Methods and Compositions for the Treatment and Diagnosis of SAGE1-Associated Conditions

Abstract

This disclosure describes methods and kits for diagnosing and determining SAGE1-positive diseases, and compositions and methods for treating SAGE1-positive diseases. This disclosure further describes the SAGE1 complex, and methods and compositions for inhibiting the SAGE1 complex and treating SAGE1-positive diseases.

Description

[Technical field]

[0001] The present disclosure relates generally to methods for diagnosing disease using SAGE1 as a biomarker, and to compositions and methods for treating disease that target SAGE1. [Background technology]

[0002] Cancer-testis antigens (CTAs) are a heterogeneous group of proteins with over 70 families and over 140 members (Fratta E. et al., Mol Oncol (2011) 5: 164). Although expression of CTA has been reported in various tumors, its expression in normal tissues is mainly restricted to the testis. Since CTA is expressed exclusively in tumors and the immune-privileged testis, it has been studied in recent years as an antigen for cancer immunotherapy and vaccine development. However, the function of CTA in tumor cell proliferation has not yet been fully elucidated.

[0003] Sarcoma antigen 1 (SAGE1) belongs to the CTA family. Nearly 20 years have passed since SAGE1 was discovered as a tumor-specific antigen, but it remains unclear if and how it plays a role in promoting tumor growth and progression. As there is a continuing need to develop effective tumor diagnostics and therapeutics, it is desirable to elucidate the role of SAGE1 in tumor cell proliferation and explore its potential to serve as a novel diagnostic marker and therapeutic target for tumors. Summary of the Invention

[0004] In one aspect, the method disclosed herein is a method for diagnosing a SAGE1-positive disease in a subject, comprising the steps of detecting SAGE1 expression in a biological sample from the subject, and assessing whether the subject has a SAGE1-positive disease based on the presence or absence or level of SAGE1 expression detected in the biological sample.

[0005] In one aspect, the method disclosed herein is a method for diagnosing a SAGE1-positive disease in a subject, comprising the step of detecting SAGE1 expression in a biological sample from the subject, and diagnosing the subject as having a SAGE1-positive disease if SAGE1 expression is detected in the biological sample.

[0006] In some embodiments, the biological sample is of a type that normally lacks detectable SAGE1 expression.

[0007] In some embodiments, the subject has been determined to have a TP53 deficiency (eg, mutation).

[0008] In some embodiments, the SAGE1 positive disease is a SAGE1 positive tumor.

[0009] In another aspect, a method disclosed herein is a method for determining the malignancy or potential for malignancy of a tumor in a subject, comprising the step of detecting SAGE1 expression in a biological sample from the subject, and determining that the tumor is malignant or has the potential to become malignant if SAGE1 expression is detected in the biological sample.

[0010] In another aspect, the disclosed method is a method for determining the aggressiveness or aggressiveness of a tumor in a subject, comprising the steps of detecting SAGE1 expression in a biological sample from the subject, and determining whether or not the tumor is aggressive based on the presence or absence or level of SAGE1 expression detected in the biological sample. and assessing whether the subject has a malignant or potentially malignant tumor based on the results of the analysis.

[0011] In some embodiments, the biological sample is of a type that normally lacks detectable SAGE1 expression.

[0012] In some embodiments, the malignancy is characterized by the following characteristics: (a) expressing one or more stem cell markers; (b) is transferable; (c) allowing uncontrolled proliferation of cells; (d) the disease is likely to progress; (e) there is a high likelihood of developing resistance to anticancer treatments; (f) a high likelihood of recurrence following anti-cancer treatment; (g) having one or more of the following characteristics: having or likely to have one or more cancer-associated driver mutations;

[0013] In some embodiments, the subject is free of clinical symptoms of malignancy.

[0014] In another aspect, a method of the disclosure is a method for predicating a likelihood that a subject has SAGE1 positive disease, comprising the step of detecting TP53 deficiency in a first biological sample from the subject, and predicating that the subject is likely to have SAGE1 positive disease if TP53 deficiency is detected.

[0015] In another aspect, a method disclosed herein is a method for predicating a possibility that a subject has SAGE1 positive disease, comprising the steps of detecting TP53 deficiency in a first biological sample from the subject, and assessing whether the subject is likely to have SAGE1 positive disease based on the presence or absence of the TP53 deficiency detected in the first biological sample.

[0016] In some embodiments, the method further comprises the step of recommending that the subject be tested for SAGE1 expression if a TP53 deficiency (eg, mutation) is detected.

[0017] In some embodiments, the method further comprises detecting SAGE1 expression in the first biological sample or in a second biological sample from the subject.

[0018] In some embodiments, if SAGE1 expression is not detected in the biological sample, the method further comprises monitoring SAGE1 expression in the subject over time.

[0019] In some embodiments, the first or second biological sample is of a type that normally lacks detectable SAGE1 expression.

[0020] In some embodiments, the method further comprises administering to the subject an effective amount of a SAGE1 inhibitor upon detecting SAGE1 expression in the first biological sample or in the additional second biological sample.

[0021] In another aspect, the method disclosed herein is a method for determining the sensitivity of a tumor sample to a SAGE1 inhibitor, comprising a step of detecting SAGE1 expression in the tumor sample, and determining that the tumor sample is sensitive to a SAGE1 inhibitor if SAGE1 expression is detected in the tumor sample.

[0022] In another aspect, the method disclosed herein is a method for determining the sensitivity of a tumor sample to a SAGE1 inhibitor, comprising the steps of detecting SAGE1 expression in the tumor sample and evaluating whether the tumor sample is sensitive to the SAGE1 inhibitor based on the presence or absence or level of SAGE1 expression detected in the tumor sample.

[0023] In another aspect, the method disclosed herein is a method for identifying a subject having a tumor that is likely to benefit from the treatment of a SAGE1 inhibitor, comprising the step of detecting SAGE1 expression in a biological sample from the subject, and identifying the subject as likely to benefit from the treatment of a SAGE1 inhibitor if SAGE1 expression is detected.

[0024] In another aspect, the method disclosed herein is a method for identifying a subject having a tumor that is likely to benefit from the treatment of a SAGE1 inhibitor, comprising the steps of detecting SAGE1 expression in a biological sample from the subject, and assessing whether the subject is likely to benefit from the treatment of a SAGE1 inhibitor based on the presence or absence or level of SAGE1 expression detected in the biological sample.

[0025] In some embodiments, the subject has been determined to have a mutation in TP53.

[0026] In some embodiments, the method further comprises providing or administering a SAGE1 inhibitor to the subject identified as likely to benefit from the treatment with a SAGE1 inhibitor.

[0027] In another aspect, the method disclosed herein is a method for monitoring the progression of SAGE1 positive disease in a subject, comprising steps (a) of detecting a level of SAGE1 expression in a first biological sample from the subject and (b) of detecting a level of SAGE1 expression in a second biological sample from the subject, the second biological sample being obtained at a time from the first sample, and determining that the tumor is progressing if the level of SAGE1 expression is higher in the second biological sample than in the first biological sample.

[0028] In another aspect, the method disclosed herein is a method for monitoring the progression of a SAGE1-positive disease in a subject, comprising the steps of (a) detecting a level of SAGE1 expression in a first biological sample from the subject, (b) detecting a level of SAGE1 expression in a second biological sample from the subject, and (c) assessing whether the SAGE1-positive disease is progressing in the subject based on the levels of SAGE1 expression detected in each of steps (a) and (b), wherein the second biological sample is obtained at a time later than the first sample.

[0029] In another aspect, the method disclosed herein is a method for monitoring the responsiveness of a subject with a SAGE1-positive disease to treatment with a SAGE1 inhibitor during a treatment period, comprising the steps of (a) detecting the level of SAGE1 expression in a biological sample from the subject after the treatment period to obtain a post-treatment SAGE1 expression level, and (b) comparing the post-treatment SAGE1 expression level with a baseline level of SAGE1 expression detected in a biological sample obtained from the subject prior to the treatment period, and identifying the subject as responsive to the treatment if the post-treatment SAGE1 expression level is lower than the baseline level.

[0030] In another embodiment, the disclosed method is a method for monitoring responsiveness to treatment with a SAGE1 inhibitor during treatment of a subject with a SAGE1 positive disease, the treatment comprising: The method includes the steps of (a) detecting the level of SAGE1 expression in the subject's biological sample after a period of time to obtain a post-treatment SAGE1 expression level, (b) comparing the post-treatment SAGE1 expression level with a baseline level of SAGE1 expression detected in a biological sample obtained from the subject before the treatment period, and (c) assessing whether or not there is responsiveness to the treatment with the SAGE1 inhibitor based on the levels of SAGE1 expression detected in each of steps (a) and (b).

[0031] In some embodiments, the SAGE1 expression is detected at the DNA level, the RNA level, or the protein level.

[0032] In some embodiments, the SAGE1 expression is (a) the presence or level of SAGE1 protein, (b) the presence or level of SAGE1 mRNA; (c) the presence or level of SAGE1 complex; (d) the level of demethylation of the SAGE1 gene; (e) the presence or level of histone acetylation in the SAGE1 gene; (f) the presence or level of transcription factor binding to the SAGE1 gene; or any combination thereof.

[0033] In some embodiments, the SAGE1 complex comprises SAGE1 and at least one component selected from INTS3, INIP, NABP1 / 2, CREBBP, TLE1, TLE2, TLE3, TLE4, TLE5, GGA3, CNOT1, TAX1BP1, SEC16A, CYLD, and PAXBP1.

[0034] In some embodiments, the SAGE1 complex comprises SAGE1 and INTS3.

[0035] In some embodiments, the methylation is detected in a region within 3 kb upstream and within 3 kb downstream of the transcription start site of the SAGE1 gene (eg, a CpG-containing portion of the SAGE1 gene).

[0036] In some embodiments, the histone acetylation is detected near an enhancer region, a promoter region, or an expressed region of the SAGE1 gene.

[0037] In some embodiments, the detection comprises an immunological test, an amplification test, a hybridization assay, or a sequencing assay.

[0038] In some embodiments, the biological sample is selected from cells, tissues, bodily fluids, and combinations thereof.

[0039] In some embodiments, the body fluid is selected from blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple fluid, lymphatic fluid, fluids of the respiratory, intestinal or genitourinary tract, breast milk, fluids of organ systems, peritoneal fluid, conditioned medium obtained from the culture of tissue explants, or combinations thereof.

[0040] In some embodiments, the tumor is selected from a solid tumor or a hematological tumor.

[0041] In some embodiments, the tumor is selected from the group consisting of adrenal gland cancer, lymphoepithelial tumor, adenoid cell carcinoma, lymphoma, acoustic neuroma, acute lymphocytic leukemia, acrolentiginous melanoma, acute myeloid leukemia, acrohidroma, chronic lymphocytic leukemia, acute eosinophilic leukemia, liver cancer, acute erythrocytic leukemia, small cell lung cancer, Acute lymphocytic leukemia, non-small cell lung cancer, acute megakaryoblastic leukemia, MALT lymphoma, acute monocytic leukemia, malignant fibrous histiocytoma, acute promyelocytic leukemia, malignant peripheral nerve sheath tumor, mantle cell lymphoma, adenocarcinoma, marginal zone B-cell lymphoma, malignant tumor of the hippocampus, adenoid cystic carcinoma, adenomatous, adenoid odontogenic tumor, mast cell leukemia, adenosquamous carcinoma, mediastinal germ cell tumor, adipose tissue tumor, medullary breast carcinoma, adrenal cortical carcinoma, medullary thyroid carcinoma, adult T-cell leukemia / lymphoma, medulloblastoma, invasive NK cell leukemia, melanoma, AIDS-related lymphoma, meningioma, pulmonary rhabdomyosarcoma, Merkel cell carcinoma, alveolar soft tissue sarcoma, mesothelioma , ameloblastoma, metastatic urothelial carcinoma, anaplastic large cell lymphoma, mixed Müllerian tumor, anaplastic thyroid carcinoma, mucinous tumor, angioimmunoblastic T-cell lymphoma, multiple myeloma, angiomyolipoma, muscle tissue tumor, angiosarcoma, mycosis fungoides, astrocytoma, myxoid liposarcoma, atypical teratoid rhabdoid tumor, myxoma, B-cell chronic lymphocytic leukemia, myxosarcoma, B-cell lymphocytic leukemia, nasopharyngeal carcinoma, B-cell lymphoma, schwannoma, basal cell carcinoma, neuroblastoma, biliary tract cancer, neurofibromatosis, bladder cancer, neuroma, blastoma, nodular melanoma, bone cancer, eye cancer, Brenner tumor, oligodendroglial, brown tumor, oligodendrocyte Dysentroglioma, Burkitt's lymphoma, eosinophilic breast cancer, brain tumor, optic nerve tumor, oral intraepithelial neoplasia, osteosarcoma, carcinosarcoma, ovarian cancer, bone and soft tissue tumor, pulmonary sulcus tumor, papillary thyroid carcinoma, myeloma, paraganglioma, chondroma, pineoblastoma, chordoma, pineocytoma, choriocarcinoma, pituitary tumor, choroid plexus papilloma, pituitary adenoma, renal clear cell sarcoma, pituitary tumor, craniopharyngioma, plasmacytoma, cutaneous T-cell lymphoma, polyembryoma, cervical cancer, precursor T-lymphoblastic lymphoma, colorectal cancer, primary central nervous system lymphoma, Degos disease, primary effusion lymphoma, proliferative small round cell tumor, primary peritoneal precancerous tumor, diffuse large B-cell lymphoma tumor, prostate cancer, germinal dysplasia of neuroepithelial tumor, pancreatic cancer, anaplastic cell tumor, pharyngeal cancer, embryonal carcinoma, pseudomyxoma peritonei, endocrine tumor, renal cell carcinoma, enteropathy-associated T-cell lymphoma, endodermal sinus tumor, renal medullary carcinoma, retinoblastoma, esophageal cancer, rhabdomyosarcoma, endodermal cell carcinoma, myoma, Richter transformation, fibrosarcoma, rectal cancer, follicular lymphoma, sarcoma, follicular thyroid carcinoma, schwannoma, ganglioneuroma, seminoma, gastrointestinal cancer, Sertoli cell tumor, germinoma, sex cord-gonadal stromal tumor, gestational choriocarcinoma, signet ring cell tumor, giant cell fibroblastoma, skin cancer, giant cell tumor of bone, small round blue cell tumor, glioma,Small cell carcinoma, glioblastoma multiforme, soft tissue sarcoma, glioma, somatostatin tumor, brain glioma, sooty wart, pancreatic hyperglucagonoma, spinal tumor, gonadoblastoma, splenic marginal lymphoma, granulosa cell tumor, squamous cell carcinoma, estrogen tumor, synovial sarcoma, gallbladder cancer, Sezary disease, gastric cancer, small intestine cancer, hairy cell leukemia, squamous cell carcinoma, hemangioblastoma, gastric cancer, head and neck cancer, T-cell lymphoma, hemangioepithelioma, testicular cancer, hematological malignancies, sarcoma, hepatoblastoma, thyroid cancer adenocarcinoma, hepatosplenic T-cell lymphoma, transitional cell carcinoma, Hodgkin's lymphoma, laryngeal cancer, non-Hodgkin's lymphoma, urinary tract cancer, invasive lobular carcinoma, genitourinary cancer, intestinal cancer, urothelial carcinoma, kidney cancer, uveal melanoma, laryngeal cancer, uterine cancer, malignant freckled sputum, verrucous carcinoma, lethal midline granuloma, optic pathway glioma, leukemia, vulvar cancer, testicular stromal tumor, vaginal cancer, liposarcoma, Waldenstrom's macroglobulinemia, lung cancer, adenolymphoma lymphangioma, Nephroblastoma, lymphosarcoma, colon adenocarcinoma (coad), liver hepatocellular carcinoma (lihc), ovarian serous cystadenocarcinoma (ov), uterine endometrial carcinoma (ucec), thyroid cancer (thca), skin cutaneous melanoma (skcm), lung adenocarcinoma (luad), head and neck squamous cell carcinoma (hnsc), glioblastoma multiforme (gbm), prostatic adenocarcinoma (prad), thymoma (thym), brain low-grade glioma (lgg), rectal adenocarcinoma (read), pheochromocytoma and Selected from paraganglioma (PCPG), esophageal carcinoma (ESCA), renal clear cell carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), renal papillary cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), renal chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), acute myeloid leukemia (LAML).

[0042] In another embodiment, a kit of the disclosure, for use in any of the methods described herein, comprises one or more reagents for detecting SAGE1 expression.

[0043] In some embodiments, the one or more reagents for detecting SAGE1 expression include: (a) a reagent for detecting the presence or level of SAGE1 protein; (b) a reagent for detecting the presence or level of SAGE1 mRNA; (c) a reagent for detecting the presence or level of a SAGE1 complex; (d) a reagent for detecting the methylation level of the SAGE1 gene; (e) a reagent for detecting the presence or level of histone acetylation in the SAGE1 gene; (f) a reagent for detecting the presence or level of binding of a transcription factor to the SAGE1 gene; and combinations thereof.

[0044] In some embodiments, the at least one reagent is selected from a SAGE1-specific oligonucleotide primer, a SAGE1-specific oligonucleotide probe, an anti-SAGE1 antibody, and an anti-SAGE1 complex antibody.

[0045] In some embodiments, the SAGE1-specific oligonucleotide primer or probe comprises an oligonucleotide comprising or specifically hybridizable to 8 or more consecutive nucleotides of SEQ ID NO:2 or 8 or more consecutive nucleotides of SEQ ID NO:1.

[0046] In another embodiment, the SAGE1 complex inhibitors of the present disclosure reduce the level or activity of the SAGE1 complex.

[0047] In some embodiments, the SAGE1 complex inhibitor comprises a polypeptide, a chemical compound, an antibody or antigen-binding fragment thereof, or a nucleic acid molecule (eg, a polynucleotide or oligonucleotide).

[0048] In some embodiments, the SAGE1 complex comprises SAGE1 in complex with at least one component selected from the group consisting of INTS3, INIP, NABP1 / 2, CREBBP, TLE1, TLE2, TLE3, TLE4, TLE5, GGA3, CNOT1, TAX1BP1, SEC16A, CYLD, and PAXBP1.

[0049] In some embodiments, the SAGE1 complex comprises SAGE1 in complex with INTS3.

[0050] In some embodiments, the SAGE1 complex inhibitor inhibits the binding of SAGE1 to INTS3.

[0051] In some embodiments, the SAGE1 complex inhibitor binds to INTS3 or prevents INTS3 from binding to one or more residues of SAGE1 selected from the group consisting of F838, F873, K874, M832, V876, R872, K828, R836 and Q840 (residue numbering according to SEQ ID NO: 3 for SAGE1).

[0052] In some embodiments, the SAGE1 complex inhibitor comprises an antibody or antigen-binding fragment thereof that (a) specifically binds to SAGE1, (b) specifically binds to a component that complexes with SAGE1 (e.g., INTS3), or (c) specifically binds to the SAGE1 complex but does not bind to SAGE1 or a component that complexes with SAGE1.

[0053] In some embodiments, the SAGE1 complex inhibitor comprises a SAGE1-binding fragment of INTS3, a mutant or derivative thereof, or a fusion polypeptide thereof.

[0054] In some embodiments, the SAGE1-binding fragment of INTS3 is SAGE1. The present invention includes the amino acid sequence of SEQ ID NO:9, a variant or fragment thereof, or a fusion polypeptide thereof, capable of binding to

[0055] In some embodiments, the SAGE1 complex inhibitor comprises an INTS3-binding fragment of SAGE1, a mutant or derivative thereof, or a fusion polypeptide thereof.

[0056] In some embodiments, the INTS3-binding fragment of SAGE1 comprises the amino acid sequence of SEQ ID NO:8, a variant or fragment thereof, or a fusion polypeptide thereof capable of binding to INTS3.

[0057] In some embodiments, the SAGE1 complex inhibitor comprises an INTS3-binding fragment of INTS6 or INTS6L capable of binding to INTS3, a mutant or derivative thereof, or a fusion polypeptide thereof.

[0058] In some embodiments, the INST3-binding fragment of INTS6 or INTS6L comprises the amino acid sequence of SEQ ID NO:10, SEQ ID NO:104, or SEQ ID NO:105, a variant or fragment thereof, or a fusion polypeptide thereof capable of binding to INTS3.

[0059] In another embodiment, a SAGE1 inhibitor of the disclosure comprises a first moiety capable of binding to a ubiquitin pathway protein and a second moiety capable of binding to SAGE1 or a SAGE1 complex.

[0060] In some embodiments, the first moiety and the second moiety are attached via a linker.

[0061] In another aspect, the chimeric antigen receptor (CAR) of the present disclosure comprises an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain specifically binds to an epitope of SAGE1 or an MHC-associated epitope of SAGE1.

[0062] In some embodiments, the CAR further comprises a costimulatory domain.

[0063] In another embodiment, the cells of the present disclosure are genetically engineered to express said CAR described herein.

[0064] In some embodiments, the cells include T cells, tumor infiltrating lymphocytes, NK-T cells, TCR expressing cells, CD4+ T cells, CD8+ T cells, or NK cells.

[0065] In another embodiment, an ex vivo activated antigen presenting cell of the disclosure is exposed to an epitope of SAGE1 or an MHC-associated epitope of SAGE1, the antigen presenting cell optionally comprising a dendritic cell.

[0066] In another aspect, the pharmaceutical composition of the disclosure comprises the SAGE1 complex inhibitor described herein, the SAGE1 inhibitor described herein, or the cell described herein, and a pharma- ceutically acceptable carrier.

[0067] In another embodiment, the pharmaceutical composition of the present disclosure comprises a SAGE1 inhibitor and a pharma- ceutically acceptable carrier.

[0068] In some embodiments, the SAGE1 inhibitor is capable of reducing the level or activity of SAGE1.

[0069] In some embodiments, the SAGE1 inhibitor inhibits SAGE1 mRNA transcription, reduces SAGE1 mRNA levels, reduces SAGE1 protein levels, or inhibits at least one SAGE1-mediated biological function.

[0070] In some embodiments, the SAGE1 inhibitor comprises a polynucleotide or oligonucleotide, a polypeptide, a chemical compound, or an antibody or antigen-binding fragment thereof.

[0071] In some embodiments, the SAGE1 inhibitor specifically binds to SAGE1 protein and includes a polypeptide, a chemical compound, an oligonucleotide, or an antibody or antigen-binding fragment thereof.

[0072] In some embodiments, the SAGE1 inhibitor comprises an oligonucleotide targeting a SAGE1 nucleic acid (eg, SAGE1 mRNA or SAGE1 gene), a polynucleotide encoding the oligonucleotide, or a nucleic acid vector comprising the oligonucleotide or polynucleotide.

[0073] In some embodiments, the oligonucleotide comprises a sequence complementary to at least a portion of a SAGE1 mRNA or a sequence complementary to at least a portion of a SAGE1 gene.

[0074] In some embodiments, the portion of SAGE1 mRNA comprises 10 or more contiguous nucleotides within the sequence between nucleotides 266-366, 866-1116, 1316-1616, 1916-2266, 2466-2666, and 2782-2946 of SEQ ID NO:2.

[0075] In some embodiments, the oligonucleotide comprises a sequence selected from SEQ ID NOs: 82-95 and 106-147.

[0076] In some embodiments, the oligonucleotide comprises a sense / antisense sequence pair selected from SEQ ID NOs: 70 / 71, 72 / 73, 74 / 75, 76 / 77, 78 / 79, or 80 / 81.

[0077] In some embodiments, the oligonucleotide is a small interfering RNA, a short hairpin RNA, an antisense oligonucleotide, or a guide RNA.

[0078] In another aspect, the method of the present disclosure is a method for preventing or treating a SAGE1 positive disease in a subject in need thereof, comprising: (a) the SAGE1 inhibitor described herein; (b) the pharmaceutical composition described herein; (c) the SAGE1 complex inhibitor described herein; (e) the cell described herein; or (f) any combination of these to said subject.

[0079] In some embodiments, the SAGE1 positive disease is a SAGE1 positive tumor.

[0080] In another aspect, the disclosed method is a method of preventing, inhibiting, or slowing the progression of a tumor in a subject having a TP53 mutation, the method comprising: (a) the SAGE1 inhibitor described herein; (b) the pharmaceutical composition described herein; (c) the SAGE1 complex inhibitor described herein; (e) the cell described herein; or (f) any combination of these to said subject.

[0081] In some embodiments, the SAGE1 inhibitor is capable of reducing the level or activity of SAGE1.

[0082] In some embodiments, the SAGE1 inhibitor inhibits SAGE1 mRNA transcription, reduces SAGE1 mRNA levels, reduces SAGE1 protein levels, or inhibits at least one SAGE1-mediated biological function.

[0083] In some embodiments, the SAGE1 inhibitor comprises a polynucleotide or oligonucleotide, a polypeptide, a chemical compound, or an antibody or antigen-binding fragment thereof.

[0084] In some embodiments, the SAGE1 inhibitor comprises an oligonucleotide targeted to SAGE1 mRNA or the SAGE1 gene, a polynucleotide encoding the oligonucleotide, or a nucleic acid vector comprising the oligonucleotide or polynucleotide.

[0085] In some embodiments, the oligonucleotide comprises a sequence complementary to at least a portion of a SAGE1 mRNA or a sequence complementary to at least a portion of a SAGE1 gene.

[0086] In some embodiments, the SAGE1 inhibitor specifically binds to SAGE1 protein and includes a polypeptide, a chemical compound, an oligonucleotide, or an antibody or antigen-binding fragment thereof.

[0087] In some embodiments, the method further comprises administering a second therapeutic agent.

[0088] In some embodiments, the second therapeutic agent comprises a chemotherapeutic agent, an anti-cancer agent, a radiation therapy, an immunotherapy agent, an anti-angiogenic agent, a targeted therapy agent, a cell therapy agent, a gene therapy agent, a hormone therapy agent, or a cytokine.

[0089] In another embodiment, the method of the disclosure is a method of screening for an agent that is a SAGE1 inhibitor, comprising the steps of: (a) contacting a SAGE1 protein or a functional equivalent thereof with a test agent and detecting binding between the test agent and the SAGE1 or functional equivalent thereof; or (b) contacting a test agent with a SAGE1-binding fragment of a component of the SAGE1 complex and detecting binding between the test agent and the SAGE1-binding fragment; or (c) contacting a test agent with SAGE1 and a SAGE1-binding fragment of a component of a SAGE1 complex, and detecting the ability of the test agent to inhibit or inhibit binding between SAGE1 and the SAGE1-binding fragment, or the ability of the test agent to inhibit formation of a SAGE1 complex.

[0090] In another aspect, the method disclosed herein is a method for screening for an agent that is a SAGE1 inhibitor, comprising the steps of contacting a SAGE1-expressing cell or a functional equivalent thereof with a test agent and determining the ability of the test agent to reduce the amount or activity of SAGE1 or its functional equivalent.

[0091] In some embodiments, the cell is a tumor cell.

[0092] In another aspect, a recombinant cell of the present disclosure comprises a first gene encoding SAGE1 and a second gene encoding a reporter, the reporter being configured to generate a detectable signal in response to expression of SAGE1 or activity mediated by SAGE1.

[0093] In another aspect, the disclosure provides a set of X-ray crystal structure coordinates of a binding interface of a SAGE1 complex, said binding interface comprising one or more amino acid residues of SAGE1 selected from the group consisting of F838, F873, K874, M832, V876, R872, K828, R836, and Q840, or equivalent residues of fragments, variants, or derivatives thereof, wherein the numbering of said residues is according to SEQ ID NO:3.

[0094] In some embodiments, the X-ray crystal structure coordinates are set forth in the Protein Data Bank (PDB) code 7C5U.

[0095] In another aspect, the disclosure provides a set of x-ray crystal structure coordinates for a binding interface of INTS3, said binding interface comprising one or more amino acid residues of INTS3 selected from the group consisting of T804, S841, S874, S769, N933, R849, Q773, C777, M781, A816, N818, E838, E850, F871, R848, F805, L845, L815, L844, Y808, C842, Q846, Q870, R877, H878, K882, E732, V766, Q771, D768, A765, Q731, E835, E803, C809, and L772, or the equivalent residues of a fragment, variant, or derivative thereof, wherein the numbering of said residues is set forth in SEQ ID NO: Same as 5.

[0096] In some embodiments, the X-ray crystal structure coordinates are set forth in the Protein Data Bank (PDB) code 7C5U.

[0097] In another aspect, the disclosure provides a set of X-ray crystal structure coordinates for the binding interface between SAGE1 and INTS3, the binding interface of SAGE1 being comprised of one or more amino acid residues selected from the group consisting of F838, F873, K874, M832, V876, R872, K828, R836, and Q840 of SAGE1 (the numbering of said residues is represented by SEQ ID NO: 3), or an equivalent residue of a fragment, variant, or derivative thereof, wherein the binding interface of INTS3 comprises one or more amino acid residues selected from the group consisting of T804, S841, S874, S769, N933, R849, Q773, C777, M781, A816, N818, E838, E850, F871, R848, F805, L845, L815, L844, Y808, C842, Q846, Q870, R877, H878, K882, E732, V766, Q771, D768, A765, Q731, E835, E803, C809, and L772 of INTS3 (the numbering of said residues is as set forth in SEQ ID NO: 5), or the equivalent residue of a fragment, variant, or derivative thereof.

[0098] In some embodiments, the X-ray crystal structure coordinates are set forth in the Protein Data Bank (PDB) code 7C5U.

[0099] In another embodiment, a machine-readable data storage medium of the present disclosure has stored thereon said set of X-ray crystallographic coordinates described herein.

[0100] In another embodiment, the method of the disclosure is a method of identifying an agent that is a potential SAGE1 complex inhibitor, comprising: (a) generating on a computer a representation of the three-dimensional structure of a binding interface based on said set of X-ray crystal structure coordinates described herein; (b) generating a representation of the drug on a computer; (c) fitting a representation of the drug in step (b) to a computer representation of the three-dimensional structure of the binding interface in step (a) such that the drug interacts with one or more residues of the binding interface; (d) evaluating the interaction of the agent with one or more residues of the binding interface in step (c); If the interaction results in a low-energy, stable complex comprising the agent and SAGE1 or INTS3, which may compete for the SAGE1-INTS3 complex, then the agent is identified as a potential SAGE1 complex inhibitor.

[0101] In another embodiment, the virtual screening method of the present disclosure is a method for identifying a potential SAGE1 complex inhibitor, comprising: (a) generating on a computer a representation of the three-dimensional structure of the binding interface based on the X-ray crystal structure coordinates described herein; (b) generating, on a computer, a representation of the drug, or accessing, from a library, a representation of the drug; (c) fitting the representation of the agent in step (b) to a computer representation of the three-dimensional structure of the binding interface in step (a) to provide a configuration of the agent interacting with one or more residues of the binding interface; (d) evaluating the interaction of the agent with one or more residues of the binding interface in step (c); If the interaction results in a low-energy, stable complex comprising the agent and SAGE1 or INTS3, which may compete for the SAGE1 complex, then the agent is identified as a potential SAGE1 complex inhibitor.

[0102] As used herein and throughout this disclosure, the articles "a," "an," and "the" refer to one or more (i.e., at least one) of the object of the article. For example, "a polypeptide" means one or more polypeptides.

[0103] Whenever a series of numerical values ​​is described in this application, it is understood that any of those numerical values ​​may be the upper or lower limit of a certain numerical range. Moreover, the present invention is understood to encompass all such numerical ranges, i.e., ranges having combinations of upper and lower values, each of which may be a numerical value described herein. Ranges presented herein are understood to include all values ​​within the range. For example, 1-10 is understood to include all values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and fractional values, as appropriate. Similarly, a range defined by "at least / greater than" is understood to include that number and values ​​greater than it.

[0104] As used herein, "about" is understood to mean within three standard deviations of the mean, or within the standard tolerance in a particular technical field. In one embodiment, the variation of about is understood to be within 0.5.

[0105] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to at least one) of the object of the article. For example, "an element" means one or more elements.

[0106] As used herein, the term "including" is used synonymously with the term "including, but not limited to." Similarly, the term "such as" is used synonymously with the term "such as, but not limited to."

[0107] In this specification, the term "or" is used inclusively to mean and / or, unless the context clearly indicates otherwise.

[0108] Terms such as "comprising" are intended to be construed as open-ended terms (i.e., "including, but not limited to") unless otherwise specified.

[0109] The accompanying drawings are incorporated in and form a part of this specification and, together with the description, further explain the principles of the disclosure and enable those skilled in the art to make and use the disclosure. [Brief description of the drawings]

[0110] [Figure 1A] FIG. 1A shows the expression of SAGE1 in normal tissues using the GTEx RNA-Seq dataset. [Figure 1B] FIG. 1B shows the expression of TCGA in a wide variety of cancer types. [Figure 1C] FIG. 1C shows overall survival (OS) and progression-free survival (PFI) of TCGA patients stratified by SAGE1 mRNA expression levels. [Figure 2A] FIG. 2A shows IHC staining demonstrating that SAGE1 is expressed in human testis and several tumor tissues. [Figure 2B] FIG. 2B shows real-time quantitative PCR (qPCR) analysis of SAGE1 expression levels in each tumor tissue. [Figure 2C] FIG. 2C shows Western blot analysis of SAGE1 expression levels in each cancer cell line and a SAGE1-negative non-cancer cell line. [Figure 2D] FIG. 2D shows real-time qPCR analysis of SAGE1 expression levels in 60 colon cancer tissues and corresponding precancerous lesions. [Figure 2E] FIG. 2E shows the pattern of SAGE1 expression levels by TCGA cancer type. [Figure 3A] FIG. 3A shows that reduction of SAGE1 inhibits the growth of HuTu80, A375, KYSE30, A549, and PLCPRF5 cells in an RTCA assay. [Figure 3B] FIG. 3B shows that reduction of SAGE1 suppresses anchorage-independent growth of HuTu80, A375, KYSE30, A549, and PLCPRF5 cells on soft agar. [Figure 3C]FIG. 3C shows that depletion of SAGE1 in HuTu80 cells prevents xenograft growth in nude mice. [Figure 3D] , [Figure 3E] Figures 3D and 3E show that shSAGE1 treatment reduced the brightness signal (Figure 3D) and extended survival (Figure 3E) of an orthotopic model derived from the uveal melanoma cell line MUM2B. [Figure 3F] FIG. 3F shows the ability of SAGE1KD in KYSE30 with different siRNAs in quantitative PCR. [Figure 4A] , [Figure 4B] , [Figure 4C] Figures 4A, 4B, and 4C show knockdown of SAGE1 in human hepatocellular carcinoma (HCC-P). Representative images show HCC-PDX tumors with (n=9) or without (n=9) treatment with shSAGE1 adenovirus (Figure 4A), and statistical analysis of tumor volume (Figure 4B) and weight (Figure 4C) are shown (P<0.01 by nonparametric Mann-Whitney test). [Figure 4D] , [Figure 4E] , [Figure 4F] Figures 4D, 4E, and 4F show knockdown of SAGE1 in human colorectal cancer (CRC-P). Representative images show CRC-PDX tumors with (n=6) or without (n=9) treatment with shSAGE1 adenovirus (Figure 4D), and statistical analysis of tumor volume (Figure 4E) and weight (Figure 4F) are shown (P<0.01 by nonparametric Mann-Whitney test). [Figure 4G] , [Figure 4H] , [Figure 4I]Figures 4G, 4H, and 4I show knockdown of SAGE1 in human lung adenocarcinoma (LUAD-P). Representative images show LUAD-PDX tumors with (n=6) or without (n=9) treatment with shSAGE1 adenovirus (Figure 4G), and statistical analysis of tumor volume (Figure 4H) and weight (Figure 4I) are shown (P<0.01 by nonparametric Mann-Whitney test). [Figure 4J] , [Figure 4K] , [Figure 4L] Figure 4J, K, and L show knockdown of SAGE1 in human squamous cell carcinoma of the lung (LUSC-P). Representative images show LUSC-PDX tumors with (n=7) or without (n=9) treatment with shSAGE1 adenovirus, and statistical analysis of tumor volume (Figure 4J) (P<0.01 by nonparametric Mann-Whitney test) and weight (Figure 4K). Hematoxylin and eosin (H&E) staining of xenograft tumor tissues and IHC staining of pan-cytokeratin, a marker that identifies cancer cells from surrounding non-epithelial cells, are shown (Figure 4L). [Figure 4M] , [Figure 4N] , [Figure 4O] Figures 4M, 4N, and 4O show that transduction of shSAGE1 adenovirus into SAGE1-negative HCC does not suppress tumor growth. Representative images show PDX tumors treated or not with shSAGE1 adenovirus, with statistical analysis of tumor weight (Figure 4M) and volume (Figure 4N) with n=7 per group (P<0.01 by nonparametric Mann-Whitney test). Representative images show the heart, liver, spleen, kidney, and lung of nude mice in both control and shSAGE-treated groups (H&E staining, scale 200 μm) (Figure 4O). [Figure 5A] , [Figure 5B] , [Figure 5C]Figure 5A, Figure 5B, and Figure 5C show that shSAGE treatment suppressed tumor metastasis in an experimental model of metastasis. Cells from HCC-P, LUAD-P, and LUSC-P PDXs were injected into the tail vein. After 7–10 days, recipients were treated with AAV control or AAV-SAGE1 at 4 × 10 genome copies per mouse by tail vein injection. After 70–90 days, the animals were evaluated by PET-CT for the presence of metastatic tumors. If the health of the mice deteriorated, the analysis was performed earlier (Figure 5A). Figure 5B shows the calculation of total tumor burden by volume × SUV-bw mean value and total tumor burden analysis by T-test. The survival time of HCC-P mice was also calculated and analyzed by Kaplan-Meier method. Figure 5C shows the evaluation of the presence of metastatic tumors by euthanizing the mice and dissecting their organs, followed by staining with hematoxylin and eosin and MHC-I. [Figure 6A] FIG. 6A shows SDS-PAGE and silver staining analysis of SAGE1-interacting proteins immunoprecipitated in HuTu80 cells. [Figure 6B] FIG. 6B shows MS analysis of immunoprecipitated SAGE1 complex composition of four cancer cell lines. [Figure 6C] , [Figure 6D] Figures 6C and 6D show Co-IP analysis of SAGE1 interaction with INTS3 (Figure 6C) and with TLE3 and CBP (Figure 6D). [Figure 6E] FIG. 6E shows the domain organization of SAGE1, INTS3, and INTS6. [Figure 6F] FIG. 6F shows a structure-based sequence alignment of SAGE1I3BD and INTS6I3BD. [Figure 6G] Figure 6G shows ITC measurements of the interaction between SAGE1I3BD and INTS3CTD, inset is the ITC titration data. [Figure 6H] FIG. 6H shows GST pull-down analysis of a mixture of purified INTS3CTD-INTS6I3BD complex and GST-tagged SAGE1I3BD. [Figure 6I] Figure 6I shows quantitative MS analysis of immunoprecipitated complexes using anti-INTS3 antibody from 293T cells (293T-1, 293T-2, 293T-3) or SAGE1-overexpressing 293T cells (293OE SAGE-1, 293OE SAGE-2, 293OE SAGE-3). [Figure 7A] , [Figure 7B] 7A and 7B show a two-view ribbon diagram of the INTS3CTD dimer (FIG. 7A) and a two-view ribbon diagram of the SAGE1I3BD-INTS3CTD complex crystal structure (FIG. 7B). [Figure 7C] FIG. 7C shows details of the interaction of SAGE1I3BD with the INTS3CTD dimer. [Figure 7D] FIG. 7D shows the surface electrostatic potential of the SAGE1I3BD binding pocket of INTS3CTD. [Figure 7E] FIG. 7E shows that the binding of SAGE1I3BD causes the side chains of Phe805 and Tyr807 in the INTS3CTD dimer to change from symmetric to asymmetric. [Figure 7F] Figure 7F shows that superimposing the structural model of INTS6I3BD onto SAGE1I3BD makes it easier to see the differences between Phe838SAGE1 and Pro823INTS6. [Figure 7G] FIG. 7G shows Co-IP analysis of the interaction of WT SAGE1, SAGE1I3BD, and SAGE1mut with INTS3. [Figure 7H] FIG. 7H shows a comparison of ITC measurements of the interaction of WT or mutant SAGE1I3BD with INTS3CTD. [Figure 7I] , [Figure 7J] , [Figure 7K] , [Figure 7L]Figures 7I, 7J, 7K, and 7L show that re-expression of WT SAGE1, but not SAGE1mut, restored cell growth of SAGE1-KD Caco2 (Figure 7I), SAGE1-KO HuTu 80 (Figure 7J), and anchorage-independent growth of SAGE1-KO HuTu 80 cells on soft agar (Figure 7K), and led to a rapid increase in tumor size and weight in xenograft assays (Figure 7L). [Figure 8A] , [Figure 8B] Figures 8A and 8B show cross sections of size-exclusion chromatography of INTS3CTD (Figure 8A) and INTS3CTD-INTS6I3BD (Figure 8B) complex on a Superdex 200 column, and the purified complex peak fractions were resolved by SDS-PAGE and stained with Coomassie Brilliant Blue. [Figure 8C] FIG. 8C shows a crystal lattice analysis indicating that two INTS3CTD molecules related by crystallographic symmetry mediate extensive interactions. [Figure 8D] Figure 8D shows a detailed view of the INTS3CTD dimer interface, with stick models of the side chains of residues important for the interaction. [Figure 8E] , [Figure 8F] Figures 8E and 8F show stereo views of electron density maps of Phe805INTS3 and Tyr808INTS3 in the apoINTS3CTD dimer (Figure 8E) and the SAGE1I3BD-INTS3CTD complex structure (Figure 8F). [Figure 8G] Figure 8G shows a close-up of the SAGE1I3BD-INTS3CTD interface, with stick models highlighting the six key interacting residues that were replaced with alanine in SAGE1mut. [Figure 9A] FIG. 9A shows that depletion of SAGE1 in HuTu80 cells affects a panel of cell signaling pathways, transcription factors, and membrane proteins in Western blot assays. [Figure 9B]FIG. 9B shows that depletion of SAGE1 in HuTu80 and KYSE30 cells results in a G0 / G1 block in flow cytometry assays. [Figure 9C] Figure 9C shows MS analysis showing that the expression of most cell cycle genes was decreased in SAGE1-KD HCC-P cells. These results indicate that SAGE1 is also essential for cell cycle control in primary liver cancer cells. [Figure 9D] Figure 9D shows that depletion of SAGE1 in HCC15 cells results in a G0 / G1 block in flow cytometry assays, and that depletion of SAGE1 in HCC15 cells results in a loss of ki67 expression in IF staining and arrest at G0 phase. [Figure 9E] FIG. 9E shows that depletion of SAGE1 in HCC15 primary cancer cells affects a panel of cell signaling pathways, transcription factors, and membrane proteins in Western blot assays. [Figure 10A] Figure 10A shows the distribution of SAGE1 expression across primary tumors, with the dotted line indicating the threshold between high SAGE1 patients (the top 6% of TCGA patients with SAGE1 expression) and low SAGE1 patients (the other 94% of TCGA patients). [Figure 10B] Figure 10B shows the enrichment of genomic alterations between high SAGE1 patients (TCGA patients with SAGE1 expression in the top 6% of patients) and low SAGE1 patients (other 94% of patients in TCGA). Odds ratios were estimated with Fisher's exact test, and the FDR method was applied to adjust the p-values. For high SAGE1 patients, somatic mutations and protein-only coding mutations of TP53 remained (Frame_Shift_Del, Frame_Shift_Ins, In_Frame_Del, In_Frame_Ins, Missense_Mutation, Nonsense_Mutation, Nonstop_Mutation, Splice_Site, and Translation_Start_Site). [Figure 11A]FIG. 11A shows SAGE1 sarcoma antigen 1 [Homo sapiens (human)] with gene ID: 55511, located at Xq26.3, GRCh38.p13 (Genome Reference Consortium Human Build 38 patch release 13), the specific location of the human gene is ChrX: 135,893,700..135,913,062. [Figure 11B] Figure 11B shows cap analysis of gene expression (CAGE) analysis indicating that the transcription start site of SAGE1 is located near ChrX: 135893700 (i.e., the 5' end of the first exon) (white arrow). ChIP analysis revealed clear H3K27Ac and H3K4Me3 modification signals (gray arrows) and numerous transcription factor binding signals (black arrows) near the site. [Figure 11C] Figure 11C shows genome-wide DNA methylation sequence (WGBS (Whole Genome Bisulfite Sequencing)) analysis showing clear differences in DNA methylation modification within 2 kb (-1 kb to 1 kb) of the transcription start site of SAGE1 (chrX: 135, 892, 700-135, 894, 700) (purple arrows), with cells with high SAGE1 expression showing low DNA methylation levels at this site, and cells with low or no SAGE1 expression showing high DNA methylation levels at this site, indicating that DNA demethylation is involved in the regulation of SAGE1 expression. [Figure 12] FIG. 12 shows the crystallographic data collection and refinement statistics. [Figure 13A] FIG. 13A shows residues involved in interactions between INTS3CTD dimers. [Figure 13B] FIG. 13B shows residues involved in the INTS3CTD-SAGE1I3BD interaction. [Figure 14A] , [Figure 14B] , [Figure 14C] , [Figure 14D]Figures 14A to 14D show the spectral counts of highly reliable SAGE1 interacting molecules in HuTu80 cells (Figure 14A), K562 cells (Figure 14B), U2OS cells (Figure 14C), and KYSE30 cells (Figure 14D) identified by IP-MS analysis. [Figure 15] FIG. 15 shows the prevalence of pan-cancer SAGE1 expression (estimated by IHC staining). [Figure 16] FIG. 16 shows the suppression of SAGE1 expression by human SAGE1 shRNA. [Figure 17-1] , [Figure 17-2] , [Figure 17-3] , [Figure 17-4] , [Figure 17-5] , [Figure 17-6] , [Figure 17-7] , [Figure 17-8] , [Figure 17-9] , [Figure 17-10] , [Figure 17-11] , [Figure 17-12] , [Figure 17-13] , [Figure 17-14] , [Figure 17-15] , [Figure 17-16] , [Figure 17-17] , [Figure 17-18] , [Figure 17-19] , [Figure 17-20] , [Figure 17-21] Figures 17-1 through 17-21 show sequences described in this disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0111] Before describing the present disclosure in further detail, it is to be understood that the disclosure is not limited to the particular embodiments described herein, as such may, of course, vary, and the scope of the present disclosure will be limited only by the appended claims, and the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0112] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are described herein.

[0113] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference and to disclose and describe the methods and / or materials in connection with which the publication is cited. The citation of any publication is for its publication prior to the filing date, but is not an admission that the present disclosure is not entitled to antedate such publication by way of prior application. Additionally, the actual publication dates provided may need to be independently confirmed and may differ in actuality.

[0114] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein may be modified without departing from the scope or spirit of the present disclosure in any manner that would deviate from its elements and features. Any features of the various embodiments may be readily separated or combined with each other to the extent that they are not included herein. Any method described may be carried out in the order of description or in any other order which is logically possible.

[0115] The term "antibody" as used herein includes immunoglobulins, monoclonal antibodies, polyclonal antibodies, monovalent antibodies, bivalent antibodies, polyvalent antibodies, bispecific antibodies, and multispecific antibodies that bind to a specific antigen. Normal natural antibodies contain two heavy (H) chains and two light (L) chains. Mammalian heavy chains are classified as alpha, delta, epsilon, gamma, and mu, each consisting of a variable region (VH) and a first, second, third, and sometimes fourth constant region (CH1, CH2, CH3, and CH4, respectively), while mammalian light chains are classified as lambda or kappa, each consisting of a variable region (VL) and a constant region. Antibodies are "Y" shaped, with the stem of the Y consisting of the second and third constant regions of two heavy chains linked by disulfide bonds. Each arm of the Y contains the variable and first constant region of one heavy chain bound to the variable and constant region of one light chain. The variable regions of the light and heavy chains are involved in antigen binding. The variable regions of both chains generally contain three hypervariable loops called complementarity determining regions (CDRs) (light chain CDRs containing LCDR1, LCDR2, and LCDR3, and heavy chain CDRs containing HCDR1, HCDR2, and HCDR3). The three CDRs are interposed between lateral extensions known as framework regions (FRs) (light chain FRs containing LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs containing HFR1, HFR2, HFR3, and HFR4), which are more constant than the CDRs and serve as a scaffold for supporting the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding, but perform various effector functions. Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains. The five major classification classes, or isotypes, of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of alpha, delta, epsilon, gamma, and mu heavy chains, respectively. Several of the major antibody classes are divided into subclasses, such as IgG1 (gamma1 heavy chain), IgG2 (gamma2 heavy chain), IgG3 (gamma3 heavy chain), IgG4 (gamma4 heavy chain), IgA1 (alpha1 heavy chain), or IgA2 (alpha2 heavy chain).

[0116] The term "biomarker" or "marker" as used herein refers to a biological molecule that is a measurable indicator of some biological state or condition. Examples of biomarkers provided herein include genes (e.g., genomic DNA, cDNA) or gene products such as mRNA transcribed from the gene, proteins encoded by the gene, and protein complexes. In some embodiments, the biomarker is a tumor marker associated with tumors, particularly malignant tumors. Tumor markers can be quantitatively measured biochemically or immunochemically to provide information for diagnostic, prognostic, or therapeutic assays in clinical oncology. Tumor markers are useful for screening tumor patients, as they can often diagnose tumors months earlier than clinical and imaging tests. Tumor markers can also be used for prognosis, monitoring and detection of tumor recurrence, and determining the effectiveness of treatment.

[0117] The term "complex" as used herein with respect to a protein or polypeptide refers to a group of two or more associated polypeptide chains. Different polypeptide chains may have different functions. Typically, the polypeptide chains in a protein complex are linked by non-covalent interactions. Different protein complexes may have different stability over time.

[0118] The term "complementary" or "complementarity" refers to the ability of a nucleic acid to form hydrogen bonds with another nucleic acid sequence, either by conventional Watson-Crick type or other novel types. Percent complementarity indicates the percentage of residues in a nucleic acid molecule that can form hydrogen bonds with another nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50% complementary, 60% complementary, 70% complementary, 80% complementary, 90% complementary, 100% complementary). Percent complementarity can be calculated using the Basic Local Sequence Search Tool (BLAST program) (Altschul (1990) J. Mol. Biol. . 215, 403-410; Zhang and Madden (1997) Genome Res. 7, 649-656).

[0119] The term "derivative" with respect to a polypeptide or polynucleotide refers to a chemically modified polypeptide or polynucleotide in which one or more specified number of substituents are covalently attached to one or more specified amino acid residues of the polypeptide or to one or more specified nucleotides of the polynucleotide (such as by conjugation with heterologous polypeptides, fatty acids, and chemical compounds or by glycosylation, acetylation, phosphorylation). The term "derivative" with respect to a compound refers to a compound synthesized from the parent compound by replacing one atom with another atom or group of atoms.

[0120] The term "diagnosis" refers to determining whether a subject has a condition (e.g., a SAGE1-positive disease) using the methods described herein. The term also includes the use of the methods to assess the level of disease in a subject.

[0121] The term "detection" refers to the act of quantitative or semi-quantitative determination, testing, or measurement, which may be performed by conventional means. The term contemplates a change in matter, such as a transformation of a biological sample, such as a blood sample or other tissue sample, from one state to another through physical testing. The phrase "detecting SAGE1 expression" and the like is used to mean that a sample may be tested (directly or indirectly) to ascertain the presence or absence or level of SAGE1 expression. It is understood that the presence or absence and level of SAGE1 expression may guide diagnostic or therapeutic decisions.

[0122] The term "high" with respect to a biomarker (e.g., SAGE1) refers to a level of the biomarker detected in a biological sample that is higher than the corresponding reference level of that biomarker. Similarly, the term "low" as used herein refers to a level of the biomarker detected in a biological sample that is lower than the corresponding reference level of that biomarker. In some embodiments, the level of SAGE1 expression can be considered "higher" or "lower" than a reference level if the level is 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, or 2-fold, 3-fold, 4-fold, 5-fold, or more higher or lower, respectively, than the reference level.

[0123] The term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmacologically active agent sufficient to obtain a desired physiological effect in an individual in need of the agent. The effective amount will vary from individual to individual depending on the health and condition of the individual being treated, the taxonomic group of the individual being treated, the formulation, the diagnosis of the individual's medical condition, and other relevant factors.

[0124] As used herein, the term "fragment" refers to a partial sequence of a reference polypeptide or polynucleotide, regardless of length, which retains at least partial biological activity of the reference polypeptide.

[0125] As used herein, the term "inhibitor" refers to an agent that inhibits (e.g., antagonizes, suppresses, reduces, blocks, reverses, alters) the effect of a target molecule (e.g., SAGE1 or the SAGE1 complex). An inhibitory effect can be achieved, for example, by reducing the amount of the target molecule or by suppressing the activity of the molecule. Such inhibitors can include any compound, protein, or nucleic acid or derivatives thereof that exhibits antagonistic activity.

[0126] As used herein, the term "isolated" refers to a molecule or complex thereof that is no longer present in its original natural environment.

[0127] The term "percent (%) sequence identity" refers to the percentage of identical amino acids (or nucleic acids) found at the maximum It is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, when the sequences are aligned and gaps are inserted, if necessary, so that the sequence identity is the same as that of the reference sequence. In other words, percent (%) sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) in the reference sequence being compared by the total number of amino acid residues (or bases) in either the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues are not considered to be identical residues. Alignment to determine percent amino acid (or nucleic acid) sequence identity can be performed using publicly available tools such as, for example, BLASTN, BLASTp (published at the US National Center for Biotechnology Information (NCBI) website; see also Altschul SF et al., J. Mol. Biol., 215: 403-410 (1990); Stephen F. et al., Nucleic Acids Res., 25: 3389-3402 (1997)), ClustalW2 published at the European Bioinformatics Institute website; see also Higgins DG et al., Methods in Enzymology, 266: 383-402 (1996); Larkin MA et al., Bioinformatics (Oxford, England), 23 (21): 2947-8 (2007).), and ALIGN or Megalign (DNASTAR) software. Those skilled in the art may use the default parameters provided by this tool, or may customize the parameters appropriately for the alignment, for example by selecting an appropriate algorithm.

[0128] As used herein, the term "integrator complex subunit 3" or "INTS3" refers to the INTS3 gene and INTS3 gene products, such as the INTS3 gene mRNA and the INTS3 gene encoded protein. Fragments, mutants, and derivatives thereof are also intended to be included. The human INTS3 gene encoded protein is a predicted 1043 amino acid protein with Uniprot accession number Q68E01-1 (the sequence of the INTS3 protein with the first methionine removed, which is incorporated herein as SEQ ID NO: 5).

[0129] As used herein, the term "integrator complex subunit 6" or "INTS6" refers to the INTS6 gene and INTS6 gene products, such as the INTS6 gene mRNA and the INTS6 gene encoded protein. Fragments, variants, and derivatives thereof are also intended to be included. The human INTS6 gene encoded protein is a predicted 887 amino acid protein with Uniprot accession number Q9UL03-1 (the sequence of which is incorporated herein as SEQ ID NO: 6).

[0130] As used herein, the term "integrator complex subunit 6-like" or "INTS6L" refers to the INTS6L gene and INTS6L gene products, such as the INTS6L gene mRNA and the protein encoded by the INTS6L gene. Fragments, mutants, and derivatives thereof are also intended to be included. The protein encoded by the human INTS6L gene is a predicted 861 amino acid protein with Uniprot accession number Q5JSJ4-1 (the sequence of which is incorporated herein as SEQ ID NO: 7).

[0131] As used herein, the term "kit" refers to an article of manufacture (e.g., a package or container) that includes at least one reagent that specifically detects or modulates the expression of SAGE1 within the scope of the present disclosure. The kit may be advertised, distributed, or sold as a unit for carrying out the methods described herein.

[0132] The term "level" with respect to a biomarker refers to the amount of the biomarker of interest present in a sample. This amount may be expressed in absolute amount, i.e., the total amount of the biomarker in the sample, or in relative amount, i.e., the concentration or percentage of the biomarker in the sample. The level of a biomarker can be measured at the DNA level (e.g., expressed as the amount of epigenetic modifications such as DNA methylation or histone acetylation), at the RNA level (e.g., mRNA abundance), or at the protein level (e.g., (amount of protein or protein complex).

[0133] The terms "possibility" and "likely" are a measure of the likelihood that an event will occur. Likelihood, as a person of ordinary skill would arrive at through common sense, training, or experience, is more likely than guess, but less certain. In some embodiments, the terms "possibility" and "likely" refer to a percentage likelihood, e.g., 50% or more, 60% or more, 80% or more, 90% or more, 95% or more, 99% or more.

[0134] The term "proximal" with respect to acetylation of histones of the SAGE1 gene refers to the location of acetylation being near a particular region or site of the SAGE1 gene (e.g., within 50 bp, 100 bp, 200 bp, 400 bp, 600 bp, 800 bp, or 1 kb upstream or downstream).

[0135] The terms "therapeutically beneficial" or "responsive" as used in the context of cancer therapy (e.g., treatment with a SAGE1 inhibitor) refer to an effective or favorable response to the treatment, and not an unfavorable response, i.e., an adverse event. In a patient, an effective response can be manifested by a number of clinical indicators, including the absence of detectable tumor (complete response), a decrease in tumor size and / or number of tumor cells (partial response), cessation of tumor growth (stable disease), a stronger anti-tumor immune response, and / or tumor regression or rejection, some relief of one or more symptoms associated with the tumor, increased survival after treatment, and / or decreased mortality at some time after treatment.

[0136] The terms "TP53" and "p53" are used interchangeably herein and refer to the tumor protein p53 and may refer to the p53 protein and the DNA (e.g., coding gene sequence) or RNA encoding p53, including all isoforms and variants. Other names for this p53 include, for example, antigen NY-CO-13, phosphoprotein p53, tumor suppressor p53, and cellular tumor antigen p53. An exemplary sequence for human p53 is available in the UniProtKB database under accession number P04637 (P53-HUMAN).

[0137] The term "prognosis" includes a prediction of the likely course and outcome of a disease or the likelihood of recovery from the disease. When used in relation to a tumor, prognosis can be, for example, the tendency for a treatment to be effective or ineffective, the development of a clinical subtype, recurrence, metastasis, complete or partial remission. A good prognosis is a good expected or likely outcome of treating the tumor, and a poor prognosis is a less good expected or likely outcome of treating the tumor.

[0138] The terms "polynucleotide" or "nucleic acid" or "oligonucleotide" are used interchangeably and refer to a chain of covalently linked nucleotides. The nucleotides may be deoxyribonucleotides or ribonucleotides, each of which may be independently modified or unmodified.

[0139] The terms "polypeptide" and "protein" are used interchangeably and refer to a chain of amino acid residues covalently linked by peptide bonds. A protein or polypeptide may contain moieties other than amino acids (e.g., may be a glycoprotein) and / or may be processed or modified. Furthermore, one of skill in the art will understand that a protein may comprise one or more polypeptide chains linked, for example, by disulfide bonds or associated in other ways.

[0140] The term "subject" refers to a human or non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). In many embodiments, the subject is a human. A subject may be a patient undergoing diagnosis or treatment for a disease by a health care provider. ... The term "subject" is used interchangeably herein with "individual" or "patient." A subject may be afflicted with or susceptible to a disease or disorder, but may or may not exhibit symptoms of the disease or disorder.

[0141] As used herein, the term "sample" or "biological sample" refers to a biological sample obtained from a subject that contains one or more biomarkers of interest to be assessed in the methods of the invention. The sample may include cells or secreted products from cells, including, but not limited to, isolated cells, tissue samples, or bodily fluid samples. In some embodiments, the sample may include tumor cells or secreted products from tumor cells. In some embodiments, the sample is a tumor cell sample.

[0142] As used herein, "sarcoma antigen 1" or "SAGE1" refers to the SAGE1 gene and SAGE1 gene products, such as SAGE1 mRNA and the protein encoded by the SAGE1 gene. Fragments, variants, and derivatives thereof are also included. Human SAGE1 gene is located on chromosome X (X: 135, 893, 700-135, 913, 026, Xq26.3, according to Genome Reference Consortium Human Build 38 patch release 13) and has a gene ID of 55511 in the NCBI database (this sequence is incorporated herein as SEQ ID NO: 1). Human SAGE1 transcript variant 1 is the canonical mRNA sequence of SAGE1, and has an NCBI accession number of NM_001381902.1 (GI: 1833303494) (this sequence is incorporated herein as SEQ ID NO: 2). SAGE1 transcript variant 1 encodes at least two SAGE1 proteins. The canonical SAGE1 protein is a predicted 904 amino acid protein with Uniprot accession number Q9NXZ1-1 (this sequence is incorporated herein as SEQ ID NO: 3). Unless otherwise stated, position information for the SAGE1 protein is determined from this sequence. The SAGE1 protein isoform is a predicted 528 amino acid protein with NCBI accession number AAI44261.1 (this sequence is incorporated herein as SEQ ID NO: 4), also within the scope of this disclosure.

[0143] The term "structural coordinates" refers to Cartesian coordinates derived from mathematical equations that describe the pattern obtained by diffraction of a monochromatic beam of X-rays by the atoms (scattering centers) of a protein or protein complex in the crystalline state. This diffraction data is used to calculate an electron density map of the repeating unit of the crystal. This electron density map is then used to establish the positions of the individual atoms of the molecule or molecular complex.

[0144] The terms "tumor" or "cancer" are used interchangeably and refer to diseases involving abnormal cell growth, including all stages and types affecting tissues, organs, or cells of the human body. The term includes all known cancers and neoplastic diseases, whether malignant, positive, soft tissue, solid, or hematologic, including pre-metastatic and post-metastatic tumors of any stage or grade. Tumors are generally classified according to the tissue or organ in which they are located or from which they originate, as well as the morphology of the cancerous tissue and cells. In some embodiments, the types of tumors include adrenal gland cancer, lymphoepithelial tumor, adenoid cell carcinoma, lymphoma, acoustic neuroma, acute lymphocytic leukemia, acrolentiginous melanoma, acute myeloid leukemia, acrohidroma, chronic lymphocytic leukemia, acute eosinophilic leukemia, liver cancer, acute erythroid leukemia, small cell lung cancer, acute lymphocytic leukemia, non-small cell lung cancer, acute megakaryoblastic leukemia, MALT lymphoma, acute monocytic leukemia, malignant fibrous histiocytoma, acute promyelocytic leukemia, malignant peripheral nerve sheath tumor, mantle cell lymphoma, adenocarcinoma, marginal zone B cell lymphoma, Malignant tumors of the hippocampus, adenoid cystic carcinoma, adenomatous, adenoid odontogenic tumor, mast cell leukemia, adenosquamous carcinoma, mediastinal germ cell tumor, adipose tissue tumor, medullary carcinoma of the breast, adrenal cortical carcinoma, medullary thyroid carcinoma, adult T-cell leukemia / lymphoma, medulloblastoma, invasive NK cell leukemia, melanoma, AIDS-related lymphoma, meningioma, pulmonary rhabdomyosarcoma, Merkel cell carcinoma, alveolar soft part sarcoma, mesothelioma, ameloblastoma, metastatic urothelial carcinoma, anaplastic large cell lymphoma, mixed Mullerian tumor, anaplastic thyroid carcinoma, mucinous tumor, angioimmunoblastic T-cell lymphoma, multiple myeloma, blood Tubulomyolipoma, Muscle tissue tumor, Angiosarcoma, Mycosis fungoides, Astrocytoma, Myxoid liposarcoma, Atypical teratoid rhabdoid tumor, Myxoma, B-cell chronic lymphocytic leukemia, Myxosarcoma, B-cell lymphocytic leukemia, Nasopharyngeal carcinoma, B-cell lymphoma, Schwannoma, Basal cell carcinoma, Neuroblastoma, Biliary tract cancer, Neurofibromatosis, Bladder cancer, Neuroma, Blastoblastoma, Nodular melanoma, Bone cancer, Eye cancer, Brenner tumor, Oligodendroglial, Brown tumor, Oligodendroglioma, Burkitt lymphoma, Eosinophilic breast cancer, Brain tumor, Optic nerve tumor, Oral intraepithelial neoplasia, Osteosarcoma, Carcinosarcoma, Ovarian cancer, Bone and soft tissue tumor, Pulmonary sulcus tumor, Thyroid papillary carcinoma, Myeloma, Paramyeloma Ganglioneuroma, chondroma, pineoblastoma, chordoma, pineocytoma, choriocarcinoma, pituitary tumor, choroid plexus papilloma, pituitary adenoma, renal clear cell sarcoma, pituitary tumor, craniopharyngioma, plasmacytoma, cutaneous T-cell lymphoma, polyembryoma, cervical cancer, precursor T-lymphoblastic lymphoma, colorectal cancer, primary central nervous system lymphoma, Degos disease, primary effusion lymphoma, proliferative small round cell tumor, primary peritoneal precancer, diffuse large B-cell lymphoma, prostate cancer, embryonal dysplasia of neuroepithelial tumors, pancreatic cancer, anaplastic cell tumor, pharyngeal cancer, embryonal carcinoma, peritoneal pseudomyxoma, endocrine tumor, renal cell carcinoma, enteropathy-associated T-cell lymphoma, endocrine tumor Germ cell sinus tumor, renal medullary carcinoma, retinoblastoma, esophageal cancer, rhabdomyosarcoma, endodermal cell tumor, rhabdomyosarcoma, myoma, Richter transformation, fibrosarcoma, rectal cancer, follicular lymphoma, sarcoma, follicular thyroid carcinoma, schwannoma, gangliocytoma, seminoma, gastrointestinal cancer, Sertoli cell tumor, germinoma, sex cord-gonadal stromal tumor, gestational choriocarcinoma, signet ring cell tumor, giant cell fibroblastoma, skin cancer, giant cell tumor of bone, small round blue cell tumor, glioma, small cell carcinoma, glioblastoma multiforme, soft tissue sarcoma, glioma, somatostatin tumor, brain glioma, sooty wart, hyperglucagonoma of the pancreas, spinal tumor, gonadoblastoma, splenic marginal lymphoma, condylar tumor Granulosa cell tumor, squamous cell carcinoma, estrogenic tumor, synovial sarcoma, gallbladder cancer, Sezary disease, gastric cancer, small intestine cancer, hairy cell leukemia, squamous cell carcinoma, hemangioblastoma, gastric cancer, head and neck cancer, T-cell lymphoma, hemangioepithelioma, testicular cancer, hematologic malignancies, sarcoma, hepatoblastoma, thyroid cancer, hepatosplenic T-cell lymphoma, transitional cell carcinoma, Hodgkin's lymphoma, laryngeal cancer, non-Hodgkin's lymphoma, urinary tract cancer, invasive lobular carcinoma, genitourinary cancer, intestinal cancer, urothelial carcinoma, kidney cancer, uveal melanoma, laryngeal cancer, uterine cancer, malignant freckled sputum, warty carcinoma, lethal midline granuloma, optic pathway glioma, leukemia, vulvar cancer, testicular stromal tumor, vaginal cancer,In some embodiments, the tumor may be selected from the group consisting of colorectal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSCC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), low grade glioma of the brain (LGG). , rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), acute myeloid leukemia (LAML).

[0145] As used herein, the term "treatment" or "treating" refers to preventing, alleviating, delaying or slowing the onset of a disease, preventing, slowing the progression of symptoms associated with a disease, suppressing or arresting symptoms associated with a disease, achieving complete or partial remission of a disease, curing a disease, or a combination thereof. With respect to tumors, "treatment" or "treating" may refer to preventing, inhibiting, slowing or slowing the development, proliferation, progression, metastasis, cancerous symptoms, or a combination thereof of neoplastic or malignant cells. It is understood that treatment does not necessarily mean a cure or complete ablation of the disease, disorder, or symptoms of the disease or disorder.

[0146] The term "vector" refers to a vehicle into which a polynucleotide is operably inserted for introduction, replication, or expression. Vectors may include a variety of regulatory elements. These include, but are not limited to, origins of replication, promoters, transcription initiation sequences, enhancers, selectable marker genes, reporter genes, etc. Vectors also include substances that aid in entry into host cells, such as, but are not limited to, virus particles, liposomes, ionic compounds, or amphipathic compounds.

[0147] In this disclosure, terms such as "comprises, comprised, comprising, contains, containing" have their meanings according to U.S. Patent Law, which are inclusive, i.e., open-ended, and do not exclude additional elements or method steps not recited. Also, terms such as "consisting essentially of, consists essentially of" have their meanings according to U.S. Patent Law, which allow for the inclusion of additional materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. The term "consists of, consisting of" has its meaning according to U.S. Patent Law, which is that these terms are close-ended.

[0148] II. SAGE1 as a novel diagnostic biomarker and therapeutic target Sarcoma antigen 1 (SAGE1) belongs to a large heterogeneous group of cancer-testis antigens (CTAs). Although SAGE1 was identified as a tumor-specific antigen nearly 20 years ago, its role in tumor growth and progression remains poorly understood.

[0149] In this disclosure, through numerous studies, it has been found that SAGE1 is expressed in various tumor tissues and cells, including intestinal cancer, lung cancer, liver cancer, breast cancer, skin cancer, head and neck cancer, and leukemia, and that SAGE1 is not detectable in normal cells other than testicular tissue.

[0150] The present disclosure unexpectedly discovers that SAGE1 promotes transcription involved in oncogenesis and upregulates multiple cancer proliferation pathways, which may have profound implications on abnormal proliferation, neoplastic and tumor progression, making SAGE1 expression a valuable biomarker for the diagnosis and prognosis of SAGE1-positive diseases, monitoring the progression of SAGE1-positive diseases, predicting therapeutic outcomes and selecting treatment regimens.

[0151] The present disclosure further identifies, for the first time, SAGE1 as a tumor-agnostic therapeutic target. Downregulation of SAGE1 effectively inhibits tumor growth in patient-derived xenograft (PDX) models of various tumors, and even leads to pathological complete remission. The present disclosure shows that regardless of the tissue of origin or genomic landscape diversity, SAGE1 expression, which may lead to oncogene addiction, defines a new diagnosis as SAGE1-positive disease (e.g., SAGE1-positive tumors), and suppressing SAGE1 levels or activity may be an effective therapeutic approach for SAGE1-positive disease.

[0152] As used herein, the term "SAGE1 positive" refers to the expression of SAGE1 in a cell or tissue. The term "SAGE1 positive disease" refers to a condition characterized by the presence of SAGE1 positive cells or tissues in which SAGE1 is not normally expressed. A SAGE1 positive disease is characterized by abnormal or unwanted expression of SAGE1 that deviates from the normal expression pattern. SAGE1 is not expressed in normal tissues, except spermatogonia and occasionally the placenta and brain.

[0153] In one embodiment, a SAGE1 positive disorder is characterized by the presence of SAGE1 positive cells that are not spermatogonial stem cells (SSCs or SPGs). In one embodiment, a SAGE1 positive disorder is characterized by the presence of SAGE1 positive cells that are cells found in the fallopian tubes, salivary glands, adipose tissue, adrenal glands, blood, bladder, blood vessels, breast, cervix, colon, esophagus, heart, kidney, liver, lung, muscle, nerves, ovaries, pancreas, prostate, skin, small intestine, spleen, stomach, thyroid, uterus, or vagina.

[0154] In one embodiment, a SAGE1 positive disease is characterized by the presence of SAGE1 positive abnormal cells. As used herein, the term "abnormal cells" refers to cells that differ from corresponding normal cells in one or more aspects of biology, physiology, or pathology. Abnormal cells may differ from corresponding normal cells, for example, in cell growth, genetic mutations, abnormal activation of certain biological pathways, abnormal expression of certain genes, abnormal migration capacity, or abnormal differentiation capacity, among others.

[0155] In some embodiments, the SAGE1 positive disease comprises a tumor (ie, a SAGE1 positive tumor).

[0156] In some embodiments, the SAGE1 positive disease is selected from a solid tumor or a hematological tumor.

[0157] In some embodiments, the SAGE1 positive disease is adrenal gland cancer, lymphoepithelial tumor, adenoid cell carcinoma, lymphoma, acoustic neuroma, acute lymphocytic leukemia, acral lentigo melanoma, acute myeloid leukemia, acrohidroma, chronic lymphocytic leukemia, acute eosinophilic leukemia, liver cancer, acute erythroid leukemia, small cell lung cancer, acute lymphocytic leukemia, non-small cell lung cancer, acute megakaryoblastic leukemia, MALT lymphoma, acute monocytic leukemia, malignant fibrous histiocytoma, acute promyelocytic leukemia, malignant peripheral nerve sheath tumor, mantle cell lymphoma, adenocarcinoma, marginal zone B cell lymphoma, malignant tumor of the hippocampus, adenoid cystic carcinoma, adenomatous, adenoid odontogenic Tumors, mast cell leukemia, adenosquamous carcinoma, mediastinal germ cell tumors, adipose tissue tumors, medullary breast carcinoma, adrenal cortical carcinoma, medullary thyroid carcinoma, adult T-cell leukemia / lymphoma, medulloblastoma, invasive NK cell leukemia, melanoma, AIDS-related lymphoma, meningioma, pulmonary rhabdomyosarcoma, Merkel cell carcinoma, alveolar soft part sarcoma, mesothelioma, ameloblastoma, metastatic urothelial carcinoma, anaplastic large cell lymphoma, mixed Müllerian tumor, anaplastic thyroid carcinoma, mucinous tumor, angioimmunoblastic T-cell lymphoma, multiple myeloma, angiomyolipoma, muscle tissue tumors, angiosarcoma, mycosis fungoides, astrocytoma, myxoid liposarcoma, atypical teratoid rhabdo id tumor, myxoma, B-cell chronic lymphocytic leukemia, myxosarcoma, B-cell lymphocytic leukemia, nasopharyngeal carcinoma, B-cell lymphoma, schwannoma, basal cell carcinoma, neuroblastoma, biliary tract cancer, neurofibromatosis, bladder cancer, neuroma, blastoma, nodular melanoma, bone cancer, eye cancer, Brenner tumor, oligodendroglial, brown tumor, oligodendroglioma, Burkitt lymphoma, eosinophilic breast cancer, brain tumor, optic nerve tumor, oral intraepithelial neoplasia, osteosarcoma, carcinosarcoma, ovarian cancer, soft tissue and bone tumor, pulmonary sulcus tumor, papillary thyroid carcinoma, myeloma, paraganglioma, chondroma, pineoblastoma, chordoma, pineocytoma, choriocarcinoma, pituitary tumor, choroid plexus papilloma, hypophysis Pituitary adenoma, renal clear cell sarcoma, pituitary tumor, craniopharyngioma, plasmacytoma, cutaneous T-cell lymphoma, polyembryoma, cervical cancer, precursor T-lymphoblastic lymphoma, colorectal cancer, primary lymphoma of the central nervous system, Degos disease, primary effusion lymphoma, proliferative small round cell tumor, primary peritoneal precancer, diffuse large B-cell lymphoma, prostate cancer, embryonal dysplasia of neuroepithelial tumors, pancreatic cancer, anaplastic cell tumor, pharyngeal cancer, embryonal carcinoma, pseudomyxoma peritonei, endocrine tumors, renal cell carcinoma, enteropathy-associated T-cell lymphoma, endodermal sinus tumor, renal medullary carcinoma, retinoblastoma, esophageal cancer, rhabdomyosarcoma, endodermal sinus tumor ...Richter transformation, fibrosarcoma, rectal cancer, follicular lymphoma, sarcoma, follicular thyroid cancer, schwannoma, gangliocytoma, seminoma, gastrointestinal cancer, Sertoli cell tumor, germinoma, sex cord-gonad stromal tumor, gestational choriocarcinoma, signet ring cell tumor, giant cell fibroblastoma, skin cancer, giant cell tumor of bone, small round blue cell tumor, glioma, small cell carcinoma, glioblastoma multiforme, soft tissue sarcoma, glioma, somatostatin tumor, brain glioma, sooty wart, hyperglucagonoma of the pancreas, spinal tumor, gonadoblastoma, splenic marginal lymphoma, granulosa cell tumor, squamous cell carcinoma, estrogenic tumor, synovial sarcoma, gallbladder cancer, Sezary disease, gastric cancer, small intestine cancer, hairy cell leukemia, squamous cell carcinoma, blood angioblastoma, gastric cancer, head and neck cancer, T-cell lymphoma, hemangioepithelioma, testicular cancer, hematological malignancies, sarcoma, hepatoblastoma, thyroid cancer, hepatosplenic T-cell lymphoma, transitional cell carcinoma, Hodgkin's lymphoma, laryngeal cancer, non-Hodgkin's lymphoma, urinary tract cancer, invasive lobular carcinoma, genitourinary cancer, intestinal cancer, urothelial carcinoma, kidney cancer, uveal melanoma, laryngeal cancer, uterine cancer, malignant freckled sputum, verrucous carcinoma, lethal midline granuloma, optic pathway glioma, leukemia, vulvar cancer, testicular stromal tumor, vaginal cancer, liposarcoma, Waldenstrom's macroglobulinemia, lung cancer, adenolymphoma lymphangioma, nephroblastoma, lymphosarcoma, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), egg, Focal serous cystadenocarcinoma (OV), endometrioid carcinoma (UCEC), thyroid cancer (THCA), cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme tumor (gbm), prostate adenocarcinoma (prad), thymoma (thym), low-grade brain glioma (lgg), rectal adenocarcinoma (read), pheochromocytoma and paraganglioma (pcpg), esophageal cancer (esc) A), selected from kidney clear cell carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), or acute myeloid leukemia (LAML).

[0158] III. Detection of SAGE1 Expression for Diagnosis, Prognosis, and Monitoring of Disease i. Diagnosis of SAGE1-positive diseases In one aspect, the method disclosed herein is a method for diagnosing a SAGE1-positive disease in a subject, comprising the steps of detecting SAGE1 expression in a biological sample from the subject, and assessing whether the subject has a SAGE1-positive disease based on the presence or absence or level of SAGE1 expression detected in the biological sample.

[0159] In one aspect, the method disclosed herein is a method for diagnosing a SAGE1-positive disease in a subject, comprising the step of detecting SAGE1 expression in a biological sample from the subject, and diagnosing the subject as having a SAGE1-positive disease if SAGE1 expression is detected in the biological sample.

[0160] In some embodiments, the SAGE1 positive disease is a SAGE1 positive tumor. In some embodiments, the subject is determined to have a defective (e.g., mutated) p53. Details of this embodiment are provided in the following section.

[0161] In some embodiments, the method further comprises the step of recommending, prescribing, or administering a SAGE1 inhibitor to a subject diagnosed with a SAGE1 positive disease.

[0162] ii. Determination of malignancy In another aspect, the method disclosed herein is a method for determining the malignancy or malignant potential of a tumor in a subject, comprising the steps of detecting SAGE1 expression in a biological sample from the subject and assessing whether the subject has a malignant or potentially malignant tumor based on the presence or absence or level of SAGE1 expression detected in the biological sample.

[0163] In one aspect, a method disclosed herein is a method for determining the malignancy or potential for malignancy of a tumor in a subject, comprising the step of detecting SAGE1 expression in a biological sample from the subject, and determining that the tumor is malignant or has the potential to become malignant if SAGE1 expression is detected in the biological sample.

[0164] The term "malignancy" is the opposite of benign and refers to the presence of a malignant tumor that has the ability to invade and destroy nearby cells (locally) and even spread (metastasize) to other parts of the body. Malignant cells tend to grow rapidly and uncontrolled, and are not usually killed by changes in their genetic makeup. Some malignant cells exhibit a stem cell-like phenotype ("stemness") that is believed to give rise to new tumors, leading to recurrence and metastasis. Thus, malignant tumors may be ineffective against treatment or may return after treatment removes or destroys detectable traces. The term "malignant potential" refers to the potential for a disease to progress to malignancy. A subject may not yet show signs of malignancy, but may become malignant in the future or may become malignant if the disease progresses to malignancy.

[0165] In some embodiments, the malignancy has one or more of the following characteristics: (a) expression of one or more stem cell markers, (b) the ability to metastasize, (c) the ability to undergo uncontrolled proliferation of cells, (d) a high likelihood of disease progression, (e) a high likelihood of developing resistance to anti-cancer therapy, (f) a high likelihood of recurrence following anti-cancer therapy treatment, and (g) the occurrence or likelihood of occurrence of one or more cancer-associated driver mutations. Tumor progression may result in decreased patient survival, increased metastasis, increased cell proliferation, or increased tumor burden. In some embodiments, the subject does not exhibit clinical symptoms of malignancy (e.g., the characteristics described above).

[0166] The term "cancer-associated driver mutations" as used herein refers to mutations that promote the development of cancer by conferring a selective growth advantage. Cancer-associated driver mutations can affect gene function, for example, by copy number alterations, mutations in coding or non-coding regions, dysregulation of microRNAs, epigenetic changes, and mutations in chromatin modifiers, among others. Cancer-associated driver mutations have been commonly observed in tumor suppressor p53, CDKN2A gene, KMT2D gene, SMARCA4 gene, NFE2L2 gene, FAT1 gene, SPTA1 gene, ARID1A gene (see, e.g., FIG. 10), MYC oncogene, BRAF oncogene in tumor patients (see, e.g., Pon, Julia R and Marra, Marco A, Annu. Rev. Pathol. Mech. Dis. 2015.10: 25-50).

[0167] In some embodiments, the SAGE1 positive tumor is likely to be malignant or has the potential to become malignant.

[0168] In some embodiments, the method further comprises providing grade-matched treatment options to the subject having a tumor determined to be malignant or likely to become malignant.

[0169] In some embodiments, the subject has been determined to have a p53 mutation, details of which are provided in the following section.

[0170] In some embodiments, the method further comprises the step of recommending, prescribing, or administering a SAGE1 inhibitor to the subject if SAGE1 expression is detected in the biological sample.

[0171] iii. TP53 deficiency (or mutation) In another aspect, a method disclosed herein is a method for predicating a possibility that a subject has SAGE1 positive disease, comprising the steps of detecting TP53 deficiency in a first biological sample from the subject, and assessing whether the subject is likely to have SAGE1 positive disease based on the presence or absence of the TP53 deficiency detected in the first biological sample.

[0172] In another aspect, a method of the disclosure is a method for predicating a likelihood that a subject has SAGE1 positive disease, comprising the step of detecting TP53 deficiency in a first biological sample from the subject, and if TP53 deficiency is detected, the subject is predicated as having a high likelihood of having SAGE1 positive disease.

[0173] As used herein, the term "deficient" or "deficient" refers to insufficient activity or level, including, for example, lower than normal activity or normal level, or no activity or level at all. For example, when TP53 activity or level is deficient, TP53 function is not functioning or does not function as well as normal, and a biological sample is The expression level of TP53 in the

[0174] Loss of TP53 is a common genetic abnormality in various cancers. In some embodiments, a deficiency in TP53 activity or level can be indicated by an inactivating mutation in TP53. As used herein, the term "inactivating mutation" with respect to TP53 refers to a mutation that results in at least partial (or complete) loss of function or activity of the TP53 gene or gene product, or a mutation that results in a non-functional gene or gene product. For example, the activity of the affected TP53 gene or gene product is significantly lower than that of the wild-type counterpart, or may be completely absent. In some embodiments, the inactivating mutation in TP53 is a translocation, deletion, insertion, substitution, or a combination thereof, that reduces the biological activity of TP53.

[0175] As used herein, the term "TP53" is intended to encompass alternative forms, including mRNA, protein, and even DNA (e.g., genomic DNA). Thus, the level and / or activity and / or mutational status of TP53 can be measured in RNA (e.g., mRNA), protein, or DNA (e.g., genomic DNA).

[0176] The mutation status or expression level of TP53 at the DNA or RNA level can be measured by any method known in the art, including but not limited to, amplification tests, hybridization assays, or sequencing assays.The mutation status or expression level of TP53 at the protein level can be measured by any method known in the art, including but not limited to, immunological tests.

[0177] In some embodiments, the method further comprises the step of recommending that the subject be tested for SAGE1 expression if a TP53 deficiency is detected, for example if an inactivating mutation in TP53 is detected.

[0178] In some embodiments, the method further comprises detecting SAGE1 expression in the first biological sample or in a second biological sample from the subject. SAGE1 expression can be detected in the first biological sample, i.e., the same sample in which TP53 is detected, or alternatively in a second biological sample from a subject of the same or different tissue type as the first biological sample. The biological sample for detecting SAGE1 expression is a sample of a type that normally lacks detectable SAGE1 expression.

[0179] In some embodiments, if SAGE1 expression is not detected in the biological sample, the method further includes a step of monitoring SAGE1 expression in the subject over time, for example, after one month, two months, three months, etc.

[0180] iv. Determination of sensitivity to SAGE1 inhibitors In another aspect, the method disclosed herein is a method for determining the sensitivity of a tumor sample to a SAGE1 inhibitor, comprising the steps of detecting SAGE1 expression in the tumor sample and evaluating whether the tumor sample is sensitive to the SAGE1 inhibitor based on the presence or absence or level of SAGE1 expression detected in the tumor sample.

[0181] In another aspect, the method disclosed herein is a method for determining the sensitivity of a tumor sample to a SAGE1 inhibitor, comprising the step of detecting SAGE1 expression in a sample tumor cell, wherein the detected SAGE1 expression indicates that the tumor cell is sensitive to a SAGE1 inhibitor.

[0182] The term "sensitivity" with respect to a tumor cell refers to the ability of the tumor cell to respond to a tumor treatment (e.g., treatment with a SAGE1 inhibitor). The sensitivity of a tumor cell can be measured, for example, in terms of inhibition of tumor cell proliferation or tumor cell death.

[0183] In some embodiments, the tumor sample is obtained from a subject. In some embodiments, the method further comprises the step of recommending treatment with a SAGE 1 inhibitor to the subject if the tumor sample is determined to be sensitive to a SAGE 1 inhibitor. In some embodiments, the method further comprises the step of providing or administering a SAGE 1 inhibitor to the subject from whom the tumor sample was obtained and who is determined to be sensitive to a SAGE 1 inhibitor.

[0184] v. Identification of subjects who are responsive to SAGE1 inhibitors In another aspect, the method disclosed herein is a method for identifying a subject having a tumor that is likely to benefit from the treatment of a SAGE1 inhibitor, comprising the steps of detecting SAGE1 expression in a biological sample from the subject, and assessing whether the subject is likely to benefit from the treatment of a SAGE1 inhibitor based on the presence or absence or level of SAGE1 expression detected in the biological sample.

[0185] In another aspect, the method disclosed herein is a method for identifying a subject having a tumor that is likely to benefit from the treatment of a SAGE1 inhibitor, comprising the step of detecting SAGE1 expression in a biological sample from the subject, and identifying the subject as likely to benefit from the treatment of a SAGE1 inhibitor if SAGE1 expression is detected.

[0186] In some embodiments, the subject has been determined to have a mutation in TP53.

[0187] In some embodiments, the method further comprises the step of recommending, prescribing, or administering a SAGE1 inhibitor to the subject identified as likely to benefit from the treatment with a SAGE1 inhibitor.

[0188] In some embodiments, the method further comprises detecting SAGE1 expression in the biological sample or another biological sample from the subject.

[0189] In some embodiments, if SAGE1 expression is not detected in the biological sample, the method further comprises monitoring SAGE1 expression in the subject over time.

[0190] vi. Monitoring the progression of SAGE1-positive disease In another aspect, the method disclosed herein is a method for monitoring the progression of a SAGE1-positive disease in a subject, comprising the steps of (a) detecting a level of SAGE1 expression in a first biological sample from the subject, (b) detecting a level of SAGE1 expression in a second biological sample from the subject, and (c) assessing whether the SAGE1-positive disease is progressing in the subject based on the levels of SAGE1 expression detected in each of steps (a) and (b), wherein the second biological sample is obtained at a time later than the first sample.

[0191] In another aspect, the disclosed method is a method of monitoring tumor progression in a subject, comprising the steps of (a) detecting a level of SAGE1 expression in a first biological sample from the subject, and (b) detecting a level of SAGE1 expression in a second biological sample from the subject, the second biological sample being separated in time from the first sample. In some embodiments, the method further comprises the step (c) of comparing the level of SAGE1 expression in the second biological sample to the level in the first biological sample to determine tumor progression. In some embodiments, the tumor is determined to be progressing if the level of SAGE1 expression in the second biological sample is higher in the second biological sample than in the first biological sample.

[0192] In some embodiments, the tumor is determined to be in regression if the level of SAGE1 expression in the second biological sample is lower in the second biological sample than in the first biological sample. In some embodiments, the tumor may or may not be treated during the time period between the first and second biological samples. In some embodiments, the method further comprises recommending, prescribing, or administering a SAGE1 inhibitor to the subject if the tumor is determined to be progressing.

[0193] vii. Monitoring Subject Response to SAGE1 Inhibitors In another aspect, the method disclosed herein is a method for monitoring responsiveness to treatment with a SAGE1 inhibitor during a treatment period of a subject with a SAGE1-positive disease, comprising the steps of (a) detecting the level of SAGE1 expression in a biological sample from the subject after the treatment period to obtain a post-treatment SAGE1 expression level, (b) comparing the post-treatment SAGE1 expression level with a baseline level of SAGE1 expression detected in a biological sample obtained from the subject before the treatment period, and (c) evaluating whether or not there is responsiveness to the treatment with the SAGE1 inhibitor based on the levels of SAGE1 expression detected in each of steps (a) and (b).

[0194] In another aspect, the method disclosed herein is a method for monitoring the responsiveness of a subject having a tumor to treatment with a SAGE1 inhibitor, comprising the steps of (a) detecting the level of SAGE1 expression in a biological sample from the subject after the treatment period to obtain a post-treatment SAGE1 expression level, and (b) comparing the post-treatment SAGE1 expression level with a baseline level of SAGE1 expression detected in a biological sample obtained from the subject prior to the treatment period.

[0195] In some embodiments, if the post-treatment level is lower than the baseline level, the subject is deemed to be responsive to the treatment.

[0196] In some embodiments, if the post-treatment level is higher than the baseline level, the subject is deemed to be non-responsive to the treatment.

[0197] Detection of SAGE1 expression In any of the methods and embodiments described herein, SAGE1 expression can be detected by any suitable method known in the art. In some embodiments, the methods described herein include contacting the biological sample with an agent capable of detecting the presence or level of SAGE1 expression in the biological sample.

[0198] SAGE1 expression can be detected based on the presence or absence of SAGE1 expression, with the presence of SAGE1 expression indicating SAGE1 positivity.

[0199] Alternatively, SAGE1 expression can be detected based on the level of SAGE1 expression, with a detected level higher than a reference level indicating positive SAGE1. The reference level can be obtained from one or more reference samples (e.g., samples obtained from healthy subjects, healthy tissues, or precancerous tissues of tumor patients). Detection of SAGE1 expression can be performed by comparing the reference samples and the biosamples of interest. The reference level can be obtained in parallel with obtaining a sample. The reference level can also be obtained from a database that includes a data set, standard, or level based on one or more reference samples. In some embodiments, the data set, standard, or level is normalized. Since SAGE1 is not typically found in most healthy cells or tissues, the reference level is understood to include a level (or noise level) that indicates no SAGE1 expression.

[0200] As used herein, SAGE1 is intended to encompass alternative forms including mRNA, protein (and complexes thereof), and even DNA (e.g., genomic DNA). Thus, SAGE1 expression can be detected at the DNA transcription level, the RNA level, or the protein level.

[0201] In some embodiments, the SAGE1 expression is indicated by (a) the presence or level of SAGE1 protein, (b) the presence or level of SAGE1 mRNA, (c) the presence or level of SAGE1 complex, (d) the methylation level of the SAGE1 gene, (e) the presence or level of histone acetylation of the SAGE1 gene, (f) the presence or level of transcription factor binding to the SAGE1 gene, or (g) a combination thereof.

[0202] In some embodiments, the SAGE1 expression is indicated by (a) the presence or a higher level than baseline of transcription of the SAGE1 gene, (b) the presence or a higher level than baseline of SAGE1 mRNA, (c) the presence or a higher level than baseline of SAGE1 protein, (d) the presence or a higher level than baseline of SAGE1 complex, or (e) a combination thereof.

[0203] The presence or level of SAGE1 mRNA can be detected by any suitable nucleic acid test known in the art, such as a nucleic acid amplification test, a nucleic acid hybridization assay, a nucleic acid sequencing assay, or other methods such as high performance liquid chromatography (HPLC) fragment analysis and capillary electrophoresis. Nucleic acid amplification tests, hybridization assays, and sequencing assays are described in the following sections. In order to detect the presence and levels of RNA molecules by amplification tests or sequencing assays, it may be necessary to reverse transcribe the RNA into cDNA prior to analysis.

[0204] The presence or level of SAGE1 protein can be detected by any assay known in the art that is suitable for detecting or quantifying polypeptides, including electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyeprdiffusion chromatography, mass spectrometry, or biochemical analytical methods such as various immunological tests. A person skilled in the art can readily adapt known protein detection methods for use in determining the presence or level of expression of SAGE1 protein.

[0205] The presence or level of SAGE1 can also be indicated by the presence or level of SAGE1 complex. In this specification, "SAGE1 complex", "SAGE1-containing complex" and "complex including SAGE1" are used synonymously and refer to a protein complex that includes one or more proteins or polypeptides in addition to SAGE1 protein or a fragment thereof. The present disclosure unexpectedly discovered that SAGE1 can form a complex with other proteins or polypeptides and exert a biological function through this complex. For example, SAGE1 interacts with INTS3 and replaces INTS6 of Integrator. Integrator is a large protein complex consisting of 14 or more subunits (INTS1 to INTS14) that play important roles in the regulation of most RNAPII-dependent genes, such as 3'-end processing of non-coding small nuclear RNAs, synthesis of enhancer RNAs, transcription pause-release and processivity, and premature transcription termination of Integrator target genes and enhancers. In some embodiments, the SAGE1 complex includes SAGE1 and INTS3. In some embodiments, the S The AGE1 complex includes a SAGE1-INTS3 complex. The SAGE1-INTS3 complex can bind to the promoter proximal region of genes bound by Integrator, and then activates the transcription of such genes. The present disclosure has discovered that expression of genes enriched in pathways essential for cell proliferation and cancer phenotypes is significantly stimulated by expression of SAGE1. In some embodiments, the SAGE1 complex further includes one or more molecules selected from INIP, NABP1 / 2, CREBBP, TLE1, TLE2, TLE3, TLE4, TLE5, GGA3, CNOT1, TAX1BP1, SEC16A, CYLD, and PAXBP1.

[0206] The presence or level of SAGE1 complexes can be detected by any suitable method for measuring protein-protein interactions. In one embodiment, protein-protein interaction analysis methods are based on immunological tests or proximity assays, such as Mesoscale Discovery (MSD) Enzyme-Linked Immunosorbent Assay (MSD-ELISA), ELSIA for standard complexes, proximity ligation assay (PLA), co-immunoprecipitation, or immunoblotting. Immunological tests for detecting SAGE1 complexes typically use antibodies that specifically bind to components of the complex or to epitopes unique to the complex to detect or measure the presence or level of the complex. Although protein-protein interactions are transient and may dissociate during sample preparation, chemical or physical crosslinking can stabilize or permanently join interacting components of protein complexes. Once components of a protein complex are covalently crosslinked, other steps (e.g., cell lysis, affinity purification, electrophoresis, or mass spectrometry) can be used to analyze protein-protein interactions while maintaining the original interacting complexes.

[0207] Transcription of the SAGE1 gene can be determined by measuring DNA methylation or DNA demethylation of the SAGE1 gene. DNA methylation is a biological process that adds a methyl group (e.g., by the action of a DNA methyltransferase enzyme) to a DNA molecule (e.g., a cytosine base or bases of a DNA molecule). In mammals, DNA methylation is observed at the 5'-terminal position of cytosine-phosphate-guanine (CpG) dinucleotides (i.e., "CpG sites"), which are often near the start site of transcription, and can lead to epigenetic inactivation of genes. Conversely, the process of DNA demethylation can remove methyl groups from methylated DNA molecules (e.g., converting a methylcytosine base to a cytosine), which can lead to epigenetic activation of genes. In some embodiments, a level of DNA methylation of the SAGE1 gene that is lower than a baseline level (or a level of DNA demethylation that is higher than a baseline level) indicates the presence or higher level of transcription of the SAGE1 gene.

[0208] The human SAGE1 gene is located on chromosome X (ChrX: 135, 893, 700-135, 913, 026, Xq26.3 according to Genome Reference Consortium Human Build 38 patch release 13) and has a gene ID of 55511 in the NCBI database (this sequence is incorporated herein as SEQ ID NO: 1). DNA methylation of the SAGE1 gene can occur in the expressed region (ChrX: 135, 893, 716-135, 913, 062) that is transcribed into SAGE1 mRNA, or in the non-expressed region 5' upstream from the expressed region (e.g., ChrX: 135, 890, 700-135, 893, 715). The expressed region includes a stretch of DNA enriched with CpG (ChrX: 135, 893, 450-135, 893, 686), designated as the CpG-containing region. In some embodiments, DNA methylation of the SAGE1 gene is detected in the CpG-containing region of the SAGE1 gene. According to the Cap Analysis of Gene Expression (CAGE) method of gene expression of the SAGE1 gene, the transcription start site is located at approximately ChrX: 135893700, and further, according to whole genome bisulfite sequencing (WGBS), there are DNA methylation differences within the region 1 kb upstream or downstream of the transcription start site of the SAGE1 gene across cell lines with different levels of SAGE1 expression (based on WGBS data K562: ENCSR765JPC, ENCSR481JIW (A549), ENCSR550RTN (HeLa-S3) at www.encodeproject.org). In some embodiments, the DNA methylation is detected in a region within 1 kb upstream and 1 kb downstream of the transcription start site of the SAGE1 gene (e.g., spanning ChrX: 135, 892, 700-135, 894, 700). In some embodiments, the DNA demethylation is detected in a region within 3 kb upstream and 3 kb downstream of the transcription start site of the SAGE1 gene (e.g., spanning ChrX: 135, 890, 700-135, 896, 700).

[0209] DNA methylation can be detected by any suitable method in the art. In some embodiments, the biological sample is treated with a reagent capable of distinguishing between unmethylated and methylated cytosines in the DNA contained in the biological sample. Such reagents include, for example, bisulfite and a methylation-sensitive restriction enzyme. Bisulfite can selectively modify unmethylated cytosines and convert them to uracil, while leaving methylated cytosines intact. After this conversion, DNA methylation at specific loci or genes can be detected and measured by nucleic acid amplification tests, sequencing assays, or hybridization assays as described below. In some embodiments, the DNA contained in the biological sample is amplified prior to detection of the DNA methylation.

[0210] The presence or level of transcription of the SAGE1 gene can also be determined by measuring the presence or level of histone acetylation of the SAGE1 gene. Histone acetylation is a biological process in which an acetyl group is added to the N-terminus of a histone protein (e.g., by the action of histone acetyltransferase). Histone acetylation alters condensed chromatin into a looser structure associated with higher gene transcription levels. In mammals, histone acetylation is typically observed at lysine residues 9-12 of histone H3 protein or lysine residue 16 of histone H4 protein (H3K9ac, H3K27ac, H4K16ac). In some embodiments, a higher level of histone acetylation of the SAGE1 gene than a baseline level indicates the presence or level of transcription of the SAGE1 gene. In some embodiments, histone acetylation is detected in non-expressed or expressed regions of the SAGE1 gene. In some embodiments, histone acetylation is detected near an enhancer or promoter region of the SAGE1 gene. In some embodiments, the histone acetylation or transcription factor binding to the SAGE1 gene is detected in a region within 1 kb upstream and within 1 kb downstream of the transcription start site of the SAGE1 gene (chrX: 135, 892, 700-135, 894, 700). In some embodiments, the histone acetylation or transcription factor binding to the SAGE1 gene is detected in a region within 3 kb upstream and within 3 kb downstream of the transcription start site of the SAGE1 gene (chrX: 135, 890, 700-135, 896, 700).

[0211] The presence or level of transcription of the SAGE1 gene can also be determined by measuring the binding of a transcription factor to the SAGE1 gene, such as to the promoter or enhancer region of the SAGE1 gene. In some embodiments, the binding of a transcription factor to the SAGE1 gene is detected in a region spanning ChrX: 135, 892, 756-135, 894, 140. Transcription of the SAGE1 gene is controlled by transcription factors such as Myc and CTCF. In some embodiments, the presence or level of transcription factor binding to the SAGE1 gene, which is greater than a baseline level, indicates the presence or level of transcription of the SAGE1 gene.

[0212] Both histone acetylation and transcription factor binding to the SAGE1 gene can be analyzed by chromatin immunoprecipitation (ChIP). ChIP allows the detection and relative quantification of specific protein-DNA interactions at a single locus or multiple loci. ChIP involves chemically or physically cross-linking a protein to a DNA sequence, followed by cleavage and fragmentation of the DNA sequence, immunoprecipitation of the cross-linked complex with an antibody specific for the protein of interest, reversal of the cross-link to release the associated DNA fragment, and immunoprecipitation of the resulting DNA fragment. are analyzed by nucleic acid amplification tests (e.g., end-point or quantitative polymerase chain reaction), hybridization assays (e.g., ChIP-chip), or sequencing assays (ChIP-seq). Nucleic acid amplification tests, hybridization assays, and sequencing assays are described above. Antibodies specific to acetylated histones, such as anti-H3K9ac antibodies, anti-H3K27ac antibodies, and anti-H4K16ac antibodies, are useful for detecting histone acetylation, and anti-Myc antibodies are useful for detecting binding of transcription factors to the SAGE1 gene. ChIP-seq analysis of multiple tumor cell lines revealed that the region near the transcription start site of the SAGE1 gene is enriched to cause H3K27ac modification and bind to an array of transcription factors.

[0213] Any suitable method can be used to detect SAGE1 expression, such as an immunological test, an amplification test, a hybridization assay, or a sequencing assay.

[0214] Amplification test

[0215] In nucleic acid amplification tests, a target nucleic acid (e.g., DNA or RNA) is copied to increase the number of copies of the amplified nucleic acid sequence. Amplification can be exponential or linear. Examples of nucleic acid amplification methods include polymerase chain reaction ("PCR", see U.S. Pat. Nos. 4,683,195 and 4,683,202, and PCR Protocol A Guide To Methods And Applications (Innis et al., eds., 1990)), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative real-time PCR (qRT-PCR), quantitative PCR such as nested PCR, ligase chain reaction (see Abravaya, K. et al., Nucleic Acids Research, 23: 675-682, (1995)), branched DNA signal amplification (see Urdea, MS et al., AIDS, 7 (suppl 2): ​​S11-S14, (1993)), amplifiable RNA reporters, Q beta replication (see Lizardi et al., Biotechnology (1988) 6: 1197), transcription-based amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA (1989) 86: 1173-1177), boomerang DNA amplification, strand displacement activity, cycling probe technology, self-sustained sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA (1990) 87: 1874-1878), rolling circle replication (U.S. Pat. No. 5,854,033), nucleic acid sequence-based isothermal amplification (NASBA), and amplification using serial analysis of gene expression (SAGE).

[0216] The level of nucleic acid can be quantified during an amplification test, also known as real-time amplification or quantitative amplification. Methods of quantitative amplification are disclosed, for example, in Gibson et al., Genome Research (1996) 6: 995-1001; DeGraves et al., Biotechniques (2003) 34 (1): 106-10, 112-5; Deiman B et al., Mol Biotechnol. (2002) 20 (2): 163-79. Quantification is usually based on monitoring a detectable signal indicative of the copies of the template during the cycles of an amplification (e.g., PCR) reaction. The detectable signal can be generated by an intercalating agent or a labeled primer or a labeled probe used during the amplification. The level of the detected biomarker can be quantified by methods known in the art. As a means of quality control, the level of an internal control biomarker may be measured. One skilled in the art will appreciate that an internal control biomarker may be inherently present in a sample and its level may be used to normalize the measured level of SAGE1 to compensate for differences in absolute amounts of samples. Alternatively, the nucleic acid levels may be quantified following the amplification test.

[0217] Hybridization assay

[0218] Nucleic acid hybridization assays allow detection of a target nucleic acid by hybridizing the target nucleic acid with a probe. In some embodiments, the probes used in hybridization assays are detectably labeled. In some embodiments, the hybridization Nucleic acid-based probes for the ELISA assay are unlabeled. Such unlabeled probes can be immobilized on a solid support, such as a microarray, and hybridized to a target nucleic acid molecule that is detectably labeled.

[0219] In one embodiment, a hybridization assay can be performed by isolating nucleic acids (e.g., RNA or DNA), separating the nucleic acids (e.g., by gel electrophoresis), and then transferring the separated nucleic acids to a suitable membrane filter (e.g., a nitrocellulose filter) where the probe hybridizes to the target nucleic acid and allows detection. See, for example, Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7. Hybridization of the probe to the target nucleic acid can be detected or measured by methods known in the art. For example, autoradiographic detection of hybridization can be achieved by exposing the hybridized filter to photographic film. Densitometric scanning of the photographic film exposure of the hybridized filter provides an accurate measurement of the target nucleic acid level. A computerized imaging system can also be used to quantitate the biomarker levels.

[0220] Sequencing assays

[0221] Sequencing assays (e.g., next-generation or third-generation sequencing methods) are useful for detecting the presence and levels of target DNA molecules, sequencing the target DNA, and counting the sequenced target DNA molecules. These sequencing methods include sequencing-by-synthesis, sequencing-by-ligation, ultra-deep sequencing, and ELISA. Sequencing by synthesis is based on principles such as single molecule sequencing, or single molecule sequencing. For example, sequencing by synthesis incorporates a labeled nucleotide or nucleotide analogue to synthesize a complementary strand of a target nucleic acid in a polymerase amplification. Immediately or at the time the labeled nucleotide is incorporated, the signal of labeling is measured and the identity of the nucleotide is recorded. The detectable label of the incorporated nucleotide is removed before the incorporation, detection, and identification steps are repeated. Sequencing by synthesis may be performed on a solid surface (or microarray or chip) using fold-back PCR and anchored primers. Target DNA fragments can be attached to the solid surface by hybridizing to the anchored primers and the bridge is amplified. Examples of sequencing methods include sequencing, pyrosequencing, SoLiD (supported oligonucleotide ligation and detection), Ion Torrent technology, SMRT (single-molecular real time) sequencing, and Helicos sequencing. sequencing, nanopore sequencing, and the like, but are not limited to these.

[0222] Immunologic testing

[0223] Immunological tests typically use antibodies that specifically bind to the protein of interest. Such antibodies can be obtained by methods known in the art (see, for example, Huse et al., Science (1989)). 246: 1275-1281; Ward et al., Nature (1989) 341: 544-546), or from commercial sources. Examples of immunological tests include, but are not limited to, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunosorbent assay (EIA), radioimmunoassay (RIA), immunoprecipitation, sandwich assays, competitive assays, immunofluorescent staining imaging, immunohistochemistry, and fluorescence-activated cell sorting (FACS). For a review of immunological methods and immunological testing methods, see Basic and Clinical Immunology (S See Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed., 1993) and Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunoassays can also be performed in any of several configurations discussed in Enzyme Immunoassay (Maggio, ed., 1980) and Harlow & Lane, supra. For a review of immunoassays in general, see Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed., 1993) and Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991).

[0224] Any of the assays and methods described herein for detecting SAGE1 expression can be adapted or optimized for use in automated and semi-automated systems or point-of-care analytical systems. When determining the level of SAGE1 expression, the level of SAGE1 expression detected by the assays described herein can be normalized by any suitable method known in the art. For example, the level can be normalized to an internal control, which can be the average or total level of multiple internal markers or an internal marker.

[0225] In some embodiments, SAGE1 is the sole biomarker detected by the methods described herein. In some embodiments, detecting SAGE1 expression by the methods described herein comprises providing a biological sample from a subject.

[0226] ix. Evaluation based on SAGE1 expression Although SAGE1 is expressed in cells with high stemness, such as human totipotent stem cells, during embryonic development, it is not expressed in most normal tissues. However, SAGE1 expression has been confirmed in various abnormal tissues, suggesting that SAGE1 can be used as a biomarker for diagnosing SAGE1-positive diseases.

[0227] Based on the detection of SAGE1 expression, methods for diagnosing tumors that have not been effectively detected in the early stages, methods for evaluating tumor malignancy, progression, metastasis, recurrence, prognosis, and the effectiveness of tumor treatment, and methods for selecting targeted therapy are realized. Compared with traditional biomarkers, SAGE1 has high specificity and is nearly 100% accurate in distinguishing between normal tissues and tumors.

[0228] In some embodiments, expression of SAGE1 in a biological sample that does not normally express SAGE1 indicates that the subject from whom the biological sample was obtained has a SAGE1-positive disease, whereas absence of SAGE1 expression indicates the absence of such a SAGE1-positive disease.

[0229] Similarly, expression of SAGE1 in a tumor biological sample indicates that the tumor is likely to be malignant or has the potential to become malignant, that the tumor is sensitive to treatment with a SAGE1 inhibitor, or that the subject is likely to be responsive to a SAGE1 inhibitor.

[0230] In some embodiments, the level of SAGE1 expression can be determined and compared to a threshold level that can be determined based on a positive reference sample known to express SAGE1 and a negative reference sample known not to express SAGE1, such that the positive reference sample can be distinguished from the negative reference sample.

[0231] In some embodiments, changes in the level of SAGE1 expression in a subject may indicate progression of a SAGE1-positive disease or the responsiveness of the subject to a SAGE1 inhibitor, for example, an increased level of SAGE1 expression indicates that a SAGE1-positive disease has progressed or that a subject administered a SAGE1 inhibitor is likely to become unresponsive to or has developed resistance to the treatment.

[0232] x. Biological samples Any suitable biological sample can be used to detect SAGE1 expression, in some embodiments, the biological sample is specifically suspected of containing SAGE1 positive cells, SAGE1 expression products, or the SAGE1 gene or fragments thereof.

[0233] In some embodiments, the biological sample comprises abnormal cells, particularly those suspected of containing a SAGE1 expression product or a SAGE1 gene or fragment thereof. In some embodiments, the biological sample is of a type that normally lacks detectable SAGE1 expression. In some embodiments, the biological sample does not contain spermatogonial stem cells (SSCs). In some embodiments, the biological sample is obtained from the fallopian tubes, salivary glands, adipose tissue, adrenal glands, blood, bladder, blood vessels, breast, cervix, colon, esophagus, heart, kidney, liver, lung, muscle, nerve, ovary, pancreas, prostate, skin, small intestine, spleen, stomach, thyroid, uterus, or vagina.

[0234] In some embodiments, the biological sample is selected from cells, tissues, bodily fluids, and combinations thereof. A suitable method known to one of skill in the art can be used to obtain a biological sample from a subject. In general, the type of sample (i.e., cells, tissues, or bodily fluids) is selected based on the availability and structure of the organ or tissue to be evaluated for tumor cell growth or the type of cancer to be evaluated. The biological sample may be any bodily fluid or cell of the subject that may harbor tumor cells or tumor cell contents (e.g., DNA, RNA, proteins, intracellular organelles). In some embodiments, the biological sample is selected from the group consisting of tissue sections, biopsies, paraffin-embedded tissues, bodily fluids, samples from surgical resections, and combinations thereof.

[0235] The body fluid may be an intracellular fluid, an extracellular fluid, or a combination of the two. In some embodiments, the body fluid is selected from the group consisting of blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple fluid, lymphatic fluid, fluids of the respiratory, intestinal and genitourinary tracts, breast milk, fluids of organ systems, peritoneal fluid, conditioned medium obtained from the culture of tissue explants, or combinations thereof.

[0236] Biological samples may be used fresh, frozen, fixed, or preserved after acquisition. The biological sample may be prepared by any method appropriate for the detection technique used. For example, the biological sample may be prepared by isolating or extracting tumor cells (such as circulating tumor cells) from a body fluid sample. In another example, the biological sample may be prepared by fixing a tissue sample in paraffin. The fixed tissue may then be sectioned and contacted with an antibody to detect binding to a target (e.g., SAGE1 protein or SAGE1 complex). In one embodiment, the biological sample is processed to isolate proteins, DNA (genomic or cell-free DNA), mRNA, or subcellular organelles for detection of SAGE1 expression.

[0237] xi.SAGE1 expression detection kit In another embodiment, a kit of the disclosure, for use in any of the methods described herein, comprises one or more reagents for detecting SAGE1 expression.

[0238] In some embodiments, the one or more reagents for detecting SAGE1 expression are selected from a reagent for detecting the presence or level of SAGE1 protein, a reagent for detecting the presence or level of SAGE1 mRNA, a reagent for detecting the presence or level of a SAGE1 complex, a reagent for detecting the methylation level of the SAGE1 gene, a reagent for detecting the presence or level of histone acetylation of the SAGE1 gene, a reagent for detecting the presence or level of transcription factor binding to the SAGE1 gene, and combinations thereof.

[0239] In some embodiments, the kit includes one or more reagents, such as primers, probes, and / or antibodies. The agent may be specific for the detection of SAGE1 genomic DNA, SAGE1 mRNA, SAGE1 protein, the SAGE1 complex, methylated or unmethylated cytosine or its converted nucleotides, or for the detection of acetylated histones or for the detection of transcription factors binding to SAGE1 genomic DNA.

[0240] In some embodiments, the kit comprises at least one reagent selected from a SAGE1-specific oligonucleotide primer, a SAGE1-specific oligonucleotide probe, an anti-SAGE1 antibody, and an anti-SAGE1 complex antibody.

[0241] The terms "primer" and "probe" as used herein with respect to nucleic acids refer to oligonucleotides capable of specifically hybridizing to a target polynucleotide sequence due to sequence complementarity with at least a portion of the primer in the sequence of the target polynucleotide sequence. Primers are useful in nucleic acid amplification tests and sequencing assays in which a new polynucleotide strand is generated by extending the primer. Probes are useful in identifying a target nucleic acid in a sample by hybridizing to the target nucleic acid. Primers and probes can be readily designed by those skilled in the art with common general knowledge so as to specifically anneal to the nucleotide sequence of the target nucleotide sequence described herein. The length of the primer or probe is at least 8 nucleotides, typically 8-70 nucleotides, and usually 18-26 nucleotides. A primer has a sequence complementarity of 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more to the hybridizing portion of the target polynucleotide sequence in order to properly hybridize to the target sequence.

[0242] In one embodiment, a primer or probe described herein comprises a polynucleotide sequence capable of hybridizing to a portion (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides) of SAGE1 genomic DNA (SEQ ID NO: 1) or mRNA (SEQ ID NO: 2). In one embodiment, a primer or probe described herein comprises a polynucleotide sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementarity to a portion (e.g., 66, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more consecutive nucleotides) of SAGE1 genomic DNA (SEQ ID NO: 1) or mRNA (SEQ ID NO: 2).

[0243] In one embodiment, the antibodies described herein comprise an antigen-binding region capable of specifically binding to an epitope within the SAGE1 protein having the sequence of SEQ ID NO:3.

[0244] The primers, probes, and / or antibodies described herein may or may not be detectably labeled. As used herein, the term "detectable label" refers to a molecule or moiety that allows for detection. The term "detectably labeled" with respect to a reagent refers to the reagent containing or being capable of being bound by a detectable label. In certain embodiments, the primers, probes, and antibodies described herein can specifically bind to a detectably labeled ligand. Detectable labels suitable for labeling primers, probes, and antibodies are, for example, chromophores, radioisotopes, fluorescent dyes, chemiluminescent moieties, particles (visible or fluorescent), nucleic acids, ligands, or catalysts such as enzymes. It is understood that a detectable label need not itself generate a detectable signal, but in certain embodiments, can react with a detectable partner or one or more additional compounds to generate a detectable signal. As another example, enzymes are useful as detectable labels due to their catalytic action on chromogenic, fluorogenic, or luminescent substrates that lead to the generation of a detectable signal.

[0245] In some embodiments, the kit may include other agents for carrying out the methods described herein. For such applications, the kit may include some or all of the following: appropriate buffers, reagents for isolating nucleic acids, reagents for amplifying nucleic acids (e.g., polymerase, dNTP mix), reagents for sequencing nucleic acids, reagents for quantifying nucleic acids (e.g., intercalating agents, detection probes), reagents for isolating proteins, and reagents for detecting proteins (e.g., secondary antibodies).

[0246] In some embodiments, the kit may include an agent for detecting methylation. For such applications, the kit includes a conversion reagent capable of distinguishing between unmethylated and methylated CpG sites in DNA for DNA manipulation. In some embodiments, the reagent is a bisulfite reagent. The bisulfite reagent may be selected from the group consisting of, for example, ammonium bisulfite, sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, hydrogen sulfite, and combinations thereof. Alternatively, the sulfite reagent is commercially available, such as MethylCode™ Bisulfite Conversion Kit, EpiMark™ Bisulfite Conversion Kit, EpiJET™ Bisulfite Conversion Kit, EZ DNA Methylation-Gold™ Kit, etc. In some embodiments, the reagent is a methylation-sensitive restriction enzyme (MSRE). In some embodiments, the MSRE is selected from the group consisting of HpaII, SalI, ScrFI, BbeI, NotI, SmaI, XmaI, MboI, BstBI, ClaI, MluI, NaeI, NarI, PvuI, SacII, HhaI, and combinations thereof.

[0247] Reagents useful in any of the methods described herein are contained in a carrier or compartmented container, e.g., a bag, box, tube, rack, or other container or support, which may be compartmented.

[0248] In one embodiment, the kit further comprises a standard negative control and / or a standard positive control.

[0249] The kits may also include instructional materials, typically, but not limited to, written or printed materials, containing directions (i.e., protocols) for the practice of the methods described herein.

[0250] In another aspect, the present disclosure provides for the use of a substance for detecting SAGE1 gene or its active fragment in the preparation of a kit for diagnosing tumors. The kit can diagnose tumors by detecting the expression level of SAGE1 gene or its active fragment, and can determine whether a subject is suitable for SAGE1-targeted therapy. The inventors have discovered that the expression of SAGE1 gene or its active fragment is closely related to whether a subject has tumors, and that tumor patients with positive or high levels of SAGE1 expression are more suitable for SAGE1-targeted therapy. The present disclosure demonstrates that inhibition of SAGE1 can significantly suppress tumor cell proliferation, migration, and anchorage-independent growth, while restoration of the blocked SAGE1 expression can restore the clonogenicity of tumor cells.

[0251] In some embodiments, SAGE1 is the only biomarker in the kit. The kit does not include materials for detecting other biomarkers other than materials for detecting the SAGE1 gene or active fragments thereof.

[0252] IV. Targeting SAGE1 and the SAGE1 Complex for Disease Treatment In another aspect, the disclosure is directed to SAGE1 inhibitors and their use in treating SAGE1 positive diseases.

[0253] Surprisingly, the present disclosure provides a method for inhibiting the expression and / or function of SAGE1, thereby inhibiting the expression and / or function of SAGE1. We found that the proliferation, migration, and anchorage-independent growth of various tumor cells expressing E1 were significantly suppressed, while the tumorigenicity of the cells could be restored when SAGE1 expression was restored, demonstrating the use of SAGE1 inhibitors as a tumor treatment. SAGE1 is specifically expressed in various tumor tissues, but not in normal tissues other than the testis. Although the SAGE1 protein is normally located in the nucleus, peptide fragments of this protein are presented on the surface of tumor cells by major histocompatibility complex (MHC), which can be used for target recognition and immune activation in tumor therapy. Therefore, SAGE1-targeted therapy is highly specific and does not affect normal tissues with SAGE1 expression.

[0254] As used herein, the term "SAGE1 inhibitor" refers to an agent that inhibits the level of SAGE1 or biological activity mediated by SAGE1. For example, a SAGE1 inhibitor can partially inhibit, i.e., suppress, or completely inhibit, i.e., eliminate, the expression and / or activity of SAGE1, including the ability of SAGE1 to bind to INTS3 and form a SAGE1-INTS3 complex. Reducing the level of SAGE1 can be achieved by inhibiting transcription of the SAGE1 gene, reducing the level of SAGE1 mRNA, or reducing the level of SAGE1 protein. Inhibiting the biological activity of SAGE1 can be achieved by preventing SAGE1 from binding to or activating one or more of its binding partners.

[0255] The type of SAGE1 inhibitor may be any suitable type of inhibitor. In some embodiments, the SAGE1 inhibitor comprises a nucleic acid molecule (e.g., a polynucleotide or oligonucleotide), a polypeptide, a compound (e.g., a chemical compound), or an antibody or antigen-binding fragment thereof. In some embodiments, the SAGE1 inhibitor is not a chimeric receptor or a T cell receptor. In some embodiments, the SAGE1 inhibitor is not a chimeric receptor or a T cell receptor that binds to a SAGE1 epitope or an MHC-associated epitope of SAGE1.

[0256] i.Nucleic acid In some embodiments, the SAGE1 inhibitor comprises a nucleic acid molecule, hi some embodiments, the SAGE1 inhibitor comprises an oligonucleotide targeted to a SAGE1 nucleic acid, such as SAGE1 mRNA or gene, a polynucleotide encoding the oligonucleotide, or a nucleic acid vector comprising the oligonucleotide or polynucleotide.

[0257] Oligonucleotides targeting SAGE1 nucleic acids can include both single-stranded and double-stranded oligonucleotides, including, but not limited to, antisense oligonucleotides, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), CRISPR / Cas9 guide RNA, and other oligomeric compounds or oligonucleotide analogs.

[0258] The oligonucleotide targets an appropriate portion of the SAGE1 nucleic acid. As used herein, the term "portion" refers to a predetermined number of contiguous nucleotides of an oligonucleotide or nucleic acid. One of skill in the art can determine an appropriate target portion of SAGE1, e.g., a portion that is sufficiently specific in sequence to suppress undesired off-target binding and / or that is sufficiently accessible to the oligonucleotide despite secondary or tertiary structure of the SAGE1 nucleic acid. Various tools exist for this purpose. For example, the BLAST algorithm can be used to compare the sequence of the SAGE1 nucleic acid to other sequences throughout the genome to avoid selecting target portions that may nonspecifically hybridize to sequences other than the selected target nucleic acid (i.e., non-target or off-target sequences). In some embodiments, the target portion of the SAGE1 nucleic acid is 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides in length, or within a range determined by any two of these lengths.

[0259] In some embodiments, the SAGE1 nucleic acid is SAGE1 mRNA. The target portion of the SAGE1 mRNA may be located in a structurally defined region of the SAGE1 mRNA, such as the 3'UTR, 5'UTR, intron, exon-intron junction, coding region, translation initiation region, or translation termination region. In some embodiments, the target portion of the SAGE1 mRNA comprises six or more contiguous nucleotides (e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides) within the sequence between nucleotides 266-366, 866-1116, 1316-1616, 1916-2266, 2466-2666, 2782-2946 of SEQ ID NO:2. In some embodiments, the target portion of SAGE1 mRNA can be selected from the group consisting of nucleotides 282-310, 333-351, 1076-1094, 880-898, 1933-1951, 2148-2166, 1471-1489, 2493-2511, 1331-1349, 2215-2233, 1559-1579, 2799-2819 of SEQ ID NO: 2. In some embodiments, the target portion of SAGE1 mRNA comprises a sequence selected from the group consisting of SEQ ID NOs: 11-67.

[0260] In some embodiments, the SAGE1 nucleic acid is SAGE1 genomic DNA. The target portion in the SAGE1 genomic DNA may be located in a non-coding region or a coding region of the SAGE1 genomic DNA. In some embodiments, the SAGE1 nucleic acid comprises a sequence selected from SEQ ID NOs: 68 and 69.

[0261] In some embodiments, the SAGE1 inhibitor comprises a sequence complementary to a target portion of a SAGE1 nucleic acid (e.g., genomic DNA or mRNA) and inhibits its expression or function. The sequence of the oligonucleotide and the target portion of the SAGE1 nucleic acid may not require 100% complementarity. In some embodiments, the SAGE1 inhibitor comprises a sequence having at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to the target portion of the SAGE1 nucleic acid. The level of complementarity is considered sufficient to produce a desired effect upon hybridization of the oligonucleotide with the SAGE1 target nucleic acid. The desired effect may include, but is not limited to, a reduction in the level of mRNA of the target nucleic acid, a reduction in the level of a protein encoded by the target nucleic acid, or a change in a phenotype associated with the target nucleic acid.

[0262] In some embodiments, the SAGE1 nucleic acid targeted oligonucleotides are at least 8-80, 10-80, 12-50, 15-30, 18-24, 19-22, or 20 nucleotides or base pairs in length. In some embodiments, the oligonucleotides are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 or 80 nucleotides or base pairs in length, or within a range determined by any two of these lengths.

[0263] In some embodiments, the oligonucleotides can be chemically modified. Modifications can include substitutions or changes to the internucleoside linkages, sugar moieties of the nucleotide, or nucleobases of the nucleotide. Modified oligonucleotides can have other properties, such as enhanced cellular uptake, They can have desirable properties such as increased affinity for a nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity. Chemically modified nucleotides can also be used to increase the binding affinity of a truncated or deleted oligonucleotide for its target nucleic acid.

[0264] The naturally occurring internucleoside linkages in RNA and DNA are phosphodiester linkages at the 3' to 5' ends. Oligonucleotides with modified internucleoside linkages include those that retain a phosphorus atom and those that do not. Exemplary phosphorus containing internucleoside linkages include, but are not limited to, phosphodiester, phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate. Exemplary non-phosphorus containing internucleoside linkages include, but are not limited to, amide backbones, morpholino backbones, and peptide nucleic acid (PNA) backbones. Phosphorus containing and non-phosphorus containing linkages are made by known methods. In one embodiment, oligonucleotides targeted to SAGE1 nucleic acids include one or more modified internucleoside linkages.

[0265] The oligonucleotides of the present disclosure may include one or more nucleosides with sugar modifications. In some embodiments, the nucleosides include chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include, but are not limited to, those with additional substituents (including 2', 3', and 5' end substituents), those bridging non-geminal rings to form bicyclic nucleic acids, those replacing the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2), where R, R1, and R2 are each independently H, C1-C2 alkyl, or a protecting group, and combinations thereof. The oligonucleotides may also have sugar analogs such as cyclobutyl instead of pentofuranosyl.

[0266] Nucleobase (or base) modifications or substitutions are structurally distinct from, but functionally interchangeable with, naturally occurring nucleobases. As used herein, "natural" nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Both natural and modified nucleobases can participate in hydrogen bonds. Such nucleobase modifications may confer nuclease stability, binding affinity, or other biologically beneficial properties to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, inosine, hypoxanthine, 6-methyladenine, 5-Me pyrimidines (e.g., 5-methylcytosine (5-me-C)), 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, and the like, including synthetic nucleobases such as, for example, 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalkylamino)adenine, or other hetero-substituted alkyl adenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6(6-aminohexyl)adenine, 2,6-diaminopurine, 5-ribosyluracil (Carlile (2014) Nature 5 15 (7525): 143-6).

[0267] Not all positions in an oligonucleotide need be uniformly modified, and in fact one or more of the aforementioned modifications may be incorporated into a single oligonucleotide, or even a single nucleoside within an oligonucleotide. In some embodiments, both the nucleobase and the backbone may be modified. In some embodiments, both the sugar and the internucleoside linkage may be modified. In some embodiments, the oligonucleotide is a chimeric oligonucleotide, comprising two or more chemically distinct regions, each of at least one nucleotide. These oligonucleotides typically contain one or more regions of modified nucleotides that confer one or more beneficial properties (e.g., increased nuclease resistance, increased cellular uptake, increased binding affinity to a target, etc.) and a region that is a substrate for an enzyme capable of cleaving RNA:DNA or RNA:RNA hybrids. Such chimeric oligonucleotides are Leotides are also known in the art as hybrids or gapmers.

[0268] The oligonucleotides may be covalently linked to one or more moieties or conjugate systems that enhance the activity, cellular distribution, or cellular uptake of the resulting antisense oligonucleotide. Exemplary conjugate groups include cholesterol moieties and lipid moieties. Other conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0269] Oligonucleotides can also be modified to provide improved properties, such as nuclease stability, typically with one or more stabilizing groups added to one or both ends of the oligonucleotide. These end modifications protect the oligonucleotide from exonuclease degradation and aid in intracellular transport and / or localization. Stabilizing groups include cap structures. The cap can be at the 5' end (5-cap), the 3' end (3'-cap), or both ends.

[0270] ii. Antisense oligonucleotides In some embodiments, the SAGE1 inhibitor is an oligonucleotide. In some embodiments, the oligonucleotide is an antisense oligonucleotide. Antisense oligonucleotides are typically designed to block expression of a DNA or RNA target by binding to the target and halting expression at the transcription, translation, or splicing level. The antisense oligonucleotides of the present disclosure are antisense nucleic acid sequences designed to hybridize to SAGE1 nucleic acids under stringent conditions. As used herein, "stringent conditions" refers to conditions under which a sequence will hybridize to its target sequence but will not hybridize to other sequences that are not complementary. Stringent conditions are sequence-dependent and will vary in each situation. For example, longer fragments may require higher hybridization temperatures for specific hybridization than shorter fragments. Other factors also affect the stringency of hybridization, so it is important to include the length and base composition of the complementary strand, the presence of organic solvents, the degree of base mismatch, and a combination of parameters rather than an absolute measure of any one parameter. Typically, stringent conditions are selected to be about 5° C. lower than the melting temperature (Tm) of the specific sequence at a given ionic strength and pH. Thus, oligonucleotides are selected that are sufficiently complementary to the target, i.e., hybridize sufficiently well and are sufficiently specific, to produce the desired effect, while striving to avoid significant off-target effects, i.e., not directly bind, not directly affect expression levels, and not transcribe to targets other than those of interest.

[0271] Antisense oligonucleotides can vary in optimal length (e.g., 12-18 nucleotides in length), but their target sequences are unique in the transcriptome (Seth (2009) J Med Chem 52: 10-13). In some embodiments, the antisense oligonucleotides include one or more modifications described herein. For example, the modified internucleoside linkages of the antisense oligonucleotide are phosphorothioate internucleoside linkages, the modified sugar is a bicyclic sugar or 2'-O-methylethyl, and the modified nucleobase is 5-methylcytosine. As another example, the antisense oligonucleotide is a gapmer comprising a gap segment comprised of linked deoxynucleosides, a 5' wing segment comprised of linked nucleosides, and a 3' wing segment comprised of linked nucleosides, the gap segment being positioned immediately adjacent between the 5' wing segment and the 3' wing segment, and the nucleosides of each wing segment comprise a modified sugar.

[0272] iii. Interfering oligonucleotides In some embodiments, the oligonucleotide is a double-stranded interfering oligonucleotide, including but not limited to a small interfering RNA ("siRNA") or a short hairpin RNA ("shRNA") Double-stranded interfering oligonucleotides are capable of translational inhibition or degradation of a target mRNA.

[0273] Interfering RNA (e.g., siRNA) can be made by combining two separate oligonucleotides, one of which is the sense strand and the other the antisense strand, where the antisense and sense strands are self-complementary to form a duplex or double-stranded structure. The antisense strand comprises a nucleotide sequence complementary to at least a portion of a target nucleic acid molecule, and the sense strand comprises a nucleotide sequence corresponding to at least a portion of the target nucleic acid sequence. In some embodiments, the duplex structure is about 15 to about 30 base pairs, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs. In some embodiments, the siRNA comprises a 3' overhang (e.g., a doublet such as dTdT) on each strand. In some embodiments, the siRNA comprises a sense / antisense RNA strand pair with or without a 3' dTdT overhang selected from the group consisting of SEQ ID NOs: 70 / 71, 72 / 73, 74 / 75, 76 / 77, 78 / 79, 80 / 81.

[0274] Alternatively, an interfering RNA (e.g., shRNA) can be a single oligonucleotide, in which the self-complementary sense and antisense regions are linked by a nucleic acid-based or non-nucleic acid-based linker. In some embodiments, the interfering RNA is an shRNA having a sense region, an antisense region, and a loop region. The loop region is generally about 2 to about 10 nucleotides in length. In some embodiments, the loop region is about 6 to about 9 nucleotides in length. In some embodiments, the sense and antisense regions are about 15 to about 30 nucleotides in length (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length). After post-transcriptional processing, the shRNA is converted to an siRNA by a cleavage event by the Dicer enzyme.

[0275] The target RNA cleavage reaction guided by siRNA has high sequence specificity. In general, siRNA containing the same nucleotide sequence as a part of the target nucleic acid is preferable for inhibition. However, in carrying out the present invention, it is not necessary that the sequence identity between the siRNA and the target gene is 100%. Therefore, the present invention has the effect of being able to tolerate the expected sequence diversity due to gene mutation, strand polymorphism, or evolutionary divergence. For example, it has been found that siRNA sequences with insertions, deletions, and single point mutations compared to the target sequence also have an inhibitory effect.

[0276] In some embodiments, shRNAs useful in the present disclosure comprise a sequence selected from the group consisting of SEQ ID NOs: 82-95 and 106-145.

[0277] In some embodiments, the double-stranded interfering oligonucleotide comprises one or more modifications as described herein.In some embodiments, the double-stranded interfering oligonucleotide is not limited to molecules that only comprise RNA, but also includes chemically modified nucleotides.For example, both strands are entirely composed of ribonucleotides, one strand is entirely composed of ribonucleotides and one strand is entirely composed of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides.

[0278] iv. CRISPR / Cas CRISPR-Cas genome editing systems typically include a non-specific CRISPR-associated endonuclease (endonuclease (Cas, e.g., Cas9)) and a guide RNA, allowing efficient genome editing in a wide variety of organisms and cell types (Doudna & Charpentier, Science 346, 1258096 (2014)). The CRISPR-Cas genome editing system can also be used to inhibit the appearance of SAGE1. In some embodiments, the oligonucleotide is a guide RNA. The terms "sgRNA" and "guide RNA" are used interchangeably herein and refer to a short RNA that includes a "scaffold" sequence required for Cas9 binding and a user-defined nucleotide "spacer" or "targeting" or "guide" sequence that is complementary to at least a portion of the target genomic DNA. The term "guide sequence" as used herein also includes DNA that encodes the corresponding DNA or RNA guide sequence. Recognition of the target site by Cas9 is programmed by both the guide sequence of the guide RNA and recognition of the adjacent short PAM. Guide RNAs can be designed using standard tools known in the art.

[0279] The genomic target of Cas9 can be altered by modifying the guide sequence present in the guide RNA. The guide sequence of the guide RNA sequence can be internal or external to the locus. In general, the guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between the guide sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 97.5%, 99%, or more. In some embodiments, the guide sequence is about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or more in length. In some embodiments, the guide sequence is about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12 nucleotides or less in length.

[0280] In some embodiments, the guide sequence is selected to reduce the degree of secondary structure in the guide sequence. The secondary structure may be determined by a suitable polynucleotide folding algorithm. Some programs are based on Gibbs free energy calculations. An example of such an algorithm is mFoId, which is described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another folding algorithm is the online web server RNAfoid, developed by the Institute for Theoretical Chemistry at the University of Vienna using a centroid structure prediction algorithm (see, e.g., AR Gruber et al., 2008, Cell 106 (1): 23-24 and PA Carr and GM Church, 2009, Nature Biotechnology 27 (12): 1151-62).

[0281] In some embodiments, the guide sequence is complementary to a portion of the SAGE1 genome comprising a sequence selected from the group consisting of SEQ ID NOs: 68 and 69. In some embodiments, the guide RNA comprises a sequence selected from the group consisting of SEQ ID NOs: 146 and 147.

[0282] v. SAGE1 complex inhibitor In some embodiments, the SAGE1 inhibitor comprises a SAGE1 complex inhibitor.

[0283] As used herein, the term "SAGE1 complex inhibitor" can reduce the level or activity of the SAGE1 complex. For example, the SAGE1 complex inhibitor can inhibit the formation of the SAGE1 complex, inactivate the SAGE1 complex, or reduce the biological activity of the SAGE1 complex. Biological activities include, but are not limited to, binding of the SAGE1 complex to DNA (eg, the promoter proximal region of a gene typically bound by an Integrator) and regulating transcription of the gene.

[0284] In some embodiments, the SAGE1 complex inhibitor inhibits the formation of SAGE1 complex. In some embodiments, the SAGE1 complex inhibitor inactivates SAGE1 complex. In some embodiments, the SAGE1 complex inhibitor reduces the biological activity of SAGE1 complex.

[0285] In the present disclosure, various SAGE1 complexes are provided. Protein-protein interactions between SAGE1 and other binding partners can be determined, for example, by immunoprecipitation (see, for example, Hamilton BJ et al., Biochem. Biophys. Res. Commun. 1999, 261: 646-51). SAGE1 can be immunoprecipitated from SAGE1-expressing cell lines using anti-SAGE1 antibodies. Alternatively, His-tag antibodies can be used for cell lines engineered to express a fusion of SAGE1 with a His-tag. Immunoprecipitated complexes can be examined for protein association by procedures such as Western blotting, 35S-methionine labeling of proteins, protein microsequencing, silver staining, and two-dimensional gel electrophoresis.

[0286] In some embodiments, the SAGE1 complex is selected from the group consisting of INTS3, INIP (UniProt Accession: Q9NRY2), NABP1 / 2 (UniProt Accession: Q96AH0, Q9BQ15), CREBBP (UniProt Accession: Q92793), TLE1 (UniProt Accession: Q04724), TLE2 (UniProt Accession: Q04725), TLE3 (UniProt Accession: Q04726), TLE4 (UniProt Accession: Q04727), TLE5 (UniProt Accession: Q08117), GGA3 (UniProt Accession: Q9NZ52), CNOT1 (UniProt Accession: A5YKK6), TAX1BP1 (UniProt Accession: Q86VP1), SEC16A (UniProt Accession: O15027), CYLD ... The present invention relates to an inhibitor that inhibits the complex formed by SAGE1 and at least one component selected from the group consisting of SAGE1 complexed with ...

[0287] In some embodiments, the SAGE1 complex comprises SAGE1 in complex with INTS3. In some embodiments, the SAGE1 complex inhibitor reduces the level of the SAGE1 complex by inhibiting the interaction between SAGE1 and INTS3. In some embodiments, the SAGE1 complex inhibitor inhibits the binding of SAGE1 to INTS3.

[0288] Surprisingly, the present disclosure finds that inhibition of the SAGE1 complex in tumor cells can suppress tumor growth, and that tumor cell proliferation can be restored by expression of exogenous wild-type SAGE1, but not by expression of mutant SAGE1 whose residues are putatively involved in interaction with INTS3. The present disclosure also finds that differential expression of genes in response to inhibition of SAGE1 expression is consistent with inhibition of INTS3. These findings demonstrate that SAGE1 can form a complex with INTS3 to function, and that disrupting the interaction of SAGE1 with INTS3 or decreasing the amount of SAGE1-INTS3 complex in SAGE1-positive cells may be useful in treating SAGE1-positive cells.

[0289] In some embodiments, the SAGE1 complex inhibitor interferes with the interaction of SAGE1 with the component (e.g., INTS3) that is complexed with SAGE1. In some embodiments, the SAGE1 complex inhibitor interferes with the interaction of SAGE1 with INTS3. The inhibition may be achieved by blocking amino acid residues of the SAGE1 protein involved in the interaction with INTS3. As used herein, the term SAGE1-INTS3 complex generally refers to a complex formed between SAGE1 (or a fragment thereof) and INTS3 (or a fragment thereof) by protein-protein interaction. INTS3 may typically form a dimer, which may further form a SAGE1-INTS3 complex with SAGE1. The C-terminal fragment of SAGE1 (SEQ ID NO: 1) may be a cytochrome P450 (C-terminal fragment) or a cytochrome P450 (C-terminal fragment) that is involved in the interaction with INTS3. According to a crystal structure of a complex comprising the C-terminal fragment of INTS3 (residues 818-904 of SEQ ID NO:3) and the C-terminal fragment of INTS3 (residues 572-978 of SEQ ID NO:5), amino acid residues of SAGE1 in the binding interface with INTS3 may include at least one or more residues selected from the group consisting of F838, F873, K874, M832, V876, R872, K828, R836, and Q840 (residues numbered according to SEQ ID NO:3). In some embodiments, the SAGE1 complex inhibitor binds to INTS3 or prevents INTS3 from binding to at least one or more residues selected from F838, F873, K874, M832, V876, R872, K828, R836, and Q840 of SAGE1. For example, a SAGE1 complex inhibitor may bind to SAGE1 or INTS3 in a manner that competes with INTS3 for binding to at least one residue in the binding interface of SAGE1 or that allosterically alters the conformation of SAGE1 or INTS3, thereby weakening or eliminating the binding between SAGE1 and INTS3.

[0290] The SAGE1 complex inhibitor may be of any suitable type of inhibitor, for example a nucleic acid molecule (eg a polynucleotide or oligonucleotide), a polypeptide, a compound (eg a chemical compound), or an antibody or antigen-binding fragment thereof.

[0291] a) Compound In some embodiments, the SAGE1 complex inhibitor comprises a compound capable of inhibiting the formation of a SAGE1 complex (e.g., a SAGE1-INTS3 complex). Compounds can be obtained using any of a number of approaches in combinatorial library technology known in the art, such as spatially addressable parallel solid-phase or solution-phase libraries, synthetic libraries requiring deconvolution, "one-bead one-compound" (OBOC) libraries, and synthetic libraries using affinity chromatography selection (Lam, KS (1997) Anticancer Drug Des. 12: 145).

[0292] Compounds suitable for the purposes of the present disclosure are those capable of binding to the active or allosteric binding site of SAGE1. An active binding site refers to a region of SAGE1 that, due to its shape and charge potential, favorably interacts with another component of the SAGE1 complex (e.g., INTS3) via various covalent and / or non-covalent forces. An allosteric binding site refers to a region of SAGE1 other than the active binding site that, when induced by a modulator, alters the conformation of SAGE1 to affect its activity. In some embodiments, the compound competes with INTS3 for binding to the active binding site of SAGE1. For example, the compound can alter the location of INTS3 protein or fragments thereof bound to SAGE1, disassembling the SAGE1 complex and inhibiting its activity. In some embodiments, the compound binds to the allosteric binding site of SAGE1 and does not compete with INTS3.

[0293] Compounds can be screened in cell-based or non-cell-based assays as disclosed herein. Compounds can also be virtually screened by the methods disclosed herein. In some embodiments, a lead compound is first identified and the pharmacophore of the lead compound required for binding to SAGE1 is then determined. Computer modeling can then be performed to generate, design, and select a set of candidate compounds that have the same pharmacophore as the lead compound but exhibit superior pharmacological properties. Compounds can be screened in pools (eg, multiple compounds per test sample) or individually.

[0294] b) Anti-SAGE1 antibody In some embodiments, the SAGE1 complex inhibitor comprises an antibody or fragment thereof.

[0295] In some embodiments, the SAGE1 complex inhibitor comprises an antibody or fragment thereof that (a) specifically binds to SAGE1, (b) specifically binds to a component that complexes with SAGE1 (e.g., INTS3), or (c) specifically binds to the SAGE1 complex but does not bind to SAGE1 or a component that complexes with SAGE1.

[0296] In some embodiments, the SAGE1 complex inhibitor comprises an antibody or fragment thereof that can specifically bind to SAGE1 or a fragment of SAGE1 that may interact with INTS3, thereby blocking the interaction between SAGE1 and INTS3.

[0297] SAGE1 protein or a fragment of SAGE1 that interacts with INTS3 (e.g., residues 818-904 of SEQ ID NO: 3) can be used as an immunogen and antibodies that bind to the immunogen can be generated using standard techniques for preparing polyclonal and monoclonal antibodies, according to methods well known in the art. The antigenic peptide typically contains at least 8 amino acid residues and encompasses an epitope present in each full-length molecule, such that antibodies against the peptide will form specific immune complexes with each full-length molecule. Preferably, the antigenic peptide contains at least 10 amino acid residues. In certain embodiments, such antigenic peptides can be specific for any polypeptide molecule of a species, such as mouse or human.

[0298] Additionally, within the scope of the present invention are recombinant polypeptide antibodies, such as chimeric and humanized monoclonal antibodies, which contain both human and non-human portions and can be produced by standard recombinant DNA techniques. Also within the scope of the present invention are fully human antibodies made against the SAGE1 protein or fragments thereof. Fully human antibodies can be made using mice into which constant regions of human immunoglobulin genes have been introduced, and the target antibodies can be mass-produced by isolating antibody-producing lymphocytes from the mouse and turning them into hybridomas. They can also be made by phage display, which uses phages that incorporate human antibody genes and display human antibodies on their surface in the form of fusion with the phage coat protein.

[0299] In some embodiments, the antibodies or antigen-binding fragments of the disclosure are monoclonal antibodies, bispecific antibodies, multispecific antibodies, recombinant antibodies, labeled antibodies, bivalent antibodies, anti-idiotypic antibodies, dimeric or multimeric antibodies, or modified antibodies (e.g., glycosylated antibodies). In some embodiments, the antibodies or antigen-binding fragments of the disclosure are diabodies, Fab, Fab', F(ab')2, Fd, Fv fragments, disulfide stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (bivalent diabodies), bispecific scFv dimers, multispecific antibodies, heavy chain antibodies, camelized single domain antibodies, nanobodies, domain antibodies, or bivalent domain antibodies.

[0300] c) Inhibitory polypeptides In some embodiments, the SAGE1 complex inhibitor comprises a polypeptide. Polypeptides useful in the present disclosure include polypeptides that are fragments, variants, or derivatives of SAGE1 that are capable of binding to a component of a SAGE1 complex (e.g., INTS3), or polypeptides that are fragments, variants, or derivatives of a SAGE1 complex that are capable of binding to SAGE1, but where the completed complex lacks the activity of the SAGE1 complex (e.g., the SAGE1-INTS3 complex) in regulating gene transcription.

[0301] In some embodiments, the SAGE1 complex inhibitor comprises a SAGE1-binding fragment of INTS3 or a variant or derivative thereof. The polypeptide fragment of INTS3 binds to SAGE1 and interferes with the binding of SAGE1 to INTS3. The term "variant" refers to a polypeptide in which one or more amino acid residues have been altered or modified compared to a naturally occurring polypeptide. The amino acid sequence of a variant polypeptide may be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of INTS3 or a fragment of INTS3. An exemplary amino acid sequence of INTS3 is shown in SEQ ID NO:5. In some embodiments, the polypeptide fragment of INTS3 comprises a C-terminal fragment of INTS3. A C-terminal fragment of INTS3 may comprise at least the last 500, 400, 300, 250, 200, or 150 residues from the C-terminus, or 500-100, 400-100, 300-100, 200-100, 500-50, 400-50, 300-50, or 200-50 residues from the C-terminus. In some embodiments, the C-terminal SAGE1-binding fragment of INTS3 comprises the amino acid sequence of SEQ ID NO:9 (i.e., residues 572-978 of SEQ ID NO:5) or a variant or fragment thereof capable of binding to SAGE1.

[0302] In some embodiments, the SAGE1 complex inhibitor comprises an INTS3-binding fragment of SAGE1 or a variant or derivative thereof. The polypeptide fragment of SAGE1 binds to INTS3 and interferes with the binding of SAGE1 to INTS3. In some embodiments, the INTS3-binding fragment of SAGE1 comprises a C-terminal fragment of SAGE1. The C-terminal fragment of SAGE1 may comprise the last 100, 90, 80, 70, 60, or 50 residues counting from the C-terminus. In some embodiments, the C-terminal fragment of SAGE1 comprises residues 818-904 of SEQ ID NO:3. In some embodiments, the SAGE1 complex inhibitor comprises a polypeptide having the sequence of SEQ ID NO:8 (i.e., residues 818-904 of SEQ ID NO:3) capable of binding to SAGE1, or a variant or fragment thereof.

[0303] Polypeptides useful in the present disclosure also include INTS3-binding fragments of INTS6 or INTS6L, or variants or derivatives thereof, which can bind to INTS3 in competition with SAGE1, thereby blocking the binding of SAGE1 to INTS3. In some embodiments, the SAGE1-binding fragment of INTS6 or INTS6L comprises the amino acid sequence of SEQ ID NO:10 (e.g., residues 803-887 of SEQ ID NO:6 of INTS6), SEQ ID NO:104 (e.g., residues 786-887 of SEQ ID NO:6 of INTS6), or SEQ ID NO:105 (e.g., residues 759-861 of SEQ ID NO:7 of INTS6L), or a variant (e.g., having at least 50% sequence identity) or fragment thereof, capable of binding to INTS3.

[0304] In some embodiments, the polypeptide fragment capable of disrupting or competitively inhibiting the interaction of SAGE1 with INTS3 is fused to a second polypeptide heterologous to the fragment to form a fusion polypeptide. The second polypeptide, e.g., an immunoglobulin constant region or a human serum albumin binding portion, can enhance the solubility of the first polypeptide. The fusion polypeptide may be modified to alter the affinity, stability, or valency of the target polypeptide or to facilitate expression, secretion, or purification of the fusion polypeptide.

[0305] vi. Proteolysis-inducing chimeric molecule (Protac) The disclosure further contemplates SAGE1 inhibitors (and SAGE1 complex inhibitors) that include bifunctional molecules that include a first portion capable of binding to a ubiquitin pathway protein and a second portion capable of binding to SAGE1 or a SAGE1 complex, the bifunctional molecules placing SAGE1 or a SAGE1 complex in proximity to the ubiquitin pathway, thereby enabling degradation of SAGE1 or a SAGE1 complex.

[0306] In some embodiments, the bifunctional molecule comprises a proteolysis-inducing chimeric molecule (PROTAC) that promotes degradation of SAGE1 or the SAGE1 complex via the ubiquitin pathway. PROTACs induce degradation of a protein of interest by hijacking the activity of an E3 ubiquitin ligase that is used to ubiquitinate the protein of interest for subsequent degradation by the 26S proteasome (see, for example, Wang Y, Acta Pharmaceutica Sinica B 2020;10 (2):207-238 for further details).

[0307] The ubiquitin pathway protein binding moiety may include any molecule that binds to an E3 ligase. Examples of E3 ligases suitable for growing bifunctional molecules such as PROTACs include von Hippel-Lindau (VHL), cereblon, MDM2 (Mouse double minute 2), beta-TrCP1, inhibitor of apoptosis protein (IAP) E3 ligase, TRIM21 (UniProtKB-P19474(RO52_HUMAN)), anaphase promoting complex (APC), UBR5 (EDD1), SOCS / BC-box / eloBC / CUL5 / RING, LNXp80, CBX4, CBLL1, HACE1, HECTD1, HECTD2, HECTD3, HECW1, HECW2, HERC1, HERC2, HERC3, HERC4, HUWE1, ITCH, NEDD4, NEDD4, L, PPIL2, PRPF19, PIAS1, PIAS2, PIAS3, PIAS4, RANBP2, RNF4, RBX1, SMURF1, SMURF2, STUB1, TOPORS, TRIP12, UBE3A, UBE3B, UBE3C, UBE4A, UB E4B, UBOX5, UBR5, WWP1, WWP2, Parkin, A20 / TNFAIP3, AMFR / gp78, ARA54, beta-TrCP1 / BTRC, BRCA1, CBL, CHIP / STUB1, E6, E6AP / UBE3A, F-box protein 15 / FBXO15, FBXW7 / Cdc4, GRAIL / RNF128, HOIP / RNF31, cIAP-1 / HIAP-2, cIAP-2 / HIAP-1, cIAP(pan), ITCH / AIP4, KAP1, MARCH8, Mind Bomb 1 / MIB1, Mind Bomb 2 / MIB2, MuRF1 / TRIM63, NDFIP1, NEDD4, NleL, Parkin, RNF2, RNF4, RNF8, RNF168, RNF43, SART1, Skp2, SMURF2, TRAF-1, TRAF-2, TRAF-3, TRAF-4, TRAF-5, TRAF-6, TRIM5, TRIM21, TRIM32, UBR5, and ZNRF3.

[0308] As an example, VHL is one of the E3 ligases with innovative therapeutic potential. VHL is the substrate recognition subunit of the E3 ligase complex VCB, which consists of elongins B and C, Cul2, and Rbx1.

[0309] As another example, cereblon is a protein encoded by the CRBN gene in humans. Cereblon forms an E3 ubiquitin ligase complex with damaged DNA binding protein 1 (DDB1), regulator of cullin 4A (CUL4A), and regulator of cullin 1 (ROC1). This complex ubiquitinates a number of other proteins.

[0310] The two parts of the compound can be connected directly or via a chemical linker. The linker can be a cleavable or non-cleavable linker. Linkers that have some flexibility are preferred, so PEG and (un)saturated alkane chains of various lengths are often used. In some embodiments, the linker is independently covalently attached to the two parts, for example via an amide, ester, thioester, keto, carbamate (urethane), carbon, or ester group, each group being inserted anywhere in the two parts to most tightly bind the ubiquitin pathway protein binding portion of the ubiquitin ligase and the SAGE1 or SAGE1 complex binding portion of the SAGE1 or SAGE1 complex to be degraded.

[0311] In certain embodiments, the compounds described herein comprise multiple ubiquitin pathway protein binding moieties, multiple SAGE1 or SAGE1 complex binding moieties, multiple linkers, or combinations thereof.

[0312] Bifunctional molecules are designed and produced by methods known in the art, and details are described, for example, in PCT patent application WO2017201449A1, U.S. patent applications US10,584,101, US10,071,164, U.S. patent application US20180147202, Ishida T, Ciulli A. E3 Ligase Ligands for PROTACs, How They Were Found and How to Discover New Ones, SLAS DISCOVERY: Advancing the Science of Drug Discovery. November 2020. doi: 10.1177 / 2472555220965528, Wang, Y. et al., Degradation of proteins by PROTACs and Other strategies can be found in Acta Pharmaceutica Sinica B, Volume 10, Issue 2, 2020, Pages 207-238, which are incorporated herein in their entirety.

[0313] V. Pharmaceutical Compositions and Combinations In another aspect, a pharmaceutical composition of the present disclosure comprises a SAGE1 inhibitor as described herein. In some embodiments, the SAGE1 inhibitor is a SAGE1 complex inhibitor. The term "pharmaceutical composition" refers to a formulation that contains a SAGE1 inhibitor of the present disclosure in a form suitable for administration to a subject. The composition can be used to treat tumors and / or modulate the expression levels of SAGE1-INTS3.

[0314] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable excipient. The term "pharma-ceutically acceptable excipient" refers to an excipient that is generally safe, non-toxic, and useful in formulating a pharmaceutical composition that is biologically or otherwise desirable for a subject. In some embodiments, the pharma-ceutically acceptable excipient comprises a pharma-ceutically acceptable carrier and / or a pharma-ceutically acceptable diluent. Suitable excipients may include, for example, antioxidants, fillers, binders, disintegrants, buffers, preservatives, lubricants, flavors, thickeners, colorants, emulsifiers, stabilizers, and the like.

[0315] Pharmaceutical compositions are typically constructed to be compatible with the intended route of administration. The particular excipients used will vary depending on the means and purpose for which the compounds of the present disclosure are applied. Solvents are generally selected based on solvents that are recognized by those skilled in the art as safe for administration to mammals, including humans. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic water-soluble or water-miscible solvents. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycol (e.g., PEG400, PEG300), and the like, and mixtures thereof.

[0316] In some embodiments, suitable excipients include buffers such as phosphates, citrates, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol alcohol, butyl alcohol, benzyl alcohol, methionyl chloride, methyl methionine ... These may include alkylparabens such as ethylparaben or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, low molecular weight (less than 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG).

[0317] In some embodiments, suitable excipients may include one or more stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifiers, glidants, processing aids, colorants, sweeteners, flavorings, flavorings, and other known additives that enhance the presentation of the drug (i.e., the compound of the present disclosure or a pharmaceutical composition thereof) or aid in the manufacture of the pharmaceutical product (i.e., drug product). The drug substance may be encapsulated in microcapsules prepared, for example, by droplet formation or interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980). Liposomes are small vesicles composed of various types of lipids, phospholipids, and / or surfactants that are useful for delivery of drugs (such as the compounds disclosed herein and optionally chemotherapeutic agents) to mammals, including humans. The components of liposomes are often arranged in a bilayer, similar to the lipid composition of biological membranes.

[0318] The pharmaceutical compositions described herein may be in any form that allows the composition to be administered to a subject, including, but not limited to, a human, and are configured to be compatible with the intended route of administration. Examples of routes of administration include parenteral (subcutaneous, intravenous, intramuscular, and intraperitoneal), oral, rectal, dermal, transdermal, pleural, intrapulmonary, and nasal (respiratory) routes. The pharmaceutical composition may be, for example, a solution, suspension, emulsion, pill, capsule, tablet, sustained release formulation, or powder.

[0319] Since various routes are contemplated for the pharmaceutical compositions described herein, the pharmaceutical compositions described herein may be provided in bulk or in dosage units depending on the intended route of administration. For example, for oral, buccal, and sublingual administration, solid dosage forms may include powders, suspensions, granules, tablets, pills, capsules, gelcaps, and caplets, while liquid dosage forms may include emulsions, syrups, elixirs, suspensions, and solutions. For injection, liquid dosage forms may include emulsions and suspensions, while solid dosage forms may include powders that are reconstituted with a suitable solution. For inhalation, dosage forms may include solutions, sprays, dry powders, and aerosols. For topical (such as buccal and sublingual) or transdermal administration, dosage forms may include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, and patches. For vaginal administration, dosage forms may include pessaries, tampons, creams, gels, pastes, foams, and sprays. Methods for formulating suitable pharmaceutical compositions are known in the art; see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

[0320] In embodiments, the pharmaceutical composition is formulated into an injectable composition. The injectable pharmaceutical composition may be prepared in any convenient form, for example, a solution, a suspension, an emulsion, or a solid form suitable for forming a solution, suspension, or emulsion. Preparations for injection include sterile and / or non-pyrogenic solutions ready for injection, sterile dry powders to be mixed with a vehicle, such as lyophilized powders, including subcutaneous tablets. These may include pre-dried soluble products, sterile suspensions ready for injection, insoluble sterile dry products for use in combination with excipients, and sterile and / or non-pyrogenic emulsions. Solutions may be aqueous or non-aqueous.

[0321] In some embodiments, the parenteral formulation is packaged in a unit dose ampule, vial, or syringe with needle. All preparations to be administered parenterally are sterile and pyrogen-free, as known and practiced in the art. In some embodiments, a sterile lyophilized powder is prepared by dissolving the polypeptide complexes disclosed herein in a suitable solvent. The lyophilized powder is reconstituted with water for injection to provide a formulation for parenteral administration. In some embodiments, sterile and / or pyrogen-free water, or other suitable liquid carrier, is added to the lyophilized powder for reconstitution. The exact amounts will vary depending on the selected treatment and can be determined empirically.

[0322] In some embodiments, the pharmaceutical compositions of the present disclosure can be formulated as short-acting, fast-releasing, long-acting, and sustained-releasing. Thus, the pharmaceutical compositions of the present disclosure can be formulated as controlled release or sustained release. In some embodiments, the pharmaceutical compositions containing SAGE1 inhibitors that are nucleic acids or SAGE1 inhibitors that contain nucleic acids can be administered by any method suitable for administering nucleic acid agents. These methods include, for example, gene guns, bio-syringes, and skin patches.

[0323] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0324] In certain embodiments, the SAGE1 inhibitors described herein may be administered in combination with any other therapeutic agent used to treat tumors. As used herein, the term "administered in combination" includes simultaneous administration as part of the same pharmaceutical composition, simultaneous administration as separate compositions, or administration at different times as separate compositions. If the pharmaceutical composition is administered before or after another agent, it is still "administered in combination" with that agent as used herein, even if the composition and the other agent are administered by different routes. The SAGE1 inhibitors described herein and the other therapeutic agent can be administered either simultaneously or staggered, provided that they have the same effect on the subject. When possible, the other therapeutic agent administered in combination with the SAGE1 inhibitors described herein will be administered according to the schedule described in the product information for that therapeutic agent, the Physicians' Desk Reference ( Physicians' Desk Reference, 70th Ed (2016)), or according to protocols known in the art.

[0325] VI. Cell therapy i. Manipulated immune cells The present disclosure also contemplates immune cells engineered to express chimeric receptors and directing them to SAGE1 positive tumors, with specificity for tumor cells being based on the antibody binding domain of the receptor that confers binding specificity to epitopes of SAGE1 or MHC-associated epitopes of SAGE1.

[0326] As used herein, "MHC" refers to a major histocompatibility complex molecule found on the cell surface that presents protein fragments from within the cell to immune cells (such as T cells). In some embodiments, the MHC molecule is an MHC class I protein that presents peptides derived from cytosolic proteins. In some embodiments, the MHC class I protein is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, or HLA-G. In some embodiments, the HLA-A is HLA-A02.

[0327] In some embodiments, the SAGE1 epitope is an MHC class I restricted peptide. In some embodiments, the epitope is about 8 to about 12 amino acids in length, such as about 8, 9, 10, 11, or 12. Suitable MHC class I restricted peptides of SAGE1 can be determined based on the presence of specific MHC (e.g., HLA-A*02:01) binding motifs and proteasomal and immunoproteasomal cleavage sites, for example, using computer prediction models known to those of skill in the art.

[0328] In some embodiments, the antibody binding domain is an Fv-like antigen binding module. In some embodiments, the antibody binding domain is an Fb-like antigen binding module comprising a first polypeptide chain and a second polypeptide chain.

[0329] In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR) comprising an antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen-binding domain specifically binds to an epitope of SAGE1 or an MHC-associated epitope of SAGE1.

[0330] The transmembrane domain of the CAR may be natural or synthetic. If natural, the domain may be derived from any membrane-bound or transmembrane protein. A transmembrane region for particular use in the present invention may be derived from (i.e., may comprise at least the transmembrane region of) the alpha, beta, delta, gamma, or zeta chain of the T cell receptor, CD28, CD3s, CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. In some embodiments, the transmembrane domain may be synthetic, in which case it may comprise primarily hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine may be found at each end of a synthetic transmembrane domain. In some embodiments, a short peptide linker, e.g., about 2 to about 10 amino acids (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, or 10) in length, may form the transmembrane and intracellular signaling domains of the CAR. In some embodiments, the linker is a glycine-serine doublet.

[0331] The intracellular signaling domain of the CAR is responsible for at least one activity of the normal effector function of the immune cell in which the CAR is placed. For example, the effector function of a T cell can be a cytolytic activity or a helper activity, including secretion of cytokines. Thus, the intracellular signaling domain can include an intracellular signaling sequence derived from the entire or cleaved portion of the intracellular signaling domain of a protein that transmits an effector function signal to cause the cell to perform a specialized function. Examples of intracellular signaling domains include the cytoplasmic sequence of the T cell receptor (TCR), the cytoplasmic sequence of a co-receptor that cooperates to initiate signaling after antigen receptor ligation, any derivative or variant of these sequences, and any synthetic sequence with the same function. Examples of intracellular signaling sequences for particular use in the present disclosure include those derived from CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD5, CD22, CD79a, CD79b, CD66d, FcR gamma, FcR beta, and TCR zeta.

[0332] In some embodiments, the intracellular signaling domain of the CAR further comprises a costimulatory domain. It is known that full activation of T cells requires a signal generated through the T cell receptor (TCR) and a secondary or costimulatory signal. Costimulatory domains useful in the present disclosure can be derived from the intracellular domain of costimulatory molecules. Examples of costimulatory molecules include, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like.

[0333] In some embodiments, the intracellular signaling domain of the CAR comprises a CD3ζ intracellular signaling sequence and a CD28 and / or 4-IBB intracellular signaling sequence.

[0334] In some embodiments, the engineered receptor is an engineered T cell receptor (TCR) comprising an antigen binding domain and a T cell receptor module (TCRM) comprising a TCR transmembrane domain, wherein the antigen binding domain specifically binds to an MHC-associated epitope of SAGE1.

[0335] In some embodiments, the engineered TCR is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, which may be linked by a covalent bond (e.g., a peptide or other chemical bond) or a non-covalent bond (e.g., a disulfide bond), etc.

[0336] In some embodiments, the TCRRM is capable of recruiting at least one TCR-associated signaling module, hi some embodiments, the TCR-associated signaling module is selected from the group consisting of CD3δε, CD3γε, and ζζ.

[0337] In some embodiments, the TCR comprises a transmembrane module of a TCR (such as an αβTCR or γδTCR). In some embodiments, the TCR further comprises one or both of a binding peptide of a TCR or a fragment thereof. In some embodiments, the engineered TCR further comprises at least one intracellular domain. For example, one or more of the at least one intracellular domain of the engineered TCR comprises a sequence from an intracellular domain of a TCR or a T cell costimulatory signaling sequence. The costimulatory signaling sequence can be a portion of an intracellular domain of a costimulatory molecule, including, for example, CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds to CD83, and the like. In some embodiments, the engineered TCR further comprises one or more peptide linkers between the antigen binding domain and the TCR, thereby optimizing the length of the extracellular domain.

[0338] Contemplated in this disclosure are nucleic acids encoding a CAR or engineered TCR and vectors into which the nucleic acids are inserted. The vector may include a sequence encoding a signal peptide so that expression of the receptor is targeted to the cell surface.

[0339] The present disclosure also provides cells engineered to express a CAR or engineered TCR. Such cells can be generated by introducing a vector containing a nucleic acid encoding a CAR or engineered TCR into a cell. In some embodiments, the cell comprises a dendritic cell, a T cell, or a natural killer cell.

[0340] ii. Manipulated antigen presenting cells In another embodiment, the ex vivo activated antigen presenting cells (APCs) of the present disclosure are loaded with SAGE1 or a fragment thereof to enhance immune responses (eg, antigen-specific T cell responses) against SAGE1-positive diseases.

[0341] APCs are cells that present antigens complexed with major histocompatibility complex (MHC) proteins on their surface. Examples of APCs include dendritic cells (DCs), macrophages, B cells, and certain activated epithelial cells. In some embodiments, the APCs are dendritic cells. APCs that have been loaded with SAGE1 can potently bind to immune cells (e.g., T cells) of a patient by presenting SAGE1-specific peptides complexed with MHC-I and / or MHC-II molecules. Generates tumor-specific immune responses.

[0342] In general, in the preparation of ex vivo activated APC, APC cells (e.g. DCs) are isolated from a patient. The isolated cells are activated ex vivo to mature and present an antigen of interest before being returned to the patient. Specific tumor antigens (e.g. SAGE1) and tumor lysates, etc. can be used to activate APC. APCs can also be activated by electroporation, transfection, or transduction using expression vectors (e.g. viral vectors or mRNAs) encoding specific tumor antigens (e.g. epitopes of SAGE1 or MHC-associated epitopes of SAGE1), total tumor cell mRNA, tumor-derived exosomes (TDE), or cells (e.g. fibroblasts) modified to express epitopes of SAGE1 or MHC-associated epitopes of SAGE1, etc.

[0343] APCs suitable for administration to a subject (e.g., a cancer patient) can be isolated and obtained from any tissue in which such cells are found, but can also be provided in culture. APCs (e.g., DCs) can be found, for example, in mammalian bone marrow or PMBC, mammalian spleen, or mammalian skin. For example, bone marrow can be harvested from a mammal and cultured in a medium that promotes the growth of DCs. The medium can include GM-CSF, IL-4 and / or other cytokines (e.g., TNF-α), growth factors, and supplements. DCs are low in number among all the cells in which they are present, necessitating the isolation and enrichment of DCs. To obtain an enriched population of isolated DCs, numerous procedures involving repeated density gradient separation, fluorescence activated cell sorting, positive selection, negative selection, or a combination thereof are routinely performed.

[0344] After a suitable culture period in medium containing the appropriate cytokines (e.g., 4, 6, 8, 10, 12, or 14 days to allow DCs to proliferate, differentiate, and mature), clusters of DCs can be cultured in the presence of one or more epitopes of SAGE1 or transfected / transduced with an expression vector encoding one or more epitopes of SAGE1 and harvested for cancer vaccines using standard techniques. In some embodiments, APCs can be fused with cells expressing an epitope of SAGE1 or an MHC-associated epitope of SAGE1. In some embodiments, the epitope of SAGE1 is an MHC class I-restricted epitope or an MHC class II-restricted epitope.

[0345] In another aspect, the vaccine composition of the disclosure comprises ex vivo activated APC as described herein.In another aspect, the method of the disclosure is a method for preventing or treating a SAGE1 positive disease comprising administering an effective amount of activated APC as described herein.

[0346] The APC-based vaccine may be administered to a subject by any suitable route of administration, including injection, infusion, inoculation, direct surgical administration, or a combination thereof, hi another embodiment, the vaccine is administered intravenously.

[0347] VII. METHODS OF TREATING SAGE1 POSITIVE DISEASES In another aspect, the disclosed method is a method of treating a SAGE1 positive disease in a subject in need thereof, comprising administering to the subject an effective amount of (a) a SAGE1 inhibitor, (b) a pharmaceutical composition described herein, (c) a SAGE1 complex inhibitor described herein, (d) a cell described herein, or (e) a combination thereof. In one embodiment, SAGE1 expression is detected in a biological sample from the subject prior to administration. Any of the methods disclosed herein can be used to detect SAGE1 expression.

[0348] In some embodiments, the SAGE1 positive disease is a tumor. Any tumor disclosed herein may be treated using the methods described herein, so long as the tumor is SAGE1 positive. In some embodiments, the treatment may be to prevent or reduce the onset, progression, metastasis, or progression of the tumor. or prevent, delay, or inhibit recurrence.

[0349] In another aspect, the disclosed method is a method for preventing, inhibiting, or slowing the progression of tumor malignancy in a subject with TP53 mutation, comprising administering to the subject an effective amount of (a) a SAGE1 inhibitor, (b) a pharmaceutical composition described herein, (c) a SAGE1 complex inhibitor described herein, (d) a cell described herein, or (e) a combination thereof. In one embodiment, SAGE1 expression is detected in a biological sample from the subject prior to administration. Any of the SAGE1 inhibitors, SAGE1 complex inhibitors, and cells described herein can be used.

[0350] In some embodiments, an effective amount is a therapeutically effective amount. In some embodiments, a therapeutically effective amount is an amount sufficient to prevent, alleviate, or ameliorate symptoms of a disease or to prolong the survival of a subject undergoing treatment. Determining a therapeutically effective amount is well within the capabilities of one of ordinary skill in the art. A therapeutically effective amount will vary with the particular treatment administered to a subject and will depend on a variety of well-known factors, such as the subject's weight, size, and health, the nature and extent of the disease, the rate of administration, the therapeutic or combination of therapeutics selected for administration, and the prescribing physician's instructions. A therapeutically effective amount for a given situation can be determined by routine experimentation within the skill and judgment of the clinician. For example, an initial dose may be higher than subsequent doses. As another example, the dose may be modified during treatment depending on the subject's response.

[0351] In one embodiment, the effective amount is a prophylactically effective amount. A prophylactically effective amount can be an amount that provides a desired prophylactic effect in a subject, such as prevention of a disease (e.g., a SAGE1 positive disease) or alleviation of symptoms associated with the disease (e.g., a SAGE1 positive disease). Alleviation of symptoms can be determined by any clinical measurement used by a physician or other medical professional to assess the severity or progression of the condition, and may include an improvement in the need for hospitalization.

[0352] Methods for introducing oligonucleotides or nucleic acids into cells include physical, biological, and chemical methods. Physical methods for introducing polynucleotides into cells include transfection, transformation, transduction, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation.

[0353] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, and beads, as well as lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. A typical colloidal system used as a delivery vehicle in vitro and in vivo is the liposome (e.g., an artificial membrane vesicle).

[0354] Biological methods for introducing a polynucleotide of interest into a cell include the use of a nucleic acid vector. The oligonucleotides disclosed herein can be inserted into a vector and expressed from a transcription unit within the vector. In some embodiments, the oligonucleotides are contained in or encoded by a nucleic acid vector. As will be apparent to those skilled in the art, there are a variety of vectors suitable for transferring the nucleic acid of the invention into a cell. It is within the skill of the art to select an appropriate vector to carry the nucleic acid and optimize the situation for inserting the selected expression vector into a cell without undue experimentation.

[0355] Viral vectors are the most widely used method for inserting genes into mammalian, e.g. human, cells. The viral vectors used are suitable for replication and, in some cases, integration in eukaryotic cells. Typical vectors contain transcription terminators. The viral vectors include a translational initiation sequence, a translation terminator and a translation terminator, an initiation sequence, a promoter useful for controlling the expression of the desired nucleic acid sequence, an origin of replication functional in at least one organism, and one or more selectable markers (4th Edition, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 2012). Viral vectors, including DNA and RNA viruses, can be derived from retroviruses, lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0356] In some embodiments, one or more vectors expressing one or more elements of the CRISPR system are introduced into a host cell, where expression of the elements of the CRISPR system results in the formation of a CRISPR complex at one or more target sites, for example, the CAS enzyme and guide sequence are operably linked to the same or separate regulatory elements on a single vector, or each is linked to separate regulatory elements on separate vectors.

[0357] A nucleic acid or polynucleotide can also be introduced into a cell using a non-viral vector, such as a plasmid, hi one embodiment, a plasmid encoding an shRNA or guide RNA is transfected into the cell.

[0358] VIII. Screening method for SAGE1 inhibitors In another aspect, the disclosure provides a method for identifying or screening for an agent that is a SAGE1 inhibitor (e.g., an agent that inhibits the level of SAGE1 or a biological activity mediated by SAGE1), e.g., a polynucleotide, a polypeptide, a chemical compound, an antibody or antigen-binding fragment thereof, or a derivative of any of these.

[0359] In some embodiments, the method includes contacting a SAGE1 protein or a functional equivalent thereof with an agent to be screened and detecting binding between the SAGE1 or functional equivalent thereof and the agent. The presence of binding indicates that the agent may be a SAGE1 inhibitor. As used herein, a "functional equivalent" refers to a fragment, variant, derivative, or fusion polypeptide of a naturally occurring polypeptide (e.g., SAGE1) that has a different chemical structure but retains at least a portion of the biological function of the naturally occurring polypeptide. In some embodiments, a functional equivalent retains at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the biological activity of the naturally occurring polypeptide. Biological functions of SAGE1 include, but are not limited to, promoting tumorigenesis, such as by upregulating the transcription of growth-promoting genes, and binding to INTS3.

[0360] In some embodiments, the method includes contacting a test agent with a SAGE1-binding fragment of a component of a SAGE1 complex and detecting binding between the SAGE1-binding fragment and the test agent, the presence of binding indicating that the agent may be a SAGE1 complex inhibitor.

[0361] In some embodiments, the method includes contacting a test agent with SAGE1 and a SAGE1-binding fragment of a component of a SAGE1 complex and detecting the ability of the test agent to inhibit or reduce binding of SAGE1 to the SAGE1-binding fragment. Alternatively, the method may include detecting the ability of the test agent to inhibit formation of a SAGE1 complex. If the test agent inhibits or reduces binding of SAGE1 to a SAGE1-binding fragment of a component of a SAGE1 complex or inhibits formation of a SAGE1 complex, the test agent may be a SAGE1 complex inhibitor. In some embodiments, the component of a SAGE1 complex is one or more selected from the group consisting of INTS3, INIP, NABP1 / 2, CREBBP, TLE1, TLE2, TLE3, TLE4, TLE5, GGA3, CNOT1, TAX1BP1, SEC16A, CYLD, and PAXBP1. In some embodiments, the component of the SAGE1 complex comprises INTS3. In some embodiments, the SAGE1-binding fragment of INTS3 comprises the amino acid sequence of SEQ ID NO:9, a variant or fragment thereof, or a fusion protein of any of these, capable of binding to SAGE1.

[0362] In some embodiments, the method comprises contacting SAGE1 with INTS3 in the presence of an agent and detecting formation of a SAGE1-INTS3 complex, where if the agent inhibits formation of the SAGE1-INTS3 complex, the agent is identified as a SAGE1 inhibitor. In some embodiments, the cell-free screening assay comprises contacting a SAGE1-INTS3 complex with an agent and detecting the amount of the complex, where if the agent reduces the amount of the complex, the agent is identified as a SAGE1 inhibitor.

[0363] Binding of an agent to SAGE1 or a functional equivalent thereof can be determined in a variety of ways. For example, SDS-PAGE and / or mass spectrometry can be used to analyze the presence and / or amount of binding. As another example, SAGE1 and a SAGE1-binding fragment of a component of the SAGE1 complex (e.g., INTS3) can be linked to a radioisotope or enzyme label, and the labeled molecule in the link can be detected to determine binding of the agent to SAGE1 or the SAGE1-binding fragment. This binding can also be determined in real time, without labeling either component, by methods such as surface plasmon resonance (SPR). In some embodiments, either SAGE1 or a SAGE1-binding fragment of a component of the SAGE1 complex (e.g., INTS3) can be immobilized to facilitate separation / detection of bound or bound molecules and to accommodate automation of the assay. For example, a SAGE1-binding fragment of SAGE1 and / or a component of the SAGE1 complex (eg, INTS3) can be provided in the form of a fusion protein (eg, a glutathione-S-transferase-based fusion protein) that contains another domain that allows it to be bound to a matrix.

[0364] For example, peptide libraries can be screened to identify molecules that interact or bind to SAGE1, its functional equivalents, or SAGE1-binding fragments of INTS3, or that block the binding of SAGE1 to a target such as INTS3. Useful peptide libraries include those that encode random or controlled sets of amino acids that can be expressed as fusion proteins with bacteriophage coat proteins and then screened against SAGE1 or its functional equivalents. Exemplary peptide libraries include those based on HSP90 or fibronectin (Anders Olson C. et al., Protein Sci., 2007; 16: 476, Wahlberg E. et al., Proc. Natl. Acad. Sci. (2003); 100: 3185). Screening methods that can be used to identify molecules that interact with the SAGE1 protein sequence are disclosed, for example, in U.S. Patent Nos. 5,723,286 and 5,733,731.

[0365] In some embodiments, the method comprises contacting a cell expressing SAGE1 or a functional equivalent thereof with an agent to be screened and determining the ability of the agent to reduce the amount or activity of SAGE1 or a functional equivalent thereof, hi some embodiments, the cell is a tumor cell.

[0366] For example, small molecules can be identified that interfere with SAGE1 function, including molecules that interfere with the ability of SAGE1 to mediate RNAPII-dependent transcriptional regulation, the ability of SAGE1 to activate the transcription of oncogenes, or with transcriptional misregulation that leads to tumorigenesis. Ligands that regulate SAGE1 function can be identified based on their ability to bind to the SAGE1 complex or their ability to interfere with the function of the SAGE1 complex.

[0367] SAGE1 activity can be monitored, for example, by detecting the expression of genes that are regulated by the SAGE1 complex. For example, activity of SAGE1 can be indicated by detecting the introduction of a reporter gene that is operably linked to a responsive regulatory element targeted by the SAGE1 complex.

[0368] In some embodiments, the method includes contacting the SAGE1 complex (e.g., a SAGE1-INTS3 complex) with a drug and detecting activation of a reporter gene under the control of the SAGE1 complex, and if the drug suppresses or prevents activation of the reporter gene, the drug is identified as a SAGE1 inhibitor.

[0369] In some embodiments, the recombinant cells of the present disclosure comprise a first gene encoding SAGE1 and a second gene encoding a reporter, the reporter being configured to generate a detectable signal in response to expression of SAGE1 or activity mediated by SAGE1.

[0370] Exemplary methods are discussed, for example, in U.S. Patent No. 5,928,868, issued July 27, 1999, and include forming hybrid ligands, where at least one ligand is a small molecule. In an illustrative embodiment, cells engineered to express a fusion protein of SAGE1 and a DNA binding protein are used to co-express the hybrid ligand / small molecule fusion protein and a cDNA library transcriptional activator protein. The cells further contain a reporter gene, the expression of which is regulated in the vicinity of the first and second fusion proteins, an event that occurs only if the hybrid ligand binds to the target sites of both hybrid proteins. Those cells expressing the reporter gene are selected to identify unknown small molecules or unknown ligands. This method provides a means to identify modulators that activate or inhibit SAGE1.

[0371] In some embodiments, the screening assay for agents that are SAGE1 inhibitors is a cell-based assay and includes contacting a cell expressing a SAGE1-expressing cell with a test agent and determining the ability of the test agent to reduce the amount or activity of SAGE1 or a SAGE1 complex. In some embodiments, the cell is a tumor cell. The tumor cell is obtained from an animal model or a tumor patient. In some embodiments, the contacting step is performed in vivo, ex vivo, or in vitro.

[0372] The amount of SAGE1 can be indicated by the level of expression of SAGE1 mRNA or its protein or fragments. The level of expression of SAGE1 mRNA or its protein or fragments in a cell can be determined by methods described herein that detect SAGE1 mRNA or its protein. If the amount of SAGE1 mRNA is reduced (statistically significantly less) in the presence of the agent than in its absence, the agent is identified as a SAGE1 inhibitor.

[0373] The activity of SAGE1 can be indicated by the amount or activity of SAGE1 complex, which can be determined as described above. The activity of SAGE1 or SAGE1 complex can also be indicated by a SAGE1-regulated cellular response, such as a decrease in tumor cell viability or proliferation compared to a control. The control can be tumor cells that have not been exposed to the drug or tumor cells that have been exposed to an anti-cancer drug. For example, cell proliferation or invasion can be determined by monitoring cell counts, cell movement, Matrigel assays, induction of proliferation and / or invasion related gene expression, etc., as described herein. Furthermore, the in vivo activity of SAGE1 or SAGE1 complex can be indicated by tumor shrinkage, for solid tumors, for example, by imaging with a PET scan.

[0374] The present disclosure further relates to agents identified in the above-described screening assays. Thus, it is within the scope of the present invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with the agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of the agent. Additionally, the present invention relates to the use of an agent identified in the above-described screening assay for treatment as described herein.

[0375] IX. Structure-based discovery of SAGE1-INTS3 complex inhibitors In another aspect, an isolated SAGE1-INST3 complex of the present disclosure comprises SAGE1, or a fragment thereof, and INST3, or a fragment thereof. In some embodiments, a fragment of SAGE1 is capable of binding to INTS3. In some embodiments, a fragment of INTS3 is capable of binding to SAGE1.

[0376] In some embodiments, the fragment of INTS3 is a C-terminal fragment of INTS3, e.g., 500, 400, 300, 250, 200, or 150 residues from the C-terminus, or 500-100, 400-100, 300-100, 200-100, 500-50, 400-50, 300-50, or 200-50 residues from the C-terminus. In some embodiments, the fragment of SAGE1 is a C-terminal fragment of SAGE1, e.g., the last 100, 90, 80, 70, 60, or 50 residues from the C-terminus. In some embodiments, SAGE1 has the amino acid sequence of SEQ ID NO:3 or a sequence at least 70% identical thereto. In some embodiments, INTS3 has the amino acid sequence of SEQ ID NO:5 or a sequence at least 70% identical thereto. In some embodiments, the SAGE1-INTS3 complex comprises a C-terminal fragment of INTS3 comprising residues 572-978 of SEQ ID NO:5, and a C-terminal fragment of SAGE1 comprising residues 818-904 of SEQ ID NO:3.

[0377] In some embodiments, the SAGE1-INTS3 complex is crystallized. The crystallized SAGE1-INTS3 complex may provide a means to obtain atomic modeling information for specific amino acids and their atoms that are linked at the interface between SAGE1 and INTS3.

[0378] In some embodiments, the crystallized SAGE1-INTS3 complex of the present disclosure is capable of diffracting X-rays to a resolution of greater than 5 Å, 4 Å, 3 Å, 2 Å, or 1.5 Å and is useful for determining the three-dimensional structure of the molecule.

[0379] iX-ray crystal structure coordinates In another aspect, the disclosure presents a set of X-ray crystal structure coordinates for a SAGE1-INTS3 complex, deposited in the Protein Data Bank (PDB) under the code 7C5U (Deposition ID D_1300015303) (Bernstein, F. C, TF Koetzle et al. (1977) J MoI Biol 112 (3):535-42), which shows the X-ray crystal structure coordinates for a SAGE1-INTS3 complex comprising a SAGE1 polypeptide comprising the amino acid sequence of residues 818-904 of SEQ ID NO: 3 and an INTS3 polypeptide having the amino acid sequence of residues 572-978 of SEQ ID NO: 5. In some embodiments, the crystals described herein have approximate dimensions of 260.112±0.1 Å, b=46.240±0.1 Å, c=103.101±0.1 Å, α=90±0.1°, β=113.280±0.1°, γ=90±0.1°, are symmetric and are in the space group C2.

[0380] The three-dimensional structure of the SAGE1-INTS3 complex, and in particular the interface between SAGE1 and INTS3, allows for the design and identification of SAGE1 inhibitors, including SAGE1-INTS3 complex inhibitors, that interfere with the interaction between SAGE1 and INTS3. For example, knowledge of the three-dimensional structure can aid in the design of molecules, preferably pharmaceutical agents, that can bind to SAGE1 or INTS3. This allows for the disruption of the SAGE1-INTS3 complex, preventing it from performing biological functions such as upregulating the expression of genes involved in cell proliferation and cancer phenotype. Reference to the interface may refer to a portion of the interface (e.g., all or a portion of the structural coordinates of the SAGE1 portion of the deposited coordinate file (PDB code: 7C5U), all or a portion of the structural coordinates of the INTS3 portion of the deposited coordinate file (PDB code: 7C5U), or both the SAGE1 portion and the INTS3 portion of the deposited coordinate file (PDB code: 7C5U). The structural coordinates were deposited in the PDB at RCSB on May 20, 2020 under accession number 7C5U. The deposit information will be made publicly available following publication of the invention disclosed herein.

[0381] In some embodiments, the disclosure provides a set of X-ray crystal structure coordinates of a SAGE1 complex, wherein the binding interface comprises one or more amino acid residues of SAGE1 selected from the group consisting of F838, F873, K874, M832, V876, R872, K828, R836, and Q840 (numbering according to SEQ ID NO: 3), or equivalent residues of fragments, variants, or derivatives thereof.

[0382] In some embodiments, the disclosure provides a set of X-ray crystal structure coordinates for a binding interface (e.g., a SAGE1 binding interface) of INTS3, the binding interface comprising: T804, S841, S874, S769, N933, R849, Q773, C777, M781, A816, N818, E838, E850, F871, R848, F805, L845, L815, L844, Y808, C842, Q846, Q870, R877, H878, K882, E732, V766, Q771, D768, A765, Q731, E835, E803, C809, and L772 (numbering is provided by SEQ ID NO: 5), or the equivalent residues of a fragment, variant, or derivative thereof.

[0383] In some embodiments, the disclosure provides a set of X-ray crystal structure coordinates for the binding interface between SAGE1 and INTS3, the SAGE1 binding interface being determined by one or more amino acid residues selected from the group consisting of F838, F873, K874, M832, V876, R872, K828, R836, and Q840 of SAGE1 (the numbering of such residues is set forth in SEQ ID NO: 3), or an equivalent residue of a fragment, variant, or derivative thereof, wherein the binding interface of INTS3 comprises one or more amino acid residues selected from the group consisting of T804, S841, S874, S769, N933, R849, Q773, C777, M781, A816, N818, E838, E850, F871, R848, F805, L845, L815, L844, Y808, C842, Q846, Q870, R877, H878, K882, E732, V766, Q771, D768, A765, Q731, E835, E803, C809, and L772 of INTS3 (the numbering of said residues is as set forth in SEQ ID NO: 5), or the equivalent residue of a fragment, variant, or derivative thereof.

[0384] As can be understood by those skilled in the art, a set of structural coordinates of a protein complex or its proteins is a relative set of points that defines a three-dimensional shape. Therefore, it is possible to define similar or identical shapes with completely different sets of coordinates, and this is within the scope of the present invention. Furthermore, small variations in individual coordinates have little effect on the overall shape. In terms of a binding interface, such variations are not expected to significantly change the atoms or residues that are connected at the interface.

[0385] It should be noted that variations in the structural coordinates may occur due to changes in the crystal structure caused by amino acid mutations, additions, substitutions, and / or deletions or other changes in any of the components that make up the crystal. Such variations may be considered to be within an acceptable error threshold compared to the original coordinates, such as a root mean square deviation (rmsd) of about 2 Å or less from the conserved main-chain atoms, and may be included in the resulting 3D coordinates. The original shapes are considered to be the same. In some embodiments, the mean square deviation is less than or equal to about 1.5 Å, 1.0 Å, or 0.5 Å. As used herein, the term "mean square deviation" refers to the square root of the arithmetic mean of the squares of the deviations from the mean value. In terms of atomic objects, these numbers are given in Angstroms.

[0386] The structural coordinates of the SAGE1-INTS3 complex may be mathematically manipulated to produce the above changes. For example, the structural coordinates shown in the archived coordinate file (PDB code: 7C5U) may be manipulated by crystallographic permutation of the original structural coordinates, fractionalization of the original structural coordinates, integer additions or subtractions to the original set of structural coordinates, inversion of the original structural coordinates, or a combination thereof. For example, the SAGE1-INTS3 complex of the present disclosure preferably comprises a binding interface characterized by a mean square deviation of the amino acid residues shown in the archived coordinate file (PDB code: 7C5U) from the conserved backbone atoms of the amino acids ±2 Å or less (more preferably 1.5 Å or less, even more preferably 1 Å or less, and most preferably 0.5 Å or less).

[0387] Thus, the present disclosure further provides X-ray crystal structure coordinates that include the binding interface of the SAGE1-INTS3 complex described herein (e.g., archived coordinate file (PDB code: 7C5U)), in which the root mean square deviations of the amino acids that make up the binding interface from the main chain atoms are within about 2 Å or less, about 1.5 Å or less, about 1 Å or less, or about 0.5 Å or less.

[0388] ii. Computer-Readable Storage Medium In order to use the structural coordinates generated from the SAGE1-INTS3 complex described herein, it may be necessary to display, transform, or otherwise manipulate the relevant coordinates as a three-dimensional shape or graphical representation. Such three-dimensional representations of structural coordinates are commonly used in rational drug design, molecular replacement methods, homology modeling, and mutational analysis. This is typically done using a variety of commercially available software programs that can generate a three-dimensional graphical representation of a molecule or a portion thereof from a set of structural coordinates. There are many existing software programs in this scientific and technical field, which are incorporated herein by reference in their entirety. For example, GRID (Oxford University, Oxford, UK), AUTODOCK (Scripps Research Institute, La Jolla, Calif.), Flo99 (Thistlesoft, Morris Township, NJ.), etc.

[0389] Thus, in another aspect, a computer readable storage medium of the present disclosure has stored therein crystal structure coordinates according to the present disclosure, such as those shown in the stored coordinate file (PDB code: 7C5U).

[0390] Machines, such as computers, for storing, transferring, and using such programs are also contemplated for generating a three-dimensional representation of the SAGE1-INTS3 complex, including the binding interface. The machine includes a machine-readable data storage medium on which the crystal structure coordinates of the present disclosure are stored. Computer-readable storage media are well known to those skilled in the art, and include, for example, hard disks, CD-ROMs, floppy disks, DVDs, USB sticks, and the like, as well as other magnetic, magneto-optical, optical, floptical, and other computer-usable media. The machine further includes a working memory for storing instructions for processing the machine-readable data, and a central processing unit (CPU) coupled to the working memory and the machine-readable data storage medium for processing the machine-readable data into a desired three-dimensional representation. The machine of the present invention also includes a display coupled to the CPU so that the three-dimensional representation can be viewed by a user. Thus, when used with a machine programmed with instructions for using the data, such as a computer loaded with one or more programs of the type described above, the machine described herein can generate the three-dimensional representation of the SAGE1-INTS3 complex, including the three-dimensional representation of the SAGE1-INTS3 complex, ... The configured machine is capable of producing a three-dimensional representation of the SAGE1-INTS3 complex described herein.

[0391] iii. Methods for identifying and screening potential SAGE1 complex inhibitors In another embodiment, the method of the disclosure is a method of identifying an agent that is a potential SAGE1 complex inhibitor, comprising the steps of: (a) generating on a computer a representation of the three-dimensional structure of a binding interface based on said set of X-ray crystal structure coordinates described herein; (b) generating a representation of the drug on a computer; (c) fitting a representation of the drug in step (b) to a computer representation of the three-dimensional structure of the binding interface in step (a) such that the drug interacts with one or more residues of the binding interface; (d) evaluating the interaction of the agent with one or more residues of the binding interface in step (c); If the interaction results in a low-energy, stable complex comprising the agent and SAGE1 or INTS3, which may compete for the SAGE1-INTS3 complex, then the agent is identified as a potential SAGE1 complex inhibitor.

[0392] In another embodiment, the virtual screening method of the present disclosure is a method for identifying a potential SAGE1 complex inhibitor, comprising: (a) generating on a computer a representation of the three-dimensional structure of the binding interface based on the X-ray crystal structure coordinates described herein; (b) generating a representation of the drug on a computer or accessing a representation of the drug from a library; (c) fitting a representation of the drug in step (b) to a computer representation of the three-dimensional structure of the binding interface in step (a) such that the drug interacts with one or more residues of the binding interface; (d) evaluating the interaction of the agent with one or more residues of the binding interface in step (c); If the interaction results in a low-energy, stable complex comprising the agent and SAGE1 or INTS3, which may compete for the SAGE1 complex, then the agent is identified as a potential SAGE1 complex inhibitor.

[0393] The representation of the binding interface on a computer can be a graphical representation, i.e., a depiction of the coordinates of amino acid residues in three-dimensional space. Software for generating the three-dimensional graphical representation of the binding interface in step (a) and / or the three-dimensional graphical representation of the agent in step (b) is well known and commercially available. Examples include Quanta, WebLite Viewer, Schrodinger Suite, AUTODDOCK, and DOCK.

[0394] The fitting (i.e., docking) in the above step (c) is a process of examining the complementarity between the representation of the drug in step (b) and the representation of the three-dimensional structure of the binding interface in step (a). Fitting can be performed by a method known in the art, and can use various calculation methods for evaluating the "fit" between the binding interface and the drug, such as ultra-precise Glide docking (Glide XP).

[0395] The interactions of an agent with one or more residues of the binding interface of the SAGE1-INTS3 complex of the present disclosure may be examined and optimized by computational evaluation, for example, shape, size, and electrostatic complementarity, which may be qualitatively judged visually or quantitatively using scoring functions such as LUDI, PLP, PMF, SCORE, GOLD, FlexX, Emodel score, and Glide XP score. These qualitative and quantitative evaluations may include, but are not limited to, the following: The scoring methods may be used individually or in combination, such as in a consensus scoring format.

[0396] Alternatively, the interaction of the drug with one or more residues of the binding interface can be evaluated based on the interaction energy of a complex formed by binding or linking the drug with a single SAGE1 or INTS3 molecule. As used herein, the term "low energy stable complex" refers to a complex in which the energy of interaction, including hydrophobic enclosure, hydrogen bond interactions, and internal energy (such as van der Waals interactions) between the drug and SAGE1 or INTS3, is below a predetermined threshold that can be set by a computer program. The energy of interaction can be determined using software known in the art, such as, for example, the ultra-precise Glide docking (Glide XP) of the Glide program.

[0397] The comparison of the energy with the predefined threshold may be performed manually or computer-based. In the computer-based comparison, the interaction energy value may be compared with the predefined threshold stored in a database by a computer program, which may further evaluate the comparison result and automatically provide the desired assessment in a suitable output format. A person skilled in the art may easily identify a potential SAGE1-INTS3 complex inhibitor based on the comparison. In some embodiments, the predefined threshold is a Glide XP score, which is determined by hydrophobic enclosure, hydrogen bond interactions, internal energy (such as van der Waals interactions), electrostatic interactions, and two XP penalties (i.e., desolvation penalty and ligand-distortion penalty). In some embodiments, the predefined threshold is a Glide XP score of -10, compared to a reference Glide XP score of -12.

[0398] The effect of potential SAGE1 complex inhibitors identified as described above may be further evaluated in silico or experimentally (e.g., competitive binding experiments or measuring the effect of the drug on the biological activity of the SAGE1-INTS3 complex, etc.).

[0399] In some embodiments, the method for identifying or screening for potential SAGE1-INTS3 complex inhibitors further comprises a step (e) of modifying the drug based on the results obtained in step (d). Modifications can be made to the drug to improve or change the drug's binding properties and selectivity, i.e. affinity with SAGE1 or INTS3. Generally, the initial modifications are conservative, i.e. the replacing groups are approximately the same size, shape, hydrophobicity, and charge as the original groups. Drugs so modified can be analyzed for interactions with one or more residues of the binding interface of the SAGE1-INTS3 complex by the same computational methods detailed above. In some embodiments, the method further comprises repeating steps (b)-(e) in the method for identifying or screening for potential SAGE1-INTS3 complex inhibitors.

[0400] Various molecular analysis techniques and rational drug design techniques are further disclosed in US Pat. Nos. 5,834,228 and 7,930,109 and PCT Publication WO 99 / 09148, the contents of which are incorporated herein by reference.

[0401] In some embodiments, the agent is a polynucleotide, a polypeptide, a chemical compound, an antibody or an antigen-binding fragment thereof, or a derivative of any of these.

[0402] X. INCORPORATION OF EMBODIMENTS DISCLOSED IN THE PRIORITY APPLICATION i. Use of SAGE1 as a biomarker for tumors In another aspect, the present disclosure provides a method for detecting the SAGE1 gene or an active fragment thereof in the manufacture of a kit for evaluating the therapeutic effect of tumor treatment and / or determining the prognosis of tumors. This paper illustrates the use of substances to

[0403] In some embodiments, the material is for detecting the SAGE1 gene or an active fragment thereof in a cell, tissue, or body fluid, the body fluid being preferably selected from the group consisting of intracellular fluid, extracellular fluid, interstitial fluid, plasma, lymphatic fluid, and combinations thereof; and / or

[0404] said substance being for detecting the protein expression level of the SAGE1 gene or the molecular expression level of the SAGE1 gene; and / or

[0405] said material for detecting the SAGE1 gene or an active fragment thereof comprises a specific probe for detecting the SAGE1 gene or an active fragment thereof; and / or

[0406] The material for detecting the SAGE1 gene or an active fragment thereof includes primers for specifically amplifying the SAGE1 gene.

[0407] In some embodiments, the tumor is a solid tumor or a hematological tumor, preferably selected from the group consisting of intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain tumor, and / or thyroid cancer. is selected from: high grade glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell carcinoma (KIRC), cervical squamous cell and adenocarcinoma (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), acute myeloid leukemia (LAML).

[0408] In some embodiments, said kit for assessing the therapeutic effect of a tumor treatment and / or determining the prognosis of a tumor is based on the expression level of the SAGE1 gene in a sample.

[0409] In some embodiments, the SAGE1 gene is the only biomarker in the kit.

[0410] In another aspect, the disclosure provides for the use of an agent for detecting the SAGE1 gene or an active fragment thereof in the manufacture of a kit for diagnosing tumors.

[0411] In some embodiments, the material is for detecting the SAGE1 gene or an active fragment thereof in a cell, tissue, or body fluid, the body fluid being preferably selected from the group consisting of intracellular fluid, extracellular fluid, interstitial fluid, plasma, lymphatic fluid, and combinations thereof; and / or

[0412] said substance being for detecting the protein expression level of the SAGE1 gene or the molecular expression level of the SAGE1 gene; and / or

[0413] said material for detecting the SAGE1 gene or an active fragment thereof comprises a specific probe for detecting the SAGE1 gene or an active fragment thereof; and / or

[0414] The material for detecting the SAGE1 gene or an active fragment thereof includes primers for specifically amplifying the SAGE1 gene.

[0415] In some embodiments, the tumor is a solid tumor or a hematological tumor, preferably selected from the group consisting of intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain tumor, and / or thyroid cancer. is selected from: high grade glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell carcinoma (KIRC), cervical squamous cell and adenocarcinoma (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), acute myeloid leukemia (LAML).

[0416] In some embodiments, the diagnosis of tumor or suitability of the subject for SAGE1 targeted therapy is based on the expression level of the SAGE1 gene in the sample.

[0417] In some embodiments, the SAGE1 gene is the only biomarker in the kit.

[0418] Cancer testis antigens, tumor-associated antigens, mainly expressed in cells of testicular and germinal tissues, are not expressed or expressed at low levels in other normal tissues. However, this antigen is expressed at high levels in different proportions in different tissue types of malignant tumor cells, making it a potential antigen target in terms of early tumor detection and immunotherapy. Its role in tumorigenesis has been revealed in recent studies, and it may be responsible for regulating important cellular processes during growth, stem cell differentiation, and carcinogenesis. However, whether and how these reactivated testis proteins act to support carcinogenesis has yet to be explained. Through many studies, the present disclosure unexpectedly discovered that SAGE1, a cancer testis antigen located on the X chromosome (X:135895410-135913726, Xq26.3), is expressed in various tumor tissues and cells, such as intestinal cancer, lung cancer, liver cancer, breast cancer, skin cancer, head and neck cancer, and leukemia, and that the expression of SAGE1 is not detected in other normal cells other than testicular tissue. Thus, it is contemplated that SAGE1 (e.g., the SAGE1 gene or its expression product) may be used to analyze whether a subject is prone to tumor formation, diagnose whether a subject has a tumor, analyze the therapeutic efficacy of a tumor treatment, and / or determine the prognosis of a tumor.

[0419] In another aspect, the present disclosure provides for the use of a substance for detecting SAGE1 gene or an active fragment thereof in the preparation of a kit for evaluating the therapeutic effect of tumor treatment and / or determining the prognosis of a tumor. Generally, the kit is capable of detecting the expression level of SAGE1 gene or an active fragment thereof in a sample by a substance for detecting SAGE1 gene or an active fragment thereof, so that the expression level of SAGE1 gene or an active fragment thereof in the sample can be used to evaluate the malignancy of a tumor, evaluate the therapeutic effect of a tumor treatment, and / or determine the prognosis of a tumor. The present disclosure has discovered that the expression of SAGE1 gene or an active fragment thereof is closely related to the malignancy of a tumor in a patient, the therapeutic effect of the treatment in a patient who has received the treatment, and / or the prognosis of a tumor.

[0420] In particular, in the case of solid tumors or hematological tumors, SAGE1-positive tumors generally exhibit higher malignancy, and patients with SAGE1-positive tumors generally respond less well to tumor treatments, have a worse prognosis, and develop new tumors in a shorter time period. For example, patients with SAGE1-positive tumors have a shorter overall survival and / or progression-free survival. As another example, positive SAGE1 expression is associated with lymph node metastasis, and patients with SAGE1-positive tumors are more likely to develop lymph node metastasis or develop new tumors. SAGE1 Expression may also refer to the expression level of SAGE1 mRNA or the expression level of SAGE1 protein. Positive SAGE1 expression (or SAGE1 positivity) generally refers to the presence of SAGE1 expression or a high level of SAGE1 expression relative to a baseline. In some embodiments, positive SAGE1 expression is indicated by detectable expression of SAGE1 mRNA in tumor tissue. In some embodiments, positive SAGE1 expression is indicated by detectable expression of SAGE1 protein in tumor tissue. In some embodiments, positive SAGE1 expression is indicated by detecting higher SAGE1 mRNA expression in the sample compared to surrounding healthy tissue. In some embodiments, positive SAGE1 expression is indicated by detecting higher SAGE1 protein expression in the sample compared to surrounding healthy tissue.

[0421] For patients with solid tumors or hematological tumors, the higher the SAGE1 positive level, the more aggressive the tumor, the lower the response after tumor treatment, the worse the prognosis, and the shorter the time for new tumor growth.For example, the higher the SAGE1 expression level, the shorter the overall survival and / or progression-free survival of the patient.In another example, high SAGE1 expression level is associated with lymph node metastasis, and the higher the SAGE1 expression level, the more likely the patient is to develop lymph node metastasis or develop new tumors.

[0422] The therapeutic effect of tumor treatment or tumor prognosis can be evaluated by assessing the overall survival and / or progression-free survival of tumor patients. Overall survival (OS) usually refers to the time from a certain point in time (e.g., when the tumor is diagnosed or when the tumor is surgically removed) until the patient dies from any cause. Progression-free survival (PFS) generally refers to the time from a certain point in time (e.g., when the tumor is diagnosed or when the tumor is surgically removed) until another point in time, when local or partial tumor regression is detected, when distant metastasis is detected, or when the patient dies.

[0423] In another aspect, the present disclosure provides a use of a substance for detecting SAGE1 gene or its active fragment in the preparation of a kit for diagnosing tumors, which can diagnose tumors by detecting the expression level of SAGE1 gene or its active fragment, or determine whether a subject is suitable for SAGE1-targeted therapy. The present disclosure finds that the expression of SAGE1 gene or its active fragment is closely related to whether a subject has tumors, and that tumor patients with positive SAGE1 expression or high levels of SAGE1 expression are more suitable for SAGE1-targeted therapy.

[0424] Specifically, in patients with solid tumors and / or hematological tumors, the SAGE1 gene is specifically positively expressed in tumor tissue. Therefore, the expression level of the SAGE1 gene can determine whether a patient has a tumor. For example, patients with positive SAGE1 expression are usually more likely to have a tumor. Alternatively, the expression level of the SAGE1 gene can be used to determine whether a subject is suitable for SAGE1-targeted therapy. The present disclosure demonstrates that inhibition of SAGE1 can significantly suppress tumor cell proliferation, migration, and anchorage-independent growth, while the restoration of blocked SAGE1 expression can restore tumor cell clonogenicity, indicating that patients with positive SAGE1 expression and / or high levels of SAGE1 expression are generally suitable for SAGE1-targeted therapy.

[0425] In the present disclosure, a substance for detecting the SAGE1 gene or an active fragment thereof is well known to those skilled in the art, and a method for preparing the substance is also well known to those skilled in the art. Specifically, the substance may generally detect the expression of the SAGE1 gene or an active fragment thereof, and specifically detect the protein expression level of the SAGE1 gene or the molecule (e.g., SAGE1 mRNA) expression level of the SAGE1 gene. For example, the substance may be a substance capable of specifically binding to a target molecule (e.g., SAGE1 mRNA, SAGE1 protein, etc.); Binding of the agent is detected. In one embodiment, the agent can be a probe capable of specifically binding to the SAGE1 gene, and in another embodiment, the agent can be an antibody capable of specifically targeting the SAGE1 protein. As another example, the agent can be an agent capable of specifically amplifying the SAGE1 gene, and expression of the SAGE1 gene or active fragments thereof can be detected by detecting gene fragments resulting from this specific amplification. In one embodiment, the agent can be primers capable of specifically amplifying the SAGE1 gene and / or a label (e.g., a dye) suitable for detecting the product obtained by amplification, etc.

[0426] In the present disclosure, the method of using the kit generally differs depending on the substance for detecting the SAGE1 gene or its active fragment. The kit can generally be used for detection performed on cells, tissues, and body fluids of a patient. Specifically, the substance for detecting the SAGE1 gene or its active fragment can be used to detect the SAGE1 gene or its active fragment in the cells, tissues, and body fluids of a subject. The body fluid is, for example, a fluid selected from intracellular fluid (including nucleus) and extracellular fluid, etc., or a combination thereof. The extracellular fluid is, for example, a fluid selected from interstitial fluid, plasma, lymph, etc., or a combination thereof. In one embodiment, the sample to be detected by the kit is selected from cells, blood samples (e.g., plasma samples and / or whole blood samples, etc.), and tissues (e.g., tumor tissues or precancerous tissues, etc.) or pathological sections.

[0427] In some embodiments, the tumor is a solid tumor or a hematological tumor, such as, for example, intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain low grade malignant tumors. The cancer is selected from hepatic glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), and acute myeloid leukemia (LAML), etc.

[0428] The SAGE1 gene may be the sole biomarker in the kit, i.e. the kit does not contain materials for detecting other biomarkers in addition to materials for detecting the SAGE1 gene or active fragments thereof, and the SAGE1 gene may be used as the sole biomarker in the kit to assess the therapeutic effect of tumor treatment and / or to determine the prognosis of tumors or to diagnose tumors.

[0429] The SAGE1 gene or its active fragments of the present disclosure can serve as a biomarker for evaluating tumor malignancy, metastasis, recurrence, prognosis, and selecting targeted therapy. Compared with conventional biomarkers, SAGE1 has high specificity (SAGE1 is expressed in tumor tissues but not in normal tissues), with close to 100% accuracy in distinguishing tumors from normal tissues, and can also serve as a therapeutic target for tumor treatment and can be used in tumor-free imaging. The expression profile of SAGE1 can also provide a basis for evaluating the therapeutic effect of tumor treatment, for monitoring recurrence and metastasis, and for determining the malignancy level of tumors.

[0430] ii. Use of SAGE1 inhibitors in the preparation of medicines or kits In another embodiment, the present disclosure provides for the use of a SAGE1 inhibitor in the manufacture of a medicament or kit for 1) treating a tumor and / or 2) modulating the expression level of the SAGE1-INTS3 complex.

[0431] In some embodiments, the SAGE1 inhibitor inhibits the expression and / or function of SAGE1.

[0432] In some embodiments, the SAGE1 inhibitor is the sole active ingredient.

[0433] In some embodiments, the SAGE1 inhibitor is selected from a nucleic acid molecule, a protein molecule, or a chemical compound.

[0434] In some embodiments, the nucleic acid molecule is selected from the group consisting of interfering RNA for SAGE1, antisense oligonucleotides for SAGE1, and agents for knocking out or knocking down SAGE1 expression, and / or the protein molecule is an anti-SAGE1 antibody, preferably a monoclonal antibody.

[0435] In some embodiments, the nucleic acid molecule targets a target sequence comprising a sequence set forth in any of SEQ ID Nos. 59-67, and the polynucleotide sequence of the nucleic acid molecule comprises a sequence set forth in any of SEQ ID Nos. 87-95 and 96-99.

[0436] In some embodiments, the SAGE1 inhibitor competes with SAGE1 for binding to INTS3, and preferably the SAGE1 inhibitor is INTS6, INTS6L, or a combination thereof.

[0437] In some embodiments, the tumor is a SAGE1 positive tumor.

[0438] In some embodiments, the tumor is a solid tumor or a hematological tumor, preferably selected from the group consisting of intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain tumor, and / or thyroid cancer. is selected from: high grade glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell carcinoma (KIRC), cervical squamous cell and adenocarcinoma (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), acute myeloid leukemia (LAML).

[0439] In another embodiment, a composition of the disclosure comprises a SAGE1 inhibitor disclosed herein, the composition comprising:

[0440] Treating the tumor; and / or

[0441] Regulates the expression level of the SAGE1-INTS3 complex.

[0442] A family of cancer / testis genes (cancer testis antigens or CTAs) is usually expressed only in adult testis, with low or no expression in other normal tissues, but is also expressed in most types of malignant tumors and hematological tumors. These CTAs are considered to be major biomarkers of cancer and are also excellent antigen targets for cancer immunotherapy. Several cancer testis antigens, such as NY-ESO-1, have been developed for cancer vaccines that have entered phase 3 clinical trials. Their role in tumorigenesis has been elucidated in recent studies and may play a role in regulating important cellular processes during development, stem cell differentiation, and carcinogenesis. Numerous cancer testis antigens are Although selective expression in cancer stem cells (CSCs) or cancer initiating cells (CICs) has been demonstrated, whether and how reactivated testis proteins act to support oncogenic activity remains largely unexplained. Through extensive research, the present disclosure unexpectedly discovered that CT-X cancer testis antigen, located on chromosome X (X:135895410-135913726, Xq26.3), is aberrantly activated as a tumor-specific antigen in various types of human tumors outside of normal testis tissue. SAGE1 can form a functional complex with INTS3, and the SAGE1-INTS3 complex is specifically found in various tumor cells and tumor tissues. The present disclosure further demonstrates that specifically targeting the SAGE1 gene / protein or SAGE1-INTS3 complex gene / protein significantly suppresses the proliferation and anchorage-independent growth of SAGE1-positive cancer cells as well as the growth of in vivo xenografts, thereby providing an effective new target for tumor treatment.

[0443] In one aspect, the present invention provides the use of SAGE1 inhibitors in the manufacture of drugs or kits for the treatment of tumors. It has been found that SAGE1 is not only positively expressed in various types of tumors, but also that patients with positive SAGE1 expression have a relatively poor prognosis. SAGE1 is specifically positively expressed in tumor tissues, but is not expressed in healthy tissues (e.g., pre-cancerous tissues). The present disclosure shows that inhibition of SAGE1 can significantly suppress tumor cell proliferation, migration, and anchorage-independent growth, while restoration of the blocked SAGE1 expression can restore the clonogenicity of tumor cells, demonstrating that SAGE1 inhibitors can be used to treat tumors.

[0444] The term "treatment" as used herein refers to preventive, curative, or palliative measures capable of producing a desired medicinal and / or physiological effect. Such an effect preferably refers to the medical suppression of one or more symptoms of a disease, or the complete elimination of a disease, or the prevention or delay of the onset of a disease, and / or the reduction of the risk of progression or worsening of a disease.

[0445] In another embodiment, the present disclosure provides for the use of a SAGE1 inhibitor in the manufacture of a medicament or kit for modulating the expression level of SAGE1-INTS3 complex. The present disclosure has discovered that SAGE1, a tumor-specific antigen, can form a complex with INTS3 after abnormal activity. The SAGE1-INTS3 complex is specifically found in various tumor cells and tumor tissues. When SAGE1 is inhibited, expression of wild-type SAGE1 can promote tumor cell proliferation, but expression of mutant SAGE1 with point mutations in key residues that interact with INTS3 (F838A, F873A, K874A, R872A, M832A, Q840A) does not. According to this discovery, SAGE1 needs to form a complex with INTS3 to function, and reducing the amount of SAGE1-INTS3 complex by preventing the interaction between SAGE1 and INTS3 can suppress tumor cell growth.

[0446] The term SAGE1-INTS3 complex generally refers to a complex of SAGE1 and INTS3 formed by protein-protein interaction. In general, INTS3 can form a dimer (e.g., in solution), which can further form a complex with SAGE1 through interactions such as hydrogen bonds and salt bridges. The parameters of the unit cell that the crystal structure of the SAGE1-INST3 complex can take are 260.112±0.1 Å, b=46.240±0.1 Å, c=103.101±0.1 Å, α=90±0.1° Å, β=113.280±0.1° Å, and γ=90±0.1° Å. In the SAGE1-INST3 complex, the main amino acid residues involved in the formation of the SAGE1-INST3 complex typically include F838, F873, K874, R872, M832, and Q840.

[0447] The SAGE1 inhibitors disclosed herein can inhibit the expression and / or function of SAGE1. For example, the SAGE1 inhibitors can partially inhibit the expression and / or function of SAGE1. The expression and / or function of SAGE1 can be inhibited, i.e., suppressed, or completely inhibited, i.e., eliminated, including the ability of SAGE1 to bind to INTS3 and form a SAGE1-INTS3 complex. The types of agents that can function as SAGE1 inhibitors are known to those of skill in the art. For example, inhibitors can be antagonists, blockers, and the like. As another example, an inhibitor can inhibit SAGE1 expression at the nucleic acid (e.g., mRNA) level or at the protein level. As yet another example, an inhibitor can be an agent that competes with SAGE1 for binding to INTS3. In some embodiments, the SAGE1 inhibitor can be a nucleic acid molecule, a protein molecule, or a compound, and the like. The nucleic acid molecule can be selected from an interfering RNA against SAGE1, an antisense oligonucleotide against SAGE1, or an agent for knocking out or knocking down the expression of SAGE. In particular, the SAGE1 inhibitor can be an siRNA, miRNA, shRNA, a gene knockout vector, or a gene expression vector capable of expressing an siRNA, miRNA, or an interfering RNA, and the like. The protein molecule may be selected from anti-SAGE1 antibodies, which may be monoclonal or polyclonal. As an example, the agent capable of competing with SAGE1 for binding to INTS3 may be INTS6 (DDX26, Accession: NP_036273.1) or INTS6L (DDX26b, Accession: Q8BND4.1) or a combination thereof. In some embodiments, the nucleic acid molecule targets a target sequence comprising a sequence set forth in any of SEQ ID Nos. 59-67. In some embodiments, the polynucleotide sequence of the nucleic acid molecule comprises a sequence set forth in any of SEQ ID Nos. 87-95. In some embodiments, the polynucleotide sequence of the nucleic acid molecule comprises a sequence set forth in any of SEQ ID Nos. 96-99.

[0448] In the medicament or kit disclosed herein, the SAGE1 inhibitor may be used as the sole active ingredient or in combination with other active ingredients for the treatment of tumors.

[0449] Tumors treated with the medicaments or kits disclosed herein are most often SAGE1 positive tumors. SAGE1 positivity or positive SAGE1 expression generally refers to the presence of SAGE1 expression or a high level of SAGE1 expression relative to a baseline. In some embodiments, positive SAGE1 expression is indicated by detectable expression of SAGE1 mRNA in tumor tissue. In some embodiments, positive SAGE1 expression is indicated by detectable expression of SAGE1 protein in tumor tissue. In some embodiments, positive SAGE1 expression is indicated by detecting higher expression of SAGE1 mRNA in tumor tissue compared to surrounding healthy tissue. In some embodiments, positive SAGE1 expression is indicated by detecting higher expression of SAGE1 protein in tumor tissue compared to surrounding healthy tissue. In some embodiments, the tumor is a solid tumor or a hematological tumor, such as, for example, intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain low grade malignant tumors. The cancer is selected from hepatic glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), and acute myeloid leukemia (LAML), etc.

[0450] In another embodiment, a composition of the disclosure comprises a SAGE1 inhibitor as disclosed herein, which is used to treat tumors and / or modulate the expression levels of the SAGE1 complex.

[0451] In another aspect, the present disclosure provides methods for detecting SAGE1-INTS in, e.g., individuals and cells. The present invention provides a method for modulating the expression level of the SAGE1 complex, comprising, for example, administering to a subject an effective amount of a SAGE1 inhibitor or composition disclosed herein.

[0452] In another aspect, the disclosure provides a method of treatment comprising administering to a subject an effective amount of a SAGE1 inhibitor or composition disclosed herein. The method can be used to treat diseases including, but not limited to, tumors. In some embodiments, the tumor is a solid tumor or a hematological tumor, such as intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain low grade malignant tumors, and the like. The cancer is selected from hepatic glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), and acute myeloid leukemia (LAML), etc.

[0453] As used herein, the term "subject" generally refers to humans, non-human mammals, and primates, such as dogs, cats, horses, sheep, pigs, and cows, that may benefit from treatment with the agents, kits, or compounds described herein.

[0454] As used herein, the term "therapeutically effective amount" generally refers to an amount effective over an appropriate administration period for the treatment of the diseases described herein.

[0455] The SAGE1 gene described herein is used as a target for antitumor drugs. The present disclosure has discovered that by blocking the expression of the SAGE1 gene, the proliferation, migration, etc. of various tumor cells that show positive SAGE1 expression (e.g., melanoma cells A375, duodenal adenocarcinoma cells HUTO80, colorectal adenocarcinoma cells Caco-2, and esophageal cancer cells TE1) can be significantly inhibited, which means that SAGE1 functions as a tumor promoter for various types of tumors. In addition, by blocking the expression of the SAGE1 gene, the growth of solid tumors derived from humans can be significantly inhibited, and the death of tumor cells in solid tumors is promoted, indicating that this target has an important physiological function. Furthermore, SAGE1 is specifically expressed in various tumor cells, but not in normal tissues other than the testis. Although the SAGE1 protein is usually present in nucleic acids, peptide fragments of the protein are presented on the surface of tumor cells by the major histocompatibility complex (MHC), which can be used for target recognition in targeted therapy and / or immune activation in cancer immunotherapy. Therefore, gene interference therapy targeting SAGE1 is highly specific and does not affect normal tissues that do not express SAGE1, so it does not harm normal physiological functions or health. SAGE1 inhibitors that inhibit the expression and / or function of SAGE1 have great potential for clinical tumor treatment.

[0456] iii. Use of substances that modulate the expression and / or function of the SAGE1-INTS3 complex In another aspect, the present disclosure provides for the use of an agent for modulating the expression and / or function of SAGE1-INTS3 complex in the manufacture of a medicament or kit for the treatment of tumors.

[0457] In some embodiments, the agent for modulating expression and / or function of SAGE1-INTS3 complex is a single active ingredient.

[0458] In some embodiments, the agent is for use in modulating the expression and / or function of the SAGE1-INTS3 complex.

[0459] In some embodiments, the agent for modulating the expression and / or function of SAGE1-INTS3 complex is selected from a nucleic acid molecule, a protein molecule, and a chemical compound.

[0460] In some embodiments, the agent for modulating expression and / or function of a SAGE1-INTS3 complex is a SAGE1 inhibitor.

[0461] In some embodiments, the SAGE1 inhibitor is selected from the group consisting of an interfering RNA for SAGE1, an antisense oligonucleotide for SAGE1, and an agent for knocking out or knocking down SAGE1 expression.

[0462] In some embodiments, the SAGE1 inhibitor is an agent that competes with SAGE1 for binding to INTS3, and / or the SAGE1 inhibitor is selected from INTS6 and INTS6L.

[0463] In some embodiments, the tumor is a SAGE1 positive tumor.

[0464] In some embodiments, the tumor is a solid tumor or a hematological tumor, preferably selected from the group consisting of intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain tumor, and / or thyroid cancer. is selected from: high grade glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell carcinoma (KIRC), cervical squamous cell and adenocarcinoma (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), acute myeloid leukemia (LAML).

[0465] In another aspect, the present disclosure provides a composition for use in treating a tumor, said composition comprising an agent for use in modulating the expression and / or function of the SAGE1-INTS3 complex.

[0466] Through extensive research, the present disclosure unexpectedly discovered that SAGE1, i.e., CT-X cancer-testis antigen located on chromosome X (X:135895410-135913726, Xq26.3), is expressed as a tumor-specific antigen in various tumor tissues and cells, such as intestinal cancer, lung cancer, liver cancer, breast cancer, skin cancer, head and neck cancer, leukemia, etc., and forms a novel functional complex with INTS3 in various types of human tumors after abnormal activation. The present disclosure found that by specifically targeting SAGE1 gene / protein or SAGE1-INTS3 complex gene / protein, the proliferation and anchorage-independent growth of SAGE1-positive cancer cells as well as the growth of in vivo xenografts can be significantly suppressed, thereby providing an effective new target for tumor treatment.

[0467] In another aspect, the present disclosure provides for the use of a substance for regulating the expression and / or function of the SAGE1-INTS3 complex in the manufacture of a medicament or kit for the treatment of tumors. The present disclosure has discovered that the SAGE1-INTS3 complex is present only in cells in which SAGE1 is expressed and binds to INTS3, but is absent in cells negative for SAGE1 expression. The predominant differential gene expression due to decreased SAGE1 expression is consistent with that due to decreased INTS3 expression. That is, genes that are upregulated in response to decreased SAGE1 expression are upregulated by INTS3. SAGE1 was also upregulated in response to decreased expression of INTS3 and vice versa, suggesting that SAGE1 regulates and modulates the expression of tumor-related genes by forming a complex with INTS3. INTS3 and SAGE1 are synergistic and function as a single complex unit. Disrupting the INTS3-SAGE1 interaction can completely abolish the ability of SAGE1 to promote tumor cell growth and significantly inhibit xenograft growth. This disclosure demonstrates that the SAGE1-INTS3 complex can function as a target to inhibit tumor growth.

[0468] The term SAGE1-INTS3 complex generally refers to a complex of SAGE1 and INTS3 formed by protein-protein interaction. In general, INTS3 can form a dimer (e.g., in solution), which can further form a complex with SAGE1 through interactions such as hydrogen bonds and salt bridges. The parameters of the unit cell that the crystal structure of the SAGE1-INST3 complex can take are 260.112±0.1 Å, b=46.240±0.1 Å, c=103.101±0.1 Å, α=90±0.1° Å, β=113.280±0.1° Å, and γ=90±0.1° Å. In the SAGE1-INST3 complex, the main amino acid residues involved in the formation of the SAGE1-INST3 complex typically include F838, F873, K874, R872, M832, and Q840.

[0469] The term "treatment" as used herein refers to preventive, curative, or palliative measures capable of producing a desired medicinal and / or physiological effect. Such an effect preferably refers to the medical suppression of one or more symptoms of a disease, or the complete elimination of a disease, or the prevention or delay of the onset of a disease, and / or the reduction of the risk of progression or worsening of a disease.

[0470] The substance for regulating the expression and / or function of the SAGE1-INTS3 complex may be a substance that inhibits the expression and / or function of the SAGE1-INTS3 complex. For example, the substance for inhibiting the expression and / or function of the SAGE1-INTS3 complex may partially inhibit, i.e., suppress, the expression and / or function of the SAGE1-INTS3 complex, or may completely inhibit, i.e., completely eliminate, the expression and / or function of the SAGE1-INTS3 complex. The substance for inhibiting the expression and / or function of the SAGE1-INTS3 complex may be any substance (e.g., a nucleic acid molecule, a protein molecule, or a compound, etc.) that can perform the above-mentioned functions.

[0471] The substance for modulating the expression and / or function of the SAGE1-INTS3 complex can be a SAGE1 inhibitor. The SAGE1 inhibitor can partially inhibit, i.e., suppress, or completely inhibit, i.e., eliminate, the expression and / or function of SAGE1, including the ability of SAGE1 to bind to INTS3 and form the SAGE1-INTS3 complex. The types of agents that can function as SAGE1 inhibitors are known to those of skill in the art. For example, inhibitors can be antagonists, blockers, and the like. As another example, the inhibitor can inhibit SAGE1 expression at the nucleic acid (e.g., mRNA) level or protein level. As yet another example, the inhibitor can be an agent that competes with SAGE1 for binding to INTS3. In some embodiments, the SAGE1 inhibitor can be a nucleic acid molecule, a protein molecule, or a compound, and the like. The nucleic acid molecule can be selected from an interfering RNA against SAGE1, an antisense oligonucleotide against SAGE1, and an agent for knocking out or knocking down the expression of SAGE. Specifically, the SAGE1 inhibitor can be an siRNA, miRNA, shRNA, gene knockout vector, or a gene expression vector capable of expressing siRNA, miRNA, or interfering RNA, etc. The protein molecule can be selected from anti-SAGE1 antibodies, which can be monoclonal or polyclonal. As an example, an agent capable of competing with SAGE1 for binding to INTS3 can be INTS6 (DDX26, Accession: NP_036273.1) or INTS6L (DDX26b, Accession: Q8BND4.1), or a combination thereof. In some embodiments, the nucleic acid molecule targets a target sequence comprising a sequence set forth in any of SEQ ID Nos. 59-67. In some embodiments, the polynucleotide sequence of the nucleic acid molecule comprises a sequence set forth in any of SEQ ID Nos. 87-95. In some embodiments, the polynucleotide sequence of the nucleic acid molecule comprises a sequence set forth in any of SEQ ID Nos. 96-99.

[0472] In the medicament or kit disclosed herein, the SAGE1 inhibitor may be used as the sole active ingredient or in combination with other active ingredients for the treatment of tumors.

[0473] Tumors treated with the medicaments or kits disclosed herein are most often SAGE1 positive tumors. SAGE1 positivity or positive SAGE1 expression generally refers to the presence of SAGE1 expression or a high level of SAGE1 expression relative to a baseline. In some embodiments, positive SAGE1 expression is indicated by detectable expression of SAGE1 mRNA in tumor tissue. In some embodiments, positive SAGE1 expression is indicated by detectable expression of SAGE1 in tumor tissue. In some embodiments, positive SAGE1 expression is indicated by detecting higher SAGE1 mRNA expression in tumor tissue compared to surrounding healthy tissue. In some embodiments, positive SAGE1 expression is indicated by detecting higher SAGE1 protein expression in tumor tissue compared to surrounding healthy tissue. In some embodiments, the tumor is a solid tumor or a hematological tumor, such as, for example, intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial cancer (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain low grade malignant tumors. The cancer is selected from hepatic glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), and acute myeloid leukemia (LAML), etc.

[0474] In another aspect, the present disclosure provides a composition for use in treating a tumor, said composition comprising a substance disclosed herein for use in modulating the expression and / or function of the SAGE1-INTS3 complex.

[0475] In another aspect, the disclosed method of treatment comprises administering to a subject an effective amount of an agent capable of modulating expression and / or function of the SAGE1-INTS3 complex or a complex disclosed herein. The method can be used to treat diseases including, but not limited to, tumors. In some embodiments, the tumor is a solid tumor or a hematological tumor, such as, for example, intestinal cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, colon adenocarcinoma (COAD), liver hepatocellular carcinoma (LIHC), ovarian serous cystadenocarcinoma (OV), endometrial carcinoma (UCEC), thyroid cancer (THCA), skin cutaneous melanoma (SKCM), lung adenocarcinoma (LUAD), head and neck squamous cell carcinoma (HNSC), glioblastoma multiforme (GBM), prostate adenocarcinoma (PRAD), thymoma (THM), brain low grade malignant tumors (LLD), and / or brain tumors. The cancer is selected from hepatic glioma (LGG), rectal adenocarcinoma (READ), pheochromocytoma and paraganglioma (PCPG), esophageal carcinoma (ESCA), kidney clear cell renal carcinoma (KIRC), cervical squamous cell carcinoma and adenocarcinoma of the cervix (CESC), bladder urothelial carcinoma (BLCA), kidney papillary renal cell carcinoma (KIRP), pancreatic adenocarcinoma (PAD), gastric adenocarcinoma (STAD), kidney chromophobe adenoma (KICH), invasive carcinoma of the breast (BRCA), lung squamous cell carcinoma (LUSC), sarcoma (SARC), and acute myeloid leukemia (LAML), etc.

[0476] In the present invention, a "subject" generally refers to a person who is receiving treatment with a formulation, kit, or combination formulation. This includes humans, non-human mammals, and primates such as dogs, cats, horses, sheep, pigs and cows that may benefit.

[0477] In the present invention, the term "therapeutically effective amount" generally refers to an amount that provides an effect of treating the above-mentioned disease after an appropriate administration period.

[0478] The SAGE1-INTS3 complex described herein is used as a target for antitumor drugs. SAGE1 is expressed on the cell surface of various tumors, including melanoma, bladder cancer, liver cancer, epidermoid carcinoma, non-small cell lung cancer, and squamous cell carcinoma, but is not usually expressed in normal tissues other than the testis. The SAGE1-INTS3 complex can effectively serve as a therapeutic target for any type of tumor that is positive for SAGE1 expression, and a relatively high percentage of tumors are positive for SAGE1 expression. The SAGE1-INTS3 complex is different from other targets that have been used for targeted therapy. The interaction between SAGE1 and INTS3 in the novel complex, rather than genetic mutation, provides the basis for the mechanism of action in targeted therapy. This is also based on the fact that under physiological conditions, SAGE1 expression is usually only found in the reproductive system, such as the testis, and at low levels in the brain but not in other tissues, while SAGE1 is specifically expressed at different frequencies in various tumor tissues. Due to internal barriers in the testis and brain, specific targeting of the SAGE1 / SAGE1-INST3 gene / protein does not affect normal tissues. EXAMPLES

[0479] While the present disclosure has been particularly shown and described with reference to specific embodiments, some of which are preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure disclosed herein.

[0480] Materials and methods used in the examples are described at the end of this section.

[0481] Example 1: Distribution of SAGE1 expression in normal and cancer tissues

[0482] SAGE1 expression is restricted to the testis, with little or no expression in the majority of somatic tissues (Figure 1A and Figure 1B). The TPM values ​​of each gene used for visualization on the GTEx website (https: / / gtexportal.org / home / gene / ) were downloaded from the GTEx website (https: / / gtexportal.org / home / datasets).

[0483] In the case of cancer tissues, they were abnormally expressed in almost all malignant tumors (Figure 1B). The FPKM value of each gene in the TCGA project was obtained from the GDC website (https: / / portal. gdc. cancer. gov) and was calculated as exp(log(FPKM) -log(sum(FPKM)) + log(10 6 )) is used to convert it to TPM.

[0484] Example 2: Overall survival analysis by cancer type and patterns of SAGE1 expression levels in TCGA

[0485] SAGE1 has been identified as a cancer proliferation gene whose expression promotes cancer cell proliferation and is associated with poor prognosis in cancer patients (Figure 1C). To understand the biological significance of SAGE1 expression in cancer, we performed survival curve analysis using the TCGA dataset. The presence of SAGE1 in pan-cancer was associated with poor prognosis. We also found that higher levels of SAGE1 expression in pan-cancer were associated with shorter overall survival, suggesting that SAGE1 expression tends to be associated with advanced disease stage and poor prognosis.

[0486] SAGE1 expression levels were analyzed for each cancer type in TCGA and classified into three groups according to normalized counts: negative (0RESM), low expression (0.1857-11.2969RESM), and high expression (>11.30RESM).

[0487] Example 3: SAGE1 expression levels in various tumor tissues and treatment effect in PDX mice using IHC staining

[0488] With limited expression in normal tissues, SAGE1 was found to be highly expressed in many cancer types and cancer cell lines other than testicular cancer, compared to healthy tissues or pre-malignant lesions.

[0489] Histologically, both SAGE1 expression and cell proliferation levels were substantially suppressed in SAGE1 knockdown tumors, as shown by IHC staining (Fig. 4L). Notably, of the seven tumors in LUS-P after shSAGE1 treatment, four were undetectable at the end point, and the remaining three tumors showed residual masses of fibroinflammation but no cancer remnants as determined by pathology and IHC examination.

[0490] Example 4: SAGE1-positive cancer cells are dependent on SAGE1

[0491] To investigate the function of SAGE1 in cancer, we first identified SAGE1-positive cancer cell lines (Figure 2C) by Western blotting and qPCR analysis of tumor tissues (Figure 2B). Knockdown of SAGE1 (SAGE1-KD) in five cancer cell lines (Figure 3A) by lentivirus carrying short hairpin RNA (shRNA Y12682 (SEQ ID NO: 128) targeting the sequence shown in SEQ ID NO: 33 GCTGCAGTCACTCACAACA) slowed cell growth in both real-time cell analysis (RTCA) and anchorage-independent growth assays (Figure 3B). To further investigate the in vivo function of SAGE1, we examined xenograft growth in nude mice by subcutaneously injecting HuTu80 parental and SAGE1-KO cells in parallel. Compared to parental xenografts, SAGE1-KO HuTu80 xenografts showed significant growth suppression (Figure 3C). Moreover, in an orthotopic model derived from a SAGE1-positive uveal melanoma cell line (MUM2B), knockdown of SAGE1 resulted in a significant decrease in brightness signal compared to the control group (P<0.001, Figure 3D), but baseline imaging 5 days after intraocular seeding showed the same brightness (Figure 3D). Furthermore, a significantly better survival time was observed in the SAGE1-KD group (Figure 3E). Collectively, these results demonstrated that SAGE1 is important for the tumorigenic growth of SAGE1-positive cancer cells. We tested the ability of SAGE1KD in KYSE30 using different siRNAs (SEQ ID NOs: 70-81) by quantitative PCR (Figure 3F).

[0492] Example 5: SAGE1 regulates multiple pathways involved in cancer cell proliferation and cell cycle

[0493] To further investigate the function of SAGE1 in cancer cells at the protein level, we monitored the protein products of receptor tyrosine kinases (RTKs) activated by these growth factors, which would be phosphorylated at lower levels in SAGE1-deficient cancer cells. Indeed, phosphorylation of EGFR, FGFR1, FGFR3, Met, Kit, FLT3, and PDGFα was obviously suppressed in SAGE1-KD Hutu80 cells, strongly suggesting that SAGE1 plays an important role in regulating RTK-mediated pathways. We also observed that phosphorylation of key signaling molecules in the Wnt, TGF-β, JAK-STAT, and Notch pathways was significantly suppressed, confirming that these pathways are also regulated by SAGE1. Western blotting analysis also showed that the proteins of SAGE1-regulated transcription factors were obviously decreased in SAGE1-KD cells (Figure 9A).

[0494] The main downstream effect of all these SAGE1-regulated pathways is cell proliferation. To directly assess the effect of SAGE1 on cancer cell proliferation, we performed fluorescence-activated cell sorting (FACS) analysis of multiple SAGE1-positive cancer cell lines in both WT and SAGE1-KD conditions. Quantitative measurements showed that knockdown of SAGE1 arrested cells at the Go / G1 phase of the cell cycle and blocked them from entering the G2 / M phase (Figure 9B), suggesting that SAGE1 plays an important role in cell proliferation in SAGE1-positive cancer cells.

[0495] To further explore the function of SAGE1 in primary patient-derived tumor cells, we performed WB and TMT-labeled MS-bac on HCC-P control and SAGE1-KD in primary HCC-P cells (Figure 9C and Figure 9E). The results showed that the expression of RTKs, cell cycle-related proteins, and transcription factors was dependent on SAGE1, indicating that the gene expression program in HCC-P cells was controlled by SAGE1, similar to that in HuTu80 cells. In line with this, FACS and Ki67 staining data showed a clear arrest at Go / G1 phase (Figure 9D), and MS analysis revealed that the expression of most cell cycle genes was reduced in SAGE1-KD HCC-P cells. These results indicate that SAGE1 is also essential for cell cycle control in primary liver cancer cells. Taken together, our analysis demonstrates that SAGE1 is a pan-cancer master regulator that drives tumor cell proliferation by activating multiple cancer pathways.

[0496] Example 6

[0497] To further explore the function of SAGE1 in patient-derived tissues and primary tumor cells, we performed qPCR and Western blotting analysis of SAGE1 (Figure 2B and 2C). With limited expression in normal tissues, we found high expression of SAGE1 in a number of cancer cell lines and tumor tissues compared to healthy tissues or precancerous tissues. Furthermore, qPCR analysis of 60 colorectal cancer (CRC) tissues and corresponding precancerous tissues confirmed this cancer-specific expression profile of SAGE1 (Figure 2D).

[0498] Example 7. Targeting SAGE1 enhances a novel approach to disrupt cancer progression

[0499] We next directly assessed whether targeting SAGE1 would be an effective anti-cancer therapeutic strategy, given that SAGE1 is required for the proliferation of SAGE1-positive cancer cells. We screened a large panel of early passage patient-derived xenografts (PDXs) by immunohistochemistry (IHC) staining for SAGE1-positive tumors (Figure 15). Seven SAGE1-positive PDXs derived from different cancer types, namely hepatocellular carcinoma (HCC-P), lung squamous cell carcinoma (LUS-P), lung adenocarcinoma (LUA-P), colon carcinoma (CRC-P), oropharyngeal squamous cell carcinoma (OSC-P), gallbladder carcinoma (GBC-P), and esophageal squamous cell carcinoma (ESC-P), were prepared for in vivo SAGE1-KD treatment. Two SAGE1-negative PDXs, namely HCC-N and LUS-N, were used as controls.

[0500] shRNA82 (SEQ ID NO: 80) was introduced into adenovirus and administered to PDX mice. This treatment significantly reduced tumor volume and weight with a mean growth inhibition rate of more than 85% (P<0.001), indicating that SAGE1 plays an important role in tumor growth (Figure 4A-L). Notably, of the seven tumors in LUS-P after shSAGE1 treatment, four were undetectable at the endpoint, and the remaining three residual masses showed fibrotic inflammation but no cancer remnants were visible by pathology or IHC examination. In stark contrast, the same adenoviral treatment in two SAGE1-negative tumors, HCC-N and LUS-N, had no noticeable effect on tumor growth during the treatment period, with an average growth inhibition rate of 3.08%, strongly suggesting that the tumor inhibition observed in SAGE1-positive PDXs was specific to SAGE1 (Fig. 4M-O). Notably, animals treated with shSAGE1 showed no significant changes in body weight, appearance, or behavioral patterns, and no signs of organ damage (Fig. 4O), indicating that no off-target effects were detected with shSAGE1 treatment at either the organ or in vivo levels. Taken together, our preclinical data indicates that SAGE1 is critical for the tumorigenic growth of multiple cancer types. Knockdown of SAGE1 slows tumor growth, suggesting that such tumors have become dependent on SAGE1 and that targeting SAGE1 may be a novel therapeutic strategy for SAGE1-positive tumors.

[0501] Example 8. SAGE1 competes with INTS6 for binding to INTS3 in cancer cells

[0502] To gain mechanistic insight into the function of SAGE1 in tumorigenesis, we performed proteomic analysis of endogenous SAGE1 complexes recruited into HuTu80 cells by anti-SAGE1 antibodies. The complexes were resolved by SDS-PAGE, visualized by silver staining, and subjected to MS analysis (Fig. 6A and Fig. 14A-D). We reproducibly identified a number of proteins that have been implicated in transcriptional regulation as reliable SAGE1 interactors, including CBP (CRBP-binding protein), TLE (Groucho / Transducin-Like Enhancer of Split) proteins, INTS3 (a component of the Integrator complex), and the INTS3 binding partners NABP (nucleic acid-binding protein) and INIP (INTS3-NABP interacting protein) (Fig. 6B). Notably, we identified the same group of proteins in proteomic analysis of SAGE1 complexes in other SAGE1-expressing cancer cell lines, strongly suggesting that these proteins are bona fide SAGE1 interactors in cancer cells (Fig. 6B). The interaction of SAGE1 with these proteins was further confirmed by co-immunoprecipitation (co-IP) analysis (FIGS. 6C and 6D).

[0503] INTS3(INTS3 CTD Previous studies have suggested that the C-terminal domain of INTS3 (residues 572-978, SEQ ID NO: 9) mediates a direct interaction with a small domain at the C-terminus of INTS6 (residues 803-887, SEQ ID NO: 10). CTD and INTS6 803-887 We co-purified and found that they formed a stable binary complex that could be analyzed by size exclusion chromatography (Figures 8A and 8B). Interestingly, a small C-terminal region of SAGE1 (residues 818-904, SEQ ID NO: 8) binds to INTS6. 803-887 Since the sequence is very similar to that of SAGE1 (43% identity and 82% similarity) (Figures 6E and 6F), 818-904 INTS6 803-887 It is likely that the three-dimensional structure of INTS3 is similar to that of CTDIndeed, SAGE1 may also mediate interactions with 818-904 has an equilibrium dissociation constant of ~39 nM and INTS3 CTD It has been shown by isothermal calorimetry (ITC) measurements that it can bind to SAGE1 (Figure 6G). 818-904 and INTS6 803-887 SAGE I3BD and INTS6 I3BD (INTS3 binding domain of SAGE1 and INTS6) ( Figure 6E ).

[0504] SAGE I3BD INTS6 I3BD These are called INTS3 because they are very similar to CTD To test this idea, we purified INTS3 CTD -INTS6 I3BD GST-tagged complexes SAGE I3BD This in vitro competitive assay allowed the detection of SAGE I3BD But indeed INTS6 I3BD Competing with INTS3 CTD Higher affinity with We clearly found that INTS3 binds to Integrator at the INTS6-INTS6 domain (Fig. 6H). To further test this in vivo competition, we examined INTS3-interacting proteins in SAGE1-negative 293T cells by IP-MS analysis. Anti-INTS3 antibodies were able to pull down INTS6 and all other components of Integrator, suggesting that INTS3 exists as a subunit of Integrator in 293T cells (Fig. 6I). In contrast, the same approach failed to pull down INTS6 in SAGE1-overexpressing 293T cells (Fig. 6I). Instead, SAGE1 was robustly detected in the pull-down assay (Fig. 6I), indicating that ectopically expressed SAGE1 in 293T cells disrupts the INTS3-INTS6 interaction and displaces INTS3 from Integrator, allowing it to form a new SAGE1-INTS3 complex.

[0505] Example 9. INTS3 CTD -SAGE1 I3BD Crystal structure of the complex

[0506] To understand how SAGE1 competes with INTS6 for interaction with INTS3, we first CTD The crystal structure of INTS3 was determined by single-wavelength anomalous dispersion (SAD) at 2.3 Å resolution (Figure 7A, coordinates deposited in the PDB under the code 7C5T). CTD It consists of 23 closely packed helices and has a long, rod-like conformation (Fig. 7A). CTD INTS3 adopts a dimeric conformation in the crystal, with both hydrophobic and electrostatic interactions at the dimer interface, resulting in a total solvent-exposed surface area of ​​1620 Å (Figures 8C, 8D, and 8E). Calibrated size-exclusion chromatography experiments have revealed that INTS3 CTD The elution peak of INTS3 corresponds to a molecular weight of ~95 kDa (Figure 8A). CTD We also purified the INTS3-INTS6 protein complex (Fig. 8B).

[0507] Next, SAGE1 I3BD -INTS3 CTD The complex was crystallized and its structure was determined by molecular replacement at 2.9 Å resolution (Figures 7B-7D and 12, coordinates deposited in the PDB under code 7C5U). I3BD -INTS3 CTD In the complex structure, SAGE1 I3BD The molecule has a 1:2 stoichiometry of INTS3. CTD The ITC measurements also showed that SAGE1 binds to the dimer (Figure 7B). I3BD -INTS3 CTD The stoichiometry of the interaction between SAGE1 and SAGE2 was found to be 1:2 (Figure 7H). I3BD is a four-helix bundle, INTS3 CTD It is located at the dimer joint (Figure 7B to Figure 7D). SAGE1 I3BDIn INTS3, the two internal loops connecting the helices are linked by both hydrophobic and electrostatic interactions. CTD Mediates contact with the dimer (Figures 7E and 8F). INTS3 CTD The dimer interface is highly acidic and interacts with SAGE1 through both charge and shape complementarity. I3BD This acidic INTS3 interacts with a panel of basic residues (Lys833, Arg836, Arg872, and Lys874) (Figures 8F and 8G). CTD There are two adjacent hydrophobic pockets near the center of the surface. SAGE1 The side chain of Phe873 enters the deeper pocket of the cavity, and the other phenylalanine Phe873 SAGE1 is placed shallower. I3BD is INTS3 CTD does not interfere with the dimeric state of INTS3 CTD By binding to the dimer, Phe805 INTS3 and Tyr808 INTS3 The structure of the aromatic side chain of SAGE1 changes from symmetric to asymmetric. I3BD Two hydrophobic pockets are formed for efficient binding to INTS3 (Figures 7E, 8C, 8E, and 8F). CTD Dimer and SAGE1 I3BD -INTS3 CTD Residues involved in the complex interactions are identified (Figures 13A and 13B).

[0508] INTS6 I3BD and SAGE1 I3BD Because of the high sequence identity, SAGE1 I3BD -INTS3 CTD Based on the complex structure, INTS3 CTD -INTS6 I3BD The interface was modeled. As expected, the proline substitution (Phe838 SAGE1 From Pr o823 INTS6 ) except INTS6 I3BD -INTS3 CTD The interface is SAGE1 I3BD and INTS3 CTDThe interface between INTS6 and the proline substitution is almost the same as that between the proline substitution and the INTS6 protein (Fig. 7F). I3BD and INTS3 CTD This is the reason for the weak interaction with INTS3 (Figure 7F). Thus, these structural data provide strong evidence that SAGE1 expression in cancer cells disrupts the interaction between INTS3 and INTS6, allowing de novo formation of the SAGE1-INTS3 complex.

[0509] Example 10: SAGE1-INTS3 interaction is essential for cancer cell proliferation

[0510] To investigate the in vivo function of the SAGE1-INTS3 interaction, we first isolated a deletion mutant of INTS3 and SAGE1 (SAGE1 ΔI3BD ) and investigated their interaction with INTS3. Co-IP experiments revealed that the I3BD domain of SAGE1 was required for the SAGE1-INTS3 interaction (Figure 7G). Next, we generated mutant SAGE1 (SAGE1mut) in which six key residues in the interface, including Phe838, were replaced with alanine, and confirmed that these mutations completely abolished the SAGE1-INTS3 interaction (Figures 7G and 7H). According to the Co-IP data, the ITC analysis showed that the I3BD domain of SAGE1 was required for the SAGE1-INTS3 interaction (Figure 7G). CTD and mutated SAGE1 I3BD No detectable interaction was observed with INTS3 ( Fig. 7H ), supporting that the observed interface between SAGE1 and INTS3 in the crystal structure is essential for SAGE1-INTS3 interaction.

[0511] To investigate whether the interaction between SAGE1 and INTS3 is important for cancer cell proliferation, wild-type (WT) SAGE1 and mutant SAGE1 were introduced into SAGE1-KO HuTu80 cells and SAGE1-KD Caco2 cells, respectively. Expression of WT SAGE1 restored cell growth in both cell lines, confirming that the suppression observed in SAGE1-KO HuTu80 cells and SAGE1-KD Caco2 cells was specific to SAGE1 (Figures 7I and 7J). In stark contrast, SAGE1mut, although expressed at higher levels than WT SAGE1, did not restore cell proliferation. We next further investigated the effect of the SAGE1-INTS3 interaction in soft agar and xenograft assays using SAGE1-KO HuTu80 cells resurrected with WT or mutant SAGE1. Consistent with the cell growth data, ectopic expression of WT, but not mutant, SAGE1 rescued anchorage-independent growth of SAGE1-KO HuTu80 cells, thereby increasing tumor volume and weight (Fig. 7K and L). Collectively, these mutational analyses demonstrated that SAGE1-INTS3 interaction is essential for cancer cell proliferation and that disrupting this interaction can attenuate the tumorigenic function of SAGE1.

[0512] Example 11: In vivo metastasis analysis by tail vein injection assay

[0513] These experiments demonstrate the importance of SAGE1 for tumorigenesis and metastasis in vivo. Knockdown of SAGE1 slows tumor metastasis, suggesting that such tumors have become dependent on SAGE1 and that targeting SAGE1 may represent a novel therapeutic strategy against SAGE1-positive tumors in vivo.

[0514] In an experimental model of metastasis, shSAGE1 treatment suppressed tumor metastasis. Cells derived from HCC-P, LUAD-P, and LUSC-P PDXs were injected via the tail vein. After 7–10 days, 4 × 10 cells / mouse were injected via tail vein. 11The recipients were treated with AAV control or AAV-SAGE1 (carrying shRNA Y12682 (SEQ ID NO: 128) targeting the sequence shown in SEQ ID NO: 33 GCTGCAGTCACTCACAACA) at 1 genome copy each. After 70–90 days, the animals were evaluated by PET-CT for the pres...

Claims

1. 1. A method for aiding in the detection of SAGE1-positive tumors in a subject having a TP53 mutation, comprising detecting SAGE1-INTS3 complex expression in a biological sample isolated from said subject, said SAGE1-INTS3 complex expression being indicated by detection of a SAGE1-INTS3 complex; The detection of the SAGE1-INTS3 complex is carried out by co-immunoprecipitation as an immunological test, wherein the SAGE1-INTS3 complex is not detected due to mutations in residues F838, F873, K874, M832, R872 and Q840 of SAGE1 (residue numbering is according to SEQ ID NO: 3 of SAGE1) such that INTS3 does not bind to the mutated SAGE1.

2. The biological sample is selected from cells, tissues, body fluids, and combinations thereof. The method of claim 1.

3. The body fluid is selected from blood, serum, plasma, ascites, cyst fluid, pleural fluid, peritoneal fluid, cerebrospinal fluid, tears, urine, saliva, sputum, nipple fluid, lymphatic fluid, fluids of the respiratory, intestinal and genitourinary tracts, breast milk, organ system fluids, conditioned medium obtained from the culture of tissue explants, or combinations thereof. The method of claim 2.

Images