELISA or RIA.
• kits is any manufacture (e.g. a package or container) comprising at least one reagent, e.g. a probe or small molecule, for specifically detecting and/or affecting the expression of a marker of the present invention.
• the kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention.
• the kit may comprise one or more reagents necessary to express a composition useful in the methods of the present invention.
• the kit may further comprise a reference standard, e.g, a nucleic acid encoding a protein that does not affect or regulate signaling pathways controlling cell growth, division, migration, survival or apoptosis.
• neoadjuvant therapy refers to a treatment given before the primary treatment.
• neoadjuvant therapy can include chemotherapy, radiation therapy, and hormone therapy.
• neoadjuvant therapy can allows patients with large breast cancer to undergo breast-conserving surgery.
• an “over-expression” or “significantly higher level of expression” of a biomarker refers to an expression level in a test sample that is greater than the standard error of the assay employed to assess expression, and is preferably at least 10%, and more preferably 1.2, 1.3, 1.4,
• control sample e.g ., sample from a healthy subject not having the biomarker associated disease
• average expression level of the biomarker in several control samples e.g ., the average expression level of the biomarker in several control samples.
• a “significantly lower level of expression” of a biomarker refers to an expression level in a test sample that is at least 10%, and more preferably 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
• control sample e.g., sample from a healthy subject not having the biomarker associated disease
• average expression level of the biomarker in several control samples e.g., 20 times or more lower than the expression level of the biomarker in a control sample (e.g., sample from a healthy subject not having the biomarker associated disease) and preferably, the average expression level of the biomarker in several control samples.
• pre-determined biomarker amount and/or activity measurement(s) may be a biomarker amount and/or activity measurement(s) used to, by way of example only, evaluate a subject that may be selected for a particular treatment, evaluate a response to a treatment such as inhibitor(s) of the regulators of one or more biomarkers listed in Tables 1-5, in combination with an immunotherapy, and/or evaluate the disease state.
• a pre-determined biomarker amount and/or activity measurement(s) may be determined in populations of patients with or without cancer.
• the pre-determined biomarker amount and/or activity measurement s) can be a single number, equally applicable to every patient, or the pre determined biomarker amount and/or activity measurement(s) can vary according to specific subpopulations of patients.
• Age, weight, height, and other factors of a subject may affect the pre-determined biomarker amount and/or activity measurement(s) of the individual.
• the pre-determined biomarker amount and/or activity can be determined for each subject individually.
• the amounts determined and/or compared in a method described herein are based on absolute measurements.
• the amounts determined and/or compared in a method described herein are based on relative measurements, such as ratios (e.g, serum biomarker normalized to the expression of housekeeping or otherwise generally constant biomarker).
• the pre-determined biomarker amount and/or activity measurement(s) can be any suitable standard.
• the pre determined biomarker amount and/or activity measurement(s) can be obtained from the same or a different human for whom a patient selection is being assessed.
• the pre-determined biomarker amount and/or activity measurement(s) can be obtained from a previous assessment of the same patient. In such a manner, the progress of the selection of the patient can be monitored over time.
• the control can be obtained from an assessment of another human or multiple humans, e.g ., selected groups of humans, if the subject is a human.
• the extent of the selection of the human for whom selection is being assessed can be compared to suitable other humans, e.g, other humans who are in a similar situation to the human of interest, such as those suffering from similar or the same condition(s) and/or of the same ethnic group.
• predictive includes the use of a biomarker nucleic acid and/or protein status, e.g, over- or under- activity, emergence, expression, growth, remission, recurrence or resistance of tumors before, during or after therapy, for determining the likelihood of response of a cancer to inhibitor(s) of one or more biomarkers listed in Tables 1-5, in combination with an immunotherapy (e.g., treatment with a combination of such inhibitor and an immunotherapy, such as an immune checkpoint inhibitor).
• an immunotherapy e.g., treatment with a combination of such inhibitor and an immunotherapy, such as an immune checkpoint inhibitor.
• Such predictive use of the biomarker may be confirmed by, e.g, (1) increased or decreased copy number (e.g, by FISH, FISH plus SKY, single-molecule sequencing, e.g, as described in the art at least at J.
• Biotechnol., 86:289-301, or qPCR overexpression or underexpression of a biomarker nucleic acid (e.g, by ISH, Northern Blot, or qPCR), increased or decreased biomarker protein (e.g, by IHC), or increased or decreased activity, e.g, in more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, or more of assayed human cancers types or cancer samples; (2) its absolute or relatively modulated presence or absence in a biological sample, e.g, a sample containing tissue, whole blood, serum, plasma, buccal scrape, saliva, cerebrospinal fluid, urine, stool, or bone marrow, from a subject, e.g. a human, afflicted with cancer; (3) its absolute or relatively modulated presence or absence in clinical subset of patients with cancer (e.
• prevent refers to reducing the probability of developing a disease, disorder, or condition in a subject, who does not have, but is at risk of or susceptible to developing a disease, disorder, or condition.
• probe refers to any molecule which is capable of selectively binding to a specifically intended target molecule, for example, a nucleotide transcript or protein encoded by or corresponding to a biomarker nucleic acid. Probes can be either synthesized by one skilled in the art, or derived from appropriate biological preparations. For purposes of detection of the target molecule, probes may be specifically designed to be labeled, as described herein. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.
• prognosis includes a prediction of the probable course and outcome of cancer or the likelihood of recovery from the disease. In some embodiments, the use of statistical algorithms provides a prognosis of cancer in an individual. For example, the prognosis can be surgery, development of a clinical subtype of cancer (e.g ., solid tumors, such as esophageal cancer and gastric cancer), development of one or more clinical factors, or recovery from the disease.
• response to immunotherapy or “response to inhibitor(s) of one or more biomarkers listed in Tables 1-5, in combination with an immunotherapy” relates to any response of the hyperproliferative disorder (e.g., cancer) to an anti-cancer agent, such as an inhibitor of one or more biomarkers listed in Tables 1-5, and an immunotherapy, preferably to a change in tumor mass and/or volume after initiation of neoadjuvant or adjuvant therapy.
• an anti-cancer agent such as an inhibitor of one or more biomarkers listed in Tables 1-5
• Hyperproliferative disorder response may be assessed, for example for efficacy or in a neoadjuvant or adjuvant situation, where the size of a tumor after systemic intervention can be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound or palpation. Responses may also be assessed by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be recorded in a quantitative fashion like percentage change in tumor volume or in a qualitative fashion like “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinical stable disease” (cSD), “clinical progressive disease” (cPD) or other qualitative criteria.
• pCR pathological complete response
• cCR clinical complete remission
• cPR clinical partial remission
• cSD clinical stable disease
• cPD clinical progressive disease
• Assessment of hyperproliferative disorder response may be done early after the onset of neoadjuvant or adjuvant therapy, e.g, after a few hours, days, weeks or preferably after a few months.
• a typical endpoint for response assessment is upon termination of neoadjuvant chemotherapy or upon surgical removal of residual tumor cells and/or the tumor bed. This is typically three months after initiation of neoadjuvant therapy.
• clinical efficacy of the therapeutic treatments described herein may be determined by measuring the clinical benefit rate (CBR).
• the clinical benefit rate is measured by determining the sum of the percentage of patients who are in complete remission (CR), the number of patients who are in partial remission (PR) and the number of patients having stable disease (SD) at a time point at least 6 months out from the end of therapy.
• the CBR for a particular cancer therapeutic regimen is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or more.
• Additional criteria for evaluating the response to cancer therapies are related to “survival,” which includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
• the length of said survival may be calculated by reference to a defined start point (e.g ., time of diagnosis or start of treatment) and end point (e.g., death, recurrence or metastasis).
• criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
• a particular cancer therapeutic regimen can be administered to a population of subjects and the outcome can be correlated to biomarker measurements that were determined prior to administration of any cancer therapy.
• the outcome measurement may be pathologic response to therapy given in the neoadjuvant setting.
• outcome measures such as overall survival and disease-free survival can be monitored over a period of time for subjects following cancer therapy for which biomarker measurement values are known.
• the doses administered are standard doses known in the art for cancer therapeutic agents. The period of time for which subjects are monitored can vary.
• Biomarker measurement threshold values that correlate to outcome of a cancer therapy can be determined using well-known methods in the art, such as those described in the Examples section.
• resistance refers to an acquired or natural resistance of a cancer sample or a mammal to a cancer therapy (i.e., being nonresponsive to or having reduced or limited response to the therapeutic treatment), such as having a reduced response to a therapeutic treatment by 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2- fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more.
• the reduction in response can be measured by comparing with the same cancer sample or mammal before the resistance is acquired, or by comparing with a different cancer sample or a mammal that is known to have no resistance to the therapeutic treatment.
• multidrug resistance A typical acquired resistance to chemotherapy is called “multidrug resistance.”
• the multidrug resistance can be mediated by P-glycoprotein or can be mediated by other mechanisms, or it can occur when a mammal is infected with a multi -drug-resistant microorganism or a combination of microorganisms.
• the term “reverses resistance” means that the use of a second agent in combination with a primary cancer therapy (e.g ., chemotherapeutic or radiation therapy) is able to produce a significant decrease in tumor volume at a level of statistical significance (e.g., p ⁇ 0.05) when compared to tumor volume of untreated tumor in the circumstance where the primary cancer therapy (e.g, chemotherapeutic or radiation therapy) alone is unable to produce a statistically significant decrease in tumor volume compared to tumor volume of untreated tumor. This generally applies to tumor volume measurements made at a time when the untreated tumor is growing log rhythmically.
• a primary cancer therapy e.g ., chemotherapeutic or radiation therapy
• response refers to an anti-cancer response, e.g. in the sense of reduction of tumor size or inhibiting tumor growth.
• the terms can also refer to an improved prognosis, for example, as reflected by an increased time to recurrence, which is the period to first recurrence censoring for second primary cancer as a first event or death without evidence of recurrence, or an increased overall survival, which is the period from treatment to death from any cause.
• To respond or to have a response means there is a beneficial endpoint attained when exposed to a stimulus. Alternatively, a negative or detrimental symptom is minimized, mitigated or attenuated on exposure to a stimulus. It will be appreciated that evaluating the likelihood that a tumor or subject will exhibit a favorable response is equivalent to evaluating the likelihood that the tumor or subject will not exhibit favorable response (i.e., will exhibit a lack of response or be non-responsive).
• RNA interfering agent is defined as any agent which interferes with or inhibits expression of a target biomarker gene by RNA interference (RNAi).
• RNA interfering agents include, but are not limited to, nucleic acid molecules including RNA molecules which are homologous to the target biomarker gene of the present invention, or a fragment thereof, short interfering RNA (siRNA), and small molecules which interfere with or inhibit expression of a target biomarker nucleic acid by RNA interference (RNAi).
• RNA interference is an evolutionally conserved process whereby the expression or introduction of RNA of a sequence that is identical or highly similar to a target biomarker nucleic acid results in the sequence specific degradation or specific post- transcriptional gene silencing (PTGS) of messenger RNA (mRNA) transcribed from that targeted gene (see Coburn and Cullen (2002) ./. Virol. 76:9225), thereby inhibiting expression of the target biomarker nucleic acid.
• mRNA messenger RNA
• dsRNA double stranded RNA
• RNAi is initiated by the dsRNA-specific endonuclease Dicer, which promotes processive cleavage of long dsRNA into double-stranded fragments termed siRNAs.
• siRNAs are incorporated into a protein complex that recognizes and cleaves target mRNAs.
• RNAi can also be initiated by introducing nucleic acid molecules, e.g., synthetic siRNAs or RNA interfering agents, to inhibit or silence the expression of target biomarker nucleic acids.
• “inhibition of target biomarker nucleic acid expression” or “inhibition of marker gene expression” includes any decrease in expression or protein activity or level of the target biomarker nucleic acid or protein encoded by the target biomarker nucleic acid.
• the decrease may be of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the expression of a target biomarker nucleic acid or the activity or level of the protein encoded by a target biomarker nucleic acid which has not been targeted by an RNA interfering agent.
• sample used for detecting or determining the presence or level of at least one biomarker is typically brain tissue, cerebrospinal fluid, whole blood, plasma, serum, saliva, urine, stool (e.g, feces), tears, and any other bodily fluid (e.g, as described above under the definition of “body fluids”), or a tissue sample (e.g, biopsy) such as a small intestine, colon sample, or surgical resection tissue.
• body fluids e.g, a tissue sample such as a small intestine, colon sample, or surgical resection tissue.
• the method of the present invention further comprises obtaining the sample from the individual prior to detecting or determining the presence or level of at least one marker in the sample.
• cancer means to alter cancer cells or tumor cells in a way that allows for more effective treatment of the associated cancer with a cancer therapy (e.g, anti-immune checkpoint, chemotherapeutic, and/or radiation therapy).
• a cancer therapy e.g, anti-immune checkpoint, chemotherapeutic, and/or radiation therapy.
• normal cells are not affected to an extent that causes the normal cells to be unduly injured by the therapies.
• An increased sensitivity or a reduced sensitivity to a therapeutic treatment is measured according to a known method in the art for the particular treatment and methods described herein below, including, but not limited to, cell proliferative assays (Tanigawa N, Kern D H, Kikasa Y, Morton D L, Cancer Res.
• the sensitivity or resistance may also be measured in animal by measuring the tumor size reduction over a period of time, for example, 6 month for human and 4-6 weeks for mouse.
• a composition or a method sensitizes response to a therapeutic treatment if the increase in treatment sensitivity or the reduction in resistance is 25% or more, for example, 30%, 40%, 50%, 60%, 70%, 80%, or more, to 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold or more, compared to treatment sensitivity or resistance in the absence of such composition or method.
• the determination of sensitivity or resistance to a therapeutic treatment is routine in the art and within the skill of an ordinarily skilled clinician. It is to be understood that any method described herein for enhancing the efficacy of a cancer therapy can be equally applied to methods for sensitizing hyperproliferative or otherwise cancerous cells (e.g., resistant cells) to the cancer therapy.
• siRNA Short interfering RNA
• small interfering RNA is defined as an agent which functions to inhibit expression of a target biomarker nucleic acid, e.g, by RNAi.
• An siRNA may be chemically synthesized, may be produced by in vitro transcription, or may be produced within a host cell.
• siRNA is a double stranded RNA (dsRNA) molecule of about 15 to about 40 nucleotides in length, preferably about 15 to about 28 nucleotides, more preferably about 19 to about 25 nucleotides in length, and more preferably about 19, 20, 21, or 22 nucleotides in length, and may contain a 3’ and/or 5’ overhang on each strand having a length of about 0, 1, 2, 3, 4, or 5 nucleotides.
• the length of the overhang is independent between the two strands, i.e., the length of the overhang on one strand is not dependent on the length of the overhang on the second strand.
• the siRNA is capable of promoting RNA interference through degradation or specific post-transcriptional gene silencing (PTGS) of the target messenger RNA (mRNA).
• PTGS post-transcriptional gene silencing
• an siRNA is a small hairpin (also called stem loop) RNA (shRNA).
• shRNAs are composed of a short (e.g, 19-25 nucleotide) antisense strand, followed by a 5-9 nucleotide loop, and the analogous sense strand.
• the sense strand may precede the nucleotide loop structure and the antisense strand may follow.
• shRNAs may be contained in plasmids, retroviruses, and lentiviruses and expressed from, for example, the pol III U6 promoter, or another promoter (see, e.g., Stewart, etal. (2003) RNA Apr;9(4):493-501 incorporated by reference herein).
• RNA interfering agents e.g., siRNA molecules
• a biomarker gene that is involved in downregulating MHC class I surface expression, such as HLA class I surface expression, in cancer and thereby treat, prevent, or inhibit cancer in the subject.
• small molecule is a term of the art and includes molecules that are less than about 1000 molecular weight or less than about 500 molecular weight. In one embodiment, small molecules do not exclusively comprise peptide bonds. In another embodiment, small molecules are not oligomeric. Exemplary small molecule compounds which can be screened for activity include, but are not limited to, peptides, peptidomimetics, nucleic acids, carbohydrates, small organic molecules (e.g, polyketides) (Cane etal. (1998) Science 282:63), and natural product extract libraries. In another embodiment, the compounds are small, organic non-peptidic compounds. In a further embodiment, a small molecule is not biosynthetic.
• the term “specific binding” refers to antibody binding to a predetermined antigen.
• the antibody binds with an affinity (KD) of approximately less than 10 '7 M, such as approximately less than 10 '8 M, 10 '9 M or 10 '10 M or even lower when determined by surface plasmon resonance (SPR) technology in a BIACORE® assay instrument using an antigen of interest as the analyte and the antibody as the ligand, and binds to the predetermined antigen with an affinity that is at least 1.1-, 1.2-, 1.3-, 1.4-, 1.5-, 1.6-, 1.7-, 1.8-, 1.9-, 2.0-, 2.5-, 3.0-, 3.5-, 4.0-, 4.5-, 5.0-, 6.0-, 7.0-, 8.0-, 9.0-, or 10.0-fold or greater than its affinity for binding to a nonspecific antigen (e.g, BSA, casein) other than the predetermined antigen or a closely- related antigen.
• an antibody recognizing an antigen and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.” Selective binding is a relative term referring to the ability of an antibody to discriminate the binding of one antigen over another.
• protein complex means a composite unit that is a combination of two or more proteins formed by interaction between the proteins.
• a "protein complex” is formed by the binding of two or more proteins together through specific non-covalent binding interactions.
• covalent bonds may also be present between the interacting partners.
• the two interacting partners can be covalently crosslinked so that the protein complex becomes more stable.
• the protein complex may or may not include and/or be associated with other molecules such as nucleic acid, such as RNA or DNA, or lipids or further cofactors or moieties selected from a metal ions, hormones, second messengers, phosphate, sugars.
• a "protein complex” encompassed by the present invention may also be part of or a unit of a larger physiological protein assembly.
• isolated protein complex means a protein complex present in a composition or environment that is different from that found in nature, in its native or original cellular or body environment.
• an isolated protein complex is separated from at least 50%, more preferably at least 75%, most preferably at least 90% of other naturally co-existing cellular or tissue components.
• an "isolated protein complex” may also be a naturally existing protein complex in an artificial preparation or a non-native host cell.
• An “isolated protein complex” may also be a "purified protein complex", that is, a substantially purified form in a substantially homogenous preparation substantially free of other cellular components, other polypeptides, viral materials, or culture medium, or, when the protein components in the protein complex are chemically synthesized, free of chemical precursors or by-products associated with the chemical synthesis.
• a “purified protein complex” typically means a preparation containing preferably at least 75%, more preferably at least 85%, and most preferably at least 95% of a particular protein complex.
• a “purified protein complex” may be obtained from natural or recombinant host cells or other body samples by standard purification techniques, or by chemical synthesis.
• modified polypeptide or “modified protein complex” refers to a polypeptide or a protein complex present in a composition that is different from that found in nature in its native or original cellular or body environment.
• modification refers to all modifications of a protein or protein complex encompassed by the present invention including cleavage and addition or removal of a group.
• the "modified polypeptide” or “modified protein complex” comprises at least one modification (e.g ., fragment, mutation, and the like) or subunit that is modified, /. e. , different from that found in nature, in its native or original cellular or body environment.
• the "modified subunit” may be, e.g., a derivative or fragment of the native subunit from which it derives.
• activity when used in connection with proteins or protein complexes means any physiological or biochemical activities displayed by or associated with a particular protein or protein complex including but not limited to activities exhibited in biological processes and cellular functions, ability to interact with or bind another molecule or a moiety thereof, binding affinity or specificity to certain molecules, in vitro or in vivo stability (e.g, protein degradation rate, or in the case of protein complexes ability to maintain the form of protein complex), antigenicity and immunogenicity, enzymatic activities, etc. Such activities may be detected or assayed by any of a variety of suitable methods as will be apparent to skilled artisans.
• interaction antagonist means a compound that interferes with, blocks, disrupts or destabilizes a protein-protein interaction; blocks or interferes with the formation of a protein complex, or destabilizes, disrupts or dissociates an existing protein complex.
• interaction agonist means a compound that triggers, initiates, propagates, nucleates, or otherwise enhances the formation of a protein interaction; triggers, initiates, propagates, nucleates, or otherwise enhances the formation of a protein complex; or stabilizes an existing protein complex.
• PRC1.1 complex refers to a complex of proteins comprising USP7, KDM2B, BCOR or BCORLl, RING1 A, RING1B, RYBP/YAF2, PCGF1, and SKP1.
• Loss of PRC1.1 has been shown to accelerate development of sonic hedgehog-driven medulloblastoma (Kutscher et al. (2020) bioRxiv 2020.02.06.938035).
• the complex has been studied in the context of the commitment of hematopoietic stem and progenitor cells (HSPCs). PRC 1.1 insufficiency in these cells induced myeloid-based differentiation, leading to the myeloid malignancies (Iwama (2016) Exp. Hematol. 64: S39).
• subject refers to any healthy animal, mammal or human, or any animal, mammal or human afflicted with a cancer, e.g ., brain, lung, ovarian, pancreatic, liver, breast, prostate, and/or colorectal cancers, melanoma, multiple myeloma, and the like.
• a cancer e.g ., brain, lung, ovarian, pancreatic, liver, breast, prostate, and/or colorectal cancers, melanoma, multiple myeloma, and the like.
• subject is interchangeable with “patient.”
• survival includes all of the following: survival until mortality, also known as overall survival (wherein said mortality may be either irrespective of cause or tumor related); “recurrence-free survival” (wherein the term recurrence shall include both localized and distant recurrence); metastasis free survival; disease free survival (wherein the term disease shall include cancer and diseases associated therewith).
• the length of said survival may be calculated by reference to a defined start point (e.g. time of diagnosis or start of treatment) and end point (e.g. death, recurrence or metastasis).
• criteria for efficacy of treatment can be expanded to include response to chemotherapy, probability of survival, probability of metastasis within a given time period, and probability of tumor recurrence.
• the term “synergistic effect” refers to the combined effect of two or more anti-cancer agents (e.g, inhibitor(s) of one or more biomarkers listed in Tables 1-5, in combination with an immunotherapy) can be greater than the sum of the separate effects of the anti-cancer agents/therapies alone.
• T cell includes CD4 + T cells and CD8 + T cells.
• T cell also includes both T helper 1 type T cells and T helper 2 type T cells.
• antigen presenting cell includes professional antigen presenting cells (e.g ., B lymphocytes, monocytes, dendritic cells, Langerhans cells), as well as other antigen presenting cells (e.g., keratinocytes, endothelial cells, astrocytes, fibroblasts, and oligodendrocytes).
• therapeutic effect refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance.
• the term thus means any substance intended for use in the diagnosis, cure, mitigation, treatment or prevention of disease or in the enhancement of desirable physical or mental development and conditions in an animal or human.
• therapeutically- effective amount means that amount of such a substance that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment.
• a therapeutically effective amount of a compound will depend on its therapeutic index, solubility, and the like.
• certain compounds discovered by the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.
• the terms “therapeutically effective amount” and “effective amount” may be that amount of a compound, material, or composition comprising an agent that is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
• Toxicity and therapeutic efficacy of subject compounds may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g, for determining the LD50 and the EDso. Compositions that exhibit large therapeutic indices are preferred.
• the LDso lethal dosage
• the EDso i.e., the concentration which achieves a half-maximal inhibition of symptoms
• the concentration which achieves a half-maximal inhibition of symptoms can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent.
• the ICso i.e., the concentration which achieves half-maximal cytotoxic or cytostatic effect on cancer cells
• the ICso can be measured and can be, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more increased for the agent relative to no administration of the agent.
• cancer cell growth in an assay can be inhibited by at least about 10%,
• a “transcribed polynucleotide” or “nucleotide transcript” is a polynucleotide (e.g . an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA) which is complementary to or homologous with all or a portion of a mature mRNA made by transcription of a biomarker nucleic acid and normal post-transcriptional processing (e.g. splicing), if any, of the RNA transcript, and reverse transcription of the RNA transcript.
• a polynucleotide e.g . an mRNA, hnRNA, a cDNA, or an analog of such RNA or cDNA
• the term “unresponsiveness” includes refractivity of cancer cells to therapy or refractivity of therapeutic cells, such as immune cells, to stimulation, e.g, stimulation via an activating receptor or a cytokine. Unresponsiveness can occur, e.g, because of exposure to immunosuppressants or exposure to high doses of antigen.
• the term “anergy” or “tolerance” includes refractivity to activating receptor-mediated stimulation. Such refractivity is generally antigen-specific and persists after exposure to the tolerizing antigen has ceased. For example, anergy in T cells (as opposed to unresponsiveness) is characterized by lack of cytokine production, e.g, IL-2.
• T cell anergy occurs when T cells are exposed to antigen and receive a first signal (a T cell receptor or CD- 3 mediated signal) in the absence of a second signal (a costimulatory signal). Under these conditions, reexposure of the cells to the same antigen (even if reexposure occurs in the presence of a costimulatory polypeptide) results in failure to produce cytokines and, thus, failure to proliferate.
• Anergic T cells can, however, proliferate if cultured with cytokines (e.g, IL-2).
• cytokines e.g, IL-2
• T cell anergy can also be observed by the lack of IL-2 production by T lymphocytes as measured by ELISA or by a proliferation assay using an indicator cell line.
• a reporter gene construct can be used.
• anergic T cells fail to initiate IL-2 gene transcription induced by a heterologous promoter under the control of the 5’ IL-2 gene enhancer or by a multimer of the API sequence that can be found within the enhancer (Kang et al. (1992) Science 257: 1134).
• Lysine (Lys, K) AAA, AAG Methionine (Met, M) ATG Phenylalanine (Phe, F_) TTC, TTT Proline (Pro, P) CCA, CCC, CCG, CCT
• Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT
• Threonine Thr, T
• ACA Threonine
• ACC ACC
• ACG ACT Tryptophan
• Trp, W TGG Tyrosine
• Tyr, Y TAC, TAT
• nucleotide triplet An important and well-known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
• nucleotide sequence of a DNA or RNA encoding a biomarker nucleic acid can be used to derive the polypeptide amino acid sequence, using the genetic code to translate the DNA or RNA into an amino acid sequence.
• polypeptide amino acid sequence corresponding nucleotide sequences that can encode the polypeptide can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
• description and/or disclosure herein of a nucleotide sequence which encodes a polypeptide should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
• description and/or disclosure of a polypeptide amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
• nucleic acid and amino acid sequence information for the loci and biomarkers of the present invention are well-known in the art and readily available on publicly available databases, such as the National Center for Information (NCBI).
• NCBI National Center for Information
• exemplary nucleic acid and amino acid sequences derived from publicly available sequence databases are provided below and include, for example, PCT Publ. WO 2014/022759, which is incorporated herein in its entirety by this reference. Table 5

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